PYRIDO[2,3-d]PYRIMIDIN-4-ONE COMPOUNDS AS TANKYRASE
INHIBITORS
Wnt signaling triggers three intracellular signaling cascades which include the -
catenin-mediated canonical pathway, the non-canonical planar cell polarity and the
Wnt/calcium pathway. The evolutionarily conserved canonical Wnt signaling pathway
regulates many cellular processes including cell proliferation, differentiation, adhesion
and maintenance. The canonical pathway, which regulates -catenin protein levels within
cells, is initiated when Wnt ligands bind to cell surface Frizzled and lipoprotein receptorrelated
protein (LRP)5/6 co-receptors, which in turn promote the displacement of the
kinase GSK3 from the APC/Axin/GSK-3 (adenomatous polyposis coli (APC), Axin,
glycogen synthase kinase 3/(GSK3)) destruction complex. In the presence of Wnt
binding (On-state), Dishevelled (a protein in the Wnt pathway) is activated which, in turn,
recruits GSK3 away from the destruction complex leading to the accumulation of
cytosolic -catenin, translocation of -catenin to the nucleus, interaction with T-cell
factor/lymphoid enhancer factor (TCF/LEF) family transcription factors and transcription
of canonical Wnt pathway responsive genes. In the absence of Wnt ligands (Off-state),
cytosolic -catenin is constitutively phosphorylated and targeted for ubiquitination and
degradation by the proteasome.
The two highly homologous human tankyrase isoforms, tankyrase 1 and 2
(TNKS 1 and TNKS2) are members of the poly(ADP-ribose)polymerase (PARP) enzyme
family that catalyze the post-translational modification of proteins using NAD+ as a
substrate to successively add ADP ribose moieties onto target proteins (parylation or
parsylation). One of the protein substrates for tankyrases is Axin, a concentrationlimiting
component of the - catenin destruction complex; parsylation marks Axin for
degradation and tankyrase inhibition leads to Axin stabilization, Wnt signaling inhibition
and catenin degradation.
Wnt signaling pathway activating mutations are found in a broad range of cancers
and are believed to contribute to tumor initiation, maintenance, and/or progression.
Therefore, inhibition of tankyrase activity appears to be a promising approach in the
treatment of cancers such as colorectal cancer, gastric cancer, liver cancer, breast cancer
(including triple negative breast cancer), ovarian cancer, medulloblastoma, melanoma,
lung cancer (including non-small cell lung cancer), pancreatic cancer, prostate cancer,
glioblastoma, T-cell lymphoma, T-lymphoblastic lymphoma, T-cell acute lymphocytic
leukemia (T-ALL)), mantle cell lymphoma, multiple myeloma, chronic myeloid
leukemia, and acute myeloid leukemia.
Considerable efforts have been made to identify pharmaceutical agents that inhibit
the canonical Wnt p-catenin signaling pathway. TNKS1 and TNKS2 inhibitors such as
WO 2013/117288 are known.
Despite WO 2013/1 17288, there is a need to find compounds having TNKS1 and
TNKS2 inhibitory activity. There is a further need to find compounds having selective
inhibition of TNKS1 and TNKS2 over other PARPs.
Figure 1 is a representative XRPD pattern for 8-Methyl-2-[4-(pyrimidin-2-
ylmethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one 4-
methylbenzenesulfonic acid salt, the compound of Example 6.
One as ect of the present invention are compounds of Formula I :
X is -CH 2-, -C(CH )H-, or-C(CH 2CH )H-;
R is hydrogen, hydroxy, or halo;
R2 is hydrogen, halo, -CN, -CH , CF , or -OCH ;
or a pharmaceutically acceptable salt thereof.
Preferably, a further aspect of the present invention provides compounds of
Formula I wherein:
Y is:
X is -CH 2-, -C(CH3)H-, or-C(CH 2CH )H-;
R is hydrogen, hydroxy, or halo;
R2 is hydrogen, halo, -CN, -CH , CF , or -OCH ;
or a pharmaceutically acceptable salt thereof.
Preferably, another aspect of the present invention provides compounds of
Formula I wherein:
Y is
X is -CH 2-, -C(CH )H-, or-C(CH 2CH )H-;
R2 is hydrogen, halo, -CN, -CH , CF , or -OCH ;
or a pharmaceutically acceptable salt thereof.
Another preferred aspect of the present invention provides compounds of Formula
I wherein:
Y is:
X is -CH 2-, -C(CH )H-, or-C(CH 2CH )H-;
R is hydrogen, hydroxy, or halo;
R2 is hydrogen, halo, -CN, -CH , CF , or -OCH ;
or a pharmaceutically acceptable salt thereof.
A preferred aspect of the present invention is a compound:
8-Methyl-2-[4-(pyrimidin-2-ylmethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof;
8-Methyl-2-[4-(l-pyrimidin-2-ylethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof;
2-[4- [(4-Chloropyrimidin-2-yl)methyl]piperazin- 1-yl] -8-methyl-3 ,5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof; or
2-[4-[(4-methoxypyrimidin-2-yl)methyl]piperazin-l-yl]-8-methyl-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof.
Another preferred aspect of the present invention is a compound:
2-[4-[(3-Bromo-2-pyridyl)methyl]piperazin-l-yl]-8-methyl-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof;
2-[4-[(3-Chloro-2-pyridyl)methyl]piperazin-l-yl]-8-methyl-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof;
2-[4-[(3-Fluoro-2-pyridyl)methyl]piperazin-l-yl]-8-methyl-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof; or
2-[[4-(8-Methyl-4-oxo-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-2-yl)piperazin-lyl]
methyl]pyridine-3-carbonitrile, or a pharmaceutically acceptable salt thereof.
A further aspect of the present invention is a pharmaceutical composition
comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, in
association with a pharmaceutically acceptable carrier.
Still a further aspect of the present invention provides a method of inhibiting
Tankyrase 1 and 2 in a cancer patient in need thereof, comprising administering a
therapeutically effective amount of a compound of Formula I, or a pharmaceutically
acceptable salt thereof, to said patient.
Another aspect of the present invention provides a method of treating a cancer
which is colorectal cancer, gastric cancer, liver cancer, breast cancer, triple negative
breast cancer, ovarian cancer, medulloblastoma, melanoma, lung cancer, non-small cell
lung cancer, pancreatic cancer, prostate cancer, glioblastoma, T-cell lymphoma, Tlymphoblastic
lymphoma, T-cell acute lymphocytic leukemia (T-ALL), mantle cell
lymphoma, multiple myeloma, chronic myeloid leukemia, or acute myeloid leukemia in a
patient comprising administering to a patient in need thereof a therapeutically effective
amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.
A still further aspect of the present invention provides a compound of Formula I,
or a pharmaceutically acceptable salt thereof, for use in therapy.
Another aspect of the present invention provides a compound of Formula I, or a
pharmaceutically acceptable salt thereof, for use in the treatment of a cancer which is
colorectal cancer, gastric cancer, liver cancer, breast cancer, triple negative breast cancer,
ovarian cancer, medulloblastoma, melanoma, lung cancer, non-small cell lung cancer,
pancreatic cancer, prostate cancer, glioblastoma, T-cell lymphoma, T-lymphoblastic
lymphoma, T-cell acute lymphocytic leukemia (T-ALL), mantle cell lymphoma, multiple
myeloma, chronic myeloid leukemia, or acute myeloid leukemia.
A further aspect of the present invention provides use of a compound of Formula
I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for
treatment of a cancer which is colorectal cancer, gastric cancer, liver cancer, breast
cancer, triple negative breast cancer, ovarian cancer, medulloblastoma, melanoma, lung
cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, glioblastoma, Tcell
lymphoma, T-lymphoblastic lymphoma, T-cell acute lymphocytic leukemia (T-ALL),
mantle cell lymphoma, multiple myeloma, chronic myeloid leukemia, or acute myeloid
leukemia.
The term "patient" means mammal and "mammal" includes, but is not limited to,
a human.
The term "triple negative breast cancer" refers to a breast cancer characterized by
a tumor sample having tested negative for estrogen receptors (ER), progesterone receptors
(PR), and hormone epidermal growth factor receptors 2 (HER2/neu).
"Therapeutically effective amount" or "effective amount" means the dosage of a
compound, or pharmaceutically acceptable salt thereof, or pharmaceutical composition
containing a compound, or pharmaceutically acceptable salt thereof, necessary to inhibit
Wnt/ -catenin signaling pathway in a cancer patient, and either destroy the target cancer
cells or slow or arrest the progression of the cancer in a patient. Anticipated dosages of a
compound or a pharmaceutically acceptable salt thereof are in the range of 0.1 to 200
mg/patient/day. Preferred dosages are anticipated to be in the range of 1 to 175
mg/patient/day. Most preferred dosages are anticipated to be in the range of 5 to 150
mg/patient/day. The exact dosage required to treat a patient and the length of treatment
time will be determined by a physician in view of the stage and severity of the disease as
well as the specific needs and response of the individual patient. Although expressed as
dosage on a per day basis, the dosing regimen may be adjusted to provide a more optimal
therapeutic benefit to a patient and to manage and/or ameliorate undesirable
pharmacodynamic effects. In addition to daily dosing, dosing every other day (Q2D);
every other day over a five day period followed by two days without dosing (T.I.W.); or
every third day (Q3D) may be appropriate.
The terms "treatment," "treat," and "treating," are meant to include the full
spectrum of intervention for the cancer from which the patient is suffering, such as
administration of the active compound to alleviate to slow or reverse one or more of the
symptoms and to delay progression of the cancer even if the cancer is not actually
eliminated. The patient to be treated is a mammal, in particular a human being.
A compound of the present invention is preferably formulated as a pharmaceutical
composition using a pharmaceutically acceptable carrier and administered by a variety of
routes. Preferably, such compositions are for oral administration. Such pharmaceutical
compositions and processes for preparing them are well known in the art. See, e.g.,
REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (A. Gennaro, et al. ,
eds., 19th ed., Mack Publishing Co., 1995). In a particular embodiment, the
pharmaceutical composition comprises 8-methyl-2-[4-(pyrimidin-2-ylmethyl)piperazinl-
yl]-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one or a pharmaceutically
acceptable salt thereof, together with a pharmaceutically acceptable carrier and optionally
other therapeutic ingredients particularly for treatment of a specific cancer type.
A compound of the present invention is capable of reaction with a number of
inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Such
pharmaceutically acceptable salts and common methodology for preparing them are well
known in the art. See, e.g., P. Stahl, et al., HANDBOOK OF PHARMACEUTICAL
SALTS: PROPERTIES, SELECTION AND USE, (VCHA/Wiley-VCH, 2002); S.M.
Berge, et al., "Pharmaceutical Salts, " Journal of Pharmaceutical Sciences, Vol. 66, No.
1, January 1977.
A compound of the present invention, such as Example 1, is named: 8-methyl-2-
[4-(pyrimidin-2-ylmethyl)piperazin-l-yl]-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-
4(3H)-one (IUPAC) ; and may also be named: pyrido[2,3-d]pyrimidin-4(3H)-one,
5,6J,8-tetrahydro-8-methyl-2-[4-(2-pyrimidinylmethyl)-l-piperazinyl]- (CAS); and other
names may be used to unambiguously identify a compound of the present invention.
It will be understood compounds of Formula I are depicted as a single
stereoisomer. A particular defined substituent may give rise to a chiral center affording a
racemic mixture or two stereoisomers. As used herein, unless otherwise designated,
references to a specific compound are meant to include individual stereoisomers and
racemic mixtures including the named compound. Specific stereoisomers can be
prepared by stereospecific synthesis using enantiomerically pure or enriched starting
materials. The specific stereoisomers of either starting materials, intermediates, or
racemic mixtures can be resolved by techniques well known in the art, such as those
found in Stereochemistry of Organic Compounds , E. I . Eliel and S. H. Wilen (Wiley
1994) and Enantiomers, Racemates, and Resolutions , J., Jacques, A. Collet, and S. H.
Wilen (Wiley 1991), including chromatography on chiral stationary phases, enzymatic
resolutions, or fractional crystallization or chromatography of diastereomers formed for
that purpose, such as diastereomeric salts. Where a chiral compound is isolated or
resolved into its isomers, but absolute configurations or optical rotations are not
determined, the isomers are arbitrarily designated as isomer 1 and isomer 2 corresponding
to the order each elutes from chiral chromatography and if chiral chromatography is
initiated early in the synthesis, the same designation is applied to subsequent
intermediates and examples.
One of ordinary skill in the art will recognize the compounds of Formula I can
exist in tautomeric equilibrium. For illustrative purposes, the equilibrium is shown
below:
For convenience, the 4-oxo form is depicted in Formula I, and the corresponding
nomenclature is used throughout this specification. However, such depictions include the
corresponding tautomeric hydroxy form.
The compounds employed as initial starting materials in the synthesis of
compounds of the present invention are well known and, to the extent not commercially
available, are readily synthesized using specific references provided, by standard
procedures commonly employed by those of ordinary skill in the art, or are found in
general reference texts.
Examples of known procedures and methods include those described in general
reference texts such as Comprehensive Organic Transformations, VCH Publishers Inc,
1989; Compendium of Organic Synthetic Methods, Volumes 1-10, 1974-2002, Wiley
Interscience; Advanced Organic Chemistry, Reactions Mechanisms, and Structure, 5th
Edition, Michael B. Smith and Jerry March, Wiley Interscience, 2001; Advanced Organic
Chemistry, 4th Edition, Part B, Reactions and Synthesis, Francis A. Carey and Richard J .
Sundberg, Kluwer Academic / Plenum Publishers, 2000, etc., and references cited therein.
Additionally, certain intermediates described in the following schemes may
contain one or more nitrogen protecting groups. The variable protecting group may be
the same or different in each occurrence depending on the particular reaction conditions
and the particular transformations to be performed. The protection and deprotection
conditions are well known to the skilled artisan and are described in the literature (See for
example "Greene's Protective Groups in Organic Synthesis", Fourth Edition, by Peter
G.M. Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2007).
Abbreviations used herein are defined according to Aldrichimica Acta, Vol. 17,
No. 1, 1984. Other abbreviations are defined as follows: "APC" refers to adenomatous
polyposis coli; "BID" refers to twice daily dosing; "biotinylated NAD+" refers to 6-
biotin-17-nicotinamide-adenine-dinucleotide; "BOC" refers to refers to tertbutyloxycarbonyl;
"DCM" refers to dichloromethane; "DMF" refers to
dimethylformamide; "DMAP" refers to 4-dimethylaminopyridine; "DMEM" refers to
Dulbecco's Modified Eagle's Medium; "DMSO" refers to dimethyl sulfoxide; "DPBS"
refers to Dulbecco's Phosphate Buffered Saline; "DTT" refers to dithiothreitol; "EDO"
refers to l-ethyl-3-(3-dimethylaminopropyl)carbodiimide; "EGFP" refers to Enhanced
Green Fluorescent Protein; "EtOAc" refers to ethyl acetate; "FBS" refers to Fetal Bovine
Serum; "Flag tag" refers to Flag peptide DYKDDDDK, N-terminus to C-terminus (SEQ
ID NO: 6); "HEK" refers to human embryonic kidney; "HEPES" refers to 4-(2-
hydroxyethyl)-l-piperazineethanesulfonic acid; "HOAc" refers to acetic acid; "HRP"
refers to horseradish peroxidase; "IPAm" refers to isopropylamine; "MEM" refers to
Minimum Essential Medium; "MeOH" refers to methanol; "MOI" refers to multiplicity
of infection; "NCBI" refers to National Center for Biotechnology Information; "PBS"
refers to Phosphate Buffered Saline; "RPMI" refers to Roswell Park Memorial Institute;
"SCX" refers to a purification column of strong cation exchange phenyl sulfonic acid
bound to silica; "SCX-2" refers to a purification column of strong cation exchange
propylsulfonic acid bound to silica; "SFC" refers to supercritical fluid chromatography;
"TBS" refers to Tris buffered saline; "THF" refers to tetrahydrofuran; "TBME" refers to
r -butyl methyl ether; "TMB peroxidase" refers to 3,3',5,5'-tetramethylbenzidine; Tris"
refers to tris(hydroxymethyl)aminomethane; and "X-Phos" refers to 2-
(dicyclohexylphosphino)-2' ,4' ,6'-triisopropylbiphenyl.
The compounds of the present invention, or salts thereof, may be prepared by a
variety of procedures known in the art, some of which are illustrated in the Schemes,
Preparations, and Examples below. The specific synthetic steps for each of the routes
described may be combined in different ways, or in conjunction with steps from different
schemes, to prepare compounds of Formula I, or pharmaceutically acceptable salts
thereof. The products of each step in the schemes below can be recovered by
conventional methods well known in the art, including extraction, evaporation,
precipitation, chromatography, filtration, trituration, and crystallization. In the schemes
below, all substituents unless otherwise indicated, are as previously defined. The
reagents and starting materials are readily available to one of ordinary skill in the art.
Scheme 1
Scheme 1 depicts the formation of 8-methyl-2-piperazin-l-yl-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one (6) as a TFA salt, the neutral material (7) and the
trifluoroboranate methyl salt of (7).
In Step A, a protected 2-piperazine-l-yl-3H-pyrido[2,3-d]pyrimidin-4-one (3) is
obtained from cyclization of a protected piperazine-l-carboxamide hydrochloride salt (1)
with ethyl 2-chloronicotinate (2). The reaction proceeds in an inert solvent, such as
DMF, in the presence of a strong base, such as potassium ieri-butoxide, at a temperature
of 50 - 120 °C. The preferred protecting group is a ieri-butyloxycarbonyl but other
carbamate protecting groups could be used.
In Step B, a protected 2-piperazine-l-yl-3H-pyrido[2,3-d]pyrimidin-4-one (3) is
methylated using methyl iodide to give the quaternary salt, protected 8-methyl-2-
piperazin-l-yl-3H-pyrido[2,3-d]pyrimidin-8ium-4-one iodide (4) (Z is I, Scheme 1). The
reaction proceeds in an autoclave in an inert solvent, such as THF or dioxane, at a
temperature of 50 - 120 °C for 4 to 24 h. Alternatively the methyl sulfate salt can be
formed by adding dimethylsulfate to compound (3) in a polar aprotic solvent such as
DMF with heating at about 60-80 °C. The solution is transferred at room temperature to a
vessel containing TMBE to precipitate the product (4) for Z is CH3OSO 3.
In Scheme 1, Step C, the quaternary salt (4, Z is I) is reduced to provide protected
8-methyl-2-piperzin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one (5). The
reduction can be accomplished using a reducing agent, such as sodium cyanoborohydride
in a mixture of DMF/TFA of about 10/1. The TFA and reducing agent are added at a
temperature of 0 - 5 °C with the temperature not allowed to rise above 15 °C. The
reaction is then allowed to go about 12 to 24 h at RT. Additional amounts of TFA and
reducing agent can be added with cooling to drive the reaction to completion.
Alternatively, Step C can be accomplished by reducing the methyl sulfate salt (4,
Z is CH3OSO 3) with a reducing agent such as platinum oxide and hydrogenating at about
155 psi in a polar solvent such as MeOH to give compound (5).
In Step D, the piperazinyl ring is deprotected to provide 8-methyl-2-piperazin-lyl-
3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one (6). Acidic conditions for removal of
protecting groups such as BOC are well known in the art. Preferred conditions use a 1/1
mixture of DCM and TFA at RT for a period of 2 to 24 h to give the product as a TFA
salt with 2-4 equivalents of TFA present. The salt form is not characterized but is
calculated by weight.
Step E shows the deprotection of the protecting group as discussed above to give
the TFA salt which can be neutralized with an ion exchange resin or an SCX column
using ammonia/MeOH to give the neutral material (7). The nitrogen of the pyrazine ring
can be converted to the methyl trifluoroboranate salt using potassium bromomethyl
trifluoroborate in a solvent such as THF to give a salt form as shown in Step F, compound
8.
Scheme 2
H
I
Scheme 2 illustrates the formation of compounds of Formula I from the salt form
(6), the neutral material (7), or the methyl trifluoro boranate salt (8).
There are many ways to form compounds of Formula I from the intermediates (6), (7),
and (8) such as alkylation, reductive alkylation, or Suzuki couplings depending on the
availability or synthesis of appropriate aldehydes or ketones for reductive alkylations,
appropriate halide reagents for alkylations or boronic acid reagents for Suzuki couplings.
The TFA amine salt (6) or neutral amine (7) can be used in reductive alkylations or
alkylation to give compounds of Formula I. Reductive alkylations are well known in the
art and involve the reaction of an aldehyde with an amine using a reducing agent such as
sodium cyanoborohydride in an appropriate solvent such as DMF at room temperature or
other reducing agents such as sodium triacetoxyborohydride in an appropriate solvent
such as DCM at room temperature. A catalytic amount of methanol can be used if
desired with DMF and sodium cyanoborohydride to push the reaction forward faster. An
appropriate aryl methyl halide can be used to alkylate the TFA amine salt (6) or neutral
material (7) using an appropriate organic base such as triethylamine or an inorganic base
such as potassium carbonate in a solvent such as acetonitrile or DCM. Sodium iodide can
be used in situ to convert a chloromethyl substrate to a more reactive iodo methyl
substrate to complete the alkylation of the appropriate pyrazine, pyridine, or pyrimidine.
The reactions can be stirred at room temperature for 1-3 days to give compounds of
Formula I .
Compound (8) can be coupled with aryl halides under Suzuki palladium catalyzed
cross coupling conditions to form N-methyl heteroaryl substituted products. Compound 8
the methyl trifluoroboranate salt, provides another variation to prepare compounds of
Formula I . The skilled artisan will recognize that there are a variety of conditions useful
for facilitating such cross-coupling reactions. Accordingly, a suitable palladium reagent
includes palladium(II) acetate and a suitable organophosphorus reagent such as X-phos
with a base such as cesium carbonate, sodium carbonate, potassium carbonate, or
potassium phosphate tribasic monohydrate. Other suitable organophosphorus and
palladium reagents include bis(triphenylphosphine)palladium(II) chloride,
tris(dibenzylideneacetone)dipalladium (0) with tricyclohexylphosphine, (1,1 ' -
bis(diphenylphosphino)ferrocene)palladium(II) chloride, or palladium
tetrakistriphenylphosphine.
In an optional step, a pharmaceutically acceptable salt of a compound of Formula
I, such as a hydrochloride salt, can be formed by reaction of a free base compound of
Formula I with an appropriate acid in a suitable solvent under standard conditions.
Additionally, the formation of such salts can occur simultaneously upon deprotection of a
nitrogen protecting group.
The following preparations and examples further illustrate the invention. Unless
noted to the contrary, the compounds illustrated herein are named and numbered using
Accelrys® Draw version 4.0 (Accelrys, Inc., San Diego, CA), IUPACNAME
ACDLABS, or ChemDraw® Ultra 12.0.
General Method Description of Reverse Phase Purification
System: Agilent 1200 LCM/MS equipped with Mass Selective Detector (MSD)
mass spectrometer and Leap autosampler/fraction collector; Column: 75 x 30 mm
Phenomenex Gemini-NX, 5particle size column with 10 x 20 mm guard; Solvent
System: A: 10 mM ammonium bicarbonate, pH 10 as the aqueous phase, B: ACN as the
organic phase; Flow rate: 85 mL/min; Method: The gradient for each method is
designated in the method name as the beginning %B-ending B (e.g. as in High pH 13-
48). The gradient is set as follows: 0-1 min (hold at begin %B), 1-8 min (gradient from
begin %B to end %B), 8-8.1 min (ramp from end %B to 100%B), 8.1-9 min (hold 100%
B).
Preparation 1
4-Methoxypyrimidine-2-carbaldehyde
Combine 4-methoxypyrimidine-2-methanol (300 mg, 2.14 mmol) and oxalyl
chloride ( 1 mL, 2.0 M in DCM) in DCM (5 mL) and cool to -78 °C. Add DMSO (0.33
mL, 4.71 mmol) and stir the mixture for 2 h. Add triethylamine (0.66 mL, 4.71 mmol)
and warm the mixture to room temperature overnight. Add water (5 mL), stir 1 h and
extract with DCM (2 x 50 mL). Dry the combined organic extracts over sodium sulfate,
filter, and concentrate the filtrate under reduced pressure to give a light brown solid and
used without further purification. H NMR(400 MHz, CDC1 ) aldehyde peak 9.94 (s,
1H), remaining peaks not assignable due to the mixture of more than one compound.
Preparation 2
4-Methylpyrimidine-2-carbaldehyde
Prepare 4-methylpyrimidine-2-carboxaldehyde essentially as described in
Preparation 1 using (4-methylpyrimidin-2-yl)methanol to obtain the title compound and
use without further purification. H NMR, 400 MHz, CDC1 ) 10.06 (s, 1H), 8.79 (d,
1H, J = 5.2 Hz), 7.33 (d, 1H, J = 4.8 Hz), 2.65 (s, 3H)
Preparation 3
4-(Trifluoromethyl)-2-vinyl-pyrimidine
Combine 2-chloro-4-(trifluoromethyl)pyrimidine (250 mg, 1.37 mmol), potassium
vinyltrifluoroborate (184 mg, 1.37 mmol), cesium carbonate (1.34 g, 4.11 mmol), THF (5
mL) and water (0.5 mL) in a vial. Degas with a stream of nitrogen for 1 min. Add
bis(triphenylphosphine)palladium(II) chloride (48 mg, 0.069 mmol), seal the vial, and
heat the mixture at 85 °C for 3 days. Cool, dilute the mixture with DCM, filter through
diatomaceous earth, and concentrate the filtrate under reduced pressure to give the title
compound (0.200 g, 84%) which is used without further purification. H NMR (400
MHz, CDC1 ) 8.93 (d, 1H, = 5.0 Hz), 7.43 (d, 1H, = 5.0 Hz), 6.94 (ABX, 1H, JAX =
17.3, JBX = 10.47), 6.74 (ABX, 1H, JAB = 1-54, AX = 17.3), 5.84 (ABX, 1H, JAB = 1-54,
JBX = 10.47).
Preparation 4
5-Methyl-2-vinyl-pyrimidine
Prepare the title compound essentially as described in Preparation 3 using 2-
choro-5-methylpyrimidine and use without further purification (220 mg, 94%). NMR
(400 MHz, CDCI 3) 8.38 (s, 2H), 6.73 (ABX, 1H, JAX = 17.4, JBX = 10.7), 6.41 (ABX,
1H, JAB = 1.8, JAX = 17.4), 5.54 (ABX, 1H, JAB = 1.8, JBx = 10.7), 2.16 (s, 3H).
Preparation 5
4-(Trifluoromethyl)pyrimidine-2-carbaldehyde
Combine 4-(trifluoromethyl)-2-vinyl-pyrimidine (200 mg, 0.84 mol) with sodium
periodate (737 mg, 3.45 mmol) and osmium tetroxide, polymer bound (292 mg, 0.057
mmol) in dioxane (3 mL) and water(l mL) and stir mixture at room temperature
overnight. Dilute the mixture with EtOAc (5 mL) and water (5 mL). Filter the mixture
through glass wool and separate the layers. Dry the organic layer over magnesium
sulfate, filter, and concentrate the filtrate to give the title compound (40 mg, 20%). Use
the crude material without further purification. H NMR (400 MHz, CDC1 ) aldehyde
10.16 (s, 1H), remaining peaks not assignable due to the mixture of more than one
compound.
Preparation 6
5-Methylpyrimidine-2-carbaldehyde
Prepare 5-methylpyrimidine-2-carbaldehyde essentially as described in
Preparation 5, using 2-chloro-5-methyl-pyrimidine to give the title compound (90 mg,
44%) and use without further purification. H NMR (400 MHz, CDC1 ) 10.06 (s, 1H),
8.79 (s, 2H), 2.42 (s, 3H).
Preparation 7
2-(Bromomethyl)-4-chloro-pyrimidine
r
Combine 2-methyl-4-chloropyrimidine (200 mg, 1.55 mmol), carbon tetrachloride
(5 mL), N-bromosuccinimide (304 mg, 1.71 mmol), and benzoyl peroxide (38 mg, 1.6
mmol) in a vial, flush with nitrogen for 2 min, seal and heat at 80 °C overnight. Use the
crude reaction mixture without further purification. GC-MS m/ e ( BR/ Br 205/207
(M+) .
Preparation 8
2-(Chloromethyl)pyrazine
I
Dissolve 2-pyrazinylmethanol (2.0 g, 18.1 mmol) in DCM (200 mL) and cool to 0
°C while stirring under nitrogen. Add thionyl chloride (4.63 mL, 63.6 mmol) drop wise
over 10 min, and allow to warm to 25 °C. Stir at room temperature for 16 h. Concentrate
the mixture and then dilute with DCM. Wash the crude solution with saturated NaHCC>3,
dry over MgSC filter, and concentrate. Dissolve the crude residue in DCM and purify
by silica gel flash chromatography (hexane/EtOAc, 95:5 to 100% EtOAc gradient) to give
the title compound as a pale yellow oil. H NMR (400 MHz, CDC1 ) 4.67 (s = 2H),
8.50-8.55 (m, 2H), 8.73 (s, 1H).
Preparation 9
2-(Bromomethyl)-3-chloro-pyrazine
Add together 2-chloro-3-methyl-pyrazine (5.0 g, 38.9 mmo
bromosuccinimide (6.92 g, 38.9 mmol), benzoyl peroxide (0.471 g, 1.94 mmol), and
carbon tetrachloride (50 mL) and heat at reflux under nitrogen for 16 h. Cool the reaction
mixture to 25 °C and dilute with hexanes. Filter the mixture, concentrate the liquid under
reduced pressure, and purify by silica gel flash chromatography (hexane/EtOAc, 95:5
with gradient to 40:60) to give the title compound as a brown oil (3.93 g, 49%). H NMR
(400 MHz, CDC1 ) 4.66 (s = 2H), 8.32 (d, J = 2.4 Hz, 1H), 8.47 (d, J = 2.4 Hz, 1H).
Preparation 10
Methyl 3,5-difluoropyridine-2-c arboxylate
Add together 3,5-difluoropyridine-2-carboxylic acid (1.4 g, 8.8 mmol) and DCM
(30 mL) and cool to 0 °C. Add MeOH (3 mL), DMAP, (1.32 g, 10.6 mmol) and EDCI
(2.1 g, 10.6 mmol). Allow mixture to warm to room temperature and stir overnight.
Concentrate the reaction mixture under reduced pressure and purify the residue by
chromatography on silica gel (elution with 10/1 petroleum ether/EtOAc) to give the title
compound (1.03 g, 72%). LC-ES/MS m/z 174 (M+H)+.
Preparation 11
(3,5-Difluoro-2-pyridyl)methanol
Add together methyl 3,5-difluoropyridine-2-carboxylate (1.0 g, 4.6 mmol), THF
(10 mL) and 2 M lithium borohydride in THF (15 mL, 23 mmoL). Stir the mixture at
room temperature for 2 days. Concentrate under reduced pressure and purify the residue
by silica gel chromatography eluting with DCM to give the title compound (0.448 g,
60%).
Preparation 12
2-(Chloromethyl)- 3,5-difluoropyridine
Add together (3,5-difluoro-2-pyridyl)methanol (200 mg, 1.4 mmol), DCM (4 mL),
DMF (0.1 mL) and thionyl chloride ( 1 mL) and stir the mixture for 4 hours. Adjust the
pH to about 7.0 with 4 M aqueous sodium bicarbonate and extract the mixture with DCM
(3 x 50 mL). Combine the organic portions, dry over anhydrous sodium sulfate, filter,
and concentrate to give the title compound (182 mg, 81%) as an orange oil.
Preparation 13
r -Butyl 4-carbamimidoylpiperazine-l-carboxylate hydrochloride
Charge a 10 L jacketed reactor with r -butyl piperazine-l-carboxylate (1500 g,
8.054 mol) and DMF (4.05 L). Stir until a solution is obtained. Add lH-pyrazole-1-
carboxamide HCl (1180 g, 8.054 mol) followed by diisopropylamine (1041 g, 8.054 mol)
over 15 min at 22 to 28 °C. Heat at 55 to 60 °C for 5 h and then cool to 20 °C. Transfer
the reaction mixture to a 50 L jacketed reactor containing TBME (30 L) over 15 to 20
minutes and rinse the transfer lines with DMF (400 mL). Stir the suspension for 1 hour at
20 °C and then filter through three separate 26 cm Buchner funnels. Wash each product
cake with TMBE (2 x 1000 mL) and dry in a vacuum oven at 60 °C overnight to give the
title compound (1898 g, 89%) as a white solid. H NMR (400 MHz, DMSO-d 6) 1.41 (s,
9H), 3.42-3.50 (m, 4H), 3.33-3.42 (m, 4H), 7.77 (s, 4H).
Preparation 14
r -Butyl 4-(4-oxo-3H-pyrido[2,3-d]pyrimidin-2-yl)piperazine-l-carboxylate
Charge a 22 L round bottom flask with r -butyl 4-carbamimidoylpiperazine-lcarboxylate
hydrochloride (1897 g, 7.165 mol), DMF (10.1 L), and ethyl 2-
chloronicotinate (1266 g, 6.824 mol). Add potassium ieri-butoxide (1293 g, 11.52 mol)
portion- wise over 55 min while allowing the temperature to rise gradually from 17 to 62
°C. Heat the reaction at 100 °C for 2.5 h. -Cool the reaction mixture to 20 °C and transfer
to a 30 L jacketed reactor containing water (15.2 L) using DMF (350 mL) to rinse the
reactor and transfer lines. Introduce 3 N hydrochloric acid (1.52 L) until a pH of 5 to 6 is
obtained and stir the suspension for 2 hours at 20 °C. Collect the product by filtration
through three separate 26 cm Buchner funnels and wash each cake with water (3 x 1.5 L).
Combine the cakes, suspend them in water (15.0 L), and stir for 60 min at 20 °C. Collect
the product by filtration through two separate 26 cm Buchner funnel and wash the cake
with water (2 x 1.5 L). Dry the solid in a vacuum oven at 60 °C for 24 hours to give the
title compound (1332 g, 59%) as a yellow solid. H NMR (400 MHz, DMSO-d 6) 1.43
(s, 9H), 3.36-3.49 (m, 4H), 3.62-3.76 (m, 4H), 7.13-7.20 (m, 1H), 8.22-8.27 (m, 1H),
8.62-8.70 (m, 1H).
Preparation 15
r -Butyl 4-(8-methyl-4-oxo-3H-pyrido[2,3-d]pyrimidin-8-ium-2-yl)piperazine-lcarboxylate,
methyl sulfate
Charge a 20 L jacketed reactor with r -butyl 4-(4-oxo-3H-pyrido[2,3-
d]pyrimidin-2-yl)piperazine-l-carboxylate (1331 g, 4.017 mol) and DMF (6.66 L). Add
dimethylsulfate (583 g, 2.83 mol) over 5-10 min and observe a slight exotherm to 38 °C.
Heat the solution to 63 to 73 °C for 30 minutesv Then introduce additional
dimethylsulfate (50.7 g, 0.402 mol) and heat at 68 to 73 °C for 30 min. Sample of the
reaction shows 2.0% starting material remaining. Add additional dimethylsulfate (50.7 g,
0.402 mol) and heat at 68 to 73 °C. Sample again to show 1.3% staring material
remaining. Cool the reaction to 20 °C and transfer over 20 to 30 minutes to a 50 L jacket
reactor containing TBME (26.6 L), r -butyl 4-(8-methyl-4-oxo-3H-pyrido[2,3-
d]pyrimidin-8-ium-2-yl)piperazine-l-carboxylate and seeds ( 1 g) using DMF (300 mL) to
rinse the reactor and transfer lines. Stir the suspension overnight at 20 °C overnight and
collect the product by filtration using three separate 26 cm Buchner funnels. Rinse each
cake with TBME (3 x 1.3 L), Combine the three portions and suspend the material in
TMBE (13.3 L), stir for 1 h at 20 °C, and collect the product on two separate 26 cm
Buchner funnel. Wash each cake with TBME (3 x 2.0 L) and dry in a vacuum oven at 55
to 60 °C for 16 hours to give the title compound (1812 g, 98%) as a yellow solid.
NMR (400 MHz, DMSO-d6) 1.44 (s, 9H), 3.37 (s, 3H), 3.45-3.55 (m, 4H), 3.80-3.95
(m, 4H), 4.08 (s, 3H), 7.40-7.45 (m, 1H), 8.72-8.76 (m, 1H), 8.86-8.91 (m, 1H)
Preparation 15 Seed Crystal Formation
Charge a 250 mL reactor with r -butyl 4-(4-oxo-3H-pyrido[2,3-d]pyrimidin-2-
yl)piperazine-l-carboxylate (15 g, 45.3 mmol) and dimethylformamide (75 mL). Add
dimethylsulfate (6.28 g, 49.8 mmol) in one portion to the stirring mixture (observe slight
exotherm to 34 °C). Heat the resulting solution at 68 to 73 °C for 3 hours. Add
dimethylsulfate (0.57 g, 4.5 mmol) and stir at 68 to 73 °C for an additional 1 hour. Cool
to 20 °C and transfer drop wise to a 1 L flask containing TBME (300 mL). Observe the
sticky solid change to stirrable suspension, stir overnight at 20 °C and filter on a Buchner
funnel. Wash the product cake with TBME (3 x 35 mL) and suspend in TBME (100 mL).
Stir the suspension for 1 hour at 20 °C, filter on a Buchner funnel and wash the cake with
TBME (2 x 35 mL). Dry the solids in a vacuum oven at 55 to 60 °C for 16 hours to give
r -Butyl 4-(8-methyl-4-oxo-3H-pyrido[2,3-d]pyrimidin-8-ium-2-yl)piperazine-lcarboxylate,
methyl sulfate (18.84 g, 91% yield) (HPLC purity 95%).
Preparation 16
r -Butyl 4-(8-methyl-4-oxo-3H-pyrido[2,3-d]pyrimidin-8-ium-2-yl)piperazine-lcarboxylate
iodide
Combine r -butyl 4-(4-oxo-3H-pyrido[2,3-d]pyrimidin-2-yl)piperazine-lcarboxylate
( 115.43 g), THF (1.4 L), and methyl iodide (24 mL) in a 2 L Parr autoclave
with mechanical stirrer. Seal the autoclave and stir the reaction with heating at 70 °C for
22 hours. Cool the reaction to room temperature and transfer the resulting bright yellow
slurry to a round bottom flask using MeOH. Concentrate the slurry and dry the resulting
solid in a vacuum oven at 50-60 °C to give the title compound as a yellow solid (167.44
g, 100%). LC-ES/MS m/z 346.2 [M+H]+, TR = 1.16 min.
Preparation 17
r -Butyl 4-(8-methyl-4-oxo-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-2-yl)piperazine-lcarboxylate
Charge a 2 gallon pressure vessel with r -butyl 4-(8-methyl-4-oxopyrido[
2,3-d]pyrimidin-8-ium-2-yl)piperazine-l-carboxylate, methyl sulfate (881 g, 1.927
mol) and MeOH (4.9 L). Add platinum oxide (1% w/w, 8.81 g) and pressurize with
hydrogen to 155 psi. Stir for 4 hours at 22-28 °C. Filter through a filter cartridge to
collect the catalyst. Set aside the filtrate to be combined with the filtrate of a second
hydrogenation. Repeat the reaction on another portion of r -butyl 4-(8-methyl-4-oxo-
3H-pyrido[2,3-d]pyrimidin-8-ium-2-yl)piperazine-l-carboxylate, methyl sulfate (925 g,
2.023 mol), MeOH (5.0 L) and platinum oxide (1% w/w, 9.25g) as before filter and
combine the two filtrates and concentrate to an oily residue. Dissolve the residue in DCM
(9.3 L) and wash with 0.1 N aqueous HOAc (2 x 2.0 L and 2 x 1.1L) and 0.5 M NaOH
(8.0 L). Add water (4.0 L) and adjust to pH 6-7 with 0.1 N aqueous HOAc (about 1.9 L).
Separate the layers and dry the organic layer over magnesium sulfate, filter, and
concentrate to a volume of about 4.5 L and add EtOAc (16.0 L) to crystallize the product.
Concentrate the suspension to a volume of about 4.5 L, cool to 0 to 5 °C and collect the
product by filtration. Wash the cake with EtOAc (2 x 2.0 L and 2 x 1.1 L) and dry in a
vacuum oven at 55 to 65 °C for 16 h to give the title compound ( 1185 g, 77%) as a white
solid (very electrostatic). H NMR (400 MHz, CDC1 ) 1.45 (s, 9H), 1.76-1.88 (m, 2H),
2.53 (t, J = 6.3 Hz, 2h), 3.04 (s, 3H), 3.20-3.26 (m, 2H), 3.40-3.52 (m, 4 H), 3.60-3.70 (m,
4H), 5.01 (s, 1H), 12.1-12.3 (br s, 1H). C NMR (101 MHz, CDC1 ) 20.00, 21.63,
28.81, 36.55, 44.99, 50.20, 80.38, 85.85, 152.38, 155.15, 160.14, 164.03. ESI/ MS m/z
350.0 [M+H]+.
Preparation 17 Alternative Synthesis
Charge a round bottom flask with r -butyl 4-(8-methyl-4-oxo-3H-pyrido[2,3-
d]pyrimidin-8-ium-2-yl)piperazine-l-carboxylate iodide (167.44 g, 353.76 mmol) and
DMF (815 mL) and cool in an ice bath to an internal temperature of 0-5 °C. Add
trifluoroacetic acid (80.25 mL, 1.06 mol) at a rate to maintain the internal temperature
below 15 °C (45 min). Cool the reaction to an internal temperature of 0-5 °C and add
sodium cyanoborohydride (66.69 g, 1.06 mol) at a rate to maintain the temperature below
15 °C (about 50 min). Stir the reaction for 18 h while warming to 25 °C. Cool the
reaction back down to 0-5 °C and add trifluoroacetic acid (80.25 mL, 1.06 mol) at a rate
to maintain the temperature below 15 °C (10 min) followed by sodium cyanoborohydride
(66.69 g, 1.06 mol) at a rate to maintain the temperature below 15 °C (10 min). Stir the
reaction overnight while warming to 25 °C. Cool the reaction back down to 0-5 °C and
add trifluoroacetic acid (26.6 mL, 0.345 mol) at a rate to maintain the temperature below
10 °C (5 min) followed by sodium cyanoborohydride (22.3 g, 0.345 mol) in two portions
over 10 min. Stir overnight while warming to 25 °C. Fit a 12 L bucket with an overhead
stirrer and charge it with sodium bicarbonate (297.18 g, 3.54 mol) and water (7.2 L). Add
the crude reaction mixture to the bicarbonate solution using a separatory funnel over a
period of 30 min and observe gas evolution. Stir the mixture for 2 hours and then let sit at
25 °C overnight. Collect the resulting solid by filtration and rinse with water and diethyl
ether. Dry the solid in a vacuum oven at about 60 °C overnight to give the title
compound ( 111.34 g, 90%) as a beige solid. LC-ES/MS m/z 350.0 [M+H]+, TR = 1.93
min.
Preparation 18
8-Methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one bis-(2,2,2-
trifluoroacetic acid) salt
Add together r -butyl 4-(8-methyl-4-oxo-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-2-yl)piperazine-l-carboxylate (46.8 g, 133.9 mmol) and DCM (100 mL).
Add trifluoroacetic acid (80 mL, 1.09 moles) over 15 min and stir at 25 °C overnight.
Concentrate under reduced pressure, add DCM (200 mL), and re-concentrate under
reduced pressure four times to give the title compound as an off white solid (69.685 g,
100% crude). LC-ES/MS m/z 250.0 [M+H]+, TR = 0.53 min.
Preparation 19
8-Methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one; tri-2,2,2-
trifluoroacetic acid
Add together r -butyl 4-(8-methyl-4-oxo-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-2-yl)piperazine-l-carboxylate (196.0 g, 560.8 mmol) and DCM (783 mL).
Cool the cloudy suspension in an ice bath to an internal temperature of 4 °C and add
trifluoroacetic acid (254 mL) over 10 min while stirring. Stir for 42 hours at room
temperature. Remove the volatiles under reduced pressure and dissolve in DCM.
Remove the volatiles under reduced pressure and dry under vacuum at 40 °C overnight to
give the title compound (431.5 g, 99%) as a white solid. H NMR (300 MHz, DMSO-d6)
8.96 (br s, 2H), 7.49 (br s, TFA/water, 9H), 3.78 (t, 4H, J = 5 Hz), 3.29 (t, 2H, J = 5
Hz), 3.15 (br s, 4H), 3.04 (s, 3H), 2.37 (t, 2H , J = 6.3 Hz), 1.76 (pt, 2H, J = 5.8 Hz
Preparation 20
8-Methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one; tetra-2,2,2-
trifluoroacetic acid
Add together r -butyl 4-(8-methyl-4-oxo-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-2-yl)piperazine-l-carboxylate (0.29 g, 0.83 mmol), DCM (3 mL) and
trifluoroacetic acid (3 mL). Stir the reaction at room temperature for 4 hrs. Concentrate
the mixture and dissolve the residue in DCM (2x) and concentrate the mixture. Dry
under vacuum for 1 hour to give the title compound (0.556 g, 95%) which is used without
purification or characterization.
Preparation 21
8-Methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one
Charge a round bottom flask with r -butyl 4-(8-methyl-4-oxo-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-2-yl)piperazine-l-carboxylate (730 mg, 1.53 mmol),
DCM (15 mL) and trifluoroacetic acid (15 mL) and stir the reaction at room temperature
for 3 hours. Concentrate under reduced pressure, dissolve the residue in methanol, add
ion-exchange resin Dowex® 50WX4-400 (5.5 g), and stir the mixture at room
temperature overnight. Filter the mixture and wash the ion exchange resin with 7 M
ammonia in methanol. Combine the filtrate and washings, and concentrate under reduce
pressure to give the title compound as an orange solid (385 mg, 95%). LC-ES/MS m/z
250 [M+H]+.
Alternate Preparation 21
Prewash a 10 g SCX-2 column with DCM (30 mL) and load with a solution of 8-
methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one bis-(2,2,2-
trifluoroacetic acid) (0.5 g, 1.05 mmol) in DCM (10 mL) and MeOH ( 1 mL). Wash the
column with DCM (30 mL), followed by MeOH (30 mL), and elute with 2 M
NH3/MeOH (60 mL). Concentrate the fractions containing the product under reduced
pressure to give the title compound (0.26 g, 100%).
Preparation 22
Potassium trifluoro-[[4-(8-methyl-4-oxo-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-2-yl)
piperazin- 1-yl]methyl]boranuide
Dissolve 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-
one (250 mg, 1.00 mmol) in THF (4 mL) and add potassium bromomethyl trifluoroborate
(208 mg, 1.00 mmol). Heat at 80 °C for 90 min and then concentrate under a stream of
nitrogen. Add acetone (30 mL) and potassium bicarbonate (100 mg, 1.00 mmol) and stir
at room temperature overnight. Remove the solids by filtration and concentrate the
filtrate under reduced pressure to give the title compound (273 mg, 73%) as an off-white
solid. ES/MS m/z 330 [M-K+]
Example 1
8-Methyl-2-[4-(pyrimidin-2-ylm
d]pyrimidin-4-one
Dissolve 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-
one; tri-2,2,2-trifluoroacetic acid (409 g, 531 mmol) in DCM (2.0 L) in a 5 L extraction
funnel. Purge the funnel with nitrogen and equip it with an overhead mechanical stirrer.
Add pyrimidine-2-carboxaldehyde (66.6 g, 616 mmol) and stir for 40 min. Then add
sodium triacetoxyborohydride (225.1 g, 1060 mmol) portion wise and observe an
exotherm from 20 to 36 °C immediately after the addition. Stir the reaction at room
temperature overnight. Pour the reaction carefully into 1M NaOH (2.32 L) at 20 °C in a
10 L reactor while controlling the resulting exotherm with a chiller. Adjust the pH to 8 to
9 with 1 N NaOH (2.55 L). Collect the organic layer and extract the aqueous layer with
DCM (2.32 L). Combine the organic layers, concentrate and dry under a stream of
nitrogen overnight to give the title compound (193 g, 100% crude product). NMR
(300 MHz, DMSO-d6) (br s, 1H), 8.78 (d, 2H, J = 5.5 Hz), 7.41 (t, 1H, J = 4.9 Hz), 3.76
(s, 2H), 3.52 (br s, 4H), 3.187 (t, 2H , J = 5.2 Hz), 2.96 (s, 3H), 2.54 (m, 2H), 2.28 (t, 2H,
J = 6Hz), 1.72(pt, 2H, J = 5.2 Hz). This lot is combined with 4 other lots for purification
(203 g). Purify by silica filtration (20 cm diameter by 8 cm high) and elute with 90%
DCM / 10% 2 M NH in MeOH to give the title compound (162 g, 84%). 'H-NMR (300
MHz, CDC1 ) : 8.74 (d, = 5.1 Hz, 2H), 7.20 (t, = 5.1 Hz, 1H), 3.85 (s, 2H), 3.78-3.69
(m, 4H), 3.27-3.19 (m, 2H), 3.03 (2, 3H), 2.68-2.59 (m, 4H), 2.47-2.38 (m, 2H), 1.88-
1.76 (m, 2H).
Example 1 Alternate Synthesis
Combine 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-
one tri-2,2,2-trifluoroacetic acid (203.23 g, 344 mmol), pyrimidine-2-carbaldehyde (40 g,
370 mmol) and DMF (343 mL) and stir until a homogeneous solution is obtained (30
min). Cool the solution in an ice bath and add sodium cyanoborohydride (25 g, 397
mmol) in a 5 g portion followed by a 10 g portion 5 minutes later. Stir for 1.5 hr and add
the final 10 g of cyanoborohydride. Stir 30 minutes while maintaining the temperature
below 25 °C. Stir the reaction overnight and allow to warm 25 °C. Pour the reaction
mixture into a beaker (4L) fitted with an overhead stirrer containing sodium bicarbonate
(115 g, 1.37 mol) and water (350 mL). Stir the mixture for 20 min and then add EtOAc
(1L) and stir for 30 min. Pour the mixture through diatomaceous earth (700 g) over about
30 min and gravity filter for about 1 hour. Then apply vacuum and rinse the
diatomaceous earth with EtOAc (2 x 1 L). Combine and concentrate the organic layer to
give a viscous yellow oil with a crude weight of 117 g. Purify the oil by silica gel flash
chromatography (DCM to 90% DCM/MeOH, gradient). Dry the resulting material in a
vacuum oven at 50 °C to give the title compound as a white foamy solid (44.22 g, 38%).
LC-ES/MS m/z 342.3 [M+H] +, TR = 0.96 min.
Example 2
2-[4-[(3-Chloro-2-pyridyl)methyl]piperazin-l-yl]-8-methyl-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one
Add together 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-
4-one; tetra 2,2,2-trifluoroacetic acid (0.556 g, 0.788 mmol), 3-chloropicolinaldehyde
(557.87 mg, 3.94 mmol) in DMF (3 mL) followed by sodium cyanoborohydride (148.6
mg, 2.36 mmol). The reaction is stirred at room temperature for 5 days. Dilute the
reaction with CHCI 3 (75 mL) and wash with saturated NaHCC>3 and brine. Dry over
Na2S0 4, filter, and concentrate to dryness. The crude product is purified by silica gel
chromatography eluting with DCM to 90% DCM/MeOH to give the title product (214
mg, 72%). LC ES/MS m/z 375.3 ( 5C1) (M+H) +.
The following Examples 3, 4 and 5 are prepared essentially following the
procedure described in Example 2, using the appropriate aldehyde or ketone, stirring at
room temperature for 2 hrs up to 48 hrs and monitoring reaction for completion, adding
more sodium cyanoborohydride if needed and stirring for a further 24 hours.
Table 1
Dichlormethane is solvent and sodium triacetoxyborohydride is used instead of sodium
borohydride.
MeOH is solvent.
Example 6
8-Methyl-2-[4-(pyri midin-2-ylmethyl)piperazm
d]pyrimidin-4-one 4-methylbenzenesulfonic acid salt
Add 8-methyl-2-[4-(pyrimidin-2-ylmethyl)piperazin-l-yl]-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one (274.5 mg, 0.804 mmol) to ethanol (0.5 mL) and
add toluenesulfonic acid monohydrate (160 mg). Sonicate to give a dark amber red
solution. Add heptane (5 mL), cap the mixture, stir, and heat the mixture to 80 °C. The
mixture solidifies to a tan-brown solid. Stir mixture for 30 minutes, collect the solid by
vacuum filtration and air dry to give the title compound (387 mg, 94%).
Example 6 X-Ray Powder Diffraction
The XRPD patterns of crystalline solids are obtained on a Bruker D4 Endeavor Xray
powder diffractometer, equipped with a CuKa source (= 1.54060 A) and a Vantec
detector, operating at 35 kV and 50 mA. The sample is scanned between 4 and 40° in 2,
with a step size of 0.0087° in 2and a scan rate of 0.5 seconds/step, and with 0.6 mm
divergence, 5.28mm fixed anti-scatter, and 9.5 mm detector slits. The dry powder is
packed on a quartz sample holder and a smooth surface is obtained using a glass slide. It
is well known in the crystallography art that, for any given crystal form, the relative
intensities of the diffraction peaks may vary due to preferred orientation resulting from
factors such as crystal morphology and habit. Where the effects of preferred orientation
are present, peak intensities are altered, but the characteristic peak positions of the
polymorph are unchanged. See, e.g. The U. S. Pharmacopeia 35 - National Formulary 30
Chapter <941> Characterization of crystalline and partially crystalline solids by X-ray
powder diffraction (XRPD) Official December 1, 2012-May 1, 2013. Furthermore, it is
also well known in the crystallography art that for any given crystal form the angular
peak positions may vary slightly. For example, peak positions can shift due to a variation
in the temperature or humidity at which a sample is analyzed, sample displacement, or the
presence or absence of an internal standard. In the present case, a peak position variability
of + 0.2 in 2will take into account these potential variations without hindering the
unequivocal identification of the indicated crystal form. Confirmation of a crystal form
may be made based on any unique combination of distinguishing peaks (in units of ° 2),
typically the more prominent peaks. The crystal form diffraction patterns, collected at
ambient temperature and relative humidity, were adjusted based on NIST 675 standard
peaks at 8.85 and 26.77 degrees 2-theta.
A prepared sample of Example 6 is characterized by an XRPD pattern using CuKa
radiation as having diffraction peaks (2-theta values) as described in Table 1 below.
Specifically the pattern contains a peak at 7.68 in combination with one or more of the
peaks selected from the group consisting of 12.02, 12.93, 15.17, 19.24 and 23.21 with a
tolerance for the diffraction angles of 0.2 degrees.
X-ray powder diffraction peaks of Example 6
Table 2
Peak Angle Relative (2-Theta °) Intensity ( )
1 7.28 20
2 7.68 100
3 9.88 15
4 12.02 46
5 12.42 16
6 12.93 69
7 13.11 36
8 13.76 33
9 15.17 45
10 17.02 32
11 17.24 17
12 19.24 83
13 19.88 19
14 20.27 27
15 21.85 36
16 22.06 47
17 22.52 23
18 23.21 75
19 23.42 27
20 24.36 38
2 1 24.80 33
22 25.76 2 1
23 25.87 23
24 28.58 28
Example 7
-Methyl-2-[4-(l-pyrimidin-2-ylethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one
Example 8
-Methyl-2-[4-(l-pyrimidin-2-ylethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, Isomer 1
Example 9
-Methyl-2-[4-(l-pyrimidin-2-ylethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, Isomer 2
Combine 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-
one bis-(2,2,2-trifluoroacetic acid) salt (1.0 g, 2.1 mmol), 2-acetylpyrimidine (0.38 g, 3.14
mmol), DMF (3 mL), MeOH ( 1 mL), sodium cyanoborohydride (0.20 g, 3.14 mmol) and
stir at room temperature for 20 hours. Add additional 2-acetylpyrimidine (0.38 g, 3.14
mmol) and sodium cyanoborohydride (0.20 g, 3.14 mmol) and heat at 80 °C overnight.
Evaporate the DMF under a stream of nitrogen for 4 days and purify by silica gel flash
chromatography eluting with DCM to 90% DCM/MeOH to give the title compound,
Example 7 (330 mg, 44%) as the racemate.
Separate 8-Methyl-2-[4-(l-pyrimidin-2-ylethyl)piperazin-l-yl]-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one using SFC on a Phenomene® Lux® Cellulose-2
column (2.1 x 25 cm, 5 ) . Mobile Phase: 40% EtOH (0.2% IPAm)/carbon dioxide.
Flow rate: 70 mL/min. Detection: 225 nm. Obtain the first eluting peak as Isomer 1 and
the second eluting peak as Isomer 2.
Example 8, Isomer 135 mg, 99.25% pure with 0.75% of Isomer 2 as an impurity (TR =
4.59 min; Phenomenex® Lux® Cellulose-2 column (2.1 x 25 cm, 5 ), mobile phase:
40% EtOH (0.2% IPAm)/carbon dioxide, flow rate: 5 mL/min. detection: 225 nm). LCES/
MS m/z 356.3 (M+H)+.
Example 9, Isomer 2 : 121 mg, 96.63% pure with 3.36% of Isomer 1 as an impurity (T R =
5.91 min, Phenomenex® Lux® Cellulose-2 column (2.1 x 25 cm, 5 ), mobile phase:
40% EtOH (0.2% IPAm)/carbon dioxide, flow rate: 5 mL/min, detection: 225 nm). LCES/
MS m/z 356.3 (M+H)+.
Example 10
8-Methyl-2- [4-(1-pyrimidin-2-ylpropyl)piperazin- 1-yl] -3,5,6,7-tetrahydropyrido [2,3 -
d]pyrimidin-4-one
O
Example 11
8-Methyl-2- [4-(1-pyrimidin-2-ylpropyl)piperazin- 1-yl] -3,5,6,7-tetrahydropyrido [2,3 -
d]pyrimidin-4-one, Isomer 1
Example 12
8-Methyl-2- [4-(1-pyrimidin-2-ylpropyl)piperazin- 1-yl] -3,5,6,7-tetrahydropyrido [2,3 -
d]pyrimidin-4-one, Isomer 2
Prepare Examples 10, 11, and 12 essentially following the procedure as described
in Examples 7, 8, and 9 using l-(2-pyrimidinyl)-l-propanone and purifying by silica gel
flash chromatography eluting with a gradient of DCM to 90% DCM/MeOH. Purify a
second time by silica gel chromatography using the same conditions. Then purify further
by high pH prep HPLC (Column: 150 g High resolution C18 Gold; Initial: 5%
acetonitrile/95% 10 mM ammonium bicarbonate w/5% MeOH with gradient to 60%
acetonitrile/40% ammonium bicarbonate w/5% MeOH over 30 min., detection
wavelengths 239, 254, 280 and 290 nm) to give the title compound, Example 10 (140 mg,
18%). Separate the resulting enantiomeric mixture using SFC using the conditions
described for Examples 8 and 9 with the exception that a Lux® Cellulose-4 column is
used.
Example 11, Isomer 1: 50 mg, >99.9% purity (T = 4.26 min; Phenomenex®
Lux® Cellulose-4 column (2.1 x 25 cm, 5 ), mobile phase: 40% EtOH (0.2%
IPAm)/carbon dioxide, flow rate: 5 mL/min, detection: 225 nm). LC-ES/MS m/z 370.2
(M+H)+.
Example 12, Isomer 2 : 47 mg, >99.9% purity (T = 5.67 min; Phenomenex®
Lux® Cellulose-4 column (2.1 x 25 cm, 5 ) . Mobile Phase: 40% EtOH (0.2%
IPAm)/carbon dioxide. Flow rate: 5 mL/min. Detection: 225 nm), LC-ES/MS m/z 370.2
(M+H)+.
Example 13
2-[4-[(4-methoxypyrimidin-2-yl)methyl]piperazin-l-yl]-8-methyl-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one
Combine 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-
one bis-(2,2,2-trifluoroacetic acid) salt (0.5 g, 1.0 mmol), 4-methoxypyrimidine-2-
carbaldehyde (150 mg, 1.08 mmol), DMF (10 mL), MeOH ( 1 mL) and sodium
cyanoborohydride (136 mg, 2.17 mmol) and stir at room temperature for 3 days. Dilute
with MeOH and absorb onto an SCX-2 column, rinse with MeOH and then elute with 2
M N¾ in MeOH, concentrate and purify by silica gel flash chromatography eluting with
a gradient of DCM to 90% DCM/MeOH to give the title compound (43 mg, 11%). LCES/
MS m/z 372.1 (M+H)+TR = 1.519 min.
The following Examples are prepared essentially following the procedure of
Example 13, using the appropriate aldehyde and the appropriate chromatography.
Table 3
Stir reaction for 3 days.
Add additional aldehyde ( 1 eq) and sodium cyanoborohydride (1.5 eq); then heat a t 8 0
°C for 6 h . Further purify b y general method o f reverse phase chromatography (High p H
5-100).
Further purify b y general method o f reverse phase chromatography (High p H 9-24).
4 Further purify b y general method o f reverse phase chromatography (Low pH 0.1%
TFA/ACN) followed b y chromatography o n an SCX-2 column.
5 SSttiirr rreeaaccttiioonn aatt rrtt ffoorr 1122 hhoouurrss aanndd further purify b y general method o f reverse phase
chromatography (High pH 13-48).
Example 20
2-[4-[(3-Fluoro-2-pyridyl)methyl]piperazin-l-yl]-8-methyl-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one
Suspend 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-
one bis-( 2,2,2-trifkioroacetic acid) (20.31 g , 34.34 mmol) in DCM (114 mL) and purge
with nitrogen. Add 3-fluoropyridine-2-carbaldehyde (5.26 g , 41.2 mmol) and stir for 6 0
minutes. Then add sodium triacetoxyborohydride (14.56 g , 68.68 mmol) in one portion
and observe an exotherm to reflux. Stir and allow the reaction to cool to room
temperature over 1 hour. Pour into 0.5 M NaOH (200 mL) and adjust the pH : 7-8 with 2
M NaOH. Collect the organic layer and extract the aqueous layer with DCM (2 x 200
mL). Combine and dry the organic layers over sodium sulfate, filter, and concentrate to
give an oil. Purify b y silica gel chromatography (400 g), eluting with 95% DCM/ 5%
MeOH for 8 column volumes and with 90% DCM/10% MeOH for 8 column volumes to
give the title compound (7.59 g , 61.7%) with >97% HPLC purity and also 2.64 g (21.5%)
o f additional product with 87% HPLC purity. L C ES/MS m/z 359. 1 (M+l) + .
Example 21
2-[4-[(2-Methoxyphenyl)methyl]piperazin-l-yl]-8-methyl-3,5,6,7-tetrahydropyrM
d]pyrimidin-4-one
Combine 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-
one bis-(2,2,2-trifluoroacetic acid) salt (0.25 g, 0.52 mmol), 2-methoxybenzaldehyde
(0.21 g, 1.57 mmol), DMF (15 mL), MeOH (5 mL) and sodium cyanoborohydride (100
mg, 1.57 mmol) and stir at room temperature forl2 hours. Dilute with methanol and
absorb onto an SCX-2 column, rinse with MeOH and then elute with 2 M NH in MeOH,
concentrate and purify by reverse phase chromatography, see general method for reverse
chromatography (High pH 21-55) to give the title compound (223 mg, 59%). LC-ES/MS
m/z 370.2 (M+H)+.
The following compounds are prepared essentially as described in Example 2 1
using the appropriate aldehyde.
Table 4
d Reverse phase chromatography (High pH 39-73).
Example 28
2-[4-[(3-Huoro-2-pyridyl)methyl]pi
d]pyrimidin-4-one, dihydrochloride
In 2 vials add to each DCM (5 mL) 4M HCI in dioxane ( 1 rriL, 4 mmol), and 2-[4-
[(3-fluoro-2-pyridyl)methyl]piperazin-l-yl]-8-methyl-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one (0.117 g, 0.65 mmol). Shake the vials for 1 hour, combine into 1 vial
and then evaporate under a stream of nitrogen overnight. Dry the material in a vacuum
oven overnight to give the title compound (0.28 g, 99%). LC-ES/MS m/z 359.2 [M+H]+,
TR = 1.04 min.
The following Examples are prepared essentially as described in Example 28
using the appropriate compound of Examples 21-27.
Table 5

Example 37
2-[[4-(8-Methyl-4-oxo-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-2-yl)piperazin-lyl]
methyl]benzonitrile; bis-2,2,2-trifluoroacetic acid
Combine a solution of 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one bis-( 2,2,2-trifluoroacetic acid) (0.364 g, 0.76 mmol) in DMF (3.2 mL)
with 2-cyanobenzaldehyde (0.499 g, 3.18 mmol) and stir at room temperature for 1 hour.
Add sodium cyanoborohydride (0.143 g, 2.28 mmol) and stir at room temperature
overnight. Pour into water and attempt collection by filtration. Observe clogging of filter
and then add 2 N NaOH and NaCl (aqueous) and extract the crude product into ethyl
acetate (3x). Combine the organic layers, dry over sodium sulfate, filter, and concentrate.
Purify by silica gel chromatography eluting with 98% DCM /2% ethanol isocratic for 10
min with step gradient to 95% DCM /5% ethanol and hold for 45 min. Purify further by
preparative reverse phase chromatography eluting with 5% acetonitrile (0. 1% TFA)/95%
water (0.1%TFA) gradient to 54% acetonitrile (0.1% TFA)/46% water (0.1%TFA) to give
the title compound (48.1 mg, 10.7%). LC-ES/MS m/z 365.2 [M+H]+
Example 38
2-[4-[(2-Chlorophenyl)methyl]piperazin-l-yl]-8-methyl-3,5,6J-tetrahydropyrido
d]pyrimidin-4-one
Prepare the title compound essentially as described in Example 37 using 2-chloro-
5-methyl-pyrimidine 2-chlorobenzaldehyde. Purify the crude material by silica gel flash
chromatography (5% EtOH/CHCl to 10% EtOH/CHCl gradient). Recrystallize the
material from DMSO/MeOH to obtain a white solid (83 mg, 29%). LC-ES/MS m/z 374.3
( 5C1) [M+H]+.
Example 39
2-[4-[(5-Fluoropyrimidin-2-yl)methyl]piperazin-l-yl]-8-methyl-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one
Combine potassium trifluoro-[[4-(8-methyl-4-oxo-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-2-yl)piperazin-l-yl]methyl]boranuide (273 mg, 0.74 mmol), 2-chloro-5-
fluoropyrimidine (98 mg, 0.74 mmol), cesium carbonate (723 mg, 2.22 mmol), X-Phos
(71 mg, 0.15 mmol), THF (10 mL) and water ( 1 mL) and degas with a stream of nitrogen
for 2 min. Add palladium (II) acetate (17 mg, 0.074 mmol) and heat at 80 °C overnight.
Partition the mixture between ethyl acetate and water and separate the organic layer. Dry
over magnesium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify
the resulting residue by silica gel flash chromatography eluting with DCM to 90%
DCM/MeOH to give the title compound (20 mg, 7.5%). LC-ES/MS m/z 360.2 [M+H]+.
Example 40
2-[[4-(8-Methyl-4-oxo-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-2-yl)piperazin-lyl]
methyl]pyridine-3-carbonitrile
Dissolve 2-[4- [(3 -bromo-2-pyridyl)methyl]piperazin- 1-yl] -8-methyl-3 ,5 ,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one (167 mg, 0.398 mmol) in DMF (10 mL) and
degas with a stream of nitrogen for 1 min. Add tetrakis(triphenylphosphine)palladium
(92 mg, 0.080 mmol) and zinc cyanide (187 mg, 1.59 mmol) and heat the mixture at 120
°C overnight. Cool the reaction and dilute with MeOH. Transfer the mixture to a 10 g
SCX-2 column and elute with 2 M N¾ in MeOH. Concentrate and further purify the
resulting residue reverse phase chromatography (High pH 9-29) to give the title
compound (35 mg, 24%). LC-ES/MS m/z 366.2 (M+H)+
Example 41
2-[4-[(4-Chloropyrimidin-2-yl)methyl]piperazin-l-yl]-8-methyl-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one
Combine 2-(bromomethyl)-4-chloro-pyrimidine (322 mg, 1.55 mmol) as a crude
solution in carbon tetrachloride (5 mL), DCM (2 mL), triethylamine (0.65 mL, 4.65
mmol), and 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one
bis-(2,2,2-trifluoroacetic acid) (739 mg, 1.55 mmol) and stir at 25 °C for 3 days. Partition
the reaction mixture between DCM and water and extract the aqueous layer with DCM.
Combine the organic layers, dry over MgS0 4, filter, and concentrate. Purify the resulting
residue by mass guided SFC (column: 4-nitrobenzene sulfonamide, Princeton
Chromatography, 150 x 30 mm, flow rate = 100 mL/min; method: 95% C0 2/14 mM
ammonia in MeOH isocratic for 30 sec. then gradient to 60% C0 2/14 mM ammonia in
MeOH gradient over 330 se , then ramp to 50% C0 2/14 mM ammonia in MeOH over 10
sec. and hold for 30 sec.) to give 7 1 mg of desired product with ammonia salt impurities.
The product is further purified by silica gel flash chromatography (CH2Cl2/MeOH, 90:10)
to give the title compound (28 mg, 5%). LC-ES/MS m/z 376.0 [M+H]+
Example 42
8-Methyl-2-[4-(pyrazin-2-ylmethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-
d] rimidin-4-one
Combine 8-methyl-2-piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-
one bis-(2,2,2-trifluoroacetic acid) (500 mg, 1.05 mmol), triethylamine (0.73 mL, 5.2
mmol), sodium iodide (78.5 mg, 0.52 mmol) and acetonitrile (7 mL). Add 2-
(chloromethyl)pyrazine (201 mg, 1.57 mmol) and stir at 25 °C for 72 h. Dilute the
reaction with DCM and water and extract the product into DCM. Wash the organic
portion with saturated NaHC0 and brine, dry over Na2S0 4, filter, and concentrate.
Purify the resulting residue by silica gel flash chromatography (DCM / 2M N¾ in
MeOH, 90: 10) to give the title compound as a yellow solid (249 mg, 0.72 mmol,70%).
LC-ES/MS m/z 342.0 [M+H]+, TR = 0.99 min.
The following Examples are prepared essentially following the procedure
described in Example 42, using the appropriate halo-methylpyrazine.
Table 6
Example 45
2-[4-[(3,5-Difluoro-2-pyridyl)methyl]piperazin- l-yl]-8-methyl-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one
Add 2-(chloromethyl)-3,5-difluoropyridine (182 mg, 1.11 mmol), 8-methyl-2-
piperazin-l-yl-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one (260 mg, 1.04 mmol),
potassium carbonate (2.2 g, 15 mmol) and acetonitrile (5 mL) and stir the mixture at room
temperature overnight. Concentrate the reaction mixture under reduced pressure and
purify the residue by silica gel chromatography (elution with MeOH/DCM, 1/20) to give
the title compound (160 mg, 43%). LC-ES/MS m/z 377.2 (M+H)+, TR = 1.1 1 min.
Cancer is increasingly recognized as a heterogeneous collection of diseases whose
initiation and progression are induced by the aberrant activation or function of one or
more genes that regulate DNA repair, genome stability, cell proliferation, cell death,
adhesion, angiogenesis, invasion, and metastasis in cell and tissue microenvironments.
Variant or aberrant function of the "cancer" genes may result from naturally occurring
DNA polymorphism, changes in genome copy number (through amplification, deletion,
chromosome loss, or duplication), changes in gene and chromosome structure (through
chromosomal translocation, inversion, or other rearrangement that leads to deregulated
gene expression), and point mutations. Cancerous neoplasms may be induced by one
aberrant gene function, and maintained by the same aberrant gene function, or
maintenance and progression exacerbated by additional aberrant gene activations or
functions.
Beyond the genetic chromosomal aberrations mentioned above, each of the
cancers may also include epigenetic modifications of the genome including DNA
methylation, genomic imprinting, and histone modification by acetylation, methylation,
or phosphorylation. An epigenetic modification may play a role in the induction and/or
maintenance of the malignancy.
Diagnosis of cancerous malignancies by biopsy, immunophenotyping and other
tests are known and routinely used. In addition to high resolution chromosome banding
and advanced chromosomal imaging technologies, chromosome aberrations in suspected
cases of cancer can be determined through cytogenetic analysis such as fluorescence in
situ hybridization (FISH), karyotyping, spectral karyotyping (SKY), multiplex FISH (MFISH),
comparative genomic hybridization (CGH), single nucleotide polymorphism
arrays (SNP Chips) and other diagnostic and analysis tests known and used by those
skilled in the art.
An important part of the Wnt/p-catenin signaling pathway is the regulated
proteolysis of the downstream effector -catenin by the -catenin destruction complex.
The principal constituents of the -catenin destruction complex are adenomatous
polyposis coli (APC), Axin, and GSK3a/p. In the absence of Wnt pathway activation,
cytosolic -catenin is constitutively phosphorylated and targeted for degradation. Upon
Wnt stimulation, the -catenin destruction complex disassociates, which leads to the
accumulation of cytosolic -catenin, translocation to the nucleus, and transcription of Wnt
canonical pathway responsive genes.
Considerable efforts have been made to identify pharmaceutical agents that inhibit
the canonical Wnt -catenin signaling pathway. TNKS1 and TNKS2 inhibitors such as
XAV939, Huang et al., Nature, 2009, 461, 614; JW55, Waaler et al., Cancer Res., 2012,
72(11), 2822; G007-LK, Lau et al., Cancer Res., 2013, 73(10), 3132; TNKS656, Shultz et
al., . Med. Chem. published online July 11, 2013, DOI: 10.1021/jm400807n; and WO
2013/1 17288 are known. Despite these efforts, no clinical TNKS1 and TNKS2 inhibitor
therapeutic agents have emerged at this time.
Aberrant activation of the pathway, mediated by over expression of Wnt proteins
or mutations affecting components of the -catenin destruction complex, thus leading to
stabilization of -catenin, has been observed in cancers. Notably, truncating mutations of
APC are the most prevalent genetic alterations in colorectal carcinomas (Miyaki, M. et al.
Cancer Res. 1994, 54, 3011-20; Miyoshi, Y. et al. Hum. Mol. Genet. 1992, 1, 229-33; and
Powell, S. M. et al. Nature 1992, 359, 235-7). In addition, Axinl and Axin2 mutations,
negative regulators of the Wnt signaling pathway, have been identified in patients with
hepatocarcinomas and colorectal cancer respectively (Taniguchi, K. et al. Oncogene
2002, 21, 4863-71; Liu, W. et al. Nat. Genet. 2000, 26, 146-7; Lammi, L. et al. Am. J.
Hum. Genet. 2004, 74, 1043-50). These somatic mutations result in Wnt-independent
stabilization of -catenin and constitutive activation of -catenin-mediated transcription.
Aberrant Wnt signaling pathway activity has been implicated in several cancers
(Waaler et al. Cancer Res. 2012, 72, 2822-2832; Busch et al. BMC Cancer 2013, 13, 211 ;
Yang et al. Oncogene 2011, 30, 4437-4446; De Robertis et al. Mol. Cancer Ther. 2013,
12, 1180-1189; Polakis, P. Curr. Opin. Genet. Dev. 2007, 17, 45-51; and Barker, N. et al.
Nat. Rev. Drug Discov. 2006, 5, 997-1014), including colorectal, gastric, liver, breast,
triple negative breast cancer, ovarian, medulloblastoma, melanoma, lung, non-small cell
lung, pancreas, prostate cancers and glioblastomas. Aberrant Wnt^-catenin pathway
signaling activity has been implicated in T-cell lymphoma, T-lymphoblastic lymphoma,
T-cell acute lymphocytic leukemia (T-ALL) Groen et al. Cancer Res. 2008, 68, 6969-
6977; multiple myeloma Qiang et al. Oncogene 2003, 22, 1536-1545, and Chim et al.
Leukemia 2007, 21, 2527-2536; mantle cell lymphoma Gelebart et al. Blood 2008, 112,
5171-5179; chronic myeloid leukemia (CML), Heidel et al. Cell Stem Cell 2012,
10(4):412-424, and acute myeloid leukemia (AML), Ysebaert et al. Leukemia 2006, 20,
1211-1216.
It has been found that -catenin degradation can be promoted by stabilizing the
Axin/APC/GSIGa/ destruction complex through the inhibition of the poly-ADP-ribose
polymerase (PARP) enzymes tankyrase 1 and tankyrase 2, Huang et al. Nature 2009, 461,
614-620.
The following in vitro and in vivo studies demonstrate the Wnt -catenin signaling
pathway inhibitory activity and efficacy of exemplified and tested compounds of Formula
I, or a pharmaceutically acceptable salt thereof, in inhibiting hTNKSl and hTNKS2,
stabilization of Axin2 in HEK293 cells, selectivity against PARP1 inhibition, reduce the
expression of Wnt-inducible genes expression, and in vivo antitumor activity. These
assays are generally recognized by those skilled in the art as indicative of human clinical
chemotherapeutic activity. Inhibition of TNKS 1 and TNKS 2 are believed to be
effective against aberrant activation of the Wnt -catenin signaling pathway. Assays
evidencing Wnt/ -catenin signaling pathway inhibitory activity and efficacy may be
carried out substantially as follows or by similar assays affording similar data.
Assays
Generally, cell lines are generated using commercially available materials and by
procedures known to and routinely used by those skilled in the art.
Biochemical assay to demonstrate compound inhibition of hTNKS enzyme activity
The enzymatic activity of hTNKSl and hTNKS2 is assessed using an enzymelinked
immunosorbent assay (ELISA) which detects poly ADP ribose incorporated into
plate-bound Telomeric repeat binding factor 1 (TRF1) protein (NCBI, Accession number
NP_059523.2 (SEQ ID NO: 1) in a 384-well format. Using recombinant hTNKSl
(NCBI, Accession number NP_003738.2 (SEQ ID NO: 2) or hTNKS2 (NCBI, Accession
number NP_07951 1.1 (SEQ ID NO: 3) enzyme, this assay uses biotinylated NAD+ and
measures its incorporation into recombinant hTRFl using a streptavidin-horseradish
peroxidase (HRP) conjugate and TMB peroxidase substrate to generate a colorimetric
signal. Recombinant Flag-tagged hTRFl protein is generated by expressing full length
human TRF1 protein with an N-terminal Flag tag in E. coli. Recombinant Flag-hTNKSl
(with a change of Q83P) protein is generated by expressing full length human TNKS1
with an N-terminal Flag tag in Baculovirus according to the manufacturer's protocol of
Bac-to-Bac Baculovirus Expression system (Invitrogen™; See also Invitrogen™ User
Manual, Version F, dated 04 September 2010; and Invitrogen™ Instruction Manual dated
27 February 2002). The Flag tags on hTRFl and hTNKl are used only for purification of
the enzymes and are not otherwise involved in the ELISA assay.
Flag-TRFl is diluted to 5 g/ml using TBS coating buffer (50 mM Tris, pH 8.0,
150 mM NaCl) and 25 ΐ is added to each well of a Corning 3700 plate (Tewksbury, MA
#CLS3700). The plates are incubated overnight at 4 °C. The next day the plates are
washed 3 times with 50 ΐ/well wash buffer (PBS (prepared from lOx concentrate using
Hyclone, Logan, UT #SH30258.01) with 0.1% Tween-20 (Sigma, St. Louis, MO #7949))
followed with blocking for 1.5 hours at room temperature using 50 ΐ 1% Casein block
buffer (Thermo Scientific, Waltham, MA #37528) in l x PBS (Roche, Indianapolis, IN
# 11666789001). After blocking, the plates are washed 3 times with 50 ΐ/well wash
buffer. The enzyme assay is set up using 2 g/ml of Flag-hTNKSl, 9.5 NAD+
(Sigma, St. Louis, MO #N0632), 0.5 biotin-NAD+ (Trevigen, Gaithersburg, MD
#4670-500-01), and compounds diluted from 10 to 4 nM final concentration in 50
mM Tris, pH 8.0 (Invitrogen™, Grand Island, NY #15568-025), 4 mM MgCl2, 0.2 mM
DTT, 0.5% Triton X-100 (Roche, Indianapolis, IN #11332481001) and 1.0 % DMSO in a
total volume of 25 ΐ . The reaction is incubated for 120 minutes at room temperature and
stopped by washing the plate 3 times with 50 ΐ/well wash buffer. Detection of the
biotin-NAD+ incorporation is done using 25 ΐ/well of Strepavidin-HRP (GE Life
Sciences Pittsburgh, PA #RPN1231V) diluted 1:3000 with wash buffer and incubated for
60 minutes at room temperature. The plate is then washed 3 times using 50 ΐ/well wash
buffer. This is followed by incubation with 25 ΐ/well TMB peroxidase substrate kit
(KPL, Gaithersburg, MD #50-76-02 and #50-65-02) for 15 minutes at room temperature
and stopping the reaction using 25 ΐ/well of 2 N H2SO4. The absorbance is read at 450
nm using an Envision model 2103.
By substantially following the procedures described above for hTNKS1, and using
hTNKS2, an essentially similar ELISA assay is prepared.
Activity of those compounds tested against both hTNKS isoforms is shown in
Table 7.
Table 7
Example No. hTNKS1 ICso (nM) hTNKS2 ICso (nM)
1 19.1 (± 17.7, n=13) 13.7 (± 7.77, n=13)
2 10.7 (±7.13, n=6/7) 6.25 (± 1.20, n=5)
3 10.9 (± 10.5, n=4) 6.58 (±4.48, n=5)
4 26.4 (± 11.2, n=5) 11.6 (±6.88, n=5)
5 85.2 (± 35.5, n=8) 69.4 (±17.6, n=8)
6 17.5 (± 6.48, n=3) 9.95
7 41.9 (± 19.9, n=5) 37.1 (±8.12, n=5)
8 94.8 (± 2.99, n=3/4) 68.7 (± 8.48, n=3/4)
9 83.3 (± 89.4, n=4) 54.9 (± 44.7, n=4)
10 158 (± 89.0, n=4) 121 (± 11.3, n=4)
11 32.7 (± 29.9, n=5) 24.7 (± 20.0, n=5)
12 27.5 (± 32.7, n=5) 22.0 (± 15.1, n=5)
13 14.9 (± 9.89, n=6/7) 12.4 (±3.67, n=6)
14 22.8 (± 13.0, n=7) 11.7 (± 7.32, n=6)
15 25.3 (± 5.05, n=6/7) 16.7 (±5. 16, n=5)
16 55.1 (± 32.5, n=6) 43.4 (± 23.8, n=4)
17 67.6 (±64.7, n=5) 81.5 (± 9.15, n=4)
18 114 (±84.2, n=6) 85.1 (± 6.72, n=5)
19 19.9 (± 6.75, n=5) 11.8 (± 6.55, n=5)
20 31.8 (±38.2, n=6) 11.3 (± 4.26, n=5)
28 29.5 (± 47.3, n=5) 35.8 (± 19.1, n=4/5)
29 18.4 (±13.3, n=5) 14.1 (± 3.57, n=5)
30 41.7 (±40.2, n=5) 24.5 (±22.6, n=5)
3 1 15.2 (±5.04, n=5) 12.2 (± 4.59, n=5)
32 37.2 (±39.7, n=6) 22.7 (±24.2, n=6)
33 35.8 (± 32.5, n=6) 26.6 (± 20.8, n=6)
34 47.6 (±36.6, n=5) 11.6 (± 4.30, n=4)
35 21.6 7.54
36 26.2 (± 2.55, n=5) 17.6 (±11.0, n=5)
37 19.8 (± 13.3, n=4) 10.8 (± 1.34, n=4)
38 45.3 (± 32.0, n=4) 30.2 (± 5.13, n=4)
39 30.3 (± 26.6, n=6) 15.9 (± 8.48, n=6)
40 14.3 (± 4.89, n=5) 8.41 (± 1.99, n=5)
4 1 14.5 (± 10.7, n=5) 10.6 (± 3.51, n=5)
42 19.8 (± 15.9, n=5) 13.0 (±7.41, n=5)
43 34.3 (± 12.7, n=4) 12.7 (± 6.54, n=4)
44 17.6 (± 8.49, n=6) 12.7 (+ 2.49, n=6)
45 37.3 (± 11.1, n=6) 19.6 (± 9.93, n=6)
Mean + SEM; SEM = standard error of the mean
Data in Table 7 provides evidence that the compounds tested have in vitro
inhibitory activity against both isoforms of human tankyrases.
EGFP-Axin2 Stabilization Assay to demonstrate cell-based activity of tankyrase
inhibitors
Enhanced Green Fluorescent Protein-Axin2 cells (EGFP-Axin2) are prepared by
stable transfection of HEK293 cells with an Axin2 construct containing an N-terminal
truncated EGFP tag (amino acids 228-466; SEQ ID NO:7). Changes in Axin2 levels,
specifically Axin2 stabilization, are monitored by quantitation of the level of EGFP in the
stable cell line after various treatments in a 384-well format. Increases in fluorescence,
reflecting the stabilization of the EGFP-Axin2 fusion protein as a consequence of
tankyrase inhibition, are monitored in an Acumen Laser Scanning Cytometer.
HEK293 cells (ATCC, Manassas, VA #CRL-1573) are maintained in complete
medium of DMEM:F12 (Invitrogen™, Grand Island, NY #93-0152DK) containing 5%
FBS (Invitrogen™, Grand Island, NY #10082-147), 20 mM HEPES (Hyclone, Logan,
UT #SH30237.01), and Glutamax (Invitrogen™, Grand Island, NY #35050-061).
EGFP-Axin2 cells are generated by transfecting HEK293 cells with full length human
Axin2 (NCBI, Accession number NP_004646.3 (SEQ ID NO: 4)) containing a truncated
(amino acids 228-466) EGFP N-terminal tag (full length EGFP, NCBI, Accession number
ABG78037.1 (SEQ ID NO: 5) in pcDNA3.1+ (according to the manufacturer's protocol,
Invitrogen™, Grand Island, NY #V79020). Stable EGFP-Axin2 cells are maintained in
HEK293 complete medium above with the addition of 800 g/ml G418 (Invitrogen™,
Grand Island, NY #10131-035). The EGFP-Axin2 stabilization assay is done by plating
2000 EGFP-Axin2 cells/well in a poly-D-lysine coated BD 384-well plate (BD
Biosciences, San Jose, CA #356663) and incubating in 30 ΐ/well complete HEK293
medium and grown overnight at 37 °C, 5% C0 2. Compounds in 100% DMSO are added
directly to the cell media at 100 nl/well. Final concentration of compounds tested in the
assay is 33 -1.7 nM with final concentration of DMSO in the assay being 0.33%.
Cells are incubated with compound for 24 hours at 37 °C, 5% C0 2 and the cells fixed
using 2% final concentration of formaldehyde for 15 minutes at room temperature. The
fixed cells are washed twice for 20 minutes each in 40 ΐ/well PBS (Hyclone, Logan, UT
#SH30264.01) containing 0.1% Triton X-100 (Thermo Fisher Scientific, Waltham, MA
#BP151-500). They are then stained using 30 ΐ/well PBS containing 10 g/ml
propidium iodide (Invitrogen™, Grand Island, NY #P3566) and 50 g/ml RNaseA
(Sigma, St. Louis, MO #R6513). EGFP intensity is measured using an Acumen model
eX3 Acumen Laser Scanning Cytometer gated to have 10% EGFP/cell.
Table 8
Example No. Axin2 Stabilization
ECso (nM)
1 65.9 (±_22.7, n=ll)
2 32.0 (± 9.34, n=4)
3 18.4 (± 15.8, n=3)
4 26.9 (± 11.5, n=3)
5 115 (± 78.3, n=4)
6 59.5 (± 6.94, n=4)
7 121 (± 21.9, n=3)
8 71.5 (± 28.7, n=5)
9 70.9 (± 30.6, n=4)
10 590 (± 228, n=3)
11 119 (± 26.6, n=4)
12 122 (± 31.2, n=4)
13 101 (± 26.8, n=4)
14 52.7 (± 23.9, n=4)
15 115 (± 17.5, n=4)
16 509 (± 130, n=3)
17 198 (± 57.4, n=3)
18 901 (± 138, n=3)
19 62.6 (± 14.6, n=4)
20 77.9 (± 29.6, n=3)
28 130 (± 79.4, n=4)
29 41.7 (± 12.9, n=3)
30 57.7 (± 27.1, n=3)
3 1 38.5 (± 6.69, n=3)
32 136 (± 84.8, n=4)
33 122 (± 73.6, n=4)
34 156 (± 141, n=3)
35 27.8
36 29.1(± 8.88, n=3)
37 23.9 (± 4.48, n=3)
38 34.8 (± 4.46, n=3)
39 181 (± 64.9, n=5)
40 39.8 (± 16.2, n=6)
4 1 61.6 (± 10.5, n=4)
42 141 (± 44.0, n=3)
43 74.5 (± 20.0, n=4)
44 118 (± 33.2, n=4)
45 144 (± 1.88 n=3)
Mean ± SEM; SEM = stand ard error of the mean
Data in Table 8 provides evidence that the compounds tested stabilize Axin2 in
HEK293 cells.
Human PARPl Enzyme Assay to assess selectivity of tankyrase inhibitors (vs.
PARPl)
The Poly ADP-ribose polymerase l(PARPl) assay is an ELISA which detects
poly ADP ribose incorporated into plate-bound histone protein in a 384- well format.
Using recombinant hPARPl enzyme, this assay uses biotinylated NAD+ and measures
the incorporation into histone using a streptavidin-horseradish peroxidase (HRP)
conjugate and TMB peroxidase substrate to generate a colorimetric signal.
Histone is diluted to 0.1 mg/ml in coating buffer (50 mM Na2CC>3, pH 9.4,
Mallinckrodt, St. Louis, MO) and 25 ΐ is added to each well of a Corning 3700 plate.
The plates are incubated overnight at 4 °C. The next day the plates are washed 3x with
50 ΐ/well wash buffer (PBS (prepared from lOx concentrate) with 0.1% Tween-20)
followed with blocking for 1.5 hours at room temperature using 50 ΐ 1% Casein block
buffer in l x PBS (Roche, Indianapolis, IN # 11666789001). After blocking, the plates are
washed 3 times with 50 ΐ/well wash buffer. The PARPl enzyme assay is set up by using
0.01 U/hPARPl (Trevigen, Gaithersburg, MD #4668-500-01), 0.5X PARP cocktail,
activated DNA (Trevigen, Gaithersburg, MD #4671-096-03 and #4671-096-06) and
compounds diluted from 10 to 4 nM final concentration in Assay buffer containing
50 mM Tris, pH 8.0, 10 mM MgCl 2, 1 mM DTT (Invitrogen™, Grand Island, NY
#15508-013), 0.5% Triton X-100 and 1.0 % DMSO. The reaction is incubated for 60
minutes at room temperature and stopped by washing the plate 3 times with 50 ΐ/well
wash buffer. Detection of the biotin-NAD+ incorporation is done using 25 ΐ/well of
Strepavidin-HRP diluted 1:3000 with wash buffer and incubated for 60 minutes at room
temperature. The plate is then washed 3 times using 50 ΐ/well wash buffer. This is
followed by incubation with 25 ΐ/well TMB peroxidase substrate kit (KPL,
Gaithersburg, MD #50-76-02 and #50-65-02) for 15 minutes at room temperature and
stopping the reaction using 25 ΐ/well of 2 N H2S0 4. The absorbance is read at 450 nm
using an Envision model 2103.
Table 9
PARP1 Inhibition
Example No.
ICso (nM)
1 4,510 (± 3030, n=2)
2 919 (n=l/2)
3 2,330 (± 234, n=2)
4 3,020 (± 197, n=2)
5 7,660 (± 1450, n=2)
6 2,670 (± 433, n=2)
7 5,930 (± 647, n=2)
8 5470
9 3940
10 33,700 (± 3130, n=2)
11 13,500
12 5,050
13 3,470 (± 318, n=2)
14 5,660 (± 698, n=2)
15 6,910 (± 746, n=2)
16 187 (± 183, n=2)
17 7,220 (± 1790, n=2)
18 77,600 (± 24000, n=2)
19 5390
20 8,240 (±10800, n=4)
29 6,070 (± 1730, n=2)
30 8,800 (± 1720, n=2)
3 1 17,400 (± 48.1, n=2)
32 21,500 (± 2340, n=2)
33 15,300 (± 2230, n=2)
34 16,200 (± 5740, n=2)
36 95,700 (± 6420, n=2)
37 2,440 (± 598, n=2)
38 46,700 (± 23700, n=2)
39 7,740
40 2,840
4 1 4,660
42 2,650 (± 143, n=2)
43 6,390 (± 1430, n=2)
44 5,830 (± 229, n=2)
45 3,530 (± 2030, n=2)
Mean + SEM; SEM = standard error of the mean
Data in Table 9 provides evidence as to each tested compound's selective
inhibition of tankyrases when compared to PARP1 inhibition.
DLD-1 TOPFlash Assay to determine the ability of tankyrase inhibitors to reduce
the expression of Wnt-inducible genes
DLD-1 cells contain a mutation in the adenoma polyposis coli (APC) gene which
encodes a truncated APC protein. This protein is incapable of binding the destruction
complex and causes a constitutively activated Wnt pathway by allowing catenin to
translocate to the nucleus and activate the TCF/LEF transcription factors. DLD-1
TOPFlash is a reporter cell line derived from DLD-1 (human colorectal adenocarcinoma)
cells by stable transfection of a TCF4 promoter linked to a luciferase reporter. The
amount of luciferase in the cell lysates is quantitated by measuring luminescence in a 96
well format.
DLD-1 cells (ATCC, Manassas, VA, #CCL-221) are maintained in complete
medium of RPMI (Invitrogen™, Grand Island, NY #11875-093) containing 10% FBS
(Hyclone, Logan, UT #SH30070.03). DLD-1 TOPFlash cells are generated according to
the manufacturer' s protocol by infecting DLD- 1 cells with Cignal Lenti TCF/LEF
Reporter (Luc) (Qiagen, Valencia, CA #CLS-018L-8, lot#BX16) at an MOI of 10.
Polybreen (Sigma, St. Louis, MO #H9268) is added to a final concentration of 8 g/ml
and the cells are incubated overnight at 37 °C, 5% C0 2. The next day the medium is
changed to fresh growth medium containing 10 g/ml puromycin (Clontech, Mountain
View, CA #631305) and the cells are incubated for an additional 3 days at 37 °C, 5% C0 2
to generate a stable cell line. The TOPFlash assay is done by plating DLD-1 TOPFlash
cells at 10,000 cells/well in a Corning 96 well white/clear plate (Corning Tewksbury, MA
#3610) and incubating in 30 ΐ/well complete medium containing RPMI (Hyclone,
Logan, UT #SH30027.01), 10% FBS and 10 g/ml puromycin and grown overnight at 37
°C, 5% C0 2. The compounds in 100% DMSO are diluted 28.5 fold in OptiMEM®
(Invitrogen™, Grand Island, NY #31985-062) containing 0.2% BSA (diluted from 7.5%
BSA Invitrogen™, Grand Island, NY #15260-037) and 5 ΐ diluted compound added to
the 30 ΐ cell culture medium/well. Final concentration of compounds tested in the assay
is 50 -1.5 nM with final concentration of DMSO in the assay being 0.48%. Cells are
incubated with compound for 24 hours at 37 °C, 5% C0 2 and the plates removed from the
incubator and placed at room temperature for 30 minutes. BugLite™ (3x) reagent is
prepared by dissolving 2.296g DTT (Sigma, St. Louis, MO #D0632), 1.152g CoA
(Sigma, St. Louis, MO #C3019), 0.248g ATP (Sigma, St. Louis, MO #A7699), and 0.42g
Luciferin (Biosynth AG, Itasca, IL #L-8240) in 1 liter of Triton-X100 lysis buffer which
contains 150 mM Tris (108.15 ml 1M Tris HC1 and 41.85 ml 1M Tris Base (Sigma, St.
Louis, MO #T3253 and T-1503)), 3 mM MgCl2 and 3% Triton X-100. After the plates
are equilibrated to room temperature, 18 ΐ of 3x BugLite reagent is added to each well
and the plates are incubated at room temperature for 30 minutes with shaking.
Luminescence is measured using an Envision model 2103.
Table 10
7 0.0146 (± 0.0128, n=3)
8 0.0184 (± 0.0110, n=3)
9 0.0282 (± 0.0257, n=4)
10 0.171 (± 0.0474, n=3)
11 0.0281 (± 0.00945, n=3)
12 0.0195 (± 0.0111, n=3)
13 0.0230 (± 0.0126, n=3)
14 0.00935 (± 0.00872, n=3)
15 0.0154 (± 0.00630, n=3)
16 0.102 (± 0.0268, n=3)
17 0.0373 (± 0.0103, n=3)
18 0.156 (± 0.0724, n=3)
19 0.0126 (± 0.00325, n=3)
20 0.0222 (± 0.00155, n=2)
28 0.0228 (± 0.0105, n=3)
29 0.0177 (± 0.0138, n=3)
30 0.0168 (± 0.0124, n=3)
3 1 0.0178 (± 0.0119, n=3)
32 0.0201 (± 0.0178, n=3)
33 0.0268 (± 0.0229, n=3)
34 0.0236 (± 0.0246, n=3)
35 0.0058
36 0.0208 (± 0.0168, n=3)
37 0.00622 (± 0.00286, n=3)
38 0.0222 (± 0.00824, n=3)
39 0.0206 (± 0.0129, n=4)
40 0.00695 (± 0.00456, n=4)
4 1 0.0138 (± 0.00253, n=3)
42 0.0220 (± 0.00397, n=3)
43 0.0110 (± 0.00217, n=3)
44 0.0112 (± 0.00315, n=3)
45 0.0188 (± 0.00510, n=4)
Mean + SEM; SEM = standard error of the mean
Data in Table 10 demonstrates that the compounds tested are inhibitors of Wnt
inducible genes as measured by the TOPFlash Wnt reporter assay.
Assessment of in vivo antitumor activity of tankyrase inhibitors:
C57BL/6J-Apc 7J strain mice carry a truncating mutation at codon 850 of the
Ape gene and develop intestinal polyps and colorectal neoplasms at the age of 3-6
months. Truncation in the APC gene leads to activation of the Wnt signaling pathway
and elevated levels of catenin are frequently detected in pre-neoplastic/neoplastic
lesions in these mice.
In order to assess the in vivo antitumor activity of tankyrase inhibitors, C57BL/6JApcM
n strain mice are purchased from Jackson Laboratories (stock number 002020) and
acclimated for 1 week. Animals are divided into 2 groups and treated with either 25
mg/kg (BID) of Example 1 or vehicle for 60 consecutive days. At the end of the
treatment period, the animals are sacrificed and the number of polyps in the small
intestine is counted under a dissection microscope. As shown in Table 11, C57BL/6JApcM
n strain mice treated with the compound of Example 1 has a statistically significant
lower number of tumors in the small intestine when compared to vehicle-treated animals.
Table 11
Data in Table 11 provides evidence that in vivo treatment with Example 1 reduces
the number of intestinal polyps in C57BL/6J-A 7J strain mice.
Colony Formation Assay
The compound of Example 1 is also tested in an in vitro colony formation assay
against four different human tumor cell lines, three derived from pancreatic tumors
(Capan-2, HPAF-II, and Pane 04.03) and one derived from non-small cell lung cancer
(A549). This assay is carried out using commercially available materials by procedures
known and routinely used by those skilled in the art.
A549 cells (ATCC, Manassas, VA # CCL-185) are maintained in complete
medium of F12K (Hyclone, Logan, UT #SH30526) containing 10% FBS (Invitrogen,
Grand Island, NY #16000-044). Capan-2 cells (ATCC, Manassas, VA # HTB-80) are
maintained in complete medium of McCoys 5A (Hyclone, Logan, UT #SH30200)
containing 10% FBS (Invitrogen, Grand Island, NY #16000-044). HPAF-II cells (ATCC,
Manassas, VA #CRL-1997) are maintained in complete medium of EMEM (Hyclone,
Logan, UT #SH30024) containing 10% FBS (Invitrogen, Grand Island, NY #16000-044).
Pane 04.03 cells (ATCC, Manassas, VA #CRL-2555) are maintained in complete medium
of RPMI (Hyclone, Logan, UT #SH30255) containing 15% FBS (Invitrogen, Grand
Island, NY #16000-044) and 20 g/ml Insulin (Sigma, St. Louis, MO #19278). Colony
formation assays are done by plating A549 cells at 250 cells/well; Capan-2 cells at 2000
cells/well; HPAF-II cells at 1000 cells/well or Pane 04.03 cells at 2000 cells/well each in
a 6-well plate (Corning Life Sciences, Tewksbury, MA #353046), incubating in 2 ml
complete medium and grown overnight at 37°C, 5% C0 2. The next day the medium is
removed and replaced with 2 ml fresh respective growth medium for each cell line. Test
compounds in 100% DMSO are diluted in 100% DMSO to 1000X concentration and 2 ΐ
added to 2 ml of medium in the well to achieve a final concentration of 0.03 to 3 .
Cells are incubated at 37°C, 5% C0 2. Every three to four days, the medium is removed
and replaced with 2 ml fresh complete medium for each cell line as before. After the
medium change, fresh compound is added as above. Cells are incubated a total of 11
days in the presence of compound. On day 11, the medium is removed and the cells
washed once with 5 ml DPBS (Hyclone, Logan, UT #SH30028). Crystal violet stain
(0.5% crystal violet (Sigma, St Louis, MO, #C3886) in 20% Methanol (EMD Millipore,
Billerica, MA #MX0490-4) is added to each well (0.4 ml)and incubated at room
temperature for 15 min. The wells are then washed twice with DPBS and the plates
photographed using the Fuji LAS4000 (FujiFilm, Tokyo, Japan). Analysis of the colony
area within each well is done using FujiFilm Colony Version 1.1 Software (FujiFilm,
Tokyo, Japan).
Table 12
Effect of Compound of Example 1 on Colony Formation
The data in Table 12 evidences the compound of Example 1 inhibits colony
formation when compared to control against each of the cell lines tested.
Table 13
Polypeptide Used in Assays Amino Acid Sequences
hTRFl (SEQ ID NO: 1)
NCBI, Accession number NP_059523.2
hTNKSl (SEQ ID NO: 2)
NCBI, Accession number NP_003738.2
hTNKS2 (SEQ ID NO: 3)
NCBI, Accession number NP_0795 11.1
hAxin2 (SEQ ID NO: 4)
NCBI, Accession number NP_004646.3
EGFP (SEQ ID NO: 5)
NCBI, Accession number ABG78037.1
Flag Peptide (SEQ ID NO: 6)
Sigma-Aldrich
EGFP (SEQ ID NO: 7)
Amino acids 228-466
NCBI, Accession number ABG78037.1
Sequences
SEP ID NO: 1 - hTRFl - protein
MAEDVSSAAPSPRGCADGRDADPTEEQMAETERNDEEQFECQELLECQVQVGA
PEEEEEEEEDAGLVAEAEAVAAGWMLDFLCLSLCRAFRDGRSEDFRRTRNSAEAI
IHGLSSLTACQLRTIYICQFLTRIAAGKTLDAQFENDERITPLESALMIWGSIEKEH
DKLHEEIQNLIKIQAIAVCMENGNFKEAEEVFERIFGDPNSHMPFKSKLLMIISQKD
TFHSFFQHFSYNHMMEKIKSYVNYVLSEKSSTFLMKAAAKVVESKRTRTITSQDK
PSGNDVEMETEANLDTRKSVSDKQSAVTESSEGTVSLLRSHKNLFLSKLQHGTQ
QQDLNKKERRVGTPQSTKKKKESRRATESRIPVSKSQPVTPEKHRARKRQAWLW
EEDKNLRSGVRKYGEGNWSKILLHYKFNNRTSVMLKDRWRTMKKLKLISSDSE
D
SEP ID NO: 2 - hTNKSl - protein
MAASRRSQHHHHHHQQQLQPAPGASAPPPPPPPPLSPGLAPGTTPASPTASGLAPF
ASPRHGLALPEGDGSRDPPDRPRSPDPVDGTSCCSTTSTICTVAAAPVVPAVSTSS
AAGVAPNPAGSGSNNSPSSSSSPTSSSSSSPSSPGSSLAESPEAAGVSSTAPLGPGA
AGPGTGVPAVSGALRELLEACRNGDVSRVKRLVDAANVNAKDMAGRKSSPLHF
AAGFGRKDVVEHLLQMGANVHARDDGGLIPLHNACSFGHAEVVSLLLCQGADP
NARDNWNYTPLHEAAIKGKIDVCIVLLQHGADPNIRNTDGKSALDLADPSAKAV
LTGEYKKDELLEAARSGNEEKLMALLTPLNVNCHASDGRKSTPLHLAAGYNRV
RIVQLLLQHGADVHAKDKGGLVPLHNACSYGHYEVTELLLKHGACVNAMDLW
QFTPLHEAASKNRVEVCSLLLSHGADPTLVNCHGKSAVDMAPTPELRERLTYEF
KGHSLLQAAREADLAKVKKTLALEIINFKQPQSHETALHCAVASLHPKRKQVTEL
LLRKGANVNEKNKDFMTPLHVAAERAHNDVMEVLHKHGAKMNALDTLGQTA
LHRAALAGHLQTCRLLLSYGSDPSIISLQGFTAAQMGNEAVQQILSESTPIRTSDV
DYRLLEASKAGDLETVKQLCSSQNVNCRDLEGRHSTPLHFAAGYNRVSVVEYLL
HHGADVHAKDKGGLVPLHNACSYGHYEVAELLVRHGASVNVADLWKFTPLHE
AAAKGKYEICKLLLKHGADPTKKNRDGNTPLDLVKEGDTDIQDLLRGDAALLD
AAKKGCLARVQKLCTPENINCRDTQGRNSTPLHLAAGYNNLEVAEYLLEHGAD
VNAQDKGGLIPLHNAASYGHVDIAALLIKYNTCVNATDKWAFTPLHEAAQKGR
TQLCALLLAHGADPTMKNQEGQTPLDLATADDIRALLIDAMPPEALPTCFKPQAT
VVSASLISPASTPSCLSAASSIDNLTGPLAELAVGGASNAGDGAAGTERKEGEVA
GLDMNISQFLKSLGLEHLRDIFETEQITLDVLADMGHEELKEIGINAYGHRHKLIK
GVERLLGGQQGTNPYLTFHCVNQGTILLDLAPEDKEYQSVEEEMQSTIREHRDG
GNAGGIFNRYNVIRIQKVVNKKLRERFCHRQKEVSEENHNHHNERMLFHGSPFIN
AIIHKGFDERHAYIGGMFGAGIYFAENSSKSNQYVYGIGGGTGCPTHKDRSCYIC
HRQMLFCRVTLGKSFLQFSTMKMAHAPPGHHSVIGRPSVNGLAYAEYVIYRGEQ
AYPEYLITYQIMKPEAPSQTATAAEQKT
SEP ID NO: 3 - hTNKS2 - protein
MSGRRCAGGGAACASAAAEAVEPAARELFEACRNGDVERVKRLVTPEKVNSRD
TAGRKSTPLHFAAGFGRKDVVEYLLQNGANVQARDDGGLIPLHNACSFGHAEV
VNLLLRHGADPNARDNWNYTPLHEAAIKGKIDVCIVLLQHGAEPTIRNTDGRTA
LDLADPSAKAVLTGEYKKDELLESARSGNEEKMMALLTPLNVNCHASDGRKSTP
LHLAAGYNRVKIVQLLLQHGADVHAKDKGDLVPLHNACSYGHYEVTELLVKHG
ACVNAMDLWQFTPLHEAASKNRVEVCSLLLSYGADPTLLNCHNKSAIDLAPTPQ
LKERLAYEFKGHSLLQAAREADVTRIKKHLSLEMVNFKHPQTHETALHCAAASP
YPKRKQICELLLRKGANINEKTKEFLTPLHVASEKAHNDVVEVVVKHEAKVNAL
DNLGQTSLHRAAYCGHLQTCRLLLSYGCDPNIISLQGFTALQMGNENVQQLLQE
GISLGNSEADRQLLEAAKAGDVETVKKLCTVQSVNCRDIEGRQSTPLHFAAGYN
RVSVVEYLLQHGADVHAKDKGGLVPLHNACSYGHYEVAELLVKHGAVVNVAD
LWKFTPLHEAAAKGKYEICKLLLQHGADPTKKNRDGNTPLDLVKDGDTDIQDLL
RGDAALLDAAKKGCLARVKKLSSPDNVNCRDTQGRHSTPLHLAAGYNNLEVAE
YLLQHGADVNAQDKGGLIPLHNAASYGHVDVAALLIKYNACVNATDKWAFTPL
HEAAQKGRTQLCALLLAHGADPTLKNQEGQTPLDLVSADDVSALLTAAMPPSAL
PSCYKPQVLNGVRSPGATADALSSGPSSPSSLSAASSLDNLSGSFSELSSVVSSSGT
EGASSLEKKEVPGVDFSITQFVRNLGLEHLMDIFEREQITLDVLVEMGHKELKEIG
INAYGHRHKLIKGVERLISGQQGLNPYLTLNTSGSGTILIDLSPDDKEFQSVEEEM
QSTVREHRDGGHAGGIFNRYNILKIQKVCNKKLWERYTHRRKEVSEENHNHANE
RMLFHGSPFVNAIIHKGFDERHAYIGGMFGAGIYFAENSSKSNQYVYGIGGGTGC
PVHKDRSCYICHRQLLFCRVTLGKSFLQFSAMKMAHSPPGHHSVTGRPSVNGLA
LAEYVIYRGEQAYPEYLITYQIMRPEGMVDG
SEP ID NO: 4 - hAxin2 - protein
MSSAMLVTCLPDPSSSFREDAPRPPVPGEEGETPPCQPGVGKGQVTKPMPVSSNT
RRNEDGLGEPEGRASPDSPLTRWTKSLHSLLGDQDGAYLFRTFLEREKCVDTLDF
WFACNGFRQMNLKDTKTLRVAKAIYKRYIENNSIVSKQLKPATKTYIRDGIKKQ
QIDSIMFDQAQTEIQSVMEENAYQMFLTSDIYLEYVRSGGENTAYMSNGGLGSLK
VVCGYLPTLNEEEEWTCADFKCKLSPTVVGLSSKTLRATASVRSTETVDSGYRSF
KRSDPVNPYHIGSGYVFAPATSANDSEISSDALTDDSMSMTDSSVDGIPPYRVGS
KKQLQREMHRSVKANGQVSLPHFPRTHRLPKEMTPVEPATFAAELISRLEKLKLE
LESRHSLEERLQQIREDEEREGSELTLNSREGAPTQHPLSLLPSGSYEEDPQTILDD
HLSRVLKTPGCQSPGVGRYSPRSRSPDHHHHHHSQYHSLLPPGGKLPPAAASPGA
CPLLGGKGFVTKQTTKHVHHHYIHHHAVPKTKEEIEAEATQRVHCFCPGGSEYY
CYSKCKSHSKAPETMPSEQFGGSRGSTLPKRNGKGTEPGLALPAREGGAPGGAG
ALQLPREEGDRSQDVWQWMLESERQSKPKPHSAQSTKKAYPLESARSSPGERAS
RHHLWGGNSGHPRTTPRAHLFTQDPAMPPLTPPNTLAQLEEACRRLAEVSKPPK
QRCCVASQQRDRNHSATVQTGATPFSNPSLAPEDHKEPKKLAGVHALQASELVV
TYFFCGEEIPYRRMLKAQSLTLGHFKEQLSKKGNYRYYFKKASDEFACGAVFEEI
WEDETVLPMYEGRILGKVERID
SEP ID NO: 5 - EGFP - full length protein
MDRKFVFLVSILSIVVASVTGETTRAPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTP
TPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPT
PTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTP
TPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTPTSMVSKGEELFTGVV
PILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYG
VQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV
NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGS
VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGI
TLGMDELYK
SEQ ID NO: 6 - Flag Peptide
DYKDDDDK
SEP ID NO: 7 - EGFP - truncated protein amino acids 228-466
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLP
VPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKT
RAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
MVLLEFVTAAGITLGMDELYK
We claim:
1. A compound having the formula:
wherein:
Y is:
X is -CH 2-, -C(CH )H-, or-C(CH 2CH )H-;
R is hydrogen, hydroxy, or halo;
R2 is hydrogen, halo, -CN, -CH , CF , or -OCH
or a pharmaceutically acceptable salt thereof.
2. A compound of claim 1 wherein:
Y is:
X is -CH 2-, -C(CH )H-, or -C(CH 2CH )H-;
R is hydrogen, hydroxy, or halo;
R2 is hydrogen, halo, -CN, -CH , CF , or -OCH ;
or a pharmaceutically acceptable salt thereof.
3. A compound of claim 1 or 2 wherein:
Y is
X is -CH 2-, -C(CH3)H-, or-C(CH 2CH )H-;
R2 is hydrogen, halo, -CN, -CH , CF , or -OCH ;
or a pharmaceutically acceptable salt thereof.
4. A compound of claim 1 or 2 wherein:
Y is:
X is -CH 2-, -C(CH )H-, or-C(CH 2CH )H-;
R is hydrogen, hydroxy, or halo;
R2 is hydrogen, halo, -CN, -CH , CF , or -OCH ;
or a pharmaceutically acceptable salt thereof.
5. A compound of any one of claims 1, 2, or 3 which is:
8-Methyl-2-[4-(pyrimidin-2-ylmethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof;
8-Methyl-2-[4-(l-pyrimidin-2-ylethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof thereof;
2-[4-[(4-Chloropyrimidin-2-yl)methyl]piperazin-l-yl]-8-methyl-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof; or
2-[4-[(4-methoxypyrimidin-2-yl)methyl]piperazin-l-yl]-8-methyl-3, 5,6,7-
tetrahydropyrido[2,3-d]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof.
6. A compound of any one of claims 1, 2, or 4 which is:
2-[4-[(3-Bromo-2-pyridyl)methyl]piperazin-l-yl]-8-methyl-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one or a pharmaceutically acceptable salt thereof;
2-[4-[(3-Chloro-2-pyridyl)methyl]piperazin-l-yl]-8-methyl-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, , or a pharmaceutically acceptable salt thereof;
2-[4-[(3-Fluoro-2-pyridyl)methyl]piperazin-l-yl]-8-methyl-3,5,6,7-tetrahydropyrido[2,3-
d]pyrimidin-4-one, , or a pharmaceutically acceptable salt thereof; or
2-[[4-(8-Methyl-4-oxo-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-2-yl)piperazin-lyl]
methyl]pyridine-3-carbonitrile, or a pharmaceutically acceptable salt thereof.
7. A compound of any one of claims 1, 2, 3 or 5 which is 8-methyl-2-[4-
(pyrimidin-2-ylmethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-
one, or a pharmaceutically acceptable salt thereof.
8. A compound of any one of claims 1, 2, 3, 5 or 7 which is 8-Methyl-2-[4-
(pyrimidin-2-ylmethyl)piperazin-l-yl]-3,5,6,7-tetrahydropyrido[2,3-d]pyrimidin-4-one 4-
methylbenzenesulfonic acid salt.
9. A pharmaceutical composition comprising a compound of any one of claims 1
to 8, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically
acceptable carrier.
10. A method of treating a cancer which is colorectal cancer, gastric cancer, liver
cancer, breast cancer, triple negative breast cancer, ovarian cancer, medulloblastoma,
melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer,
glioblastoma, T-cell lymphoma, T-lymphoblastic lymphoma, T-cell acute lymphocytic
leukemia (T-ALL), mantle cell lymphoma, multiple myeloma, chronic myeloid leukemia,
or acute myeloid leukemia in a patient comprising administering to a patient in need
thereof a therapeutically effective amount of a compound of any one of claims 1 to 8, or a
pharmaceutically acceptable salt thereof.
11. A compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt
thereof, for use in therapy.
12. A compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt
thereof, for use in the treatment of a cancer which is colorectal cancer, gastric cancer,
liver cancer, breast cancer, triple negative breast cancer, ovarian cancer,
medulloblastoma, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer,
prostate cancer, glioblastoma, T-cell lymphoma, T-lymphoblastic lymphoma, T-cell acute
lymphocytic leukemia (T-ALL), mantle cell lymphoma, multiple myeloma, chronic
myeloid leukemia, or acute myeloid leukemia.