THIENO[2,3-C]PYRROL-4-ONE DERIVATIVES AS ERK INHIBITORS
The present invention relates to thieno[2,3-c]pyrrol-4-one compounds, or
pharmaceutically acceptable salts thereof, and pharmaceutical compositions comprising
the compounds, that inhibit activity of extracellular- signal-regulated kinase (ERK) and
may be useful for treating cancer.
The ERK/MAPK pathway is important for cell proliferation and frequently
observed to be activated in many tumors. RAS genes, which are upstream of ERKl/2, are
mutated in several cancers including colorectal, melanoma, non-small cell lung cancer as
well as breast and pancreatic tumors. High RAS activity is accompanied by elevated
ERK activity in many human tumors. Studies have also shown that ERK is a critical
component of RAS signalling. These observations support the attractiveness of the
ERKl/2 signaling pathway for developing anticancer therapies in a broad spectrum of
human tumors.
ERK inhibitors are known in the art; see, for example, WO2013 130976.
Additionally, other aminopyrimidine compounds are known in the art; see, for example,
WO 2010/022121. There remains a need to provide alternative ERK inhibitors, more
particularly for the treatment of cancer. Accordingly, the present invention provides
ERKl/2 inhibitors which may be useful for treating cancer.
The present invention provides a compound of the following formula:
Formula I
wherein:
R2 and R are independently methyl or R2 and R can be taken together to
form cyclopropyl;
R is hydrogen, methyl, chloro, fluoro, or trifluromethyl; and
R5 is
or a pharmaceutically acceptable salt thereof.
The present invention also provides a compound of the following formula:
Formula I
wherein:
R is
R2 and R3 are independently methyl or R2 and R3 can be taken together to
form cyclopropyl;
R is hydrogen, methyl, chloro, fluoro, or trifluoromethyl; and
R5 is
or a pharmaceutically acceptable salt thereof.
The present invention also provides an embodiment for a compound of Formula I
wherein R2 and R3 are methyl.
The present invention also provides another embodiment for a compound of
Formula I wherein R4 is hydrogen.
The present invention also provides yet another embodiment for a compound of
Formula I wherein R1 is .
The present invention also provides yet a further embodiment for a compound of
Formula I wherein R5 is
Preferably, the present invention provides a compound which is 6,6-dimethyl-2-
{2-[(l-methyl-lH-pyrazol-5-yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-
dihydro-4H-thieno[2,3-c]pyrrol-4-one, or a pharmaceutically acceptable salt thereof.
As a particular embodiment, the present invention provides the compound which
is 6,6-dimethyl-2-{2-[(l-methyl-lH-pyrazol-5-yl)amino]pyrimidin-4-yl}-5-[2-
(morpholin-4-yl)ethyl]-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one.
The present invention provides a pharmaceutical composition comprising 6,6-
dimethyl-2-{2-[(l-methyl-lH-pyrazol-5-yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-
yl)ethyl]-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one, or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable carrier, diluent, or excipient. The present
invention provides a pharmaceutical composition comprising 6,6-dimethyl-2-{2-[(lmethyl-
lH-pyrazol-5-yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-
4H-thieno[2,3-c]pyrrol-4-one, and a pharmaceutically acceptable carrier, diluent, or
excipient.
The present invention provides a method for treating cancer comprising
administering to a patient in need thereof an effective amount of 6,6-dimethyl-2-{2-[(lmethyl-
lH-pyrazol-5-yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-
4H-thieno[2,3-c]pyrrol-4-one, or a pharmaceutically acceptable salt thereof. The present
invention provides a method for treating cancer comprising administering to a patient in
need thereof an effective amount 6,6-dimethyl-2-{2-[(l-methyl-lH-pyrazol-5-
yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4H-thieno[2,3-
c]pyrrol-4-one.
The present invention provides 6,6-dimethyl-2-{2-[(l-methyl-lH-pyrazol-5-
yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4H-thieno[2,3-
c]pyrrol-4-one, or a pharmaceutically acceptable salt thereof, for use in therapy. The
present invention provides 6,6-dimethyl-2-{2-[(l-methyl-lH-pyrazol-5-
yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4H-thieno[2,3-
c]pyrrol-4-one, or a pharmaceutically acceptable salt thereof, for use in the treatment of
cancer. The present invention provides a pharmaceutical composition for use in treating
cancer, the pharmaceutical composition comprising 6,6-dimethyl-2-{2-[(l-methyl-lHpyrazol-
5-yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4Hthieno[
2,3-c]pyrrol-4-one, or a pharmaceutically acceptable salt thereof.
The present invention also provides 6,6-dimethyl-2-{2-[(l-methyl-lH-pyrazol-5-
yl)armno]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4H-thieno[2,3-
c]pyrrol-4-one for use in therapy. The present invention provides 6,6-dimethyl-2-{2-[(lmemyl-
lH-pyrazol-5-yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-
4H-thieno[2,3-c]pyrrol-4-one for use in the treatment of cancer. The present invention
provides a pharmaceutical composition for use in treating cancer, the pharmaceutical
composition comprising 6,6-dimethyl-2-{2-[(l-methyl-lH-pyrazol-5-yl)amino]pyrimidin-
4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one.
The present invention provides the use of 6,6-dimethyl-2-{2-[(l-methyl-lHpyrazol-
5-yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4Hthieno[
2,3-c]pyrrol-4-one, or a pharmaceutically acceptable salt thereof, in the
manufacture of a medicament for the treatment of cancer. The present invention also
provides the use of 6,6-dimethyl-2-{2-[(l-methyl-lH-pyrazol-5-yl)amino]pyrimidin-4-
yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one in the
manufacture of a medicament for the treatment of cancer.
The present invention provides 6,6-dimethyl-2-{2-[(l-methyl-lH-pyrazol-5-
yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4H-thieno[2,3-
c]pyrrol-4-one in a crystalline form. The present invention also provides 6,6-dimethyl-2-
{2-[(1-methyl- 1H-pyrazol-5 -yl)amino]pyrimidin-4-yl }-5- [2-(morpholin-4-yl)ethyl] -5,6-
dihydro-4H-thieno[2,3-c]pyrrol-4-one in a crystalline form characterized by a X-ray
powder diffraction pattern having characteristic peaks, in 2+ 0.2°, occurring at 19.3° in
combination with one or more of the peaks selected from the group consisting of 15.5°,
17.1°, 18.0°, 20.2°, 21.5° and 22. .
Furthermore, the present invention provides preferred embodiments of the
methods and uses as described herein, in which cancer is selected from the group
consisting of melanoma, colorectal cancer, pancreatic cancer, and non-small cell lung
cancer. Preferred cancers are colorectal cancer, pancreatic cancer, and non-small cell
lung cancer.
The present invention also provides 6,6-dimethyl-2-{2-[(l-methyl-lH-pyrazol-5-
yl)armno]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4H-thieno[2,3-
c]pyrrol-4-one, or a pharmaceutically acceptable salt thereof, for use in simultaneous,
separate or sequential administration in combination with one or more chemotherapy
agents in the treatment of cancer. Preferred chemotherapy agents for such a combination
are a pan-RAF inhibitor compound, more particularly l-(3,3-dimethylbutyl)-3-(2-fluoro-
4-methyl-5-(7-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-6-yl)phenyl)urea), a
CDK4/6 inhibitor compound, more particularly palbociclib, ribociclib, or [5-(4-ethylpiperazin-
l-ylmethyl)-pyridin-2-yl]-[5-fluoro-4-(7-fluoro-3-isopropyl-2-methyl-3Hbenzoimidazol-
5-yl)-pyrimidin-2-yl]-amine, or a pharmaceutically acceptable salt thereof,
or an anti-VEGFR2 antibody, more particularly ramucirumab. Additional preferred
chemotherapy agents for such a combination are a TGF-beta receptor kinase inhibitor
compound, more particularly galunisertib (see WO 2004/048382), an ALK-5 kinase
inhibitor, more particularly EW-7197, a MEK inhibitor compound, more particularly
cobimetinib or trametinib, or a Notch inhibitor compound, more particularly 4,4,4-
trifluoro-N-[(lS)-2-[[(7S)-5-(2-hydroxyethyl)-6-oxo-7H-pyrido[2,3-d][3]benzazepin-7-
yl]amino]-l-methyl-2-oxo-ethyl]butanamide (see WO 2013/016081). Further additional
preferred chemotherapy agents for such a combination are a PD-Ll (Programmed deathligand
1) inhibitor or a PD-1 (Programmed death 1) inhibitor.
The present invention preferably contains compounds of Formula I with the
followin substituents:
b) R2 is methyl;
c) R3 is methyl;
R4 is hydrogen; or
More preferably, the present invention contains compounds of Formula I with the
following combinations of substituents:
a) R2 and R are methyl;
b R2 is meth l, R3 is methyl, and R4 is hydrogen;
c) R^s and R5 is
d) R2 is methyl, R3 is methyl, R4 is hydrogen, and R1 is
e) R2 is methyl, R3 is methyl, R4 is hydrogen, and R is or
f) R2 is methyl, R3 is methyl, R4 is hydrogen, and R1 is , and R5 is
As used above, and throughout the description of the invention, the following
terms, unless otherwise indicated, shall be understood to have the following meanings:
A "pharmaceutically acceptable carrier, diluent, or excipient" is a medium
generally accepted in the art for the delivery of biologically active agents to mammals,
e.g., humans.
"Pharmaceutically acceptable salts" or "a pharmaceutically acceptable salt" refers
to the relatively non-toxic, inorganic and organic salt or salts of the compound of the
present invention.
"Effective amount" means the amount of the compound, or pharmaceutically
acceptable salt thereof, of the present invention or pharmaceutical composition containing
a compound, or pharmaceutically acceptable salt thereof, of the present invention that will
elicit the biological or medical response of or desired therapeutic effect on a tissue,
system, animal, mammal or human that is being sought by the researcher, veterinarian,
medical doctor or other clinician.
The terms "treatment," "treat," "treating," and the like, are meant to include
slowing or reversing the progression of a disorder. These terms also include alleviating,
ameliorating, attenuating, eliminating, or reducing one or more symptoms of a disorder or
condition, even if the disorder or condition is not actually eliminated and even if
progression of the disorder or condition is not itself slowed or reversed.
It will be understood by the skilled artisan that compounds of the present
invention are capable of forming salts. The compounds of the present invention contain
basic heterocycles, and accordingly react with any of a number of inorganic and organic
acids to form pharmaceutically acceptable acid addition salts. Such pharmaceutically
acceptable acid addition 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, 2008); S.M.
Berge, et al, "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, Vol 66, No. 1,
January 1977.
The compounds of the present invention are preferably formulated as
pharmaceutical compositions administered by a variety of routes. Preferably, such
compositions are for oral administration. Such pharmaceutical compositions and
processes for preparing the same are well known in the art. See, e.g., REMINGTON:
THE SCIENCE AND PRACTICE OF PHARMACY (A. Gennaro, et al, 2 1s ed., Mack
Publishing Co., 2005).
The compounds of the present invention are generally effective over a wide
dosage range. For example, dosages per day normally fall within the daily range of about
1 to 2000 mg. Preferably such doses fall within the daily range of 50 to 1000 mg. More
preferably such doses fall within the daily range of 125 to 400 mg. In some instances
dosage levels below the lower limit of the aforesaid ranges may be more than adequate,
while in other cases still larger doses may be employed, and therefore the above dosage
ranges are not intended to limit the scope of the invention in any way. It will be
understood that the amount of the compound actually administered will be determined by
a physician, in the light of the relevant circumstances, including the condition to be
treated, the chosen route of administration, the actual compound or compounds
administered, the age, weight, and response of the individual patient, and the severity of
the patient's symptoms.
The skilled artisan will appreciate that certain compounds of the present invention
contain at least one chiral center. The present invention contemplates all individual
enantiomers or diastereomers, as well as mixtures of the enantiomers and diastereomers
of said compounds including racemates. It is preferred that compounds of the present
invention containing at least one chiral center exist as single enantiomers or
diastereomers. The single enantiomers or diastereomers may be prepared beginning with
chiral reagents or by stereoselective or stereospecific synthetic techniques. Alternatively,
the single enantiomers or diastereomers may be isolated from mixtures by standard chiral
chromatographic or crystallization techniques.
The designation of "isomer 1" in a compound name represents that the
corresponding intermediate or compound of the present invention is the first of two
eluting enantiomers when a mixture of a pair of enantiomers is separated by chiral
chromatography. The designation of "isomer 2" in a compound name represents that the
corresponding intermediate or compound of the present invention that is the second of
two eluting enantiomers when the mixture of a pair of enantiomers is separated by chiral
chromatography.
The compounds of the present invention can be prepared according to synthetic
methods well known and appreciated in the art. Suitable reaction conditions for the steps
of these reactions are well known in the art and appropriate substitutions of solvents and
co-reagents are within the skill of the art. Likewise, it will be appreciated by those skilled
in the art that synthetic intermediates may be isolated and/or purified by various well
known techniques as needed or desired, and that frequently, it will be possible to use
various intermediates directly in subsequent synthetic steps with little or no purification.
Furthermore, the skilled artisan will appreciate that in some circumstances, the order in
which moieties are introduced is not critical. The particular order of steps required to
produce the compounds of the present invention is dependent upon the particular
compound being synthesized, the starting compound, and the relative liability of the
substituted moieties, as is well appreciated by the skilled chemist. All substituents, unless
otherwise indicated, are as previously defined, and all reagents are well known and
appreciated in the art.
As used herein, the following terms have the meanings indicated: "ACN" refers to
acetonitrile; "DCM" refers to dichloromethane; "DMF" represents N,Ndimethylformamide;
"DMSO" refers to dimethyl sulfoxide; "DTT" refers to
dithiothreitol; "EDTA" refers to ethylenediaminetetraacetic acid; "EGTA" refers to
ethylene glycol tetraacetic acid; "ELISA" refers to enzyme-linked immunosorbent assay;
"EtOAc" refers to ethyl acetate; "EtOH" refers to ethanol; "FBS" refers to fetal bovine
serum; "HBSS" refers to Hank's Balanced Salt Solution; "IC 50" refers to half maximal
inhibitory concentration; "IVTI" refers to in vivo target inhibition; "MS" refers to mass
spectroscopy; "MeOH" refers to methanol; "NMR" refers to nuclear magnetic resonance;
"PBST" refers to phosphate buffered saline containing Tween-20; "THF" refers to
tetrahydrofuran; "UVW" refers to ultra-violet wavelength, and "XRD" refers to X-ray
diffraction.
Unless noted to the contrary, the compounds illustrated herein are named and
numbered using either ACDLABS or Accelrys Draw 4.1.
Compounds of the present invention may be synthesized as illustrated in the
following schemes, where R1, R2, R , R4, and R5 are as previously defined.
Scheme 1: Synthesis of compounds of Formula I
Scheme 1 illustrates the general synthesis of compounds of Formula I. Compound
1 is reacted with a suitably substituted Compound 2 under well-known aromatic
substitution or coupling reaction conditions to provide a compound of Formula I. More
specifically, Compound 1 is reacted with Compound 2 at elevated temperature in the
presence of a suitable base such as sodium hydride, isopropylmagnesium chloride, cesium
carbonate, potassium carbonate or i7. Separate the phases and dry the
organic phase over anhydrous sodium sulfate. Filter the mixture and concentrate the
filtrate under reduced pressure. Concentrate the aqueous phase under reduced pressure.
Combine the residues from the organic and aqueous phases and purify by reverse phase
column chromatography (Column: 130 g C18; Mobile Phase: A) water, B) ACN;
Gradient: 0-20% B). Concentrate the fractions and dissolve the residue in 25% MeOH in
DCM. Filter the mixture and concentrate the filtrate under reduced pressure. Dissolve
the residue in EtOAc and add water. Separate the layers and back extract the aqueous
layer with EtOAc (8 x 100 mL). Concentrate the combined organic extract under reduced
pressure to give the title compound 2.27 g (76%). H NMR (400.1 MHz, CD3CN) 7.02
(d, J=2 Hz, 1H), 5.33 (d, J=2 Hz, 1H), 4.28 (bs, 2H), 3.10 (m, 1H), 0.96 (m, 4H).
Preparation 36
2-(Dibenzylamino)ethanol
Treat a mixture of 2-(benzylamino)ethanol (0.95 mL, 6.6 mmol) in ACN (35 mL)
with potassium carbonate (1.83 g, 13.2 mmol) followed by benzyl bromide (1.18 mL,
9.89 mmol). Heat the reactions mixture at 80 °C for 1.5 hours. Cool the reaction to room
temperature and filter the mixture. Concentrate the filtrate under reduced pressure and
purify the residue by silica gel column chromatography eluting with a gradient from 0-
30% EtOAc in hexane to give the title compound 1.7 g (100%). MS (m/z): 242 (M+l).
Preparation 37
2-[2-(Dibenzylamino)ethoxy]-2,2-difluoro-acetic acid
Treat a solution of 2-(dibenzylamino)ethanol (1.5 g, 6.2 mmol) and sodium
chloro-2,2-difluoro-acetic acid (950 mg, 6.19 mmol) in THF (12 mL) at 0 °C with sodium
hydride (60 wt % in mineral oil, 500 mg, 12.5 mmol). Heat the reaction mixture to reflux
overnight. Add additional sodium hydride (60 wt% in mineral oil, 120 mg, 3 mmol) to
the reaction mixture and continue heating for an additional hour. Cool the reaction to
room temperature and dilute with water. Extract the mixture with diethyl ether. Separate
the layers and adjust the aqueous layer to pH 6 with 6 N hydrochloric acid. Extract the
aqueous solution with EtOAc. Combine all organic solutions and dry over anhydrous
sodium sulfate. Filter the mixture and concentrate the filtrate under reduced pressure to
give the title compound 1.03 g (49%). MS (m/z): 336 (M+l).
Preparation 38
Methyl 2-[2-(dibenzylamino)ethoxy]-2,2-difluoro-acetate
Treat a solution of 2-[2-(dibenzylamino)ethoxy]-2,2-difluoro-acetic acid (100 mg,
0.298 mmol) in toluene (9 mL) and MeOH (2 mL) with (trimethylsilyl)diazomethane (2
M in hexane, 0.16 mL, 0.32 mmol) drop wise. Stir the mixture for 15 minutes at room
temperature. Quench the reaction with acetic acid (0.1 mL) and concentrate the reaction
mixture under reduced pressure to give the title compound 102 mg (98%). MS (m/z): 350
(M+l).
Preparation 39
4-Benzyl-2,2-difluoro-morpholin-3-one
Treat a suspension of palladium (10% on carbon, 50 mg, 0.141 mmol) in EtOH
(15 mL) with methyl 2-[2-(dibenzylamino)ethoxy]-2,2-difluoro-acetate (485 mg, 1.39
mmol) in EtOH (15 mL). Stir the reaction mixture under a hydrogen atmosphere
(balloon) at room temperature overnight. Filter the reaction mixture through CELITE®
and concentrate the filtrate under reduced pressure to give the title compound 294 mg
(93%). MS (m/z): 228 (M+l).
Preparation 40
4-Benzyl-2,2-difluoro-morpholine
Treat a solution of 4-benzyl-2,2-difluoro-morpholin-3-one (290 mg, 1.28 mmol)
in THF (13 mL) with boron dimethyl sulfide complex (2 M in THF, 3.06 mL, 6.12
mmol). Heat the reaction mixture at 55 °C for 3.5 hours and then remove the heat and
continue stirring overnight. Heat the reaction mixture to 55 °C for an additional two
hours. Cool the reaction mixture to room temperature and quench by the dropwise
addition of hydrochloric acid (6 N, 3.06 mL, 18.4 mmol). Heat the reaction mixture at
100 °C for one hour. Cool the mixture to room temperature and concentrate under
reduced pressure. Dilute the mixture with water and adjust the pH to 12 with 2 N sodium
hydroxide. Extract the mixture with EtOAc. Dry the organic extracts over anhydrous
sodium sulfate, filter and concentrate the filtrate under reduced pressure to give the title
compound 120 mg (44%). MS (m/z): 214 (M+l).
Preparation 41
2-Bromo-6,6-dimethyl-5-[2-(5-oxa-8-azaspiro[2.6]nonan-8-yl)ethyl]thieno[2,3-c]pyrrol-
4-one
Heat a mixture of 2-(2-bromo-6,6-dimethyl-4-oxo-thieno[2,3-c]pyrrol-5-yl)ethyl
methanesulfonate (2.76 g, 6.9 mmol) and 5-oxa-8-azaspiro[2.6]nonane (2.18, 16.3 mmol)
in DMF (33 mL) at 80 °C overnight. Cool the mixture to room temperature and dilute
with EtOAc. Wash the organic solution with saturated NaCl (3 x 30 mL). Dry the
organic solution over anhydrous sodium sulfate, filter and concentrate the filtrate under
reduced pressure. Purify the residue by reverse phase column chromatography (Column:
lOOg Gold C18; Mobile Phase: A) 0.1% formic acid in water, B) 0.1% formic acid in
ACN; Gradient: 5% B for 5 minutes, 5%-50% B over 20 minutes; Flow Rate: 53
mL/minute) to give the title compound 3.6 g (85%). MS (m/z): 399/401 (M+l/M+3).
Preparation 42
i A, V600E) accounts for 80% of the mutations.
BRAF mutations are found, with rare exceptions, in a mutually exclusive pattern with
RAS mutations, suggesting that these genetic alterations activate common downstream
effectors.
Biological Assays
The following assays demonstrate that the exemplified compounds of the present
invention are inhibitors of ERK1 and ERK2 kinase activity. The results of the following
assays also demonstrate that the exemplified compounds of the present invention inhibit
ERK signaling in cancer cells. Additionally, the compound of Example 1 demonstrates
ERK pathway target inhibition in certain xenograft tumor models of cancer. Furthermore,
the compound of Example 1 inhibits tumor growth in certain xenograft tumor models of
cancer.
ERK1 Kinase Assay
The purpose of this assay is to measure the ability of compounds to inhibit ERK1
kinase activity. Perform the ERK1 kinase assay in vitro using a TR-FRET assay. Start
reactions (12.5 ) by adding 5 of ERK1 enzyme (Invitrogen, #PR5254B, final
concentration 100 ng/mL) plus substrate GFP-ATF2 (Invitrogen, # PV4445, final
concentration 0.2 ), 5 of ATP solution (Invitrogen, # PV3227, final concentration
10 M) prepared in kinase buffer (50 mM Hepes pH 7.4, 5 mM MgCl2, 0.1 mM EGTA,
0.01% Triton X-100, 1mM DTT) and 2.5 of testing compounds in DMSO solution
(final 4%, v/v) in a 384-well PROXIPLATE™ (Perkin Elmer, #GRN6260). Incubate the
reaction mixture at room temperature for 60 minutes. Stop the reaction by addition of
12.5 of stop buffer (10 mM EDTA, 2 nM Tb-anti-pATF2 (pThr71) antibody,
Invitrogen, #PV4448) in TR-FRET dilution buffer (Invitrogen, # PV3574). Incubate the
plates at room temperature for an additional 60 minutes and read on an ENVISION®
(PerkinElmer) plate reader at the excitation wavelength 340 nm. Calculate the TR-FRET
ratio by dividing the GFP acceptor emission signal (at 520 nm) by the Tb donor emission
signal (at 495 nm). Calculate percent inhibition using compound treated wells relative to
on-plate Max (DMSO control) and Min (No enzyme added) control wells TR-FRET ratio
data { inhibition = 100-[(test compound - median Min)/(median Max - median Min) X
100]}. Test all compounds at 10 concentrations (20 to 0.001 ) using a 1:3 dilution
scheme. Derive Abs_ICso values by fitting percent inhibition and ten-point concentration
data to a 4-parameter nonlinear logistic equation (equation 205) using
ACTIVITYBASE® 7.3 (ID Business Solutions Limited).
The exemplified compounds within the scope of the invention are tested in this
assay substantially as described above. The results of this assay demonstrate that all of
the exemplified compounds inhibit ERK1 kinase activity, with IC50 values less than 0.15
. For example, the compound of Example 1 has an IC50 value of 4.86 nM (±0.20,
n=7).
ERK2 Kinase Assay
The purpose of this assay is to measure the ability of compounds to inhibit ERK2
kinase activity. Perform the ERK2 kinase assay in vitro using a TR-FRET assay. Start
all reactions (12.5 ) by adding 5 ΐ of ERK2 enzyme (Invitrogen, #PV3595B, final
cone 50 ng/mL) plus substrate GFP-ATF2 (Invitrogen, #PV4445, final cone 0.2 ), 5
ΐ of ATP solution (Invitrogen, #PV3227, final cone 10 ) prepared in kinase buffer
(50 mM Hepes pH 7.4, 5 mM MgCl2, 0.1 mM EGTA, 0.01% Triton X-100, 1mM DTT)
and 2.5 ΐ of testing compounds in DMSO solution (final 4%, v/v) in a 384-well
PROXIPLATE™ (Perkin Elmer, #GRN6260). Incubate reactions at room temperature
for 60 minutes. Stop reactions by addition of 12.5 ΐ of stop buffer (10 mM EDTA, 2
nM Tb-anti-pATF2 (pThr71) antibody, Invitrogen, #PV4448) in TR-FRET dilution buffer
(Invitrogen, # PV3574). Incubate the plates at room temperature for an additional 60
minutes and read ON ENVISION® (PerkinElmer) plate reader at the excitation
wavelength of 340 nm. Calculate a TR-FRET ratio by dividing the GFP acceptor
emission signal (at 520 nm) by the Tb donor emission signal (at 495 nm). Calculate
percent inhibition using compound wells relative to on-plate Max (DMSO control) and
Min (No enzyme added) control wells TR-FRET ratio data {% inhibition = 100- [(test
compound - median Min)/(median Max - median Min) X 100] }. Test all compounds at
10 concentrations (20 to 0.001 ) using a 1:3 dilution scheme. Derive Abs_IC50
values by fitting percent inhibition and ten-point concentration data to a 4-parameter
nonlinear logistic equation (equation 205) using ACTIVITYBASE 7.3 (ID Business
Solutions Limited).
The exemplified compounds within the scope of the invention are tested in this
assay substantially as described above. The results of this assay demonstrate that all of
the exemplified compounds inhibit ERK2 kinase activity, with IC50 values less than 0.15
. For example, the compound of Example 1 has an IC50 value of 5.24 nM (±0.24,
n=7).
ERK1/2 Cell Mechanistic Assay (pRSKl Alphascreen Assay)
The purpose of this assay is to measure the ability of compounds to inhibit ERK
signaling in cancer cells in vitro. Carry out the pRSKl Alphascreen assay using the
HCT1 16 colorectal cancer cell line (ATCC, # CCL-247). Routinely culture HCT1 16
cells in Dulbecco's Modified Eagle's Medium (DMEM) (Hyclone, #SH30022) growth
medium containing 5% Fetal Bovine Serum (FBS) (Gibco, #16000-044) in T-150 flasks
and incubate in a 5% CO2 incubator at 37 °C. Harvest cells when they become confluent
and freeze in freezing medium at IxlOe 7 cells/mL as "assay ready frozen cells" and store
in liquid nitrogen. To run the assay, plate 40,000 HCT116 cells/well in a 96-well tissue
culture plate and incubate at 37 °C in a 5% CO2 incubator overnight. Test compounds at
10 concentrations starting at a 20 top concentration and utilize a 1:3 dilution scheme
(20 to 0.001 ) with a final DMSO concentration of 0.5% (v/v). Add compounds
in 20 ΐ serum free growth medium and incubate at 37 °C for two hours. Remove
growth medium and add 50 ΐ of l x lysis buffer [Cell Signaling Technology, #9803]
containing l x holt protease and phosphate inhibitor cocktail [Thermo, #78441] to each
well and incubate at room temperature for 10 minutes on a shaker. Transfer 4 ΐ of cell
lysate from each well to respective wells in a 384 well assay plate [Perkin Elmer,
#6006280] and add 5 ΐ of reaction mix [2000 parts l x assay buffer (Perkin Elmer,
#A1000), 1 part biotin-RSKl antibody (Santa Cruz, #sc-23 1-B-G), 4 parts pRSKl
antibody (Abeam, #ab32413), 35 parts acceptor beads (Perkin Elmer, #6760617R)]. Seal
the plate with foil plate seal (Beckman Coulter, # 538619) and incubate at room
temperature for two hours. Add 2 ΐ of donor beads [20 parts l x assay buffer, 1 part
donor beads] to each well and seal the plate with clear plate seal (Applied Biosystems,
#431 1971) and incubate at room temperature in the dark for two hours. Measure the
fluorescence intensity in each well by reading the plates in ENVISION® (PerkinElmer)
plate reader. Derive the Rel IC50 values by fitting percent pRSKl inhibition [% inhibition
=100- [(test compound - median Min)/ (median Max-median Min) X 100] and ten-point
concentration data to a 4-parameter nonlinear logistic equation (Abase equation 205)
using ACTIVITYBASE® 7.3 (ID Business Solutions Limited).
The exemplified compounds within the scope of the invention are tested in this
assay substantially as described above. The results of this assay demonstrate that all of
the exemplified compounds inhibit ERK substrate (RSK) phosphorylation in tumor cells,
with IC50 values less than 3 . For example, the compound of Example 1 has an IC50
value of 0.429 (+ 0.173, n=8).
In Vivo Target inhibition (IVTI) Assay (pRSKl ELISA Assay)
The purpose of this assay is to measure the ability of a test compound to inhibit
ERK1/2 substrate phosphorylation in an animal model. Implant female athymic nude
mice (22-25 g) from Harlan Laboratories with 5xl0e 6 HCT116 colorectal cancer cells
(ATCC, # CCL-247) subcutaneously in the right flank region in 200 L of 1:1 Hank's
Balanced Salt Solution (HBSS) + Matrigel solution. Measure tumor growth and body
weight twice per week beginning the seventh day after the implantation. When tumor
sizes reach 300-500 mm3, randomize animals and group into groups of five animals.
Dose animals with either compound at an appropriate dose in a compound specific
vehicle or vehicle alone (vehicle: 1% HEC/0.25% Tween 80/0.05% Antifoam) orally and
collect tumors and blood at desired time intervals after dosing. Sacrifice animals using
isoflurane anesthesia plus cervical dislocation. Flash freeze tumors and store at -80 °C
until processing for pRSKl levels by ELISA assay. Collect blood in EDTA tubes and
spin down for plasma and freeze at -80°C in a 96-well plate. Determine compound
exposures using standard methods.
Pulverize tumors in liquid nitrogen and lyse in l x lysis buffer (MSD, #R60TX-3)
containing l x halt protease & phosphatase inhibitor cocktail (Thermo Scientific,
#0861281), 1mM phenylmethanesulfonyl fluoride (PMSF) (Sigma, # 93482-50ML-F)
and 1 sodium metavanadate (Sigma, #590088) using Matrix D beads (MP
Biomedical, #6913-500) in a FastPrep-24™ Cell Disrupter machine (MP Biomedical) in a
cold room (4 °C). Transfer tumor lysates to fresh tubes after spinning at 14000 rpm for
20 minutes at 4 °C. Determine protein concentration of tumor or cell lysates using Pierce
BCA Protein Assay Kit (cat# 23225, Thermo Scientific). This kit contains three main
components - (1) BCA Reagent A, containing sodium carbonate, sodium bicarbonate,
bicinchoninic acid and sodium tartarate in 0.1 M sodium hydroxide, (2) BCA Reagent B,
containing 4% cupric sulfate, and (3) Albumin standard ampules, containing 2 mg/mL in
0.9% saline and 0.05% sodium azide. In a 96-well plate, add bovine serum albumin
protein standard for a concentration range of 20-2000 ug/mL in 25 in duplicate wells
to generate a standard curve. Add cell or tumor lysates diluted in 25 1 x PBS to
duplicate test wells. Prepare working BCA reagent by adding 2% Reagent B to Reagent
A (2 mL of B + 98 mL of A), mix well and add 200 to each sample or standard. Mix
well, cover the plate and incubate at 37 °C for 30 minutes. Cool plate to room
temperature and measure the absorbance at or near 562 nm on a plate reader (Envision
plate reader from Perkin Elmer). Subtract the average 562 nm absorbance measurement
of the blank standard replicates from the 562 nm measurements of all other individual
standard and unknown (cell or tumor lysate) sample replicates. Prepare a standard curve
by plotting the average blank-corrected 562 nm measurement for each bovine serum
albumin standard versus its concentration in g/mL. Use the standard curve to determine
the protein concentration of each unknown samples using curve-fit logarithms in
Microsoft Excel. Freeze remaining tumor lysates at -80 °C. Use once freeze-thawed
tumor lysates to measure pRSKl expression by sandwich ELISA.
Coat 96-well plates (Thermo, #15042) overnight at 4 °C with 40 ng of RSK1 goat
antibody (Santa Cruz, # sc-231-G) and incubate at room temperature for one hour and
then at 4 °C overnight. Wash plates three times with 300 ΐ of PBST (lx phosphate
buffered saline (PBS) containing 0.05% Tween-20), block with 100 per well of
blocking buffer (Thermo Scientific, #37532) and incubated at room temperature for two
hours. Wash plates three times with 300 PBST and transfer 20 of tumor lysate to
each well and incubate at 4 °C overnight. Wash plates three times with 300 PBST and
incubate with 100 of pRSKl (T359/S363) rabbit antibody (1:1000 dilution in blocking
buffer) at room temperature for four hours. Wash plates three times with 300 PBST
and incubate with 100 anti-rabbit HRP-conjugated secondary antibody (GE
Healthcare UK, #NA934V; diluted 1:10000 in blocking buffer) Incubate at room
temperature for one hour. Wash plates three times with 300 ΐ of PBST, add 100 ΐ of
SUPERSIGNAL® ELISA Femto maximum sensitivity substrate (Thermo, #37075) and
incubate on a shaker for one minute. Determine the luminescence signal using an
ENVISION® plate reader. Determine the pRSKl level in each tumor lysate by
considering tumor lysates from animals treated with vehicle alone as 100%. Analyze
each sample in duplicate and use average numbers for calculations. Calculate TED5 0
using Excel and XL Fit.
A compound within the scope of the invention is tested in this assay substantially
as described above. The results of this assay demonstrates that the compound of Example
1 inhibits RSK1 phosphorylation in a tumor xenograft model. For example, the
compound of Example 1 has a TED 0 value of 16 mg/kg.
Xenograft Tumor Models
The purpose of this assay is to measure reduction in tumor volume in response to
test compound administration. Expand human colorectal cancer cells HCTl 16 (ATCC, #
CCL-247) in culture, harvest and inject 5xl0e 6 cells in 200 of 1:1 HBSS+matrigel
solution subcutaneously on to the rear right flank of female athymic nude mice (22-25 g,
Harlan Laboratories). Expand human pancreatic cancer cells MIA PACA-2 (ATCC, #
CRL-1420) or human non-small cell lung cancer cells CALU-6 (ATCC, # HTB-56) or
human colorectal cancer cells COLO-205 (ATCC, # CCL-222) in culture, harvest and
inject 5xl0e 6 cells in 200 of 1:1 HBSS+matrigel solution subcutaneously on to the
rear right flank of female athymic nude mice (22-25 g, Harlan Laboratories). Measure
tumor growth and body weight twice per week beginning the seventh day after the
implantation. When tumor sizes reach 200-400 mm3, randomize animals and group into
groups of eight to ten animals. Prepare test compound in an appropriate vehicle (vehicle:
1% HEC/0.25% Tween 80/0.05% Antifoam) and administer by oral gavage for 14 to 2 1
days. Tumor response is determined by tumor volume measurement performed twice a
week during the course of treatment. Body weight is taken as a general measure of
toxicity.
A compound within the scope of invention is tested in this assay run substantially
as above. The compound of Example 1 is found to have delta T/C values as provided in
Table 2 below. These results indicate that the compound of Example 1 demonstrates
significant anti-tumor activity in several human cancer xenograft models including
HCT116, MIA PACA-2, CALU-6 and COLO-205.
Table 2 : Efficacy of Example 1 in xenograft models
Analysis for tumor volume is based on Log 10 and SpatialPower covariance structure.
*: significant (p<0.05)
NA: Not applicable
Delta T/C is calculated when the endpoint tumor volume in a treated group is at or above baseline tumor
volume. The formula is 100*(T-T )/(C-C ), where T and C are mean endpoint tumor volumes in the treated
or control group, respectively. T and C are mean baseline tumor volumes in those groups.
Regression% is calculated when the endpoint volume is below baseline. The formula is 100*(T-T )/T .
Where T is the mean baseline tumor volume for the treated group.
For HCTl 16, MIA PACA-2 and CALU-6, models, grand mean of all groups from baseline (randomization)
at day 10, day 20 and day 15, respectively was used to compute %change of T/C.
In Vivo Combination Studies
Due to tumor heterogeneity combination therapy has become essential in certain
types of cancer treatment for effective therapy or to overcome acquired resistance. It is
hypothesized that a combination of targeted therapies has the potential to be more
effective in slowing or even halting cancers. In that context, the compound of Example 1
is tested for tumor growth inhibition in combination with a pan-RAF inhibitor compound
(see WO 2013/134243, l-(3,3-dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-
(methylamino)pyrido[2,3-d]pyrimidin-6-yl)phenyl)urea, hereinafter "the pan-RAF
inhibitor compound"), a CDK4/6 inhibitor compound (see WO 2010/075074, [5-(4-ethylpiperazin-
l-ylmethyl)-pyridin-2-yl]-[5-fluoro-4-(7-fluoro-3-isopropyl-2-methyl-3Hbenzoimidazol-
5-yl)-pyrimidin-2-yl]-amine, or a pharmaceutically acceptable salt
thereof), hereinafter "the CDK4/6 inhibitor compound"), or DC101 (see, for example,
Witte L., et al Cancer Metastasis Rev., 17, 155-161, 1998, rat monoclonal antibody
directed against mouse VEGFR2 that may be used in experiments as a surrogate in mice
for an anti-VEGFR2 Ab, preferably ramucirumab (see WO 2003/075840, also known as
Cyramza®, IMC-1121b, CAS registry number 947687-13-0)). More specifically, the
compound of Example 1 is tested in combination with either the pan-RAF inhibitor
compound or the CDK4/6 inhibitor compound in HCTl 16, a KRAS mutant colorectal
cancer xenograft model. Also, the compound of Example 1 is tested in combination with
either the CDK4/6 inhibitor compound or DC101 in NCI-H441, A549, and NCI-H2122,
KRAS mutant non-small cell lung cancer (NSCLC) xenograft models.
The HCTl 16 combination efficacy study is done in athymic nude rats. Expand
human colorectal cancer cells HCTl 16 (ATCC, # CCL-247) in culture, harvest and inject
5xl0e 6 cells in 200 L of 1:1 HBSS+matrigel solution subcutaneously on to the rear right
flank of female NIH nude rats (120-145 gm, Taconic Farms). Measure tumor growth and
body weight twice per week beginning the seventh day after the implantation. When
average tumor sizes reach 200-300 mm3, randomize animals and group into groups of five
to seven animals. Prepare test compound in an appropriate vehicle (see below) and
administer by oral gavage for 2 1 to 28 days. Tumor response is determined by tumor
volume measurement performed twice a week during the course of treatment.. Vehicle
used in this study is 1% HEC (hydroxy ethyl cellulose)/0.25% Tween® 80/0.05%
Antifoam. The compound of Example 1 and the pan-RAF inhibitor compound are
formulated in 1% HEC /0.25% Tween® 80/0.05% Antifoam. The CDK4/6 inhibitor
compound is formulated in 1% HEC in 25 mM Sodium phosphate buffer, pH 2.
Administration of the compound of Example 1 at 10 mpk and 20 mpk QD results in
single agent activity of 52% and 64% tumor growth inhibition respectively (Table 3). In
contrast, administration of the pan-RAF inhibitor compound at 10 mpk and 20 mpk BID
results in single agent activity of 29% and 68%, respectively. All treatments are
statistically significant (p<0.05) from vehicle control except the pan-RAF inhibitor
compound at 10 mpk BID. Administration of the compound of Example 1 at 10 mpk, QD
in combination with the pan-RAF inhibitor compound at 10 mpk, BID results in 94%
tumor growth inhibition (p<0.001) and the combination result is "Synergistic" as
calculated by Bliss Independence method (Table 3). This combination appears to be
tolerated as there is no significant body weight loss. Administration of the compound of
Example 1 at 10 mpk, QD in combination with the pan-RAF inhibitor compound at 20
mpk, BID has also results in significant (p<0.05) tumor growth inhibition (95%) and the
combination result is "Additive" " as calculated by Bliss Independence method (Table 3).
This combination appears to be tolerated as there is no significant body weight loss. In
the same study, administration of the compound of Example 1 at 10 mpk, QD with the
CDK4/6 inhibitor compound at 20 mpk QD results in 98% tumor growth inhibition
whereas single agent efficacy of the compound of Example 1 and the CDK4/6 inhibitor
compound are 52% and 76% tumor growth inhibition, respectively. Combination of these
two agents show a statistically significant "Additive" result as calculated by Bliss
Independence method (Table 3). This combination appears to be tolerated as there is no
significant body weight loss. These results suggest that combination of the compound of
Example 1 with either the pan-RAFinhibitor compound or the CDK4/6 inhibitor
compound may provide greater benefit to patients having KRAS mutant colorectal
cancer.
Table 3 : Combination studies of the compound of Example 1 with the pan-RAF inhibitor
compound, or the CDK4/6 inhibitor compound in HCT1 16 KRAS mutant colorectal
cancer xenograft model
Analysis for tumor volume is based on Log 10 and SpatialPower covariance structure.
*: significant (p<0.05)
The statistical effect of the combination of two agents is determined by Bliss Independence method: First,
a repeated measures model is fit to log tumor volume vs. group, time and group-by-time. Then contrast
statements are used to test for an interaction effect at each time point. The expected additive response
(EAR) for the combination is calculated on the tumor volume scale as, EAR volume = VI * V2 / V0, where
V0, VI, and V2 are the estimated mean tumor volumes for the vehicle control, treatment 1 alone, and
treatment 2 alone, respectively. If the interaction test is significant (p < 0.05), the combination effect is
declared synergistic if the observed combination volume is less than the EAR volume, antagonistic if the
observed combination volume is greater than the EAR volume, or additive otherwise, at the doses and
schedules that are tested.
NA: Not applicable
Delta T/C is calculated when the endpoint tumor volume in a treated group is at or above baseline tumor
volume. The formula is 100*(T-T )/(C-C ), where T and C are mean endpoint tumor volumes in the treated
or control group, respectively. T and C are mean baseline tumor volumes in those groups.
Dose for 28 days in all studies
Grand mean of all groups from baseline (randomization) at day 11 was used to compute % change of T/C
Combination efficacy is also tested in three KRAS mutant NSCLC xenograft
models including A549 (KRAS_G12S) in SCID mice as well as NCI-H441
(KRAS_G12V) and NCI-H2122 (KRAS_G12C) in athymic nude mice. Expand human
non-small cell lung cancer cells NCI-H441 (ATCC, # CRL-5807) and NCI-H2122
(ATCC, #CRL-5985) in culture, harvest and inject 5xl0e 6 cells in 200 of 1:1
HBSS+matrigel solution subcutaneously on to the rear right flank of female athymic nude
mice (20-22 gm, Harlan Laboratories). Expand human non-small cell lung cancer cells
A549 (ATCC, # CCl-185) in culture, harvest and inject 5xl0e 6 cells in 200 of 1:1
HBSS+matrigel solution subcutaneously on to the rear right flank of female CB-17 SCID
mice (18-20 gm, Taconic Farms). For all cell lines, measure tumor growth and body
weight twice per week beginning the seventh day after the implantation. When average
tumor sizes reach 200-300 mm3, randomize animals and group into groups of five to
seven animals. Prepare test compound in an appropriate vehicle (see below) and
administer by oral gavage (compound of Example 1 and the CDK4/6 inhibitor compound)
or intraperitoneally (DC101) for 2 1 to 28 days. Tumor response is determined by tumor
volume measurement performed twice a week during the course of treatment. Vehicle
used in these studies is 1% HEC/0.25% Tween® 80/0.05% Antifoam. The compound of
Example 1 is formulated in 1% HEC /0.25% Tween® 80/0.05% Antifoam and the
CDK4/6 inhibitor compound is formulated in 1% HEC in 25 mM sodium phosphate
buffer, pH 2. The administration of the compound of Example 1 as a single agent at 50
mpk results in 41% and 91% tumor growth inhibition in NCI-H2122 and A549 tumors
respectively; and leads to 101% tumor growth inhibition (i.e., 1% tumor regression) in
NCI-H441 tumors. Administration of the CDK4/6 inhibitor compound as single agent at
50 mpk results in 53%, 51% and 81% tumor growth inhibition in NCI-H2122, A549 and
NCI-H441 models respectively. Also, the combination of the compound of Example 1
(50 mpk) with the CDK4/6 inhibitor compound (50 mpk) results in 151 % (i.e. 51%
regression) and 147 % (i.e. 47% regression) tumor growth inhibition in A549 and NCIH441
tumors respectively; and 82% tumor growth inhibition in NCI-H2122 tumors. The
combination result in all three tumor models are "Additive" as calculated by Bliss
Independence method (Table 4). In general, all treatments appear to be tolerated in these
studies as indicated by no significant body weight loss. In the same NCI-H441 xenograft
model, DC101 in phosphate buffer is also administered intraperitoneally twice per week
(BIW) as a single agent or in combination with the compound of Example 1, 50 mpk, QD
for 28 days. Administration of DC101 at 20 mpk BIW results in a single agent activity of
102% tumor growth inhibition (i.e. 2% regression). Combination of the compound of
Example 1 at 50 mpk, QD with DC101 at 20 mpk, BIW results in 146% tumor growth
inhibition (i.e. 46% regression). This combination result is "Additive" as calculated by
Bliss Independence method (Table 4). This combination appears to be tolerated as there
is no significant body weight loss. These results suggest that combination of the
compound of Example 1 with either the CDK4/6 inhibitor compound or an anti-VEGFR2
antibody may provide greater benefit to non-small cell lung cancer patients with KRAS
mutation.
Table 4 : Combination studies of the compound of Example 1 with the CDK4/6 inhibitor
compound or DC101 in KRAS mutant non-small cell lung cancer xenograft models
Analysis for tumor volume is based on Log 10 transformation and a repeated measures ANOVA with a
special power covariance structure
*: significant (p <0.05)
The statistical effect of the combination of two agents is determined by Bliss Independence method: First,
a repeated measures model is fit to log tumor volume vs. group, time and group-by-time. Then contrast
statements are used to test for an interaction effect at each time point. The expected additive response
(EAR) for the combination is calculated on the tumor volume scale as, EAR volume = VI * V2 / V0, where
V0, VI, and V2 are the estimated mean tumor volumes for the vehicle control, treatment 1 alone, and
treatment 2 alone, respectively. If the interaction test is significant (p < 0.05), the combination effect is
declared synergistic if the observed combination volume is less than the EAR volume, antagonistic if the
observed combination volume is greater than the EAR volume, or additive otherwise, at the doses and
schedules that are tested.
NA: Not applicable
Delta T/C is calculated when the endpoint tumor volume in a treated group is at or above baseline tumor
volume. The formula is 100*(T-T )/(C-C ), where T and C are mean endpoint tumor volumes in the treated
or control group, respectively. T and C are mean baseline tumor volumes in those groups.
Baseline (randomization) at day 11 (HCT1 16) , day 25 (NCI-H441), day 22 (A549), day 18 (NCI-H2122)
are used to compute % change of T/C.
Dose for 28 days in all studies.
We Claim:
1. A compound of the formula:
wherein:
R is
R and R are methyl or R and R can be taken together to form
cyclopropyl;
R is hydrogen, methyl, chloro, fluoro, or trifluoromethyl; and
R5 is
or a pharmaceutically acceptable salt thereof.
2. The compound or salt according to Claim 1 wherein R2 and R are
independently methyl.
3. The compound or salt according to Claim 2 wherein R4 is hydrogen.
4. The compound or salt according to Claim 3 wherein R is
5. The compound or salt according to Claim 3 wherein R is
6. The compound or salt according to Claim 4 which is 6,6-dimethyl-2-{2-[(lmethyl-
lH-pyrazol-5-yl)amino]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-
4H-thieno[2,3-c]pyrrol-4-one.
7. The compound according to Claim 6 which is 6,6-dimethyl-2-{2-[(l-methyllH-
pyrazol-5-yl)armno]pyrimidin-4-yl}-5-[2-(morpholin-4-yl)ethyl]-5,6-dihydro-4Hthieno[
2,3-c]pyrrol-4-one.
8. A pharmaceutical composition comprising the compound or salt according to
any one of Claims 1-7, and a pharmaceutically acceptable carrier, diluent, or excipient.
9. A method of treating cancer, comprising administering to a patient in need
thereof, an effective amount of the compound or salt according to any one of Claims 1-7.
10. The method according to Claim 9, wherein the cancer is selected from the
group consisting of melanoma, colorectal cancer, pancreatic cancer, and non-small cell
lung cancer.
11. The method according to Claim 10, wherein the cancer is colorectal cancer.
12. The method according to Claim 10, wherein the cancer is pancreatic cancer.
13. The method according to Claim 10, wherein the cancer is non-small cell lung
cancer.
14. The compound or salt according to any one of Claims 1-7 for use in therapy.
15. The compound or salt according to any one of Claims 1-7 for use in the
treatment of cancer.
16. The compound or salt for use according to Claim 15 wherein the cancer is
selected from the group consisting of melanoma, colorectal cancer, pancreatic cancer, and
non-small cell lung cancer.
17. The compound or salt for use according to Claim 16, wherein the cancer is
colorectal cancer.
18. The compound or salt for use according to Claim 16, wherein the cancer is
pancreatic cancer.
19. The compound or salt for use according to Claim 16, wherein the cancer is
non-small cell lung cancer.