COMPOUNDS USEFUL FOR INHIBITING CHK1.

The present invention relates to an aminopyrazole compound, or a
pharmaceutically acceptable salt thereof, that inhibits Chkl and is useful for treating
cancers characterized by defects in deoxyribonucleic acid (DNA) replication,
chromosome segregation, and/or cell division.
Chkl is a protein kinase that lies downstream from Atm and/or Atr in the DNA
damage checkpoint signal transduction pathway. In mammalian cells, Chkl is
phosphorylated in response to agents tha cause DNA damage including ionizing
radiation (IR), ultraviolet (UV) light, and hydroxyurea. This phosphorylation which
activates Chkl in mammalian ce ls is dependent on Air. Chkl plays a role in the Atr
dependent DNA damage checkpoint leading to arrest in S phase and at G2M. Chkl
phosphorylates and inactivates Cdc25A, the dual-specificity phosphatase that normally
dephosphorylates cyclin E/Cdk2, halting progression through S-phase. Chkl also
phosphorylates and inactivates Cdc25C, the dual specificity phosphatase that
dephosphorylates cyclin B/Cdc2 (also known as Cdkl) arresting cell cycle progression at
the boundary of G2 and mitosis (Furnari et al., Science, 277:1495-7, 1997). In both cases,
regulation of Cdk activity induces a cell cycle arrest to prevent cells from entering mitosis
in the presence of DNA damage or unreplicated DNA
Various inhibitors of C k l have been reported. In addition, WO 2005/121121
discloses certain aminopyrazole compounds asserted to be modulators of glucose
metabolism.
However, there is still a need for Chk l inhibitors that are potent inhibitors of the
cell cycle checkpoints that can act effectively as potentiators of DNA damaging agents.
The present invention provides compounds that are potent inhibitors of Chk l , which may
be beneficial for the treatment of cancer. The compounds potently abrogate a Chkl
mediated cell cycle arrest induced by treatment with DNA damaging agents in tissue
culture and in vivo. Additionally, the compounds of the present invention provide
inhibition of Chk2, which may be beneficial for the treatment of cancer. Furthermore, the
compounds of the present invention inhibit cell proliferation of cancer cells by a
mechanism dependent on Chkl inhibition. Such new compounds could address the need
for safe and effecti ve treatments of cancer.
T e present invention provides a compound which is (/?)-[5-(2-methoxy-6-
methyi-pyridm-3-yi)-2H-pyrazo or a
pharmaceutically acceptable salt thereof. Preferred embodiments are (i?)-[5-(2-methoxy-
6-methyl-pyridin-3-yl)-2H-pyrazol-3-ylj-[6-(piperidm-3-yloxy)-pyrazi ( ?)-
[5-(2-methoxy-6-methyl-pyridin-3-yl)-2H-pyrazo1-3-yl]-[6-{piperidin-3-yb
2-yl]-amine methane sulfonic acid salt, (i?)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-2Hpyrazol-
3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2-ylj-amine acetic acid salt, (/?)-[5-(2-
methoxy-6-methyl-pyrkim-3-y3)-2H-py
amine hemioxalate salt, and (^)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-
[6-(piperidm-3-yloxy)-pyrazin-2-yi]-amine hemisuccinate salt.
As a particular embodiment, the present invention provides the compound which
is (i?)-[5-(2-methoxy-6-methyl-pyridm-3-y3)-2H-pyrazo3-3-yl]-[6-(piperidin-3-yloxy)-
pyrazin-2-yl]-amine.
The present invention provides the methane sulfonic aci , acetic acid,
hemioxalate, and hemisuccinate salts of (i?)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-2Hpyrazol-
3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2-yl]-amine.
Another embodiment is a hydrate of (ii)~[5-(2-methoxy-6-methyl-pyridin-3-yl)-
2H-pyrazol-3 -yl] -[6-(piperi din-3 -yloxy)-pyrazin-2-yl] -amine.
The present invention provides (/?)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-2Hpyrazol-
3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2-yl]-amine hydrate in crystalline form.
The present invention also provides (i?)-[5-(2~methoxy-6-methyl-pyridin-3-yl)~
2 -pyrazol-3-yl]-[6-(piperidir -3-yloxy)-pyrazin-2-y ]-amine hydrate in crystalline form
characterized by a X-ray powder diffraction pattern having peaks at 2± 0.2 at 5.17 in
combination with one or more of the peaks selected from the group consisting of 5.73,
17.7 1 and 20. 12.
The present invention provides a pharmaceutical composition comprising (R}-[5-
(2-methoxy-6-methyl-pyridin-3 -yl)-2H-pyrazol-3 -yl]-[6-(piperidin-3-yloxy)-pyrazin-2-
yl] -amine, or a pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier, diluent, or excipient.
The present invention provides a pharmaceutical composition comprising (i?)-[5-
(2-methoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2-
yl]-amine, or a p armace ica v acceptable sa t thereof, together with a pharmaceutically
acceptable carrier, diluent, or excipient and optionally other therapeutic ingredients.
The present invention provides a method of treating cancer, comprising
administering to a patient in need thereof an effective amount of (i?)-[5-(2-methoxy-6-
meAyl-pyridm-3-y1)-2H-pyrazol-3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2-yl]-am or a
pharmaceutically acceptable salt thereof. In addition, the present invention also provides
a method of treating cancer, comprising administering to a patient in need thereof an
effective amount of (/?)-[5-(2-meihoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-[6-
(piperidin-3-yloxy)-pyrazin-2-yl]-amine, or a pharmaceutically acceptable salt thereof,
and ionizing radiation. Furthermore, the present invention provides a method of treating
cancer, comprising administering to a patient in need thereof an effective amount of (R)-
[5-(2-methoxy-6-methyl-pyridin-3-yi)-2H-pyrazo1-3-yl]-[6-(pi
2-yl]-amine, or a pharmaceutically acceptable salt thereof and one or more chemotherapy
agents.
The present invention provides the use o i ?)-[5-(2-methoxy-6-methyi-pyridin-3-
yl)-2H-pyrazoI-3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2-yl]-amine, or a pharmaceutically
acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer.
In addition, the present invention also provides the use of (^)-[5-(2-methoxy-6-methylpyridin-
3-yl)-2H-pyrazol-3-yl]-[6-(piperidin-3-yloxy)-pyraz:in-2-yl]-amine, or a
pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the
treatment of cancer wherein said treatment comprises combination therapy with ionizing
radiation. Furthermore, the present invention provides the use of (7?)-[5-(2-methoxy-6-
methyI-pyridm-3-yl)-2H-pyrazQ]-3-yl] or a
pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the
treatment of cancer by combination therapy wherein said combination therapy treatment
comprises administration of said medicament and administration of one or more
chemotherapy agents to the same patient.
The present invention provides (i?)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-2Hpyrazol-
3-y3]-[6-(piperidin-3-yloxy)-pyraz:in-2-yl]-amine, or a pharmaceutically
acceptable salt thereof, for use in therapy. In addition, the present invention also provides
( )-[5-(2-methoxy-6-methyl pyridin 3-yl)-2H-pyrazol-3-yl]-[6-(piperidii -3-yloxy)-
pyrazin-2-yl]-amine, or a pharmaceutically acceptable salt thereof, and ionizing radiation
for use i therapy. Furthermore, the present invention provides (fl)-[5-(2-meihoxy-6-
methyi-pyridm-3-yl)-2H-pyrazol-3-yl]-[6-^ or a
pharmaceutically acceptable salt thereof, and one or more chemotherapy agents for use in
therapy.
The present invention provides (/?)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-2Hpyrazol-
3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2-yl]-amme, or a pharmaceutically
acceptable salt thereof, for use in the treatment of cancer. In addition, the present
invention also provides ( )-[5-(2-met oxy-6-methyl-pyridin-3-yl)-2 -pyrazol-3-yl]-[6-
(piperidm-3-yloxy)-pyrazin-2-yl]-amine, or a pharmaceutically acceptable salt thereof,
and ionizing radiation for use in the treatment of cancer. Furthermore, the present
invention provides (i?)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-[6-
(piperidm-3-yloxy)-pyrazm-2-yl]-amme, or a pharmaceutically acceptable salt thereof
and one or more chemotherapy agents for use in the treatment of cancer.
The present invention provides use of (^)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-
2 -pyrazol-3-yl]-[6-(piperi dir -3-yloxy)-pyrazin-2-yl]-amine, or a pharmaceutically
acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer,
wherein the medicament is to be administered simultaneously, separately, or sequentially
with ionizing radiation.
The present invention provides use of (i?)-[5-(2-meihoxy-6-methyl-pyridin-3-yl)-
2H-pyrazol-3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2-yl]-amine, or a pharmaceutically
acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer,
wherein the medicament also comprises one or more chemotherapy agents or is to be
administered simultaneously, separately, or sequentially with one or more chemotherapy
agents.
The present invention provides (i?)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-2Hpyrazo3-
3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2-yl]-amme, or a pharmaceutically
acceptable salt thereof, for use i simultaneous, separate, or sequential combination with
ionizing radiation in the treatment of cancer.
The present invention provides (i?)-[5-(2-methoxy-6-methyl-pyridin-3-yi)-2Hpyrazol-
3-yl]-[6-(piperidiri-3-yloxy)-pyrazin-2-yl]-amine, or a pharmaceutically
acceptable salt thereof, for use in simultaneous, separate, or sequential combination with
one or more chemotherapy agents in the treatment of cancer.
Furthermore, the present invention provides preferred embodiments of the
methods and uses as described herein, in which the one or more chemotherapy agents is
selected from the group consisting of 5-fiuorouracil, hydroxyurea, gemcitabine,
methotrexate, pemeirexed, doxorubicin, etoposide, cisplatin, and taxol. Additionally, the
present invention provides more preferred embodiments of the methods and uses as
described herein, in which two chemotherapy agents are selected from the group
consisting of 5-fluorouracil, hydroxyurea, gemcitabine, methotrexate, pemeirexed,
doxorubicin, etoposide, cisplatin, and taxol Also, the present invention provides even
more preferred embodiments of the methods and uses as described herein, i which the
chemotherapy agent is selected from the group consisting of 5-fluorouracil, hydroxyurea,
gemcitabine, methotrexate, pemeirexed, doxorubicin, etoposide, cisplatin, and taxol
Preferred embodiments of the methods and uses described herein are cancers selected
from the group consisting of bladder cancer, colon cancer, gastric cancer, liver cancer,
lung cancer, mammary cancer, melanoma, ovarian cancer, pancreatic cancer,
mesothelioma, renal cancer, and uterine cancer.
As used above, and throughout the description of the invention, the following
terms, unless otherwise indicated, shall be understood to have the following meanings:
"Pharmaceutically acceptable salt" or "pharmaceutically acceptable salts" refers to
the relatively non-toxic, inorganic and organic salts of compounds of the present
invention.
The compounds of the present invention are capable of reaction, for example, with
a number of inorganic and organic acids to form pharmaceutically acceptable salts. Such
pharmaceutically acceptable salts and common methodology for preparing t em are well
known in the art. See, e.g., P. Stahl, et aL, Handbook of Pharmaceutical Salts: Properties,
Selection, and Use, (VCHA/Wiiey-VCH, 2002); .M. Berge, ei 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 using one or more pharmaceutically acceptable carriers,
diluents, or excipie s and administered by a variety of routes. Preferably, such
compositions are for oral, subcutaneous, or intravenous 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, el aL, eds.,
st ed., Mack Publishing Co., 2005).
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.
"Therapeutically effective amount" or "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 amount of compound of the present invention actually administered will be
determined by a physician under the relevant circumstances, including the condition to be
treated, the chosen route of administration, the actual compound of the present invention
administered, the age, weight, and response of the individual patient, and the severity of
the patient's symptoms. Dosages per day normally fa l within the range of about 0.1 to
about 10 mg kg of body weight. In some instances dosage levels below the lower limit of
the aforesaid range may be more than adequate, while in other cases still larger doses may
be employed.
The compounds of the present invention may be prepared by a variety of
procedures known in the art, as well as those described in the Preparations and Examples
below. The specific synthetic steps for each of the routes described may be combined in
different ways to prepare the compounds of the present invention.
The reagents and starting materials are generally readily available to one of
ordinary skill in the art. Others may be made by standard techniques of organic an d
heterocyclic chemistry, techniques which are analogous to the syntheses of known
structurally similar compounds and the procedures described in the Preparations and
Examples which follow, including any novel procedures. The following Preparations and
Examples are provided to illustrate the invention in further detail and represent typical
syntheses of the compounds. The names of the compounds of the present invention are
generally provided by ISIS Draw 2.5 SP2 with Autonom add-in.
As used herein, the following terms have the meanings indicated: "BCA" refers to
bicmchoninic acid; "BSA" refers to bovine serum albumin; "DMSO" refers to
dimethylsulfoxide; "DPBS" refers to dibasic phosphate-buffered saline; "DTT" refers to
dithiothreitol; "EtOAc" refers to ethyl acetate;"FBS" refers to fetal bovine serum;
"HEPES" refers to N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; "MEM" refers
to minimum essential medium; "MeOH" refers to methanol; "PBS" refers to phosphatebuffered
saline; "PI" refers to propid u iodide; "RNAase" refers to ribonuelease A;
"RPMI" refers to Rosweli Park Memorial Institue; "TBST" refers to tris-buffered saline
Tween-20; "THF" refers to tetrahydrofuran; "TR-FRET" refers to time resolved
fluorescent energy transfer; "Tris" refers to tris(hydroxymethyl) ammomethane; "Triton-
X" refers to 4-(l,l,3 3-tetramethyibutyi)phenyi-poiyethylene glycol toctylphenoxypolyethoxyethanol
polyethylene glycol -oeiy pheny ether; and "Tween-
20" refers to poiysorbate 20.
ier/-Butyl (i?)-3-(6-chloropyrazin-2-yl)oxypiperidine-l-carboxylate
Sodium hydride (225.6 g, 5.64 mol) is dispersed into THF (3 L) and the
temperature is lowered to 0 - 5 °C. A solution of (i?)-3-hydroxy-l-boc piperidine (891.6
g, 4.43 mol) in THF (3 L) is added over 1 h while maintaining the temperature between 0
- 5 °C. The reaction is stirred for I h. 2,6-Dichloropyrazine (600 g, 4.03 mol) as a
solution in THF (3 L) is added dropwise over 1.5 h maintaining the same temperature.
The reaction is stirred for 2 h at 25 - 30 °C, and then poured onto ice. The mixture is
diluted with water and extracted with ethyl acetate. The extracts are dried over anhydrous
sodium sulfate, filtered, arsd concentrated. The residual oil is triturated with 5%
dichloromethane in hexane to give the product as a white solid. The solid is collected by
filtration and dried to give 38 g crude material. The crude product is retriturated with
5% dichloromethane in hexanes to give a white solid in quantitative yield. ES/MS m 'z
Preparation 2
2-Methoxy-6-methyl-nicotimc acid methyl ester
To a stirred solution of 2-chloro-6-methyl-nicotinic acid methyl ester ( 10 4 g,
56.52 mmol) in MeOH under nitrogen is added a solution of sodium (2.58 g, 3 04
mmol) in methanol (80.0 mL) (sodium metal is dissolved in methanol under a nitrogen
atmosphere) at room temperature. The reaction mixture is refluxed overnight. The
reaction is cooled to room temperature an the pH is adjusted to pH = 7 with acetic acid
The reaction mixture is diluted with ethyl acetate ( 00 mL) and water (30 mL). The
organic layer is separated and the aqueous layer is extracted with ethyl acetate ( x 75
mL). The combined orgaiiic extracts are dried over a SC>4, filtered, an concentrated to
give crude product. Yield: 7.25 g (71%). MR (400 MHz, CDCI 3) , 8.066 - 8.047
(d, ./ 7.6 Hz, 1H), 6.782 - 6.764 (d, J 7.2 Hz, i . 4.029 is. 3H), 3.879 (s, 3H), 2.483
(s, 3H); ES/MS m/z 82.2 • i .
Preparation 3
5-(2-Methoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-ylamine
«-BuLi (1.2 M, 96.0 mL, 5.6 mmol) is added to a solution of acetonitrile (6.08
mL, 5.4 mmol) in THF (300 mL) at - 78 °C and allowed to stir for 30 min at - 78 °C.
2-Methoxy-6-methyl-nicotmic acid methyl ester (20 g, 105.1 mmol) in THF (200 mL) is
added and stirred at - 78 °C for another 30 rrrin. The reaction mixture is quenched at -78
°C with water (500 mL) and washed with EtOAc (2 x 250 mL). The aqueous layer is
separated and evaporated. This is co-distilled twice with toluene to obtain 3-(2-methoxy-
6-methyl-pyridm-3-y1)-3-oxo-propioniirile. Yield = 2 1.4 g (crude). ES/MS m z 191.1
[M+Hf.
A solution of 3-(2-methoxy-6-niethyl-pyridin-3-yl)-3-oxo-propionitrile (21 g,
1 0.4 mmol) in ethanol (200 mL) is placed in a sealed tube. Hydrazine hydrate (32.1 mL,
662.4 mmol) and acetic acid (2 1 0 mL) are added and the reaction heated at 100 C for 2
h. The solvent is evaporated off and the reaction mixture is diluted with EtOAc (500 mL)
and saturated sodium bicarbonate solution (100 mL). The organic layer is separated and
the aqueous layer is extracted with EtOAc (2 x 250 mL). The combined organic extracts
are dried over a2S0 4, filtered, and concentrated to give the crude product which is taken
into the next step without any further purification. Yield 16.5 g (73%). Ή NMR (400
MHz, DMSO- 6 11.50 (bs, ), 7.90 (d, J 7.6 Hz, H), 6.86 (d, J = 7.6 Hz, 1H), 5.88
(s, i f ! . 4.64 (s, 2H), 3.91 (s, 3H), 2.38 (s, 3H).
5-Ainino-3 -(2 -methoxy-6-methyl-pyridin-3-yl)-pyrazole- 1-carboxylic acid r -butyl
ester
A solution of 5-(2-methoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-ylamine (16.0 g
78.3 mmol) in THF (200 mL) is added slowly to a stirred suspension of NaH (60% in
mineral oil, 3.4 g, 85.0 mmol) in THF (200 mL) at 0 °C. After 5 min at 0 °C, ά -tertbutyldicarbonate
(19.8 mL, 86 mmol) is added slowly to the reaction mixture and stirred
at 0 °C for 30 mm. The reaction mixture is quenched with ice-water (approximately 250
mL) and the product is extracted into ethyl acetate (2 500 mL) The combined organic
portions are washed with water and saturated NaCl solution (200 mL), dried over
anhydrous a2S0 4, filtered, and concentrated under vacuum to afford crude material.
This material is triturated with hexane twice to obtain 8.5 g (78%) of the title compound.
NMR (400 MHz, DMSO- d6 8.05 (d, J = 7.6 Hz, . 6.90 id. ./ 7.2 Hz, i f •. 6.28
(s, 2H), 5 85 (s, 1H), 3 90 (s, 3H), 2 40 (s, 3H), 1.56 (s, 9H).
Preparation 5
( ?)-3- {6-[2- r t-Butoxycarbonyl-5-(2-memoxy-6-
ylamino]-pyrazin-2-yloxy}-piperidme-l-carboxylic acid r t-butyl ester
A mixture of 5-ammo-3-(2-methoxy-6-metliy3-pyridin-3-yl)-pyrazole-l -
carboxylic acid tert-bxityl ester (50.0 g, 164.5 mmol). tert-b y (J¾)-3-(6-chIoropyrazin-2-
yl)oxypiperidine- -carboxylate (56.6 g, 180.9 mmol), 4,5-bis-diphenylphosphanyl-9,9-
dimethyl-9H-xantbene (14.2 g, 24.6 mmol) and Cs2C0 3 (85.5 g, 263 mmol) in 1,4-
dioxane (1.4 L) is equally divided into two side by side round bottom flasks and both are
purged with argon for 2 h . Pd(OAc) 2 (5.4 g 24.6 mmol) is added (half to each vessel)
and purging continues for 1 h. The reactions are then heated at 90-95 C for 1 h. The
reaction mixtures are cooled to room temperature, combined, and diluted with ethyl
acetate ( 1 L). The mixture is then filtered through diatomaeeous earth, washed with ethyl
acetate and the filtrate is concentrated. The crude product is purified on silica gel with
15% EtOAc/hexane as eluent to provide 55 g (57% yield) of a white powder. The 55 g of
purified product is combined with 15 g of similarly prepared and purified material
(obtained from 20 g of 5-ammo-3-(2-methoxy-6-methyl~pyridin-3-yl)-pyrazole-l -
carboxylic acid ter/-buty l ester). The combined 70 g of material is dissolved in a 4:1
mixture of THF and methanol ( 1.4 L) and treated with QuadraSil™ AP ( 140 g) for 2 h.
The reaction mixture is filtered through diatomaeeous earth and washed with ethyl acetate
(4 00 mL). The filtrate is again stirred with QuadraSil™ AP (140 g) for h and
fi ltered as above. The solvent is evaporated to give the title compound as a white solid.
Yield = 70 g (47%). ES/MS m/z 582.5 [M+H]+.
Example 1
(/?)-r5~(2-Methoxy~6~methy]-pyri
razin-2-y ] -amine
To a stirred solution of (i?)-3-{6-[2-^ri-butoxycarbonyl-5-(2~methoxy-6-methylpyridin-
3-yl)-2H-pyrazol-3-ylamino]-pyrazin-2-yloxy}-piperidine-l-carboxylic acid iertbutyl
ester ( 3.0 g, 22.3 mmol) in dichloromethane (150 ml.) is added a solution of
trifluoroaeetic acid (12.4 mL, 167 mmol) in dichloromethane (20 mL) over a period of 5
mi at 0 °C. The reaction is allowed to warm to room temperature and stirred for 3 h.
The reaction is diluted with dichloromethane (1000 mL), followed by addition of
saturated sodium bicarbonate solution (250 mL) and then stirred for 4 h. The organic
portion is separated an d dried over anhydrous sodium sulfate, filtered, and evaporated.
The resulting material is ciystallized from isopropanol to obtain the desired product.
Yield = 7.2 g (85%). ί MR (400 MHz, DMSO- ) 12.40 (s, H ). 9.71 (s, H ). 8.02
(d, 7.6 Hz, I}. 7 97 (s, . 7 46 (s, 1H), 6.93 (d, 7.6 Hz, I). 6.91 (s, I). 4 94-
4.86 (m, 1H), 3.97 (s, 3H), 3.20-3.13 (m, 1H), 2.83-2.75 (m, 1H), 2.57 (dd, j 2.0. 8.4
Hz, IH), 2.53-2.45 (m obscured, 1H), 2.42 (s, 3H), 2.15-2.05 (m, 1H), 1.71-1.63 (m, 1H),
1.60-1 .49 (m, HI). 1.49-1.40 (m,lH); ES/MS m/z 382.5 [V! • IS .
(i?)-[5-(2-Methoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-y3]-[6-(piperidin-3-yloxy)-
pyrazin--2--yl] -amine methane sulfonic acid salt
Methane sulfonic acid (0.247 g, 2.57 mmol) is added to a stirred solution of (i?)-
[5-(2-methoxy-6-methyl-pyridm-3-yl)-2H-py^
2-yl]-amine (0.982 g, 2.57 mmol) in dichloromethane (25 mL) at 0 °C, The reaction is
aliowed to warm to room temperature and agitated for 45 min. The solvent is evaporated,
and the resulting salt is washed with ether ( 0 mL) and peniane ( 10 mL) sequentially to
obtain the desired product. Yield 1.13 9 g (92.6%). Ή NMR (400 MHz, DM80- <¾), 
12.5 (bs, 1H), 9.8 1 (s, 1H), 8.73 (bs, 1H), 8.54 (bs, 1H), 8.07 (s, 1H), 7.96 (d, J 7.6 Hz,
1H), 7.56 (s, 1H), 6.95 (d, J 7.6 Hz, 1H), 6.8 1 (s, 1H), 5.3 1-5.24 (rn, 1H), 3.97 (s, 3H),
3.48-3.39 (m, i). 339-3.30 (m, 1H), 3.18-3. 10 ( n . l}. 3 10-3.01 (m H), 2.43 (s, 3H),
2.32 (s, 3H), 2.03- 1.85 (m, 3H), 1.73 - 1.65 (m, 1H); ES/MS m z 382.4 VI f i .
(ii)-[5-(2-Methoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-[6-(piperidinpyrazin-
2-yl]-amine acetic acid salt
- CH3C0 H
To a solutio of (/?)-[5-(2-meiiioxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-[6-
(piperidm-3-yloxy)-pyrazin-2-yl]-amine (0.100 g, 0.26 mmol) in dichloromethane (10
mL) is added acetic acid (0.015 mL, 0.26 mmol) dissolved in dichloromethane ( mL) at
0 °C. The reaction mixture is stirred for 60 min at room temperature and then the solvent
is evaporated to obtain a residue. The residue is triturated with diethyl ether (20 mL)
followed by w-pentane (20 mL). The material is dried under high vacuum for 4 h to
obtain the desired product. Yield 0.060 g (51.8 %). !NMR (400 MHz, DM8G- ΰ¾) 
2.40 (bs, US). 9.70 (s, 1H), 8.02 (d, J = 7.6 Hz, H), 7 98 (s, 1H), 7.46 (s, 1H), 6.94 (d, J
= 7.6 Hz, . 6.90 (s, ) . 4.97 4 38 (m, 1H), 3.98 (s, 3H), 3.20-3.13 (m, i). 2.83-
2.74 (m, 1H), 2.66-2.56 ( , II). 2.42 (s, 3H), 2.14-2.03 (m, IH), 1.89 (s, 3H), 1.74-1.62
(m, 1H), 1.61-1.51 (m, . 1.50-1.40 (m, 1H), 1.30-1.20 (m, 1H); ES/MS m/z 382.5
Example 4
(/?)-[5-(2-Methoxy-6-methyl-py^^
razin-2-yl]-amine hemioxalate salt
To a solution of (i?)-[5-(2~methoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-[6-
(piperidin-3-yloxy)-pyrazin-2-yl]-amine (0.100 g, 0.26 mmol) in dichloromethane (10
mL) is added oxalic acid (0.01 2 mg, 0. 3 mmol) dissolved in MeO (0. 1 mL) at 0 °C.
The reaction mixture is stirred for 60 min at room temperature and then the solvent
evaporated to obtain a residue. The residue is triturated with diethyl ether (20 mL)
followed by «-pentane (20 mL). The material is dried under high vacuum for 4 h to
obtain the titled compound. Yield 0.095 g (77%). Ή NMR (400 MHz, DMSO- d ) 
9.77 (s, H), 8.07 (s, H), 7.95 (d, J=7.6 Hz, 1H), 7.55 (s, 1H), 6.95 (d, J=7.6 Hz, ),
6.79 (s, H), 5.33-5.24 (m, H), 3.97 (s, 3H), 3.45-3.30 (m, 2H), 3 18-3.09 (m, IH), 3.08-
2.98 (m, IH), 2.42 (s, 3H), 2.05 - 1.85 (tn, 2H), 1.74 - 1.63 (m, 1H), 1.18 - 1.10 ( n, 1H);
ES/MS m z 382.4 j.Y • | .
Example 5
(/?)-[5-(2-Methoxy-6-methy1-pyridin^
razm-2-yl -amme hemisuccinate salt
To a solution of (i?)-[5-(2~methoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-[6-
(piperidin-3-yloxy)-pyrazm-2-yl]-amine (0. 1 g, 0.26 mmol) in dichloromethane (10 mL)
is added succinic acid (0.0 5 g, 0.13 mmol) dissolved in ethanol ( 1 mL, dissolved at 50
°C) at room temperature. The reaction mixture is stirred for 2 h a room temperature.
The solvent is evaporated and the residue obtained is triturated with diethyl ether (20 mL)
followed by n-pentane (20 mL). The material is dried under high vacuum for 8 h to
obtain the title compound. Yield 0. 02 g (78%). Ή NMR (400 MHz, DMSO-aV) 
12.4 (bs, ). 9.72 (s, 1H), 8.05 - 7.96 (m, 1 ). 7.48 (s, I). 6.93 (d, J 7.6 Hz, l i b . 6.86
(s, IH), 5.06 - 4.97 (tn, IH), 3.97 (s, 3H), 2.90 - 2.8 1 (m, IH), 2.74 - 2.62 (tn, 1H), 2.42
!s . 3H), 2.30 s . 2H), 2.09 - 2.0 1 (m, H), 1.80 - 1.60 (m, 2H), 1.57 - 1.46 (m, H I;, 1.14-
1.10 (m, H), 1.10-1.00 ( , IH); ES/MS m z 382.4 [M+H]+.
Example 6
(/?)-[5-(2-Methoxy-6-methy1-pyri$
pyrazin-2-yl]-amine hydrate
Suspend (i?)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-[6-
(piperidin-3-yloxy)-pyrazin-2-yl]-amine (52. 1 g; ES/MS z 382.2 [M+H]+) in 5:95
water-ethaiiol mixture ( 0 mL) and slurry at ambient temperature for 48 hours. A white
crystalline solid is recovered by vacuum filtration.
X-Ray powder diffraction (XRD) patterns of crystalline solids are obtained on a
Bruker D4 Endeavor X-ray 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 0.009° in 2. The dry powder is packed on a
quartz sample holder and a smooth surface is obtamed using a glass slide. I the present
case, a peak position variability of ± 0.2 in 2takes into account 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.
Thus, a sample crystalline form of the compound is characterized by an XRD
pattern using CuKa radiation as having diffraction peaks (2-theta values) as described in
Table 1 below. Specifically the pattern contains a peak at 5.17 in combination with one or
more of the peaks selected from the group consisting of 15.73, 17.71 and 20.12 with a
tolerance for the diffraction angles of 0 2 degrees.
Table : X-ray powder diffraction peaks of Example 6.
Chkl Biochemical Assay
The effect of compounds on Chkl biochemical activity can be determined using a
CHK1/substrate peptide filter binding assay. In this assay, a synthetic peptide based on
the amino acid sequence residues 206-225 of Cdc25, is used as a phospho-acceptor
substrate for recombinant Chkl protein kinase. Using - -as the phospho-donor
substrate, Chkl transfers the radioactive -JJphosphate group to the synthetic peptide.
The reaction is measured by capturing the peptide substrate on a cation exchange paper
filter plate and scintillation counting of emitted beta particles.
The kinase reactions (40 L• reaction volumes) are performed in 96-weil V-bottom
polystyrene plates. Reactions are initiated with the addition of Chkl enzyme. Final
reaction conditions are 67 r M HEPES sodium salt p 7 4, 0.007% (v/v) TRITON™ X-
100, 2.7 M DTT, 2.7 mM MgCl 2, 12 peptide substrate, 60 ATP disodium salt,
0.75 -33-, 0.75 M active Chkl enzyme, 4% (v/v) DMSO and serial dilution of
the compound (1:3 serial dilution, starting at 20 , 10 points}.
Following Chkl enzyme addition, the reactions are in bated at room temperature
for 90 min, and then terminated with the addition of 40 of phosphoric acid. The
reaction mixture is transferred to the corresponding wells of a phosphoceilulose cation
exchange paper opaque filter plate to sit for 30 min. The filter plate is washed in a
vacuum manifold with five washes of 200 of 0.5% phosphoric acid (v/v). The filter
plate is dried overnight prior to the addition of 40 of Microscint™~20 to each well of
the plate. After sitting for 4 h at room temperature, the radioactivity in the plate is
measured using a MicroBeta Trilux microplate scintillation counter (Perkin Elmer).
For IC 0 determination, the percent inhibition for each concentration is calculated
using the scintillation count ratio from controls run on each plate. The ten-point
compound concentration data is subsequently fit to a four-parameter logistic equation
using ActivityBase 4.0. Absolute IC 0 values are calculated from the resulting curve.
Compounds of the invention are tested in this assay run substantially as above. For
example, the compound of Example 1 is tested and found to have an IC5 of <0.001 
(n = 6). Furthermore, the compound of Example 2 is tested and found to have a XC 5 of
<0.001 (n = 3). These results indicate tha compounds within the scope of the present
invention are potent inhibitors of Chkl.
Chk2 Biochemical Assay
The effect of compounds on Chk2 biochemical activity can be determined using a
CHK2/substrate peptide filter binding assay. n this assay, a synthetic peptide based on
the amino acid sequence residues 206-225 of Cdc25C, is used as a phospho-acceptor
substrate for recombinant Chk2 protein kinase. Using - 'P-ATP as the phospho-donor
substrate, Chk2 transfers the radioactive -"phosphate group to the synthetic peptide.
The reaction is measured by capturing the peptide substrate o a cation exchange paper
filter plate and scintillation counting of emitted beta particles.
The kinase reactions (40 _. reaction volumes) are performed in 96-weil V-bottom
polystyrene plates. Reactions are initiated with the addition of Chk2 enzyme. Final
reaction conditions are 67 mM HEPES sodium salt pH 7.4, 0.007% (v/v) TRITON™ X-
100, 2.7 mM DTT, 2.7 mM MgCl2, 12 peptide substrate, 60 ATP disodium salt,
0.75 - -, 1.4 M active Chk2 enzyme, 4% (v/v) DMSO and serial dilution of
the compound (1:3 serial dilution, starting at 20 , 0 points).
Following Chk2 enzyme addition, the reactions are incubated at room temperature
for 90 min, and then terminated with the addition of 140 of phosphoric acid. The
reaction mixture is transferred to the corresponding wells of a phosphoceilulose cation
exchange paper opaque filter plate to sit for 30 min. The filter plate is washed in a
vacuum manifold with five washes of 200 of 0.5% phosphoric acid (v/v). The filter
plate is dried overnight prior to the addition of 40 of Microscint™-20 to each well of
the plate. After sitting for 4 h at room temperature, the radioactivity in the plate is
measured using a MicroBeta Trilux micropiate scintillation counter (Perkin Elmer).
For IC determination, the percent inhibition for each concentration is calculated
using the TR-FRET ratio from controls mn on each plate. The ten-point compound
concentration data is subsequently fit to a four-parameter logistic equation usin
ActrvityBase 4.0. Absolute IC50 values are calculated from the resulting curve.
Compounds of the invention are tested in this assay run substantially as above. For
example, the compound of Example 1 is tested and found to have an IC50 of 0.0 
(SE = 0.002, n = 6). Furthermore, the compound of Example 2 is tested and found to
have an IC50 of 0.012 (SE = 0.008, n = 3). These results indicate that compounds
within the scope of the present invention are potent inhibitors of Chk2.
C k Autophosphorylation Cell Based Assay
An inhibitor of Chkl will prevent the kinase activity of the protein from
phosphorylating substrates in cells in which the NA damage response has been
activated. An easily detectable substrate for Chkl is an autophosphorylation site on Chkl
itself, serine 296. The following immunoblot assay can be used to measure the amount of
phosphorylation of serine 296 on Chkl and indirectly the activity level of the Chkl
protein kinase. HeLa cells are cultured in MEM w/ Earle's Balanced Salt Solution with
L-glutamine supplemented with 10% (v/v) heat inactivated fetal bovine serum, l x MEM
non-essential amino acids, l x sodium pyruvate an 1 O cells plated in 600 of
MEM culture media per wel of a 24 well cell culture plate. Cells are incubated for 24 h
at 37 °C, 5% C0 2 and 95%- 100% humidity. Sixteen . of a 4 stock of doxorubicin
in culture media are added to each appropriate well to make a final concentration of 00
nM doxorubicin. Plates are returned to the incubator for 24 additional hours prior to
Chkl inhibitor compound addition. Compounds are solubiiized at 10 niM in 100%
DMSO, then diluted to 2 M in 40% (v/v) DMSO and then diluted to 00 with
culture media plus 4% (v/v) DMSO. Subsequently, serial dilutions of the compounds
(1:3) are prepared over a 100 to 0.005 range. Sixty-six of compound stock is
added to the appropriate wells in the plate to produce a final DMSO concentration of
0.4% (v/v) and a final compound concentration range between 1 and 0.0005 .
The plates are return ed to the incubator for an additional 2 h and then removed for cell
lysis and processing. The media is then removed from the plate, each well washed once
with 0.5 mL of ice cold Dulbecco's Phosphate-Buffered Saline (DPBS), all liquid is
removed, and the plate is placed on ice for the remainder of the procedure. To each well
is added 75 of ice cold lysis buffer, consisting of Cell Extraction Buffer containing
phosphatase inhibitor cocktail (Sigma, cat# P0044 + P5725) and protease inhibitor
cocktail tablets (Roche Diagnostics, cat# 836 153001) After 10 min each well is
scraped and the lysate transferred into a 1.5 mL polypropylene microcentrifuge tube on
ice. Each lysate is sonicated for 45 sec with a plate cuphorn sonicator (Misonix) while
suspended in a water/ice bath. Fifty ΐ of each sample is transferred into a 0.5 mL
polypropylene microcentrifuge tube containing 25 i of 4X Laemmii Sample Buffer,
heated at 95 °C for 5 m a d stored frozen at -80 °C. The remaining iysate is used for
determination of protein concentration (BCA protein assay kit, Thermo Scientific). Five
of each cell iysate in sample buffer is applied to an E-Page 96 well gel and subjected
to electrophoresis. Proteins are electrotransferred from the ge to Immobilon-P membrane
PVDF (0.45 ) according to procedures well understood in the art (Towbin et al,
PNAS (1979) 76(9), 4350-4). The membrane is rinsed briefly with 10 mM Tris/HCI p
8.0, 50 mM NaC and 0.05% (v/v) T een 20 (TBST) and soaked for one hour at 25 °C
in TBST/5% (v/v) reconstituted Carnation® instant milk. The membrane is washed four
times with TBST for 5 min, then soaked at 4 °C for 24 h in TBST/5% (w/v) bovine serum
albumin with an appropriate dilution of rabbit anti-phospho-Clikl (serine 296). The
membrane is washed 4 with TBST for 5 min at 25 °C and then soaked at 25 C for 2 h in
TBST/5% milk containing an appropriate dilution of donkey anti-rabbit gG conjugated
to horseradish peroxidase (GE Healthcare, cat# NA9340) to detect autophosphorylated
Chkl protein. The membrane is washed again 4x with TBST for 5 min at 25 °C.
Antigen-antibody -reporter conjugates immobilized on the membrane are detected with
the Super Signal Western Femto HRP-detection reagent using a FUJI LAS-4000 imaging
system. Phospho-Chkl (ser296) band intensities are calculated using "Total Lab"
software (Nonlinear Dynamics). The percent inhibition of the doxorubicin induced Chkl
autophosphorylation is calculated by using the following formula: % inhibition (sample
-phospho-Chkl band intensity no doxorubicin negative control -phospho-Chkl band
intensity) /(doxorubicin positive control -phospho-Chkl band intensity- no doxorubicin
negative control -phospho-Chkl band intensity) x 00. Compounds of the invention are
tested in this assay run substantially as above. The compound of Example 1 is tested in
this assay and found to have an EC5.3 of <0.001 (n = ). The compound of Example 3
is tested in this assay and found to have an EC50 of <0.Q01 (n 1). These results
indicate that compouiids within the scope of the present invention are potent inhibitors of
Chkl.
Doxorubicin-Induced G2M Checkpomt Abrogation eLa Cell-Based Acumen Assay
An inhibitor of Chkl will disable the G2M DNA damage checkpoint in p53-minus
tumor ceils treated with the topoisomerase II inhibitor, doxorubicin. A measurement of
G2M checkpoint abrogation is the phosphorylation of histone H3 on serine 10 that occurs
after cells traverse e G2M checkpoint and enter mitosis. The following high content
imaging assay can be used to measure the phosphorylation of histone H3 in cells. HeLa
cells are cultured in MEM Media supplemented with 0% (v/v) FBS and plated at 2000
cells per well in poly D-lysine coated clear bottom black plates, 00 volume per well.
Plates are then incubated in a cell culture incubator for 18-24 h (37 °C, 5% C0 2 and 95%
relative humidity). Following the initial incubation, 20 of MEM Media plus 0% FBS
containing 625 nM doxorubicin are added to the appropriate wells of the plates resulting
in a final concentration of 25 nM. The plates are returned to the incubator for 24 h,
sufficient to arrest the cells at the G2M checkpoint. The next day the cells are treated
with compounds. Compounds are sofublized at 0 M in 00% DMSO and then diluted
to a 10x stock starting at 50 in MEM plus 4% (v/v) DMSO. Subsequently, serial
dilutions of the compounds ( 1:2) are prepared over a 50 to 0.39 range. Thirteen
of compound stock is added to the appropriate wells in the plate to produce a final
DMSO concentration of 0.4% and a final compound concentration range between 5 
and 0.039 . The plates are returned to the incubator for an additional 7 h and then
removed for fixation. Liquid is carefully removed from each wel and 00 of
PREFER™ fixative is added. Plates are retained at room temperature for 20 mm, the
fixative removed an the cells are then permeablized by the addition of 100 of
0.1% (v/v) Triton® X 00 in DPBS for 10 min. The solution is removed and the plate
washed twice with 100 , DPBS per well followed by the addition of 100 of DPBS
containing 50 ug/mL Ribonuclease A (RNAase, from bovine pancreas) for one hour at
room temperature. The RNAase solution is removed and the cells stained for the
presence of histone H3 phosphorylated on serine 10 (pHH3) by adding to each well 50 
of RNAase solution containing a 1:500 dilution of rabbit anti-pHH3 (serlO) plus 1%
(w/v) BSA. Plates are sealed and kept at 4 °C overnight. The primary antibody is
removed by washing each plate twice with 100 DPBS per well and replaced with 50
of a 1:750 dilution of Alexa Fluor© 488 goat anti-rabbit gG (H+L) (2 mg/mL) in
DPBS plus 1% (w/v) BSA. Plates are kept for one hour at room temperature covered
with aluminum foil to protect from light. The plates are again washed twice with 100
per well DPBS and replaced with 00 of 5 nM propidiurn iodide ( 1: 00 dilution with
PBS from the original solution). The plates are sealed with a black seal to protect the
plates from light. Plates are incubated for 30 in to stain nuclei. Plates are scanned with
ACUMEN EXPLORER™ Laser-scanning fluorescence micropfate cytometers using 488
nm excitation (TTP LABTECH LTC) to measure pHH3 and DNA content including 2N,
and 4N. The pHH3 positive cells are identified by mean intensity at 5 9 nm from Alexa
488. Total intensity at 655-705 nm from propidium iodide/DNA is used to identify
individual cells and subpopulations in cell cycle (2N ceils, 4N cells). The final readout
for each population is determined by normalizing to the % of total cells producing a final
assay output of %pHH3, %2N, and %4N. 0% activity s then determined by treating
cells with the maximum concentration of an inhibitor control compound at 100 nM to
determine the final % activity of each compound. 0% activity is based on no compound
treatment. The Relative EC50 is determined by using ACTIVITY BASE™, excel fit,
curve fitting using a four parameter logistic fit, equation 205, to determine the %pHH3
relative to control max at 100%. Compounds of the invention are tested in this assay run
substantially as above. The compound of Example 1 is tested and found to have an EC50
of 0.029 (n 1). The compounds of Example 2 and Example 3 are tested and found
to have EC50 results of 0.033 (n = 1} and 0.019 (n = 1) respectively. These
results indicate that compounds within the scope of the present invention will disable the
G2M DNA damage checkpoint.
s (Two-Fold Sensitization) Assay
An inhibitor of Chkl can potentiate the anti-proliferative activity of gemcitabine
(or other cytotoxics) through abrogation of the intra-S phase checkpoint, resulting in
sustained and increased DNA damage. The capacity for continued tumor cell
proliferation after DNA damage can be analyzed by determining the ability of cells to
replicate their DNA. This assay assesses the ability of ceils to replicate their DNA after
cells have had an opportunity to repair DNA damage. In this assay, cells are treated with
a dilution series of gemcitabine, and then 22 h later with the compound of Example 3.
After an additional 44 h, the relative cell number is assessed by a MTS (3-(4,5-
dimethyUhiazol-2-yI)-5-(3-carboxymethoxyphenyl)-2-(4-su fophenyl)-2H-terrazolium)
dye reduction assay. The ECt¾parameter is a measure of the concentration of a Chkl
inhibitor necessary to reduce by half the GI90 concentration of gemcitabine, measured in
this assay in the absence of Chkl inhibition. HT-29 cells (obtained from A' CC), are
grown in RPM 1640 plus 0% (v/v) heat inactivated FBS. The cells are plated at 2 5 x
03 per well, in a volume of 00 , ors 96-well tissue culture plates and incubated for 24
h. Gemcitabine dilutions are prepared at 6x concentrations in McCoy's 5A medium
(modified) (lx) and added to wells at 20 per well. Gemcitabine dilutions were set up
with the highest final concentration of gemcitabine being 1.0 and dilutions made by
three-fold steps to 0.5 nM.
Chkl Inhibitor is prepared by dilutions in DMSO to 4000x final concentration,
and then diluted 666-fold into McCoy's medium to generate 6x stocks. Chkl inhibitor
dilutions proceed by 2.5-fold steps starting at 25 nM down to 0.3 nM. Twenty-two hours
after gemcitabine addition, Chkl inhibitor is added in a volume of 24 to wells
containing 120 of medium plus gemcitabine. Each gemcitabine dilution receives a
single Chkl inhibitor dilution. Control wells received DMSO, gemcitabine, or Chkl
inhibitor alone. Forty-four hours after addition of Chkl inhibitor. 30 of CeilTiter 96®
Q o s assay reagent, is added to each well and held at room temperature for 1 hour and
45 minutes. Absorbance is read on a SpectraMax 250 (Molecular Devices)
spectrophotometer at 490 nm. Data from the SpectraMax spectrophotometer are analyzed
with GraphPad Prism 4.0. First, an averaged no cell control absorbance is subtracted
from all other values in the matrix of data from each plate. Next, duplicate data points are
averaged. Data are normalized for each Chkl inhibitor concentration, with 0% cell
number set as corrected A = 0, and 100% ceil number set as the 0 nM gemcitabine
mean value. These results are then transformed. Gemcitabine concentrations are
converted to log concentrations, and normalized cell number values are converted to
percent inhibition (percent inhibition = 00- normalized value). Transformed data are
plotted, and a non- linear regression is nm to estimate an IC50 value for gemcitabine at
each Chkl inhibitor concentration. The nonlinear regression is calculated allowing the
slope to vary, and without constraints for the top or bottom of the dose-response curves.
The E C f value is calculated as follows; GI50 values for gemcitabine for each Chkl
inhibitor concentration are determined, plotted, and the concentration of Chk l inhibitor
necessary to decrease the gemcitabine alone GI5.3 by two-fold is determined by
interpolation.
Compounds within the scope of the invention are tested in this assay run
substantially as above. For example, the compou d of Example 3 is tested and found to
have an E &value of 1.0 nM (SE = 0.1, n = 3). Furthermore. 25 nM of the compound
decreases the EC50 of gemcitabine 7-fold from 22 nM to 3 nM in HT-29 colon carcinoma
cells. Alone, 25 nM of the compound of Example 3 has little effect on the proliferation of
HT-29 cells. These results indicate that compounds within the scope of the present
invention effectively potentiate the anti-proliferative activity of gemcitabine at low
concentrations.
Gemcitabine IC50 values obtained with treatment of various concentrations of Example 3
[Example 3], nM ICso (nM)
0 22
0.256 23
0.64 19
1.60 14
4.0 10
10 4
5
Chkl in vivo Target Inhibition Assay
Calu-6 cells are cultured in growth media (MEM with Earle's Balanced Sa t
Solution with L-glutamine supplemented with 0% (v/v) heat inactivated FBS, l x MEM
non-essential amino acids, sodium pyruvate) and expanded. Ceils are harvested and
washed twice with phosphate buffered saline and 1 0 cells in growth media (without
serum) are mixed with equal volume of BD Matrigel™ matrix, then inj ected
subcutaneously into the flank of pre-irradiated (4.5 Gy) nude mice (athymic nude). At
day 5 after implant (tumor size = 150-200 mm'), gemcitabine formulated fresh in saline
daily is administered to animals by intraperitoneal route at 50 mg kg dose. Six hours
later animals are orally administered Chkl compound formulated in 0 2% Tween-
80/0.5% methylcelMose p adjusted to 6.8 by addition of dilute NaOH. Animals are
sacrificed 2 h post Ch inhibitor dose, tumors harvested and immediately processed in
ice cold Cell Extraction buffer containing phosphatase inhibitor cocktail (Sigma, cat#
P0044 + P5725) and protease inhibitor cocktail tablets (Roche Diagnostics, cat#
1836153001) Tumors are processed in 1.5-2.0 mL of lysis buffer in an iced 15 111L
polypropylene conical tube using a motorized tissue homogenizer set to high for 5 sec.
With the sample kept on ice, the lysate is drawn four times through a 1 n L syringe with a
25 gauge needle. 0.35 m of tumor lysate is transferred into a 1.5 n L polypropylene
microcentrifuge tube containing 0. 5 L of 4x Laemmli sample buffer. Sample is then
mixed and heated for 5 min at 95 C and sonicated for i rain using high power on a
Misonix 3000 plate horn sonicator. Samples are then stored on ice, or stored at -80 °C for
target inhibition assessment by western blot. Five of each tumor lysate in sample
buffer is applied to E-Page 96 well gels and subj ected to electrophoresis. Proteins are
transferred to nitrocellulose BA83 Protean membrane (Whatman, Cat# 10402405)
according to procedures well understood in the art (Towbin et , P AS (1979) 76(9),
4350-4). The membrane is then processed to measure Chk! protein autophosphorylated
on serine 296. The membrane is rinsed briefly with water, then 10 mM Tris/HCi p 8.0,
150 mMNaCl and 0.05% (v/v) Tween 20 ( TBST) and soaked for one hour at 25 °C in
TBST/ 5% (w/v) reconstituted Carnation instant milk. The membrane is then washed
four times with TBST for 5 min. The membrane is soaked at 4 °C for 16 h i TBST/5%
(w/v) BSA in an appropriate dilution of rabbit -phospho -Chkl anti-phospho -Chkl (serine
296). Then the membrane is washed four times with TBST for 5 min at 25 °C and then
soaked at 25 °C for 2 h m TBST/ 5% m k coiitaining an appropriate dilution of donkey
anti-rabbit IgG conjugated to horseradish peroxidase to detect phospho-Chkl (ser 296).
The membrane is washed again four times with TBST for 5 min at 25 °C. Antigenantibody-
reporter conjugates immobilized on the membrane are detected with the Super
Signal Western Pernio HRP-detection reagent.
Signals are detected and captured usin the FUJI LAS-4000 imaging system.
Phospho-Chkl (ser296) band intensities are calculated using "Total Lab" software
(Nonlinear Dynamics). The percent inhibition of the gemcitabine induced Chkl
autophosphorylation is calculated by using the following formula: % inhibition = (sample
-phospho-Chkl band intensity - average gemcitabine (Max) positive control -phospho-
Chkl band intensity) / (average negative control (Min) -phosphor-Chk! band intensity -
average gemcitabine (Max) positive control -phospho-Chkl band intensity) x 100
Compounds within the scope of the invention are tested in this assay run
substantially as above. For example, the compound of Example 3 is tested and found to
have a Target Modulatory Effective Dose 50 (TMED 0) for Chkl autophosphorylation of
1.3 mg kg (n = ). This result indicates that compounds within the scope of the present
invention potently inhibit the activation of the Chkl protein kinase in vivo.
Human Tumor Xenograft Models
The ability of Chkl inhibitors to potentiate tumor killing by DNA damaging
agents can be determined i vivo using the Calu-6 lung and HT-29 colon tumor xenograft
efficacy models. Calu-6 lung cancer cells are cultured in growth media (MEM with
Earle's Balanced Salt Solution with L-glutamine supplemented with 10% (v/v) heat
inactivated FBS, MEM non-essential amino acids, x sodium pyruvate ) and HT-29
colon cancer cells (ATCC) are cultured in growth media, (McCoy's 5A medium
supplemented with 10% FBS} and expanded.
Cells are harvested and washed twice with phosphate buffered saline and 5 0°
ceils (HT-29) or 0° cells (Calu-6) in growth media (without serum) are mixed with
equal volume of BD Matrigel Mmatrix then injected subcutaneously into the flank of
nude mice (CD-I nu n ).
Subcutaneous Administration of Chkl Inhibitor
At about day 16 after implant (150-200 mm5), gemcitabine is formulated fresh in
saline daily and administered to animals by intraperitoneal route at 60 mg/kg dose.
Twenty four hours later animals are administered the compound of Example 3, in 0.2%
Tween-80/0.5% methvlceilulose subcutaneously BID. After two days of rest, dosing is
repeated for three additional cycles (Q4Dx4 with the compound of Example 3 offset +24
hours). Tumor growth inhibition (TGI) is calculated as the percent reduction in mean
tumor size of a compound treated group from the mean rumor size of the vehicle-treated
control group. Compounds within the scope of the invention are tested in this assay run
substantially as above. For example, the compound of Example 3 dosed in combination
with gemcitabine is found to demonstrate excellent dose dependent anti-tumor activity in
both the HT-29 and Calu-6 tumor xenograft models, with up to a six-fold increase in
tumor growth inhibition over gemcitabine alone. This result indicates that compounds
within the scope of the present invention administered subcutaneously significantly
increase the anti-tumor activity of gemcitabine in human tumor xenograft models.
HT29 subcutaneous
ns = not statistically significant
Oral Administration ofChkl Inhibitor
At about day 16 after implant (150-200 mm3), gemcitabine is formulated fresh in saline
daily and administered to animals by intraperitoneal route at 40 mg kg dose. Twent four
hours later animals are administered Chk compound, in 0 2% Tween-80/0.5%
methylcellulose by the oral route BID. After three days of rest, dosing was repeated for
three additional cycles (Q5Dx4 with the compound of Example 3 offset +24 hours).
Tumor growth inhibition (TGI) is calculated as described in the previous paragraph.
Compounds within the scope of the invention are tested in this assay run substantially as
above. For example, the compound of Example 3 is dosed in combination with
gemcitabine and found to demonstrate excellent dose dependent anti-tumor activity in
both the HT-29 and Calu-6 tumor xenograft models, with up to a 2.Si-fold increase in
tumor growth inhibition over gemcitabine alone. This result indicates that compounds
within the scope of the present invention administered orally significantly increase the
anti-tumor activity of gemcitabine in human tumor xenograft models.
Calu6 oral
ns not statistically significant
WE CLAIM:
. A compound which is (^)-[5-(2-methoxy-6-methyl-pyridin-3-yl)-2Hpyrazol-
3-yl]-[6-(piperidin-3-yloxy)-pyrazin~2-yl]-amine, or a pharmaceutically
acceptable salt thereof.
2 The compound according to Claim 1 which is (/?)-[5-(2-methoxy-6-
methyl~p>TicUn~3-yl)-2H^yrazol-3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2~yl]-amine.
3. The compound according to Claim 1 which is (i?)-[5-(2-methoxy-6-
methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-[6-{piperidm-3-yloxy)-pyrazin-2-yl]-a^
methane sulfonic acid salt.
4. The compound according to Claim 1 which is (ii)-[5-(2-methoxy-6-
methyl-pyridin-3-yl)-2H-pyrazol-3-yl]-[6-(piperidm-3-yloxy)-pyrazin-2-yl]-am acetic
acid salt.
5. The compound according to Claim 1 which is (i?)-[5-(2-methoxy-6-
methyl-pyridin-3-yl)-2H-pyTazol-3-yl]-[6-(piperidin-3-yloxy)-pyrazin-2-yl]-amine
hemioxalate sail
6. The compound according to Claim 1 which is (i?)-[5-(2-meihoxy-6-
methyi-pyridin-3-yl)-2H-pyrazoI-3-yl]-[6-(p^
hemisuccinaie salt.
7. A pharmaceutical composition comprising the compoursd or salt according
to any one of Claims 1-6, and a pharmaceutically acceptable carrier, diluent, or excipient.
8. 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 1-6.
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 1-6
and ionizing radiation.
10. 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 1-6
and one or more chemotherapy agents.
. The method according to Claim 0, wherein the one or more
chemotherapy agents is selected from the group consisting of 5-fluorouracil, hydroxyurea,
gemcitabine, methotrexate, pemetrexed, doxorubicin, etoposide, cisplatin, and taxol.
2. The method according to any one of Claims 8- 1, wherein the cancer is
selected from the group consisting of bladder cancer, colon cancer, gastric cancer, liver
cancer lung cancer, mammary cancer melanoma, ovarian cancer, pancreatic cancer,
mesothelioma, renal cancer, and uterine cancer.
13. The compound or salt according to any one of Claims 1-6 for use in
therapy.
14. The compound or salt according to any one of Claims 1-6 for use in the
treatment of cancer.
5. The compound or salt according to any one of Claims 1-6 for use in
simultaneous, separate or sequential combination with ionizing radiation in the treatment
of cancer.
16. The compound or salt according to any one of Claims 1-6 for use in
simultaneous, separate, or sequential combination with one or more chemotherapy agents
in the treatment of cancer.
17. The compound or salt for use according to Claim 16, wherein the one or
more chemotherapy agents is selected from the group consisting of 5-fluorouracil,
hydroxyurea, gemcitabine, methotrexate, pemetrexed, doxorubicin, etoposide. cisplatm,
and taxol.
18. The compound or salt for use according to any one of Claims 14-17,
wherein the cancer is selected from the group consisting of bladder cancer, colon cancer,
gastric cancer, liver cancer, lung cancer, mammary cancer, melanoma, ovarian cancer,
pancreatic cancer, mesothelioma, renal cancer, and uterine cancer.
19. A pharmaceutical composition comprising the compound or salt according
to any one of Claims 1-6, together with a pharmaceutically acceptable carrier, diluent, or
excipient and optionally other therapeutic ingredients.