U.S. patent application number 15/221649 was filed with the patent office on 2017-02-09 for jak1 inhibitors.
The applicant listed for this patent is Eli Lilly and Company. Invention is credited to Joshua Ryan Clayton.
Application Number | 20170037034 15/221649 |
Document ID | / |
Family ID | 56618268 |
Filed Date | 2017-02-09 |
United States Patent
Application |
20170037034 |
Kind Code |
A1 |
Clayton; Joshua Ryan |
February 9, 2017 |
JAK1 Inhibitors
Abstract
The present invention relates to certain benzimidazole
compounds, or pharmaceutically acceptable salts thereof, that
inhibit Janus kinase 1 (JAK1), pharmaceutical compositions
comprising the compounds, and methods of using the compounds to
treat certain types of cancer.
Inventors: |
Clayton; Joshua Ryan;
(Fishers, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Eli Lilly and Company |
Indianapolis |
IN |
US |
|
|
Family ID: |
56618268 |
Appl. No.: |
15/221649 |
Filed: |
July 28, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62200684 |
Aug 4, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 43/00 20180101; A61K 31/4184 20130101; C07D 403/12
20130101 |
International
Class: |
C07D 403/12 20060101
C07D403/12 |
Claims
1.-18. (canceled)
19. A compound of Formula I: ##STR00020## or a pharmaceutically
acceptable salt thereof.
20. The compound according to claim 19 wherein the compound is
selected from the group consisting of: ##STR00021## or a
pharmaceutically acceptable salt thereof, and ##STR00022## or a
pharmaceutically acceptable salt thereof.
21. The compound according to claim 20 which is ##STR00023## or a
pharmaceutically acceptable salt thereof.
22. The compound according to claim 20 which is ##STR00024##
23. A pharmaceutical composition comprising a compound of Formula
I: ##STR00025## or a pharmaceutically acceptable salt thereof, and
a pharmaceutically acceptable carrier, diluent, or excipient.
24. The pharmaceutical composition according to claim 23 wherein
the compound is selected from the group consisting of: ##STR00026##
or a pharmaceutically acceptable salt thereof, and ##STR00027## or
a pharmaceutically acceptable salt thereof.
25. The pharmaceutical composition according to claim 24 wherein
the compound is ##STR00028## or a pharmaceutically acceptable salt
thereof.
26. The pharmaceutical composition according to claim 24 wherein
the compound is ##STR00029##
27. A method of treating cancer, comprising administering to a
patient in need thereof, an effective amount of a compound of
Formula I: ##STR00030## or a pharmaceutically acceptable salt
thereof, wherein the cancer is selected from the group consisting
of lung cancer, including non-small cell lung cancer and lung
adenocarcinoma, adenocarcinoma, hepatocellular carcinoma, including
Asian hepatocellular carcinoma, colorectal cancer, breast cancer,
and leukemia, including acute lymphocyte leukemia.
28. The method according to claim 27, wherein the compound is
selected from the group consisting of: ##STR00031## or a
pharmaceutically acceptable salt thereof, and ##STR00032## or a
pharmaceutically acceptable salt thereof.
29. The method according to claim 28 wherein the compound is
##STR00033## or a pharmaceutically acceptable salt thereof.
30. The method according to claim 28 wherein the compound is
##STR00034##
31. The method according to claim 27, wherein the cancer is
non-small cell lung cancer.
32. The method according to claim 27, wherein the cancer is lung
adenocarcinoma.
33. The method according to claim 27, wherein the cancer is breast
cancer.
34. The method according to claim 27, wherein the cancer is Asian
hepatocellular carcinoma.
35. The method according to claim 28, wherein the cancer is
non-small cell lung cancer.
36. The method according to claim 28, wherein the cancer is lung
adenocarcinoma.
37. The method according to claim 28, wherein the cancer is breast
cancer.
38. The method according to claim 28, wherein the cancer is Asian
hepatocellular carcinoma.
39. The method according to claim 29, wherein the cancer is
non-small cell lung cancer.
40. The method according to claim 29, wherein the cancer is lung
adenocarcinoma.
41. The method according to claim 29, wherein the cancer is breast
cancer.
42. The method according to claim 29, wherein the cancer is Asian
hepatocellular carcinoma.
43. The method according to claim 30, wherein the cancer is
non-small cell lung cancer.
44. The method according to claim 30, wherein the cancer is lung
adenocarcinoma.
45. The method according to claim 30, wherein the cancer is breast
cancer.
46. The method according to claim 30, wherein the cancer is Asian
hepatocellular carcinoma.
Description
[0001] The present invention relates to certain benzimidazole
compounds, or pharmaceutically acceptable salts thereof, that
inhibit Janus kinase 1 (JAK1), pharmaceutical compositions
comprising the compounds, and methods of using the compounds to
treat certain types of cancer.
[0002] The JAK kinases are a tyrosine kinase family that regulate
tyrosine phosphorylation of various effectors and initiate
activation of downstream signaling pathways. JAK1 is a member of
this family that mediates the activation of signal transducer and
activator of transcription 3, STAT3. Persistent STAT3 activation is
tumorigenic and promotes cancer cell survival and proliferation.
Aberrations in STAT3 activation have been also shown to disrupt
tumor immune surveillance in the tumor microenvironment. Therefore,
inhibition of JAK1 can block STAT3 activation resulting in tumor
growth inhibition and tumor immune surveillance. In addition,
activating JAK1 mutations have been identified in both T-lineage
acute lymphoblastic leukemia and Asian hepatocellular carcinoma and
have been demonstrated as oncogenic. These results suggest JAK1 is
a viable oncology target.
[0003] JAK2 is known to form homodimers that mediate EPO and TPO
receptor-signaling to the STATS pathway, which regulates red blood
cells and platelet production. Inhibition of JAK2 can result in
anemia and thrombocytopenia. JAK1, however, does not exhibit these
activities and, thus, suggests that compounds that selectively
inhibit JAK1 may have a better hematotoxicity and/or immunogenicity
profile than compounds that selectively inhibit JAK2 or JAK1/2 dual
inhibitors.
[0004] JAK kinase inhibitor compounds are known in the literature.
For example, US 2015/0203455 discloses certain benzimidazole
compounds that are JAK inhibitors.
[0005] There remains a need to provide alternative JAK1 inhibitors
for treatment of cancer. Also, there remains a need to provide
selective JAK1 inhibitors that reduce or avoid JAK2 inhibition.
Accordingly, the present invention provides certain inhibitors of
JAK1 which may be useful for treating cancer. Additionally, the
present invention provides certain selective JAK1 inhibitors that
may reduce JAK2 inhibition.
[0006] The present invention provides a compound of Formula I:
##STR00001##
or a pharmaceutically acceptable salt thereof.
[0007] Preferably, the present invention provides a compound which
is selected from the group consisting of
(2S)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol:
##STR00002##
or a pharmaceutically acceptable salt thereof, and
[0008]
(2R)-3-{[cis-4({4[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H--
benzimidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol
:
##STR00003##
or a pharmaceutically acceptable salt thereof.
[0009] Preferably, the present invention provides a compound which
is
(2S)-3-{[({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benzimidaz-
ol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol:
##STR00004##
[0010] or a pharmaceutically acceptable salt thereof. More
preferably, the present invention provides a compound which is
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1
-trifluoropropan-2-ol:
##STR00005##
or a pharmaceutically acceptable salt thereof.
[0011] As a particular embodiment, the present invention provides a
compound which is
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol.
[0012] As a particular embodiment, the present invention also
provides a compound which is
(2S)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol.
[0013] The present invention provides a pharmaceutical composition
comprising
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol, or
a pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier, diluent, or excipient. The present invention
provides a pharmaceutical composition comprising
(2R)-3-{[cis-4-({4(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benzim-
idazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol, and a
pharmaceutically acceptable carrier, diluent, or excipient.
[0014] The present invention provides a method for treating cancer
comprising administering to a patient in need thereof an effective
amount of a compound of Formula I, 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 of
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-b-
enzimidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol,
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 of
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol.
[0015] The present invention provides a compound of Formula I, or a
pharmaceutically acceptable salt thereof, for use in therapy. The
present invention provides
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol, or
a pharmaceutically acceptable salt thereof, for use in therapy. The
present invention provides
(2R)-3-{[cis-4({4-[1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benzim-
idazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol, or a
pharmaceutically acceptable salt thereof, for use in the treatment
of cancer.
[0016] The present invention provides a compound of Formula I for
use in therapy. The present invention also provides
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol for
use in therapy. The present invention provides
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol for
use in the treatment of cancer.
[0017] The present invention provides the use of a compound of
Formula I, or a pharmaceutically acceptable salt thereof, in the
manufacture of a medicament for the treatment of cancer. The
present invention provides the use of
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol, 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
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol in
the manufacture of a medicament for the treatment of cancer.
[0018] The present invention provides the freebase of
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol in a
crystalline form. The present invention also provides the freebase
of
(2R)-3-{[cis-4-({4-](1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol in a
crystalline form characterized by an X-ray powder diffraction
pattern having characteristic peaks, in 2.theta..+-.0.2, occurring
at 19.5.degree. in combination with one or more of the peaks
selected from the group consisting of 11.9.degree.,
15.4.degree.,and 17.6.degree..
[0019] The present invention provides the dimethane sulfonic acid
salt of
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol in a
crystalline form. The present invention also provides the dimethane
sulfonic acid salt of
(2R)-3-{[cis-4({4[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benzim-
idazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol in a
crystalline form characterized by an X-ray powder diffraction
pattern having characteristic peaks, in 2.theta..+-.0.2.degree.,
occurring at 21.7.degree. in combination with one or more of the
peaks selected from the group consisting of 21.2.degree.,
18.0.degree., and 15.7.degree..
[0020] 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 lung cancer,
including non-small cell lung cancer, small cell lung cancer, and
lung adenocarcinoma, adenocarcinoma, hepatocellular carcinoma,
including Asian hepatocellular carcinoma, colorectal cancer, breast
cancer, lymphoma, and leukemia, including acute lymphocyte leukemia
and T-lineage acute lymphoblastic leukemia. Preferred cancers are
lung cancer, including non-small cell lung cancer and lung
adenocarcinoma, adenocarcinoma, hepatocellular carcinoma, including
Asian hepatocellular carcinoma, colorectal cancer, breast cancer,
and leukemia, including acute lymphocyte leukemia. More preferred
cancers are non-small cell lung cancer, lung adenocarcinoma, breast
cancer, and Asian hepatocellular carcinoma.
[0021] As used above, and throughout the description of the
invention, the following terms, unless otherwise indicated, shall
be understood to have the following meanings:
[0022] 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
[0023] "Pharmaceutically acceptable salts" or "a pharmaceutically
acceptable salt" refers to the relatively non-toxic, inorganic and
organic salt or salts of a compound of the present invention.
[0024] "Effective amount" means the amount of a 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.
[0025] 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.
[0026] A compound of the present invention is 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 them are well known in
the art. See, e.g., P. Stahl, et al., HANDBOOK OF PHARMACEUTICAL
SALTS: PROPERTIES, SELECTION AND USE, (VCHA/Wiley-VCH, 2002); S. M.
Berge, et al., "Pharmaceutical Salts, "Journal of Pharmaceutical
Sciences, Vol 66, No. 1, January 1977.
[0027] A compound of the present invention is preferably formulated
as a pharmaceutical composition using a pharmaceutically acceptable
carrier, diluent, or excipient and administered by a variety of
routes. Preferably, such compositions are for oral administration.
Such pharmaceutical compositions and processes for preparing them
are well known in the art. See, e.g., Remington: The Science and
Practice of Pharmacy (A. Gennaro, et al., eds., 21st ed., Mack
Publishing Co., 2005).
[0028] The amount of a 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 fall within the range of about 50 to 1000
mg per day, preferably 80 to 600 mg per day, most preferably 300 mg
per day. 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. Dosage levels can be determined
by one of skill in the art.
[0029] A compound of the present invention, or pharmaceutically
acceptable salt thereof, 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 a
compound of the invention, or pharmaceutically acceptable salt
thereof.
[0030] 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 and heterocyclic chemistry,
techniques which are known to one of ordinary skill in the art, and
the procedures described in the Examples which follow including any
novel procedures. The following Preparations and Examples further
illustrate the invention. Unless noted to the contrary, the
compounds illustrated herein are named and numbered using IUPACNAME
ACDLABS.
[0031] Individual isomers, enantiomers, or diastereomers may be
separated or resolved by one of ordinary skill in the art at any
convenient point in the synthesis of compounds by methods such as
selective crystallization techniques or chiral chromatography (See,
e.g., Enantiomers, Racemates, and Resolutions (J. Jacques, et al.,
John Wiley and Sons, Inc., 1981)).
[0032] The skilled artisan will appreciate a compound of the
present invention contains 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 a compound of
the present invention exists as a single enantiomer or
diastereomer. The single enantiomer or diastereomer may be prepared
beginning with chiral reagents or by stereoselective or
stereospecific synthetic techniques. Alternatively, the single
enantiomer or diastereomer may be isolated from mixtures by
standard chiral chromatographic or crystallization techniques.
[0033] A compound 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 a compound 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.
[0034] As used herein, the following terms have the meanings
indicated: "ATP" refers to adenosine 5.degree.-triphosphate; "BSA"
refers to bovine serum albumin; "DMSO" refers to dimethyl
sulfoxide; "EDTA" refers to ethylenediaminetetraacetic acid; "EGTA"
refers to ethylene glycol tetraacetic acid; "FBS" refers to fetal
bovine serum; "GFP" refers to green fluorescent protein; "HEPES"
refers to 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; "HWB"
refers to human whole blood; "IC.sub.50" refers to the
concentration of compound that reduces a given response (ligand
binding or enzyme response) by 50%; "IC.sub.50 relative" refers to
the relative concentration giving half the compound's maximum
response; "IVTI" refers to in vivo target inhibition; "JAK" refers
to Janus kinase; "MS" refers to mass spectroscopy; "NMR" refers to
nuclear magnetic resonance; "NSCLC" refers to non-small cell lung
cancer; "PBS" refers to phosphate buffered saline; "RNase" refers
to ribonuclease; "RT" refers to room temperature; "SCLC" refers to
small cell lung cancer; "STAT" refers to signal transducers and
activators of transcription; "TED" refers to threshold effective
dose; "TR-FRET" refers to time resolved fluorescence resonance
energy transfer.
PREPARATION 1
(2R)-2-(Trifluoromethyl)oxirane
##STR00006##
[0036] Add acetic acid (0.89 mL, 0.052 eq) to a solution of
(1S,2S)-(+)-1,2-cyclohexanediamino-N,N'-bis(3,5-di-t-butylsalicylidene)co-
balt (II) (0.90 g, 0.0050 eq) in toluene (16.65 mL). Stir at room
temperature for 30 minutes. Remove the solvent in vacuo. Add
toluene (20 mL) and concentrate in vacuo. Cool to 0.degree. C. and
add 2-(trifluoromethyl)oxirane (37.00 g, 330 mmol; 80.0% ee, (2R)
is the major enantiomer). Stir for five minutes and add water (0.80
mL, 0.15 eq) dropwise. Allow to slowly warm to room temperature and
stir overnight. Vacuum distill at room temperature, collecting the
title compound in a cooled flask as a light yellow oil (28.10 g,
76%; 99.8% ee). .sup.1H NMR (CDC13) .delta. 2.92-2.94 (m, 1H),
2.98-3.01 (m, 1H), 3.41-3.46 (m, 1H).
[0037] Combine the title compound (0.13 g, 1.16 mmol) and methanol
(1.3 mL). Cool to 0.degree. C. and add triethylamine (0.17 mL, 1.10
eq) and thiophenol (0.12 mL, 1.05 eq). Stir for 30 minutes. GCMS of
an aliquot shows formation of
1,1,1-trifluoro-3-phenylsulfanyl-propan-2-ol (from ring-opening of
the title compound); m/z=222. Chiral LC-MS shows 99.8% ee,
(2S)-1,1,1-trifluoro-3-phenylsulfanyl-propan-2-ol is the major
enantiomer.
PREPARATION 2
1-Bromo-3,5-difluoro-2-nitrobenzene
##STR00007##
[0039] Add nitric acid (fuming, 20 mL) dropwise to a solution of
1-bromo-3,5-difluorobenzene (35.00 mL, 304 mmol) in sulfuric acid
(50 mL) at 0.degree. C. Allow to slowly warm to room temperature
and stir overnight. Pour the reaction mixture into a mix of ice and
water (600 mL). Allow to slowly warm to room temperature. Add ethyl
acetate (200 mL) and hexanes (100 mL). Stir until all solids
dissolve. Separate the layers. Wash the organics with saturated
aqueous sodium chloride, dry over anhydrous sodium sulfate, filter,
and concentrate in vacuo to give the title compound as a yellow oil
(57.37 g, 79%). GCMS m/z=237,239 (Br).
PREPARATION 3
3 -Bromo-5-fluoro-N-methyl-2-nitroaniline
##STR00008##
[0041] Add 2 M monomethylamine in tetrahydrofuran (92 mL, 2.00 eq)
to a solution of 1-bromo-3,5-difluoro-2-nitrobenzene (21.90 g, 92
mmol) in 1,4-dioxane (92 mL). Stir at room temperature for 45
minutes. Add water and extract with ethyl acetate. Wash the
organics with saturated aqueous sodium chloride, dry over anhydrous
sodium sulfate, filter, and concentrate in vacuo. Purify by normal
phase chromatography, eluting with a 20-40% methylene chloride in
hexanes gradient, to give the title compound as an orange solid
(16.95 g, 74%). MS (ES) m/z=(.sup.79Br/.sup.81Br) 249/251
(M+H).
PREPARATION 4
tert-Butyl{cis-4-[3-bromo-5-(methylamino)-4-nitrophenoxy]cyclohexyl}carbam-
ate
##STR00009##
[0043] Combine 3-bromo-5-fluoro-N-methyl-2-nitroaniline (75.04 g,
301 mmol), tert-butyl (cis-4-hydroxycyclohexyl)carbamate (89.52 g,
1.38 eq), and tetra(n-butyl)ammonium bisulfate (15.58 g, 0.15 eq)
in dichloromethane (975 mL) and 5 M aqueous sodium hydroxide (241
mL). Stir rapidly at 37.degree. C. under nitrogen for five days.
Cool to room temperature. Dilute with dichloromethane (200 mL) and
water (400 mL). Separate the layers. Extract the aqueous with
dichloromethane (3.times.100 mL). Wash the combined organics with
saturated aqueous sodium chloride, dry over anhydrous sodium
sulfate, filter, and concentrate in vacuo. Purify by normal phase
chromatography, eluting with a 0-40% ethyl acetate in hexanes
gradient, to give the title compound as an orange solid (68.57 g,
51%). MS (ES) m/z=(.sup.79Br/.sup.81Br) 42/444 (M-H).
PREPARATION 5
tert-Butyl{cis-4-[(4-bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl}ca-
rbamate
##STR00010##
[0045] Combine tert-butyl
{cis-4-[3-bromo-5-(methylamino)-4-nitrophenox]cyclohexyl}carbamate
(76.92 g, 173 mmol) and platinum 5% on carbon (sulfided, 3.85 g) in
tetrahydrofuran (923 mL) in a Parr reactor. Stir at room
temperature under 414 kPa hydrogen for three days. Filter through
diatomaceous earth. Wash with tetrahydrofuran. Add
trimethylorthoformate (165 mL, 8.70 eq) to the combined
tetrahydrofuran filtrates. Stir for 22 hours at 63.degree. C.
Concentrate the majority of the reaction mixture in vacuo. Dilute
with water (400 mL) and ethyl acetate (400 mL). Basify with aqueous
sodium carbonate to adjust pH to 9. Separate the layers. Extract
the aqueous with ethyl acetate (2.times.200 mL). Dry the combined
organics over anhydrous sodium sulfate, filter, and concentrate in
vacuo. Dilute with methyl tert-butyl ether (400 mL) and sonicate
for 30 minutes. Filter, wash with methyl tert-butyl ether, and dry
under vacuum to give the title compound as a light brown solid
(52.02 g, 71%). MS (ES) m/z=(.sup.79Br/.sup.81Br) 424/426
(M+H).
PREPARATION 6
cis-4-[(4-Bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexanamine
##STR00011##
[0047] Add trifluoroacetic acid (666 mL) slowly via addition funnel
to a solution of
tert-butyl{cis-4-[(4-bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl}c-
arbamate (222 g, 497 mmol) in dichloromethane (1110 mL) at
0.degree. C. Allow to slowly warm to room temperature and stir
overnight. Concentrate the reaction mixture in vacuo. Add water
(250 mL) and basify with 50% aqueous sodium hydroxide to adjust pH
to 10. Add water (250 mL). Extract with 20% methanol in
dichloromethane (1500 mL, then 500 mL, then 250 mL). Wash the
combined organics with 2 M aqueous sodium hydroxide, dry over
anhydrous magnesium sulfate, filter, and concentrate to give the
title compound as a brown solid (155 g, 91%). MS (ES)
m/z=(.sup.79Br/.sup.81Br) 324/326 (M+H).
PREPARATION 7
(2R)-3-({cis-4-[(4-Bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl
}amino)-1,1,1-trifluoropropan-2-ol
##STR00012##
[0049] Add (2R)-2-(trifluoromethyl)oxirane (73.29 g, 1.50 eq) to a
solution of
cis-4[(4-bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexanamine
(150.4 g, 436 mmol) in methanol (1053 mL). Stir at room temperature
overnight. Concentrate the reaction mixture in vacuo. Purify by
normal phase chromatography, eluting with a 0-10% ethanol in
dichloromethane gradient, to give the title compound as an
off-white solid (98.10 g, 52%). MS (ES) m/z=(.sup.79Br/.sup.81Br)
436/438 (M+H).
PREPARATION 8
3({cis-4-[(4-Bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl}amino)-1,1-
,1-trifluoropropan-2-ol
##STR00013##
[0051] Add 2-(trifluoromethyl)oxirane (2.13 mL, 1.02 eq; 80.0% ee,
(2R) is the major enantiomer) to a solution of
cis-4-[(4-bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexanamine
(7.90 g, 24.37 mmol) in isopropanol (130 mL). Heat at 70.degree. C.
overnight. Concentrate the reaction mixture in vacuo. Purify by
normal phase chromatography, eluting with a stepwise gradient from
100% ethyl acetate to 2.5% to 5% to 7.5% to 10% methanol in ethyl
acetate, to give the title compound (7.86 g, 74%). MS (ES)
m/z=(.sup.79Br/.sup.81Br) 436/438 (M+H).
PREPARATION 9
(5S)-3
-{cis-4-[(4-Bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl}-5-(-
trifluoromethyl)-1,3-oxazolidin-2-one
##STR00014##
[0053] Combine
3-({cis-4-[(4-bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl}amino)-1-
,1,1-trifluoropropan-2-ol (3.34 g, 7.66 mmol; 80.0% ee, (2R) is the
major enantiomer), 1,1'-carbonyldiimidazole (2.48 g, 2.00 eq), and
4-dimethylaminopyridine (0.094 g, 0.10 eq) in dichloromethane (38.3
mL). Stir at room temperature under nitrogen overnight. Concentrate
the reaction mixture in vacuo. Purify by normal phase
chromatography, eluting with a 0-5% methanol in dichloromethane
gradient, to give
3-{cis-4-[(4-bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl}-5-(trifl-
uoromethyl)-1,3-oxazolidin-2-one (3.28 g; 80.0% ee, (5R) is the
major enantiomer).
[0054] Separate the above with the following chiral chromatography
conditions to give the title compound (0.32 g, 9%). MS (ES)
m/z=(.sup.79Br/.sup.81Br) 462/464 (M+H): Enantiomer 1, >99% ee,
75%/25% CO.sub.2/MeOH, 5 mL/min, 4.6.times.150 mm, Chiralpak
AD-H.
PREPARATION 10
(2S)-3-({cis-4-[(4-Bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl}amin-
o)-1,1,1-trifluoropropan-2-ol
##STR00015##
[0056] Combine
(5S)-3-{cis-4-1(4-bromo-l-methyl-1H-benzimidazol-6-yl)oxylcyclohexyl}-5-(-
trifluoromethyl)-1,3-oxazolidin-2-one (0.32 g, 0.69 mmol) and
potassium trimethylsilanolate (0.36 g, 4.00 eq) in tetrahydrofuran
(7 mL). Stir at room temperature under nitrogen for six days.
Dilute with water. Filter, wash with water, and dry under vacuum to
give the title compound as a white solid (0.22 g, 73%). MS (ES)
m/z=(.sup.79Br/.sup.81Br) 436/438 (M+H).
EXAMPLE 1
(2R)-3-{[cis-4-({[4-[(1,5-Dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol
##STR00016##
[0058] Combine (2R)-3
-({cis-4-[(4-bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl}amino)-1,-
1,1-trifluoropropan-2-ol (0.20 g, 0.46 mmol),
1,5-dimethylpyrazol-3-amine (0.056 g, 1.1 eq), potassium carbonate
(0.158 g, 2.5 eq),
2-(di-tert-butylphosphino)-2',4',6'-triisopropyl-3,6-dimethoxy-1,1'-biphe-
nyl (0.046 g, 0.20 eq), tris(dibenzylideneacetone)dipalladium(0)
(0.042 g, 0.10 eq), and acetic acid (0.01 mL) in tert-butyl alcohol
(5 mL). Seal with a crimp cap. Heat in a microwave reactor at
120.degree. C. for 45 minutes. Concentrate the reaction mixture in
vacuo. Purify by normal phase chromatography, eluting with a
stepwise gradient from 100% ethyl acetate to 1% to 2.5% to 5%
methanol in ethyl acetate, to give the title compound (0.071 g,
33%). MS (ES) m/z=467 (M+H).
ALTERNATE EXAMPLE 1
(2R)-3-{[cis-4-({4[(1,5-Dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benzim-
idazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol
##STR00017##
[0060] Combine
(2R)-3-({cis-4-[(4-bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl}ami-
no)-1,1,1-trifluoropropan-2-ol (50 g, 115 mmol),
1,5-dimethylpyrazol-3-amine (18.25 g, 1.43 eq), and potassium
carbonate (50 g, 3.16 eq) in 2-methylbutan-2-ol (400 mL). Stir and
degas with a bubbling nitrogen line for 15 minutes. Add
2-(dicyclohexylphosphino)3,6-dimethoxy-2',4',6'-triisopropyl-1,1'biphenyl
(2.20 g, 0.034 eq) and tris(dibenzylideneacetone)dipalladium(0)
(1.60 g, 0.015 eq). Stir and degas with a bubbling nitrogen line
for five minutes. Add acetic acid (3 mL). Heat at reflux under
nitrogen for twenty hours. Add
2-(dicyclohexylphosphino)3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-bip-
henyl (1.10 g, 0.017 eq) and
tris(dibenzylideneacetone)dipalladium(0) (0.80 g, 0.0075 eq). Heat
at reflux under nitrogen for three hours. Concentrate the reaction
mixture in vacuo. Dilute with water (500 mL). Acidify with 35%
aqueous hydrochloric acid to adjust pH to 1. Add ethyl acetate (100
mL) and stir for five minutes. Treat the stirring mixture with
activated charcoal (5 g) and filter through diatomaceous earth.
Separate the layers and discard the organics. Basify the aqueous
layer with 30% w/w aqueous ammonium hydroxide to adjust pH to 10.
Filter to obtain a solid. Purify by normal phase chromatography,
eluting with 10% 2 M ammonia/methanol in dichloromethane, to give
the title compound (52 g, 95%). MS (ES) m/z=467 (M+H).
SECOND ALTERNATE EXAMPLE 1
(2R)-3-{[cis-4-({4-[(1,5-Dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benzi-
midazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol,
Crystalline Form I
[0061] A: Dissolve a portion of the normal phase chromatography
product of Alternate Example 1 (1.60 g) in 9:1 acetone:water (45
mL). Stir at room temperature for 15 minutes and add SiliaBond.RTM.
DMT (0.42 g). Filter after five hours at room temperature.
Concentrate the filtrate to remove all acetone, then dilute with
water (10 mL). Filter and dry under vacuum to give crystalline
material (1.37 g). MS (ES) m/z=467 (M+H).
[0062] B: Slurry the normal phase chromatography product of
Alternate Example 1 (47.0 g) in isopropanol (1.15 L). Heat the
mixture to reflux to obtain a solution. Add glassy carbon (6 g).
After one hour at reflux, filter through diatomaceous earth. Wash
with isopropanol (50 mL) and seed the filtrate with crystalline
material from Subsection A as provided directly above (0.20 g,
portionwise). Stir and allow to cool to room temperature over two
hours. Filter and dry under vacuum to give crystalline material
(37.8 g). MS (ES) m/z=467 (M+H).
EXAMPLE 2
(2S)-3-{[cis-4-({4-[(1,5-Dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benzi-
midazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol
##STR00018##
[0064] Combine
(2S)-3-({cis-4-[(4-Bromo-1-methyl-1H-benzimidazol-6-yl)oxy]cyclohexyl}ami-
no)-1,1,1-trifluoropropan-2-ol (0.077 g, 0.18 mmol),
1,5-dimethylpyrazol-3-amine (0.030 g, 1.45 eq), potassium carbonate
(0.060 g, 2.46 eq),
2-(di-tert-butylphosphino)-2',4',6'-triisopropyl-3,6-dimethoxy-1,1'-biphe-
nyl (0.022 g, 0.25 eq), tris(dibenzylideneacetone)dipalladium(0)
(0.0080 g, 0.050 eq), and acetic acid (0.01 mL) in tert-butyl
alcohol (2.5 mL). Seal with a crimp cap. Heat at 95.degree. C.
overnight. Dilute with ethyl acetate and filter through
diatomaceous earth. Concentrate the filtrate in vacuo. Purify by
normal phase chromatography, eluting with a 5-100% B gradient (A:
dichloromethane; B: 15% 0.75 M ammoniated methanol in
dichloromethane). Further purify by reverse phase chromatography,
eluting with a 5-100% B gradient (A: 10 nM aqueous ammonium
bicarbonate with 10% methanol; B: acetonitrile). Concentrate clean
fractions from ethanol, then again from dichloromethane, to give
the title compound (0.054 g, 65%). MS (ES) m/z=467 (M+H).
EXAMPLE 3
(2R)-3
-{[cis-4-({4-[(1,5-Dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol;
dimethane sulfonic acid
##STR00019##
[0066] Combine
(2R)-3-{[cis-4-({4-[(1,5-dimethyl-1H-pyrazol-3-yl)amino]-1-methyl-1H-benz-
imidazol-6-yl}oxy)cyclohexyl]amino}-1,1,1-trifluoropropan-2-ol
(1.00 g, 2.15 mmol) and acetone (35 mL). Heat at 51.degree. C. and
add a solution of methanesulfonic acid (0.30 mL, 2.10 eq) in
acetone (5 mL) dropwise. Stir at 51.degree. C. for one hour, and
then cool to room temperature. Filter the resulting solid and dry
in a vacuum oven at 70.degree. C. overnight to give the title
compound (1.25 g, 88%).
X-Ray Powder Diffraction
[0067] Obtain the XRD patterns of crystalline solids on a Bruker D4
Endeavor X-ray powder diffractometer, equipped with a CuKa source
.lamda.=1.54060 .ANG.) and a Vantec detector, operating at 35 kV
and 50 mA. Scan the sample between 4 and 40.degree. in 2.theta.,
with a step size of 0.009.degree. in 2.theta. and a scan rate of
0.5 seconds/step, and with 0.6 mm divergence, 5.28 fixed
anti-scatter, and 9.5 mm detector slits. Pack the dry powder on a
quartz sample holder and obtain a smooth surface using a glass
slide. Collect the crystal form diffraction patterns at ambient
temperature and relative humidity. It is well known in the
crystallography art that, for any given crystal form, the relative
intensities of the diffraction peaks may vary due to preferred
orientation resulting from factors such as crystal morphology and
habit. Where the effects of preferred orientation are present, peak
intensities are altered, but the characteristic peak positions of
the polymorph are unchanged. See, e.g., The United States
Pharmacopeia #23, National Formulary #18, pages 1843-1844, 1995.
Furthermore, it is also well known in the crystallography art that
for any given crystal form the angular peak positions may vary
slightly. For example, peak positions can shift due to a variation
in the temperature or humidity at which a sample is analyzed,
sample displacement, or the presence or absence of an internal
standard. In the present case, a peak position variability of
.+-.0.2.degree. in 2.theta. will take into account these potential
variations without hindering the unequivocal identification of the
indicated crystal form. Confirmation of a crystal form may be made
based on any unique combination of distinguishing peaks (in units
of .degree. 2.theta.), typically the more prominent peaks. Adjust
the crystal form diffraction patterns, collected at ambient
temperature and relative humidity, based on NIST 675 standard peaks
at 8.853.degree. and 26.774.degree. 2.theta..
[0068] Characterize a prepared sample of the compound of Second
Alternate Example 1 by an XRD pattern using CuKa radiation as
having diffraction peaks (2.theta. values) as described in Table 1
below, and in particular having peaks at 19.5.degree. in
combination with one or more of the peaks selected from the group
consisting of 11.9.degree., 15.4.degree., and 17.6.degree.; with a
tolerance for the diffraction angles of 0.2.degree..
TABLE-US-00001 TABLE 1 X-ray powder diffraction peaks of Second
Alternate Example 1 Angle (.degree.2-Theta) +/- Relative Intensity
Peak 0.2.degree. (% of most intense peak) 1 6.4 17.0% 2 11.9 46.6%
3 14.7 11.7% 4 15.4 41.9% 5 16.2 27.5% 6 16.9 24.9% 7 17.6 40.1% 8
19.5 100.0% 9 20.8 32.1% 10 21.5 19.6%
[0069] Characterize a prepared sample of the compound of Example 3
by an XRD pattern using CuKa radiation as having diffraction peaks
(2.theta. values) as described in Table 2 below, and in particular
having peaks at 21.7.degree. in combination with one or more of the
peaks selected from the group consisting of 21.2.degree.,
18.0.degree., and 15.7.degree.; with a tolerance for the
diffraction angles of 0.2.degree..
TABLE-US-00002 TABLE 2 X-ray powder diffraction peaks of Example 3
Angle (.degree.2-Theta) +/- Relative Intensity Peak 0.2.degree. (%
of most intense peak) 1 5.6 26.2% 2 10.0 25.7% 3 13.1 28.7% 4 15.7
70.0% 5 18.0 90.5% 6 19.6 64.5% 7 20.6 41.8% 8 21.2 99.3% 9 21.7
100.0% 10 22.3 59.9%
JAK1, JAK2 and JAK3 In Vitro Enzyme Assays
[0070] The JAK LanthaScreen.TM. Kinase Assay (Invitrogen) is used
to determine the ability of test compounds to inhibit JAK1, JAK2
and JAK3 kinase activity. These are TR-FRET assay formats that use
long-lifetime terbium labeled antibody as the donor species and
GFP-STAT1 as the acceptor species. Use the TR-FRET ratio to monitor
JAK kinase activity where an increase in phosphorylation of the
GFP-STAT1 results in an increase in the TR-FRET ratio. Perform the
kinase reaction using a 12.5 .mu.l reaction volume in shallow black
384-well Proxiplate. Add reagents to obtain final reaction
conditions of 50 ml HEPES pH, 1.76 mM Triton X-100, ATP (20.0 .mu.M
for JAK1 and JAK3 or 5 .mu.M for JAK2) enzyme assays, 10.0 mM
MgCl.sub.2, 1 mM EGTA and 0.01% Brij-35, 0.05 mM GFP-STAT1, 14 nM
JAK1 enzyme for JAK1, 1.0 nM for JAK2 or 2.5 nM for JAK3 enzyme
assays, and 4% DMSO and serial dilutions of test compound (diluted
1:3 from 20,000 to 1 nM). Following ATP/GFP-STAT1 addition,
centrifuge the assay plates for 1 minute at 1000 revolutions per
minute (RPM). Allow the plates to incubate at RT for 60 minutes and
then add 12.5 .mu.l of a stopping buffer containing 20 mM EDTA, 2
nM Terbium-anti-phosphorylated Signal Transducers and Activators of
Transcription [phosphorylation Tyrosine 701 amino acid] Antibody
(Tb-anti-pSTAT1[pTyr701], 0.67 mM tris(hydroxymethyl)aminoethane
hydrochloride (Trizma.RTM.) pH 7.5, 0.02% NaN.sub.3 and 0.01%
nonylphenylpolyethylene glycol (Nonidet.RTM. P40). Incubate at RT
for 90 min and read in an EnVision plate reader with 340 nm
wavelength excitation filter and emission filters of 520 nm and 495
nm wavelengths. Derive the ratio from the emission wavelength for
the GFP-STAT1 which is measured at 520 nm versus the emission at
495 nm for the (Tb-anti-pSTAT1[pTyr701]. Derive the IC.sub.50 value
for each compound using percent inhibition data which is calculated
from the reaction data relative to on-plate controls (active enzyme
versus enzyme inhibited at 2.0 mM with tofacitinib). Use
ACTIVITYBASE 4.0 to fit the percent inhibition and ten-point
compound concentration data to a four-parameter logistic
equation.
[0071] A compound within the scope of the invention is tested in
this assay substantially as described above. The compound of
Example 1 shows a JAK1 IC.sub.50 of 4.0.+-.0.8 nM (n=6), a JAK2
IC.sub.50 of 557.+-.337 nM (n=6), and a JAK3 IC.sub.50 of
1910.+-.786 nM (n=6). The results show that the compound of Example
1 inhibits JAK1 enzyme in vitro. These results also show that the
compound of Example 1 is a more potent inhibitor of JAK1 enzyme and
selective over JAK2 and JAK3 in vitro.
BaF3 Mutant JAK-1 (S729C) Clone 12 Proliferation Assay
[0072] The purpose of this assay is to determine the inhibitory
activity of JAK-1 specific inhibitors using the BaF3 mutant JAK-1
(5729C) Clone 12 proliferation assay.
[0073] mtJAK1 (S729C) expressing cell line is created by
transducing Ba/F3 cells (Murine pro-B-cells) with a retrovirus
expressing human mtJAK1 (5729C)-pQCXIN vector DNA. Single cell
cloning is performed to select Ba/F3 cells expressing highest
levels of mtJAK1 (S729C) by serial dilution. BaF3 mutant JAK-1
(S729C) suspension cells are cultured and maintained in (Gibco RPMI
1640 cat #A10491-01) containing 10% Hi-FBS (Hyclone cat
#10082-047), 1 .mu.g/ml puromycin dihydrochloride (Sigma cat
#9620), 1.0 mg/mL G418 sulfate (Corning ref #30-234-CI), referred
to as R10+ from now on.
[0074] Proliferation assays are performed in culture medium without
selection and from now on will be referred to as (R10-). Briefly,
cultured cells are decanted, centrifuged and reconstituted in 10 mL
of R10- and counted using a Beckman Coulter Vi-Cell. Cells are
further diluted to 2e4/mL in R10- and plated in a 96 well white
opaque assay plates (Costar cat #3610) at 1.times.10.sup.3 cells
(50 .mu.ls) per well. In corresponding 96 well polypropylene
plates, test compounds are diluted in 100% DMSO followed by an
additional dilution in R10- to yield a 2.times.test compound
template. Diluted test compounds are added to the corresponding
cell plate to yield a CRC from 20 .mu.M to 0.001 .mu.M. Minimum and
maximum controls are included on each plate. Staurosporine (1 .mu.M
final) is used for the minimum control and R10- containing
equivalent DMSO levels as the CRC is used as the maximum control.
Plates are covered with water filled Microcline evaporation lids
(cat #LLS-0310) and incubated at 37.degree. C. in 5% CO.sub.2 for 3
days. Following the 3 day incubation, plates are removed from the
incubator and allowed to cool to ambient temperature. Once cooled,
the plates are developed by adding 100 .mu.L of Cell Titer Glo
(Promega cat #G7571), mixing 2 minutes on a plate mixer and
incubating an additional 10 mins at room temperature. Luminescence
is then read on a Perkin Elmer Victor2 using a preset program for
luminescence at 0.1 second. IC.sub.50 curves are generated using an
internal STATS TMAG program and GraphPad Prism version 4.03.
[0075] A compound within the scope of the invention is tested in
this assay substantially as described above. The compound of
Example 1 shows an IC.sub.50 of 956 nM .+-.217 nM (n=4). The result
shows that the compound of Example 1 is active against mtJAK1
(S729C) expressing in Ba/F3 cells.
[0076] AlphaScreen SureFire Protocol
p-STAT3-(p-Tyr705)-IL6-TF-1-JAK1 Cell-Based Assay
[0077] The JAK1 cell based assay described below is used to
determine the JAK1 cellular potency of test compounds.
Cell Preparation:
[0078] Starve TF-1 cells in DMEM medium with 0.5% 26400 (FBS) and
1X Pen/Strep at 37.degree. C. Plate 100K cells per well in BD 96
well black plates with clear bottoms. Maintain the plates at RT for
30-60 minutes before incubating overnight at 37.degree. C. and 5%
CO.sub.2. Count cells using Vi-Cell counter, using a cell
suspension at 100 cells/mL and plated 100 .mu.L/well in Beckman
Dickinson Biocoat plates (Catalog #354640).
Test Compound Preparation and Treatment:
[0079] Prepare compounds at 1:3 serial dilutions in DMSO and
further dilute into the medium. Test compounds in a range of 10
point concentrations from 20,000 to 1 nM. Add diluted compound to
corresponding cell plates. Incubate the plates at 37.degree. C. for
20 mm. Add IL6 solution at the final concentration 30 ng/mL to
corresponding cell plates and continue to incubate at 37.degree. C.
for 30 mm Remove media and add 50 .mu.L 1.times. lysis buffer to
each well.
pSTAT3 Detection:
[0080] Perform the following steps sequentially: make acceptor mix
(activation buffer/reaction buffer/acceptor beads); transfer 4
.mu.L lysate from 96 well plates to 384 well-Proxiplates; add 5
.mu.L acceptor mix to 384 proxiplate plate(s) and seal plates with
aluminum seal; shake 1-2 minutes on plate shaker; incubate plate at
RT for 2 hr with gentle shaking; make donor mix (donor beads in
dilution buffer); add 2 .mu.L donor mix to assay plates; seal
plates with aluminum seal; shake 1-2 minutes on plate shaker;
incubate at RT for 2 hr with gentle shaking; read plate with
Envision; protocol AlphaScreen Surefire 384.
[0081] A compound within the scope of the invention is tested in
this assay substantially as described above. The compound of
Example 1 shows an IC.sub.50 of 87 nM .+-.75 (n=10). This result
shows that the compound of Example 1 inhibits JAK1 enzyme in a cell
based assay.
Phospho-STAT3 (Tyr705) ACUMEN Protocol--H1975JAK1 Cell-Based
Assay
[0082] An H1975-JAK1 cellular assay is used to confirm the potency
of test compounds in NSCLC cells in which the STAT3 pathway is
activated by autocrine IL-6 loop.
Cell Preparation:
[0083] Perform the following steps sequentially: examine cells
under the microscope; aspirate media off the cells with a sterile
vacuum-driven pipette; wash cells with approximately 5 mL PBS;
aspirate off PBS with a sterile vacuum-driven pipette; add 5 mL of
0.25% Trypsin-EDTA to 150 cm.sup.2 flask; gently rock flask to
cover cells with trypsin and let sit in hood for 3 minutes or place
flask in incubator for 3 minutes to loosen cells from flask;
agitate flask a few times to ensure cells are loose; add 10 mL of
growth media to flask, gently dispense suspension over the cell
growth side of the flask and pipette up and down to triturate
cells; spin (1300 rpm 5 min), resuspend in 10 mL of growth media;
strain through a cell strainer (BD Falcon 352350, 70.sub.1.tm cell
strainer); collect the cells in a sterile 50 mL sterile conical
tube; count cells using Vi-Cell counter (0.5 mL cells).
[0084] Day 1: Plate 30000 cells per well in BD 96 well black plates
with clear bottoms. Count cells using Vi-Cell counter, use a cell
suspension at 300000 cells/mL and plated 100 .mu.L/well in Beckman
Dickinson Biocoat plates Catalog #354640. Maintain the plates RT
for 30-60 minutes before putting them into the incubator. Then
incubate overnight at 37.degree. C. and 5% CO.sub.2.
Compound Preparation and Treatment:
[0085] Prepare a deep well plate containing 1 mL media without FBS
with 0.6% DMSO, then add 2 .mu.L of compound (10mM DMSO solution),
making a 20 .mu.M stock plate. Perform 1:3 serial dilutions of the
compounds in media without FBS with 0.6% DMSO in the dilution
plates. Test compounds in a range of 10 point concentrations from
20 .mu.M to 1 nM. Remove media from the assay plates with cells.
Transfer 100 .mu.L of compounds from the dilution plate to the
assay plates (Beckman Dickinson Biocoat plates Catalog #356440)
containing the cell attached with 100 .mu.L media, and use TEMO
program: SAMPLE TRANSFER CELL BASED ASSAY from 3797 slow
dispense.gem. Then incubate the cells for 4 hr at 37.degree. C.
pSTAT3 Detection:
[0086] On Day 1, perform the following steps sequentially: remove
media; add 100 .mu.L of 3.7% para-formaldehyde; incubate for 30
minutes in the dark; wash with 100 .mu.L PBS; add 100 .mu.L cold
methanol; incubate 15 minutes in the dark; wash 2 times with 100
.mu.L PBS; add 100 .mu.L blocking solution (1% BSA in PBS);
incubate for 30 minutes in the dark; add 50 .mu.L primary antibody
pSTAT3 1:500 (in blocking solution: 1% BSA; Mouse
Anti-phospho-STAT3(Tyr705)); seal plates with aluminum foil and
incubate at 4.degree. C. with gentle shaking overnight.
[0087] On Day 2, perform the following steps sequentially: wash 2
times with 100 .mu.L PBS; add 50 .mu.L of secondary antibody
(1:1000; Ab2.degree. goat anti-mouse IgG); incubate for 1 hour at
RT in the dark; wash 2 times with 100 .mu.L PBS; add 100 .mu.L of
Propidium Iodide (1:1000 in PBS from commercial solution)
containing RNAse (50 .mu.g/mL in PBS); seal plates with transparent
film and incubate at RT for 2 hours before reading; read on Acumen
Explorer (set parameter based on positive and negative well). Laser
(Scan 1) 488 nm.
Data Processing:
[0088] Process data through Activity Base and analyze using a
4-parameter nonlinear logistic equation (four-parameter logistic
concentration-response curve):
Y=bot+[(top-bot)/1+(.times./IC50)slope]
where Y=% inhibition, X=concentration yielding y% inhibition,
Bottom=minimum value of y attained by curve, Top=maximum value of y
attained by curve and Slope=steepness of curve at IC50.
%Inh=[(median Max-x/median Max-median Min)]100
[0089] A compound within the scope of the invention is tested in
this assay substantially as described above. The compound of
Example 1 shows an IC.sub.50 of 297 nM (n=1) The result shows that
the compound of Example 1 is active against JAK1 in NSCLC cells in
which the STAT3 pathway is activated by autocrine IL-6 loop.
AlphaScreen SureFire Protocol p-STAT5 (p-Tyr694/699)-EPO-JAK2
Cell-Based Assay
[0090] The fold selectivity of test compounds for JAK1 over JAK2 is
determined by a cellular assay, in which EPO is used to activate
JAK2-STAT5 pathway, and pSTAT5 is used as the readout.
Cell Preparation:
[0091] Starve UT-7 cells overnight in DMEM medium with 0.5% 1600
(PBS) and 1.times. Pen/Strep at 37.degree. C. (without GM-CSF).
Plate 50000 cells per well in BD 96 well black plates with clear
bottoms (in medium without FBS and GM-CSF). Maintain the plates at
RT for 30-60 minutes before incubating overnight at 37.degree. C.
and 5% CO.sub.2. Count cells using Vi-Cell counter, use a cell
suspension at 500000 cells/mL and plated 100 .mu.L/well in Beckman
Dickinson Biocoat plates Catalog #354640.
Compound Dilution and Treatment:
[0092] Test compounds in a range of 10 point concentrations from
20,000 to 1 nM. Prepare compounds at 1:3 serial dilutions in DMSO
and further dilute into the medium. Add diluted compound to
corresponding cell plates and incubate at 37.degree. C. for 20 mm,
then add 20 .mu.L EPO (295 ng/mL. Final concentration 45 ng /mL) to
corresponding cell plates, continue to incubate at 37.degree. C.
for 30 mm.
Lyse Cells:
[0093] Dump media (carefully) and add 50 .mu.L 1.times. lysis
buffer to each well (freeze lysate ON). (lysis buffer:5.times.;
diluted in water to a final concentration of 1.times.).
p-STATS Detection:
[0094] Follow the following steps sequentially: make acceptor mix
(activation buffer/reaction buffer/acceptor beads); transfer 4
.mu.L lysate from 96 well plates to 384 well-Proxiplate; add 5
.mu.L acceptor mix to 384 proxiplate plate(s) with multidrop combi;
seal plates with aluminum seal; shake 1-2 minutes on plate shaker;
incubate plate at RT for 2 hr with gentle shaking; make donor mix
(donor beads in dilution buffer); add 2 .mu.l donor mix to assay
plates with multidrop combi; seal plates with aluminum seal; shake
1-2 minutes on plate shaker; incubate at RT for 2 hr with gentle
shaking; read plate with Envision; protocol AlphaScreen Surefire
384.
[0095] A compound within the scope of the invention is tested in
this assay substantially as described above. The compound of
Example 1 exhibited an IC.sub.50 of 11.8 .mu.M .+-.5.4 (n=5) The
result shows that the compound of Example 1 is selective for JAK1
over JAK2 in a cellular assay, in which EPO is used to activate
JAK2-STATS pathway
Human Whole Blood Assays Determination of pSTAT3 (JAK1) and pSTAT5
(JAK2) in Lymphocytes and Monocytes
[0096] Human whole blood (HWB) assays were developed and validated
to determine the JAK1 and JAK2 selectivity of test compounds.
[0097] Dilute the test compounds, 10 points, (1:3) in DMSO 100% and
a step down in PBS+0.1% BSA. Use tofacitinib as a reference
compound in each plate, as well as a maximum signal (stimulated
wells) and a minimum signal (no stimulated wells) in order to
normalize data. Obtain a pool of HWB from 4 different healthy
donors. Plate the blood in a 96 well plate using a Tecan Evo 96w
and incubate with test compounds for 1 h at RT. After this time of
incubation, stimulate HWB with both IL6 (206-IL, R&D System)
and GM-CSF (PHC2015, Life Technologies) for 15 more minutes. Add a
viability dye (65-0865, eBiosicience) (1:1000) using a Tecan Evo
96w (5.times.mix)
[0098] The final concentrations in the assay are the following: 100
.mu.M for compounds, 50 .mu.M for tofacitinib, 0.1 .mu.g/mL IL6,
0.038 ug/mL GM-CSF and 1% DMSO. Lyse and fix HWB using a Lyse/fix
buffer (558049, Becton Dickinson) by adding 900 .mu.L of lysis
buffer using Tecan Evo 96w (mix 10.times. high speed). Incubate HWB
in bath at 37.degree. C. for 10 minutes. Centrifuge HWB at 500G, 8
mm and discard supernatant. Add cold methanol using a Tecan Exo 96w
in order to permeabilize cells. Incubate blood cells in ice during
30 mm. After this, wash cells 2.times. using Staining buffer
(554656, Becton Dickinson), spin at 3000 rpm, 2 min, discard
supernatant, and add the following antibodies: Anti-Human CD4 PE,
1:100 (12-0048, eBioscience), Anti-Human CD33 eFluor.RTM. 450,1:50
(48-0337, eBioscience), Phospho-STAT5 (Tyr694) (C71E5) Rabbit mAb,
1:100 (Alexa Fluor.RTM. 488 Conjugate) (3939, Cell Signalling) and
Phospho-STAT3 (Tyr705) (D3A7) XP.TM. Rabbit mAb 1:200, (Alexa
Fluor.RTM. 647 Conjugate)(4324, Cell Signalling). Incubate the
antibodies for 1 h in dark at RT, then wash cells 2.times. and read
on Cytometer Macsquant (Miltenyi Biotec). Gate the data on CD4+
(lymphocytes) and CD4Low CD33Hi (monocytes), to measure the
fluorescence from cells expressing pSTAT3 and pSTAT5 respectively.
Analyze the data using FlowJo v_10 and the normalize the median of
fluorescence versus maximum and minimum signal to determine the
IC.sub.50s. Use Graph Pad prism 5 to represent the dose response
curves.
[0099] A compound within the scope of the invention is tested in
this assay substantially as described above. The compound of
Example 1 shows an IC.sub.50 of 2.40.+-.0.64 .mu.M (n=3) for JAK1
and an IC.sub.50 of 13.0.+-.3.2 .mu.M (n=3) for JAK2. The results
demonstrate that the compound of Example 1 shows approximately
5.times. more potency for JAK1 over JAK2 in a human whole blood
assay.
pSTAT3 Mouse IVTI (JAK1) Assay
[0100] The purpose of this assay is to measure the ability of test
compounds to inhibit the phosphorylation of STAT3 (STAT3
activation) in the H1975 xenograft mouse model. Grow H1975 cells
according to ATCC specifications at the lowest possible passage
number available. Culture and maintain cells in RPMI-1640
supplemented with 10% FBS and incubated at 37.degree. C. in 5%
CO.sub.2. Harvest cells by standard techniques and mix with BD
Biosciences basement membrane matrix (MATRIGEL.RTM.) to cell
suspension to achieve a 1:1 cell/matrix ratio yielding an
inoculation volume of 0.2 mL cell/matrix suspension containing 5e6
cells. Keep cell suspensions on ice throughout the inoculation
procedure and start implantations within one hour after cell
culture harvest. Administer all implantations subcutaneously in the
right rear flank of female Athymic Nude mice obtained from Harlan.
Feed all mice Harlan Teklad #2920X ad libitum and provide water by
gel packs. Allow the implanted tumor cells to grow as a solid tumor
and measure twice a week along with body weight, beginning 5-9 days
post implant. Determine tumor volumes using the calculation:
0.536*L*W 2. After tumor volume reaches approximately 200-250
mm.sup.3, randomize animals using the multi task block
randomization tool and place into a vehicle group containing 6-10
animals and multiple treatment groups containing 6 animals each.
Formulate the test compounds in 1% HEC vehicle containing 0.25%
Tween 80 and 0.05% antifoam with 1.1 molar equivalent of
methanesulfonic acid to form an in situ salt. Do not add acid to
the vehicle control group. Calculate doses based on the most recent
group mean body weight. Administer compounds by oral gavage for
both the dose response and time course studies. Dose response
studies are a single dose administered for 2 hours prior to tumor
removal, processing and freezing. Time course studies are a single
dose administered at the TED.sub.70 determined from the dose
response study.
Tumor Tissue Processing:
[0101] Harvest and cut tumors to yield approximately 150-250
mm.sup.3 size fragments and immediately drop into a 12.times.75 mm
tube containing 1 mL of ice cold MSD Tris Lysis buffer (Meso Scale
Discovery cat #R6OTX-2) and 1.times. HALT (Thermo Scientific
product #1861281). Homogenize samples for approximately 15 seconds
using a disposable hard tissue omni tip homogenizer probe (Omni
International cat #3_750H) and allow to sit on wet ice for an
additional 15-25 minutes prior to transferring to a -60.degree. C.
freezer overnight. Remove samples from the freezer and allow to sit
at RT to initiate thawing. Once samples begin to thaw, transfer to
wet ice and continue thawing. After thawing is completed, vortex
samples and transfer to a 1.8 mL microfuge tube. Centrifuge lysates
at 14,000.times.g for 30-60 minutes at 4.degree. C. Transfer
lysates (200 .mu.L) to a 96 well polypropylene plate (Costar cat
#3879). Determine protein concentrations using the Pierce BCA
Protein Assay kit (cat #23225) as indicated below. Briefly, a
standard curve is generated using a BSA standard diluted in RIPA
lysis buffer containing 1XHALT to yield a working range of 2,000
.mu.g/mL to 25 .mu.g/mL. All lysates are diluted 1:10 in RIPA lysis
buffer containing 1.times.HALT . Pipette 25 .mu.L of the standards
and samples into a 96 well plate (Falcon cat# 353072) and add 200
.mu.L of the working reagent to each well and mix plate thoroughly
on a plate shaker for 30 seconds. Cover plate and incubate at
37.degree. C. for 30 minutes. Cool plate to RT and measure
absorbance at or near 562 nm on Molecular Devices Spectra Max.
Protein concentrations for each sample are determined using SoftMax
Pro 6.3 software program. After tumor samples are quantitated,
freeze lysates at -80.degree. C. in the 96 well polypropylene plate
and assay at a later date.
pSTAT3 Measurement:
[0102] Perform the pSTAT3 (Tyr705) assay as follows. The MSD pSTAT3
assay kit is from Meso Scale Discovery (Cat #K150DID-2). The 100X
HALT protease and phosphatase inhibitor may be substituted for the
kit phosphatase and protease inhibitors based on ease of use and
performance Prepare the MSD Tris Lysis buffer by adding 100.times.
HALT protease and phosphatase inhibitor to a 1.times. final
concentration, referred to as MSD complete lysis buffer. Remove
tumor samples from the -80.degree. C. freezer and allow to sit at
RT to initiate thawing. During the sample thaw, block the pSTAT3
Tyr705 capture plate (cat #K150DID-2) with 150 .mu.L of a blocking
solution for a minimum of 1 hr at RT on a plate shaker with
vigorous shaking (300-1000 rpm). Seal plates with an adhesive plate
seal prior to shaking. Blocking solution contains the ratio of
Blocker A (cat #R93BA-4) 600 mgs: 20 mL of 1.times. MSD Tris wash
buffer (cat #R61TX-2). Once samples begin to thaw, transfer to wet
ice to continue thawing. After thawing is completed, carefully mix
by pipetting up and down several times using a multichannel pipet.
Normalize samples in ice cold MSD complete lysis buffer to a
protein concentration of 0.4 .mu.g/.mu.L .times.100 .mu.L. Maintain
all normalized tumor samples on ice in a 96 well polypropylene
plate until they are added to the pSTAT3 capture plate. After the
pSTAT3 capture plate is blocked, wash 4.times. with 250-300 .mu.l
of 1.times. MSD Tris wash buffer using a Thermo Labsystems
Multidrop 384 plate washer. After the final wash, tap the plate
lightly to remove any remaining wash buffer. Add a total volume of
25 .mu.L (10 pig) of normalized tumor lysate per well and incubate
an additional 2-3 hours at RT on a plate shaker with vigorous
shaking (300-1000 rpm). During this incubation, dilute the
Sulfo-TAG anti Phospho-STAT 3 detection antibody provided in the
kit in the antibody dilution buffer (1 ml of blocking solution
combined with 2 mL of 1.times. MSD Tris wash buffer) by adding 60
.mu.L of Sulfo-TAG anti Phospho-STAT 3 detection antibody to 2.94
mL of antibody dilution buffer. Keep this antibody solution on wet
ice until needed. Following tumor lysate incubation, wash the plate
4.times. with 250-300 .mu.L of 1.times. MSD Tris wash buffer. After
the last wash, tap the plate to remove any remaining wash buffer
and add 25 .mu.L/well of the Sulfo-TAG anti Phospho-STAT 3
detection antibody. Incubate the plate an additional 1 hour at RT
on a plate shaker with vigorous shaking (300-1000 rpm). During this
incubation, dilute the 4.times. MSD Read Buffer T with surfactant
(cat #R92TC-3) to 1.times. with deionized water and keep at RT.
After the detection antibody incubation is complete, wash the plate
4.times. with 250-300 .mu.L of 1.times. MSD Tris wash buffer. After
the final wash, tap the plate lightly to remove any remaining wash
buffer and add a total volume of 150 .mu.L/well of 1.times. MSD
Read Buffer T with surfactant. Read the plate on the Meso Quick
Plex SQ 120. Analyze data as indicated below.
Percent pSTAT3 Inhibition Calculation:
[0103] Copy and paste raw plate data from the Meso Quick Plex SQ
120 directly into a Microsoft Excel version 2010 worksheet.
Organize data into the appropriate format (Dose Response or Time
Course), copied and pasted directly into JMP version 11 for pSTAT3
percent inhibition calculations (see formula below).
[1-(Treatment sample Signal/Mean Signal of Vehicle
Control)]*100.
Determine a Oneway Anova with treatment group mean compared to
vehicle control mean using Dunnett's.
TED Calculations:
[0104] Determine the TED.sub.50 and TEC.sub.50 values from a dose
response study. The TED.sub.50 and is the dose necessary to achieve
50% pSTAT3 inhibition and TEC.sub.50 is the plasma concentration to
achieve 50% pSTAT3 inhibition, respectively, at two hours.
[0105] A compound within the scope of the invention is tested in
this assay substantially as described above. The compound of
Example 1 shows a TED.sub.50 of 35 mg/kg and a TEC.sub.50 of 11.1
.mu.M (n=1). These results show that the compound of Example 1
effectively inhibits JAK1-STAT3 signaling (using pSTAT3 as a
readout), in vivo at 2 hours after orally dosing. These results
show that the compound of Example 1 also demonstrates PK/PD
correlated activity with TEC.sub.50
pSTAT3 Rat IVTI (JAK1) Assay
[0106] The purpose of this assay is to measure the ability of test
compounds to inhibit the pSTAT3 expression in the H1975 xenograft
rat model. Grow H1975 cells according to ATCC specifications at the
lowest possible passage number available. Culture and maintain
cells in RPMI-1640 supplemented with 10% FBS and incubated at
37.degree. C. in 5% CO.sub.2. Harvest cells by standard techniques
and mix with BD Biosciences basement membrane matrix
(MATRIGEL.RTM.) to achieve a 1:1 cell/matrix ratio yielding an
inoculation volume of 0.2 mL cell/matrix suspension containing 2e6
cells. Keep cell suspensions on ice throughout the inoculation
procedure and start implantations within one hour after cell
culture harvest. Administer all implantations subcutaneously in the
right rear flank of female NIH Nude rat obtained from Taconic. Feed
all rats Harlan Teklad Global Diets #2920X ad libitum and provide
water bottles. Allow the implanted tumor cells to grow as a solid
tumor and measure twice a week along with body weight. Begin
measuring body weight and tumor volumes 5-9 days post implant.
Determine tumor volumes using the calculation: 0.536*L*W 2. After
tumor volume reaches approximately 350-400 mm.sup.3, randomize
animals using the multi task block randomization tool and place
into a vehicle group containing 6 animals and multiple treatment
groups containing 6 animals each. Formulate the compounds in 1% HEC
vehicle containing 0.25% Tween 80 and 0.05% antifoam with 1.1 molar
equivalent of methanesulfonic acid to form an in situ salt (1.1 mL
of 1N methanesulfonic acid per mg of compound). Do not add acid to
the vehicle control group. Calculate doses based on the most recent
group mean body weight. Administer all compounds by oral gavage for
both the dose response and time course studies. Dose response
studies are a single dose administered for 2 hours prior to tumor
removal, processing and freezing. Time course studies are a single
dose administered at the TED.sub.70 determined from the dose
response study. Perform tumor removal, processing and freezing
occurs at multiple time points outlined below.
Tumor Tissue Processing
[0107] Harvest and cut tumors to yield approximately 200-250
mm.sup.3 size fragments and immediately drop into a 12.times.75 mm
tube containing 1 mL of ice cold MSD Tris Lysis buffer (Meso Scale
Discovery cat #R60TX-2) and 1.times. HALT (Thermo Scientific
product #1861281). Homogenize samples for approximately 15 seconds
using a disposable hard tissue omni tip homogenizer probe (Omni
International cat #3_750H) and allow to sit on wet ice for an
additional 15-25 minutes prior to transferring to a -60.degree. C.
freezer overnight. Remove samples from the freezer and allow to sit
at RT to initiate thawing. Once samples begin to thaw, transfer to
wet ice to continue thawing. After thawing is completed, vortex
samples and transfer to a 1.8 mL microfuge tube. Centrifuge the
lysates at 14,000.times.g for 30-60 minutes at 4.degree. C.
Transfer lysates (200 .mu.L) to a 96 well polypropylene plate
(Costar cat #3879) corresponding to the 96 well sample template
design. Determine protein concentrations using the Pierce BCA
Protein Assay kit (cat #23225) as indicated below. Briefly, a
standard curve is generated using a BSA standard diluted in RIPA
lysis buffer containing 1.times. HALT to yield a working range of
2,000 .mu.g/ml to 25 .mu.g/mL. All lysates are diluted 1:10 in RIPA
lysis buffer containing 1.times. HALT. Pipette 25 .mu.L of the
standards and samples into a 96 well plate (Falcon cat #353072) and
add 200 .mu.L of the working reagent to each well and mix plate
thoroughly on a plate shaker for 30 seconds. Cover plate and
incubate at 37.degree. C. for 30 minutes. Cool plate to RT and
measure absorbance at or near 562 nm on Molecular Devices Spectra
Max. Protein concentrations for each sample are determined using
SoftMax Pro 6.3 software program. After tumor samples are
quantitated, freeze at -80.degree. C. in the 96 well polypropylene
plate and assay at a later date.
pSTAT3 Measurement
[0108] The pSTAT3 (Tyr705) assay is performed as follows. The MSD
pSTAT3 assay kit is from Meso Scale Discovery (Cat #K150DID-2). The
100.times. HALT protease and phosphatase inhibitor may be
substituted for the kit phosphatase and protease inhibitors based
on ease of use and performance Utilize all other reagents from the
kit. Prepare the MSD Tris Lysis buffer by adding 100.times. HALT
protease and phosphatase inhibitor to a 1.times. final
concentration (referred to as MSD complete lysis buffer). Remove
tumor samples from the -80.degree. C. freezer and allow to sit at
RT to initiate thawing. During the sample thaw, block the pSTAT3
Tyr705 capture plate (cat #K150DID-2) with 150 .mu.L of a blocking
solution for a minimum of 1 hr at RT on a plate shaker with
vigorous shaking (300-1000 rpm). Seal plates with an adhesive plate
seal prior to shaking. Blocking solution should contain the ratio
of Blocker A (cat #R93BA-4) 600 mg5: 20 mL of 1.times. MSD Tris
wash buffer (cat #R61TX-2). Once samples begin to thaw, transfer to
wet ice to continue thawing. After thawing is completed, carefully
mix by pipetting up and down several times using a multichannel
pipet. Normalize samples in ice cold MSD complete lysis buffer to a
protein concentration of 0.4 .mu.g/.mu.L.times. 100 .mu.L Maintain
all normalized tumor samples on ice in a 96 well polypropylene
plate until they are added to the pSTAT3 capture plate. After the
pSTAT3 capture plate is blocked, wash 4.times. with 250-300 .mu.L
of 1.times. MSD Tris wash buffer using a Thermo Labsystems
Multidrop 384 plate washer. After the final wash, lightly tap to
remove any remaining wash buffer. Add a total volume of 25 .mu.L
(10 .mu.g) of normalized tumor lysate per well and incubate an
additional 2-3 hours at RT on a plate shaker with vigorous shaking
(300-1000 rpm). During this incubation, dilute the Sulfo-TAG anti
Phospho-STAT 3 detection antibody provided in the kit in the
antibody dilution buffer (1 mL of blocking solution combined with 2
mL of 1.times. MSD Tris wash buffer) by adding 60 .mu.L of
Sulfo-TAG anti Phospho-STAT 3 detection antibody to 2.94 mL of
antibody dilution buffer. Keep this antibody solution on wet ice
until needed. Following tumor lysate incubations, wash the plate
4.times. with 250-300 .mu.L of 1.times. MSD Tris wash buffer. After
the last wash, tap lightly to remove any remaining wash buffer and
add 25 .mu.L/well of the Sulfo-TAG anti Phospho-STAT 3 detection
antibody. Incubate the plate an additional 1 hour at RT on a plate
shaker with vigorous shaking (300-1000 rpm). During this
incubation, dilute the 4.times. MSD Read Buffer T with surfactant
(cat #R92TC-3) to 1.times. with deionized water and keep at RT.
After the detection antibody incubation is complete, wash the plate
4.times. with 250-300 .mu.L of 1.times. MSD Tris wash buffer. After
the final wash, tap lightly to remove any remaining wash buffer and
add a total volume of 150 .mu.l/well of 1.times. MSD Read Buffer T
with surfactant. Read the plate on the Meso Quick Plex SQ 120.
Analyze data as indicated below.
Percent pSTAT3 Inhibition Calculation:
[0109] Copy and paste raw plate data from the Meso Quick Plex SQ
120 directly into a Microsoft Excel version 2010 worksheet.
Organize data into the appropriate format (Dose Response or Time
Course). Copy and paste directly into JMP version 11 for pSTAT3
percent inhibition calculations (see formula below).
[1-(Treatment sample Signal/Mean Signal of Vehicle
Control)]*100.
Determine a Oneway Anova with treatment group mean compared to
vehicle control mean using Dunnett's.
TED Calculations:
[0110] The TED.sub.50 and TEC.sub.50 values are determined from a
dose response study. The TED.sub.50 is the the dose necessary to
achieve 50% pSTAT3 inhibition and TEC.sub.50 is the plasma
concentration required to achieve 50% pSTAT3 inhibition,
respectively, at two hours.
[0111] A compound within the scope of the invention is tested in
this assay substantially as described above. The compound of
Example 1 shows a TED.sub.50 of 9 mg/kg and a TEC.sub.50 of 3.9
.mu.M (n=1). These results show that the compound of Example 1
demonstrates target engagement in rat H1975 IVTI model.
Efficacy Study in HCC827 Xenograft Tumor Model
[0112] The purpose of this assay is to measure the ability of
compounds to inhibit tumor growth in the HCC827 xenograft mouse
model. Grow HCC827 cells (ATCC#CRL-2868, Lot #59392891) according
to ATCC specifications at the lowest possible passage number
available. Culture cells and maintain in RPMI-1640 supplemented
with 10% PBS and incubate at 37.degree. C. in 5% CO.sub.2. Harvest
cells from flasks using TryPLE, rinse twice in DPBS, resuspend in
HBSS, and mix with BD Biosciences basement membrane matrix
(MATRIGEL.RTM.) to cell suspension to achieve a 1:1 cell/matrix
ratio yielding an inoculation volume of 0.2 ml cell/matrix
suspension containing 5e.sup.6 cells. Maintain cell suspensions on
ice throughout the inoculation procedure and start implantations
within one hour after cell culture harvest. Administer
implantations subcutaneously in the right rear flank of female CB17
SCID mice obtained from Harlan (18-20 g). Feed mice Harlan Teklad
Protein Extruded 2920X ad libitum and provide water with the rack
watering system. Allow the implanted tumor cells to grow as a solid
tumor and measure twice a week along with body weight beginning the
seventh day after implantation. Determine tumor volumes using the
calculation: 0.536*L*W 2. After tumor volume reaches approximately
150-200 mm.sup.3, randomize animals and place into groups
containing 5-8 animals each. Formulate the compounds in 1% HEC
vehicle containing 0.25% Tween 80 and 0.05% antifoam with 1.1 molar
equivalent of methanesulfonic acid to form an in situ salt (1.1 mL
of 1N methanesulfonic acid per (insert molecular weight) mg of
compound). Store test compound at RT. Formulate compounds once per
week, and store at RT. Do not add acid to the vehicle control
group. Calculate doses based on the most recent group mean body
weight. Administer compounds by oral gavage for 28 days either BID
or QD depending on dosage.
[0113] A compound within the scope of the invention is tested in
this assay substantially as described above. The compound of
Example 1 shows 31% tumor growth inhibition at 30 mg/kg BID, 22%
tumor regression at 60 mg/kg BID, and 22% tumor regression at 120
mg/kg QD. These results show that the compound of Example 1
demonstrates tumor regression in the HCC827 xenograft tumor
model.
* * * * *