U.S. patent application number 17/587070 was filed with the patent office on 2022-07-28 for substituted imidazo[1,2-a]-pyridines as irak 1/4 and flt3 inhibitors.
The applicant listed for this patent is CHILDREN'S HOSPITAL MEDICAL CENTER, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPT. OF HEALTH AND HUMAN SERVICES, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPT. OF HEALTH AND HUMAN SERVICES. Invention is credited to Jian-Kang Jiang, Katelyn Melgar, Garrett Rhyasen, Daniel T. STARCZYNOWSKI, Craig J. Thomas, Morgan MacKenzie Walker.
Application Number | 20220235042 17/587070 |
Document ID | / |
Family ID | 1000006242545 |
Filed Date | 2022-07-28 |
United States Patent
Application |
20220235042 |
Kind Code |
A1 |
STARCZYNOWSKI; Daniel T. ;
et al. |
July 28, 2022 |
Substituted Imidazo[1,2-a]-pyridines as IRAK 1/4 and FLT3
Inhibitors
Abstract
Some embodiments of the invention include inventive compounds
(e.g., compounds of Formula (I)) and compositions (e.g.,
pharmaceutical compositions) which can be used for treating, for
example, certain diseases. Some embodiments include methods of
using the inventive compound (e.g., in compositions or in
pharmaceutical compositions) for administering and treating (e.g.,
diseases such as head and neck squamous cell carcinoma (HNSCC),
cancer, blood disorders, etc.). Additional embodiments provide
synergistic combinations of a BCL2 inhibitor with an IRAK
inhibiting compound, and methods of using same. ##STR00001##
Inventors: |
STARCZYNOWSKI; Daniel T.;
(Cincinnati, OH) ; Thomas; Craig J.;
(Gaithersburg, MD) ; Rhyasen; Garrett;
(Burlington, MA) ; Melgar; Katelyn; (Cincinnati,
OH) ; Walker; Morgan MacKenzie; (New Haven, CT)
; Jiang; Jian-Kang; (Columbia, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHILDREN'S HOSPITAL MEDICAL CENTER
THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY,
DEPT. OF HEALTH AND HUMAN SERVICES |
Cincinnati
Bethesda |
OH
MD |
US
US |
|
|
Family ID: |
1000006242545 |
Appl. No.: |
17/587070 |
Filed: |
January 28, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16804518 |
Feb 28, 2020 |
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17587070 |
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16326571 |
Feb 19, 2019 |
11254667 |
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PCT/US2017/047088 |
Aug 16, 2017 |
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16804518 |
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62375965 |
Aug 17, 2016 |
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62812948 |
Mar 1, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 471/04 20130101;
A61K 31/496 20130101 |
International
Class: |
C07D 471/04 20060101
C07D471/04; A61K 31/496 20060101 A61K031/496 |
Goverment Interests
GOVERNMENT RIGHTS
[0003] This invention was made with government support under
HL111103 awarded by the National Institutes of Health, and in the
performance of a Cooperative Research and Development Agreement
with the National Institutes of Health, an Agency of the Department
of Health and Human Services. The Government of the United States
has certain rights in this invention.
Claims
1.-61. (canceled)
62. A method of treating a disease or disorder in an individual in
need thereof, the method comprising administering to the individual
a therapeutically effective amount of: (i) a B-cell lymphoma 2
(BCL2) inhibitor selected from: navitoclax, venetoclax, obatoclax,
gossypol, apogossypol,
4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dim-
ethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzam-
ide (AT-737), ethyl
2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate
(HA14-1),
5-[3-[4-(aminomethyl)phenoxy]propyl]-2-[8-[2-(2-benzothiazolyl)-
hydrazinylidene]-5,6,7,8-tetrahydro-2-naphthalenyl]-4-thiazolecarboxylic
acid (WEHI-539),
2-(8-(benzo[d]thiazol-2-ylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-5-(-
3-(4-(3-(dimethylamino)prop-1-yn-1-yl)-2-fluorophenoxy)propyl)thiazole-4-c-
arboxylic acid (A-1155463),
N-[4-(2-tert-butylphenyl)sulfonylphenyl]-2,3,4-trihydroxy-5-[(2-propan-2--
ylphenyl)methyl]benzamide (TW37),
1-oxo-6-thiomorpholino-1H-phenalene-2,3-dicarbonitrile (S1), (2R,3
S,6 S,7R, 8R)-8-butyl-3 -(3
-formamido-2-methoxybenzamido)-2,6-dimethyl-4,9-dioxo-1,5-dioxonan-7-yl3--
methylbutanoate (2-methoxy antimycin A3), 1,1',6,6',
7,7'-hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-[2,2'-binaphthalene]-8,8'-
-dicarbaldehyde (AT-101),
(E)-3-((9-amino-7-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine
(BXI-61), 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-1H,
1'H-2,5'-bibenzo[d]imidazole (BXI-72),
4-((E)-(((Z)-2-(cyclohexylimino)-4-methylthiazol-3(2H)-yl)imino)methyl)be-
nzene-1,2,3-triol (MIM1), and
2-((4-((4-bromophenyl)sulfonamido)-1-hydroxynaphthalen-2-yl)thio)acetic
acid (UMI-77); and (ii) an interleukin receptor-associated kinase
(IRAK) and/or FLT3 inhibitor of Formula (I) ##STR00186## or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein
in Formula (I): R.sup.1 is H, halogen, C.sub.1-C.sub.7 alkyl,
C.sub.2-C.sub.7 alkenyl, C.sub.2-C.sub.7 alkynyl, OH, or
C.sub.1-C.sub.6 alkoxy, wherein the C.sub.1-C.sub.7 alkyl,
C.sub.2-C.sub.7 alkenyl, C.sub.2-C.sub.7 alkynyl, or
C.sub.1-C.sub.6 alkoxy is optionally substituted with one or more
substituents independently selected from the group consisting of
halogen, NO.sub.2, CN, CH.sub.3, CH.sub.2CH.sub.3, C.ident.CH,
C(O)H, C(O)OH, OH, SO.sub.3H, and morpholinyl; R.sup.2 is H,
halogen, CN, C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl,
C.sub.2-C.sub.7 alkynyl, C(O)H, C(O)OH, OH, C.sub.1-C.sub.6 alkoxy,
cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the
C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl, C.sub.2-C.sub.7
alkynyl, C(O)H, C(O)OH, C.sub.1-C.sub.6 alkoxy, cycloalkyl,
heterocyclyl, aryl, or heteroaryl is optionally substituted with
one or more substituents independently selected from the group
consisting of halogen, NO.sub.2, CN, C.sub.1-C.sub.3 alkyl,
C.sub.1-C.sub.3 perfluorinated alkyl, C.ident.CH,
CH.sub.2C.ident.CH, C.ident.CCH.sub.3, C(O)H, C(O)NH.sub.2,
C(O)N(CH.sub.3).sub.2, C(O)OH, C(O)-morpholin-4-yl, NH.sub.2,
N(CH.sub.3).sub.2, OH, C.sub.1-C.sub.3 alkoxy, SO.sub.3H,
heterocyclyl, aryl, and heteroaryl; R.sup.3 is H, halogen,
C.sub.1-C.sub.3 alkyl, C.sub.2-C.sub.3 alkenyl, C.sub.2-C.sub.3
alkynyl, OH, or C.sub.1-C.sub.2 alkoxy, wherein the C.sub.1-C.sub.3
alkyl, C.sub.2-C.sub.3 alkenyl, C.sub.2-C.sub.3 alkynyl, or
C.sub.1-C.sub.2 alkoxy is optionally substituted with one or more
substituents independently selected from the group consisting of
halogen, CN, CH.sub.3, CH.sub.2CH.sub.3, C.ident.CH, C(O)H, C(O)OH,
OH, and SO.sub.3H; R.sup.4 is H, halogen, CN, NO.sub.2,
C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4
alkynyl, C(0)H, C(O)OH, OH, C.sub.1-C.sub.3 alkoxy or SO.sub.3H,
wherein the C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl,
C.sub.2-C.sub.4 alkynyl, or C.sub.1-C.sub.3 alkoxy is optionally
substituted with one or more substituents independently selected
from the group consisting of halogen, NO.sub.2, CN, CH.sub.3,
CH.sub.2CH.sub.3, CCH, C(O)H, C(O)OH, OH, and SO.sub.3H; R.sup.5 is
H, halogen, CN, NO.sub.2, C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4
alkenyl, C.sub.2-C.sub.4 alkynyl, C(O)H, C(O)OH, OH,
C.sub.1-C.sub.3 alkoxy, or SO.sub.3H, wherein the C.sub.1-C.sub.4
alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4 alkynyl, or
C.sub.1-C.sub.3 alkoxy is optionally substituted with one or more
substituents independently selected from the group consisting of
halogen, NO.sub.2, CN, CH.sub.3, CH.sub.2CH.sub.3, C.ident.CH,
C(O)H, C(O)OH, OH, and SO.sub.3H; R.sup.6 is H, halogen, CN,
NO.sub.2, C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl,
C.sub.2-C.sub.4 alkynyl, C(O)H, C(O)OH, OH, C.sub.1-C.sub.3 alkoxy,
or SO.sub.3H, wherein the C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4
alkenyl, C.sub.2-C.sub.4 alkynyl, or C.sub.1-C.sub.3 alkoxy is
optionally substituted with one or more substituents independently
selected from the group consisting of halogen, NO.sub.2, CN,
CH.sub.3, CH.sub.2CH.sub.3, CCH, C(O)H, C(O)OH, OH, and SO.sub.3H;
R.sup.7 is ##STR00187## R.sup.8 is H, CN, NO.sub.2, C.sub.1-C.sub.4
alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4 alkynyl, C(O)H,
C(O)CH.sub.3, C(O)CH.sub.2CN, C(O)-phenyl, C(O)OH, or
phenyl--CH.sub.3; Y is --NH--, --N(CH.sub.3)--,
--N(CH.sub.2CH.sub.3)--, --N(CH.sub.2CH.sub.2CH.sub.3)--,
--N[CH(CH.sub.3).sub.2]--, or --O--; m is 0, 1, 2, 3, 4, or 5; n is
0, 1, 2, 3, 4, or 5; and wherein the sum of m and n is at least
1.
63. The method of claim 62, wherein R.sup.1 is selected from H,
halogen, C.sub.1-C.sub.7 alkyl, and C.sub.1-C.sub.6 alkoxy, wherein
C.sub.1-C.sub.6 alkoxy is optionally substituted with morpholinyl;
R.sup.2 is selected from H, halogen, C.sub.2-C.sub.7 alkynyl,
C.sub.1-C.sub.6 alkoxy, CN, C(O)-morpholin-4-yl,
C(O)N(CH.sub.3).sub.2, C(O)NH.sub.2, heterocyclyl, and heteroaryl,
wherein heterocyclyl or heteroaryl are each independently
optionally substituted with one or more substituents independently
selected from C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 alkoxy, and
morpholinyl; R.sup.3 is selected from H and C.sub.1-C.sub.2 alkoxy;
R.sup.4, R.sup.5, and R.sup.6 are each independently selected from
H, halogen, C.sub.1-C.sub.3 alkyl, and C.sub.1-C.sub.2 alkoxy;
R.sup.7 is ##STR00188## R.sup.8 is selected from H, C(O)CH.sub.3,
C(O)-phenyl, and C(O)CH.sub.2CN; and Y is selected from --O--,
--NH--, and --N(CH.sub.3)--.
64. The method of claim 62, wherein R.sup.1 is selected from Cl,
CH.sub.3, OCH.sub.3, and OCH.sub.2CH.sub.2-morpholinyl.
65. The method of claim 62, wherein R.sup.2 is selected from Cl,
Br, CN, C.ident.CH, OCH.sub.3, C(O)-morpholin-4-yl,
C(O)N(CH.sub.3).sub.2, C(O)NH.sub.2, 1H-pyrrol-2-yl,
1H-pyrrol-3-yl, pyrazol-3-yl, 1H-pyrazol-4-yl,
1-methylpyrazol-4-yl, 1-(morpholin-4-yl)pyrazol-4-yl,
3,5-dimethylpyrazol-4-yl, 3,5-dimethylisoxazol-4-yl, tetrazol-5-yl,
pyridin-3-yl, pyridin-4-yl, 2-methoxypyridin-5-yl,
4-ethylpiperazin-1-yl, and ##STR00189##
66. The method of claim 62, wherein R.sup.3 is OCH.sub.3.
67. The method of claim 62, wherein at least one of (i)-(v)
applies: (i) R.sup.4, R.sup.5, and R.sup.6 are each H; (ii) R.sup.4
and R.sup.6 are each F and R.sup.5 is H; (iii) R.sup.4 is CH.sub.3
and each of R.sup.5 and R.sup.6 is H; (iv) R.sup.4 and R.sup.6 are
each H and R.sup.5 is selected from CH.sub.3, OCH.sub.3, and Br;
(v) R.sup.4 and R.sup.5 are each H and R.sup.6 is CH.sub.3.
68. The method of claim 62, wherein R.sup.7 is selected from
##STR00190##
69. The method of claim 62, wherein R.sup.8 is selected from
(O)CH.sub.3, C(O)-phenyl, and C(O)CH.sub.2CN.
70. The method of claim 62, wherein R.sup.8 is H.
71. The method of claim 62, wherein at least one applies: (i) n is
1, 2, or 3; or (ii) m is 1, 2, or 3.
72. The method of claim 62, wherein the IRAK and/or FLT3 inhibitor,
or a stereoisomer thereof, is selected from the group consisting
of: ##STR00191## ##STR00192## ##STR00193## ##STR00194##
##STR00195## ##STR00196## ##STR00197## ##STR00198## ##STR00199##
##STR00200## ##STR00201## ##STR00202## ##STR00203## or a
pharmaceutically acceptable salt or stereoisomer thereof.
73. The method of claim 62, wherein R.sup.1, R.sup.3, R.sup.4,
R.sup.5, and R.sup.6 are each H; R.sup.2 is selected from H, F, Br,
I, C.sub.2-C.sub.7 alkynyl, C.sub.2-C.sub.6 alkoxy,
C(O)-morpholin-4-yl, C(O)N(CH.sub.3).sub.2, C(O)NH.sub.2,
heterocyclyl, and heteroaryl, wherein heterocyclyl or heteroaryl is
optionally substituted with one or more substituents independently
selected from C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 alkoxy, and
mornholinvl: R.sup.7 is ##STR00204## R.sup.8 is H; m is 0, 1, 2, 3,
4, or 5; n is 0, 1, 3, 4, or5; Y is --NH--; and wherein the sum of
m and n is at least 1.
74. The method of claim 62, wherein venetoclax comprises a
pharmaceutically acceptable salt or stereoisomer thereof.
75. The method of claim 62, wherein the IRAK and/or FLT3 inhibitor
is administered to the individual in a therapeutically effective
amount of 0.005 mg/kg to 50 mg/kg body weight.
76. The method of claim 62, wherein the IRAK and/or FLT3 inhibitor
is administered to the individual via parenteral administration,
mucosal administration, intravenous administration, subcutaneous
administration, topical administration, intradermal administration,
oral administration, sublingual administration, intranasal
administration, or intramuscular administration.
77. The method of claim 62, wherein the disease or disorder is a
cancer or a blood disorder.
78. The method of claim 77, wherein the cancer is selected from
head and neck squamous cell carcinoma, acute myeloid leukemia,
lymphoma, leukemia, bone marrow cancer, non-Hodgkin lymphoma, and
Waldenstrom's macroglobulinemia.
79. The method of claim 78, wherein the cancer is acute myeloid
leukemia.
80. The method of claim 77, wherein the blood disorder is
myelodysplastic syndrome (MDS).
81. The method of claim 80, wherein the MDS is selected from MDS
with a splicing factor mutation, MDS with a mutation in isocitrate
dehydrogenase 1, and MDS with a mutation in isocitrate
dehydrogenase 2.
82. The method of claim 62, wherein the BCL2 inhibitor and the IRAK
inhibitor are administered to the individual in one or more
administrations.
83. The method of claim 62, wherein the disease or disorder is
associated with a FLT3 mutation, an IRAK4 mutation, or an IRAK1
mutation.
84. The method of claim 83, wherein the FLT3 mutation comprises a
mutation in the juxtamembrane region of FLT3, a mutation in the
kinase domain of FLT3, a FLT3-ITD mutation, a D835Y FLT3 mutation,
a D835V FLT3 mutation, a F691L FLT3 mutation, or a R834Q FLT3
mutation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 16/804,518, filed on Feb. 28, 2020, now
allowed, which is a continuation-in-part of U.S. application Ser.
No. 16/326,571, filed on Feb. 19, 2019, now allowed, which is a
U.S. National Stage entry under 35 U.S.C. .sctn. 371 of
International Application No. PCT/US2017/047088, filed on Aug. 16,
2017, designating the United States of America and published in
English on Mar. 1, 2018, which in turn claims priority to U.S.
Provisional Application No. 62/375,965, filed on Aug. 17, 2016,
each of which is hereby incorporated by reference in its
entirety.
[0002] The present application is also a non-provisional of and
claims priority to U.S. Provisional Application No. 62/812,948,
filed on Mar. 1, 2019, which application is hereby incorporated by
reference in its entirety.
BACKGROUND
[0004] Several compounds are known to treat cancer, but do so
inadequately. Some known compounds, such as Quizartinib and
Cremolanib, can be used to treat Acute Myeloid Leukemia (AML). Some
of these treatments do not result in complete remission or partial
remission. In some instances, treatment can result in mutations
that are resistant to inhibitors. Several compounds are known to
treat blood disorders (e.g., Myelodysplastic syndromes (MDS)), but
do so inadequately. Certain embodiments of the invention can
address one or more of these deficiencies.
[0005] Some embodiments of the invention include inventive
compounds (e.g., compounds of Formula (I)). Other embodiments
include compositions (e.g., pharmaceutical compositions) comprising
the inventive compound. Still other embodiments of the invention
include compositions (e.g., pharmaceutical compositions) for
treating, for example, certain diseases using the inventive
compounds. Some embodiments include methods of using the inventive
compound (e.g., in compositions or in pharmaceutical compositions)
for administering and treating (e.g., diseases such as cancer or
blood disorders). Further embodiments include methods for making
the inventive compounds. Additional embodiments of the invention
are also discussed herein.
SUMMARY
[0006] Some embodiments of the present invention include methods of
treating a disease or disorder in an individual, wherein the
disease or disorder is responsive to interleukin-1
receptor-associated kinase (IRAK) inhibition, by administering to
said individual a composition comprising a BCL2 inhibitor and a
composition comprising an IRAK inhibiting compound, or a
composition comprising a BCL2 inhibitor in combination with an IRAK
inhibiting compound, wherein the IRAK inhibiting compound is
selected from Formula (I)
##STR00002##
salts, optical isomers, geometric isomers, salts of isomers, and
derivatives thereof. In some embodiments, R.sup.1 is H, halogen,
hydroxy, C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl,
C.sub.2-C.sub.7 alkynyl, or C.sub.1-C.sub.6 alkoxy, which
C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl, C.sub.2-C.sub.7
alkynyl, or C.sub.1-C.sub.6 alkoxy is optionally substituted with
one or more of halogen, hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), ethynyl (--CCH),
sulfo (--SO.sub.3H), methyl, ethyl, or morpholinyl; R.sup.2 is H,
halogen, hydroxy, --CN, methanoyl (--COH), carboxy (--CO.sub.2H),
C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl, C.sub.2-C.sub.7
alkynyl, C.sub.1-C.sub.6 alkoxy, cycloalkyl, heterocyclyl, aryl, or
heteroaryl, which methanoyl (--COH), carboxy (--CO.sub.2H),
C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl, C.sub.2-C.sub.7
alkynyl, C.sub.2-C.sub.6 alkoxy, cycloalkyl, heterocyclyl, aryl, or
heteroaryl is optionally substituted with one or more of halogen,
hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H), nitro
(--NO.sub.2), --NH.sub.2, --N(CH.sub.3).sub.2, cyano (--CN),
ethynyl (--CCH), propynyl, sulfo (--SO.sub.3H), heterocyclyl, aryl,
heteroaryl, pyrrolyl, piperidyl, piperazinyl, morpholinyl,
--CO-morpholin-4-yl, --CONH.sub.2, --CON(CH.sub.3).sub.2,
C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 perfluoronated alkyl, or
C.sub.1-C.sub.3 alkoxy; R.sup.3 is H, halogen, hydroxy,
C.sub.1-C.sub.3 alkyl, C.sub.2-C.sub.3 alkenyl, C.sub.2-C.sub.3
alkynyl, or C.sub.1-C.sub.2 alkoxy, which C.sub.1-C.sub.3 alkyl,
C.sub.2-C.sub.3 alkenyl, C.sub.2-C.sub.3 alkynyl, or
C.sub.1-C.sub.2 alkoxy is optionally substituted with one or more
of halogen, hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H),
cyano (--CN), ethynyl (--CCH), sulfo (--SO.sub.3H), methyl, or
ethyl; R.sup.4 is H, halogen, hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), sulfo
(--SO.sub.3H), C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl,
C.sub.2-C.sub.4 alkynyl, or C.sub.1-C.sub.3 alkoxy, which
C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4
alkynyl, or C.sub.1-C.sub.3 alkoxy is optionally substituted with
one or more of halogen, hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), ethynyl (--CCH),
sulfo (--SO.sub.3H), methyl, or ethyl; R.sup.5 is H, halogen,
hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H), nitro
(--NO.sub.2), cyano (--CN), sulfo (--SO.sub.3H), C.sub.1-C.sub.4
alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4 alkynyl, or
C.sub.1-C.sub.3 alkoxy, which C.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4 alkynyl, or
C.sub.1-C.sub.3 alkoxy is optionally substituted with one or more
of halogen, hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H),
nitro (--NO.sub.2), cyano (--CN), ethynyl (--CCH), sulfo
(--SO.sub.3H), methyl, or ethyl; R.sup.6 is H, halogen, hydroxy,
methanoyl (--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano
(--CN), sulfo (--SO.sub.3H), C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4
alkenyl, C.sub.2-C.sub.4 alkynyl, or C.sub.1-C.sub.3 alkoxy, which
C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4
alkynyl, or C.sub.1-C.sub.3 alkoxy is optionally substituted with
one or more of halogen, hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), ethynyl (--CCH),
sulfo (--SO.sub.3H), methyl, or ethyl; Y is
##STR00003##
R.sup.7 is
##STR00004##
[0007] R.sup.8 is H, C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4
alkenyl, C.sub.2-C.sub.4 alkynyl, methanoyl (--COH), ethanoyl
(--COCH.sub.3), benzoyl (--COC.sub.6H.sub.5), toluoyl, carboxy
(--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), or --COCH.sub.2CN;
n is 0, 1, 2, 3, 4, or 5; m is 0, 1, 2, 3, 4, or 5; and n+m is at
least 1. In some embodiments, R.sup.1 is H, halogen, hydroxy,
C.sub.1-C.sub.7 alkyl, or C.sub.1-C.sub.6 alkoxy, which
C.sub.1-C.sub.7 alkyl or C.sub.1-C.sub.6 alkoxy is optionally
substituted with one or more of halogen, hydroxy, methanoyl
(--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN),
ethynyl (--CCH), sulfo (--SO.sub.3H), methyl, or ethyl, or
morpholinyl. In other embodiments, R.sup.1 is Cl, methyl,
2-(morpholinyl)ethoxy, or --OCH.sub.3. In yet other embodiments,
R.sup.1 is not H. In some embodiments, R.sup.2 is H, halogen,
hydroxy, --CN, methanoyl (--COH), carboxy (--CO.sub.2H),
C.sub.1-C.sub.7 alkyl, C.sub.1-C.sub.6 alkoxy, cycloalkyl,
heterocyclyl, aryl, or heteroaryl, which methanoyl (--COH), carboxy
(--CO.sub.2H), C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.6 alkoxy,
cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally
substituted with one or more of halogen, hydroxy, methanoyl
(--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2), --NH.sub.2,
--N(CH.sub.3).sub.2, cyano (--CN), ethynyl (--CCH), propynyl, sulfo
(--SO.sub.3H), heterocyclyl, aryl, heteroaryl, pyrrolyl, piperidyl,
piperazinyl, morpholinyl, --CO-morpholin-4-yl, --CONH.sub.2,
--CON(CH.sub.3).sub.2, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
perfluoronated alkyl, or C.sub.1-C.sub.3 alkoxy. In certain
embodiments, R.sup.2 is --CO-morpholin-4-yl, --CON(CH.sub.3).sub.2,
Cl, methyl, --CN, ethynyl, --CONH.sub.2, --CON(CH.sub.3).sub.2,
2-(morpholinyl)ethoxy, ethoxy, methoxy, 1H-pyrazol-4-yl,
1-methyl-pyrazol-4-yl, 1-(morpholin-4-yl)-pyrazol-4-yl,
pyridin-3-yl, 2-methoxy-pyridin-5-yl, pyridin-4-yl,
3,5-dimethylisoxazol-4-yl, 1H-pyrrol-3-yl,
3,5-(di-methyl)-pyrazolyl, pyrazol-3-yl, 5-tetrazolyl,
1H-pyrazol-4-yl, 4-ethyl-piperazin-1-yl, perfluorinated methyl, or
perfluorinated ethyl. In still other embodiments, R.sup.2 is not H.
In certain embodiments, R.sup.3 is H, halogen, hydroxy,
C.sub.1-C.sub.3 alkyl, or C.sub.1-C.sub.2 alkoxy, which
C.sub.1-C.sub.3 alkyl or C.sub.1-C.sub.2 alkoxy can optionally be
substituted with one or more of halogen, hydroxy, methanoyl
(--COH), carboxy (--CO.sub.2H), cyano (--CN), ethynyl (--CCH),
sulfo (--SO.sub.3H), methyl, or ethyl. In other embodiments,
R.sup.3 is H, methoxy, which methoxy is optional substituted with
one, two, or three halogen. In still other embodiments, R.sup.3 is
H or methoxy. In some embodiments, R.sup.4 is H, halogen, hydroxy,
methanoyl (--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano
(--CN), sulfo (--SO.sub.3H), C.sub.1-C.sub.4 alkyl, or
C.sub.1-C.sub.3 alkoxy, which C.sub.1-C.sub.4 alkyl or
C.sub.1-C.sub.3 alkoxy is optionally substituted with one or more
of halogen, hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H),
nitro (--NO.sub.2), cyano (--CN), ethynyl (--CCH), sulfo
(--SO.sub.3H), methyl, or ethyl. In other embodiments, R.sup.4 is
F, Cl, Br, methyl, perfluorinated methyl, or methoxy. In some
embodiments, R.sup.5 is H, halogen, hydroxy, methanoyl (--COH),
carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), sulfo
(--SO.sub.3H), C.sub.1-C.sub.4 alkyl, or C.sub.1-C.sub.3 alkoxy,
which C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.3 alkoxy is optionally
substituted with one or more of halogen, hydroxy, methanoyl
(--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN),
ethynyl (--CCH), sulfo (--SO.sub.3H), methyl, or ethyl. In other
embodiments, R.sup.5 is F, Cl, Br, methyl, ethyl, or methoxy. In
some embodiments, R.sup.6 is H, halogen, hydroxy, methanoyl
(--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN),
sulfo (--SO.sub.3H), C.sub.1-C.sub.4 alkyl, or C.sub.1-C.sub.3
alkoxy, which C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.3 alkoxy can
optionally be substituted with one or more of halogen, hydroxy,
methanoyl (--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano
(--CN), ethynyl (--CCH), sulfo (--SO.sub.3H), methyl, or ethyl. In
other embodiments, R.sup.6 is F, Cl, Br, methyl, perfluorinated
methyl, or methoxy. In some embodiments, Y is
##STR00005##
In other embodiments, Y is
##STR00006##
In some embodiments, R.sup.7 is piperid-2-yl, piperid-3-yl,
piperid-4-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, or azetidyl. In
certain embodiments, R.sup.8 is H, ethanoyl (--COCH.sub.3), benzoyl
(--COC.sub.6H.sub.5), ethynyl (--CCH), or --COCH.sub.2CN. In some
instances, n is 1, 2, or 3. In other instances, m is 1, 2, or
3.
[0008] In some embodiments, the compound is I-1, I-2, I-3, I-4,
I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15, I-16,
I-17, I-18, I-19, I-20, I-21, I-22, I-23, I-24, I-25, I-26, I-27,
I-28, I-29, I-30, I-31, I-32, I-33, I-34, I-35, I-36, I-37, I-38,
I-39, I-40, I-41, I-42, I-43, I-44, I-45, I-46, I-47, I-48, I-49,
I-50, I-51, I-52, I-53, I-54, I-55, I-56, I-57, I-58, I-59, I-60,
I-61, I-62, I-63, I-64, I-65, I-66, I-67, or I-68. In other
embodiments, the compound is I-20, I-21, I-22, I-23, I-24, I-25,
I-26, I-27, I-28, I-29, I-30, I-31, I-32, I-33, I-34, I-35, I-36,
I-37, I-38, I-39, I-40, I-41, I-42, I-43, I-44, I-45, I-46, I-47,
I-48, I-49, I-50, I-51, I-52, I-53, I-54, I-55, I-56, I-57, I-58,
I-59, I-60, I-61, I-62, I-63, I-64, I-65, I-66, I-67, or I-68. In
yet other embodiments, the compound is I-2, I-15, I-20, I-22, I-24,
I-26, I-27, I-42, I-53, or I-54. In still other embodiments, the
compound is I-2, I-15, I-20, I-22, I-24, I-26, I-27, I-42, I-43,
I-44, I-53, or I-54. In some instances, the compound is I-20, I-22,
I-24, I-26, I-27, I-42, I-53, or I-54. In yet other instances, the
compound is I-20, I-22, I-24, I-26, I-27, I-42, I-43, I-44, I-53,
or I-54.
[0009] In some embodiments, if Y is --NH--; R.sup.1 is H; R.sup.3
is H; R.sup.4 is H; R.sup.5 is H; R.sup.6 is H; and R.sup.8 is H,
then (a) R.sup.2 is not H, Cl, methoxy, or CN, and (b) R.sup.7 is
not
##STR00007##
In other embodiments, if Y is --NH--; R.sup.1 is H; R.sup.3 is H;
R.sup.4 is H; R.sup.5 is H; R.sup.6 is H; and R.sup.8 is H, then
(a) R.sup.2 is not H, Cl, F, Br, I, methoxy, ethoxy, or CN, and (b)
R.sup.7 is not
##STR00008##
In still other embodiments, if Y is --NH--; R.sup.1 is H; R.sup.3
is H; R.sup.4 is H; R.sup.5 is H; R.sup.6 is H; and R.sup.8 is H,
then (a) R.sup.2 can be hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl,
C.sub.2-C.sub.7 alkynyl, C.sub.3-C.sub.6 alkoxy, cycloalkyl,
heterocyclyl, aryl, or heteroaryl, which methanoyl (--COH), carboxy
(--CO.sub.2H), C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl,
C.sub.2-C.sub.7 alkynyl, C.sub.3-C.sub.6 alkoxy, cycloalkyl,
heterocyclyl, aryl, or heteroaryl can optionally be substituted
with one or more of halogen, hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), --NH.sub.2, --N(CH.sub.3).sub.2,
cyano (--CN), ethynyl (--CCH), propynyl, sulfo (--SO.sub.3H),
heterocyclyl, aryl, heteroaryl, pyrrolyl, piperidyl, piperazinyl,
morpholinyl, --CO-morpholin-4-yl, --CONH.sub.2,
--CON(CH.sub.3).sub.2, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
perfluoronated alkyl, or C.sub.1-C.sub.3 alkoxy, and (b) R.sup.7 is
not
##STR00009##
In yet other embodiments, if Y is --NH--; R.sup.1 is H; R.sup.3 is
H; R.sup.4 is H; R.sup.5 is H; R.sup.6 is H; and R.sup.8 is H, then
(a) R.sup.2 can be hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl,
C.sub.2-C.sub.7 alkynyl, C.sub.5-C.sub.6 alkoxy, cycloalkyl,
heterocyclyl, aryl, or heteroaryl, which methanoyl (--COH), carboxy
(--CO.sub.2H), C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl,
C.sub.2-C.sub.7 alkynyl, C.sub.5-C.sub.6 alkoxy, cycloalkyl,
heterocyclyl, aryl, or heteroaryl can optionally be substituted
with one or more of halogen, hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), --NH.sub.2, --N(CH.sub.3).sub.2,
cyano (--CN), ethynyl (--CCH), propynyl, sulfo (--SO.sub.3H),
heterocyclyl, aryl, heteroaryl, pyrrolyl, piperidyl, piperazinyl,
morpholinyl, --CO-morpholin-4-yl, --CONH.sub.2,
--CON(CH.sub.3).sub.2, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
perfluoronated alkyl, or C.sub.1-C.sub.3 alkoxy, and (b) R.sup.7 is
not
##STR00010##
In certain embodiments, one or more of compounds I-1, I-2, I-3,
I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15,
I-16, I-17, I-18, or I-19 are not included in Formula (I). In still
other embodiments, compounds I-1, I-2, I-3, I-4, I-5, I-6, I-7,
I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15, I-16, I-17, I-18, and
I-19 are not included in Formula (I).
[0010] Some embodiments of the invention include a composition
comprising a compound, as disclosed herein (e.g., Formula (I)). In
certain embodiments, the amount of the compound is from about
0.0001% (by weight total composition) to about 99%. In other
embodiments, the composition further comprises a formulary
ingredient, an adjuvant, or a carrier.
[0011] Some embodiments of the invention include a pharmaceutical
composition comprising a compound, as disclosed herein (e.g.,
Formula (I)). In certain embodiments, the amount of the compound is
from about 0.0001% (by weight total composition) to about 50%. In
other embodiments, the pharmaceutical composition further comprises
a formulary ingredient, an adjuvant, or a carrier.
[0012] Some embodiments of the invention include a method for
providing an animal with a compound comprising one or more
administrations of one or more compositions comprising a compound
as disclosed here (e.g., Formula (I)), where the compositions may
be the same or different if there is more than one administration.
In other embodiments, at least one of the one or more compositions
further comprises a formulary ingredient. In certain embodiments,
at least one of the one or more compositions comprises any
composition disclosed herein or any pharmaceutical composition
disclosed herein. In still other embodiments, at least one of the
one or more administrations comprises parenteral administration, a
mucosal administration, intravenous administration, subcutaneous
administration, topical administration, intradermal administration,
oral administration, sublingual administration, intranasal
administration, or intramuscular administration. In some
embodiments, if there is more than one administration at least one
composition used for at least one administration is different from
the composition of at least one other administration. In other
embodiments, the compound of at least one of the one or more
compositions is administered to the animal in an amount of from
about 0.01 mg/kg animal body weight to about 15 mg/kg animal body
weight. In certain embodiments, the animal is a human, a rodent, or
a primate.
[0013] Some embodiments, of the invention include a method for
treating an animal for a disease, comprising one or more
administrations of one or more compositions comprising any compound
disclosed herein (e.g., Formula (I)), wherein the compositions may
be the same or different if there is more than one administration.
In some instances, at least one of the one or more compositions
further comprises a formulary ingredient. In other embodiments, at
least one of the one or more compositions comprises any composition
disclosed herein or any pharmaceutical composition disclosed
herein. In certain embodiments, at least one of the one or more
administrations comprises parenteral administration, a mucosal
administration, intravenous administration, subcutaneous
administration, topical administration, intradermal administration,
oral administration, sublingual administration, intranasal
administration, or intramuscular administration. In other
embodiments, if there is more than one administration at least one
composition used for at least one administration is different from
the composition of at least one other administration. In still
other embodiments, the compound of at least one of the one or more
compositions is administered to the animal in an amount of from
about 0.005 mg/kg animal body weight to about 50 mg /kg animal body
weight. In yet other embodiments, the animal is a human, a rodent,
or a primate. In certain embodiments, the animal is in need of the
treatment. In some embodiments, the method is for treating a head
and neck squamous cell carcinoma (HNSCC), a blood disorder, MDS,
cancer, or AML. In other embodiments, the method is for treating
acute myeloid leukemia, lymphoma, leukemia, bone marrow cancer,
non-Hodgkin lymphoma, or Waldenstrom's macroglobulinemia. In yet
other embodiments, the method is for treating MDS, MDS with a
splicing factor mutation, MDS with a mutation in isocitrate
dehydrogenase 1, or MDS with a mutation in isocitrate dehydrogenase
2. In certain embodiments, the animal is susceptible to AML or MDS.
In other embodiments, the method prevents or ameliorates future AML
or MDS. In some embodiments, the method occurs after one or more of
having a blood disorder, having myelodysplastic syndrome, having
myeloproliferative disease, an occurrence of chemical exposure, an
exposure to ionizing radiation, or a treatment for cancer.
[0014] Some embodiments of the invention include a method for
preparing any compound disclosed herein (e.g., Formula (I))
comprising,
[0015] (a) reacting a compound of Formula (II) with a compound of
Formula (III) to result in a mixture comprising a compound of
Formula (IV);
[0016] (b) reacting a compound of Formula (IV) with a compound of
Formula (V) to result in a mixture comprising a compound of Formula
(VI);
[0017] (c) optionally reacting a compound of Formula (VI) with a
compound of Formula (VII) to result in a mixture comprising a
compound of Formula (VIII);
[0018] (d) removing one or more protecting groups from a compound
of Formula (VI) or from a compound of Formula (VIII); and
[0019] (e) recovering Formula (I),
where Formula (II) is
##STR00011##
Formula (III) is
##STR00012##
[0020] Formula (IV) is
##STR00013##
[0021] Formula (V) is
##STR00014##
[0022] Formula (VI) is
##STR00015##
[0023] Formula (VII) is R.sup.2Bpin (VII); and
Formula (VIII) is
##STR00016##
[0024] In some embodiments, R.sup.1 is an C.sub.1-C.sub.6 alkoxy
and the method further comprises (i) the step of reacting Formula
(IV) to convert the C.sub.1-C.sub.6 alkoxy at R.sup.1 to hydroxy
and (ii) the step of reacting the product of (i) to convert the
hydroxy at R.sup.1 to a morpholino-C.sub.1-C.sub.6-alkoxy; and
steps (i) and (ii) occur after step (a) and before step (b). In
other embodiments, in step (b), Y is not O. In yet other
embodiments, in step (b), Y is O. In some embodiments, during step
(d), at least one of the one or more protecting groups removed is
-Boc. In still other embodiments, R.sup.2 is a halogen and the
method further comprises the step of reacting Formula (I) to
convert the halogen at R.sup.2 to C.sub.2-C.sub.7 alkynyl, after
step (d).
[0025] Other embodiments of the invention are also discussed
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the description of specific embodiments presented
herein.
[0027] FIG. 1: Inhibitory activities of compounds I-2, I-22, I-24,
and quizartinib were determined by measuring the IC50 against human
cord blood CD34+ cells transduced with MLL-AF9 and FLT3-ITD
(designated as MA9-FLT3-ITD and also referred to as MLL-AF9/FLT3
ITD).
[0028] FIGS. 2A-2C: Some compounds of Formula (I) can suppress
activation of FLT3. FIG. 2A: Immunoblot analysis of MV4; 11 cells
treated with AC220 (50 nM), compound I-20 (50 nM), or compound I-24
(50 nM) for 12 or 24 hours. FIG. 2B: Immunoblot analysis of MDSL
cells treated with the indicated concentrations of I-24 or IRAK-Inh
(Amgen) for 24 hours. FIG. 2C: Phospho (P)-STATS activity was
measured by AlphaLISA assay in MV4; eleven cells treated with the
indicated concentrations of I-15, I-20, I-43, or AC220 for 5
hours.
[0029] FIGS. 3A-3D: FLT3 inhibition can result in a compensatory
activation of IRAK1/4in FLT3-ITD AML. FIG. 3A: Immunoblot analysis
of MA9-FLT3-ITD treated with AC220 (50 nM) for the indicated times.
FIG. 3B: Immunoblot analysis of MV4; eleven cells treated with
AC220 (1 or 50 nM) for the indicated times. FIG. 3C: Immunoblot
analysis MA9-FLT3-ITD treated with AC220 (50 nM), AC220 (50 nM) and
IRAK-Inh (10 .mu.M), I-20 (50 nM), or IRAK-Inh alone (10 .mu.M).
FIG. 3D: Immunoblot analysis of human cord blood CD34+ cells
transduced with MLL-AF9 and Nras (MA9-NRas) treated with AC220 (50
nM), AC220 (50 nM) and IRAK-Inh (10 .mu.M), I-20 (50 nM), or
IRAK-Inh alone (10 .mu.M).
[0030] FIGS. 4A-4D: Synergistic inhibition of FLT3-ITD AML with
FLT3 and IRAK1/4inhibitors. FIGS. 4A-4B: Heatmap response profile
(left panel) and Delta Bliss analysis (right panel) for AC220 and
IRAK-Inh (Amgen) combination treatment of MA9-FLT3-ITD cells. FIG.
4A: Cell-titer glow (CTG) percent response values represent
normalized growth, relative to controls based on SybrGreen
fluorescence intensities. FIG. 4B: Caspase activation values,
relative to controls based on caspase-glo fluorescence intensities.
FIG. 4C: The IC10 of AC220 was established in MA9-FLT3-ITD cells
after 48 hour treatment using cell-titer glow relative response
values normalized to growth compared to control cells (DMSO). FIG.
4D: MA9-FLT3-ITD cells were treated with IRAK-Inh (Amgen) alone or
in combination with 0.3 nM of AC220 (IC10) for 72 hours. Cell-titer
glow relative response values represent normalized growth compared
to control cells (DMSO).
[0031] FIGS. 5A-5G: Some compounds of Formula (I) can suppress
FLT3-ITD AML. FIG. 5A: Generation of two independent clones (#3 and
#6) derived from human cord blood CD34+ cells transduced with
MLL-AF9 and then either FLT3-ITD (MA9-FLT3-ITD) or NRas (MA9-NRas).
FIGS. 5B-5G: MA9.3 or MA9.6 clones expressing FLT3-ITD or NRas were
treated with the indicated compounds for 72 hours. Cell-titer glow
relative response values represent normalized growth compared to
control cells (DMSO) based on luminescence intensities. Cellular
IC50 values (nM) are shown for each experiment.
[0032] FIG. 6: Some compounds of Formula (I) can suppress FLT3-ITD
AML. Cell viability was determined in MLL-AF9/FLT3-ITD cells
treated with compound I-15 (1 .mu.M), compound I-43 (1 .mu.M), or
compound I-20 (1 .mu.M) for 72 hours by flow cytometric analysis of
AnnexinV.
[0033] FIGS. 7A-7E: Cell studies using compound I-20, compound
I-17, compound I-22, and compound I-24. FIGS. 7A-7B: cells were
treated with the indicated compounds for 72 hours. Cell-titer glow
relative response values represent normalized growth compared to
control cells (DMSO) based on luminescence intensities. Cellular
IC50 values (nM) are shown for each experiment. FIG. 7C: Cell
viability was determined in MA9-FLT3-ITD cells treated with 1 .mu.M
of the indicated compounds for 72 hours by flow cytometric analysis
of AnnexinV. FIG. 7D: Leukemic colony formation of MA9-FLT3-ITD
cells was determined in methylcellulose supplemented with 1 .mu.M
of the indicated compounds. Colony formation was determined after
10 days. FIG. 7E: Colony formation of normal cord blood CD34+ cells
was determined in methylcellulose supplemented with 1 .mu.M of the
indicated compounds. Colony formation was determined after 10
days.
[0034] FIGS. 8A-8G: Some compounds of Formula (I) can prevent
emergence of resistant FLT3-ITD AML. FIG. 8A Overview of
experimental design: MA9-FLT3-ITD cells were cultured in cytokines
and then treated with AC220 or compound I-20 (1, 2.5, or 5 .mu.M)
for 72 hours. Cell viability was assessed by AnnexinV staining.
Remaining cells were washed and replated in fresh media with
cytokines. Recovery of MA9-FLT3-ITD cell growth was determined
after 7 days by AnnexinV staining or Trypan Blue exclusion. FIG. 8B
Cell viability was determined in MA9-FLT3-ITD cells after 72 hours
following treatment with the indicated compounds, or after 7 days
of recovery. FIG. 8C: Overview of experimental design: MA9-FLT3-ITD
cells were cultured in cytokines and then treated with AC220,
compound I-20, or compound I-24 (5 .mu.M) for 72 hours. Cell
viability was assessed by AnnexinV staining. Remaining cells were
washed and replated in fresh media with cytokines. Recovery of
MA9-FLT3-ITD cell growth was monitored every 2 days by AnnexinV
staining. FIG. 8D: Cell viability was determined in MA9-FLT3-ITD
cells after 72 hours following treatment (Day 0) with the indicated
compounds, or every 2 days post recovery by AnnexinV staining.
Compound I-24 treated cells were not monitored past Day 2 as no
viable cells remained. FIG. 8E: Overview of experimental design:
MA9-FLT3-ITD cells were cultured in cytokines and then treated with
AC220 or compound I-24 (5 .mu.M) for 72 hours. Cell viability was
assessed by AnnexinV staining. Remaining cells were washed and
replated in fresh media with cytokines. After AC220-treated cells
recovered (Day 7), they were subsequently treated with AC220 (5
.mu.M) or 1-24 (5 .mu.M) ("i") and monitored every 2 days by
AnnexinV staining. This step was repeated once more at Day 16
("ii"). FIGS. 8F-8G: Cell viability was determined in MA9-FLT3-ITD
cells after 72 hours following treatment (Day 0) with the indicated
compounds, or every 2 days post recovery by AnnexinV staining (FIG.
8F) or Trypan Blue exclusion (FIG. 8G).
[0035] FIGS. 9A-9C: Some compounds of Formula (I) can be effective
against FLT3-ITD AML xenograft mouse models. FIG. 9A: Overview of
in vivo experimental design: NRGS mice were injected i.v. with
MA9-FLT3-ITD cells (2.times.105 cells/mouse). After 10 days, PBS or
compound I-24 (30 mg/kg) was injected i.p. for 5 daily treatments,
followed by a 2 day rest. After the 2nd treatment, one mouse from
each group was sacrificed and MA9-FLT3-ITD (GFP+) cells were
isolated from the BM by flow sorting for immunoblotting of FLT3 and
IRAK4. A second cycle of daily injections of PBS or compound I-24
for 5 days, followed by daily monitoring of morbidity. FIG. 9B:
Immunoblot analysis of sorted (GFP+) MA9-FLT3-ITD BM cells from
xenografted mice after 2 doses of compound I-24. FIG. 9C: Overall
survival of NRGS mice xenografted with MA9-FLT3-ITD treated with
compound I-24 or PBS.
[0036] FIGS. 10A-10B: Some compounds of Formula (I) can be
effective against MDS cell function and viability. FIG. 10A: Colony
formation of MDSL cells was determined in methylcellulose
supplemented with 1 .mu.M or 10 .mu.M of the indicated compounds.
Colony formation was determined after 10 days. FIG. 10B: MDSL cells
were treated with the indicated compounds for 72 hours. Cell-titer
glow relative response values represent normalized growth compared
to control cells (DMSO) based on luminescence intensities.
[0037] FIG. 11: Raw data is shown for the AF9 cells.
[0038] FIG. 12: Bliss Score data is shown for the AF9 cells.
[0039] FIG. 13: IRAK inhibitors have synergy with apoptosis
inhibitors. FIG. 13 illustrates the drug synergy, showing that
compound I-24 (also referred to herein interchangeably as
"NCGC1481", "I-24/1481", or simply "1481") disclosed herein
synergizes with venetoclax in AF9 cells. This experiment was done
in MLL-AF9 FLT3-ITD cells. The experiment shows that when a
minimally active concentration of 1481 (0.2 nM) is combined with
each concentration of Venetoclax the IC50 of Venetoclax increases
(is shifted to the left by 50-fold). IC50 of Venetoclax alone=3159
nM; IC50 of Venetoclax in the presence of 0.2 nM 1481=63.81 nM. The
IC50 of 1481 alone is 0.297 nM. The data were analyzed in GraphPad
Prism, v 8.0.2.
[0040] FIGS. 14A-14C: Compound I-24/1481 demonstrates synergy with
venetoclax in the THP-1 cell line. Data were analyzed by Prism v 8.
FIG. 14A depicts the change in IC50, fold potency of venetoclax,
and combination index (CI) when Compound I-24/1481 is administered
synergistically with venetoclax in THP-1 cells. FIG. 14B depicts
the change in percent viability when Compound I-24/1481 is
administered synergistically with venetoclax in THP-1 cells. FIG.
14C depicts the relevant heat maps; data in the upper heat map
indicates the Bliss Scores, and the lower heat map indicates the
individual data points for the various dose response curves.
Methods: Day 0: Cell Line Plating in growth media recommended by
ATCC using Tecan Fluent in 96 well plate (Eppendorf 0030730011);
Day 1: Drug Addition. Venetoclax obtained from Selleckchem (S8048);
Day 1->4: Incubate with Drug for 72 Hrs at 37C, 5% CO2; Day 5:
Add 20% original volume MTS (Promega G5440) and incubate for 4 Hrs;
Add 25% original volume 10% SDS (Invitrogen 24730-020) to lyse
cells and stop conversion of MTS to formazan. Read out is
colorimetric at 490 nm using a BioTek Synergy Neo Plate Reader.
[0041] FIGS. 15A-15C: Compound I-24/1481 demonstrates synergy with
venetoclax in the Kasumi-1 cell line. Data were analyzed by Prism v
8. FIG. 15A depicts the change in IC50, fold potency of venetoclax,
and combination index (CI) when Compound I-24/1481 is administered
synergistically with venetoclax in Kasumi-1 cells. FIG. 15B depicts
the change in percent viability when Compound I-24/1481 is
administered synergistically with venetoclax in Kasumi-1 cells.
FIG. 15C depicts the relevant heat maps; data in the upper heat
maps indicates the Bliss Scores, and the lower heat map indicates
the individual data points for the various dose response curves.
Methods: Day 0: Cell Line Plating in growth media recommended by
ATCC using Tecan Fluent in 96 well plate (Eppendorf 0030730011);
Day 1: Drug Addition. Venetoclax obtained from Selleckchem (S8048);
Day 1->4: Incubate with Drug for 72 Hrs at 37C, 5% CO2; Day 5:
Add 20% original volume MTS (Promega G5440) and incubate for 4
Hrs.; Add 25% original volume 10% SDS (Invitrogen 24730-020) to
lyse cells and stop conversion of MTS to formazan. Read out is
colorimetric at 490 nm using a BioTek Synergy Neo Plate Reader.
[0042] FIGS. 16A-16C: Compound I-24/1481 demonstrates synergy with
venetoclax in the TF-1 cell line. Data were analyzed by Prism v 8.
FIG. 16A depicts the change in IC50, fold potency of venetoclax,
and combination index (CI) when Compound I-24/1481 is administered
synergistically with venetoclax in TF-1 cells. FIG. 16B depicts the
change in percent viability when Compound I-24/1481 is administered
synergistically with venetoclax in TF-1 cells. FIG. 16C depicts the
relevant heat maps; data in the upper heat maps indicates the Bliss
Scores, and the lower heat map indicates the individual data points
for the various dose response curves. Methods: Day 0: Cell Line
Plating in growth media recommended by ATCC using Tecan Fluent in
96 well plate (Eppendorf 0030730011); Day 1: Drug Addition.
Venetoclax obtained from Selleckchem (S8048); Day 1->4: Incubate
with Drug for 72 Hrs at 37C, 5% CO2; Day 5: Add 20% original volume
MTS (Promega G5440) and incubate for 4 Hrs; Add 25% original volume
10% SDS (Invitrogen 24730-020) to lyse cells and stop conversion of
MTS to formazan. Read out is colorimetric at 490 nm using a BioTek
Synergy Neo Plate Reader.
[0043] FIG. 17: Summary of results from Reaction Biology assay with
full kinase selectivity panel, as an isolated kinase domain panel,
for compound I-24/1481. The compound was tested in 10-dose IC50
mode with 3-fold serial dilution starting at 10 .mu.M. The control
compound, Staurosporine, was tested in 10-dose IC50 mode with
4-fold serial dilution starting at 20 .mu.M or 100 .mu.M. Alternate
control compounds were tested in 10-dose IC50 mode with 3-fold or
4-fold serial dilution starting at 10 .mu.M, 20 .mu.M, or 100
.mu.M. Reactions were carried out at 10 .mu.M ATP. Data sets
generated for each kinase generated include raw data, % Enzyme
activity (relative to DMSO controls), and curve fits (not shown).
Curve fits were performed where the enzyme activities at the
highest concentration of compounds were less than 65%. An IC50
value less than 0.508 nM or higher than 10 .mu.M was estimated
based on the best curve fitting available.
[0044] FIGS. 18A-18C: Summary of results from KiNativ kinase
selectivity panel for compound I-24/1481. FIG. 18A depicts the
labeling site key. FIG. 18B depicts estimated IC50 values. FIG. 18C
depicts the full IC50 list for the various kinases. This is an in
situ kinase profiling assay; details and methodology are available
at www <dot>kinativ<dot>com. The assay was performed in
lysates of MV-4-11 cells which were pretreated for 15 minutes with
the inhibitor before the ATP probe was added. The MV4-11 cells are
FLT3-ITD+, but the assay does not measure FLT3 activity, possibly
because they do not have a fluorescent peptide substrate that picks
up FLT3 activity.
[0045] FIGS. 19A-19B: Dendrogram depiction and legend of the
KiNative results, which is a graphical representation of the data.
FIG. 19A depicts the dendrogram of the KiNative results. FIG. 19B
depicts the legend for the KiNative results.
[0046] FIG. 20: Summary of studies on compound I-24/1481. FIG. 20
(A) shows compound I-24/1481 structure and inhibition of IRAK1,
IRAK4, and FLT3. FIG. 20 (B) shows the interaction between compound
I-24/1481 and the IRAK4 binding site. FIGS. 20 (C)-20 (D) show the
Reaction Biology and KiNative Profile results and further list 12
compounds (compound I-24/1481 and 11 analogs) that show a range of
MV4; 11 cytotoxicity from 5 nM to >10,000 nM. FIG. 20 (E) shows
selected MV4; 11 Western blots. FIGS. 20 (F)-20 (G) show further
results.
DETAILED DESCRIPTION
[0047] While embodiments encompassing the general inventive
concepts may take diverse forms, various embodiments will be
described herein, with the understanding that the present
disclosure is to be considered merely exemplary, and the general
inventive concepts are not intended to be limited to the disclosed
embodiments.
[0048] Some embodiments of the invention include inventive
compounds (e.g., compounds of Formula (I)). Other embodiments
include compositions (e.g., pharmaceutical compositions) comprising
the inventive compound. Still other embodiments of the invention
include compositions for treating, for example, certain diseases
using the inventive compounds. Some embodiments include methods of
using the inventive compound (e.g., in compositions or in
pharmaceutical compositions) for administering and treating.
Further embodiments include methods for making the inventive
compound.
[0049] As used herein (unless otherwise specified), the term
"alkyl" means a monovalent, straight or branched hydrocarbon chain.
For example, the terms "C.sub.1-C.sub.7 alkyl" or "C.sub.1-C.sub.4
alkyl" refer to straight- or branched-chain saturated hydrocarbon
groups having from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7), or 1 to 4
(e.g., 1, 2, 3, or 4), carbon atoms, respectively. Examples of
C.sub.1-C.sub.7 alkyl groups include, but are not limited to,
methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl,
n-pentyl, s-pentyl, n-hexyl, and n-septyl. Examples of
C.sub.1-C.sub.4 alkyl groups include, but are not limited to,
methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, and
t-butyl.
[0050] As used herein (unless otherwise specified), the term
"alkenyl" means a monovalent, straight or branched hydrocarbon
chain that includes one or more (e.g., 1, 2, 3, or 4) double bonds.
Examples of alkenyl groups include, but are not limited to, vinyl,
allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl,
1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl,
2-hexenyl, 3-hexenyl, 4-hexenyl, and 5-hexenyl.
[0051] As used herein (unless otherwise specified), the term
"alkoxy" means any of the above alkyl groups which is attached to
the remainder of the molecule by an oxygen atom (alkyl-O-).
Examples of alkoxy groups include, but are not limited to, methoxy
(sometimes shown as MeO--), ethoxy, isopropoxy, propoxy, and
butyloxy.
[0052] As used herein (unless otherwise specified), the term
"alkynyl" means a monovalent, straight or branched hydrocarbon
chain that includes one or more (e.g., 1, 2, 3, or 4) triple bonds
and that also may optionally include one or more (e.g. 1, 2, 3, or
4) double bonds in the chain. Examples of alkynyl groups include,
but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,
2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,
4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, and
5-hexynyl.
[0053] As used herein (unless otherwise specified), the term "aryl"
means a monovalent, monocyclic or bicyclic, 5, 6, 7, 8, 9, 10, 11,
or 12 member aromatic hydrocarbon group which, when unsubstituted.
Examples of aryl groups include, but are not limited to, phenyl,
naphthyl, tolyl, and xylyl. For an aryl that is bicyclic, one or
both rings can be substituted.
[0054] As used herein (unless otherwise specified), the term
"cycloalkyl" means a monovalent, monocyclic or bicyclic, 3, 4, 5,
6, 7, 8, 9, 10, 11, or 12 membered hydrocarbon group. The rings can
be saturated or partially unsaturated. Examples of cycloalkyl
groups include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and bicycloalkyls
(e.g., bicyclooctanes such as [2.2.2]bicyclooctane or
[3.3.0]bicyclooctane, bicyclononanes such as [4.3.0]bicyclononane,
and bicyclodecanes such as [4.4.0]bicyclodecane (decalin), or spiro
compounds). For a monocyclic cycloalkyl, the ring is not aromatic.
For a bicyclic cycloalkyl, if one ring is aromatic, then the other
is not aromatic. For a bicyclic cycloalkyl, one or both rings can
be substituted.
[0055] As used herein (unless otherwise specified), the term
"halogen" means monovalent Cl, F, Br, or I.
[0056] As used herein (unless otherwise specified), the term
"heteroaryl" means a monovalent, monocyclic or bicyclic, 5, 6, 7,
8, 9, 10, 11, or 12 membered, hydrocarbon group, where 1, 2, 3, 4,
5, or 6 carbon atoms are replaced by a hetero atom independently
selected from nitrogen, oxygen, or sulfur atom, and the monocyclic
or bicyclic ring system is aromatic. Examples of heteroaryl groups
include, but are not limited to, thienyl (or thiophenyl), furyl,
indolyl, pyrrolyl, pyridinyl, pyrazinyl, oxazolyl, thiaxolyl,
quinolinyl, pyrimidinyl, imidazolyl, triazolyl, tetrazolyl,
1H-pyrazol-4-yl, 1-Me-pyrazol-4-yl, pyridin-3-yl, pyridin-4-yl,
3,5-dimethylisoxazolyl, 1H-pyrrol-3-yl, 3,5-di-Me-pyrazolyl, and
1H-pyrazol-4-yl. For a bicyclic heteroaryl, if one ring is aryl,
then the other is heteroaryl. For a bicyclic heteroaryl, one or
both rings can have one or more hetero atoms. For a bicyclic
heteroaryl, one or both rings can be substituted.
[0057] As used herein (unless otherwise specified), the term
"heterocyclyl" means a monovalent, monocyclic or bicyclic, 5, 6, 7,
8, 9, 10, 11, or 12 membered, hydrocarbon, where 1, 2, 3, 4, 5, or
6 carbon atoms are replaced by a hetero atom independently selected
from nitrogen atom, oxygen atom, or sulfur atom, and the monocyclic
or bicyclic ring system is not aromatic. Examples of heterocyclyl
groups include, but are not limited to, tetrahydropyran,
pyrolidinyl (e.g., pyrrolidin-1-yl, pyrrolidin-2-yl,
pyrrolidin-3-yl, or pyrrolidin-4-yl), piperazinyl (e.g.,
piperazin-1-yl, piperazin-2-yl, piperazin-3-yl, or piperazin-4-yl),
piperidinyl (e.g., piperadin-1-yl, piperadin-2-yl, piperadin-3-yl,
or piperadin-4-yl), and morpholinyl (e.g., morpholin-1-yl,
morpholin-2-yl, morpholin-3-yl, or morpholin-4-yl,). For a bicyclic
heterocyclyl, if one ring is aromatic (e.g., monocyclic aryl or
heteroaryl), then the other ring is not aromatic. For a bicyclic
heterocyclyl, one or both rings can have one or more hetero atoms.
For a bicyclic heterocyclyl, one or both rings can be
substituted.
[0058] As used herein (unless otherwise specified), the term
"hetero atom" means an atom selected from nitrogen atom, oxygen
atom, or sulfur atom.
[0059] As used herein (unless otherwise specified), the terms
"hydroxy" or "hydroxyl" means a monovalent --OH group.
[0060] As used herein (unless otherwise specified), the term
"substituted" (e.g., as in substituted alkyl) means that one or
more hydrogen atoms of a chemical group (with one or more hydrogen
atoms) can be replaced by one or more non-hydrogen substituents
selected from the specified options. The replacement can occur at
one or more positions. The term "optionally substituted" means that
one or more hydrogen atoms of a chemical group (with one or more
hydrogen atoms) can be, but is not required to be substituted.
[0061] Some compounds of the invention can have one or more chiral
centers and can exist in and be isolated in optically active and
racemic forms, for any of the one or more chiral centers. Some
compounds can exhibit polymorphism. The compounds of the present
invention (e.g., Formula I) encompass any optically active,
racemate, stereoisomer form, polymorphism, or mixtures thereof. If
a chiral center does not provide an indication of its configuration
(i.e., R or S) in a chemical structure, it should be considered to
represent R, S or a racemate.
Synergy of IRAK Inhibitors with Apoptosis Inhibitors
[0062] As described herein, IRAK inhibitors have been demonstrated
to have synergistic effects when administered in combination with
an apoptosis modulator/inhibitor, such as a BCL2 inhibitor. As
demonstrated in FIGS. 11-16, an exemplary apoptosis/BCL2 inhibitor
has been shown to synergize with an exemplary IRAK inhibitor in
multiple AML cell lines. Venetoclax was used as a representative
apoptosis/BCL2 inhibitor. These figures demonstrate the synergistic
effect of administering venetoclax in combination with compound
I-24 (NCGC ID: NCGC00371481, listed in Table 1 below; referred to
interchangeably herein as "I-24", "NCGC00371481", "I-24/1481", or
"1481") in multiple AML cell lines, namely MLL-AF9-FLT3-ITD, THP-1,
Kasumi-1, and TF-1.
[0063] Surprisingly, this has been shown to be a synergistic,
rather than additive effect. This is evidenced by the Bliss scores
(FIG. 12), and the nature of the relationship of the dose ratio
data (FIGS. 13-15). FIG. 13 shows that when an inactive
concentration of I-24/1481 is combined with venetoclax, the potency
of venetoclax is increased by an unexpectedly high .about.50-fold.
According to particular aspects of the invention, this combination
allows for increased efficacy of venetoclax at lower doses, to
provide for avoiding some of the toxicity observed in the clinic.
Further, the synergy is not dependent on the presence of the FLT3
receptor, as shown in FIGS. 14A-14C and 15A-15C.
[0064] In FIGS. 14A-14C, the THP-1 cells are not FLT3 positive. The
cell line does, however, express high amounts of TLR.sub.2, a
receptor that signals through IRAK, and FIGS. 14A-14C demonstrate
that there is still a synergistic interaction with compound
I-24/1481 and venetoclax. According to particular aspects, the
degree of interaction is dependent on the dose ratio combination
that is used, with higher concentrations of 1-24/1481 providing
larger shifts in the venetoclax IC50. When an
[0065] IC50 concentration of 1-24/1481 is combined with venetoclax,
the IC50 of venetoclax is shifted by 15,000 times. This unexpected
and dramatic shift in the venetoclax IC50 is substantially more
than an additive response, and demonstrates the unexpected
synergistic interaction of the two drugs in a cell line that does
not express FLT3.
[0066] In FIGS. 15A-15C, the Kasumi-1 cell line is an AML cell line
that does not express FLT3. It does, however, express high amounts
of TLR.sub.2, a receptor that signals through IRAK. Venetoclax
inhibits cell viability in these cells, and when combined with a
minimally active concentration of I-24/1481 the IC50 is shifted
.about.15-fold, with complete inhibition of the viability response
observed. The lower heat map shows that 55% inhibition is given at
three different dose ratios of 25 nM 1481 : 100 nM Venetoclax
[DR=25/143: 100/3605=0.175: 0.0277 or 6:1]; 6 nM 1481 : 400 nM
Venetoclax [DR=6/143:400/3605=0.042: 0.11 or 1:2.6], and 6 nM 1481
: 1600 nM Venetoclax 6/143: 1600/3605=0.042: 0.444 or 1:10.6], with
the dose ratio defined as the observed concentration of drug
relative to the concentration of drug giving the IC50 response when
administered alone. Since the same drug effect (55% response) is
not obtained at the same dose ratios, the drugs do not have a
constant potency ratio, evidenced also by the non-parallel nature
of the two dose-response curves for Venetoclax and I-24/1481. Since
the Venetoclax curve shifts more to the left than would be
predicted from additive response combinations, this is a
synergistic response.
[0067] Accordingly, the present invention encompasses methods for
treating a disease or disorder which is responsive to inhibition of
IRAK, comprising administration of a composition comprising an IRAK
inhibiting compound.
[0068] In some embodiments, the method can further involve
administration of an apoptotic modulator. The apoptotic modulator
may comprise a BTK and/or a BCL2 inhibitor. BTK and BCL2 inhibitors
may be, for example, those known in the art. In some embodiments,
the method may comprise the step of administering to the individual
an apoptotic modulator. In some embodiments, the apoptotic
modulator may comprise a BCL2 inhibitor selected from ABT-263
(Navitoclax), ABT-737, ABT-199 (venetoclax), GDC-0199, GX15-070
(Obatoclax) (all available from Abbott
[0069] Laboratories), HA14-1, S1, 2-methoxy antimycin A3, gossypol,
AT-101, apogossypol, WEHI-539, A-1155463, BXI-61, BXI-72, TW37,
MIM1, UMI-77, and the like, and combinations thereof. One skilled
in the art would appreciate that there are many known BCL2
inhibitors which can be used in accordance with the present
invention. In some embodiments, the BCL2 inhibitor comprises
venetoclax.
[0070] In some embodiments, the administration step comprises
administration of a composition comprising an IRAK inhibiting
compound and a BCL2 inhibitor. In some embodiments, the
administration step comprises administration of a composition
comprising an IRAK inhibiting compound in combination with a
composition comprising a BCL2 inhibitor.
[0071] In some embodiments, the IRAK inhibiting compound is
I-24/1481, or a salt, isomer, derivative or analog thereof, and the
BCL2 inhibitor is venetoclax, or a a salt, isomer, derivative or
analog thereof. In some embodiments, the IRAK inhibiting compound
is selected from I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10,
I-11, I-12, I-13, I-14, I-15, I-16, I-17, I-18, I-19, I-20, I-21,
I-22, I-23, I-24, I-25, I-26, I-27, I-28, I-29, I-30, I-31, I-32,
I-33, I-34, I-35, I-36, I-37, I-38, I-39, I-40, I-41, I-42, I-43,
I-44, I-45, I-46, I-47, I-48, I-49, I-50, I-51, I-52, I-53, I-54,
I-55, I-56, I-57, I-58, I-59, I-60, I-61, I-62, I-63, I-64, I-65,
I-66, I-67, or I-68, or a salt, isomer, derivative or analog
thereof, and the BCL2 inhibitor is venetoclax, or a a salt, isomer,
derivative or analog thereof
[0072] In some embodiments, the method can further involve
administration of an immune modulator. The immune modulator can
include, for example, Lenalidomide (Revlamid; Celgene Corporation).
In some embodiments, the method can involve administration of an
epigenetic modulator. The epigenetic modulator can include, for
example, a hypomethylating agent such as azacitidine, decitabine,
or a combination thereof.
Multi-Kinase Inhibition Demonstrated by IRAK Inhibitors
[0073] In addition to their ability to inhibit IRAK, IRAK
inhibitors have been demonstrated to have selectivity for multiple
kinases. As described herein, the kinase selectivity of compound
I-24/1481 (NCGC ID: NCGC00371481, listed in Table 1 below) was
measured in two different types of assays.
[0074] The Reaction Biology assay is described in Nature
Biotechnology, 2011, 29:1039-46 (Anastassiadis et al.),
incorporated by reference herein in its entirety. This assay uses
the isolated kinase enzyme. This assay is very useful for
determining competition of the inhibitor for ATP and/or substrates
and for measuring the kinetics of enzyme inhibition. It is also
allows for measuring the relative affinity of binding to the
isolated enzyme protein, and hence determines selectivity. This
assay uses the form of the various enzymes that are easiest to
express, which may not necessarily be the form of the enzyme that
exist in the cell. (Sometimes the carboxy terminus has been
truncated to aid in expression, or, if it is a receptor kinase, the
enzyme itself is isolated from the other parts of the receptor that
are involved in regulating kinase activity.)
[0075] The KiNativ.RTM. assay is an in situ kinase profiling assay;
details and methodology are available at www <dot> kinativ
<dot> com. This assay uses the form of the enzyme that is
found in actual cells. The assay is run either in cell lysates or
in whole cells, with the drug of interest added either to the cell
lysates (i.e. cells that have been popped open) or to the outside
of the cell in a whole cell assay prior to making the cell lysates.
The whole cell assay is optimal because the drug must pass through
the cell membrane to get to the cytoplasm where the kinase resides
if the kinase is a cytosolic kinase, as is the case for IRAK, or
the location of the kinase domain for the FLT3 receptor kinase
domain. In the case of FIGS. 18A-18C, the KiNativ assay was run on
cell lysates from the MV4:11 cells. The MV4:11 cells are a FLT3-ITD
positive cell line.
[0076] High potency inhibition was observed in a subset of kinases
in the KiNativ assay vs the Reaction Biology assay. The data show a
total of 12 kinases that are expressed and active in MV4:11 cells
and are being inhibited by compound I-24/1481 below 250 nM, namely
ABL, CDK7, GAK, HGK, IRAK1, IRAK4, LYN, MINK, PCTAIRE1, PCTAIRE2,
PCTAIRE3 and TNIK. FLT3 is not captured by the KiNativ technology.
The KiNativ assay is done in the actual cellular milieu, where the
kinase domains are in their more natural states and subject to more
native regulatory elements (e.g. other protein domains within the
kinase itself that might not be present in the Reaction Biology
assay, and which might otherwise obscure access to the active site
by our ligand). The KiNativ assay does not measure activity at FLT3
even though those cell lysates do have FLT3 activity; this result
is likely due to lack of a substrate for FLT3 that works on the
assay platform.
[0077] FIGS. 20 (C) and 20 (D) list 12 compounds (compound
I-24/1481 and 11 analogs) that show a range of MV4:11 cytotoxicity
from 5 nM to >10,000 nM.
Compounds and Compositions including Pharmaceutical
Compositions
[0078] Some embodiments of the invention include compounds of
Formula (I):
##STR00017##
[0079] In other embodiments, R.sup.1 can be a monovalent H, halogen
(e.g., F, Cl, Br, or I), hydroxy, C.sub.1-C.sub.7 alkyl (e.g.,
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, or C.sub.7
alkyl), C.sub.2-C.sub.7 alkenyl (e.g., C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, or C.sub.7 alkenyl), C.sub.2-C.sub.7 alkynyl
(e.g., C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, or C.sub.7
alkynyl), or C.sub.1-C.sub.6 alkoxy (C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6, or C.sub.7 alkoxy), which
C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl, C.sub.2-C.sub.7
alkynyl, or C.sub.1-C.sub.6 alkoxy can optionally be substituted
with one or more (e.g., 0, 1, 2, 3, 4, 5, or 6) of halogen (e.g.,
F, Cl, Br, or I), hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), ethynyl (--CCH),
sulfo (--SO.sub.3H), methyl, ethyl, or morpholinyl. In certain
embodiments, R.sup.1 can be a monovalent H, halogen (e.g., F, Cl,
Br, or I), hydroxy, C.sub.1-C.sub.7 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, C.sub.6, or C.sub.7 alkyl), or
C.sub.1-C.sub.6 alkoxy (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, or C.sub.7 alkoxy), which C.sub.1-C.sub.7 alkyl
or C.sub.1-C.sub.6 alkoxy can optionally be substituted with one or
more (e.g., 0, 1, 2, 3, 4, 5, or 6) of halogen (e.g., F, Cl, Br, or
I), hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H), nitro
(--NO.sub.2), cyano (--CN), ethynyl (--CCH), sulfo (--SO.sub.3H),
methyl, ethyl, or morpholinyl. In some embodiments, R.sup.1 is Cl,
methyl, 2-(morpholinyl)ethoxy, or --OCH.sub.3. In other
embodiments, R.sup.1 is not H.
[0080] In some embodiments, R.sup.2 can be monovalent H, halogen
(e.g., F, Cl, Br, or I), hydroxy, --CN, methanoyl (--COH), carboxy
(--CO.sub.2H), C.sub.1-C.sub.7 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, C.sub.6, or C.sub.7 alkyl),
C.sub.2-C.sub.7 alkenyl (e.g., C.sub.2, C.sub.3, C.sub.4, C.sub.5,
C.sub.6, or C.sub.7 alkenyl), C.sub.2-C.sub.7 alkynyl (e.g.,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, or C.sub.7 alkynyl),
C.sub.1-C.sub.6 alkoxy (e.g., C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6 alkoxy), cycloalkyl, heterocyclyl, aryl, or
heteroaryl, which methanoyl (--COH), carboxy (--CO.sub.2H),
C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.7 alkenyl, C.sub.2-C.sub.7
alkynyl, C.sub.2-C.sub.6 alkoxy, cycloalkyl, heterocyclyl, aryl, or
heteroaryl can optionally be substituted with one or more (e.g., 0,
1, 2, 3, 4, 5, or 6) of halogen (e.g., F, Cl, Br, or I), hydroxy,
methanoyl (--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2),
--NH.sub.2, --N(CH.sub.3).sub.2, cyano (--CN), ethynyl (--CCH),
propynyl, sulfo (--SO.sub.3H), heterocyclyl, aryl, heteroaryl,
pyrrolyl, piperidyl, piperazinyl, morpholinyl, --CO-morpholin-4-yl,
--CONH.sub.2, --CON(CH.sub.3).sub.2, C.sub.1-C.sub.3 alkyl,
C.sub.1-C.sub.3 perfluoronated alkyl, or C.sub.1-C.sub.3 alkoxy. In
other embodiments, R.sup.2 can be monovalent H, halogen (e.g., F,
Cl, Br, or I), hydroxy, --CN, methanoyl (--COH), carboxy
(--CO.sub.2H), C.sub.1-C.sub.7 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, C.sub.6, or C.sub.7 alkyl),
C.sub.1-C.sub.6 alkoxy (e.g., C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6 alkoxy), cycloalkyl, heterocyclyl, aryl, or
heteroaryl, which methanoyl (--COH), carboxy (--CO.sub.2H),
C.sub.1-C.sub.7 alkyl, C.sub.2-C.sub.6 alkoxy, cycloalkyl,
heterocyclyl, aryl, or heteroaryl can optionally be substituted
with one or more (e.g., 0, 1, 2, 3, 4, 5, or 6) of halogen (e.g.,
F, Cl, Br, or I), hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), --NH.sub.2, --N(CH.sub.3).sub.2,
cyano (--CN), ethynyl (--CCH), propynyl, sulfo (--SO.sub.3H),
heterocyclyl, aryl, heteroaryl, pyrrolyl, piperidyl, piperazinyl,
morpholinyl, --CO-morpholin-4-yl, --CONH.sub.2,
--CON(CH.sub.3).sub.2, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
perfluoronated alkyl, or C.sub.1-C.sub.3 alkoxy. In some
embodiments, R.sup.2 can be --CO-morpholin-4-yl,
--CON(CH.sub.3).sub.2, Cl, methyl, --CN, ethynyl, --CONH.sub.2,
--CON(CH.sub.3).sub.2, 2-(morpholinyl)ethoxy, ethoxy, methoxy,
1H-pyrazol-4-yl, 1-methyl-pyrazol-4-yl,
1-(morpholin-4-yl)-pyrazol-4-yl, pyridin-3-yl,
2-methoxy-pyridin-5-yl, pyridin-4-yl, 3,5-dimethylisoxazol-4-yl,
1H-pyrrol-3-yl, 3,5-(di-methyl)-pyrazolyl, pyrazol-3-yl,
5-tetrazolyl, 1H-pyrazol-4-yl, 4-ethyl-piperazin-1-yl,
perfluorinated methyl, or perfluorinated ethyl. In some
embodiments, R.sup.2 can be --CO-morpholin-4-yl,
--CON(CH.sub.3).sub.2, Cl, methyl, --CN, ethynyl,
2-(morpholinyl)ethoxy, ethoxy, or methoxy. In certain embodiments,
R.sup.2 can be 1H-pyrazol-4-yl, 1-methyl-pyrazol-4-yl,
1-(morpholin-4-yl)-pyrazol-4-yl, pyridin-3-yl,
2-methoxy-pyridin-5-yl, pyridin-4-yl, 3,5-dimethylisoxazol-4-yl,
1H-pyrrol-3-yl, 3,5-(di-methyl)-pyrazolyl, pyrazol-3-yl,
5-tetrazolyl, 1H-pyrazol-4-yl, or 4-ethyl-piperazin-1-yl. In some
embodiments, R.sup.2 can be perfluorinated methyl or perfluorinated
ethyl. In other embodiments, R.sup.2 is not H.
[0081] In some embodiments, R.sup.2 can be isochromanyl (e.g.,
3-isochromanyl), chromanyl (e.g., 7-chromanyl), pyrrolidinyl (e.g.,
2- pyrrolidinyl), pyrrolinyl (e.g., 2-pyrroline-3-yl),
imidazolidinyl (e.g., 2-imidazolidinyl), imidazolinyl (e.g.,
2-imidazolin-4-yl), pyrazolidinyl (e.g., 2-pyrazolidinyl),
pyrazolinyl (e.g., 3-pyrazoline-2-yl), piperidyl (e.g., 2-
piperidyl), piperazinyl (e.g., 1-piperazinyl), indolinyl (e.g.,
1-indolinyl), isoindolinyl (e.g., 1-isoindolinyl), quinuclidinyl
(e.g., 2-quinuclidinyl), or morpholinyl (e.g., 3- morpholinyl),
where each can be optionally substituted as described for R.sup.2
(e.g., optionally substituted with one or more (e.g., 0, 1, 2, 3,
4, 5, or 6) of halogen (e.g., F, Cl, Br, or I), hydroxy, methanoyl
(--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2), --NH.sub.2,
--N(CH.sub.3).sub.2, cyano (--CN), ethynyl (--CCH), propynyl, sulfo
(--SO.sub.3H), heterocyclyl, aryl, heteroaryl, pyrrolyl, piperidyl,
piperazinyl, morpholinyl, --CO-morpholin-4-yl, --CONH.sub.2,
--CON(CH.sub.3).sub.2, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
perfluoronated alkyl, or C.sub.1-C.sub.3 alkoxy). In other
embodiments, R.sup.2 can be thienyl (e.g., 2-thienyl), thianthrenyl
(e.g., 2-thianthrenyl), furyl (e.g., 3- furyl), pyranyl (e.g.,
2H-pyran-3-yl), isobenzofuranyl (e.g., 1-isobenzofuranyl),
chromenyl (e.g., 2H-cromen-3-yl), xanthenyl (e.g., 2-xanthenyl),
phenoxathiinyl (e.g., 2-phenoxathiinyl), 2H-pyrrolyl (e.g.,
2H-pyrrol-3-yl), pyrrolyl (e.g., 3-pyrrolyl), imidazolyl (e.g.,
2-imidazolyl), pyrazolyl (e.g., 1-pyrazolyl), isothiazolyl (e.g.,
3-isothiazolyl), isoxazolyl (e.g., 3-isoxazolyl), pyridyl (e.g.,
3-pyridyl), pyrazinyl, pyrimidinyl (e.g., 2-pyrimidinyl),
pyridazinyl (e.g., 3-pyridazinyl), indolizinyl (e.g.,
2-indolizinyl), isoindolyl (e.g., 2-isoindolyl), 3H-indolyl (e.g.,
3H-indol-2-yl), indolyl (e.g., 1-indolyl), indazolyl (e.g.,
1H-indazol-3-yl), purinyl (e.g., 8-purinyl), 4H-quinolizinyl (e.g.,
4H-quinolizin-2-yl), isoquinolyl (e.g., 3-isoquinolyl), quinolyl
(e.g., 2-quinolyl), phthalazinyl (e.g., 1-phthalazinyl),
naphthyridinyl (e.g., 1,8-naphthyridin-2-yl), quinoxalinyl (e.g.,
2-quinoxalinyl), quinazolinyl (2-quinazolinyl), cinnolinyl (e.g.,
3-cinnolinyl), pteridinyl (e.g., 2-pteridinyl), 4aH-carbazolyl
(e.g., 4aH-Carbazol-2-yl), carbazolyl (e.g., 2-carbazolyl),
carbolinyl (e.g., carbolin-3-yl), phenanthridinyl (e.g.,
3-phenanthridinyl), acridinyl (2-acridinyl), perimidinyl (e.g.,
2-perimidinyl), phenanthrolinyl (e.g., 1,7-phenanthrolin-3-yl),
phenazinyl (1-phenazinyl), phenarsazinyl (e.g., 2-phenarsazinyl),
phenothiazinyl (2-phenothiazinyl), furazanyl (e.g., 3-furazanyl),
or phenoxazinyl (e.g., 2-phenoxazinyl), where each can be
optionally substituted as described for R.sup.2 (e.g., optionally
substituted with one or more (e.g., 0, 1, 2, 3, 4, 5, or 6) of
halogen (e.g., F, Cl, Br, or I), hydroxy, methanoyl (--COH),
carboxy (--CO.sub.2H), nitro (--NO.sub.2), --NH.sub.2,
--N(CH.sub.3).sub.2, cyano (--CN), ethynyl (--CCH), propynyl, sulfo
(--SO.sub.3H), heterocyclyl, aryl, heteroaryl, pyrrolyl, piperidyl,
piperazinyl, morpholinyl, --CO-morpholin-4-yl, --CONH.sub.2,
--CON(CH.sub.3).sub.2, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
perfluoronated alkyl, or C.sub.1-C.sub.3 alkoxy).
[0082] In some embodiments, R.sup.3 can be monovalent H, halogen
(e.g., F, Cl, Br, or I), hydroxy, C.sub.1-C.sub.3 alkyl (e.g.,
C.sub.1, C.sub.2, or C.sub.3 alkyl), C.sub.2-C.sub.3 alkenyl (e.g.,
C.sub.2 or C.sub.3 alkenyl), C.sub.2-C.sub.3 alkynyl (e.g., C.sub.2
or C.sub.3 alkynyl), or C.sub.1-C.sub.2 alkoxy (e.g., C.sub.1 or
C.sub.2 alkoxy), which C.sub.1-C.sub.3 alkyl, C.sub.2-C.sub.3
alkenyl, C.sub.2-C.sub.3 alkynyl, or C.sub.1-C.sub.2 alkoxy can
optionally be substituted with one or more (e.g., 0, 1, 2, 3, 4, 5,
or 6) of halogen (e.g., F, Cl, Br, or I), hydroxy, methanoyl
(--COH), carboxy (--CO.sub.2H), cyano (--CN), ethynyl (--CCH),
sulfo (--SO.sub.3H), methyl, or ethyl. In other embodiments,
R.sup.3 can be monovalent H, halogen (e.g., F, Cl, Br, or I),
hydroxy, C.sub.1-C.sub.3 alkyl (e.g., C.sub.1, C.sub.2, or C.sub.3
alkyl), or C.sub.1-C.sub.2 alkoxy (e.g., C.sub.1 or C.sub.2
alkoxy), which C.sub.1-C.sub.3 alkyl or C.sub.1-C.sub.2 alkoxy can
optionally be substituted with one or more (e.g., 0, 1, 2, 3, 4, 5,
or 6) of halogen (e.g., F, Cl, Br, or I), hydroxy, methanoyl
(--COH), carboxy (--CO.sub.2H), cyano (--CN), ethynyl (--CCH),
sulfo (--SO.sub.3H), methyl, or ethyl. In some embodiments, R.sup.3
can be perfluorinated methyl or perfluorinated ethyl. In some
embodiments, R.sup.3 can be H, methoxy, which methoxy is optional
substituted with one, two, or three halogen (e.g., Cl, F, Br, or
I). In some embodiments, R.sup.3 can be H or methoxy.
[0083] In some embodiments, R.sup.4 can be monovalent H, halogen
(e.g., F, Cl, Br, or I), hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), sulfo
(--SO.sub.3H), C.sub.1-C.sub.4 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, or C.sub.4 alkyl), C.sub.2-C.sub.4 alkenyl (e.g., C.sub.2,
C.sub.3, or C.sub.4 alkenyl), C.sub.2-C.sub.4 alkynyl (e.g.,
C.sub.2, C.sub.3, or C.sub.4 alkynyl), or C.sub.1-C.sub.3 alkoxy
(e.g., C.sub.1, C.sub.2, or C.sub.3 alkoxy), which C.sub.1-C.sub.4
alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4 alkynyl, or
C.sub.1-C.sub.3 alkoxy can optionally be substituted with one or
more (e.g., 1, 2, 3, 4, 5, or 6) of halogen (e.g., F, Cl, Br, or
I), hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H), nitro
(--NO.sub.2), cyano (--CN), ethynyl (--CCH), sulfo (--SO.sub.3H),
methyl, or ethyl. In other embodiments, R.sup.4 can be monovalent
H, halogen (e.g., F, Cl, Br, or I), hydroxy, methanoyl (--COH),
carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), sulfo
(--SO.sub.3H), C.sub.1-C.sub.4 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, or C.sub.4 alkyl), or C.sub.1-C.sub.3 alkoxy (e.g.,
C.sub.1, C.sub.2, or C.sub.3 alkoxy), which C.sub.1-C.sub.4 alkyl
or C.sub.1-C.sub.3 alkoxy can optionally be substituted with one or
more (e.g., 1, 2, 3, 4, 5, or 6) of halogen (e.g., F, Cl, Br, or
I), hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H), nitro
(--NO.sub.2), cyano (--CN), ethynyl (--CCH), sulfo (--SO.sub.3H),
methyl, or ethyl. In some embodiments, R.sup.4 can be F, Cl, Br,
methyl, perfluorinated methyl, or methoxy.
[0084] In some embodiments, R.sup.5 can be monovalent H, halogen
(e.g., F, Cl, Br, or I), hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), sulfo
(--SO.sub.3H), C.sub.1-C.sub.4 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, or C.sub.4 alkyl), C.sub.2-C.sub.4 alkenyl (e.g., C.sub.2,
C.sub.3, or C.sub.4 alkenyl), C.sub.2-C.sub.4 alkynyl (e.g.,
C.sub.2, C.sub.3, or C.sub.4 alkynyl), or C.sub.1-C.sub.3 alkoxy
(e.g., C.sub.1, C.sub.2, or C.sub.3 alkoxy), which C.sub.1-C.sub.4
alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4 alkynyl, or
C.sub.1-C.sub.3 alkoxy can optionally be substituted with one or
more (e.g., 1, 2, 3, 4, 5, or 6) of halogen (e.g., F, Cl, Br, or
I), hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H), nitro
(--NO.sub.2), cyano (--CN), ethynyl (--CCH), sulfo (--SO.sub.3H),
methyl, or ethyl. In other embodiments, R.sup.5 can be monovalent
H, halogen (e.g., F, Cl, Br, or I), hydroxy, methanoyl (--COH),
carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), sulfo
(--SO.sub.3H), C.sub.1-C.sub.4 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, or C.sub.4 alkyl), or C.sub.1-C.sub.3 alkoxy (e.g.,
C.sub.1, C.sub.2, or C.sub.3 alkoxy), which C.sub.1-C.sub.4 alkyl
or C.sub.1-C.sub.3 alkoxy can optionally be substituted with one or
more (e.g., 1, 2, 3, 4, 5, or 6) of halogen (e.g., F, Cl, Br, or
I), hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H), nitro
(--NO.sub.2), cyano (--CN), ethynyl (--CCH), sulfo (--SO.sub.3H),
methyl, or ethyl. In some embodiments, R.sup.5 can be F, Cl, Br,
methyl, ethyl, or methoxy. In some embodiments, R.sup.5 is not F,
Cl, Br, or perfluorinated methyl, or is not substituted with
halogen (e.g., F, Cl, Br, or I), hydroxy, methanoyl (--COH),
carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), ethynyl
(--CCH), or sulfo (--SO.sub.3H).
[0085] In some embodiments, R.sup.6 can be monovalent H, halogen
(e.g., F, Cl, Br, or I), hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), sulfo
(--SO.sub.3H), C.sub.1-C.sub.4 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, or C.sub.4 alkyl), C.sub.2-C.sub.4 alkenyl (e.g., C.sub.2,
C.sub.3, or C.sub.4 alkenyl), C.sub.2-C.sub.4 alkynyl (e.g.,
C.sub.2, C.sub.3, or C.sub.4 alkynyl), or C.sub.1-C.sub.3 alkoxy
(e.g., C.sub.1, C.sub.2, or C.sub.3 alkoxy), which C.sub.1-C.sub.4
alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4 alkynyl, or
C.sub.1-C.sub.3 alkoxy can optionally be substituted with one or
more (e.g., 1, 2, 3, 4, 5, or 6) of halogen (e.g., F, Cl, Br, or
I), hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H), nitro
(--NO.sub.2), cyano (--CN), ethynyl (--CCH), sulfo (--SO.sub.3H),
methyl, or ethyl. In other embodiments, R.sup.6 can be monovalent
H, halogen (e.g., F, Cl, Br, or I), hydroxy, methanoyl (--COH),
carboxy (--CO.sub.2H), nitro (--NO.sub.2), cyano (--CN), sulfo
(--SO.sub.3H), C.sub.1-C.sub.4 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, or C.sub.4 alkyl), or C.sub.1-C.sub.3 alkoxy (e.g.,
C.sub.1, C.sub.2, or C.sub.3 alkoxy), which C.sub.1-C.sub.4 alkyl
or C.sub.1-C.sub.3 alkoxy can optionally be substituted with one or
more (e.g., 1, 2, 3, 4, 5, or 6) of halogen (e.g., F, Cl, Br, or
I), hydroxy, methanoyl (--COH), carboxy (--CO.sub.2H), nitro
(--NO.sub.2), cyano (--CN), ethynyl (--CCH), sulfo (--SO.sub.3H),
methyl, or ethyl. In some embodiments, R.sup.6 can be F, Cl, Br,
methyl, perfluorinated methyl, or methoxy.
[0086] In certain embodiments, Y can be a bivalent
##STR00018##
In some embodiments, Y can be
##STR00019##
In other embodiments, Y can be
##STR00020##
In other embodiments, Y can be
##STR00021##
[0087] The wavy bond from Y to R.sup.7 (i.e., ) indicates that, in
some instances, there is a chiral center at the R.sup.7 attachment
carbon. In some embodiments, where there is a chiral center at the
R.sup.7 attachment carbon, the wavy bond can indicate an R chiral
center, an S chiral center, or a racemate (e.g., compounds I-43,
I-44, and I-54). In certain embodiments, can be , , , or .
[0088] In some embodiments, R.sup.7 can be
##STR00022##
In other embodiments R.sup.7 can be piperid-2-yl, piperid-3-yl,
piperid-4-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, or azetidyl. In
some embodiments, R.sup.8 can be H, C.sub.1-C.sub.4 alkyl (e.g.,
C.sub.1, C.sub.2, C.sub.3, or C.sub.4 alkyl), C.sub.2-C.sub.4
alkenyl (e.g., C.sub.2, C.sub.3, or C.sub.4 alkenyl),
C.sub.2-C.sub.4 alkynyl (e.g., C.sub.2, C.sub.3, or C.sub.4
alkynyl), methanoyl (--COH), ethanoyl (--COCH.sub.3), benzoyl
(--COC.sub.6H.sub.5), toluoyl, carboxy (--CO.sub.2H), nitro
(--NO.sub.2), cyano (--CN), or --COCH.sub.2CN. In some embodiments,
R.sup.8 can be H, ethanoyl (--COCH.sub.3), benzoyl
(--COC.sub.6H.sub.5), ethynyl (--CCH), or --COCH.sub.2CN.
[0089] In certain embodiments, n can be 0, 1, 2, 3, 4, or 5. In
some embodiments, n can be 1, 2, or 3. In other embodiments, m can
be 0, 1, 2, 3, 4, or 5. In some embodiments, m can be 1, 2, or 3.
In some instances, n+m can be at least 1.
[0090] In some embodiments, the compounds of Formula (I) can be
those specified in Table 1.
TABLE-US-00001 TABLE 1 Compound Number Structure NCGC ID I-1
##STR00023## None I-2 ##STR00024## NCGC00249350 I-3 ##STR00025##
None I-4 ##STR00026## None I-5 ##STR00027## None I-6 ##STR00028##
None I-7 ##STR00029## None I-8 ##STR00030## None I-9 ##STR00031##
None I-10 ##STR00032## None I-11 ##STR00033## None I-12
##STR00034## NCGC00262328 I-13 ##STR00035## None I-14 ##STR00036##
None I-15 ##STR00037## NCGC00241410 I-16 ##STR00038## NCGC00249372
I-17 ##STR00039## NCGC00249373 I-18 ##STR00040## None I-19
##STR00041## None I-20 ##STR00042## NCGC00262327 I-21 ##STR00043##
NCGC00262326 I-22 ##STR00044## NCGC00371479 I-23 ##STR00045##
NCGC00371480 I-24 ##STR00046## NCGC00371481 I-25 ##STR00047##
NCGC00371482 I-26 ##STR00048## NCGC00371483 I-27 ##STR00049##
NCGC00371484 I-28 ##STR00050## NCGC00371488 I-29 ##STR00051##
NCGC00371485 I-30 ##STR00052## NCGC00371486 I-31 ##STR00053##
NCGC00371487 I-32 ##STR00054## NCGC00371852 I-33 ##STR00055##
NCGC00371853 I-34 ##STR00056## See I-24 I-35 ##STR00057## See I-24
I-36 ##STR00058## NCGC00371850 I-37 ##STR00059## NCGC00371857 I-38
##STR00060## NCGC00371858 I-39 ##STR00061## NCGC00371859 I-40
##STR00062## NCGC00371957 I-41 ##STR00063## NCGC00371958 I-42
##STR00064## NCGC00262331 I-43 ##STR00065## NCGC00262376 I-44
##STR00066## NCGC00262377 I-45 ##STR00067## NCGC00249356 I-46
##STR00068## NCGC00249357 I-47 ##STR00069## NCGC00249846 I-48
##STR00070## NCGC00262329 I-49 ##STR00071## NCGC00262330 I-50
##STR00072## NCGC00249829 I-51 ##STR00073## NCGC00249832 I-52
##STR00074## NCGC00249354 I-53 ##STR00075## NCGC00249838 I-54
##STR00076## NCGC00249841 I-55 ##STR00077## NCGC00249842 I-56
##STR00078## NCGC00249846 I-57 ##STR00079## NCGC00249371 I-58
##STR00080## NCGC00249374 I-59 ##STR00081## NCGC00249370 I-60
##STR00082## NCGC00249366 I-61 ##STR00083## NCGC00249375 I-62
##STR00084## NCGC00249368 I-63 ##STR00085## NCGC00249362 I-64
##STR00086## NCGC00249363 I-65 ##STR00087## See I-22 I-66
##STR00088## See I-22 I-67 ##STR00089## NCGC00249349 I-68
##STR00090## NCGC00378320
[0091] In some embodiments, one or more of compounds I-1, I-2, I-3,
I-4, I-5, 1-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15,
I-16, I-17, I-18, or I-19 are excluded from the compounds of the
invention. In other embodiments, all of compounds I-1, I-2, I-3,
I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15,
I-16, I-17, I-18, and I-19 are excluded from the compounds of the
invention. In certain embodiments, compounds I-2 and 1-15 are
excluded from the compounds of the invention.
[0092] In some embodiments, the compounds of the invention include
one or more of I-2, I-15, I-20, I-22, I-24, I-26, I-27, I-42, I-53,
and I-54. In some embodiments, the compounds of the invention
include one or more of I-2, I-15, I-20, I-22, I-24, I-26, I-27,
I-42, I-43, I-44, I-53, and I-54. In some embodiments, the
compounds of the invention include one or more of I-20, I-22, I-24,
I-26, I-27, I-42, I-53, and I-54. In some embodiments, the
compounds of the invention include one or more of I-20, I-22, I-24,
I-26, I-27, I-42, I-43, I-44, I-53, and I-54. In other embodiments,
the compounds of the invention include one or more of I-1, I-2,
I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14,
I-15, I-16, I-17, I-18, I-19, I-20, I-21, I-22, I-23, I-24, I-25,
I-26, I-27, I-28, I-29, I-30, I-31, I-32, I-33, I-34, I-35, I-36,
I-37, I-38, I-39, I-40, I-41, I-42, I-43, I-44, I-45, I-46, I-47,
I-48, I-49, I-50, I-51, I-52, I-53, I-54, I-55, I-56, I-57, I-58,
I-59, I-60, I-61, I-62, I-63, I-64, I-65, I-66, I-67, and I-68. In
other embodiments, the compounds of the invention include one or
more of I-20, I-21, I-22, I-23, I-24, I-25, I-26, I-27, I-28, I-29,
I-30, I-31, I-32, I-33, I-34, I-35, I-36, I-37, I-38, I-39, I-40,
I-41, I-42, I-43, I-44, I-45, I-46, I-47, I-48, I-49, I-50, I-51,
I-52, I-53, I-54, I-55, I-56, I-57, I-58, I-59, I-60, I-61, I-62,
I-63, I-64, I-65, I-66, I-67, and I-68.
[0093] In some embodiments, if Y is --NH--; R.sup.1 is H; R.sup.3
is H; R.sup.4 is H; R.sup.5 is H; R.sup.6 is H; and R.sup.8 is H,
then [0094] (a) R.sup.2 is not H, Cl, methoxy, or CN, and [0095]
(b) R.sup.7 is not
##STR00091##
[0096] In some embodiments, if Y is --NH--; R.sup.1 is H; R.sup.3
is H; R.sup.4 is H; R.sup.5 is H; R.sup.6 is H; and R.sup.8 is H,
then [0097] (a) R.sup.2 is not H, Cl, F, Br, I, methoxy, ethoxy, or
CN, and [0098] (b) R.sup.7 is not
##STR00092##
[0099] In some embodiments, if Y is --NH--; R.sup.1 is H; R.sup.3
is H; R.sup.4 is H; R.sup.5 is H; R.sup.6 is H; and R.sup.8 is H,
then [0100] (a) R.sup.2 can be hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), C.sub.1-C.sub.7 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, C.sub.6, or C.sub.7 alkyl),
C.sub.2-C.sub.7 alkenyl (e.g., C.sub.2, C.sub.3, C.sub.4, C.sub.5,
C.sub.6, or C.sub.7 alkenyl), C.sub.2-C.sub.7 alkynyl (e.g.,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, or C.sub.7 alkynyl),
C.sub.3-C.sub.6 alkoxy (e.g., C.sub.3, C.sub.4, C.sub.5, or C.sub.6
alkoxy), cycloalkyl, heterocyclyl, aryl, or heteroaryl, which
methanoyl (--COH), carboxy (--CO.sub.2H), C.sub.1-C.sub.7 alkyl,
C.sub.2-C.sub.7 alkenyl, C.sub.2-C.sub.7 alkynyl, C.sub.3-C.sub.6
alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl can
optionally be substituted with one or more (e.g., 0, 1, 2, 3, 4, 5,
or 6) of halogen (e.g., F, Cl, Br, or I), hydroxy, methanoyl
(--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2), --NH.sub.2,
--N(CH.sub.3).sub.2, cyano (--CN), ethynyl (--CCH), propynyl, sulfo
(--SO.sub.3H), heterocyclyl, aryl, heteroaryl, pyrrolyl, piperidyl,
piperazinyl, morpholinyl, --CO-morpholin-4-yl, --CONH.sub.2,
--CON(CH.sub.3).sub.2, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
perfluoronated alkyl, or C.sub.1-C.sub.3 alkoxy, and [0101] (b)
R.sup.7 is not
##STR00093##
[0102] In some embodiments, if Y is --NH--; R.sup.1 is H; R.sup.3
is H; R.sup.4 is H; R.sup.5 is H; R.sup.6 is H; and R.sup.8 is H,
then [0103] (a) R.sup.2 can be hydroxy, methanoyl (--COH), carboxy
(--CO.sub.2H), C.sub.1-C.sub.7 alkyl (e.g., C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, C.sub.6, or C.sub.7 alkyl),
C.sub.2-C.sub.7 alkenyl (e.g., C.sub.2, C.sub.3, C.sub.4, C.sub.5,
C.sub.6, or C.sub.7 alkenyl), C.sub.2-C.sub.7 alkynyl (e.g.,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, or C.sub.7 alkynyl),
C.sub.5-C.sub.6 alkoxy (e.g., C.sub.5 or C.sub.6 alkoxy),
cycloalkyl, heterocyclyl, aryl, or heteroaryl, which methanoyl
(--COH), carboxy (--CO.sub.2H), C.sub.1-C.sub.7 alkyl,
C.sub.2-C.sub.7 alkenyl, C.sub.2-C.sub.7 alkynyl, C.sub.5-C.sub.6
alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl can
optionally be substituted with one or more (e.g., 0, 1, 2, 3, 4, 5,
or 6) of halogen (e.g., F, Cl, Br, or I), hydroxy, methanoyl
(--COH), carboxy (--CO.sub.2H), nitro (--NO.sub.2), --NH.sub.2,
--N(CH.sub.3).sub.2, cyano (--CN), ethynyl (--CCH), propynyl, sulfo
(--SO.sub.3H), heterocyclyl, aryl, heteroaryl, pyrrolyl, piperidyl,
piperazinyl, morpholinyl, --CO-morpholin-4-yl, --CONH.sub.2,
--CON(CH.sub.3).sub.2, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
perfluoronated alkyl, or C.sub.1-C.sub.3 alkoxy, and [0104] (b)
R.sup.7 is not
##STR00094##
[0105] In some embodiments, the compounds of Formula (I) can be in
the form of salts, optical and geometric isomers, and salts of
isomers. In other embodiments, the compounds can be in various
forms, such as uncharged molecules, components of molecular
complexes, or non-irritating pharmacologically acceptable salts,
including but not limited to hydrochloride, hydrobromide, sulphate,
phosphate, nitrate, borate, acetate, maleate, tartrate, and
salicylate. In some instances, for acidic compounds, salts can
include metals, amines, or organic cations (e.g. quaternary
ammonium). In yet other embodiments, simple derivatives of the
compounds (e.g., ethers, esters, or amides) which have desirable
retention and release characteristics but which are easily
hydrolyzed by body pH, enzymes, or other suitable means, can be
employed.
[0106] In some embodiments, the compounds of the invention having a
chiral center and can exist in and be isolated in optically active
and racemic forms. In other embodiments, compounds may exhibit
polymorphism. Some embodiments of the present invention encompass
any racemic, optically active, polymorphic, or stereoisomeric form,
or mixtures thereof, of a compound described herein. The
preparation of optically active forms can be accomplished by any
suitable method, including but not limited to, resolution of the
racemic form by recrystallization techniques, synthesis from
optically-active starting materials, chiral synthesis, or
chromatographic separation using a chiral stationary phase.
[0107] In some embodiments, the compounds of the invention can
inhibit the activity of one or more of FLT3 (FMS-Like Tyrosine
kinase 3), mutations of FLT3 (e.g., mutations in the juxamembranal
region of FLT3, mutations in the kinase domain of FLT3, FLT3 point
mutations, FLT3 internal tandem duplication mutations, the FLT3-ITD
mutation, the D835Y FLT3 mutation, the D835V FLT3 mutation, the
F691L FLT3 mutation, or the R834Q FLT3 mutation), IRAK4
(Interleukin-1 Receptor Associated Kinase 4), mutations of IRAK4,
IRAK1 (Interleukin-1 Receptor Associated Kinase 1), or mutations of
IRAK1. In some embodiments, the compounds of the invention can
inhibit the activity of one or both of FLT3 and mutations of FLT3
(e.g., mutations in the juxamembranal region of FLT3, mutations in
the kinase domain of FLT3, FLT3 point mutations, FLT3 internal
tandem duplication mutations, the FLT3-ITD mutation, the D835Y FLT3
mutation, the D835V FLT3 mutation, the F691L FLT3 mutation, or the
R834Q FLT3 mutation) and optionally inhibits one or more of IRAK4,
mutations of IRAK4, IRAK1, or mutations of IRAK1. In some
embodiments, the compounds of the invention can inhibit the
activity of one or both of FLT3 and mutations of FLT3 (e.g.,
mutations in the juxamembranal region of FLT3, mutations in the
kinase domain of FLT3, FLT3 point mutations, FLT3 internal tandem
duplication mutations, the FLT3-ITD mutation, the D835Y FLT3
mutation, the D835V FLT3 mutation, the F691L FLT3 mutation, or the
R834Q FLT3 mutation) and optionally inhibits one or both of IRAK4
and IRAK1.
[0108] In certain embodiments, one or more compounds of the
invention (e.g., Formula (I)) can be part of a composition and can
be in an amount (by weight of the total composition) of at least
about 0.0001%, at least about 0.001%, at least about 0.10%, at
least about 0.15%, at least about 0.20%, at least about 0.25%, at
least about 0.50%, at least about 0.75%, at least about 1%, at
least about 10%, at least about 25%, at least about 50%, at least
about 75%, at least about 90%, at least about 95%, at least about
99%, at least about 99.99%, no more than about 75%, no more than
about 90%, no more than about 95%, no more than about 99%, or no
more than about 99.99%, from about 0.0001% to about 99%, from about
0.0001% to about 50%, from about 0.01% to about 95%, from about 1%
to about 95%, from about 10% to about 90%, or from about 25% to
about 75%.
[0109] In some embodiments, one or more compounds of the invention
(e.g., Formula (I)) can be purified or isolated in an amount (by
weight of the total composition) of at least about 0.0001%, at
least about 0.001%, at least about 0.10%, at least about 0.15%, at
least about 0.20%, at least about 0.25%, at least about 0.50%, at
least about 0.75%, at least about 1%, at least about 10%, at least
about 25%, at least about 50%, at least about 75%, at least about
90%, at least about 95%, at least about 99%, at least about 99.99%,
no more than about 75%, no more than about 90%, no more than about
95%, no more than about 99%, no more than about 99.99%, from about
0.0001% to about 99%, from about 0.0001% to about 50%, from about
0.01% to about 95%, from about 1% to about 95%, from about 10% to
about 90%, or from about 25% to about 75%.
[0110] Some embodiments of the present invention include
compositions comprising one or more compounds of the invention
(e.g., Formula (I)). In certain embodiments, the composition is a
pharmaceutical composition, such as compositions that are suitable
for administration to animals (e.g., mammals, primates, monkeys,
humans, canine, feline, porcine, mice, rabbits, or rats). In some
instances, the pharmaceutical composition is non-toxic, does not
cause side effects, or both. In some embodiments, there may be
inherent side effects (e.g., it may harm the patient or may be
toxic or harmful to some degree in some patients).
[0111] "Therapeutically effective amount" means an amount effective
to achieve a desired and/or beneficial effect. An effective amount
can be administered in one or more administrations. For some
purposes of this invention, a therapeutically effective amount is
an amount appropriate to treat an indication. By treating an
indication is meant achieving any desirable effect, such as one or
more of palliate, ameliorate, stabilize, reverse, slow, or delay
disease progression, increase the quality of life, or to prolong
life. Such achievement can be measured by any suitable method, such
as measurement of tumor size or blood cell count.
[0112] In some embodiments, one or more compounds of the invention
(e.g., Formula (I)) can be part of a pharmaceutical composition and
can be in an amount of at least about 0.0001%, at least about
0.001%, at least about 0.10%, at least about 0.15%, at least about
0.20%, at least about 0.25%, at least about 0.50%, at least about
0.75%, at least about 1%, at least about 10%, at least about 25%,
at least about 50%, at least about 75%, at least about 90%, at
least about 95%, at least about 99%, at least about 99.99%, no more
than about 75%, no more than about 90%, no more than about 95%, no
more than about 99%, no more than about 99.99%, from about 0.001%
to about 99%, from about 0.001% to about 50%, from about 0.1% to
about 99%, from about 1% to about 95%, from about 10% to about 90%,
or from about 25% to about 75%. In some embodiments, the
pharmaceutical composition can be presented in a dosage form which
is suitable for the topical, subcutaneous, intrathecal,
intraperitoneal, oral, parenteral, rectal, cutaneous, nasal,
vaginal, or ocular administration route. In other embodiments, the
pharmaceutical composition can be presented in a dosage form which
is suitable for parenteral administration, a mucosal
administration, intravenous administration, subcutaneous
administration, topical administration, intradermal administration,
oral administration, sublingual administration, intranasal
administration, or intramuscular administration. The pharmaceutical
composition can be in the form of, for example, tablets, capsules,
pills, powders granulates, suspensions, emulsions, solutions, gels
(including hydrogels), pastes, ointments, creams, plasters,
drenches, delivery devices, suppositories, enemas, injectables,
implants, sprays, aerosols or other suitable forms.
[0113] In some embodiments, the pharmaceutical composition can
include one or more formulary ingredients. A "formulary ingredient"
can be any suitable ingredient (e.g., suitable for the drug(s), for
the dosage of the drug(s), for the timing of release of the
drugs(s), for the disease, for the disease state, or for the
delivery route) including, but not limited to, water (e.g., boiled
water, distilled water, filtered water, pyrogen-free water, or
water with chloroform), sugar (e.g., sucrose, glucose, mannitol,
sorbitol, xylitol, or syrups made therefrom), ethanol, glycerol,
glycols (e.g., propylene glycol), acetone, ethers, DMSO,
surfactants (e.g., anionic surfactants, cationic surfactants,
zwitterionic surfactants, or nonionic surfactants (e.g.,
polysorbates)), oils (e.g., animal oils, plant oils (e.g., coconut
oil or arachis oil), or mineral oils), oil derivatives (e.g., ethyl
oleate , glyceryl monostearate, or hydrogenated glycerides),
excipients, preservatives (e.g., cysteine, methionine, antioxidants
(e.g., vitamins (e.g., A, E, or C), selenium, retinyl palmitate,
sodium citrate, citric acid, chloroform, or parabens, (e.g., methyl
paraben or propyl paraben)), or combinations thereof.
[0114] In certain embodiments, pharmaceutical compositions can be
formulated to release the active ingredient (e.g., one or more
compounds of the invention such as Formula (I)) substantially
immediately upon the administration or any substantially
predetermined time or time after administration. Such formulations
can include, for example, controlled release formulations such as
various controlled release compositions and coatings.
[0115] Other formulations (e.g., formulations of a pharmaceutical
composition) can, in certain embodiments, include those
incorporating the drug (or control release formulation) into food,
food stuffs, feed, or drink.
[0116] Other embodiments of the invention can include methods of
administering or treating an organism, which can involve treatment
with an amount of at least one compound of the invention (e.g.,
Formula (I)) that is effective to treat the disease, condition, or
disorder that the organism has, or is suspected of having, or is
susceptible to, or to bring about a desired physiological effect.
In some embodiments, the composition or pharmaceutical composition
comprises at least one compound of the invention (e.g., Formula
(I)) which can be administered to an animal (e.g., mammals,
primates, monkeys, or humans) in an amount of about 0.005 to about
50 mg/kg body weight, about 0.01 to about 15 mg/kg body weight,
about 0.1 to about 10 mg/kg body weight, about 0.5 to about 7 mg/kg
body weight, about 0.005 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg,
about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 3 mg/kg,
about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg,
about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 10 mg/kg,
about 12 mg/kg, or about 15 mg/kg. In regard to some conditions,
the dosage can be about 0.5 mg/kg human body weight or about 6.5
mg/kg human body weight. In some instances, some animals (e.g.,
mammals, mice, rabbits, feline, porcine, or canine) can be
administered a dosage of about 0.005 to about 50 mg/kg body weight,
about 0.01 to about 15 mg/kg body weight, about 0.1 to about 10
mg/kg body weight, about 0.5 to about 7 mg/kg body weight, about
0.005 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg,
about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about
30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 80 mg/kg, about 100
mg/kg, or about 150 mg/kg. Of course, those skilled in the art will
appreciate that it is possible to employ many concentrations in the
methods of the present invention, and using, in part, the guidance
provided herein, will be able to adjust and test any number of
concentrations in order to find one that achieves the desired
result in a given circumstance. In other embodiments, the compounds
of the invention (e.g., Formula (I)) can be administered in
combination with one or more other therapeutic agents for a given
disease, condition, or disorder.
[0117] In some embodiments, the compositions can include a unit
dose of one or more compounds of the invention (e.g., Formula (I))
in combination with a pharmaceutically acceptable carrier and, in
addition, can include other medicinal agents, pharmaceutical
agents, carriers, adjuvants, diluents, and excipients. In certain
embodiments, the carrier, vehicle or excipient can facilitate
administration, delivery and/or improve preservation of the
composition. In other embodiments, the one or more carriers,
include but are not limited to, saline solutions such as normal
saline, Ringer's solution, PBS (phosphate-buffered saline), and
generally mixtures of various salts including potassium and
phosphate salts with or without sugar additives such as glucose.
Carriers can include aqueous and non-aqueous sterile injection
solutions that can contain antioxidants, buffers, bacteriostats,
bactericidal antibiotics, and solutes that render the formulation
isotonic with the bodily fluids of the intended recipient;
[0118] and aqueous and non-aqueous sterile suspensions, which can
include suspending agents and thickening agents. In other
embodiments, the one or more excipients can include, but are not
limited to water, saline, dextrose, glycerol, ethanol, or the like,
and combinations thereof. Nontoxic auxiliary substances, such as
wetting agents, buffers, or emulsifiers may also be added to the
composition. Oral formulations can include such normally employed
excipients as, for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
and magnesium carbonate.
[0119] Administration Routes and Treatments of Disease
[0120] The compounds of the invention (e.g., Formula (I)) can be
administered to animals by any number of suitable administration
routes or formulations. The compounds of the invention (e.g.,
Formula (I)) of the invention can also be used to treat animals for
a variety of diseases. Animals include but are not limited to
mammals, primates, monkeys (e.g., macaque, rhesus macaque, or pig
tail macaque), humans, canine, feline, bovine, porcine, avian
(e.g., chicken), mice, rabbits, and rats. As used herein, the term
"subject" refers to both human and animal subjects.
[0121] The route of administration of the compounds of the
invention (e.g., Formula (I)) can be of any suitable route.
Administration routes can be, but are not limited to the oral
route, the parenteral route, the cutaneous route, the nasal route,
the rectal route, the vaginal route, and the ocular route. In other
embodiments, administration routes can be parenteral
administration, a mucosal administration, intravenous
administration, subcutaneous administration, topical
administration, intradermal administration, oral administration,
sublingual administration, intranasal administration, or
intramuscular administration. The choice of administration route
can depend on the compound identity (e.g., the physical and
chemical properties of the compound) as well as the age and weight
of the animal, the particular disease (e.g., HNSCC, cancer, MDS,
and the like), and the severity of the disease (e.g., stage or
severity of HNSCC, cancer, or MDS, and the like). Of course,
combinations of administration routes can be administered, as
desired.
[0122] Some embodiments of the invention include a method for
providing a subject with a composition comprising one or more
compounds of the invention (e.g., Formula (I)) described herein
(e.g., a pharmaceutical composition) which comprises one or more
administrations of one or more such compositions; the compositions
may be the same or different if there is more than one
administration.
[0123] Diseases that can be treated in an animal (e.g., mammals,
porcine, canine, avian (e.g., chicken), bovine, feline, primates,
rodents, monkeys, rabbits, mice, rats, and humans) using a compound
of the invention (e.g., Formula (I)) include, but are not limited
to head and neck squamous cell carcinoma (HNSCC), cancers, blood
disorders (e.g., disorders of hematopoietic stem cells in the bone
marrow or disorders related to myeloid lineage), myelodysplastic
syndromes ("MDS"), myeloproliferative disease, and diseases (e.g.,
cancers) related to mutations in FLT3 (e.g., mutations in the
juxamembranal region of FLT3, mutations in the kinase domain of
FLT3, FLT3 point mutations, FLT3 internal tandem duplication
mutations, the FLT3-ITD mutation, the D835Y FLT3 mutation, the
D835V FLT3 mutation, the F691L FLT3 mutation, or the R834Q FLT3
mutation).
[0124] In certain embodiments, MDS that can be treated in an animal
(e.g., mammals, porcine, canine, avian (e.g., chicken), bovine,
feline, primates, rodents, monkeys, rabbits, mice, rats, and
humans) using a compound of the invention (e.g., Formula (I))
include but are not limited to MDS with a splicing factor mutation,
MDS with a mutation in isocitrate dehydrogenase 1, MDS with a
mutation in isocitrate dehydrogenase 2, refractory cytopenia with
unilineage dysplasia (e.g., refractory anemia, refractory
neutropenia, and refractory thrombocytopenia), refractory anemia
with ring sideroblasts, refractory cytopenia with multilineage
dysplasia (e.g., refractory cytopenia with multilineage dysplasia
and ring sideroblasts and animals with pathological changes not
restricted to red cells such as prominent white cell precursor and
platelet precursor (megakaryocyte) dysplasia), refractory anemias
with excess blasts I and II, 5q-syndrome, megakaryocyte dysplasia
with fibrosis, and refractory cytopenia of childhood. In some
embodiments, MDS that can be treated include, but are not limited
to, MDS that is inherited, MDS with an increased risk of occurrence
due to an inherited predisposition, MDS with an increased risk of
occurrence due to other blood disorders, MDS with an increased risk
of occurrence due to chemical exposure, MDS with an increased risk
of occurrence due to ionizing radiation, MDS with an increased risk
of occurrence due to cancer treatment (e.g., a combination of
radiation and the radiomimetic alkylating agents such as busulfan,
nitrosourea, or procarbazine (with a latent period of 5 to 7 years)
or DNA topoisomerase inhibitors), MDS evolving from acquired
aplastic anemia following immunosuppressive treatment and Fanconi's
anemia, MDS with an increased risk due to an mutation in splicing
factors, MDS with an increased risk due to a mutation in isocitrate
dehydrogenase 1, and MDS with an increased risk due to a mutation
in isocitrate dehydrogenase 2. Animals that can be treated include
but are not limited to mammals, rodents, primates, monkeys (e.g.,
macaque, rhesus macaque, pig tail macaque), humans, canine, feline,
porcine, avian (e.g., chicken), bovine, mice, rabbits, and rats. As
used herein, the term "subject" refers to both human and animal
subjects. In some instances, the animal is in need of the treatment
(e.g., by showing signs of disease or MDS, or by having a low blood
cell count).
[0125] In some embodiments, MDS that can be treated in an animal
(e.g., mammals, porcine, canine, avian (e.g., chicken), bovine,
feline, primates, rodents, monkeys, rabbits, mice, rats, and
humans) using a compound of the invention (e.g., Formula (I))
include, but are not limited to MDS that can be treated by
inhibiting one or more of FLT3 (e.g., using FLT3 inhibitors),
mutations of FLT3 (e.g., using inhibitors of FLT3 mutants), IRAK4
(e.g., using IRAK4 inhibitors), mutations of IRAK4 (e.g., using
inhibitors of IRAK4 mutants), IRAK1 (e.g., using IRAK 1
inhibitors), or mutations of IRAK1 (e.g., using inhibitors of IRAK1
mutant). In certain embodiments, MDS that can be treated include,
but are not limited to MDS that can be treated by inhibiting IRAK4
(or its mutations), MDS that can be treated by inhibiting and IRAK1
(or its mutations), or MDS that can be treated by inhibiting IRAK4
(or its mutations) and IRAK1 (or its mutations).
[0126] In some embodiments, cancers that can be treated in an
animal (e.g., mammals, porcine, canine, avian (e.g., chicken),
bovine, feline, primates, rodents, monkeys, rabbits, mice, rats,
and humans) using a compound of the invention (e.g., Formula (I))
include, but are not limited to cancers of the myeloid line of
blood cells, cancerous tumors (e.g., chloroma which can be found on
any tissue or organ outside the bone marrow, such but not limited
to skin, gums, lymph nodes, small intestine, mediastinum, lungs,
epidural sites, uterus, ovaries, and the orbits of the eyes),
cancers that are inherited, cancers with an increased risk of
occurrence due to an inherited predisposition (e.g., Down
syndrome), cancers with an increased risk of occurrence due to
other blood disorders, cancers with an increased risk of occurrence
due to chemical exposure (e.g., anti-cancer therapies or
occupational chemical exposure), cancers with an increased risk of
occurrence due to ionizing radiation (e.g., anti-cancer therapies),
cancers evolving from myelodysplastic syndromes, cancers evolving
from myeloproliferative disease, and cancers of the B cells.
[0127] In some embodiments, cancers that can be treated include,
but are not limited to, acute myeloid leukemia (AML), lymphoma,
leukemia, bone marrow cancer, non-Hodgkin lymphoma (e.g., diffuse
large B-cell lymphoma), Waldenstrom's macroglobulinemia,
glioblastoma multiforme, endometrial cancer, melanoma, prostate
cancer, lung cancer, breast cancer, kidney cancer, bladder cancer,
basal cell carcinoma, thyroid cancer, squamous cell carcinoma,
neuroblastoma, ovarian cancer, renal cell carcinoma, hepatocellular
carcinoma, colon cancer, pancreatic cancer, chronic lymphocytic
leukemia (CLL), acute lymphoblastic leukemia, rhabdomyosarcoma,
meningioma, gastric cancer, Glioma, oral cancer, nasopharyngeal
carcinoma, rectal cancer, stomach cancer, and uterine cancer. In
some embodiments, cancers that can be treated include, but are not
limited to, acute myeloid leukemia, lymphoma, leukemia, bone marrow
cancer, non-Hodgkin lymphoma (e.g., diffuse large B-cell lymphoma),
and Waldenstrom's macroglobulinemia. In some embodiments, cancers
that can be treated include, but are not limited to, acute myeloid
leukemia (AML), AML that is inherited, AML with an increased risk
of occurrence due to an inherited predisposition, AML with a
recurrent genetic abnormality (e.g., with inversions or
translocations, such as MLLT3/MLL which is a translocation between
chromosome 9 and 11 ("MLL")), AML with an increased risk of
occurrence due to other blood disorders, AML with an increased risk
of occurrence due to chemical exposure, AML with an increased risk
of occurrence due to ionizing radiation, AML evolving from
myelodysplastic syndromes, AML evolving from myeloproliferative
disease, AML with an increased risk due to an FLT3 mutation, AML
with an increased risk due to an FLT3 mutation in the juxamembranal
region of FLT3, AML with an increased risk due to an FLT3 mutation
of an internal tandem duplication in the juxamembranal region of
FLT3, AML with an increased risk due to an FLT3 mutation in the
kinase domain of FLT3, AML with an increased risk due to the FLT3
mutation D835Y, AML with an increased risk due to the FLT3 mutation
D835V, AML with an increased risk due to the FLT3 mutation F691L,
and AML with an increased risk due to the FLT3 mutation R834Q.
Animals that can be treated include but are not limited to mammals,
rodents, primates, monkeys (e.g., macaque, rhesus macaque, pig tail
macaque), humans, canine, feline, porcine, avian (e.g., chicken),
bovine, mice, rabbits, and rats. As used herein, the term "subject"
refers to both human and animal subjects. In some instances, the
animal is in need of the treatment (e.g., by showing signs of
disease or cancer, or by having a cancerous tumor).
[0128] In some embodiments, cancers that can be treated in an
animal (e.g., mammals, porcine, canine, avian (e.g., chicken),
bovine, feline, primates, rodents, monkeys, rabbits, mice, rats,
and humans) using a compound of the invention (e.g., Formula (I))
include, but are not limited to cancers that can be treated by
inhibiting (e.g., reducing the activity or expression of) one or
more of FLT3 (e.g., using FLT3 inhibitors), mutations of FLT3
(e.g., using inhibitors of FLT3 mutants), IRAK4 (e.g., using IRAK4
inhibitors), mutations of IRAK4 (e.g., using inhibitors of IRAK4
mutants), IRAK1 (e.g., using IRAK 1 inhibitors), or mutations of
IRAK1 (e.g., using inhibitors of IRAK1 mutants). In certain
embodiments, cancers that can be treated include, but are not
limited to cancers that can be treated by inhibiting (e.g.,
reducing the activity or expression of) FLT3 (or its mutations) and
IRAK4 (or its mutations), cancers that can be treated by inhibiting
(e.g., reducing the activity or expression of) FLT3 (or its
mutations) and IRAK1 (or its mutations), or cancers that can be
treated by inhibiting (e.g., reducing the activity or expression
of) FLT3 (or its mutations), IRAK4 (or its mutations), and IRAK1
(or its mutations).
[0129] As used herein, the term "treating" (and its variations,
such as "treatment") is to be considered in its broadest context.
In particular, the term "treating" does not necessarily imply that
an animal is treated until total recovery. Accordingly, "treating"
includes amelioration of the symptoms, relief from the symptoms or
effects associated with a condition, decrease in severity of a
condition, or preventing, preventively ameliorating symptoms, or
otherwise reducing the risk of developing a particular condition.
As used herein, reference to "treating" an animal includes but is
not limited to prophylactic treatment and therapeutic treatment.
Any of the compositions (e.g., pharmaceutical compositions)
described herein can be used to treat an animal.
[0130] As related to treating MDS (e.g., MDS with a splicing factor
mutation, MDS with a mutation in isocitrate dehydrogenase 1, or MDS
with a mutation in isocitrate dehydrogenase 2), treating can
include but is not limited to prophylactic treatment and
therapeutic treatment. As such, treatment can include, but is not
limited to: preventing MDS (e.g., MDS with a splicing factor
mutation, MDS with a mutation in isocitrate dehydrogenase 1, or MDS
with a mutation in isocitrate dehydrogenase 2); reducing the risk
of MDS (e.g., MDS with a splicing factor mutation, MDS with a
mutation in isocitrate dehydrogenase 1, or MDS with a mutation in
isocitrate dehydrogenase 2); ameliorating or relieving symptoms of
MDS (e.g., MDS with a splicing factor mutation, MDS with a mutation
in isocitrate dehydrogenase 1, or MDS with a mutation in isocitrate
dehydrogenase 2); eliciting a bodily response against MDS (e.g.,
MDS with a splicing factor mutation, MDS with a mutation in
isocitrate dehydrogenase 1, or MDS with a mutation in isocitrate
dehydrogenase 2); inhibiting the development or progression of MDS
(e.g., MDS with a splicing factor mutation, MDS with a mutation in
isocitrate dehydrogenase 1, or MDS with a mutation in isocitrate
dehydrogenase 2); inhibiting or preventing the onset of symptoms
associated with MDS (e.g., MDS with a splicing factor mutation, MDS
with a mutation in isocitrate dehydrogenase 1, or MDS with a
mutation in isocitrate dehydrogenase 2); reducing the severity of
MDS (e.g., MDS with a splicing factor mutation, MDS with a mutation
in isocitrate dehydrogenase 1, or MDS with a mutation in isocitrate
dehydrogenase 2); causing a regression of MDS (e.g., MDS with a
splicing factor mutation, MDS with a mutation in isocitrate
dehydrogenase 1, or MDS with a mutation in isocitrate dehydrogenase
2) or one or more of the symptoms associated with MDS (e.g., an
increase in blood cell count); causing remission of MDS (e.g., MDS
with a splicing factor mutation, MDS with a mutation in isocitrate
dehydrogenase 1, or MDS with a mutation in isocitrate dehydrogenase
2); causing remission of MDS (e.g., MDS with a splicing factor
mutation, MDS with a mutation in isocitrate dehydrogenase 1, or MDS
with a mutation in isocitrate dehydrogenase 2) by preventing or
minimizing FLT3 mutations (e.g., internal tandem duplication
mutations or the D835Y mutation); preventing relapse of MDS (e.g.,
MDS with a splicing factor mutation, MDS with a mutation in
isocitrate dehydrogenase 1, or MDS with a mutation in isocitrate
dehydrogenase 2); or preventing relapse of MDS (e.g., MDS with a
splicing factor mutation, MDS with a mutation in isocitrate
dehydrogenase 1, or MDS with a mutation in isocitrate dehydrogenase
2) in animals that have intrinsic or acquired resistance to other
MDS treatments. In some embodiments, treating does not include
prophylactic treatment of MDS (e.g., preventing or ameliorating
future MDS).
[0131] As related to treating cancer (e.g., acute myeloid leukemia,
lymphoma, leukemia, bone marrow cancer, non-Hodgkin lymphoma, or
Waldenstrom's macroglobulinemia), treating can include but is not
limited to prophylactic treatment and therapeutic treatment. As
such, treatment can include, but is not limited to: preventing
cancer (e.g., acute myeloid leukemia, lymphoma, leukemia, bone
marrow cancer, non-Hodgkin lymphoma, or Waldenstrom's
macroglobulinemia); reducing the risk of cancer (e.g., acute
myeloid leukemia, lymphoma, leukemia, bone marrow cancer,
non-Hodgkin lymphoma, or Waldenstrom's macroglobulinemia);
ameliorating or relieving symptoms of cancer (e.g., acute myeloid
leukemia, lymphoma, leukemia, bone marrow cancer, non-Hodgkin
lymphoma, or Waldenstrom's macroglobulinemia); eliciting a bodily
response against cancer (e.g., acute myeloid leukemia, lymphoma,
leukemia, bone marrow cancer, non-Hodgkin lymphoma, or
Waldenstrom's macroglobulinemia); inhibiting the development or
progression of cancer (e.g., acute myeloid leukemia, lymphoma,
leukemia, bone marrow cancer, non-Hodgkin lymphoma, or
Waldenstrom's macroglobulinemia); inhibiting or preventing the
onset of symptoms associated with cancer (e.g., acute myeloid
leukemia, lymphoma, leukemia, bone marrow cancer, non-Hodgkin
lymphoma, or Waldenstrom's macroglobulinemia); reducing the
severity of cancer (e.g., acute myeloid leukemia, lymphoma,
leukemia, bone marrow cancer, non-Hodgkin lymphoma, or
Waldenstrom's macroglobulinemia); causing a regression of cancer
(e.g., acute myeloid leukemia, lymphoma, leukemia, bone marrow
cancer, non-Hodgkin lymphoma, or Waldenstrom's macroglobulinemia)
or one or more of the symptoms associated with cancer (e.g., a
decrease in tumor size); causing remission of cancer (e.g., acute
myeloid leukemia, lymphoma, leukemia, bone marrow cancer,
non-Hodgkin lymphoma, or Waldenstrom's macroglobulinemia); causing
remission of cancer (e.g., acute myeloid leukemia, lymphoma,
leukemia, bone marrow cancer, non-Hodgkin lymphoma, or
Waldenstrom's macroglobulinemia) by preventing or minimizing FLT3
mutations (e.g., internal tandem duplication mutations or the D835Y
mutation); causing remission of acute myeloid leukemia by
preventing or minimizing FLT3 mutations (e.g., internal tandem
duplication mutations or the D835Y mutation); preventing relapse of
cancer (e.g., acute myeloid leukemia, lymphoma, leukemia, bone
marrow cancer, non-Hodgkin lymphoma, or Waldenstrom's
macroglobulinemia); preventing relapse of cancer (e.g., acute
myeloid leukemia, lymphoma, leukemia, bone marrow cancer,
non-Hodgkin lymphoma, or Waldenstrom's macroglobulinemia) in
animals that have intrinsic or acquired resistance to other cancer
treatments (e.g., from some FLT3 inhibitors or from MLL); or
preventing relapse of acute myeloid leukemia in animals that have
intrinsic or acquired resistance to other cancer treatments (e.g.,
from some FLT3 inhibitors or from MLL). In some embodiments,
treating does not include prophylactic treatment of cancer (e.g.,
preventing or ameliorating future cancer).
[0132] Treatment of an animal can occur using any suitable
administration method (such as those disclosed herein) and using
any suitable amount of a compound of the invention (e.g., Formula
(I)). In some embodiments, methods of treatment comprise treating
an animal for MDS (e.g., MDS with a splicing factor mutation, MDS
with a mutation in isocitrate dehydrogenase 1, or MDS with a
mutation in isocitrate dehydrogenase 2). In some embodiments,
methods of treatment comprise treating an animal for cancer (e.g.,
acute myeloid leukemia, lymphoma, leukemia, bone marrow cancer,
non-Hodgkin lymphoma, or Waldenstrom's macroglobulinemia). Other
embodiments include treatment after one or more of having a blood
disorder, having myelodysplastic syndrome, having
myeloproliferative disease, an occurrence of chemical exposure, an
exposure to ionizing radiation, or a treatment for cancer (e.g.,
with chemotherapy, ionizing radiation, or both). Some embodiments
of the invention include a method for treating a subject (e.g., an
animal such as a human or primate) with a composition comprising a
compound of the invention (e.g., Formula (I)) (e.g., a
pharmaceutical composition) which comprises one or more
administrations of one or more such compositions; the compositions
may be the same or different if there is more than one
administration.
[0133] In some embodiments, the method of treatment includes
administering an effective amount of a composition comprising a
compound of the invention (e.g., Formula (I)). As used herein, the
term "effective amount" refers to a dosage or a series of dosages
sufficient to affect treatment (e.g., to treat MDS such as but not
limited to MDS (e.g., MDS with a splicing factor mutation, MDS with
a mutation in isocitrate dehydrogenase 1, or MDS with a mutation in
isocitrate dehydrogenase 2); or to treat cancer, such as but not
limited to acute myeloid leukemia, lymphoma, leukemia, bone marrow
cancer, non-Hodgkin lymphoma, or Waldenstrom's macroglobulinemia)
in an animal. In some embodiments, an effective amount can
encompass a therapeutically effective amount, as disclosed herein.
In certain embodiments, an effective amount can vary depending on
the subject and the particular treatment being affected. The exact
amount that is required can, for example, vary from subject to
subject, depending on the age and general condition of the subject,
the particular adjuvant being used (if applicable), administration
protocol, and the like. As such, the effective amount can, for
example, vary based on the particular circumstances, and an
appropriate effective amount can be determined in a particular
case. An effective amount can, for example, include any dosage or
composition amount disclosed herein. In some embodiments, an
effective amount of at least one compound of the invention (e.g.,
Formula (I) such as but not limited to compounds I-2, I-15, I-20,
I-22, I-24, I-26, I-27, I-42, I-53, or I-54) (which can be
administered to an animal such as mammals, primates, monkeys or
humans) can be an amount of about 0.005 to about 50 mg/kg body
weight, about 0.01 to about 15 mg/kg body weight, about 0.1 to
about 10 mg/kg body weight, about 0.5 to about 7 mg/kg body weight,
about 0.005 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1
mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 3 mg/kg, about 5
mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7
mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 10 mg/kg, about 12
mg/kg, or about 15 mg/kg. In regard to some embodiments, the dosage
can be about 0.5 mg/kg human body weight or about 6.5 mg/kg human
body weight. In some instances, an effective amount of at least one
compound of the invention (e.g., Formula (I) such as but not
limited to compounds I-2, I-15, I-20, I-22, I-24, I-26, I-27, I-42,
I-53, or I-54) (which can be administered to an animal such as
mammals, rodents, mice, rabbits, feline, porcine, or canine) can be
an amount of about 0.005 to about 50 mg/kg body weight, about 0.01
to about 15 mg/kg body weight, about 0.1 to about 10 mg/kg body
weight, about 0.5 to about 7 mg/kg body weight, about 0.005 mg/kg,
about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 1 mg/kg,
about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg,
about 40 mg/kg, about 50 mg/kg, about 80 mg/kg, about 100 mg/kg, or
about 150 mg/kg. In some embodiments, an effective amount of at
least one compound of the invention (e.g., Formula (I) such as but
not limited to compounds I-2, I-15, I-20, I-22, I-24, I-26, I-27,
I-42, I-53, or 1-54) (which can be administered to an animal such
as mammals, primates, monkeys or humans) can be an amount of about
1 to about 1000 mg/kg body weight, about 5 to about 500 mg/kg body
weight, about 10 to about 200 mg/kg body weight, about 25 to about
100 mg/kg body weight, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg,
about 10 mg/kg, about 25 mg/kg, about 50 mg/kg, about 100 mg/kg,
about 150 mg/kg, about 200 mg/kg, about 300 mg/kg, about 400 mg/kg,
about 500 mg/kg, about 600 mg/kg, about 700 mg/kg, about 800 mg/kg,
about 900 mg/kg, or about 1000 mg/kg. In regard to some conditions,
the dosage can be about 20 mg/kg human body weight or about 100
mg/kg human body weight. In some instances, an effective amount of
at least one compound of the invention (e.g., Formula (I) such as
but not limited to compounds I-2, I-15, I-20, I-22, I-24, I-26,
I-27, I-42, I-53, or I-54) (which can be administered to an animal
such as mammals, rodents, mice, rabbits, feline, porcine, or
canine) can be an amount of about 1 to about 1000 mg/kg body
weight, about 5 to about 500 mg/kg body weight, about 10 to about
200 mg/kg body weight, about 25 to about 100 mg/kg body weight,
about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about
25 mg/kg, about 50 mg/kg, about 100 mg/kg, about 150 mg/kg, about
200 mg/kg, about 300 mg/kg, about 400 mg/kg, about 500 mg/kg, about
600 mg/kg, about 700 mg/kg, about 800 mg/kg, about 900 mg/kg, or
about 1000 mg/kg.
[0134] "Therapeutically effective amount" means an amount effective
to achieve a desired and/or beneficial effect (e.g., decreasing
tumor size or increasing blood cell count). A therapeutically
effective amount can be administered in one or more
administrations. For some purposes of this invention, a
therapeutically effective amount is an amount appropriate to treat
an indication (e.g., to treat cancer, AML, or MDS). By treating an
indication is meant achieving any desirable effect, such as one or
more of palliate, ameliorate, stabilize, reverse, slow, or delay
disease (e.g., cancer, AML, or MDS) progression, increase the
quality of life, or to prolong life. Such achievement can be
measured by any suitable method, such as but not limited to
measurement of tumor size or blood cell count.
[0135] In some embodiments, the treatments can also include one or
more of surgical intervention, chemotherapy, radiation therapy,
hormone therapies, immunotherapy, and adjuvant systematic
therapies. Adjuvants may include but are not limited to
chemotherapy (e.g., temozolomide), radiation therapy,
antiangiogenic therapy (e.g., bevacizumab), and hormone therapies,
such as administration of LHRH agonists; antiestrogens, such as
tamoxifen; high-dose progestogens; aromatase inhibitors; and/or
adrenalectomy. Chemotherapy can be used as a single-agent or as a
combination with known or new therapies.
[0136] In some embodiments, the administration of at least one
compound of the invention (e.g., Formula (I)) is an adjuvant cancer
therapy or part of an adjuvant cancer therapy. Adjuvant treatments
include treatments by the mechanisms disclosed herein and of
cancers as disclosed herein, including, but not limited to tumors.
Corresponding primary therapies can include, but are not limited
to, surgery, chemotherapy, or radiation therapy. In some instances,
the adjuvant treatment can be a combination of chemokine receptor
antagonists with traditional chemotoxic agents or with
immunotherapy that increases the specificity of treatment to the
cancer and potentially limits additional systemic side effects. In
still other embodiments, a compound of the invention (e.g., Formula
(I)) can be used as adjuvant with other chemotherapeutic agents.
The use of a compound of the invention (e.g., Formula (I)) may, in
some instances, reduce the duration of the dose of both drugs and
drug combinations reducing the side effects.
[0137] In some embodiments, the treatments disclosed herein can
include use of other drugs (e.g., antibiotics) or therapies for
treating disease. For example, antibiotics can be used to treat
infections and can be combined with a compound of the invention to
treat disease (e.g., infections). In other embodiments, intravenous
immunoglobulin (IVIG) therapy can be used as part of the treatment
regime (i.e., in addition to administration of the compound(s) of
the invention).
[0138] Methods for Preparing Compounds of Formula (I)
[0139] Some embodiments of the present invention include methods
for the preparation of compounds of Formula (I). In certain
embodiments, a compound of Formula (I) can be prepared comprising
the step of reacting a compound of Formula (II) with a compound of
Formula (III) to result in Formula (IV), which is later made into
Formula (I) (e.g., using one or more synthetic steps).
##STR00095##
[0140] R.sup.1 and R.sup.3 of Formula (II) are the same as that
defined in Formula (I). Formula (II) can be prepared using any
suitable method or can be purchase where available (e.g., from
Aldrich). R.sup.4, R.sup.5, and R.sup.6 of Formula (III) are the
same as that defined in Formula (I). Formula (III) can be prepared
using any suitable method or can be purchase where available (e.g.,
from Aldrich). R.sup.1, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 of
Formula (IV) are the same as that defined in Formula (I). In
certain embodiments, the reaction of Formula (II) with Formula
(III) can be performed using direct arylation through C--H bond
activation. In some embodiments, the reaction can be carried out
under an atmosphere of dry nitrogen in dried glassware. In other
embodiments, solvents used are of anhydrous quality (e.g.,
purchased from Aldrich Chemical Co.) and/or can be used as
received.
[0141] In some embodiments, Formula (II) can be reacted with
Formula (III) under the following conditions: Formula (II) and
Formula (III) are in a mixture comprising triphenylphosphine,
diacetoxypalladium, potassium carbonate, ethanol, and 1,4-dioxane,
and is heated (e.g., with a microwave) at a certain temperature
(e.g., at about 130.degree. C.) for a certain amount of time (e.g.,
about 1 hour).
[0142] In certain embodiments, a microwave vial can be equipped
with a magnetic stir bar and can be charged with Formula (II)
(e.g., about 46 mg or about 0.25 mmol), Formula (III) (e.g., about
89 mg or about 0.38 mmol), diacetoxypalladium (e.g., about 3 mg or
about 0.01 mmol), potassium carbonate (e.g., about 69 mg or about
0.50 mmol), and triphenylphosphine (e.g., about 7 mg or about 0.025
mmol). To this 1,4-dioxane (e.g., about 0.4 mL) and ethanol (e.g.,
about 0.2 mL) can be added, in some instances. The mixture can then
be subjected to heating (e.g., microwave irradiation), such as, for
example, from about 90.degree. C. to about 180.degree. C. (e.g.,
about 130.degree. C.) for from about 30 minutes to about 1.5 hours
(e.g., about 1 h). The mixture can then be diluted with, for
example, dichloromethane (DCM) (e.g., about 10 mL) and H.sub.2O
(e.g. about 10 mL). The layers can then be separated and the
aqueous layer extracted with, for example, (3.times.10 mL) DCM. The
organic extracts can be combined and can then be washed (e.g., with
brine (1.times.10 mL)), dried (e.g., over sodium sulfate), filtered
and concentrated (e.g., in vacuo). The product can then be purified
(e.g., via ISCO chromatography (0-5% methanol/DCM)).
[0143] In some embodiments, where R.sup.1 is alkoxy (e.g., methoxy)
in Formula (IV), a morpholino-alkoxy (e.g., morpholino-ethoxy) can
be substituted for the alkoxy (e.g., methoxy) as a step to
preparing Formula (I). This can occur in two steps: (a) by
converting the alkoxy to --OH and (b) by converting the --OH to the
morpholino-alkoxy. The synthesis below is exemplary for
methoxy-to-morphilinoethoxy, but can be used to convert any
alkoxy-to-morpholinoalkoxy, where that starting alkoxy can be the
same or different as the alkoxy in the morpholinoalkoxy.
##STR00096##
[0144] R.sup.3, R.sup.4, R.sup.5, and R.sup.6 of Formula (IV) are
the same as that defined in Formula (I). In some embodiments, the
reaction can be carried out under an atmosphere of dry nitrogen in
dried glassware. In other embodiments, solvents used are of
anhydrous quality (e.g., purchased from Aldrich Chemical Co.)
and/or can be used as received.
[0145] For step (a), in some embodiments, Formula (IV) (e.g., with
R.sup.1 as a C.sub.1-C.sub.6 alkoxy) can be reacted under the
following conditions: Formula (IV) (e.g., with R.sup.1 as a
C.sub.1-C.sub.6 alkoxy) is in a mixture comprising
4-methylbenzenesulfonic acid hydrate, lithium chloride, and DMF,
and can be then heated (e.g., with a microwave) at a certain
temperature (e.g., about 120.degree. C.) for a certain amount of
time (e.g., for about 2 h).
[0146] In other embodiments for step (a), a microwave vial equipped
with a stir bar can be charged with Formula (IV) (e.g., with
R.sup.1 as a C.sub.1-C.sub.6 alkoxy) (e.g., about 85 mg or about
0.25 mmol), 4-methylbenzenesulfonic acid hydrate (e.g., about 239
mg or about 1.3 mmol) and lithium chloride (e.g., about 53 mg or
about 1.3 mmol). DMF (e.g., about 1.3 mL) can then be added and the
vial can be subjected to microwave irradiation at from about
90.degree. C. to about 150.degree. C. (e.g., about 120.degree. C.)
for from about 1 h to about 3 h (e.g., about 2 h). In some
instances, the resulting product can be purified (e.g., by reverse
phase ISCO chromatography (1-100% acetonitrile/H.sub.2O)).
[0147] In some embodiments for step (b), the product of step (a)
can be reacted under the following conditions: the product of step
(a) can be in a mixture comprising di-tert-butyl azodicarboxylate,
morpholinoalkanol (e.g., 2-morpholinoethanol), THF, and
triphenylphosphine, and can be then heated (e.g., with a microwave)
or cooled at a certain temperature (e.g., from about 20.degree. C.
to about 30.degree. C.) or can be room temperature (about
25.degree. C.) for a certain amount of time (e.g., about 1.5
h).
[0148] In some embodiments for step (b), a 25 mL round bottomed
flask, equipped with a stir bar, can be charged with the product of
step (a) (e.g., about 70 mg or about 0.22 mmol), di-tert-butyl
azodicarboxylate (e.g., about 89 mg or about 0.39 mmol),
morpholinoalkanol (e.g., 2-morpholinoethanol) (e.g., about 51 mg or
about 0.39 mmol), THF (e.g., from about 2 mL to about 50 mL or from
about 10 mL to about 15 mL), and triphenylphosphine (e.g., about
102 mg or about 0.39 mmol). The reaction mixture can be stirred at
from about 20.degree. C. to about 30.degree. C. (e.g., room
temperature or about 25.degree. C.) for from about 1 h to about 2 h
(e.g., about 1.5 h). The THF can then be removed (e.g., in vacuo)
and the resulting product can then be purified (e.g., by ISCO
chromatography (1-10% methanol/DCM)) to provide Formula (IV) with
an alkoxy-morpholino (e.g., ethoxy-morpholino).
[0149] In certain embodiments, a compound of Formula (I) can be
prepared comprising the step of reacting a compound of Formula (IV)
with a compound of Formula (V) to result in Formula (VI), which is
later made into Formula (I) (e.g., using one or more synthetic
steps).
##STR00097##
[0150] R.sup.1, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 of Formula
(IV) are the same as that defined in Formula (I). Formula (IV) can
be prepared using any suitable method (e.g., see above) or can be
purchase where available. Y (e.g., Y is not O), n, and m of Formula
(V) are the same as that defined in Formula (I). Formula (V) can be
prepared using any suitable method or can be purchase where
available (e.g., from Aldrich). R.sup.1, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, Y (e.g., Y is not O), n, and m of Formula (VI) are the
same as that defined in Formula (I). In certain embodiments, the
reaction of Formula (IV) with Formula (V) can be performed via SNAr
displacement, such as, for example, by a Buchwald-Hartwig
amination. In some embodiments, the reaction can be carried out
under an atmosphere of dry nitrogen in dried glassware. In other
embodiments, solvents used are of anhydrous quality (e.g.,
purchased from Aldrich Chemical Co.) and/or can be used as
received.
[0151] In some embodiments, Formula (IV) can be reacted with
Formula (V) under the following conditions: Formula (IV) and
Formula (V) are in a mixture comprising 2-dicyclohexylphosphino-2',
4', 6'-triisopropylbiphenyl (XPhos), diacetoxypalladium, potassium
carbonate, and t-butanol, and heated (e.g., with a microwave) at a
certain temperature (e.g., at about 110.degree. C.) for a certain
amount of time (e.g., about 3 h).
[0152] In other embodiments, Formula (IV) (e.g., about 40 mg or
about 0.12 mmol) in tert-butanol (e.g., about 1 mL) in a flame
dried microwave vial equipped with a magnetic stir bar can be added
to tert-butyl 3-aminopyrrolidine-1-carboxylate (e.g., about 51 mg
or about 0.27 mmol), diacetoxypalladium (e.g., about 5 mg or about
0.02 mmol), 2-dicyclohexylphosphino-2', 4', 6'-triisopropylbiphenyl
(e.g., about 28 mg or about 0.06 mmol) and potassium carbonate
(e.g., about 57 mg or about 0.41 mmol). The mixture can then be
purged with nitrogen, sealed and subjected to microwave irradiation
at, for example, from about 80.degree. C. to about 140.degree. C.
(e.g., about 110.degree. C.) for from about 1 h to about 5 h (e.g.,
about 3 h). The mixture can then, in some instances, be diluted
with DCM (e.g., about 10 mL) and H.sub.2O (e.g., about 10 mL). The
layers can be separated and the aqueous layer can be extracted
(e.g., with (e.g., 3.times.10 mL) DCM). The organic extracts can be
combined and washed (e.g., with brine (1.times.10 mL)), dried
(e.g., over sodium sulfate), filtered and concentrated (e.g., in
vacuo). In some instances, further purification can be
accomplished, for example via ISCO chromatography (0-3%
methanol/DCM).
[0153] When Y is O, in some embodiments, a compound of Formula (I)
can be prepared comprising the step of reacting a compound of
Formula (IV) with a compound of Formula (V) to result in Formula
(VI), which is later made into Formula (I) (e.g., using one or more
synthetic steps).
##STR00098##
[0154] R.sup.1, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 of Formula
(IV) are the same as that defined in Formula (I). Formula (IV) can
be prepared using any suitable method (e.g., see above) or can be
purchase where available. For Formula (V), n and m are the same as
that defined in Formula (I); Y is O in Formula (V). Formula (V) can
be prepared using any suitable method or can be purchase where
available (e.g., from Aldrich). R.sup.1, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, n, and m of Formula (VI) are the same as that defined in
Formula (I); Y is O. In certain embodiments, the reaction can be
carried out under an atmosphere of dry nitrogen in dried glassware.
In other embodiments, solvents used are of anhydrous quality (e.g.,
purchased from Aldrich Chemical Co.) and/or can be used as
received.
[0155] In some embodiments, Formula (IV) is reacted with Formula
(V) under the following conditions: Formula (IV) and Formula (V)
are in a mixture comprising copper(I) iodide, cesium carbonate,
3,4,7,8-tetramethyl-1,10-phenanthroline, and toluene, and
sonicated, and then heated (e.g., with a microwave) at a certain
temperature (e.g., at about 120.degree. C.) for a certain amount of
time (e.g., about 3 h).
[0156] In other embodiments, an oven dried microwave vial can be
charged with Formula (IV) (e.g., about 100 mg or about 0.30 mmol),
Formula (V) (e.g., about 332 mg or about 1.77 mmol), copper(I)
iodide (e.g., about 14 mg or about 0.07 mmol), cesium carbonate
(e.g., about 722 mg or about 2.22 mmol) and
3,4,7,8-tetramethyl-1,10-phenanthroline (e.g., about 35 mg or about
0.15 mmol). Toluene (e.g., about 0.83 mL) can be added and the vial
purged with nitrogen. The vial can then be sonicated before
subjecting to heating (e.g., microwave irradiation) at from about
90.degree. C. to about 150.degree. C. (e.g., about 120.degree. C.)
for from about 1 h to about 5 h (e.g., about 3 h). The mixture can
then be diluted with DCM (e.g., about 20 mL) and H.sub.2O (e.g.,
about 20 mL). The layers can then be separated and the aqueous
layer extracted (e.g., with (3.times.20 mL) DCM). The organic
extracts can be combined and washed (e.g., with brine (1.times.20
mL)), dried (e.g., over sodium sulfate), filtered and concentrated
(e.g., in vacuo). The residue can then be purified (e.g., via ISCO
chromatography (70-100% EtOAc/hexanes)).
[0157] In certain embodiments, a compound of Formula (I) can be
prepared comprising the step of reacting a compound of Formula (VI)
with a compound of Formula (VII) to result in Formula (VIII), which
is later made into Formula (I) (e.g., using one or more synthetic
steps).
##STR00099##
[0158] R.sup.1, R.sup.3, R.sup.4, R.sup.5, R.sup.6, Y, n, and m of
Formula (VI) are the same as that defined in Formula (I). Formula
(VI) can be prepared using any suitable method (e.g., see above) or
can be purchase where available. R.sup.2 of Formula (VII) is the
same as that defined in Formula (I), and in some instances R.sup.2
can have a group protected (e.g., via Boc) during the reaction
step. Formula (VII) can be prepared using any suitable method or
can be purchase where available (e.g., from Aldrich). R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, Y, n, and m of Formula (VIII)
are the same as that defined in Formula (I). In certain
embodiments, the reaction of Formula (VI) with Formula (VII) can be
performed via a Suzuki-Miyaura coupling strategy (e.g., for
heterocyclic boronic derivatives). In some embodiments, the
reaction can be carried out under an atmosphere of dry nitrogen in
dried glassware. In other embodiments, solvents used are of
anhydrous quality (e.g., purchased from Aldrich Chemical Co.)
and/or can be used as received.
[0159] In some embodiments, Formula (VI) can be reacted with
Formula (VII) under the following conditions: Formula (VI) and
Formula (VII) (e.g., with or without a group on R.sup.2 being
protected, such as with a Boc) are in a mixture comprising
tricyclohexylphosphine (PCy.sub.3),
tris(dibenzylideneacetone)dipalladium(0) (Pd.sub.2(dba).sub.3),
potassium carbonate, water and 1,4 dioxane, and heated (e.g., with
a microwave) at a certain temperature (e.g., at about 110.degree.
C.) for a certain amount of time (e.g., about 3 h).
[0160] In other embodiments, to an oven dried microwave vial
equipped with a stir bar can be added Formula (VI) (e.g., about 150
mg or about 0.34 mmol), Formula (VII) (e.g.,
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-R.sub.2 or
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-R.sub.2 with a group
on R.sub.2 protected, such as with Boc) (e.g., about 298 mg or
about 1.01 mmol), tricyclohexylphosphine (e.g., about 131 .mu.L or
about 0.08 mmol, about 20 wt. % in toluene),
tris(dibenzylideneacetone)dipalladium(0) (e.g., about 31 mg or
about 0.034 mmol) and aqueous potassium phosphate (e.g., about 0.7
mL of about 1.3 M). 1,4-dioxane (e.g., about 2.5 mL) can then added
and the microwave vial can be purged with nitrogen and sealed. The
mixture can then be subjected to heat (e.g., microwave irradiation)
at, for example, from about 80.degree. C. to about 140.degree. C.
(e.g., about 110.degree. C.) for from about 1 h to about 5 h (e.g.,
about 3 h). The mixture can then be diluted with ethyl acetate
(EtOAc) (e.g., about 20 mL) and H.sub.2O (e.g., about 20 mL). The
layers can then be separated and the aqueous layer can be extracted
(e.g., 3.times.20 mL) with, for example, EtOAc. The organic
extracts can then be combined and washed, for example with brine
(e.g., 1.times.20 mL), dried (e.g., over sodium sulfate), filtered,
and concentrated (e.g., in vacuo). The product can then be purified
(e.g., via ISCO chromatography (e.g., 0-10% methanol/DCM)).
[0161] In some embodiments, a compound of Formula (I) can be
prepared comprising the step of reacting a compound of Formula
(VIII) to result in Formula (I).
##STR00100##
[0162] R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, Y, n,
and m of Formula (VIII) are the same as that defined in Formula
(I). In certain embodiments, the reaction of Formula (VIII) can be
performed via a deprotection strategy (e.g., for removing one or
more protecting groups, such as one or more Boc protecting groups
or one or more of a combination of different protecting groups). In
some embodiments, the reaction can be carried out under an
atmosphere of dry nitrogen in dried glassware. In other
embodiments, solvents used are of anhydrous quality (e.g.,
purchased from Aldrich Chemical Co.) and/or can be used as
received.
[0163] In some embodiments, Formula (VIII) can be reacted under the
following conditions: Formula (VIII) is in a mixture comprising
trifluoroacetic acid (TFA) and dichloromethane (DCM), and is
optionally heated (e.g., with a microwave) or cooled at a certain
temperature (e.g., from about 20.degree. C. to about 30.degree. C.)
or can be at room temperature (e.g., about 25.degree. C.). The
product can then be purified (e.g., via ISCO chromatography
(50-100% EtOAc/hexanes)).
[0164] In some embodiments, when R.sup.2 is a halogen (e.g., CO in
Formula (I), R.sup.2 can be altered to a C.sub.2-C.sub.7 alkynyl
(e.g., ethynyl or a C.sub.2-C.sub.7 alkynyl where a triple bond is
at an end-carbon position (e.g., a 1-alkynyl)). For example, an
oven dried microwave vial can be charged with Formula (I) (e.g.,
about 30 mg or about 0.068 mmol), tert-butyl(ethynyl)dimethylsilane
(e.g., about 0.03 mL or about 0.17 mmol), tri-tert-butylphosphine
(e.g., about 0.135 mL or about 0.14 mmol, about 1 M solution in
toluene), 1,8-diazabicyclo[5.4.0]undec-7-ene (e.g., about 2 .mu.L
or about 0.014 mmol), cesium carbonate (e.g., about 44 mg or about
0.14 mmol) and dichlorobis(triphenylphosphine)palladium(II) (e.g.,
about 5 mg or about 0.007 mmol). DMF (e.g., about 0.5 mL) can then
be added and the microwave vial can be purged with nitrogen and
sealed. The mixture can then be subjected to heat (e.g., microwave
irradiation) at, for example, from about 100.degree. C. to about
200.degree. C. (e.g., about 150.degree. C.) for from about 0.5 h to
about 2 h (e.g., about 1 h). The product can then be purified
(e.g., via ISCO chromatography (50-100% EtOAc/hexanes)).
[0165] In some embodiments, Formula (I) (or any other formula
recited above) can be recovered. Recovery can occur using any
suitable method including but not limited to HPLC (e.g., reverse
phase), LC, precipitation, centrifugation, column chromatography
(e.g., size exclusion chromatography or ion exchange
chromatography), use of silica gel, or combinations thereof. In
some embodiments, a method for the preparation of a compound of
Formula (I) can comprise one or more of the above-mentioned steps.
In certain embodiments, a method for preparing a compound of
Formula (I) comprises [0166] (a) reacting a compound of Formula
(II) with a compound of Formula (III) to result in a mixture
comprising a compound of Formula (IV); [0167] (b) reacting a
compound of Formula (IV) with a compound of Formula (V) to result
in a mixture comprising a compound of Formula (VI); [0168] (c)
optionally reacting a compound of Formula (VI) with a compound of
Formula (VII) to result in a mixture comprising a compound of
Formula (VIII);
[0169] (d) removing one or more protecting groups from a compound
of Formula (VI) or from a compound of Formula (VIII); and [0170]
(e) recovering Formula (I).
[0171] The presently-disclosed subject matter is further
illustrated by the following specific but non-limiting examples.
The following examples may include compilations of data that are
representative of data gathered at various times during the course
of development and experimentation related to the present
invention.
EXAMPLES
Example Set A--Synthetic Methods and Compound Characterization
[0172] Synthesis of compound C was performed using direct arylation
through C--H bond activation (Scheme 1). With compound C in hand,
the SNAr displacement was accomplished through a Buchwald-Hartwig
amination to give compound E and the installation of aryl group at
6-position of imidazo[1,2-a]pyridine ring used a Suzuki-Miyaura
coupling strategy for heterocyclic boronic derivatives to give
compound F. For compounds not encompassed by this general scheme,
preparation procedures and spectral characterizations are further
depicted below.
##STR00101##
General Methods
[0173] Unless otherwise stated, all reactions were carried out
under an atmosphere of dry nitrogen in dried glassware. Indicated
reaction temperatures refer to those of the reaction bath, while
room temperature (rt) is noted as 25.degree. C. All solvents were
of anhydrous quality purchased from Aldrich Chemical Co. and used
as received. Commercially available starting materials and reagents
were purchased from Aldrich and were used as received.
[0174] Analytical thin layer chromatography (TLC) was performed
with
[0175] Sigma Aldrich TLC plates (5.times.20 cm, 60 .ANG., 250
.mu.m). Visualization was accomplished by irradiation under a 254
nm UV lamp. Chromatography on silica gel was performed using forced
flow (liquid) of the indicated solvent system on Biotage KP-Sil
pre-packed cartridges and using the Biotage SP-1 automated
chromatography system. .sup.1HNMR spectra were recorded on a Varian
Inova 400 MHz spectrometer. Chemical shifts are reported in ppm
with the solvent resonance as the internal standard (DMSO-d.sub.6
2.50 ppm for .sup.1H). Data are reported as follows: chemical
shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet,
br=broad, m=multiplet), coupling constants, and number of protons.
Low resolution mass spectra (electrospray ionization) were acquired
on an Agilent Technologies 6130 quadrupole spectrometer coupled to
the HPLC system. High resolution mass spectral data was collected
using and Agilent 6210 time-of-flight (TOF) mass spectrometer, also
coupled to an Agilent Technologies 1200 series HPLC system. If
needed, products were purified via a Waters semi-preparative HPLC
equipped with a Phenomenex Luna.RTM. C18 reverse phase (5 micron,
30.times.75 mm) column having a flow rate of 45 mL/min. The mobile
phase was a mixture of acetonitrile and H.sub.2O each containing
0.1% trifluoroacetic acid. Samples were analyzed for purity on an
Agilent 1200 series LC/MS equipped with a Luna.RTM. C18 reverse
phase (3 micron, 3.times.75 mm) column having a flow rate of
0.8-1.0 mL/min over a 7-minute gradient and an 8.5 minute run time
(Method 1). The mobile phase was a mixture of acetonitrile (0.025%
TFA) and H.sub.2O (0.05% TFA), with temperature maintained at
50.degree. C. Purity of final compounds was determined to be
>95%, using a 3 .mu.L injection with quantitation by AUC at 220
and 254 nm (Agilent Diode Array Detector).
[0176] Method A. Used for the synthesis of many of the compounds.
This procedure is exemplified below for compound I-20.
##STR00102##
[0177] A microwave vial equipped with a magnetic stir bar was
charged with 6-chloro-7-methoxyimidazo[1,2-a]pyridine (46 mg, 0.25
mmol), 2,6-dibromopyridine (89 mg, 0.38 mmol), diacetoxypalladium
(3 mg, 0.01 mmol), potassium carbonate (69 mg, 0.50 mmol), and
triphenylphosphine (7 mg, 0.025 mmol). To this was added 0.4 mL of
1,4-dioxane and 0.2 mL of ethanol. The mixture was subjected to
microwave irradiation at 130.degree. C. for 1 h. The mixture was
diluted with dichloromethane (DCM) (10 mL) and H.sub.2O (10 mL).
The layers were separated and the aqueous layer was extracted with
(3.times.10 mL) DCM. The organic extracts were combined and washed
with brine (1.times.10 mL), dried over sodium sulfate, filtered and
concentrated in vacuo. The crude residue was then purified via ISCO
chromatography (0-5% methanol/DCM) to deliver product (85 mg, 70%)
as an off-white solid.
##STR00103##
[0178] To a suspension of
3-(6-bromopyridin-2-yl)-6-chloro-7-methoxyimidazo[1,2-a]pyridine
(40 mg, 0.12 mmol) in tert-butanol (1 mL) in a flame dried
microwave vial equipped with a magnetic stir bar was added
tert-butyl 3-aminopyrrolidine-1-carboxylate (51 mg, 0.27 mmol),
diacetoxypalladium (5 mg, 0.02 mmol),
2-dicyclohexylphosphino-2',4', 6'-triisopropylbiphenyl (28 mg, 0.06
mmol) and potassium carbonate (57 mg, 0.41 mmol). The mixture was
purged with nitrogen then sealed and subjected to microwave
irradiation at 110.degree. C. for 3 h. The mixture was diluted with
DCM (10 mL) and H.sub.2O (10 mL). The layers were separated and the
aqueous layer was extracted with (3.times.10 mL) DCM. The organic
extracts were combined and washed with brine (1.times.10 mL), dried
over sodium sulfate, filtered and concentrated in vacuo. The crude
residue was then purified via ISCO chromatography (0-3%
methanol/DCM) to deliver product (52 mg, 53%) as an off-white
solid.
##STR00104##
[0179] To an oven dried microwave vial equipped with a stir bar was
added
6-(6-chloro-7-methoxyimidazo[1,2-a]pyridin-3-yl)-N-(pyrrolidin-3-yl)pyrid-
in-2-amine (150 mg, 0.34 mmol),
tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-c-
arboxylate (298 mg, 1.01 mmol), tricyclohexylphosphine (131 .mu.L,
0.08 mmol, 20 wt. % in toluene),
tris(dibenzylideneacetone)dipalladium(0) (31 mg, 0.034 mmol) and
aqueous potassium phosphate (0.7 mL, 1.3 M). 1,4-dioxane (2.5 mL)
was then added and the microwave vial was purged with nitrogen and
sealed. The mixture was subjected to microwave irradiation at
110.degree. C. for 3 h. The mixture was diluted with ethyl acetate
(EtOAc) (20 mL) and H.sub.2O (20 mL). The layers were separated and
the aqueous layer was extracted (3.times.20 mL) EtOAc. The organic
extracts were combined and washed with brine (1.times.20 mL), dried
over sodium sulfate, filtered and concentrated in vacuo. The crude
residue was then purified via ISCO chromatography (0-10%
methanol/DCM) to deliver product (161 mg, 69%) as an off-white
solid.
##STR00105##
[0180]
N-(azetidin-3-yl)-6-(6-chloroimidazo[1,2-a]pyridin-3-yl)pyridin-2-a-
mine (I-15):
[0181] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
8.80-8.60 (br.s, 2H), 8.43 (d, J=0.8 Hz, 1H), 8.34 (dd, J=9.6, 1.2
Hz, 1H), 7.73 (d, J=8.0 Hz, 1H), 7.66 (t, J=8.0 Hz, 1H), 7.50-7.44
(m, 2H), 6.57 (d, J=8.0 Hz, 1H), 4.86-4.76 (m, 1H), 4.37-4.26 (m,
2H), 4.06-3.96 (m, 2H); LC/MS: Method 1, retention time: 1.534 min;
HRMS: m/z (M+H).sup.+=299.0938 (Calculated for
C.sub.15H.sub.14ClN.sub.5=299.0938).
##STR00106##
[0182]
N-(azetidin-3-yl)-6-(imidazo[1,2-a]pyridin-3-yl)pyridin-2-amine
(I-12):
[0183] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6).delta. 9.83
(dt, J=7.1, 1.2 Hz, 1H), 8.90-8.70 (br.s, 2H), 8.52 (s, 1H), 7.88
(dt, J=9.1, 1.2 Hz, 1H), 7.71 (ddd, J=8.9, 6.9, 1.3 Hz, 1H),
7.67-7.58 (m, 2H), 7.32 (td, J=7.0, 1.3 Hz, 1H), 7.24 (dd, J=7.5,
0.7 Hz, 1H), 6.53 (dd, J=8.3, 0.7 Hz, 1H), 4.90-4.77 (m, 1H),
4.30-4.16 (m, 2H), 3.96-3.86 (m, 2H); LC/MS: Method 1, retention
time: 1.476 min; HRMS: m/z (M+H).sup.+=265.1327 (Calculated for
C.sub.15H.sub.15N.sub.5=265.1327).
##STR00107##
[0184]
N-(azetidin-3-yl)-6-(7-chloroimidazo[1,2-a]pyridin-3-yl)pyridin-2-a-
mine (I-63):
[0185] Method A. 1H NMR (400 MHz, DMSO-d6) .delta. 9.79 (dd, J=7.6,
0.8 Hz, 1H), 9.00-8.85(br.s, 1H), 8.85-8.75 (br.s, 1H), 8.39 (s,
1H), 7.97 (dd, J=2.4, 0.8 Hz, 1H), 7.64-7.58 (m, 2H), 7.27-7.20 (m,
2H), 6.50 (dd, J=8.0, 0.8 Hz, 1H), 4.92-4.80 (m, 1H), 4.33-4.22 (m,
2H), 4.00-3.90 (m, 2H); LC/MS: Method 1, retention time: 1.303 min;
HRMS: m/z (M+H).sup.+=299.0938 (Calculated for
C.sub.15H.sub.14ClN.sub.5=299.0938).
##STR00108##
[0186]
N-(azetidin-3-yl)-6-(7-methylimidazo[1,2-a]pyridin-3-yl)pyridin-2-a-
mine (1-61):
[0187] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.81
(d, J=7.2 Hz, 1H), 9.15-9.00 (br.s, 1H), 8.95-8.80 (br.s, 1H), 8.69
(s, 1H), 7.84-7.82 (m, 1H), 7.74 (d, J=6.0 Hz, 1H), 7.67 (dd,
J=8.4, 7.6 Hz, 1H), 7.41 (dd, J=7.2, 0.4 Hz, 1H), 7.26 (dd, J=7.6,
0.4 Hz, 1H), 6.61 (dd, J=8.4, 0.4 Hz, 1H), 4.95-4.80 (m, 1H),
4.30-4.20 (m, 2H), 4.00-3.90 (m, 2H), 2.57 (s, 3H); LC/MS: Method
1, retention time: 1.634 min; HRMS: m/z (M+H).sup.+=279.1484
(Calculated for C.sub.16H.sub.17N.sub.5=279.1484).
##STR00109##
[0188]
N-(azetidin-3-yl)-6-(7-methoxyimidazo[1,2-a]pyridin-3-yl)pyridin-2--
amine (I-50):
[0189] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.77
(d, J=8.0 Hz, 1H), 9.20-9.05 (br.s, 1H), 8.95-8.70 (br.s, 1H), 8.55
(s, 1H), 7.71 (d, J=6.0 Hz, 1H), 7.65 (dd, J=8.4, 7.6 Hz, 1H), 7.35
(d, J=2.4 Hz, 1H), 7.23 (dd, J=8.0, 0.8 Hz), 7.19 (dd, J=8.0, 2.4
Hz, 1H), 6.58 (dd, J=8.0, 0.8 Hz, 1H), 4.92-4.82 (m, 1H), 4.32-4.22
(m, 2H), 4.01 (s, 3H), 3.98-3.90 (m, 2H); LC/MS: Method 1,
retention time: 1.627 min; HRMS: m/z (M+H).sup.+=295.1433
(Calculated for C.sub.16H.sub.17N.sub.5O=295.1433).
##STR00110##
[0190]
3-(6-(azetidin-3-ylamino)pyridin-2-yl)imidazo[1,2-a]pyridine-6-carb-
onitrile (I-60):
[0191] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.22
(d, J=0.8 Hz, 1H), 9.00-8.80 (br.s, 2H), 8.42 (s, 1H), 7.87 (dd,
J=9.6, 0.8 Hz, 1H), 7.66-7.60 (m, 3H), 7.27 (d, J=8.0 Hz, 1H), 6.51
(d, J=8.0 Hz, 1H), 4.82-4.70 (m, 1H), 4.37-4.26 (m, 2H), 4.06-3.90
(m, 2H); LC/MS: Method 1, retention time: 1.772 min; HRMS: m/z
(M+H).sup.+=290.1280 (Calculated for
C.sub.16H.sub.14N.sub.6=290.1280).
##STR00111##
[0192]
N-(azetidin-3-yl)-6-(6-chloro-8-methoxyimidazo[1,2-a]pyridin-3-yl)p-
yridin-2-amine (1-42):
[0193] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.45
(s, 1H), 8.90-8.75 (br.s, 2H), 8.36 (s, 1H), 7.65-7.55 (m, 2H),
7.25 (d, J=8.0 Hz, 1H), 6.84 (s, 1H), 6.52 (d, J=8.0 Hz, 1H),
4.83-4.70 (m, 1H), 4.37-4.25 (m, 2H), 4.02 (s, 3H), 4.01-3.94 (m,
2H); LC/MS: Method 1, retention time: 1.058 min; HRMS: m/z
(M+H).sup.+=330.1105 (Calculated for
C.sub.16H.sub.17ClN.sub.5O=330.1122).
##STR00112##
[0194]
N-(azetidin-3-yl)-6-(6-chloroimidazo[1,2-a]pyridin-3-yl)-4-methoxyp-
yridin-2-amine (1-67):
[0195] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.96
(dd, J=2.4, 0.8 Hz, 1H), 9.00-8.80 (br.s, 2H), 8.48 (s, 1H), 7.83
(dd, J=9.6, 0.8 Hz, 1H), 7.58 (dd, J=9.6, 2.4 Hz, 1H), 7.55 (d,
J=4.4 Hz, 1H), 6.95 (d, J=2.4 Hz, 1H), 6.02 (d, J=2.4 Hz, 1H),
4.80-4.70 (m, 1H), 4.36-4.26 (m, 2H), 4.03-3.92 (m, 2H), 3.84 (s,
3H); LC/MS: Method 1, retention time: 1.821 min; HRMS: m/z
(M+H).sup.+=329.1043 (Calculated for
C.sub.16H.sub.16ClN.sub.50O=329.1043).
##STR00113##
[0196]
N-(azetidin-3-yl)-6-(6-chloroimidazo[1,2-a]pyridin-3-yl)-4-methylpy-
ridin-2-amine (I-55):
[0197] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.92
(d, J=1.0 Hz, 1H), 8.90-8.70 (br.s, 2H), 8.35 (s, 1H), 7.78 (dd,
J=9.6, 0.8 Hz, 1H), 7.50 (dd, J=9.6, 2.0 Hz, 1H), 7.44 (dd, J=9.6,
2.0 Hz, 1H), 7.15 (t, J=1.0 Hz, 1H), 6.32 (t, J=1.0 Hz, 1H),
4.80-4.70 (m, 1H), 4.37-4.25 (m, 2H), 4.05-3.92 (m, 2H), 2.27 (s,
3H); LC/MS: Method 1, retention time: 1.816 min; HRMS: m/z
(M+H).sup.+=313.1094 (Calculated for
C.sub.16H.sub.16ClN.sub.5=313.1094).
##STR00114##
[0198]
N-(azetidin-3-yl)-6-(6-chloroimidazo[1,2-a]pyridin-3-yl)-3,5-difluo-
ropyridin-2-amine (I-56):
[0199] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.58
(dd, J=2.0, 0.8 Hz, 1H), 9.00-8.80 (br.s, 2H), 8.16 (d, J=3.6 Hz,
1H), 7.99 (t, J.sub.HF=10.0 Hz, 1H), 7.83 (dd, J=9.6, 0.8 Hz, 1H),
7.71 (d, J=5.6 Hz, 1H), 7.56 (dd, J=9.6, 2.0 Hz, 1H), 4.90-4.82 (m,
1H), 4.34-4.24 (m, 2H), 4.18-4.08 (m, 2H); LC/MS: Method 1,
retention time: 1.739 min; HRMS: m/z (M+H).sup.+=335.0749
(Calculated for C.sub.15H.sub.12ClF.sub.2N.sub.5=335.0749).
##STR00115##
[0200]
6-(6-chloroimidazo[1,2-a]pyridin-3-yl)-N-(pyrrolidin-3-yl)pyridin-2-
-amine (1-16):
[0201] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.04
(s, 1H), 9.00-8.80 (br.s, 2H), 8.38 (s, 1H), 7.77 (dd, J=9.6, 0.8
Hz, 1H), 7.56 (dd, J=8.3, 7.5 Hz, 1H), 7.48 (dd, J=9.6, 2.1 Hz,
1H), 7.22 (dd, J=7.5, 0.7 Hz, 1H), 7.11 (d, J=5.1 Hz, 1H), 6.47
(dd, J=8.3, 0.7 Hz, 1H), 4.55-4.45 (m, 1H), 3.50-3.40 (m, 1H),
3.40-3.25 (m, 3H), 2.38-2.24 (m, 1H), 2.09-1.97 (m, 1H); LC/MS:
Method 1, retention time: 1.750 min; HRMS: m/z (M+H).sup.+=313.1094
(Calculated for C.sub.16H.sub.16ClN.sub.5=313.1094).
##STR00116##
[0202]
6-(6-chloroimidazo[1,2-a]pyridin-3-yl)-N-(piperidin-3-yl)pyridin-2--
amine (I-17)
[0203] Method A. LC/MS: Method 1, retention time: 1.793 min.
##STR00117##
[0204]
6-(6-chloroimidazo[1,2-a]pyridin-3-yl)-N-(piperidin-4-yl)pyridin-2--
amine (I-2):
[0205] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.15
(dd, J=2.0, 0.8 Hz, 1H), 8.70-8.60 (br.s, 1H), 8.60-8.55 (br.s,
1H), 8.54 (s, 1H), 7.89 (dd, J=9.6, 0.8 Hz, 1H), 7.67 (dd, J=9.6,
2.0 Hz, 1H), 7.55 (dd, J=8.4, 7.6 Hz, 1H), 7.20 (dd, J=7.6, 0.8 Hz,
1H), 7.18-7.05 (br.s, 1H), 6.51 (dd, J=8.4, 0.8 Hz, 1H), 4.10-3.95
(m, 1H), 3.46-3.36 (m, 2H), 3.14-3.02 (m, 2H), 2.24-2.14 (m, 2H),
1.80-1.66 (m, 2H); LC/MS: Method 1, retention time: 1.862 min;
HRMS: m/z (M+H).sup.+=327.1251 (Calculated for
C.sub.17H.sub.18ClN.sub.5=327.1251).
##STR00118##
[0206]
6-(6-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-yl)-N-(pyrrolidin-3-y-
l)pyridin-2-amine (I-54):
[0207] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.94
(s, 1H), 9.10-8.95 (br.s, 1H), 8.90-8.80 (br.s, 1H), 8.53 (s, 1H),
8.13 (s, 1H), 7.64 (dd, J=8.6, 7.5 Hz, 1H), 7.43 (s, 1H), 7.26 (d,
J=5.1 Hz, 1H), 7.23 (d, J=5.1 Hz, 1H), 6.59 (d, J=8.0 Hz, 1H),
4.70-4.53 (m, 1H), 4.10 (s, 3H), 3.40-3.27 (m, 1H), 3.26-3.10 (m,
3H), 2.20-2.10 (m, 1H), 2.10-2.00 (m, 1H); LC/MS: Method 1,
retention time: 1.665 min; HRMS: m/z (M+H).sup.+=345.1702
(Calculated for C.sub.19H.sub.19N.sub.7=345.1702).
##STR00119##
[0208] 6-(6-chloro-7-methoxyimidazo [1,2-a]
pyridin-3-yl)-N-(pyrrolidin-3-yl)pyridin-2-amine (I-21):
[0209] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.14
(s, 1H), 9.05-8.75 (br.s, 2H), 8.40 (s, 1H), 7.58 (t, J=8.0 Hz,
1H), 7.39 (s, 1H), 7.22 (d, J=8.0 Hz, 1H), 7.16 (d, J=4.8 Hz, 1H),
6.50 (d, J=8.0 Hz, 1H), 4.55-4.45 (m, 1H), 4.04 (s, 3H), 3.55-3.45
(m, 1H), 3.45-3.25 (m, 3H), 2.40-2.25 (m, 1H), 2.12-2.00 (m, 1H);
LC/MS: Method 1, retention time: 1.896 min; HRMS: m/z
(M+H).sup.+=343.1200 (Calculated for C.sub.17H.sub.18ClN.sub.5O32
343.1200).
##STR00120##
[0210]
6-(7-methoxy-6-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-yl)-N-(pyrr-
olidin-3-yl)pyridin-2-amine (I-20):
[0211] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.93
(s, 1H), 9.02 (br.s, 1H), 8.86 (br.s, 1H), 8.52 (s, 1H), 8.13 (s,
2H), 7.64 (dd, J=8.4, 7.5 Hz, 1H), 7.42 (s, 1H), 7.26-7.21 (m, 2H),
6.59 (dd, J=8.4, 0.7 Hz, 1H), 4.60-4.56 (m, 1H), 4.10 (s, 3H),
3.39-3.31 (m, 1H), 3.19-3.14 (m, 3H), 2.19-2.10 (m, 1H), 2.07-1.99
(m, 1H); LC/MS: Method 1, retention time: 2.829 min; HRMS: m/z
(M+H).sup.+=375.1808 (Calculated for
C.sub.20H.sub.21N.sub.7O=375.1808).
##STR00121##
[0212]
6-(7-methoxy-6-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-yl-
)-N-(pyrrolidin-3-yl)pyridin-2-amine (I-24):
[0213] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.88
(s, 1H), 8.90 (br.s, 1H), 8.78 (br.s, 1H), 8.44 (s, 1H), 8.22 (s,
1H), 7.90 (d, J=0.8 Hz, 1H), 7.62 (dd, J=8.4, 7.4 Hz, 1H), 7.36 (s,
1H), 7.22-7.17 (m, 2H), 6.56 (d, J=8.3 Hz, 1H), 4.59-4.55 (m, 1H),
4.08 (s, 3H), 3.91 (s, 3H), 3.25-3.17 (m, 2H), 2.20-2.11 (m, 1H),
2.08-1.99 (m, 1H); LC/MS: Method 1, retention time: 3.005 min;
HRMS: m/z (M+H).sup.+=389.1964 (Calculated for
C.sub.21H.sub.23N.sub.7O=389.1964).
##STR00122##
[0214]
6-(7-methoxy-6-(pyridin-3-yl)imidazo[1,2-a]pyridin-3-yl)-N-(pyrroli-
din-3-yl)pyridin-2-amine (I-22):
[0215] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.92
(s, 1H), 8.98 (br.s, 1H), 8.90 (br.s, 1H), 8.82 (d, J=2.2 Hz, 1H),
8.69 (dd, J=4.9, 1.6 Hz, 1H), 8.63 (s, 1H), 8.08 (dt, J=8.0, 1.9
Hz, 1H), 7.65-7.57 (m, 2H), 7.52 (s, 1H), 7.27-7.22 (m, 2H), 6.57
(d, J=8.4 Hz, 1H), 4.45 (s, 1H), 4.04 (s, 3H), 3.31-3.23 (m, 1H),
3.16-3.05 (m, 2H), 2.00-1.88 (m, 2H); LC/MS: Method 1, retention
time: 2.723 min; HRMS: m/z (M+H).sup.+=386.1855 (Calculated for
C.sub.22H.sub.22N.sub.6O=386.1855).
##STR00123##
[0216]
6-(7-methoxy-6-(pyridin-4-yl)imidazo[1,2-a]pyridin-3-yl)-N-(pyrroli-
din-3-yl)pyridin-2-amine (I-23):
[0217] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.94
(s, 1H), 8.91 (s, 1H), 8.82 (s, 1H), 8.76 (d, J=4 Hz, 2H), 8.59 (s,
1H), 7.71-7.70 (m, 2H), 7.62 (dd, J=8.4, 7.5 Hz, 1H), 7.49 (s, 1H),
7.25 (dd, J=7.4, 0.7 Hz, 1H), 7.18 (d, J=5.6 Hz, 1H), 6.56 (dd,
J=8.4, 0.7 Hz, 1H), 4.44 (s, 1H), 4.04 (s, 3H), 3.30-3.22 (m, 1H),
3.19-3.13 (m, 3H), 2.01-1.91 (m, 2H); LC/MS: Method 1, retention
time: 2.581 min; HRMS: m/z (M+H).sup.+=386.1855 (Calculated for
C.sub.22H.sub.22N.sub.6O=386.1855).
##STR00124##
[0218]
6-(6-(3,5-dimethylisoxazol-4-yl)-7-methoxyimidazo[1,2-a]pyridin-3-y-
l)-N-(pyrrolidin-3-yl)pyridin-2-amine (I-28):
[0219] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.67
(s, 1H), 8.87 (br.s, 2H), 8.45 (s, 1H), 7.62-7.58 (m, 1H), 7.38 (s,
1H), 7.23-7.21 (m, 1H), 7.11-7.09 (m, 1H), 6.98 (s, 1H), 6.51 (d,
J=8.3 Hz, 1H), 4.35-4.33 (m, 1H), 3.98 (s, 3H), 3.20-3.15 (m, 3H),
2.34 (s, 3H), 2.11 (s, 3H), 2.07-1.91 (m, 2H); LC/MS: Method 1,
retention time: 3.066 min; HRMS: m/z (M+H).sup.+=404.1961
(Calculated for C.sub.22H.sub.24N.sub.6O.sub.2=404.1961).
##STR00125##
[0220]
6-(7-methoxy-6-(1H-pyrrol-3-yl)imidazo[1,2-a]pyridin-3-yl)-N-(pyrro-
lidin-3-yl)pyridin-2-amine (I-27):
[0221] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 11.19
(s, 1H), 9.88 (s, 1H), 9.02 (br.s, 1H), 8.89 (br.s, 1H), 8.52 (s,
1H), 7.63 (dd, J=8.4, 7.5 Hz, 1H), 7.37-7.35 (m, 2H), 7.27-7.22 (m,
2H), 6.91-6.90 (m, 1H), 6.59 (dd, J=8.4, 0.7 Hz, 1H), 6.48-6.47 (m,
1H), 4.65-4.60 (m, 1H), 4.09 (s, 3H), 3.26-3.16 (m, 3H), 2.24-2.15
(m, 1H), 2.05-1.97 (m, 1H); LC/MS: Method 1, retention time: 3.104
min; HRMS: m/z (M+H).sup.+=374.1855 (Calculated for
C.sub.21H.sub.22N.sub.6O=374.1855).
[0222] Method C. Used for the Synthesis of Compound I-68.
##STR00126##
[0223] An oven dried microwave vial was charged with tert-butyl
3-((6-(6-chloro-7-methoxyimidazo[1,2-a]pyridine-3-yl)pyridine-2-yl)amino)-
pyrrolidine-1-carboxylate (30 mg, 0.068 mmol),
tert-butyl(ethynyl)dimethylsilane (0.03 mL, 0.17 mmol),
tri-tert-butylphosphine (0.135 mL, 0.14 mmol, 1 M solution in
toluene), 1,8-diazabicyclo[5.4.0]undec-7-ene (2 .mu.L, 0.014 mmol),
cesium carbonate (44 mg, 0.14 mmol) and
dichlorobis(triphenylphosphine)palladium(II) (5 mg, 0.007 mmol).
DMF (0.5 mL) was then added and the microwave vial was purged with
nitrogen and sealed. The mixture was subjected to microwave
irradiation at 150.degree. C. for 1 h. The crude mixture was
purified via ISCO chromatography (50-100% EtOAc/hexanes) to deliver
product (15 mg, 41%) as an off-white solid.
##STR00127##
[0224]
6-(6-ethynyl-7-methoxyimidazo[1,2-a]pyridin-3-yl)-N-(pyrrolidin-3-y-
l)pyridin-2-amine (1-68):
[0225] Method C. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.04
(s, 1H), 8.91 (br. s, 2H), 8.30 (s, 1H), 7.58-7.54 (m, 1H),
7.21-7.18 (m, 2H), 7.13-7.12 (m, 1H), 6.57 (br. s, 1H), 6.46 (d,
J=8.3 Hz, 1H), 4.49 (s, 1H), 4.38 (s, 1H), 3.96 (s, 3H), 2.34-2.29
(m, 1H), 2.08-2.05 (m, 1H). LC/MS: Method 1, retention time: 3.084
min; HRMS: m/z (M+H).sup.+=333.1590 (Calculated for
C.sub.19H.sub.19N.sub.5O=333.1590).
##STR00128##
[0226]
6-(6-(3,5-dimethyl-1H-pyrazol-4-yl)-7-methoxyimidazo[1,2-a]pyridin--
3-yl)-N-(pyrrolidin-3-yl)pyridin-2-amine (1-25):
[0227] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.64
(s, 1H), 8.93 (br.s, 2H), 8.62 (s, 1H), 7.64 (dd, J=8.4, 7.5 Hz,
1H), 7.43 (s, 1H), 7.25 (dd, J=7.5, 0.7 Hz, 1H), 7.19 (d, J=5.6 Hz,
1H), 6.58 (dd, J=8.4, 0.7 Hz, 1H), 4.34-4.30 (m, 1H), 4.01 (s, 3H),
3.32-3.26 (m, 1H), 3.17-3.03 (m, 2H), 2.08 (s, 6H), 2.00-1.90 (m,
2H); LC/MS: Method 1, retention time: 2.872 min; HRMS: m/z
(M+H).sup.+=403.2121 (Calculated for
C.sub.22H.sub.25N.sub.7O=403.2121).
##STR00129##
[0228]
3,5-difluoro-6-(7-methoxy-6-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridin--
3-yl)-N-(pyrrolidin-3-yl)pyridin-2-amine (1-33)
[0229] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.58
(s, 1H), 8.87-8.81 (br.m, 2H), 8.34 (d, J=2.6 Hz, 1H), 8.12 (s,
2H), 8.02 (dd, J=10.5, 9.7 Hz, 1H), 7.42 (s, 1H), 7.25 (d, J=8 Hz,
1H), 4.63-4.60 (m, 1H), 4.11 (s, 3H), 3.38-3.26 (m, 2H), 3.24-3.16
(m, 1H), 3.14-3.06 (m, 1H), 2.17-2.01 (m, 2H); LC/MS: Method 1,
retention time: 2.872 min; HRMS: m/z (M+H).sup.+=411.1619
(Calculated for C.sub.20H.sub.19F.sub.2N.sub.7O=411.1619).
##STR00130##
[0230]
6-(7-methoxy-6-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-yl)-N-(pipe-
ridin-3-yl)pyridin-2-amine (I-29):
[0231] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.98
(s, 1H), 8.50 (s, 1H), 8.15 (br.s, 2H), 7.59 (dd, J=8.4, 7.4 Hz,
1H), 7.38 (s, 1H), 7.23 (s, 1H), 7.15 (dd, J=7.4, 0.7 Hz, 1H),
7.10-7.08 (m, 1H), 6.97 (s, 1H), 6.56 (d, J=8.3 Hz, 1H), 4.09 (s,
3H), 4.00 (s, 1H), 3.19-3.16 (m, 2H), 2.59-2.52 (m, 2H), 2.08-2.04
(m, 2H), 1.68-1.59 (m, 3H); LC/MS: Method 1, retention time: 2.943
min; HRMS: m/z (M+H).sup.+=389.1964 (Calculated for
C.sub.21H.sub.23N.sub.7O=389.1964).
[0232] Method D. Used for the synthesis of compound I-32.
##STR00131##
[0233] An oven dried microwave vial was charged with
3-(6-bromopyridin-2-yl)-6-chloro-7-methoxyimidazo[1,2-a]pyridine
(100 mg, 0.30 mmol), tert-butyl 3-hydroxypyrrolidine-1-carboxylate
(332 mg, 1.77 mmol), copper(I) iodide (14 mg, 0.07 mmol), cesium
carbonate (722 mg, 2.22 mmol) and
3,4,7,8-tetramethyl-1,10-phenanthroline (35 mg, 0.15 mmol). Toluene
(0.83 mL) was added and the vial was purged with nitrogen. The vial
was sonicated before subjecting to microwave irradiation at
120.degree. C. for 3 h. The mixture was diluted with DCM (20 mL)
and H.sub.2O (20 mL). The layers were separated and the aqueous
layer was extracted with (3.times.20 mL) DCM. The organic extracts
were combined and washed with brine (1.times.20 mL), dried over
sodium sulfate, filtered and concentrated in vacuo. The crude
residue was then purified via ISCO chromatography (70-100%
EtOAc/hexanes) to deliver product (67 mg, 51%) as an off-white
solid.
##STR00132##
[0234]
7-methoxy-6-(1H-pyrazol-4-yl)-3-(6-(pyrrolidin-3-yloxy)pyridin-2-yl-
)imidazo[1,2-a]pyri-dine (1-32):
[0235] Method D. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.74
(s, 1H), 9.27 (br.s, 1H), 9.04 (br.s, 1H), 8.58 (s, 1H), 8.14 (s,
2H), 7.95 (t, J=7.9 Hz, 1H), 7.67 (d, J=7.5 Hz, 1H), 7.44 (s, 1H),
6.88 (d, J=8.2 Hz, 1H), 5.78-5.75 (m, 1H), 4.10 (s, 3H), 3.55-3.51
(m, 1H), 3.37-3.30 (m, 2H), 2.34-2.29 (m, 1H), 2.19-2.09 (m, 1H);
LC/MS: Method 1, retention time: 2.812 min; HRMS: m/z
(M+H).sup.+=376.1648 (Calculated for
C.sub.20H.sub.20N.sub.6O.sub.2=376.1648).
##STR00133##
[0236]
6-(7-methoxy-6-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-yl)-N-methy-
l-N-(pyrrolidin-3-yl)pyridin-2-amine (I-31):
[0237] Method A. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.92
(s, 1H), 8.94 (br.s, 1H), 8.81 (br.s, 1H), 8.53 (s, 1H), 8.09 (s,
2H), 7.75 (dd, J=8.6, 7.5 Hz, 1H), 7.39 (s, 1H), 7.29 (d, J=7.5 Hz,
1H), 7.24 (s, 1H), 7.11 (s, 1H), 6.98 (s, 1H), 6.84 (d, J=8.6 Hz,
1H), 5.30-5.26 (m, 1H), 4.09 (s, 3H), 3.20-3.17 (m, 1H), 3.03 (s,
3H), 3.00-2.93 (m, 1H), 2.23-2.15 (m, 1H), 2.10-2.02 (m, 1H);
LC/MS: Method 1, retention time: 3.014 min; HRMS: m/z
(M+H).sup.+=389.1964 (Calculated for
C.sub.21H23N.sub.7O=389.1964).
[0238] Method E. Used for the synthesis of compound I-41.
##STR00134##
[0239] A microwave vial equipped with a stir bar was charged with
3-(6-bromopyridin-2-yl)-6-chloro-7-methoxyimidazo[1,2-a]pyridine
(85 mg, 0.25 mmol), 4-methylbenzenesulfonic acid hydrate (239 mg,
1.3 mmol) and lithium chloride (53 mg, 1.3 mmol). DMF (1.3 mL) was
then added and the vial was subjected to microwave irradiation at
120.degree. C. for 2 h. The crude mixture was purified by reverse
phase ISCO chromatography (1-100% acetonitrile/H.sub.2O) to deliver
product (24 mg, 29%) as an off-white solid.
[0240] A 25 mL round bottomed flask, equipped with a stir bar, was
charged with
3-(6-bromopyridin-2-yl)-6-chloroimidazo[1,2-a]pyridine-7-ol (70 mg,
0.22 mmol), di-tert-butyl azodicarboxylate (89 mg, 0.39 mmol),
2-morpholinoethanol (51 mg, 0.39 mmol), THF (10 mL to 15 mL), and
triphenylphosphine (102 mg, 0.39 mmol). The reaction mixture was
stirred at room temperature for 1.5 h. The THF was removed in vacuo
and the crude mixture was purified by ISCO chromatography (1-10%
methanol/DCM) to deliver product (64 mg, 67%) as an off-white
solid.
##STR00135##
[0241]
6-(7-(2-morpholinoethoxy)-6-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridin--
3-yl)-N-(pyrrolidin-3-yl)pyridin-2-amine (I-41):
[0242] Method E. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.93
(s, 1H), 8.99 (br.s, 1H), 8.87 (br.s, 1H), 8.49 (s, 1H), 8.16 (s,
2H), 7.62 (t, J=7.9 Hz, 1H),7.48 (s, 1H), 7.23-7.20 (m, =2H), 6.57
(d, J=8.4 Hz, 1H), 4.64-4.55 (m, 3H), 3.78-3.12 (m, 14H), 2.18-2.09
(m, 1H), 2.06-1.98 (m, 1H); LC/MS: Method 1, retention time: 2.406
min; HRMS: m/z (M+H).sup.+=474.2492 (Calculated for
C.sub.25H.sub.30N.sub.8O.sub.2=474.2492).
Example Set B-IC.sub.50 Assays and Properties for Some
Compounds
[0243] Methods--Cell Culture
[0244] THP-1 cells were purchased from the American Type Culture
Collection (ATCC) and cultured in RPMI-1640 Medium (ATCC),
2-mercaptoethanol to a final concentration of 0.05 mM and 10% FBS.
Hep G2 were purchased from ATCC and cultured in DMEM+sodium
pyruvate with 10% FBS and 1% penicillin-streptomycin.
[0245] The MA9-FLT3-ITD cell line (also referred to as MLL-AF9/FLT3
ITD) is a leukemia cell line derived from CD34+ cord blood
harboring both MLL-AF9 chromosomal translocation and stably
expressed FLT3 ITD mutation. The MA9-FLT3-ITD cell line was
provided by Dr. James Mulloy (Cincinnati Children's Hospital) and
was cultured in IMDM with 20% FBS (Stemcell Technologies) and 1%
penicillin-streptomycin.
[0246] Methods--Cell Titer Glo Assay Protocol
TABLE-US-00002 TABLE 2 Final 1536-well assay protocol Step
Parameter Value Description 1 Cells 5 .mu.L 2500 cells/well 2
Controls 23 nl DMSO, Bortezomib 3 Library compounds 23 nl 57 .mu.M
to 0.4 nM dilution series 4 Incubation time 48 hr 37.degree. C. 5
Reagent 3 .mu.L Cell Titer Glo 6 Assay readout luminescence ViewLux
Step Notes 1 Greiner white solid plates; 4 tips dispense to all
wells 2 Column 1 media and DMSO only, Column 2 and 3 Bortezomib,
column 4 DMSO 3 Pintool transfer (tip wash sequence; DMSO, iPA,
MeOH, 3-s vacuum dry) 4 Plates covered with stainless steel rubber
gasket-lined lids containing pin holes for gas exchange 5 Cell
Titer Glo detection. Luciferase-based detection of ATP product 6
Perkin Elmer ViewLux, clear filter luminescent read
[0247] Methods--HotSpot Assay
[0248] The HotSpot.RTM. kinase profiling and screening assays were
carried out using the method of Anastassiadis et al., Nat.
Biotechnol. (2011) Vol. 29, No. 11, pp. 1039-1045 (which is herein
incorporated by reference in its entirety); 10 .mu.M was the
starting concentration with a 3-fold series dilution (10 doses) and
the final ATP concentration was 10 .mu.M.
[0249] Results
[0250] IC.sub.50 determinations were measured against MA9-FLT3-ITD
cells using Cell Titer Glo assay (Table 3).
TABLE-US-00003 TABLE 3 IC.sub.50 determination. Compound
IC.sub.50.sup.a No. R.sup.1 R.sup.2 R.sup.3 R.sup.4 R.sup.5 R.sup.6
Y m n (nM) I-15 H Cl H H H H NH 1 1 45 I-12 H H H H H H NH 1 1 260
I-63 Cl H H H H H NH 1 1 973 I-61 Me H H H H H NH 1 1 442 I-50 OMe
H H H H H NH 1 1 120 I-60 H CN H H H H NH 1 1 313 I-42 H Cl OMe H H
H NH 1 1 953 I-67 H Cl H H OMe H NH 1 1 892 I-55 H Cl H H Me H NH 1
1 291 I-56 H Cl H F H F NH 1 1 157 I-16 H Cl H H H H NH 1 2 30 I-17
H Cl H H H H NH 1 3 22 I-2 H Cl H H H H NH 2 2 16 I-54 H
1H-pyrazol-4-yl H H H H NH 1 2 34 I-21 OMe Cl H H H H NH 1 2 6 I-20
OMe 1H-pyrazol-4-yl H H H H NH 1 2 5 I-24 OMe 1-Me-pyrazol-4-yl H H
H H NH 1 2 <5 I-22 OMe pyridin-3-yl H H H H NH 1 2 26 I-23 OMe
pyridin-4-yl H H H H NH 1 2 15 I-28 OMe 3,5-dimethylisoxazolyl H H
H H NH 1 2 140 I-27 OMe 1H-pyrrol-3-yl H H H H NH 1 2 30 I-68 OMe
Ethynyl H H H H NH 1 2 15 I-25 OMe 3,5-di-Me-pyrazolyl H H H H NH 1
2 380 I-33 OMe 1H-pyrazol-4-yl H F H F NH 1 2 53 I-29 OMe
1H-pyrazol-4-yl H H H H NH 1 3 15 I-32 OMe 1H-pyrazol-4-yl H H H H
O 1 2 166 I-31 OMe 1H-pyrazol-4-yl H H H H N--Me 1 2 298 I-41
2-(morpholino) 1H-pyrazol-4-yl H H H H NH 1 2 68 ethoxy quizartinib
<5 .sup.aThe IC.sub.50 values represent the average of three
runs against Mll-AF9/FLT3-ITD cells using the Cell Titer Glow
assay.
[0251] Compound I-50 (when R.sup.1 was methoxy) exhibited the best
cell-killing activity for MA9-FLT3-ITD cells of the tested
compounds. With regard to R.sup.2, compound I-12 and compound I-61
showed a decrease in activity compared to compound I-15.
Introducing a small heterocycle at R.sup.2, such as
1-H-pyrazol-4-yl (e.g., compound I-54) slightly improved the
activity. Tested compounds with substitutions at the 8-position led
to a decrease of activity as shown, for example, by compound I-42;
a decrease in activity is also observed for the tested compounds
with substitutions at the 2- or 5-position (data not shown). The
tested compounds with substitution on the pyridine ring (e.g.,
compounds I-55, I-56, and I-67,) led to a decreased activity
analogues; small size groups like 3,5-difluoro substitution (e.g.,
compound I-56) appeared to show modest decrease of activity. Among
the tested compounds, it was found that the compounds with either
5-membered (compound I-16) or 6-membered cyclic amine (compound
I-17 and compound I-2) showed .about.2-fold improvement of potency
compared to compound I-15. Compounds tested that had any
derivatization of the free --NH of cyclic amine led to an apparent
complete loss of cell-killing activity (data not shown).
[0252] Compound I-21 exhibited an IC.sub.50 value of 6 nM, which
was almost 8-fold more potent than compound I-15. Of the compounds
tested, replacing the --Cl with -ethynyl (e.g., compound I-68)
resulted in slightly decreased potency. Some tested compounds
included heterocycles substituted at R.sup.2 while keeping methoxy
at the 7-position unchanged; the tested compounds without
substitution on this heterocyclic ring (e.g., compounds I-20, I-22,
I-23, I-24, and 1-68) had a similar potency compared to compound
I-54, while the tested compounds that had a substitution on the
heterocylic ring had variable changes in activity (e.g., compounds
I-25 and I-28 decreased in activity while compound I-24 increased
in activity).
[0253] Of the compounds tested, replacing the pyridine ring with
3,5-difluoropyridyl (compound I-33) resulted in 10-fold decrease of
potency compared to compound I-20. The tested compounds which
replaced the pyridine with a phenyl (data not shown) or a
1,3-pyrimidinyl (data not shown) exhibited micromolar range potency
for MA9-FLT3-ITD cells. Also, replacing the --NH linkage at
2-position of pyridine ring with either ether (compound I-32) or
N--Me (compound I-31) linkage led to a loss of potency for the
compounds tested. Compound I-29 (with 6-membered cyclic amine) was
3-fold less potent than compound I-20. Compound I-41 was
synthesized by attaching the solubilizing group
2-(morpholino)ethoxy at the 1-position of 1H-pyrazoyl ring;
compound I-41 exhibited an almost 14-fold decrease of potency
compared to compound I-20.
[0254] Compounds I-2, I-22, and I-24 and quizartinib were re-tested
with lower starting concentration. Compound I-24 exhibited had an
IC50 value of 0.8 nM, which was 2-fold more potent than quizartinib
(FIG. 1).
[0255] Selectivity towards FLT3 and IRAK1/4: Utilizing the HotSpot
assay, we ascertained the IC.sub.50 values for some compounds
versus IRAK1, IRAK4, and FLT3 along with a panel of other kinases
(Table 4).
TABLE-US-00004 TABLE 4 Inhibitory activities of compound I-2, I-22,
and I-24 and quizartinib. Compound IC.sub.50 (nM) Kinase I-2 I-24
I-22 quizartinib BLK <0.5 <0.5 104 >10000 CDK7/cyclin H
177 174 >10000 >10000 FLT3 <0.5 <0.5 <0.5 2.23 FLT3
(D835Y) <0.5 <0.5 <0.5 108 FLT3 (ITD) <0.5 <0.5 1
1.93 IRAK1 31.7 22.6 1940 >10000 IRAK4 40 0.8 299 >10000 LCK
0.9 <0.5 148 >10000 PDGFR.beta. 4.19 2.67 3.57 98.2 RET 0.6
<0.5 1280 17.5 SRPK1 >10000 >10000 >10000 >10000
[0256] Compounds I-2, I-22, and 1-24 inhibited FLT3 and FLT3 (ITD),
and also showed sub-nano molar potency for FLT3 (D835Y).
Quizartinib exhibited weaker activity (IC.sub.50=108 nM),
indicating compounds I-2, I-22, and 1-24 might overcome the
quizartinib-resistant AML cell lines carrying FLT3 point mutation
in TKD. Generally, compounds I-2, I-22, and I-24 exhibited better
inhibitory activities for IRAK4 than IRAK1 but compound I-24 stood
out with sub-nano molar potency for IRAK4, which, without being
bound by theory, might be the cause that compound 1-24 is
.about.27-fold more potent than compound I-2 for MA9-FLT3-ITD cells
considering their similar inhibitory profiles for other 10 kinases
screened. Quizartinib appeared inactive for both IRAK family
kinases.
[0257] Compounds I-2, I-22, and I-24 were tested against THP-1
cells (a type of FLT3 wild type AML cells) and Hep G2 cells (a type
of liver cancer cells). None of the compounds tested showed any
inhibitory activity against THP-1 or Hep G2 cells even at >10
.mu.M concentration (data not shown), suggesting high selectivity
for MA9-FLT3-ITD cells and low toxicity due to off-target
inhibition.
[0258] Compounds I-2, I-22, and I-24 showed subnamolar potency for
both IRAK4 and FLT3, but also inhibited other kinases such as LCK,
RET and PDGFR.beta..
[0259] ADME profile: Early ADME profiling was performed using
standard methods and included rat microsomal stability, PAMPA
permeability and kinetic aqueous solubility. Overall, compounds
I-2, I-22, and I-24 showed both good rat microsomal stability (RLM)
and aqueous solubility (Table 5).
TABLE-US-00005 TABLE 5 In vitro ADME properties of compounds I-2,
I-22, and I-24. IC.sub.50.sup.a RLM T.sub.1/2.sup.b
Permeability.sup.c Kinetic Solubility.sup.d Compound (nM) (min)
(10.sup.-6 cm/s) (.mu.M) I-2 22 >30.0 806.9 >48.0 I-24 0.8
>30.0 <1.3 >58.0 I-22 26 >30.0 <3.4 29
.sup.aIC.sub.50 against MLL-AF9 FLT3-ITD cells using a Cell Titer
Glo Assay. .sup.bT.sub.1/2 in rat liver microsomes (RLM) in the
presence of NADPH. .sup.cPAMPA permeability at pH 7.4.
.sup.dKinetic aqueous solubility in PBS buffer (pH 7.4) as measured
by UV quanitification.
[0260] Compound I-2 also showed good PAMPA permeability. Compounds
I-22 and I-24 might mitigate their PAMPA permeability through
intraperitoneal (IP) administration in animal studies. Compounds
I-2, I-22, and I-24 were evaluated for their in vivo
pharmacokinetic properties in NRG/NRGS mice (the strain of mice to
be used in our MA9-FLT3-ITD AML disease model). The compounds were
dosed through IP injection at 30 mg/kg and the plasma samples were
collected for analysis (Table 6).
TABLE-US-00006 TABLE 6 Pharmacokinetic evaluations of compounds
I-2, I-22, and I-24 in NRG/NRGS mice with IP injection at 30
mg/kg..sup.a, b Compound AUC.sub.inf. (hr*ng/mL) T.sub.1/2 (hr)
T.sub.max (hr) C.sub.max (ng/mL) I-2 4,630 3.3 0.5 1,710 I-24 6,800
4.2 0.083 3,570 I-22 4,850 3.9 0.5 1,540 .sup.aCompounds I-2, I-22,
and I-24 were all formulated as a solution in saline. .sup.bPlasma
samples were collected at time point of 0.083, 0.25, 0.5, 1, 2, 4,
7, and 24 h after dosing.
[0261] Compounds I-2, I-22, and I-24 showed good and similar in
vivo pharmacokinetic properties. Compound I-24 had better plasma
exposure (AUC.sub.inf.=6,800 hr*ng/mL), higher concentration in
plasma (C.sub.max=3,570 ng/mL) and longer half-life (T.sub.1/2=4.2
hr) compared to compounds I-2 and I-22.
Example Set C--Kd Kinase Assays
[0262] Kinase assays. For most assays, kinase-tagged T7 phage
strains were prepared in an E. coli host derived from the BL21
strain. E. coli were grown to log-phase and infected with T7 phage
and incubated with shaking at 32.degree. C. until lysis. The
lysates were centrifuged and filtered to remove cell debris. The
remaining kinases were produced in HEK-293 cells and subsequently
tagged with DNA for qPCR detection. Streptavidin-coated magnetic
beads were treated with biotinylated small molecule ligands for 30
minutes at room temperature to generate affinity resins for kinase
assays. The liganded beads were blocked with excess biotin and
washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween
20, 1 mM DTT) to remove unbound ligand and to reduce non-specific
binding. Binding reactions were assembled by combining kinases,
liganded affinity beads, and test compounds in 1.times. binding
buffer (20% SeaBlock, 0.17.times. PBS, 0.05% Tween 20, 6 mM DTT).
All reactions were performed in polystyrene 96-well plates in a
final volume of 0.135 ml. The assay plates were incubated at room
temperature with shaking for 1 hour and the affinity beads were
washed with wash buffer (1.times. PBS, 0.05% Tween 20). The beads
were then re-suspended in elution buffer (1.times. PBS, 0.05% Tween
20, 0.5 .mu.M non-biotinylated affinity ligand) and incubated at
room temperature with shaking for 30 minutes. The kinase
concentration in the eluates was measured by qPCR.
[0263] An 11-point 3-fold serial dilution of each test compound was
prepared in 100% DMSO at 100.times. final test concentration and
subsequently diluted to 1.times. in the assay (final DMSO
concentration=1%). Most Kds were determined using a compound top
concentration of 30,000 nM. If the initial Kd determined was
<0.5 nM (the lowest concentration tested), the measurement was
repeated with a serial dilution starting at a lower top
concentration (Table 7).
TABLE-US-00007 TABLE 7 Kd determination. Target Gene Quizartinib
I-17 Crenolinib I-22 I-24 Symbol Kd (nM) FLT3 1.4 0.61 0.2 0.065
0.025 FLT3(D835H) 2.1 0.64 0.2 0.11 0.27 FLT3(D835V) 4.2 0.093
0.016 0.021 0.0099 FLT3(D835Y) 7.6 0.4 0.19 0.12 0.29 FLT3(ITD) 7
0.31 0.26 0.17 0.36 FLT3(ITD, D835V) 480 0.036 0.023 0.014 0.01
FLT3(ITD, F691L) 160 0.41 0.11 0.12 0.012 FLT3(K663Q) 0.57 5 0.82
0.58 5.4 FLT3(N841I) 1.5 1.2 0.39 0.24 0.33 FLT3(R834Q) 12 3.4 1.3
0.42 0.3 FLT3-autoinhibited 580 11 17 1.9 0.96 IRAK1 >30000 150
260 3.5 2.9 IRAK4 >30000 3.4 62 0.29 0.3
Example Set D--IC50 Kinase Assays and ADME
[0264] Compounds were tested against 11 kinases. Compounds were
tested in 10-dose IC50 mode with 3-fold serial dilution starting at
10 .mu.M, and are relative to DMSO, the negative control. The
positive control, Staurosporine, was tested in a 10-dose IC50 mode
with 4-fold serial dilution starting at 20 .mu.M. Reactions were
carried out at 10 .mu.M ATP.
[0265] Curve fits were performed to determine IC50 where the enzyme
activities at the highest concentration of compounds were less than
65%. IC50 values less than 5.08 E-10 M or higher than 1.00E-5 M is
estimated based on the best curve fitting available (Table 8 and
Table 9).
TABLE-US-00008 TABLE 8 IC50 determination. Compound IC50 (M)
Staurosporine Kinase: I-17 I-20 I-22 I-24 IC50 (M) BLK 4.07E-08
<5.08E-10 <5.08E-10 <5.08E-10 1.45E-09 CDK7/cyclin H
5.88E-07 1.16E-07 1.77E-07 1.74E-07 1.06E-07 FLT3 <5.08E-10
<5.08E-10 <5.08E-10 <5.08E-10 1.58E-09 FLT3 (D835Y)
<5.08E-10 <5.08E-10 <5.08E-10 <5.08E-10 7.21E-11 FLT3
(ITD) <5.08E-10 <5.08E-10 <5.08E-10 <5.08E-10 1.46E-09
IRAK1 1.48E-06 5.34E-09 3.17E-08 2.26E-08 4.60E-08 IRAK4 1.12E-08
<5.08E-10 6.53E-10 8.08E-10 3.11E-09 LCK 1.67E-07 <5.08E-10
9.38E-10 <5.08E-10 2.17E-09 PDGFRb 6.70E-08 1.25E-09 4.19E-09
2.67E-09 2.72E-09 RET 1.05E-08 <5.08E-10 6.31E-10 <5.08E-10
2.17E-09 SRPK1 7.67E-06 >1.00E-05 NI NI 3.34E-08 NI--indicates
no inhibition or that compound activity data could not be fit to an
IC50 curve.
TABLE-US-00009 TABLE 9 IC50 determination. Compound IC50 (M)
Kinase: Crenolinib I-2 Staurosporine BLK 2.07E-08 1.04E-07 8.03E-10
CDK7/cyclin H 1.26E-06 NI 5.99E-08 FLT3 2.36E-10 3.56E-10 7.88E-10
FLT3 (D835Y) 1.77E-11 9.34E-12 6.21E-11 FLT3 (ITD) 6.23E-10
9.96E-10 1.23E-09 IRAK1 1.16E-07 1.94E-06 1.89E-08 IRAK4 2.06E-09
2.99E-07 2.75E-09 LCK 5.78E-08 1.48E-07 1.48E-09 PDGFRb 3.78E-08
3.57E-09 1.50E-09 RET 1.38E-08 1.28E-06 1.80E-09 SRPK1 2.17E-05 NI
1.40E-08 NI--indicates no inhibition or that compound activity data
could not be fit to an IC50 curve.
TABLE-US-00010 TABLE 10 ADME and IC50 data for selected compounds.
Stability Permeability Cmpd No. Structure NCGC ID (min) (1e-6 cm/s)
Solubility (.mu.g/mL) NRAS IC50 (.mu.M) FLT3 ITD IC50 (.mu.M) I-20
##STR00136## NCGC00262327 11.6 <6.9 4.5 33.170 0.040 I-21
##STR00137## NCGC00262326 27 29.9 7.7 I-22 ##STR00138##
NCGC00371479 >30.0 <3.4 29 9.920 0.026 I-23 ##STR00139##
NCGC00371480 >30.0 <3.6 11.6 1.830 0.015 I-24 ##STR00140##
NCGC00371481 >30.0 <1.3 >58.0 0.004 I-25 ##STR00141##
NCGC00371482 >30.0 <1.7 >60.0 15.720 0.380 I-26
##STR00142## NCGC00371483 >30.0 <1.5 >72.0 15.720 0.020
I-27 ##STR00143## NCGC00371484 >30.0 <2.3 34.3 4.430 0.030
I-28 ##STR00144## NCGC00371488 15.7 <4.7 >60.0 14.820 0.140
I-29 ##STR00145## NCGC00371485 9.8 <3.6 35.6 9.350 0.015 I-30
##STR00146## NCGC00371486 24.7 <3.1 12.3 I-31 ##STR00147##
NCGC00371487 11.5 <3.3 14.1 16.630 0.298 I-32 ##STR00148##
NCGC00371852 7.7 <3.9 8.2 3.720 0.166 I-33 ##STR00149##
NCGC00371853 9.3 <10.9 3.9 29.760 0.053 I-36 ##STR00150##
NCGC00371850 >30.0 <3.8 >50.0 4.430 0.017 I-37
##STR00151## NCGC00371857 2.3 <1.6 >55.0 16.630 2.221 I-38
##STR00152## NCGC00371858 >30.0 <2.4 >56.0 0.002 6.255
I-39 ##STR00153## NCGC00371859 8.8 <1.8 10.6 5.580 0.033 I-40
##STR00154## NCGC00371957 >30.0 <1.5 >65.0 0.626 0.008
I-41 ##STR00155## NCGC00371958 >30.0 <1.1 >70.0 17.640
0.068 I-15 ##STR00156## NCGC00241410 13 181.6 >44.0 16.630 1.177
I-42 ##STR00157## NCGC00262331 >30 <6.1 >49.0 20.930 0.953
I-43 ##STR00158## NCGC00262376 5.7 <1.5 40.1 0.076 I-44
##STR00159## NCGC00262377 6.7 <1.6 42.6 0.017 I-45 ##STR00160##
NCGC00249356 N/A <2.0 >49.0 4.290 0.460 I-46 ##STR00161##
NCGC00249357 >30.0 <2.0 43.1 16.630 0.213 I-16 ##STR00162##
NCGC00249372 25.6 332.2 >46.0 1.760 0.030 I-47 ##STR00163##
NCGC00249846 >30.0 1164.1 >50.0 1.880 0.157 I-12 ##STR00164##
NCGC00262328 >30.0 <1.3 >39.0 0.260 I-48 ##STR00165##
NCGC00262329 11.8 34.5 >46.0 2.787 I-49 ##STR00166##
NCGC00262330 26.2 39.3 >46.0 0.892 I-50 ##STR00167##
NCGC00249829 >30.0 <1.6 >44.0 0.120 I-51 ##STR00168##
NCGC00249832 9.0 >1314.0 >50.0 0.028 I-52 ##STR00169##
NCGC00249354 19.8 35.5 41.8 0.085 I-53 ##STR00170## NCGC00249838
>30.0 <19.3 19.5 4.956 I-54 ##STR00171## NCGC00249841 7.6
<1.6 >51.0 0.107 I-55 ##STR00172## NCGC00249842 N/A 89.9
>46.0 0.291 I-56 ##STR00173## NCGC00249846 >30.0 1164.1
>50.0 0.095 I-57 ##STR00174## NCGC00249371 >30.0 412.3
>50.0 0.399 I-58 ##STR00175## NCGC00249374 8.0 92.2 <1.0
0.709 I-59 ##STR00176## NCGC00249370 >30.0 401.3 18.3 0.205 I-60
##STR00177## NCGC00249366 16.4 <2.3 >43.0 0.313 I-17
##STR00178## NCGC00249373 >30.0 2018.5 >48.0 0.022 I-2
##STR00179## NCGC00249350 >30.0 806.9 >48.0 0.016 I-61
##STR00180## NCGC00249375 >30.0 <3.0 >41.0 0.442 I-62
##STR00181## NCGC00249368 >30.0 85.6 41.5 0.154 I-63
##STR00182## NCGC00249362 N/A 3.5 >44.0 0.973 I-64 ##STR00183##
NCGC00249363 N/A <5.2 >56.0 1.998 I-67 ##STR00184##
NCGC00249349 >30.0 <3.5 >49.0 0.892 I-68 ##STR00185##
NCGC00378320 11.6 12.9 >49.0 0.015
Example Set E--Cell Data
[0266] General Methods: The methods below are used in Example Set
E, unless otherwise indicated.
[0267] Cell Culture: MLL-AF9 FLT3-ITD and MLL-AF9 NRas cell lines
were provided by Dr. James Mulloy (Cincinnati Children's Hospital
Medical Center, Cincinnati, Ohio) (PMID:19277588) were cultured in
Isocov's DMEM medium with 20% FBS and 1% penicillin-streptomycin.
MV4;11 cell line was provided by Dr. Lee Grimes (CCHMC, Cincinnati,
Ohio) were cultured in RPMI 1640 medium with 10% FBS and 1%
penicillin-streptomycin. MDSL cells were provided by Dr. Kaoru
Tohyama (Kawasaki Medical School, Okayama, Japan) (PMID: 20130600).
MDSL cells were cultured in RPMI 1640 medium with 10% FBS, 1%
penicillin-streptomycin, and 10 ng/mL recombinant human
Interleukin-3 (Stemcell Technologies). Human CD34+ umbilical cord
blood was obtained from the Translational Research Development
Support Laboratory of Cincinnati Children's Hospital under an
approved Institutional Review Board protocol. These cells were
maintained in StemSpan Serum-Free Expansion Media (Stemcell
Techologies) supplemented with 10 ng/mL of recombinant human stem
cell factor (SCF) (Stemcell Technologies), recombinant human
thrombopoietin (TPO) (Stemcell Technologies), recombinant human
interleukin-3 (IL-3) (Stemcell Technologies), and recombinant human
interleukin-6 (IL-6) (Stemcell Technologies).
[0268] Reagents: IRAK1/4 inhibitor (Amgen Inc.) was purchased from
Sigma-Aldrich (I5409). AC220 was purchased from Selleckchem.
[0269] Mice: NRGS (NOD.Rag.sup.-/-; yc.sup.null; hIL-3, hGM-CSF,
hSF) mice were provided by Dr. James Mulloy (Cincinnati Children's
Hospital Medical Center, Cincinnati, OH) (PMID: 25762176).
[0270] Immunoblot: Protein lysates were made by lysing cells in
cold RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1%
Titon X-100, and 0.1% SDS), in the presence of sodium
orthovanadate, PMSF, and protease and phosphatase inhibitors.
Protein concentration was quantified using BCA assay (Pierce).
Protein lysates were separated by SDS-polyacrylamide gel
electrophoresis (BIO-RAD), transferred to nitrocellulose membranes
(BIO-RAD), and immunoblotted. The following antibodies were used
for western blot analysis: GAPDH (D16H11, Cell Signaling, 1:1000
milk) FLT3 (3462, Cell Signaling, 1:1000 BSA), phospho-FLT3
(Tyr591) (3461, Cell Signaling, 1:500 BSA), IRAK4 (4363, Cell
Signaling, 1:1000 BSA), phospho-IRAK4 (Thr345/Ser346) (11927, Cell
Signaling, 1:500 BSA), peroxidase-conjugated AffiniPure Goat
Anti-rabbit IgG (111-035-003, Jackson ImmunoResearch Laboratories,
Inc., 1:10000 milk). Blots were visualized using ECL Western
Blotting Substrate (Pierce) and imaged on autoradiography film
(HyBlot CL) or BIO-RAD ChemiDoc Touch Imaging system.
[0271] Colony formation: Cells were suspended at 1000 cells/mL
(MLL-AF9 FLT3-ITD, MLL-AF9 NRas, and human CD34+ cord blood) or
2000 cells/mL (MDSL) in methylcellulose (MethylCult H4434 Classic)
supplemented with the indicated drug. Colonies were counted 10 days
after plating.
[0272] Viability and Cell Growth: For AnnexinV analysis cell were
washed in AnnexinV Binding Buffer (eBioscience) and resuspended in
AnnexinV binding bufferwith AnnexinV-conjugated antibody (1:100,
eBioscience). A 15-minute, room temperature incubation was followed
by flow cytometric analysis. Analysis was performed using BD
FACSCanto flow cytometer with Diva software. Trypan Blue (MP
Biomedicals LLC) exclusion was done using an automated cell counter
(BioRad TC10).
[0273] CellTiter-Glo Luminescent Viability Assay (Promega): In a
96-well culture plate (Corning Inc. Costar), 25000 cells were
plated in 200 .mu.L media per well. In triplicate, 2 .mu.L of the
indicated inhibitor (100.times. in DMSO) was added to each well and
cells treated with 2 .mu.L DMSO alone was used as the control.
Treated cells were incubated at 37.degree. C. for 72 hours. In a
white 96-well assay plate (Corning Inc. Costar), 100 .mu.L of the
treated cells were transferred and brought to room temperature. An
equal volume of CellTiter-Glo Reagent at room temperature was added
to the cells and the plate was rocked for 2 minutes, followed by 10
minutes of rest. Analysis was performed using GloMax 96 microplate
Luminometer (Promega) with GloMax Software.
[0274] AlphaLISA Assay: AlphaScreen SureFire STATS (pTyr694;Tyr699)
Assay kit (Perkin Elmer). Assay was performed according to
manufacturer protocol.
[0275] FIGS. 2A-2C--Methods: Some compounds of Formula (I) can
suppress activation of FLT3. (FIG. 2A) Immunoblot analysis of MV4;
11 cells (a human FLT3-ITD-AML cell line) treated with AC220 (50
nM), compound I-20 (50 nM), or compound I-24(50 nM) for 12 or 24
hours. (FIG. 2B) Immunoblot analysis of MDSL cells (a human MDS
line with high basal levels of P-IRAK4) treated with the indicated
concentrations of compound I-24 or IRAK-Inh (Amgen; CAS Reg. No.
509093-47-4) for 24 hours. (FIG. 2C) Phospho (P)--STAT5 activity
was measured by AlphaLISA assay in MV4;eleven cells treated with
the indicated concentrations of I-15, I-20, I-43, or AC220 for 5
hours.
[0276] FIGS. 2A-2C--Results & Discussion: Some compounds of
Formula (I) can suppress activation of FLT3. To assess the ability
of the compounds to inhibit FLT3 activity in relevant human cells,
we treated MV4; eleven cells (a human FLT3-ITD-AML cell line) with
AC220, compound I-20, or compound I-24 at 50 nM for 24 hours and
evaluated phosphorylated (P)-FLT3 by immunoblotting (FIG. 2A).
Compound I-20 and compound I-24 inhibited FLT3 phosphorylation
comparably to AC220. MV4; eleven cells exhibit low basal levels of
IRAK4 phosphorylation, therefore we chose MDSL cells (a human MDS
line with high basal levels of P-IRAK4) to examine the activity of
compound I-24 on IRAK4 phosphorylation. Compound I-24 treatment of
MDSL cells effectively inhibited pIRAK4, and was comparable to
IRAK-Inh (FIG. 2B). To determine inhibition of downstream FLT3
signaling, MV4; eleven cells were treated with compound I-15,
compound I-20, compound I-43, or AC220 for 5 hours and P-STATS
activity was measured by AlphaLISA (FIG. 2C). All three compounds
inhibit STATS phosphorylation equivalent to or slightly better than
inhibition by AC220 (IC50 =1.7, 3.6, and 6.6 nM versus 11.7 nM for
AC220). These results show that compound I-15, compound I-20,
compound I-43 are just as effective at inhibiting FLT3
phosphorylation and downstream signaling as AC220 and additionally,
can inhibit IRAK1/4 phosphorylation.
[0277] FIGS. 3A-3D--Methods: FLT3 inhibition results in a
compensatory activation of IRAK1/4 in FLT3-ITD AML. (FIG. 3A)
Immunoblot analysis of human cord blood CD34+ cells transduced with
MLL-AF9 and FLT3-ITD (MA9-FLT3-ITD) treated with AC220 (50 nM) for
the indicated times. (FIG. 3B) Immunoblot analysis of MV4; eleven
cells treated with AC220 (1 or 50 nM) for the indicated times.
(FIG. 3C) Immunoblot analysis MA9-FLT3-ITD treated with AC220 (50
nM), AC220 (50 nM) and IRAK-Inh (10 .mu.M), compound I-20 (50 nM),
or IRAK-Inh alone (10 .mu.M). (FIG. 3D) Immunoblot analysis of
human cord blood CD34+ cells transduced with MLL-AF9 and Nras
(MA9-NRas) treated with AC220 (50 nM), AC220 (50 nM) and IRAK-Inh
(10 .mu.M), compound I-20 (50 nM), or IRAK-Inh alone (10
.mu.M).
[0278] FIGS. 3A-3D--Results & Discussion: FLT3 inhibition
results in a compensatory activation of IRAK1/4 in FLT3-ITD AML.
When FLT3-ITD AML cells are treated with AC220, P-IRAK4 levels
increase after 24 hour exposure to AC220. The compensatory
phosphorylation of IRAK4 is observed across several FLT3-ITD AML
cell lines, and even at AC220 concentrations as low as 1 nM (FIGS.
3A-C). An increase in P-IRAK4 was not observed in AML cells with
wild-type FLT3 (FIG. 3D). Compound I-20 did not result in
compensatory phosphorylation of IRAK4 (FIG. 3C). Without being
bound by theory, these data might suggest that IRAK signaling could
act as a compensatory pathway for FLT3-dependent AML cells to
survive FLT3 inhibition and that the compounds of Formula (I) are
able to inhibit this response.
[0279] FIGS. 4A-4D--Methods: Inhibition of FLT3-ITD AML. (FIGS.
4A-4B) Heatmap response profile (left panel) and Delta Bliss
analysis (right panel) for AC220 and IRAK-Inh (Amgen) combination
treatment of MA9-FLT3-ITD cells. (FIG. 4A) Cell-titer glow (CTG)
percent response values represent normalized growth, relative to
controls based on SybrGreen fluorescence intensities. (FIG. 4B)
Caspase activation values, relative to controls based on
caspase-glo fluorescence intensities. (FIG. 4C) The IC.sub.10 of
AC220 was established in MA9-FLT3-ITD cells after 48 hour treatment
using cell-titer glow relative response values normalized to growth
compared to control cells (DMSO). (FIG. 4D) MA9-FLT3-ITD cells were
treated with IRAK-Inh (Amgen) alone or in combination with 0.3 nM
of AC220 (IC.sub.10) for 72 hours. Cell-titer glow relative
response values represent normalized growth compared to control
cells (DMSO). Further details of these methods can be found in
Mathews Griner et al., "High-throughput combinatorial screening
identifies drugs that cooperate with ibrutinib to kill activated
B-cell--like diffuse large B-cell lymphoma cells" (2014) PNAS, Vol.
111, No. 6, pp. 2349-2354.
[0280] FIGS. 4A-4D--Results & Discussion: Inhibition of
FLT3-ITD AML. To assess whether inhibition of FLT3 and IRAK4 can
suppress FLT3-ITD AML cells, we performed a drug matrix analysis. A
score of less than -1 suggests synergy between two compounds. The
analysis with AC220 and a commercially-available selective IRAK1/4
compound indicated that FLT3 and IRAK signaling inhibition
synergize to inhibit proliferation (-2.77 as determined by
cell-titer glow) and viability (-1.63 as determined by caspase 8
cleavage) of FLT3-ITD AML (FIGS. 4A-4B). To confirm the observed
synergy, we evaluated proliferation of MA9 FLT3-ITD cells in the
presence of AC220 (0.3 nM=IC50, FIG. 4C) and increasing
concentrations of the IRAK1/4 inhibitor (FIG. 4D). Consistent with
the matrix analysis, treatment of FLT3-ITD AML cells with a FLT3
inhibitor and IRAK1/4 inhibitor results in a synergistic inhibitory
effect. Without being bound by theory, synergism between FLT3 and
IRAK inhibition might suggest that simultaneous inhibition of these
pathways would be an effective therapeutic strategy for FLT3-ITD
AML.
[0281] FIGS. 5A-5G and 6--Methods: Some compounds of Formula (I)
can suppress FLT3-ITD AML. (FIG. 5A) Generation of two independent
clones (#3 and #6) derived from human cord blood CD34+ cells
transduced with MLL-AF9 and then either FLT3-ITD (MA9-FLT3-ITD) or
NRas (MA9-NRas). (FIGS. 5B-5G) MA9.3 or MA9.6 clones expressing
FLT3-ITD or NRas were treated with the indicated compounds for 72
hours. Cell-titer glow relative response values represent
normalized growth compared to control cells (DMSO) based on
luminescence intensities. Cellular IC50 values (nM) are shown for
each experiment. In FIG. 6, cell viability was determined in
MA9-FLT3-ITD cells treated with compound I-15 (1 .mu.M), compound
I-20 (1 .mu.M), or compound I-43 (1 .mu.M) for 72 hours by flow
cytometric analysis of AnnexinV. Further details of these methods
can be found in Mathews Griner et al., "High-throughput
combinatorial screening identifies drugs that cooperate with
ibrutinib to kill activated B-cell-like diffuse large B-cell
lymphoma cells" (2014) PNAS, Vol. 111, No. 6, pp. 2349-2354.
[0282] FIGS. 5A-5G and 6--Results & Discussion: Some compounds
of Formula (I) can suppress FLT3-ITD AML. To assess the selectivity
of the inhibitors to FLT3-ITD AML, we used FLT3-ITD-containing and
NRAS-containing AML cells created from isogenic human CD34+ MLL-AF9
parental cells (FIG. 5A). Analysis of proliferation via CellTiter
Glo revealed that FLT3-ITD AML cells are more sensitive to all of
the compounds tested as compared to NRAS AML cells (FIGS. 5B-5F).
Therefore, these compounds appear to be selective for FLT3-ITD
positive cells, rather than broadly cytotoxic, and are equivalently
effective against two independent clones of MLL-AF9 AML.
Additionally, the efficacy of compounds I-15, I-20, and I-43 at
inhibiting AML cell growth (FIG. 5G) and viability (FIG. 6)
correlated with the relative potency IRAK1/4 inhibition (FIG. 5G).
Without being bound by theory, the correlation between the degree
of IRAK1/4 inhibition and cell growth inhibition might suggest that
the inhibitors' activity against IRAK1/4 is a contributor to the
efficacy of the inhibitors.
[0283] FIGS. 7A-7E--Methods: (FIGS. 7A-7B) MA9-FLT3-ITD cells were
treated with the indicated compounds for 72 hours. Cell-titer glow
relative response values represent normalized growth compared to
control cells (DMSO) based on luminescence intensities. Cellular
IC50 values (nM) are shown for each experiment. (FIG. 7C) Cell
viability was determined in MA9-FLT3-ITD cells treated with 1 .mu.M
of the indicated compounds for 72 hours by flow cytometric analysis
of AnnexinV. (FIG. 7D) Leukemic colony formation of MA9-FLT3-ITD
cells was determined in methylcellulose supplemented with 1 .mu.M
of the indicated compounds. Colony formation was determined after
10 days. (FIG. 7E) Colony formation of normal cord blood CD34+
cells was determined in methylcellulose supplemented with 1 .mu.M
of the indicated compounds. Colony formation was determined after
10 days.
[0284] FIGS. 7A-7E-Results & Discussion: Compounds I-17, I-22,
and 1-24 had subnanomolar activity against FLT3-ITD cells. In
particular, compound I-24 exhibited increased potency at inhibiting
cell growth, at inducing apoptosis and at inhibiting leukemic
colony formation, as compared to compound I-20 and AC220 (FIGS.
7A-7D). Compound I-24 inhibited colony formation of normal CD34+
cells (FIG. 7E).
[0285] FIGS. 8A-8G--Methods: (FIG. 8A) Overview of experimental
design: MA9-FLT3-ITD cells were cultured in cytokines and then
treated with AC220 or compound I-20 (1, 2.5, or 5 .mu.M) for 72
hours. Cell viability was assessed by AnnexinV staining. The
remaining cells were washed and replated in fresh media with
cytokines. Recovery of MA9-FLT3-ITD cell growth was determined
after 7 days by AnnexinV staining or Trypan Blue exclusion. (FIG.
8B) Cell viability was determined in MA9-FLT3-ITD cells after 72
hours following treatment with the indicated compounds, or after 7
days of recovery. (FIG. 8C) Overview of experimental design:
MA9-FLT3-ITD cells were cultured in cytokines and then treated with
AC220, compound I-20, or compound I-24 (5 .mu.M) for 72 hours. Cell
viability was assessed by AnnexinV staining. The remaining cells
were washed and replated in fresh media with cytokines. Recovery of
MA9-FLT3-ITD cell growth was monitored every 2 days by AnnexinV
staining. (FIG. 8D) Cell viability was determined in MA9-FLT3-ITD
cells after 72 hours following treatment (Day 0) with the indicated
compounds, or every 2 dayspost recovery by AnnexinV staining.
Compound I-24-treated cells were not monitored past Day 2 as no
viable cells remained. (FIG. 8E) Overview of experimental design:
MA9-FLT3-ITD cells were cultured in cytokines and then treated with
AC220 or compound I-24 (5 04) for 72 hours. Cell viability was
assessed by AnnexinV staining. The remaining cells were washed and
replated in fresh media with cytokines. After AC220-treated cells
recovered (Day 7), they were subsequently treated with AC220 (5
.mu.M) or compound 1-24 (5 04) ("i") and monitored every 2 days by
AnnexinV staining. This step was repeated once more at Day 16
("ii"). (FIGS. 8F-G) Cell viability was determined in MA9-FLT3-ITD
cells after 72 hours following treatment (Day 0) with the indicated
compounds, or every 2 days post recovery by AnnexinV staining (FIG.
8F) or Trypan Blue exclusion (FIG. 8G).
[0286] FIGS. 8A-8G--Results & Discussion: Some compounds of
Formula (I) can prevent emergence of resistant FLT3-ITD AML. AML
relapse is a clinical problem following FLT3 inhibitor treatment.
Therefore, we assessed AML cells recovery and emergence of
resistant cells after AC220 treatment in vitro. To permit emergence
of resistant AML cells and better recapitulate the cytokine
conditions in vivo, we cultured MA9 FLT3-ITD cells in the presence
of the cytokines (IL-3, IL-6, Stem Cell Factor (SCF), FL, and
thrombopoietin (TPO) at 10 ng/mL). The AML cells were treated with
AC220 or compound I-20 at 1 .mu.M, 2.5 .mu.M, or 5 .mu.M for 72
hours. After initial treatment with the inhibitors, the cells were
washed and replated in fresh media containing cytokines (day 0).
Viability was assessed by AnnexinV staining following 7 days of
recovery (FIG. 8A). While AC220-treated cells showed increased
viability by 7 days, compound I-20-treated cells remained
apoptotic, particularly at the higher doses (FIG. 8B). To evaluate
the kinetics of this recovery, we evaluated viability on days 2 and
4 after washing (FIG. 8C). Viability of the AML cells with 5 .mu.M
AC220 treatment results in 15-20% viability (day 0). However after
removal of the compounds, the AC220-treated cells reached 50%
viability at day 2.7, while compound I-20-treated cells didn't
reach 50% viability until day 3.4 (FIG. 8D). Thus, compound I-20
treatment appeared to reduce AML cell emergence after treatment.
And compound I-24-treated cells did not recover. To determine
whether AC220-resistant FLT3-ITD AML cells remained sensitive to
compound I-24, we treated cells with AC220 (5 .mu.M) for 72 hours,
washed the cells, replated in fresh media, and monitored viability
and cell number as described above. Once the cells recovered on day
7, we treated them with either AC220 or compound I-24 (5 .mu.M) for
72 hours and then allowed them to recover for 7 days (FIGS. 8E-8F).
Upon the second exposure to AC220, the cells were less sensitive,
with viability increasing from 20% to 35% after the first exposure.
However, these cells remained sensitive to compound I-24 (FIGS.
8E-8F). This pattern was repeated upon a third exposure to
[0287] AC220, with viability after treatment rising to 55% while
resistance to compound I-24 after two rounds of AC220 treatment was
not observed. Sequencing of the TK domain of FLT3 in the cells that
had recovered from AC220-treated revealed no mutations in the TK
domain, indicating (without wishing to be bound by theory) that
AC220 resistance does not appear to be due to diminished binding to
FLT3-ITD.
[0288] FIGS. 9A-9C--Methods: (FIG. 9A) Overview of in vivo
experimental design: NRGS mice were injected i.v. with MA9-FLT3-ITD
cells (2.times.10.sup.5 cells/mouse). After 10 days, PBS or
compound I-24 (30 mg/kg) was injected i.p. for 5 daily treatments,
followed by a 2 day rest. After the 2nd treatment, one mouse from
each group was sacrificed and MA9-FLT3-ITD (GFP+) cells were
isolated from the bone marrow ("BM") by flow sorting for
immunoblotting of FLT3 and IRAK4. A second cycle of daily
injections of PBS or compound I-24 for 5 days, followed by daily
monitoring of morbidity. (FIG. 9B) Immunoblot analysis of sorted
(GFP+) MA9-FLT3-ITD BM cells from xenografted mice after 2 doses of
compound I-24. (FIG. 9C) Overall survival of NRGS mice xenografted
with MA9-FLT3-ITD treated with compound I-24 or PBS.
[0289] FIGS. 9A-9C--Results & Discussion: Some compounds of
Formula (I) can be effective against FLT3-ITD AML xenograft mouse
models. We next assessed the in vivo efficacy of compound I-24
using a human xenograft model of FLT3-ITD AML in NOD.Rag1-/-;
.gamma.cnull mice that express human IL-3, human
granulocyte/macrophage-stimulating factor (GM-CSF) and human stem
cell factor (SCF) (NRGS). NRGS mice are radioresistant and have
been shown to be a model for AML when engrafted with MA9 FLT3-ITD
cells. MLL-AF9 FLT3-ITD cells (2.times.105 cells per mouse) were
injected via tail vein into NRGS mice (n=12). The cells were
allowed to engraft for 10 days. The mice were then treated with PBS
(n=6) or compound I-24 (30 mg/kg) (n=6) once daily,
intraperitoneally (IP), on days 10-14 and 17-21 post-xenograft
(FIG. 9A). On day 12, total bone marrow was collected from the PBS
and compound I-24 group and GFP+cells were isolated by flow
cytometry. FLT3 and IRAK4 phosphorylation was evaluated by
immunoblotting (FIG. 9B). IP delivery of compound I-24 resulted in
reduction of FLT3 and IRAK phosphorylation. To determine the
efficacy of compound I-24, NRGS xenografted mice were continually
monitored for evidence of AML. As expected, the PBS-treated mice
developed AML, as evident by infiltration of leukemic cells into
the BM, spleen, and lungs, beginning at Day 38 (median survival of
40 days). In contrast, compound I-24 treatment extended median
survival to 49 days (p=0.004) (FIG. 9C).
[0290] FIGS. 10A-10B--Methods: (FIG. 10A) Colony formation of MDSL
cells was determined in methylcellulose supplemented with 1 .mu.M
or 10 .mu.M of the indicated compounds. Colony formation was
determined after 10 days. (FIG. 10B) MDSL cells were treated with
the indicated compounds for 72 hours. Cell-titer glow relative
response values represent normalized growth compared to control
cells (DMSO) based on luminescence intensities.
[0291] FIGS. 10A-10B--Results & Discussion: Some compounds of
Formula (I) can be effective against MDS cell function and
viability. IRAK1 and IRAK4 are hyperactivated in MDS patients. The
consequences of treating MDS cells with compounds of Formula (I)
were performed by measuring viability and function of a
patient-derived MDS cell line (MDSL), which exhibits activated
IRAK1 and IRAK4. The effect of the IRAK inhibitors on MDS
progenitor function was evaluated in methylcellulose. All four
compounds tested inhibited colony formation of MDSL cells, at 10
.mu.nM, all are more potent than the commercially-available IRAK1/4
inhibitor. Compound I-24 showed increased ability to inhibit MDSL
growth compared to IRAK-Inh. Therefore in addition to being an
effective therapeutic for FLT3-ITD AML, the compounds of Formula
(I) also indicate use in MDS.
[0292] The headings used in the disclosure are not meant to suggest
that all disclosure relating to the heading is found within the
section that starts with that heading. Disclosure for any subject
may be found throughout the specification.
[0293] It is noted that terms like "preferably," "commonly," and
"typically" are not used herein to limit the scope of the claimed
invention or to imply that certain features are critical,
essential, or even important to the structure or function of the
claimed invention. Rather, these terms are merely intended to
highlight alternative or additional features that may or may not be
utilized in a particular embodiment of the present invention.
[0294] As used in the disclosure, "a" or "an" means one or more
than one, unless otherwise specified. As used in the claims, when
used in conjunction with the word "comprising" the words "a" or
"an" means one or more than one, unless otherwise specified. As
used in the disclosure or claims, "another" means at least a second
or more, unless otherwise specified. As used in the disclosure, the
phrases "such as", "for example", and "e.g." mean "for example, but
not limited to" in that the list following the term ("such as",
"for example", or "e.g.") provides some examples but the list is
not necessarily a fully inclusive list. The word "comprising" means
that the items following the word "comprising" may include
additional unrecited elements or steps; that is, "comprising" does
not exclude additional unrecited steps or elements.
[0295] In certain instances, sequences disclosed herein are
included in publicly-available databases, such as GENBANK.RTM. and
SWISSPROT. Unless otherwise indicated or apparent the references to
such publicly-available databases are references to the most recent
version of the database as of the filing date of this
Application.
[0296] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as reaction conditions,
and so forth used in the specification and claims are to be
understood as being modified in all instances by the term "about".
Accordingly, unless indicated to the contrary, the numerical
parameters set forth in this specification and claims are
approximations that can vary depending upon the desired properties
sought to be obtained by the presently-disclosed subject
matter.
[0297] As used herein, the term "about," when referring to a value
or to an amount of mass, weight, time, volume, concentration or
percentage is meant to encompass variations of in some embodiments
.+-.20%, in some embodiments .+-.10%, in some embodiments .+-.5%,
in some embodiments .+-.1%, in some embodiments .+-.0.5%, and in
some embodiments .+-.0.1% from the specified amount, as such
variations are appropriate to perform the disclosed method.
[0298] Detailed descriptions of one or more embodiments are
provided herein. It is to be understood, however, that the present
invention may be embodied in various forms. Therefore, specific
details disclosed herein (even if designated as preferred or
advantageous) are not to be interpreted as limiting, but rather are
to be used as an illustrative basis for the claims and as a
representative basis for teaching one skilled in the art to employ
the present invention in any appropriate manner. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
* * * * *