U.S. patent application number 15/973110 was filed with the patent office on 2018-09-13 for indolinone compounds and uses thereof.
The applicant listed for this patent is Oncternal Therapeutics, Inc.. Invention is credited to Jean-Michel Vernier.
Application Number | 20180256546 15/973110 |
Document ID | / |
Family ID | 54337916 |
Filed Date | 2018-09-13 |
United States Patent
Application |
20180256546 |
Kind Code |
A1 |
Vernier; Jean-Michel |
September 13, 2018 |
INDOLINONE COMPOUNDS AND USES THEREOF
Abstract
Indolinone derivative compounds that act as EWS-FLI1
transcription factor inhibitors are provided. Also provided are
pharmaceutical compositions of the indolinone derivatives, methods
of synthesizing the same, methods of treating using same, and
assays for identifying the inhibitors of EWS-FLI1 oncoprotein.
Inventors: |
Vernier; Jean-Michel; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Oncternal Therapeutics, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
54337916 |
Appl. No.: |
15/973110 |
Filed: |
May 7, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15357876 |
Nov 21, 2016 |
9987251 |
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15973110 |
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14877708 |
Oct 7, 2015 |
9604927 |
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15357876 |
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62062086 |
Oct 9, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/5377 20130101;
A61P 35/00 20180101; C07D 401/10 20130101; C07D 209/34 20130101;
A61K 31/4155 20130101; A61P 21/00 20180101; A61P 35/02 20180101;
A61K 31/404 20130101; C07D 403/10 20130101; A61P 15/00 20180101;
A61P 19/00 20180101; A61P 17/00 20180101; A61K 31/4439 20130101;
A61P 7/00 20180101; C07D 401/06 20130101; C07D 209/38 20130101;
A61P 11/00 20180101; A61P 25/00 20180101; A61P 43/00 20180101 |
International
Class: |
A61K 31/404 20060101
A61K031/404; C07D 401/06 20060101 C07D401/06; C07D 401/10 20060101
C07D401/10; C07D 403/10 20060101 C07D403/10; C07D 209/34 20060101
C07D209/34; C07D 209/38 20060101 C07D209/38; A61K 31/4155 20060101
A61K031/4155; A61K 31/4439 20060101 A61K031/4439; A61K 31/5377
20060101 A61K031/5377 |
Claims
1. A compound having a structure of Formula (I): ##STR00088## or a
stereoisomer, a pharmaceutically acceptable salt, or solvate
thereof, wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently selected from the group consisting of H, Cl, --CN and
--CF.sub.3; wherein A is selected from the group consisting of H
and C.sub.1-6 alkyl; wherein D is selected from the group
consisting of --OH and --O(C.sub.1-6 alkyl); wherein R.sub.5 and
R.sub.6 are independently selected from the group consisting of H,
F, and C.sub.1-6 alkyl, or wherein R.sub.5 and R.sub.6 taken
together form a substituted or unsubstituted cycloalkyl ring;
wherein R.sub.12 is independently selected from the group
consisting of C.sub.3-8 cycloalkyl and ##STR00089## wherein
R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are independently
selected from the group consisting of H, halogen, CN, CF.sub.3,
C.sub.1-6 alkyl, aryl, heteroaryl, --O(aryl), --O(heteroaryl),
--CO.sub.2H, --CO.sub.2(C.sub.1-6 alkyl), --NHSO.sub.2(C.sub.1-6
alkyl), --NHSO.sub.2(aryl), --NHCONH(C.sub.1-6 alkyl),
--NHCON(C.sub.1-6 alkyl).sub.2, --N(C.sub.1-6 alkyl)CONH.sub.2,
--N(C.sub.1-6 alkyl)CONH(C.sub.1-6 alkyl), --N(C.sub.1-6
alkyl)CON(C.sub.1-6 alkyl).sub.2, --SO.sub.2(C.sub.1-6 alkyl),
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-6 alkyl),
--SO.sub.2N(C.sub.1-6 alkyl).sub.2, C.sub.3-8 cycloalkyl, and
C.sub.3-8 heterocyclo alkyl.
2. The compound of claim 1, wherein R.sub.9 is selected from the
group consisting of aziridinyl, azetidinyl, pyrrolidinyl, and
morpholinolyl.
3. The compound of claim 1, wherein R.sub.9 is selected from the
group consisting of isopropyl and cyclopropyl.
4. The compound of claim 1, having a structure of Formula (Ia):
##STR00090## or a stereoisomer, a pharmaceutically acceptable salt,
or solvate thereof, wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4
are independently selected from the group consisting of H and Cl;
wherein R.sub.7, R.sub.8, R.sub.10 and R.sub.11 are independently
selected from the group consisting of H and halogen; and wherein
R.sub.9 is independently selected from the group consisting
C.sub.3-8 cycloalkyl and C.sub.3-8 heterocycloalkyl.
5. The compound of claim 1, wherein R.sub.1 and R.sub.4 are Cl and
R.sub.2 and R.sub.3 are H.
6. The compound of claim 1, selected from the group consisting of:
##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095## or
a stereoisomer, a pharmaceutically acceptable salt, ester, or
solvate thereof.
7. The compound of claim 6, selected from the group consisting of:
##STR00096## ##STR00097## or a stereoisomer, a pharmaceutically
acceptable salt, ester, or solvate thereof.
8. The compound of claim 1, selected from the group consisting of:
##STR00098## or a stereoisomer, a pharmaceutically acceptable salt,
ester, or solvate thereof.
9. The compound of claim 1, selected from the group consisting of:
##STR00099## or a stereoisomer, a pharmaceutically acceptable salt,
ester, or solvate thereof.
10. The compound of claim 1, having the structure: ##STR00100## or
a stereoisomer, a pharmaceutically acceptable salt, ester, or
solvate thereof.
11. The compound of claim 1, having the structure: ##STR00101## or
a stereoisomer, a pharmaceutically acceptable salt, ester, or
solvate thereof.
12. A pharmaceutical composition comprising the compound of claim 1
and a pharmaceutically acceptable carrier.
13. A method for inhibiting proliferation of a cell, wherein the
cell overexpresses an ETS gene or comprises an ETS fusion gene,
comprising contacting the cell with an effective amount of the
compound of claim 1.
14. The method of claim 13, wherein the ETS gene or the ETS fusion
gene is selected from the group consisting of FLI1, ERG, ETV1, and
ETV4.
15. The method of claim 13, wherein the cell is a cancer cell,
wherein the cancer is selected from the group consisting of Ewing's
sarcoma, prostate cancer, glioblastoma, acute myeloid leukemia,
breast cancer, head cancer, neck cancer, melanoma, non-small cell
lung cancer, ovarian cancer, and uterine cancer.
16. The method of claim 13, wherein the cell is mammalian.
17. A method of killing or inhibiting the growth of a neoplastic
cell, comprising contacting the cell with an effective amount of
the compound of claim 1.
18. The method of claim 17, wherein the cell is a cancer cell,
wherein the cancer is selected from the group consisting of Ewing's
sarcoma, prostate cancer, glioblastoma, acute myeloid leukemia,
breast cancer, head cancer, neck cancer, melanoma, non-small cell
lung cancer, ovarian cancer, and uterine cancer.
19. The method of claim 17, wherein the cell is mammalian.
Description
INCORPORATION BY REFERENCE TO RELATED APPLICATIONS
[0001] Any and all priority claims identified in the Application
Data Sheet, or any correction thereto, are hereby incorporated by
reference under 37 CFR 1.57. This application is a continuation of
U.S. application Ser. No. 15/357,876, filed Nov. 21, 2016, which is
a continuation of U.S. application Ser. No. 14/877,708, filed on
Oct. 7, 2015, now U.S. Pat. No. 9,604,927, which claims the benefit
of U.S. Provisional Application No. 62/062,086, filed Oct. 9, 2014.
Each of the aforementioned applications is incorporated by
reference herein in its entirety, and each is hereby expressly made
a part of this specification.
BACKGROUND
Field
[0002] Indolinone derivative compounds that act as EWS-FLI1
transcription factor inhibitors are provided. Also provided are
pharmaceutical compositions of the indolinone derivatives, methods
of synthesizing the same, methods of treating using same, and
assays for identifying the inhibitors of EWS-FLI1 oncoprotein.
DESCRIPTION
[0003] The EWS-FLI transcription factor present in vast variety of
Ewing's sarcoma family of tumors (ESFT) was characterized over ten
years ago. Progress in the treatment of Ewing's sarcoma the second
most common bone tumor in children and adolescents, has improved
survival for patients with localized tumors. However, patients with
metastases still fare badly and the therapy carries short and
long-term toxicities. The Ewing sarcoma family of tumors (ESFT) is
characterized by a chromosomal translocation that generates
EWS-FLI1, on oncogenic fusion transcription factor whose continued
expression is believed to be critical for ESFT cell survival
(Balamuth, N J, Womer, R B., Lancet Oncology 11, 184-192
(2010)).
[0004] In vitro and in vivo studies have demonstrated that the
inhibition of the binding of the oncoprotein, EWS-FLI1, to RNA
Helicase A (RHA) leads to a decrease in proliferation of ESFT cell
lines and a decrease of tumor volume. EWS-FLI1 lacks enzymatic
activity, however, the protein-protein interaction between RNA
helicase A (RHA) and EWS-FLI1-modulates oncogenesis, and is
therefore required for the maintenance of the tumor growth (Hyariye
N Erkizan et al. Nature Medicine 15(7) 750-756 (2009)). The
paradigm of disrupting key protein interactions may have utility in
treatment of diseases including sarcomas with similar
translocations, and leukemias with MLL translocations ((Helman L J,
Meltzer P. Mechanisms of sarcoma development. Nat Rev Cancer 2003;
3(9):685-94); and Pui C H, et al., N Engl J Med 2004;
350(15):1535-48). Moreover, disordered proteins may be excellent
therapeutic targets based on their intrinsic biochemical properties
(Cheng Y, LeGall T, Oldfield C J, et al., Trends Biotechnol 2006;
24(10):435-42).
SUMMARY
[0005] Despite years of in vitro and xenograft studies with
antisense and siRNA directed towards EWS-FLI1, none of these is
heretofore practical as a human therapy based on inadequate
delivery and stability. Accordingly, there is a need for improved
therapies to treat disorders such as ESFTs.
[0006] FLI-1 is a member of the ETS family transcription factors
which are normally active in the developing embryo, but not after
birth. There are 29 members of this family of transcription
factors, four of which, FLI-1, ETV1, ETV4 and ERG, have been
associated with a wide range of cancers.
[0007] Therapeutic compounds targeting the inhibition of the
binding of oncogenic fusion proteins of FLI1, ETV1, ETV4 or ERG or
the transcription factors themselves will have utility in treatment
of cancers including the Ewing's sarcoma family of tumors,
pancreatic cancer, prostate cancer, glioblastoma, non-small cell
lung cancer, and several other cancers. The preferred embodiments
fulfill these needs, and provide other advantages as well.
[0008] Some embodiments disclosed herein relate to a compound of
Formula (I) including forms such as stereoisomers, free forms,
pharmaceutically acceptable salts or esters thereof, solvates, or
combinations of such forms, wherein A, D, R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.12 are as defined
herein.
##STR00001##
[0009] Some embodiments disclosed herein relate to methods for
treating cancer in a mammal, comprising administering to the mammal
an effective amount of one or more compounds of Formula (I)
including forms such as stereoisomers, free forms, or a
pharmaceutically acceptable salt thereof, or a pharmaceutical
composition that includes one or more compounds of Formula (I)
including forms such as stereoisomers, free forms, or a
pharmaceutically acceptable salt thereof. Other embodiments
described herein relate to using one or more compounds of Formula
(I) including forms such as stereoisomers, free forms, or a
pharmaceutically acceptable salt thereof, in the manufacture of a
medicament for treatment of cancer.
[0010] Still other embodiments described herein relate to a
compound of Formula (I) including forms such as stereoisomers, free
forms, or a pharmaceutically acceptable salt thereof, for treatment
of cancer wherein the cancer is selected from the group consisting
of Ewing's sarcoma, glioblastoma, acute myeloid leukemia, breast
cancer, head & neck cancer, melanoma, non-small cell lung
cancer, ovarian cancer, prostate cancer, and uterine cancer. These
and other embodiments are described in greater detail below.
DETAILED DESCRIPTION
[0011] The following description and examples illustrate a
preferred embodiment of the present invention in detail. Those of
skill in the art will recognize that there are numerous variations
and modifications of this invention that are encompassed by its
scope. Accordingly, the description of a preferred embodiment
should not be deemed to limit the scope of the present
invention
[0012] Chromosomal translocations generating oncogenic
transcription factors are the hallmark of a variety of tumors,
including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are
characterized by the t(11;22)(q24;q12) translocation that generates
the Ewing sarcoma breakpoint region 1 and Friend leukemia virus
integration 1 (EWS-FLI1) fusion transcription factor responsible
for the highly malignant phenotype of this tumor. Continued
expression of EWS-FLI1 is believed to be critical for ESFT cell
survival. EWS-FLI1 is an attractive treatment target for Ewing
sarcoma because of its malignant cell specificity. Furthermore,
experimental evidence indicates that EWS/FLI expression is
essential for Ewing sarcoma tumor cells. In vitro targeting of
EWS-FLI1 with antisense oligodeoxynucleotides and RNA interference
(RNAi) inhibits Ewing sarcoma cell viability, growth, and oncogenic
transformation, supporting EWS-FLI1 attenuation as a potential
treatment modality. The therapeutic agents of the preferred
embodiments have broad applicability to a larger group of tumors,
and are useful as therapeutics for treatment for other oncogenic
transcription factor related malignancies such as
chemotherapy-resistant sarcomas and leukemias and difficult to
treat tumors such as Ewing's sarcoma.
Definitions
[0013] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art. All patents, applications, published
applications and other publications referenced herein are
incorporated by reference in their entirety unless stated
otherwise. In the event that there is a plurality of definitions
for a term herein, those in this section prevail unless stated
otherwise.
[0014] As used herein, any "R" group(s) such as, without
limitation, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11, and R.sub.12
represent substituents that can be attached to the indicated atom.
An R group may be substituted or unsubstituted. If two "R" groups
are described as being "taken together" the R groups and the atoms
they are attached to can form a cycloalkyl, cycloalkenyl, aryl,
heteroaryl or heterocycle. For example, without limitation, if
R.sup.a and R.sup.b of an NR.sup.aR.sup.b group are indicated to be
"taken together," it means that they are covalently bonded to one
another to form a ring:
##STR00002##
In addition, if two "R" groups are described as being "taken
together" with the atom(s) to which they are attached to form a
ring as an alternative, the R groups may not be limited to the
variables or substituents defined previously.
[0015] As used herein, "alkyl" refers to a straight or branched
hydrocarbon chain that comprises a fully saturated (no double or
triple bonds) hydrocarbon group. The alkyl group may have 1 to 20
carbon atoms (whenever it appears herein, a numerical range such as
"1 to 20" refers to each integer in the given range; e.g., "1 to 20
carbon atoms" means that the alkyl group may consist of 1 carbon
atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20
carbon atoms, although the present definition also covers the
occurrence of the term "alkyl" where no numerical range is
designated). The alkyl group may also be a medium size alkyl having
1 to 10 carbon atoms. The alkyl group could also be a lower alkyl
having 1 to 6 carbon atoms. The alkyl group of the compounds may be
designated as "C.sub.1-C.sub.6 alkyl" or similar designations. By
way of example only, "C.sub.1-C.sub.6 alkyl" indicates that there
are one to six carbon atoms in the alkyl chain, i.e., the alkyl
chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl,
iso-butyl, sec-butyl, and t-butyl, pentyl (straight and branched)
and hexyl (straight and branched). Typical alkyl groups include,
but are in no way limited to, methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, tertiary butyl, pentyl (straight and branched) and
hexyl (straight and branched). The alkyl group may be substituted
or unsubstituted.
[0016] As used herein, "cycloalkyl" refers to a completely
saturated (no double or triple bonds) mono- or multi-cyclic
hydrocarbon ring system. When composed of two or more rings, the
rings may be joined together in a fused fashion. Cycloalkyl groups
can contain 3 to 10 atoms in the ring(s) or 3 to 8 atoms in the
ring(s). A cycloalkyl group may be unsubstituted or substituted.
Typical cycloalkyl groups include, but are in no way limited to,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and
cyclooctyl.
[0017] As used herein, "aryl" refers to a carbocyclic (all carbon)
mono-cyclic or multi-cyclic aromatic ring system (including fused
ring systems where two carbocyclic rings share a chemical bond)
that has a fully delocalized pi-electron system throughout all the
rings. The number of carbon atoms in an aryl group can vary. For
example, the aryl group can be a C.sub.6-C.sub.14 aryl group, a
C.sub.6-C.sub.10 aryl group, or a C.sub.6 aryl group. Examples of
aryl groups include, but are not limited to, benzene, naphthalene
and azulene. An aryl group may be substituted or unsubstituted.
[0018] As used herein, "heteroaryl" refers to a mono-cyclic or
multi-cyclic aromatic ring system (a ring system with fully
delocalized pi-electron system) that contain(s) one or more
heteroatoms (for example, 1 to 5 heteroatoms), that is, an element
other than carbon, including but not limited to, nitrogen, oxygen
and sulfur. The number of atoms in the ring(s) of a heteroaryl
group can vary. For example, the heteroaryl group can contain 4 to
14 atoms in the ring(s), 5 to 10 atoms in the ring(s) or 5 to 6
atoms in the ring(s). Furthermore, the term "heteroaryl" includes
fused ring systems where two rings, such as at least one aryl ring
and at least one heteroaryl ring, or at least two heteroaryl rings,
share at least one chemical bond. Examples of heteroaryl rings
include, but are not limited to, furan, furazan, thiophene,
benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole,
1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole,
1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole,
indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole,
isothiazole, triazole, benzotriazole, thiadiazole, tetrazole,
pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine,
quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline and
triazine. A heteroaryl group may be substituted or
unsubstituted.
[0019] As used herein, heterocycloalkyl refers to three-, four-,
five-, six-, seven-, eight-, nine-, ten-, up to 18-membered
mono-cyclic, bicyclic, and tricyclic ring system wherein carbon
atoms together with from 1 to 5 heteroatoms constitute said ring
system. A heterocycle may optionally contain one or more
unsaturated bonds situated in such a way, however, that a fully
delocalized pi-electron system does not occur throughout all the
rings. The heteroatom(s) is an element other than carbon including,
but not limited to, oxygen, sulfur, and nitrogen. A heterocycle may
further contain one or more carbonyl or thiocarbonyl
functionalities, so as to make the definition include oxo-systems
and thio-systems such as lactams, lactones, cyclic imides, cyclic
thioimides and cyclic carbamates. When composed of two or more
rings, the rings may be joined together in a fused fashion.
Additionally, any nitrogens in a heterocycloalky may be
quaternized. Heterocycloalkyl groups may be unsubstituted or
substituted. Examples of such heterocycloalkyl groups include but
are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane,
1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane,
1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiole, 1,3-dithiolane,
1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide,
succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine,
hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine,
imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline,
oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine,
oxirane, piperidine N-Oxide, piperidine, piperazine, pyrrolidine,
pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine,
2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran,
thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone,
and their benzo-fused analogs (e.g., benzimidazolidinone,
tetrahydroquinoline and 3,4-methylenedioxyphenyl).
[0020] The term "pharmaceutically acceptable salt" refers to a salt
of a compound that does not cause significant irritation to an
organism to which it is administered and does not abrogate the
biological activity and properties of the compound. In some
embodiments, the salt is an acid addition salt of the compound.
Pharmaceutical salts can be obtained by reacting a compound with
inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or
hydrobromic acid), sulfuric acid, nitric acid and phosphoric acid.
Pharmaceutical salts can also be obtained by reacting a compound
with an organic acid such as aliphatic or aromatic carboxylic or
sulfonic acids, for example formic, acetic, succinic, lactic,
malic, tartaric, citric, ascorbic, nicotinic, methane sulfonic,
ethane sulfonic, p-toluensulfonic, salicylic or naphthalenesulfonic
acid. Pharmaceutical salts can also be obtained by reacting a
compound with a base to form a salt such as an ammonium salt, an
alkali metal salt, such as a sodium or a potassium salt, an
alkaline earth metal salt, such as a calcium or a magnesium salt, a
salt of organic bases such as dicyclohexylamine,
N-methyl-D-glucamine, tris(hydroxymethyl)methylamine,
C.sub.1-C.sub.7 alkylamine, cyclohexylamine, triethanolamine,
ethylenediamine, and salts with amino acids such as arginine and
lysine.
[0021] It is understood that, in any compound described herein
having one or more chiral centers, if an absolute stereochemistry
is not expressly indicated, then each center may independently be
of R-configuration or S-configuration or a mixture thereof. Thus,
the compounds provided herein may be enantiomerically pure,
enantiomerically enriched, racemic mixture, diastereomerically
pure, diastereomerically enriched, or a stereoisomeric mixture. In
addition it is understood that, in any compound described herein
having one or more double bond(s) generating geometrical isomers
that can be defined as E or Z, each double bond may independently
be E or Z a mixture thereof.
[0022] It is to be understood that where compounds disclosed herein
have unfilled valencies, then the valencies are to be filled with
hydrogens or isotopes thereof, e.g., hydrogen-1 (protium) and
hydrogen-2 (deuterium).
[0023] It is understood that the compounds described herein can be
labeled isotopically. Substitution with isotopes such as deuterium
may afford certain therapeutic advantages resulting from greater
metabolic stability, such as, for example, increased in vivo
half-life or reduced dosage requirements. Each chemical element as
represented in a compound structure may include any isotope of said
element. For example, in a compound structure a hydrogen atom may
be explicitly disclosed or understood to be present in the
compound. At any position of the compound that a hydrogen atom may
be present, the hydrogen atom can be any isotope of hydrogen,
including but not limited to hydrogen-1 (protium) and hydrogen-2
(deuterium). Thus, reference herein to a compound encompasses all
potential isotopic forms unless the context clearly dictates
otherwise.
[0024] It is understood that the methods and combinations described
herein include crystalline forms (also known as polymorphs, which
include the different crystal packing arrangements of the same
elemental composition of a compound), amorphous phases, salts,
solvates, and hydrates. In some embodiments, the compounds
described herein exist in solvated forms with pharmaceutically
acceptable solvents such as water, ethanol, or the like. In other
embodiments, the compounds described herein exist in unsolvated
form. Solvates contain either stoichiometric or non-stoichiometric
amounts of a solvent, and may be formed during the process of
crystallization with pharmaceutically acceptable solvents such as
water, ethanol, or the like. Hydrates are formed when the solvent
is water, or alcoholates are formed when the solvent is alcohol. In
addition, the compounds provided herein can exist in unsolvated as
well as solvated forms. In general, the solvated forms are
considered equivalent to the unsolvated forms for the purposes of
the compounds and methods provided herein.
[0025] Where a range of values is provided, it is understood that
the upper and lower limit, and each intervening value between the
upper and lower limit of the range is encompassed within the
embodiments.
Compounds
[0026] In a first aspect a compound is provided having Formula
(I):
##STR00003##
[0027] or a stereoisomer, a pharmaceutically acceptable salt, or
solvate thereof, wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently selected from the group consisting of H, Cl, --CN and
--CF.sub.3; wherein A is selected from the group consisting of H
and C.sub.1-6 alkyl; wherein D is selected from the group
consisting of --OH and --O(C.sub.1-6 alkyl); wherein R.sub.5 and
R.sub.6 are independently selected from the group consisting of H,
F, and C.sub.1-6 alkyl, or wherein R.sub.5 and R.sub.6 taken
together form a substituted or unsubstituted cycloalkyl ring;
wherein R.sub.11 is independently selected from the group
consisting of C.sub.3-8 cycloalkyl and
##STR00004##
wherein R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are
independently selected from the group consisting of H, halogen, CN,
CF.sub.3, C.sub.1-6 alkyl, aryl, heteroaryl, --O(aryl),
--O(heteroaryl), --CO.sub.2H, --CO.sub.2(C.sub.1-6 alkyl),
--NHSO.sub.2(C.sub.1-6 alkyl), --NHSO.sub.2(aryl),
--NHCONH(C.sub.1-6 alkyl), --NHCON(C.sub.1-6 alkyl).sub.2,
--N(C.sub.1-6 alkyl)CONH.sub.2, --N(C.sub.1-6 alkyl)CONH(C.sub.1-6
alkyl), --N(C.sub.1-6 alkyl)CON(C.sub.1-6 alkyl).sub.2,
--SO.sub.2(C.sub.1-6 alkyl), --SO.sub.2NH.sub.2,
--SO.sub.2NH(C.sub.1-6 alkyl), --SO.sub.2N(C.sub.1-6 alkyl).sub.2,
C.sub.3-8 cycloalkyl, and C.sub.3-8 heterocycloalkyl.
[0028] In an embodiment of the first aspect, R.sub.1, R.sub.2,
R.sub.3, and R.sub.4 are independently selected from the group
consisting of hydrogen and Cl.
[0029] In an embodiment of the first aspect, R.sub.5 and R.sub.6
taken together form a substituted or unsubstituted cycloalkyl
ring.
[0030] In an embodiment of the first aspect, A is H.
[0031] In an embodiment of the first aspect, D is OH.
[0032] In an embodiment of the first aspect, A is H and D is
OH.
[0033] In an embodiment of the first aspect, R.sub.9 is selected
from the group consisting of aziridinyl, azetidinyl, pyrrolidinyl,
and morpholinyl.
[0034] In an embodiment of the first aspect, R.sub.9 is selected
from the group consisting of isopropyl and cyclopropyl.
[0035] In an embodiment of the first aspect, a compound having a
structure of Formula (Ia):
##STR00005##
[0036] or a stereoisomer, a pharmaceutically acceptable salt, or
solvate thereof, wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently selected from the group consisting of H and Cl;
wherein R.sub.7, R.sub.8, R.sub.10 and R.sub.11 are independently
selected from the group consisting of H and halogen; and wherein
R.sub.9 is independently selected from the group consisting
C.sub.3-8 cycloalkyl and C.sub.3-8 heterocycloalkyl.
[0037] In an embodiment of the first aspect, R.sub.1 and R.sub.4
are C.sub.1 and R.sub.2 and R.sub.3 are H.
[0038] In an embodiment of the first aspect, the compound of
Formula (I) is selected from the group consisting of:
##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010##
[0039] or a stereoisomer, or a pharmaceutically acceptable salt,
ester, or solvate thereof.
[0040] In an embodiment of the first aspect, the compound selected
from the group consisting of:
##STR00011##
[0041] or a stereoisomer, a pharmaceutically acceptable salt,
ester, or solvate thereof.
[0042] In a second aspect, a pharmaceutical composition comprising
the compound of any embodiment of the first aspect or any
embodiment thereof and a pharmaceutically acceptable carrier is
provided.
[0043] In a third aspect, a pharmaceutical composition comprising
the compound of any embodiment of the first aspect or any
embodiment thereof and a pharmaceutically acceptable excipient is
provided.
[0044] In a fourth aspect, a pharmaceutical composition comprising
the compound of any embodiment of the first aspect or any
embodiment thereof and at least one additional pharmaceutically
active agent.
[0045] In a fifth aspect, a method for treating cancer is provided
comprising administering an effective amount of the compound of the
first aspect or any embodiment thereof to a subject in need
thereof.
[0046] In an embodiment of the fifth aspect, the subject is
mammalian.
[0047] In an embodiment of the fifth aspect, the subject is
human.
[0048] In an embodiment of the fifth aspect, the cancer is selected
from the group consisting of Ewing's sarcoma, prostate cancer,
glioblastoma, acute myeloid leukemia, breast cancer, head &
neck cancer, melanoma, non-small cell lung cancer, ovarian cancer,
and uterine cancer.
[0049] In a sixth aspect, a method of killing or inhibiting the
growth of a neoplastic cell is provided, comprising contacting the
cell with an effective amount of the compound of the first aspect
or any embodiment thereof.
[0050] In an embodiment of the sixth aspect, the cell is
mammalian.
[0051] In an embodiment of the sixth aspect, the cell is human.
[0052] In an embodiment of the sixth aspect, the cell is in
vitro.
[0053] In an embodiment of the sixth aspect, the cell is in
vivo.
[0054] In an embodiment of the sixth aspect, the cell is a cancer
cell, the cancer being selected from the group consisting of
Ewing's sarcoma, prostate cancer, glioblastoma, acute myeloid
leukemia, breast cancer, head & neck cancer, melanoma,
non-small cell lung cancer, ovarian cancer, and uterine cancer.
[0055] In a seventh aspect, a method for inhibiting proliferation
of a cell, wherein the cell overexpresses an ETS gene or comprises
an ETS fusion gene, comprising contacting the cell with an
effective amount of the compound of the first aspect or any
embodiment thereof.
[0056] In an embodiment of the seventh aspect, the ETS gene or the
ETS fusion gene is selected from the group consisting of FLI1, ERG,
ETV1, and ETV4.
[0057] In an embodiment of the seventh aspect, the cell is
mammalian.
[0058] In an embodiment of the seventh aspect, the cell is
human.
[0059] In an embodiment of the seventh aspect, the cell is in
vitro.
[0060] In an embodiment of the seventh aspect, the cell is in
vivo.
[0061] In an embodiment of the seventh aspect, the cell is a cancer
cell, the cancer being selected from the group consisting of
Ewing's sarcoma, prostate cancer, glioblastoma, acute myeloid
leukemia, breast cancer, head & neck cancer, melanoma,
non-small cell lung cancer, ovarian cancer, and uterine cancer.
Synthetic Methods
##STR00012##
[0063] Compounds of Formula (I) described herein may be prepared in
various ways. General synthetic routes to compounds of Formula (I)
are shown and described herein. The routes shown and described
herein are illustrative only and are not intended, nor are they to
be construed, to limit the scope of the claims in any manner
whatsoever. Those skilled in the art will be able to recognize
modifications of the disclosed syntheses and to devise alternate
routes based on the disclosures herein; all such modifications and
alternate routes are within the scope of the claims.
[0064] Depending upon the substituents present, the small molecule
inhibitors can be in a form of a pharmaceutically acceptable salt.
The terms "pharmaceutically acceptable salt" as used herein are
broad terms, and is to be given its ordinary and customary meaning
to a person of ordinary skill in the art (and is not to be limited
to a special or customized meaning), and refers without limitation
to salts prepared from pharmaceutically acceptable, non-toxic acids
or bases. Suitable pharmaceutically acceptable salts include
metallic salts, e.g., salts of aluminum, zinc, alkali metal salts
such as lithium, sodium, and potassium salts, alkaline earth metal
salts such as calcium and magnesium salts; organic salts, e.g.,
salts of lysine, N,N'-dibenzylethylenediamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, meglumine
(N-methylglucamine), procaine, and tris; salts of free acids and
bases; inorganic salts, e.g., sulfate, hydrochloride, and
hydrobromide; and other salts which are currently in widespread
pharmaceutical use and are listed in sources well known to those of
skill in the art, such as, for example, The Merck Index. Any
suitable constituent can be selected to make a salt of the
therapeutic agents discussed herein, provided that it is non-toxic
and does not substantially interfere with the desired activity.
[0065] The compounds of preferred embodiments can include isomers,
racemates, optical isomers, enantiomers, diastereomers, tautomers,
and cis/trans conformers. All such isomeric forms are included
within preferred embodiments, including mixtures thereof. As
discussed above, the compounds of preferred embodiments may have
chiral centers, for example, they may contain asymmetric carbon
atoms and may thus exist in the form of enantiomers or
diastereoisomers and mixtures thereof, e.g., racemates. Asymmetric
carbon atom(s) can be present in the (R)- or (S)-configuration, or
can be present as mixtures of the (R)- and (S)-forms. The following
are isomeric forms of the compounds of Formula (I):
##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017##
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023##
[0066] The compounds can be in amorphous form, or in crystalline
forms. The crystalline forms of the compounds of preferred
embodiments can exist as polymorphs, which are included in
preferred embodiments. In addition, some of the compounds of
preferred embodiments may also form solvates with water or other
organic solvents. Such solvates are similarly included within the
scope of the preferred embodiments.
Certain Pharmaceutical Compositions
[0067] It is generally preferred to administer the inhibitors of
preferred embodiments in an intravenous or subcutaneous unit dosage
form; however, other routes of administration are also
contemplated. Contemplated routes of administration include but are
not limited to oral, parenteral, intravenous, and subcutaneous. The
inhibitors of preferred embodiments can be formulated into liquid
preparations for, e.g., oral administration. Suitable forms include
suspensions, syrups, elixirs, and the like. Particularly preferred
unit dosage forms for oral administration include tablets and
capsules. Unit dosage forms configured for administration once a
day are particularly preferred; however, in certain embodiments it
can be desirable to configure the unit dosage form for
administration twice a day, or more.
[0068] The pharmaceutical compositions of preferred embodiments are
preferably isotonic with the blood or other body fluid of the
recipient. The isotonicity of the compositions can be attained
using sodium tartrate, propylene glycol or other inorganic or
organic solutes. Sodium chloride is particularly preferred.
Buffering agents can be employed, such as acetic acid and salts,
citric acid and salts, boric acid and salts, and phosphoric acid
and salts. Parenteral vehicles include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's
or fixed oils. Intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like.
[0069] Viscosity of the pharmaceutical compositions can be
maintained at the selected level using a pharmaceutically
acceptable thickening agent. Methylcellulose is preferred because
it is readily and economically available and is easy to work with.
Other suitable thickening agents include, for example, xanthan gum,
carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the
like. The preferred concentration of the thickener will depend upon
the thickening agent selected. An amount is preferably used that
will achieve the selected viscosity. Viscous compositions are
normally prepared from solutions by the addition of such thickening
agents.
[0070] A pharmaceutically acceptable preservative can be employed
to increase the shelf life of the pharmaceutical compositions.
Benzyl alcohol can be suitable, although a variety of preservatives
including, for example, parabens, thimerosal, chlorobutanol, or
benzalkonium chloride can also be employed. A suitable
concentration of the preservative is typically from about 0.02% to
about 2% based on the total weight of the composition, although
larger or smaller amounts can be desirable depending upon the agent
selected. Reducing agents, as described above, can be
advantageously used to maintain good shelf life of the
formulation.
[0071] The inhibitors of preferred embodiments can be in admixture
with a suitable carrier, diluent, or excipient such as sterile
water, physiological saline, glucose, or the like, and can contain
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, gelling or viscosity enhancing additives,
preservatives, flavoring agents, colors, and the like, depending
upon the route of administration and the preparation desired. See,
e.g., "Remington: The Science and Practice of Pharmacy", Lippincott
Williams & Wilkins; 20th edition (Jun. 1, 2003) and
"Remington's Pharmaceutical Sciences," Mack Pub. Co.; 18.sup.th and
19.sup.th editions (December 1985, and June 1990, respectively).
Such preparations can include complexing agents, metal ions,
polymeric compounds such as polyacetic acid, polyglycolic acid,
hydrogels, dextran, and the like, liposomes, microemulsions,
micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts
or spheroblasts. Suitable lipids for liposomal formulation include,
without limitation, monoglycerides, diglycerides, sulfatides,
lysolecithin, phospholipids, saponin, bile acids, and the like. The
presence of such additional components can influence the physical
state, solubility, stability, rate of in vivo release, and rate of
in vivo clearance, and are thus chosen according to the intended
application, such that the characteristics of the carrier are
tailored to the selected route of administration.
[0072] For oral administration, the pharmaceutical compositions can
be provided as a tablet, aqueous or oil suspension, dispersible
powder or granule, emulsion, hard or soft capsule, syrup or elixir.
Compositions intended for oral use can be prepared according to any
method known in the art for the manufacture of pharmaceutical
compositions and can include one or more of the following agents:
sweeteners, flavoring agents, coloring agents and preservatives.
Aqueous suspensions can contain the active ingredient in admixture
with excipients suitable for the manufacture of aqueous
suspensions.
[0073] Formulations for oral use can also be provided as hard
gelatin capsules, wherein the active ingredient(s) are mixed with
an inert solid diluent, such as calcium carbonate, calcium
phosphate, or kaolin, or as soft gelatin capsules. In soft
capsules, the inhibitors can be dissolved or suspended in suitable
liquids, such as water or an oil medium, such as peanut oil, olive
oil, fatty oils, liquid paraffin, or liquid polyethylene glycols.
Stabilizers and microspheres formulated for oral administration can
also be used. Capsules can include push-fit capsules made of
gelatin, as well as soft, sealed capsules made of gelatin and a
plasticizer, such as glycerol or sorbitol. The push-fit capsules
can contain the active ingredient in admixture with fillers such as
lactose, binders such as starches, and/or lubricants such as talc
or magnesium stearate and, optionally, stabilizers.
[0074] Tablets can be uncoated or coated by known methods to delay
disintegration and absorption in the gastrointestinal tract and
thereby provide a sustained action over a longer period of time.
For example, a time delay material such as glyceryl monostearate
can be used. When administered in solid form, such as tablet form,
the solid form typically comprises from about 0.001 wt. % or less
to about 50 wt. % or more of active ingredient(s), preferably from
about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 wt. % to about 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 wt. %.
[0075] Tablets can contain the active ingredients in admixture with
non-toxic pharmaceutically acceptable excipients including inert
materials. For example, a tablet can be prepared by compression or
molding, optionally, with one or more additional ingredients.
Compressed tablets can be prepared by compressing in a suitable
machine the active ingredients in a free-flowing form such as
powder or granules, optionally mixed with a binder, lubricant,
inert diluent, surface active or dispersing agent. Molded tablets
can be made by molding, in a suitable machine, a mixture of the
powdered inhibitor moistened with an inert liquid diluent.
[0076] Preferably, each tablet or capsule contains from about 1 mg
or less to about 1,000 mg or more of an inhibitor of the preferred
embodiments, more preferably from about 10, 20, 30, 40, 50, 60, 70,
80, 90, or 100 mg to about 150, 200, 250, 300, 350, 400, 450, 500,
550, 600, 650, 700, 750, 800, or 900 mg. Most preferably, tablets
or capsules are provided in a range of dosages to permit divided
dosages to be administered. A dosage appropriate to the patient and
the number of doses to be administered daily can thus be
conveniently selected. In certain embodiments it can be preferred
to incorporate two or more of the therapeutic agents to be
administered into a single tablet or other dosage form (e.g., in a
combination therapy); however, in other embodiments it can be
preferred to provide the therapeutic agents in separate dosage
forms.
[0077] Suitable inert materials include diluents, such as
carbohydrates, mannitol, lactose, anhydrous lactose, cellulose,
sucrose, modified dextrans, starch, and the like, or inorganic
salts such as calcium triphosphate, calcium phosphate, sodium
phosphate, calcium carbonate, sodium carbonate, magnesium
carbonate, and sodium chloride. Disintegrants or granulating agents
can be included in the formulation, for example, starches such as
corn starch, alginic acid, sodium starch glycolate, Amberlite,
sodium carboxymethylcellulose, ultramylopectin, sodium alginate,
gelatin, orange peel, acid carboxymethyl cellulose, natural sponge
and bentonite, insoluble cationic exchange resins, powdered gums
such as agar, karaya or tragacanth, or alginic acid or salts
thereof.
[0078] Binders can be used to form a hard tablet. Binders include
materials from natural products such as acacia, tragacanth, starch
and gelatin, methyl cellulose, ethyl cellulose, carboxymethyl
cellulose, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose,
and the like.
[0079] Lubricants, such as stearic acid or magnesium or calcium
salts thereof, polytetrafluoroethylene, liquid paraffin, vegetable
oils and waxes, sodium lauryl sulfate, magnesium lauryl sulfate,
polyethylene glycol, starch, talc, pyrogenic silica, hydrated
silicoaluminate, and the like, can be included in tablet
formulations.
[0080] Surfactants can also be employed, for example, anionic
detergents such as sodium lauryl sulfate, dioctyl sodium
sulfosuccinate and dioctyl sodium sulfonate, cationic such as
benzalkonium chloride or benzethonium chloride, or nonionic
detergents such as polyoxyethylene hydrogenated castor oil,
glycerol monostearate, polysorbates, sucrose fatty acid ester,
methyl cellulose, or carboxymethyl cellulose.
[0081] Controlled release formulations can be employed wherein the
amifostine or analog(s) thereof is incorporated into an inert
matrix that permits release by either diffusion or leaching
mechanisms. Slowly degenerating matrices can also be incorporated
into the formulation. Other delivery systems can include timed
release, delayed release, or sustained release delivery
systems.
[0082] Coatings can be used, for example, nonenteric materials such
as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose,
methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,
providone and the polyethylene glycols, or enteric materials such
as phthalic acid esters. Dyestuffs or pigments can be added for
identification or to characterize different combinations of
inhibitor doses
[0083] When administered orally in liquid form, a liquid carrier
such as water, petroleum, oils of animal or plant origin such as
peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic
oils can be added to the active ingredient(s). Physiological saline
solution, dextrose, or other saccharide solution, or glycols such
as ethylene glycol, propylene glycol, or polyethylene glycol are
also suitable liquid carriers. The pharmaceutical compositions can
also be in the form of oil-in-water emulsions. The oily phase can
be a vegetable oil, such as olive or arachis oil, a mineral oil
such as liquid paraffin, or a mixture thereof. Suitable emulsifying
agents include naturally-occurring gums such as gum acacia and gum
tragacanth, naturally occurring phosphatides, such as soybean
lecithin, esters or partial esters derived from fatty acids and
hexitol anhydrides, such as sorbitan mono-oleate, and condensation
products of these partial esters with ethylene oxide, such as
polyoxyethylene sorbitan mono-oleate. The emulsions can also
contain sweetening and flavoring agents.
[0084] Pulmonary delivery can also be employed. The compound is
delivered to the lungs while inhaling and traverses across the lung
epithelial lining to the blood stream. A wide range of mechanical
devices designed for pulmonary delivery of therapeutic products can
be employed, including but not limited to nebulizers, metered dose
inhalers, and powder inhalers, all of which are familiar to those
skilled in the art. These devices employ formulations suitable for
the dispensing of compound. Typically, each formulation is specific
to the type of device employed and can involve the use of an
appropriate propellant material, in addition to diluents,
adjuvants, and/or carriers useful in therapy.
[0085] The compound and/or other optional active ingredients are
advantageously prepared for pulmonary delivery in particulate form
with an average particle size of from 0.1 .mu.m or less to 10 .mu.m
or more, more preferably from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, or 0.9 .mu.m to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5,
5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 .mu.m.
Pharmaceutically acceptable carriers for pulmonary delivery of
inhibitor include carbohydrates such as trehalose, mannitol,
xylitol, sucrose, lactose, and sorbitol. Other ingredients for use
in formulations can include DPPC, DOPE, DSPC, and DOPC. Natural or
synthetic surfactants can be used, including polyethylene glycol
and dextrans, such as cyclodextran. Bile salts and other related
enhancers, as well as cellulose and cellulose derivatives, and
amino acids can also be used. Liposomes, microcapsules,
microspheres, inclusion complexes, and other types of carriers can
also be employed.
[0086] Pharmaceutical formulations suitable for use with a
nebulizer, either jet or ultrasonic, typically comprise the
inhibitor dissolved or suspended in water at a concentration of
about 0.01 or less to 100 mg or more of inhibitor per mL of
solution, preferably from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, or 90 mg per mL of solution. The formulation can also
include a buffer and a simple sugar (e.g., for protein
stabilization and regulation of osmotic pressure). The nebulizer
formulation can also contain a surfactant, to reduce or prevent
surface induced aggregation of the inhibitor caused by atomization
of the solution in forming the aerosol.
[0087] Formulations for use with a metered-dose inhaler device
generally comprise a finely divided powder containing the active
ingredients suspended in a propellant with the aid of a surfactant.
The propellant can include conventional propellants, such as
chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons,
and hydrocarbons. Preferred propellants include
trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, 1,1,1,2-tetrafluoroethane, and
combinations thereof. Suitable surfactants include sorbitan
trioleate, soya lecithin, and oleic acid.
[0088] Formulations for dispensing from a powder inhaler device
typically comprise a finely divided dry powder containing
inhibitor, optionally including a bulking agent, such as lactose,
sorbitol, sucrose, mannitol, trehalose, or xylitol in an amount
that facilitates dispersal of the powder from the device, typically
from about 1 wt. % or less to 99 wt. % or more of the formulation,
preferably from about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt.
% to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. % of the
formulation.
[0089] When a compound of the preferred embodiments is administered
by intravenous, parenteral, or other injection, it is preferably in
the form of a pyrogen-free, parenterally acceptable aqueous
solution or oleaginous suspension. Suspensions can be formulated
according to methods well known in the art using suitable
dispersing or wetting agents and suspending agents. The preparation
of acceptable aqueous solutions with suitable pH, isotonicity,
stability, and the like, is within the skill in the art. A
preferred pharmaceutical composition for injection preferably
contains an isotonic vehicle such as 1,3-butanediol, water,
isotonic sodium chloride solution, Ringer's solution, dextrose
solution, dextrose and sodium chloride solution, lactated Ringer's
solution, or other vehicles as are known in the art. In addition,
sterile fixed oils can be employed conventionally as a solvent or
suspending medium. For this purpose, any bland fixed oil can be
employed including synthetic mono or diglycerides. In addition,
fatty acids such as oleic acid can likewise be used in the
formation of injectable preparations. The pharmaceutical
compositions can also contain stabilizers, preservatives, buffers,
antioxidants, or other additives known to those of skill in the
art.
[0090] The duration of the injection can be adjusted depending upon
various factors, and can comprise a single injection administered
over the course of a few seconds or less, to 0.5, 0.1, 0.25, 0.5,
0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, or 24 hours or more of continuous
intravenous administration.
[0091] The compounds of the preferred embodiments can additionally
employ adjunct components conventionally found in pharmaceutical
compositions in their art-established fashion and at their
art-established levels. Thus, for example, the compositions can
contain additional compatible pharmaceutically active materials for
combination therapy (such as supplementary antimicrobials,
antipruritics, astringents, local anesthetics, anti-inflammatory
agents, reducing agents, chemotherapeutics and the like), or can
contain materials useful in physically formulating various dosage
forms of the preferred embodiments, such as excipients, dyes,
thickening agents, stabilizers, preservatives or antioxidants.
Anti-cancer agents that can be used in combination with the
compounds of preferred embodiments include, but are not limited to,
vinca alkaloids such as vinblastine and vincristine; anthracyclines
such as doxorubicin, daunorubicin, epirubicin; anthracenes such as
bisantrene and mitoxantrone; epipodophyllo-toxins such as etoposide
and teniposide; and other anticancer drugs such as actinomyocin D,
mithomycin C, mitramycin, methotrexate, docetaxel, etoposide
(VP-16), paclitaxel, docetaxel, and adriamycin); and
immunosuppressants (e.g., cyclosporine A, tacrolimus). In some
embodiments, the compounds, compositions and methods provided
herein may be in combination with histone deacetylase inhibitors
(HDAC), aurora kinase inhibitors, demethylating agents (such as
5-AZA cytidine), immunotherapy with natural killer cells, IGF-IR
antibodies, Ewing antigen antibodies, immunosuppressive drugs, and
hydroxyurea. Examples of histone deacetylase inhibitors include
vorinostat, romidepsin, panobinostat, valproic acid, belinostat,
mocetinostat, givinostat, and trichostatin A. Examples of aurora
kinase inhibitors include ZM447439, hesperadin, and VX-680.
Examples of demethylating agents include 5-azacytidine,
5-azadeoxycytidine, and procaine. Examples of immunosuppressive
drugs include 6-mercaptopurine, and azathioprine.
Certain Kits
[0092] The compounds of the preferred embodiments can be provided
to an administering physician or other health care professional in
the form of a kit. The kit is a package which houses a container
which contains the compounds in a suitable pharmaceutical
composition, and instructions for administering the pharmaceutical
composition to a subject. The kit can optionally also contain one
or more additional therapeutic agents, e.g., chemotherapeutics
currently employed for treating the sarcomas described herein. For
example, a kit containing one or more compositions comprising
compounds of the preferred embodiments in combination with one or
more additional chemotherapeutic agents can be provided, or
separate pharmaceutical compositions containing an inhibitor of the
preferred embodiments and additional therapeutic agents can be
provided. The kit can also contain separate doses of a compound of
the preferred embodiments for serial or sequential administration.
The kit can optionally contain one or more diagnostic tools and
instructions for use. The kit can contain suitable delivery
devices, e.g., syringes, and the like, along with instructions for
administering the inhibitor(s) and any other therapeutic agent. The
kit can optionally contain instructions for storage, reconstitution
(if applicable), and administration of any or all therapeutic
agents included. The kits can include a plurality of containers
reflecting the number of administrations to be given to a
subject.
Methods of Use
[0093] Some embodiments provided herein relate to methods of
treating the Ewing's sarcoma family of tumors (ESFT). ESFT contains
the unique fusion protein EWS-FLI1. ESFT affects patients between
the ages of 3 and 40 years, with most cases occurring in the second
decade. Although the embryologic cell type from which ESFT are
derived is unknown, the tumor often grows in close proximity to
bone, but can occur as a soft-tissue mass. Over 40% of patients who
present with localized tumors will develop recurrent disease and
the majority of these will die from ESFT, while 75-80% of patients
who present with metastatic ESFT will die within 5 years despite
high-dose chemotherapy (Grier H E, Krailo M D, Tarbell N J, et al.
Addition of ifosfamide and etoposide to standard chemotherapy for
Ewing's sarcoma and primitive neuroectodermal tumor of bone. N Engl
J Med 2003; 348(8):694-701). These survival rates have not improved
for the past 20 years, even after dose-intensifying chemotherapy.
To improve survival and reduce therapy-related morbidity, novel
targeted strategies for treating ESFT patients, as provided in the
preferred embodiments, can be employed.
[0094] ESFT are characterized by a translocation, occurring in 95%
of tumors, between the central exons of the EWS gene (Ewing
Sarcoma) located on chromosome 22 to the central exons of an ets
family gene; either FLI1 (Friend Leukemia Insertion) located on
chromosome 11, t(11;22), or ERG located on chromosome 21, t(21;22).
The EWS-FLI1 fusion transcript encodes a 55 kDa protein
(electrophoretic motility of approximately 68 kD) with two primary
domains. The EWS domain is a potent transcriptional activator,
while the FLI1 domain contains a highly conserved ets DNA binding
domain (May W A, Lessnick S L, Braun B S, et al. The Ewing's
sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional
activator and is a more powerful transforming gene than FLI-1. Mol
Cell Biol 1993; 13(12):7393-8); the resulting EWS-FLI1 fusion
protein acts as an aberrant transcription factor. EWS-FLI1
transformation of mouse fibroblasts requires both the EWS and FLI1
functional domains to be intact (May W A, Gishizky M L, Lessnick S
L, et al. Ewing sarcoma 11;22 translocation produces a chimeric
transcription factor that requires the DNA-binding domain encoded
by FLI1 for transformation. Proc Natl Acad Sci USA 1993;
90(12):5752-6).
[0095] EWS-FLI1 is an outstanding therapeutic target, in that it is
expressed only in tumor cells and is required to maintain the
growth of ESFT cell lines. Reduced expression levels of EWS-FLI1
using either antisense oligodeoxynucleotides (ODN) (Toretsky J A,
Connell Y, Neckers L, Bhat N K. Inhibition of EWS-FLI-1 fusion
protein with antisense oligodeoxynucleotides. J Neurooncol 1997;
31(1-2):9-16; Tanaka K, Iwakuma T, Harimaya K, Sato H, Iwamoto Y.
EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of
human Ewing's sarcoma and primitive neuroectodermal tumor cells. J
Clin Invest 1997; 99(2):239-47) or small interfering RNAs (siRNA)
(Ouchida M, Ohno T, Fujimura Y, Rao V N, Reddy E S. Loss of
tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to
EWS-fusion transcripts. Oncogene 1995; 11(6):1049-54; Maksimenko A,
Malvy C, Lambert G, et al. Oligonucleotides targeted against a
junction oncogene are made efficient by nanotechnologies. Pharm Res
2003; 20(10):1565-7; Kovar H, Aryee D N, Jug G, et al. EWS/FLI-1
antagonists induce growth inhibition of Ewing tumor cells in vitro.
Cell Growth Differ 1996; 7(4):429-37) cause decreased proliferation
of ESFT cell lines and regression of tumors in nude mice. Recent
advances in nanotechnology have improved the delivery and
controlled release of siRNA, yet neither antisense ODN nor siRNA
reduction of EWS-FLI1 in humans is possible with current
technologies (Maksimenko A, Malvy C, Lambert G, et al.
Oligonucleotides targeted against a junction oncogene are made
efficient by nanotechnologies. Pharm Res 2003; 20(10):1565-7;
Lambert G, Bertrand J R, Fattal E, et al. EWS FLI-1 antisense
nanocapsules inhibits Ewing sarcoma-related tumor in mice. Biochem
Biophys Res Commun 2000; 279(2):401-6). One interesting approach to
EWS-FLI1 targeting used comparative expression between siRNA
reduced EWS-FLI1 and a library of small molecules, which led to a
clinical trial with Ara-C(Stegmaier K, Wong J S, Ross K N, et al.
Signature-based small molecule screening identifies cytosine
arabinoside as an EWS/FLI modulator in Ewing sarcoma. PLoS medicine
2007; 4(4):e122). This method of identifying Ara-C also indicated
doxorubicin and puromycin would reduce EWS-FLI1 levels. Doxorubicin
is currently used as standard therapy for ESFT patients and yet,
survival is far from acceptable (Grier H E, Krailo M D, Tarbell N
J, et al. Addition of ifosfamide and etoposide to standard
chemotherapy for Ewing's sarcoma and primitive neuroectodermal
tumor of bone. N Engl J Med 2003; 348(8):694-701). The use of Ara-C
in ESFT patients is currently being evaluated in a Phase II trial.
While it is hoped that this represents a needed clinical
breakthrough, it certainly demonstrates the importance of small
molecule targeting of EWS-FLI1. The preferred embodiments provide
small molecule protein-protein interaction inhibitors (SMPPII) that
disrupt EWS-FLI1 from critical protein partners, thereby achieving
tumor specificity and more precise targeting of EWS-FLI1.
[0096] EWS-FLI1 is a great therapeutic target since it is only
expressed in tumor cells; however, the ability to target this
tumor-specific oncogene has previously not been successful. One of
the challenges towards small molecule development is that EWS-FLI1
lacks any known enzymatic domains, and enzyme domains have been
thought to be critical for targeted therapeutics. In addition,
EWS-FLI1 is a disordered protein, indicating that it does not
exhibit a rigid structure that can be used for structure based drug
design (Uren A, Tcherkasskaya O, Toretsky J A. Recombinant EWS-FLI1
oncoprotein activates transcription. Biochemistry 2004;
43(42):13579-89). In fact, the disordered nature of EWS-FLI1 is
critical for its transcriptional regulation (Ng K P, Potikyan G,
Savene R O, Denny C T, Uversky V N, Lee K A. Multiple aromatic side
chains within a disordered structure are critical for transcription
and transforming activity of EWS family oncoproteins. Proc Natl
Acad Sci USA 2007; 104(2):479-84). Disordered proteins are
considered as more attractive targets for small molecule
protein-protein interaction inhibitors specifically because of
their biochemical disordered properties (Cheng Y, LeGall T,
Oldfield C J, et al. Rational drug design via intrinsically
disordered protein. Trends Biotechnol 2006; 24(10):435-42)
[0097] EWS-FLI1 binds RNA helicase A in vitro and in vivo. It is
believed that protein-protein interactions of EWS-FLI1 may
contribute to its oncogenic potential; therefore, novel proteins
have been sought that directly interact with and functionally
modulate EWS-FLI1. Recombinant EWS-FLI1 that is transcriptionally
active (Uren A, Tcherkasskaya O, Toretsky J A. Recombinant EWS-FLI1
oncoprotein activates transcription. Biochemistry 2004;
43(42):13579-89) was used as a target for screening a commercial
peptide phage display library. Twenty-eight novel peptides that
differentially bind to EWS-FLI1 were identified from phage
sequencing. A National Center for Biotechnology Information
database search for human proteins homologous to these peptides
identified a peptide that was homologous to aa 823-832 of the human
RNA helicase A, (RHA, gene bank accession number A47363) (Toretsky
J A, Erkizan V, Levenson A, et al. Oncoprotein EWS-FLI1 activity is
enhanced by RNA helicase A. Cancer Res 2006; 66(11):5574-81).
[0098] While EWS-FLI1 is quite specific to ESFT cells, EWS and RHA
are ubiquitously expressed. The region between EWS-FLI1 and RHA are
targeted by molecular therapeutics that may have specificity; since
EWS-FLI1 is expressed only in tumors and the interaction points
with RHA may be unique. Therapeutic agents, namely, small molecule
protein-protein interaction inhibitors, are provided herein to
inhibit EWS-FLI1 function.
[0099] Most translocation-fusion protein sarcomas portend a poor
prognosis, including ESFT. The chromosomal translocation t(11;22),
leading to the unique and critical fusion protein EWS-FLI1, is a
perfect cancer target. Many other sarcomas share similar
translocation variants (Table 2. from Helman L J, Meltzer P.
Mechanisms of sarcoma development. Nat Rev Cancer 2003;
3(9):685-94).
[0100] EWS-FLI1 translocations have been reported in solid
pseudopapillaryneoplasms of the pancreas (Maitra A., et al.,
Detection of t(11;22)(q24;q12) translocation and EWS-FLI-1 fusion
transcript in a case of solid pseudopapillary tumor of the
pancreas. Pediatr Dev Pathol 2000; 3:603-605), however the role of
EWS-FLI1 in all solid pseudopapillary neoplasms remains to be
resolved (Katharina Tiemann et al., Solid pseudopapillary neoplasms
of the pancreas are associated with FLI-1 expression, but not with
EWS/FLI-1 translocation).
[0101] EWS or FLI1 homologues are partners in translocations that
occur in a wide range of sarcomas and leukemias. EWS, or its
homologue TLS or FUS, is involved in chromosomal translocations of
clear cell sarcoma, myxoid liposarcoma, desmoplastic small round
cell tumor, chondrosarcoma and acute myeloid leukemia. FLI1 belongs
to the ets family of genes. The FM homologue ERG is translocated in
approximately 10% of Ewing's sarcomas and 20% of acute myeloid
leukemias. This suggests that EWS-FLI1 can serve as model system
that might impact upon a family of diseases (related by
translocation partners) that affect a large number of patients
(Uren A., Tcherkasskaya O. and Toretsky J. A. Recombinant EWS-FLI1
oncoprotein activates transcription. Biochemistry 43(42) 13579-89
(2004)).
[0102] ERG is also translocated in prostate cancer, where the
TMPRSS2:ERG fusion suggests a distinct molecular subtype that may
define risk for disease progression (F. Demichelis et al.,
TMPRSS2:ERG gene fusion associated with lethal cancer in a watchful
waiting cohort. Oncogene (2007)26, 4596-4599). Other diseases where
translocations of EWS or FLI1 family members have been observed
include prostate cancer, glioblastoma, acute myeloid leukemia,
breast cancer, head & neck cancer, melanoma, non-small cell
lung cancer, ovarian cancer, and uterine cancer (Janknecht, Ralf;
Shin, Sook, and Oh, Sangphil, ETV1, 4 and 5: An Oncogenic Subfamily
of ETS Transcription Factors. Biochim. Biophys. Acta 1826 (1), 1-12
(2012)).
[0103] Therefore, the therapeutic agents of the preferred
embodiments have potential for application in many other tumors.
More broadly, some of the most difficult leukemias also have
translocation-generated fusion proteins involving the mixed-lineage
leukemia gene (MLL,11q23), and our work could serve as a paradigm
for a very treatment-resistant group of cancers (Pui C H, Chessells
J M, Camitta B, et al. Clinical heterogeneity in childhood acute
lymphoblastic leukemia with 11q23 rearrangements. Leukemia 2003;
17(4):700-6.). Thus embodiments include cancers where
translocations have occurred. Translocation fusion genes are listed
in TABLE 1.
TABLE-US-00001 TABLE 1 Ewing's sarcoma Translocation Genes Type of
fusion gene t(11; 22)(q24; q12) EWSR1-FLI1 Transcription factor
t(21; 22)(q22; q12) EWSR1-ERG Transcription factor t(7; 22)(p22;
q12) EWSR1-ETV1 Transcription factor t(17; 22)(q21; q12) EWSR1-ETV4
Transcription factor t(2; 22)(q33; q12) EWSR1-FEV Transcription
factor
[0104] A number of disorders include overexpression of an ETS gene,
or an ETS gene fusion, that is, a gene translocation that includes
an ETS gene. Examples of such ETS genes include FLI1, ERG, ETV1,
and ETV4. Examples of fusion genes include EWS-FLI, TMPRSS2-ERG.
TABLE 1A lists several cancers in which one or more ETS gene family
members are overexpressed, and/or are rearranged.
TABLE-US-00002 TABLE 1A Tumors with ETS overexpression or ETS
member Cancer gene fusion FLI1 ERG ETV1 ETV4 Prostate 41% 2% 25%
10% 6% Melanoma 34% 8% 8% 20% 5% Non-small-cell lung 33% 12% 8% 12%
5% carcinoma Uterine 25% 6% 9% 11% 6% Head and Neck 24% 6% 4% 7% 9%
Ovarian 21% 7% 3% 10% 3% Glioblastoma 19% 7% 4% 7% 4% multiforme
Acute myeloid 19% 8% 8% 4% 2% leukemia Breast 18% 5% 4% 5% 7%
Indications
[0105] Certain compounds, compositions and methods provided herein
can be used to treat a number of disorders such as a tumor or tumor
cell comprising a translocation gene fusion, such as those listed
in TABLE 1, Ewing's sarcoma, prostate cancer, glioblastoma, acute
myeloid leukemia, breast cancer, head & neck cancer, melanoma,
non-small cell lung cancer, ovarian cancer, and uterine cancer.
Some embodiments of the methods provided herein include a method
for inhibiting proliferation of a cell. In some embodiments, the
cell overexpresses an ETS gene. In some embodiments, the
overexpressed ETS gene can include FLI1, ERG, ETV1, or ETV4. In
some embodiments, the cell comprises an ETS fusion gene. In some
embodiments, the ETS fusion gene can include an ETS gene such as
FLI1, ERG, ETV1, and ETV4.
EXAMPLES
Preparation of Compounds of Formula (I)
##STR00024##
[0107] Compounds of Formula (I) were prepared according to the
synthetic schemes presented herein. A ketone of Formula (II) (4.0
equiv.) was condensed with an isatin derivative of Formula (III)
(1.0 equiv.) in the presence of diethylamine (10 drops) in methanol
(5 mL) and the mixture was stirred at room temperature for 24
hours. The reaction mixture was concentrated and purified using
flash chromatography using dichloromethane/methanol as eluent to
yield the pure product. Further purification was done by
recrystallization with methanol. The substituents on the ketone and
on the isatin derivative were selected so as to yield compounds of
Formula (I) labeled as EXAMPLES 1-23 below. NMR Spectra were
recorded for the compounds of EXAMPLES 1-26 thus obtained using a
Varian-400 spectrometer for 1H (400 MHz). Chemical shifts are given
in ppm downfield from tetramethylsilane as internal standard, and
coupling constants (J-values) are in hertz (Hz).
[0108] The chiral separation was done by dissolving the isomeric
mixtures in methanol/methylene chloride (4/1) mixture or neat
methanol and the separation was performed by Supercritical Fluid
Chromatography (SFC) using a chiralpak IA column (250 mm.times.4.6
mm; particle size 5 .mu.m) and eluted using a mixture of
methanol/CO.sub.2 (50/50). The solvent was removed under vacuum to
obtain the pure enantiomers.
[0109] In some embodiments, compounds can be prepared according to
the following synthesis schemes.
##STR00025##
[0110] In these schemes, ketone (4.0 equiv.) and a catalytic amount
of diethylamine (10 drops) are added to a solution of substituted
isatin (1.0 equiv.) in methanol (5 mL). The mixture is stirred at
room temperature until starting material (substituted isatin)
disappears completely. The resulting solution is concentrated and
applied to flash chromatography eluting with hexane/ethyl acetate
to afford pure product in quantitative yield. Further purification
is done by recrystallization with hexane/ethyl acetate.
[0111] An example compound synthesized by the above scheme
includes:
4,7-Dichloro-3-hydroxy-3-[2-(4-methoxyphenyl-2-oxoethyl)]-1,3-dihydroindo-
l-2-one: white solid; mp 149-151.degree. C.; .sup.1H NMR (DMSO, 400
MH 1 z) .delta. 10.93 (s, 1H), 7.86 (d, 2H, J=9.2 Hz), 7.26 (d, 1H,
J=8.8 Hz), 6.98 (d, 2H, J=8.8 Hz), 6.86 (d, 1H, J=8.4 Hz), 6.39 (s,
1H), 4.31 (d, 1H, J=18.0 Hz), 3.80 (s, 3H), 3.61 (d, 1H, J=18.0
Hz).
##STR00026##
[0112] In some embodiments, compounds can be prepared according to
the following synthesis scheme.
##STR00027##
[0113] In such embodiments, an appropriate acetophenone and
4,7-dichloroisatin can be condensed in the presence of a catalytic
amount of diethylamine to prepare the desired compound in
quantitative yield. An example synthesis includes the
following:
##STR00028##
[0114] 4,7-Dichloroisotin (30.05 g, 139.1 mmol, 1.0 equiv, Alfa
Aesar lot #10173559) and MeOH (450 mL, 15 vol) were charged to a
2-L, three-neck, round-bottom flask equipped with nitrogen line,
overhead mechanical stirrer, and a temperature probe. Diethylamine
(3.25 g, 0.32 equiv, Sigma-Aldrich lot # SHBD5313V) was added over
3 min (the slurry becomes dark red). A very slight increase in
temperature (from 17.5.degree. C. to 18.8.degree. C.) was observed.
1-[4-(Dimethylamino)phenyl]ethanone 2 (44.3 g, 1.95 equiv, ArkPharm
lot #0000197-130717000) was then added via a plastic funnel and the
funnel was rinsed with MeOH (75 mL, 2.5 vol). A decrease in the
reaction temperature to 15.1.degree. C. was observed. Upon stirring
for a few minutes, a dark red solution with a few undissolved
particles was obtained. The solution was stirred at ambient
temperature and periodically sampled for in-process control (IPC)
by HPLC. After 23 h of reaction, additional diethylamine was added
via syringe (1.42 g, 0.14 equiv) and the stirring continued at
ambient temperature. After 40.5 h, a light slurry formed. Solid 2
was added in portions (54.1 g, 2.38 equiv, ArkPharm lot
#0000197-130717000 and 3.2 g, 0.14 equiv, TCI lot # GK01-BRAH) for
a total of 4.47 equiv of acetophenone 2. After 88 h of reaction,
IPC by HPLC showed less than 1% AUC of isatin 1 present in the
reaction mixture. A heavy precipitate had formed. After 4.5 days,
the reaction mixture was concentrated under reduced pressure (water
bath <40.degree. C.), then under high vacuum to afford
approximately 84 g of a solid mixture, lot # BIO-W-22-11. The solid
was dissolved in a mixture of dichloromethane (385 mL) and MeOH
(140 mL) and adsorbed over 100 g of silica gel. The solvent was
removed under reduced pressure and the dry product/silica mixture
was loaded onto a column containing silica gel (1 kg, pre-packed
with heptanes) for a flash chromatographic purification. Elution
was started with 10% ethyl acetate in heptanes and a gradient up to
100% ethyl acetate was applied, and then switched to 10% methanol
in ethyl acetate. Fractions of 500 mL and up to 2 L were collected.
The product containing fractions, where product had started to
precipitate, were combined and concentrated down to approximately 1
L. The resulting precipitate was filtered out, reslurried in
EtOAc/MeOH (75:25 ratio, 200 mL), filtered, and washed with MeOH to
afford a first crop of compound. The first filtrate was
concentrated to a low volume, added MeOH to precipitate a second
crop of compound. Filtrates from isolation of both crops were
combined, concentrated to a low volume, taken up in 25 mL of MeOH,
and the resulting solid was filtered to afford a third crop of
compound. All three crops were dried under high vacuum at ambient
temperature for a day and at 40.degree. C. for four days. The total
combined weight was 40.03 g, corresponding to 76% yield of compound
(uncorrected by purity or solvent content). Solid is off-white
(with a very pale yellow to peach shade.
[0115] Another example synthesis includes the following:
##STR00029##
[0116] 4,7-Dichloroisotin (4.26 g, 19.7 mmol, 1 equiv, Alfa Aesar
lot #10173559) and MeOH (70 mL, 16.4 vol) were charged to a 250-mL,
three-neck, round-bottom flask equipped with nitrogen line,
overhead mechanical stirrer, and a temperature probe. Diethylamine
(0.43 g, 0.30 equiv, Sigma-Aldrich lot # SHBD5313V) was added over
1 min via syringe (the slurry becomes dark red).
1-[4-(Methylamino)phenyl]ethanone 3 (11.4 g, 3.9 equiv,
Sigma-Aldrich lot #01129HHV) was added in portions via a plastic
funnel, over 15 min. The funnel was rinsed with MeOH (2.times.15
mL, 7.0 vol). The reaction was stirred at ambient temperature
(approximately 18-20.degree. C.) and periodically sampled for
in-process control (IPC) by HPLC. After 40 h of reaction,
additional diethylamine was charged to the reaction via syringe
(0.16 g, 0.11 equiv) and the stirring continued at ambient
temperature. After 64 h, a light slurry formed. Additional
diethylamine was charged to the reaction via syringe (0.13 g, 0.09
equiv) and the stirring continued at ambient temperature. After 92
h of reaction IPC by HPLC analysis showed 2.1% AUC of isatin 1
present in the reaction mixture. Additional diethylamine was
charged to the reaction via syringe (0.07 g, 0.05 equiv) for a
total of 0.55 equiv of base and the stirring continued at ambient
temperature over the weekend. After a total of seven days, the
reaction was concentrated under reduced pressure (water bath
<40.degree. C.), the solid residue was dissolved in a mixture of
dichloromethane (450 mL) and MeOH (50 mL) at 30.degree. C., and
adsorbed over 20 g of silica gel. Purification was carried out in a
Combiflash Companion.TM. XL system with a RediSep disposable flash
220 g silica gel column (catalog #69-2203-422). Elution of the
residual starting material 3 was accomplished with dichloromethane
(approximately 20 column vol, while the product TK-202 was eluted
with 10% methanol in dichloromethane. The product containing
fractions, where product had already started to precipitate, were
combined in two different lots and partially concentrated under
reduced pressure. The resulting slurries were filtered and the
solid cakes were washed with MeOH to afford two fractions that were
dried under high vacuum at ambient temperature for 24 h, then
50.degree. C. for 24 h, to afford lot # BIO-W-30-17 and lot #
BIO-W-30-18. The filtrate from both crystallizations were combined
and subjected to a second chromatographic purification (on a 40-g
RediSep Gold column, catalog #69-2203-347) using a gradient from
dichloromethane to 5% methanol in dichloromethane for elution. The
product containing fractions (purity higher than 99% AUC by HPLC)
were combined and the product was allowed to precipitate over 2 h.
The solid was filtered off, washed with methanol, and dried under
high vacuum for 24 h at 50.degree. C. to afford lot # BIOW-30-19. A
second set of fractions containing product of approximately 95% AUC
purity by HPLC were combined, the solid was filtered off,
re-dissolved in dichloromethane, and added 20% methanol to
precipitate overnight. The precipitated TK-202 was filtered and
washed with methanol, then dried under high vacuum for 24 h at
50.degree. C. to afford lot # BIO-W-30-16. The total combined
weight of 5.99 g corresponds to 83% yield of compound. Solid is
off-white (with a very pale peach to tan shade).
4,7-Dichloro-3-hydroxy-3-(2-(4-(methylsulfonyl)phenyl)-2-oxoethyl)indolin--
2-one (Example 1)
[0117] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.28
(s, 3H), 3.81 (d, 1H, J=16 Hz), 4.42 (d, 1H, J=16 Hz), 6.53 (s,
1H), 6.92 (d, 1H, J=8 Hz), 7.32 (d, 1H, J=8 Hz), 8.05 (d, 2H, J=8
Hz), 8.17 (d, 2H, J=8 Hz), 11.04 (s, 1H).
3-(2-(4-(Aziridin-1-yl)phenyl)-2-oxoethyl)-4,7-dichloro-3-hydroxyindolin-2-
-one (Example 2)
##STR00030##
[0119] To 4,7-dichloroindoline-2,3-dione (A) (300 mg, 1.39 mmol) in
15 mL of methanol were 1-(4-(aziridin-1-yl)phenyl)ethanone (B) (0.9
g, 5.5 mmol) and 10 drops of diethylamine (2). The reaction was
stirred at 50.degree. C. for 24 hours. The solvent was removed and
the residue was purified with flash chromatography (0-5%
Methanol/CH2C12) to get an off white solid.
3-(2-(4-(Aziridin-1-yl)phenyl)-2-oxoethyl)-4,7-dichloro-3-hydroxyindolin--
2-one (Example 2): off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz)
.delta. 2.16 (s, 4H), 3.64 (d, 1H, J=16 Hz), 4.32 (d, 1H, J=16 Hz),
6.41 (s, 1H), 6.89 (d, 1H, J=8 Hz), 7.05 (d, 2H, J=8 Hz), 7.30 (d,
1H, J=8 Hz), 7.80 (d, 2H, J=8 Hz), 10.95 (s, 1H).
4,7-Dichloro-3-hydroxy-3-(2-(4-isopropylphenyl)-2-oxoethyl)indolin-2-one
(Example 3)
[0120] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 1.21
(d, 6H, J=4 Hz), 2.95 (m, 1H), 3.69 (d, 1H, J=16 Hz), 4.39 (d, 1H,
J=16 Hz), 6.45 (s, 1H), 6.90 (d, 1H, J=8 Hz), 7.29 (d, 1H, J=8 Hz),
7.38 (d, 2H, J=8 Hz), 7.85 (d, 2H, J=8 Hz), 10.98 (s, 1H).
4,7-Dichloro-3-(1-(4-(dimethylamino)phenyl)-1-oxopropan-2-yl)-3-hydroxyind-
olin-2-one (Example 4)
[0121] .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 1.48 (d, 3H, J=8 Hz),
3.00 (s, 6H), 4.78 (m, 1H), 6.35 (s, 1H), 6.66 (d, 2H, J=8 Hz),
6.76 (d, 1H, J=8 Hz), 7.17 (d, 1H, J=8 Hz), 7.69 (d, 2H, J=8 Hz),
10.74 (s, 1H).
4,7-Dichloro-3-(2-(4-cyclopropylphenyl)-2-oxoethyl)-3-hydroxyindolin-2-one
(Example 7)
##STR00031##
[0123] To 4,7-dichloroindoline-2,3-dione (A) (300 mg, 1.39 mmol) in
15 mL of methanol were added 1-(4-cyclopropylphenyl)ethanone (B)
(0.9 g, 5.5 mmol) and 10 drops of diethylamine (2). The reaction
was stirred at 50.degree. C. for 24 hours. The solvent was removed
and the residue was purified with flash chromatography (0-5%
Methanol/CH2C12) to get an off white solid.
4,7-Dichloro-3-(2-(4-cyclopropylphenyl)-2-oxoethyl)-3-hydroxyindolin-2-on-
e (Example 7): off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz)
.delta. 0.76 (m, 2H), 1.06 (m, 2H), 2.0 (m, 1H), 3.65 (d, 1H, J=16
Hz), 4.35 (d, 1H, J=16 Hz), 6.43 (s, 1H), 6.89 (d, 1H, J=8 Hz),
7.19 (d, 2H, J=8 Hz), 7.30 (d, 1H, J=8 Hz), 7.79 (d, 2H, J=8 Hz),
10.97 (s, 1H).
3-(2-(4-(1H-Pyrazol-1-yl)phenyl)-2-oxoethyl)-4,7-dichloro-3-hydroxyindolin-
-2-one (Example 8)
[0124] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.72
(d, 1H, J=16 Hz), 4.41 (d, 1H, J=16 Hz), 6.47 (s, 1H), 6.62 (d, 1H,
J=4 Hz), 6.90 (d, 1H, J=8 Hz), 7.31 (d, 1H, J=8 Hz), 7.84, (d, 1H,
J=4H), 7.99 (d, 2H, J=8 Hz), 8.06 (d, 2H, J=8 Hz), 8.66 (d, 1H, J=4
Hz), 10.98 (s, 1H).
4,7-Dichloro-3-hydroxy-3-(2-oxo-2-(4-(pyrrolidin-1-yl)phenyl)ethyl)indolin-
-2-one (Example 10)
##STR00032##
[0126] To 4,7-dichloroindoline-2,3-dione (A) (12.5 g, 0.06 mol) in
800 mL of methanol were added 1-(4-(pyrrolidin-1-yl)phenyl)ethanone
(B) (45 g, 0.24 mol) and 0.5 mL of diethylamine (2). The reaction
was stirred at rt for 24 hours. The solvent was removed and the
residue was purified with flash chromatography (0-5%
Methanol/CH.sub.2C.sub.12) to get 13.5 g of brown solid. It was
purified again with flash chromatography using ethyl acetate/hexane
to give 11.5 g of an off white solid. Repeating the reaction at the
same scale to give another 11.5 g of product. Two batches of
product were combined and recrystallized from methanol to get 20.5
g as an off white solid.
4,7-Dichloro-3-hydroxy-3-(2-oxo-2-(4-(pyrrolidin-1-yl)phenyl)ethyl)indoli-
n-2-one (Example 10): off-white solid; .sup.1H NMR (DMSO-d6, 400
MHz) .delta. 1.96 (m, 4H), 3.30 (m, 4H), 3.65 (d, 1H, J=16 Hz),
4.29 (d, 1H, J=16 Hz), 6.34 (s, 1H), 6.53 (d, 2H, J=8 Hz), 6.88 (d,
1H, J=8 Hz), 7.28 (d, 1H, J=8 Hz), 7.72 (d, 2H, J=8 Hz), 10.97 (s,
1H). Chiral separation was performed on under the following
conditions. Preparatory method utilized the following:a RegisCell
column L: 250 mm, IS: 50 mm, particle size: 5 .mu.m; mobile phase:
methanol/CO.sub.2, ratio: 35/65, detection wavelength: 254 nm, flow
rate: 325 g/min, co-solvent flow rate 113.75 ml/min. Dissolved
19.72 g in 1000 ml of methanol, for a concentration of 0.020 g/ml.
The injection volume was 25.00 ml for a total amount 0.500
g/injection. Yield was (+): 9.73 g, with optical rotation+247 at
20.degree. C. and (-): 9.26 g.
4-(2-(4,7-Dichloro-3-hydroxy-2-oxoindolin-3-yl)acetyl)benzenesulfonamide
(Example 11)
[0127] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.78
(d, 1H, J=16 Hz), 4.41 (d, 1H, J=16 Hz), 6.51 (s, 1H), 6.90 (d, 1H,
J=8 Hz), 7.31 (d, 1H, J=8 Hz), 7.56 (s, 2H), 7.91 (d, 2H, J=8 Hz),
8.11 (d, 2H, J=8 Hz), 10.98 (s, 1H).
4,7-Dichloro-3-(2-(3-fluoro-4-(pyrrolidin-1-yl)phenyl)-2-oxoethyl)-3-hydro-
xyindolin-2-one (Example 12)
[0128] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 1.91
(m, 4H), 3.46 (m, 4H), 3.57 (d, 1H, J=16 Hz), 4.27 (d, 1H, J=16
Hz), 6.36 (s, 1H), 6.71 (t, 1H, J=4 Hz, J=8 Hz), 7.29 (d, 1H, J=8
Hz), 7.46 (d, 1H, J=8 Hz), 7.61 (d, 1H, J=4 Hz), 7.64 (d, 1H, J=8
Hz), 11.01 (s, 1H).
3-(2-(4-(Azetidin-1-yl)phenyl)-2-oxoethyl)-4,7-dichloro-3-hydroxyindolin-2-
-one (Example 13)
##STR00033##
[0130] To 4,7-dichloroindoline-2,3-dione (A) (300 mg, 1.39 mmol) in
15 mL of methanol were added 1-(4-(azetidin-1-yl)phenyl)ethan-1-one
(B) (972 mg, 5.5 mmol) and a few drops of diethylamine (2). The
reaction was stirred at rt for 24 hours. The solvent was removed
and the residue was purified with flash chromatography (0-5%
Methanol/CH2C12) to get an off white solid.
3-(2-(4-(Azetidin-1-yl)phenyl)-2-oxoethyl)-4,7-dichloro-3-hydroxyindolin--
2-one (Example 13): off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz)
.delta. 2.32 (m, 2H), 3.51 (d, 1H, J=16 Hz), 3.95 (m, 4H), 4.30 (d,
1H, J=16 Hz), 6.35 (s, 1H), 6.36 (d, 2H, J=8 Hz), 6.87 (d, 1H, J=8
Hz), 7.28 (d, 1H, J=8 Hz), 7.73 (d, 2H, J=8 Hz), 10.89 (s, 1H).
4,7-Dichloro-3-hydroxy-3-(2-(4-methoxycyclohexyl)-2-oxoethyl)indolin-2-one
(Example 14)
[0131] off-white solid; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta.
1.24 (m, 4H), 1.92 (m, 2H), 2.08 (m, 2H), 2.32 (m, 1H), 3.06 (m,
1H), 3.26 (s, 3H), 3.33 (d, 1H, J=16 Hz), 3.69 (s, 1H), 3.70 (d,
1H, J=16 Hz), 6.91 (d, 1H, J=8 Hz), 7.20 (d, 1H, J=8 Hz), 7.61 (s,
1H).
4,7-Dichloro-3-hydroxy-3-(2-(4-methoxybicyclo
[2.2.2]octan-1-yl)-2-oxoethyl)indolin-2-one (Example 15)
[0132] off-white solid; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta.
1.59 (m, 12H), 3.16 (s, 3H), 3.22 (d, 1H, J=16 Hz), 3.58 (s, 1H),
4.14 (d, 1H, J=16 Hz), 6.90 (d, 1H, J=8 Hz), 7.23 (d, 1H, J=8 Hz),
7.67 (s, 1H).
4,7-Dichloro-3-(2-(4-(dimethylamino)bicyclo
[2.2.2]octan-1-yl)-2-oxoethyl)-3-hydroxyindolin-2-one (Example
16)
[0133] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 1.59
(m, 12H), 2.5 (s, 3H), 3.18 (d, 1H, J=16 Hz), 3.85 (d, 1H, J=16
Hz), 6.31 (s, 1H), 6.90 (d, 1H, J=8 Hz), 7.30 (d, 1H, J=8 Hz),
10.95 (s, 1H).
4,7-Dichloro-3-(2-(4-cyclopropyl-3-fluorophenyl)-2-oxoethyl)-3-hydroxyindo-
lin-2-one (Example 17)
##STR00034##
[0135] To 4,7-dichloroindoline-2,3-dione (A) (261 mg, 1.21 mmol) in
15 mL of methanol were 1-(4-cyclopropyl-3-fluorophenyl)ethanone (B)
(280 mg, 1.57 mmol) and 10 drops of diethylamine (2). The reaction
was stirred at 50.degree. C. for 24 hours. The solvent was removed
and the residue was purified with flash chromatography (0-5%
Methanol/CH2C12) to get an off white solid.
4,7-Dichloro-3-(2-(4-cyclopropyl-3-fluorophenyl)-2-oxoethyl)-3-hydroxyind-
olin-2-one (Example 17): off-white solid; .sup.1H NMR (DMSO-d6, 400
MHz) .delta. 0.83 (m, 2H), 1.08 (m, 2H), 2.11 (m, 1H), 3.68 (d, 1H,
J=16 Hz), 4.34 (d, 1H, J=16 Hz), 6.43 (s, 1H), 6.90 (d, 1H, J=8
Hz), 7.11 (m, 1H), 7.30 (d, 1H, J=8 Hz), 7.60 (d, 1H, J=8 Hz), 7.68
(d, 1H, J=8 Hz), 10.96 (s, 1H). Chiral separation was performed by
a method substantially similar to the method described above. LC
screening was performed with: column: AD-H, 250 mm.times.4.6 mm, 5
.mu.m, hexane/ethanol (65/35), 1.5 ml/min, injection volume: 10.0
.mu.l, pressure: 102.9 bar. Peak 1: retention time: 5.40 min,
width: 0.171 min, area: 4502.21, area %: 50.08. Peak 2: retention
time: 7.23 min, width: 0.239 min, area: 4488.43, area %: 49.92.
4,7-Dichloro-3-(2-(4-cyclopropyl-2-fluorophenyl)-2-oxoethyl)-3-hydroxyindo-
lin-2-one (Example 18)
##STR00035##
[0137] To 4,7-dichloroindoline-2,3-dione (A) (261 mg, 1.21 mmol) in
15 mL of methanol were 1-(4-cyclopropyl-2-fluorophenyl)ethanone (B)
(280 mg, 1.57 mmol) and 10 drops of diethylamine (2). The reaction
was stirred at 50.degree. C. for 24 hours. The solvent was removed
and the residue was purified with flash chromatography (0-5%
Methanol/CH2C12) to get an off white solid.
4,7-Dichloro-3-(2-(4-cyclopropyl-2-fluorophenyl)-2-oxoethyl)-3-hydroxyind-
olin-2-one (Example 18): off-white solid; .sup.1H NMR (DMSO-d6, 400
MHz) .delta. 0.82 (m, 2H), 1.07 (m, 2H), 2.01 (m, 1H), 3.66 (d, 1H,
J=16 Hz), 4.26 (d, 1H, J=16 Hz), 6.44 (s, 1H), 6.91 (d, 1H, J=8
Hz), 7.01 (m, 2H), 7.31 (d, 1H, J=8 Hz), 7.57 (m, 1H), 10.96 (s,
1H). Chiral separation was performed by a method substantially
similar to the method described above. LC screening was performed
with: column: RegisCell, 250 mm.times.4.6 mm, 5 .mu.m, hexane/IPA
(80/20), 1.5 ml/min, injection volume: 2.0 .mu.l, pressure: 51.5
bar. Peak 1: retention time: 5.16 min, width: 0.238 min, area:
3716.20, area %: 49.78. Peak 2: retention time: 6.49 min, width:
0.324 min, area: 3749.55, area %: 50.22.
4-Chloro-7-fluoro-3-hydroxy-3-(2-(4-methoxyphenyl)-2-oxoethyl)indolin-2-on-
e (Example 19)
[0138] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.63
(d, 1H, J=16 Hz), 3.84 (s, 3H), 4.36 (d, 1H, J=16 Hz), 6.38 (s,
1H), 6.88 (d, 1H, J=8 Hz), 7.02 (d, 2H, J=8 Hz), 7.16 (m, 1H), 7.89
(d, 2H, J=8 Hz), 11.01 (s, 1H).
4,7-dichloro-3-hydroxy-3-(2-(4-morpholinophenyl)-2-oxoethyl)indolin-2-one
(Example 20)
[0139] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.28
(m, 4H), 3.53 (d, 1H, J=16 Hz), 3.71 (m, 4H), 4.33 (d, 1H, J=16
Hz), 6.37 (s, 1H), 6.87 (d, 1H, J=8 Hz), 6.97 (d, 2H, J=8 Hz), 7.30
(d, 1H, J=8 Hz), 7.77 (d, 2H, J=8 Hz), 10.95 (s, 1H).
4,7-dichloro-3-hydroxy-3-(2-oxo-2-(pyridin-4-yl)ethyl)indolin-2-one
(Example 21)
[0140] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.78
(d, 1H, J=16 Hz), 4.39 (d, 1H, J=16 Hz), 6.53 (s, 1H), 6.92 (d, 1H,
J=8 Hz), 7.32 (d, 1H, J=8 Hz), 7.79 (d, 2H, J=4 Hz), 8.80 (d, 2H,
J=4 Hz), 11.03 (s, 1H).
4,7-dichloro-3-hydroxy-3-(2-oxo-2-(pyridin-3-yl)ethyl)indolin-2-one
(Example 22)
[0141] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.78
(d, 1H, J=16 Hz), 4.39 (d, 1H, J=16 Hz), 6.50 (s, 1H), 6.90 (d, 1H,
J=8 Hz), 7.30 (d, 1H, J=8 Hz), 7.57 (m, 1H), 8.28 (d, 1H, J=4 Hz),
8.80 (d, 1H, J=4 Hz), 9.09 (s, 1H), 11.03 (s, 1H).
4,7-dichloro-3-hydroxy-3-(2-oxo-2-(pyridin-2-yl)ethyl)indolin-2-one
(Example 23)
[0142] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.78
(d, 1H, J=16 Hz), 4.68 (d, 1H, J=16 Hz), 6.50 (s, 1H), 6.90 (d, 1H,
J=8 Hz), 7.30 (d, 1H, J=8 Hz), 7.70 (d, 1H, J=4 Hz), 7.81 (d, 1H,
J=4 Hz), 7.98 (m, 1H), 8.76 (s, 1H), 11.01 (s, 1H).
4-(2-(4,7-dichloro-3-hydroxy-2-oxoindolin-3-yl)acetyl)benzamide
(Example 24)
[0143] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.74
(d, 1H, J=16 Hz), 4.42 (d, 1H, J=16 Hz), 6.49 (s, 1H), 6.92 (d, 1H,
J=8 Hz), 7.31 (d, 1H, J=8 Hz), 7.57 (s, 1H), 7.95 (d, 2H, J=8 Hz),
7.97 (d, 2H, J=8 Hz), 8.10 (s, 1H), 11.01 (s, 1H).
4,7-dichloro-3-hydroxy-3-(2-(4-hydroxyphenyl)-2-oxoethyl)indolin-2-one
(Example 25)
[0144] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.60
(d, 1H, J=16 Hz), 4.30 (d, 1H, J=16 Hz), 6.39 (s, 1H), 6.82 (d, 2H,
J=8 Hz), 6.90 (d, 1H, J=8 Hz), 7.30 (d, 1H, J=8 Hz), 7.80 (d, 2H,
J=8 Hz), 10.45 (s, 1H), 10.93 (s, 1H).
4,7-dichloro-3-(2-(3,4-difluorophenyl)-2-oxoethyl)-3-hydroxyindolin-2-one
(Example 26)
[0145] off-white solid; .sup.1H NMR (DMSO-d6, 400 MHz) .delta. 3.70
(d, 1H, J=16 Hz), 4.37 (d, 1H, J=16 Hz), 6.48 (s, 1H), 6.91 (d, 1H,
J=8 Hz), 7.30 (d, 1H, J=8 Hz), 7.56 (m, 1H), 7.96 (m, 1H), 8.01 (m,
1H), 11.01 (s, 1H).
Biological Activity of Compounds
[0146] Biological activities of certain compounds listed in TABLE 2
were determined.
TABLE-US-00003 TABLE 2 Compound (optical rotation) Structure Name 1
(+/-) ##STR00036## 4,7-Dichloro-3-hydroxy-3-[2-(4-
methoxyphenyl-2-oxoethyl)]-1,3- dihydroindol-2-one 2 (-)
##STR00037## 4,7-Dichloro-3-hydroxy-3-[2-(4-
methoxyphenyl-2-oxoethyl)]-1,3- dihydroindol-2-one 3 (+)
##STR00038## 4,7-Dichloro-3-hydroxy-3-[2-(4-
methoxyphenyl-2-oxoethyl)]-1,3- dihydroindol-2-one 4 (+/-)
##STR00039## 4,7-Dichloro-3-hydroxy-3-(2-(4-
(methylsulfonyl)phenyl)-2- oxoethyl)indolin-2-one 5 (-)
##STR00040## 4,7-dichloro-3-hydroxy-3-[2-[4-
(methylamino)phenyl]-2-oxoethyl]- 1H-indol-2-one 6 (+) ##STR00041##
4,7-dichloro-3-hydroxy-3-[2-[4- (methylamino)phenyl]-2-oxoethyl]-
1H-indol-2-one 7 (+/-) ##STR00042## 4,7-dichloro-3-hydroxy-3-[2-[4-
(methylamino)phenyl]-2-oxoethyl]- 1H-indol-2-one 8 (+/-)
##STR00043## 3-(2-(4-(Aziridin-1-yl)phenyl)-2-
oxoethyl)-4,7-dichloro-3- hydroxyindolin-2-one 9 (+/-) ##STR00044##
4,7-Dichloro-3-hydroxy-3-(2-(4- isopropylphenyl)-2-
oxoethyl)indolin-2-one 10 (+) ##STR00045## 4,7-Dichloro-3-(1-(4-
(dimethylamino)phenyl)-1- oxopropan-2-yl)-3-hydroxyindolin- 2-one
11 (-) ##STR00046## 4,7-Dichloro-3-(1-(4- (dimethylamino)phenyl)-1-
oxopropan-2-yl)-3-hydroxyindolin- 2-one 12 (+/-) ##STR00047##
4,7-Dichloro-3-(1-(4- (dimethylamino)phenyl)-1-
oxopropan-2-yl)-3-hydroxyindolin- 2-one 13 (+) ##STR00048##
4,7-Dichloro-3-(2-(4- cyclopropylphenyl)-2-oxoethyl)-3-
hydroxyindolin-2-one 14 (-) ##STR00049## 4,7-Dichloro-3-(2-(4-
cyclopropylphenyl)-2-oxoethyl)-3- hydroxyindolin-2-one 15 (+/-)
##STR00050## 4,7-Dichloro-3-(2-(4-
cyclopropylphenyl)-2-oxoethyl)-3- hydroxyindolin-2-one 16 (+/-)
##STR00051## 3-(2-(4-(1H-Pyrazol-1-yl)phenyl)-2-
oxoethyl)-4,7-dichloro-3- hydroxyindolin-2-one 17 (+) ##STR00052##
4,7-Dichloro-3-hydroxy-3-(2-oxo-2- (4-(pyrrolidin-1-
yl)phenyl)ethyl)indolin-2-one 18 (-) ##STR00053##
4,7-Dichloro-3-hydroxy-3-(2-oxo-2- (4-(pyrrolidin-1-
yl)phenyl)ethyl)indolin-2-one 19 (+/-) ##STR00054##
4,7-Dichloro-3-hydroxy-3-(2-oxo-2- (4-(pyrrolidin-1-
yl)phenyl)ethyl)indolin-2-one 20 (+/-) ##STR00055##
4-(2-(4,7-Dichloro-3-hydroxy-2- oxoindolin-3-
yl)acetyl)benzenesulfonamide 21 (+/-) ##STR00056##
4,7-Dichloro-3-(2-(3-fluoro-4- (pyrrolidin-1-yl)phenyl)-2-
oxoethyl)-3-hydroxyindolin-2-one 22 (+/-) ##STR00057##
3-(2-(4-(Azetidin-1-yl)phenyl)-2- oxoethyl)-4,7-dichloro-3-
hydroxyindolin-2-one 23 (+/-) ##STR00058##
4,7-Dichloro-3-hydroxy-3-(2-(4- methoxycyclohexyl)-2-
oxoethyl)indolin-2-one 24 (+/-) ##STR00059##
4,7-Dichloro-3-hydroxy-3-(2-(4- methoxybicyclo[2.2.2]octan-1-yl)-2-
oxoethyl)indolin-2-one 25 (+/-) ##STR00060## 4,7-Dichloro-3-(2-(4-
(dimethylamino)bicyclo[2.2.2]octan-
1-yl)-2-oxoethyl)-3-hydroxyindolin- 2-one 26 (+/-) ##STR00061##
4,7-Dichloro-3-(2-(4-cyclopropyl-3- fluorophenyl)-2-oxoethyl)-3-
hydroxyindolin-2-one 27 (+/-) ##STR00062##
4,7-Dichloro-3-(2-(4-cyclopropyl-2- fluorophenyl)-2-oxoethyl)-3-
hydroxyindolin-2-one 27 (-) ##STR00063##
4,7-Dichloro-3-(2-(4-cyclopropyl-2- fluorophenyl)-2-oxoethyl)-3-
hydroxyindolin-2-one 27 (+) ##STR00064##
4,7-Dichloro-3-(2-(4-cyclopropyl-2- fluorophenyl)-2-oxoethyl)-3-
hydroxyindolin-2-one 28 (+/-) ##STR00065##
4-Chloro-7-fluoro-3-hydroxy-3-(2- (4-methoxyphenyl)-2-
oxoethyl)indolin-2-one 29 (+/-) ##STR00066##
4,7-dichloro-3-hydroxy-3-(2-(4- morpholinophenyl)-2-
oxoethyl)indolin-2-one 30 (+/-) ##STR00067##
4,7-dichloro-3-hydroxy-3-(2-oxo-2-
(pyridin-4-yl)ethyl)indolin-2-one 31 (+/-) ##STR00068##
4,7-dichloro-3-hydroxy-3-(2-oxo-2-
(pyridin-3-yl)ethyl)indolin-2-one 32 (+/-) ##STR00069##
4,7-dichloro-3-hydroxy-3-(2-oxo-2-
(pyridin-2-yl)ethyl)indolin-2-one 33 (+/-) ##STR00070##
4-(2-(4,7-dichloro-3-hydroxy-2- oxoindolin-3-yl)acetyl)benzamide 34
(+/-) ##STR00071## 4,7-dichloro-3-hydroxy-3-(2-(4-
hydroxyphenyl)-2-oxoethyl)indolin- 2-one 35 (+/-) ##STR00072##
4,7-dichloro-3-(2-(3,4- difluorophenyl)-2-oxoethyl)-3-
hydroxyindolin-2-one 36 (-) ##STR00073##
4,7-Dichloro-3-(2-(4-cyclopropyl-3- fluorophenyl)-2-oxoethyl)-3-
hydroxyindolin-2-one 37 (+) ##STR00074##
4,7-Dichloro-3-(2-(4-cyclopropyl-3- fluorophenyl)-2-oxoethyl)-3-
hydroxyindolin-2-one 38 (-) ##STR00075##
4,7-Dichloro-3-(2-(4-cyclopropyl-2- fluorophenyl)-2-oxoethyl)-3-
hydroxyindolin-2-one 39 (+) ##STR00076##
4,7-Dichloro-3-(2-(4-cyclopropyl-2- fluorophenyl)-2-oxoethyl)-3-
hydroxyindolin-2-one
Activities of Compounds
[0147] A modified tetrazolium salt assay using the CCK-8 kit
(Sigma-Aldrich; St Louis, Mo.) was used to measure the inhibition
of human tumor cell growth. Tumor cells (5000-7500 per well) were
added to 96 well plates and allowed to attach for 4-5 hours.
Compounds were serially diluted and added in triplicate at a
concentration of 0.02 to 5 .mu.M. DMSO was included as a vehicle
control. Cells were incubated in the presence of compound for 3
days. After incubation CCK-8 reagent was added to each well and
incubated for 2-4 hours. Viable cells were quantitated
spectrophotometrically at a wavelength of 450 nm. Percent viability
of each sample was calculated from the A450 values as follows: %
viability=(A450 nm sample/A450 nm DMSO-treated cells.times.100).
The IC.sub.50 was defined as the concentration that gave rise to
50% inhibition of cell viability. IC.sub.50 activities of
particular compounds were determined using SKES cells (Ewing
Sarcoma cell line). Results are summaries in TABLE 3. IC.sub.50
activities of particular compounds were determined using the cell
lines listed in TABLE 4.
TABLE-US-00004 TABLE 3 Cmpd IC.sub.50 1 B 2 B 3 A 4 A 5 C 6 C 7 C 8
B 9 A 10 A 11 B 12 C 13 A 14 B 15 C 16 A 17 A 18 B 19 C 20 A 21 A
22 B 23 A 24 A 25 A 26 B 27 B 28 A 29 A 30 A 31 A 32 A 33 A 34 A 35
A 36 C 37 C 38 C 39 C A is IC.sub.50 >5 .mu.M; B is IC.sub.50
<5 .mu.M; and C is not determined.
TABLE-US-00005 TABLE 4 Cell Line Tumor Type ETS family
rearrangement SKES ES EWS-FLI1 Type 2 A4573 ES EWS-FLI1 Type 3 TC71
ES EWS-FLI1 Type 1 LNCap Prostate rearranged ETV1 PC3 Prostate none
MDA-MB-231 Breast increased ETV1 MCF7 Breast none BxPC3 Pancreas
increased FLI1 PANC1 Pancreas none
[0148] Results for IC.sub.50 of certain compounds are summarized as
follows: Cmpd 1: SKES: C A4573: C; TC71: C; LNCap: C; PC3: C;
MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 2: SKES: B;
A4573: B; TC71: B; LNCap: B; PC3: A; MDA-MB-231: B; MCF7: A; BxPC3:
B; and PANC1: A. Cmpd 3: SKES: C; A4573: C; TC71: C; LNCap: C; PC3:
C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 4: SKES: A;
A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3:
C; and PANC1: C. Cmpd 5: SKES: C; A4573: C; TC71: C; LNCap: C; PC3:
C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 6: SKES: C;
A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3:
C; and PANC1: C. Cmpd 7: SKES: C; A4573: C; TC71: C; LNCap: C; PC3:
C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 8: SKES: B;
A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3:
C; and PANC1: C. Cmpd 9: SKES: A; A4573: C; TC71: C; LNCap: C; PC3:
C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 10: SKES:
A; A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C;
BxPC3: C; and PANC1: C. Cmpd 11: SKES: B; A4573: C; TC71: C; LNCap:
C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 12:
SKES: C; A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7:
C; BxPC3: C; and PANC1: C. Cmpd 13: SKES: A; A4573: C; TC71: C;
LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C.
Cmpd 14: SKES: B; A4573: B; TC71: B; LNCap: B; PC3: A; MDA-MB-231:
B; MCF7: A; BxPC3: B; and PANC1: A. Cmpd 15: SKES: C; A4573: C;
TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and
PANC1: C. Cmpd 16: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;
MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 17: SKES: A;
A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3:
C; and PANC1: C. Cmpd 18: SKES: B; A4573: C; TC71: C; LNCap: C;
PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 19:
SKES: C; A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7:
C; BxPC3: C; and PANC1: C. Cmpd 20: SKES: A; A4573: C; TC71: C;
LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C.
Cmpd 21: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231:
C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 22: SKES: B; A4573: C;
TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and
PANC1: C. Cmpd 23: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;
MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 24: SKES: A;
A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3:
C; and PANC1: C. Cmpd 25: SKES: A; A4573: C; TC71: C; LNCap: C;
PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 26:
SKES: B; A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7:
C; BxPC3: C; and PANC1: C. Cmpd 27: SKES: B; A4573: C; TC71: C;
LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C.
Cmpd 28: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231:
C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 29: SKES: A; A4573: C;
TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and
PANC1: C. Cmpd 30: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C;
MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 31: SKES: A;
A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3:
C; and PANC1: C. Cmpd 32: SKES: A; A4573: C; TC71: C; LNCap: C;
PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 33:
SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7:
C; BxPC3: C; and PANC1: C. Cmpd 34: SKES: A; A4573: C; TC71: C;
LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C.
Cmpd 35: SKES: A; A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231:
C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 36: SKES: C; A4573: C;
TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and
PANC1: C. Cmpd 37: SKES: C; A4573: C; TC71: C; LNCap: C; PC3: C;
MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. Cmpd 38: SKES: C;
A4573: C; TC71: C; LNCap: C; PC3: C; MDA-MB-231: C; MCF7: C; BxPC3:
C; and PANC1: C. Cmpd 39: SKES: C; A4573: C; TC71: C; LNCap: C;
PC3: C; MDA-MB-231: C; MCF7: C; BxPC3: C; and PANC1: C. A is
IC.sub.50>5 .mu.M; B is IC.sub.50<5 .mu.M; and C is not
determined. In a xenograft study, A4573 tumor cells were implanted
into mice. The mice were treated with certain compounds (oral bid).
The mean volume of A4573 tumors was measured at various times.
Relative to vehicle control, cmp 14 showed a 57% tumor growth
inhibition (TGI) at 100 mg/kg bid, cmp 2 did not show TGI at 200
mg/kg bid. In a similar rat xenograft study, cmpd 2 showed an 87%
TGI, and cmpd 14 showed a 53% TGI, each compared to the vehicle
control.
Metabolic Activities of Certain Compounds
[0149] Metabolic activities of certain compounds were assayed with
liver microsomes using a NADPH regenerating system and standard
protocols. Briefly, compounds were incubated with isolated human,
rat, dog, or mouse liver microsomes. Reactions were initiated in
96-well plates by addition of NADPH regenerating system
((3-nicotinamide adenine dinucleotide phosphate; isocitric acid;
and isocitric dehydrogenase). Reactions were quenched with cold
acrylonitrile at 5, 10, 20, 30 and 60 min, shaken and centrifuged
at 4000 rpm for 20 min. Supernatants containing detected analyte
compounds were analysed using LC/MS/MS with LC: Shimadzu LC 20-AD,
MS: API4000, autosampler: CTC PAL; columns used included CHIRALPAK
AS-RH 150*4.6 mm, 5 .mu.m Part No:ASRHCD-KK008, and Ace 5 Phenyl,
50.times.2.1 mm, Part No.ACE-125-0502. Data analysis: T.sub.1/2 and
CL were calculated use equations of first order kinetics:
C.sub.t=C.sub.0*e.sup.-kt;
C.sub.t=(1/2)*C.sub.0;t.sub.1/2=In2/k=0.693/k; CL=V.sub.d*k; and
V.sub.d=2 mL/mg. TABLES 5-8 summarise the results. In TABLES 5-8:
R.sup.2 is the correlation coefficient of the linear regression for
the determination of kinetic constant; T.sub.1/2 is half life; NCF
is no cofactor.
TABLE-US-00006 TABLE 5 Human liver microsome Remaining Remaining
T.sub.1/2 (T = 60 (NCF = 60 Cmpd R.sup.2 (min) min) min) 2 0.9771
20.2 12.7% 112.1% 3 0.7345 >145 77.1% 112.6% 18 0.9869 23.0
17.8% 99.4% 13 0.9268 103.4 68.5% 105.1% 14 0.9901 27.7 22.8%
104.5% 37 0.9659 85.6 62.8% 103.4% 36 0.9972 23.8 17.9% 104.0% 39
0.9910 79.7 60.4% 100.0% 38 0.9970 18.7 11.2% 99.5% Testosterone
0.9928 18.9 10.9% 88.1% Diclofenac 0.9855 7.3 0.3% 77.8%
Propafenone 0.9315 6.8 0.2% 92.5% Liver wt: 22 g/kg relative liver
weight for human
TABLE-US-00007 TABLE 6 Rat liver microsome Remaining Remaining
T.sub.1/2 (T = 60 (NCF = 60 Cmpd R.sup.2 (min) min) min) 2 0.9991
3.8 2.3% 92.7% 3 0.9989 7.1 2.7% 92.6% 18 0.9995 1.3 0.0% 88.5% 13
0.9885 3.6 0.2% 97.0% 14 0.9971 2.8 0.1% 97.6% 37 0.9968 3.5 0.3%
95.4% 36 0.9943 2.6 0.2% 97.8% 39 0.9849 3.0 0.2% 90.5% 38 0.9137
2.9 0.2% 94.7% Testosterone 1.0000 1.0 0.0% 87.3% Diclofenac 0.9985
13.3 4.1% 94.1% Propafenone 0.9926 2.0 0.0% 89.2% Liver wt: 40 g/kg
relative liver weight for rat
TABLE-US-00008 TABLE 7 Mouse liver microsome Remaining Remaining
T.sub.1/2 (T = 60 (NCF = 60 Cmpd R.sup.2 (min) min) min) 2 0.9919
16.2 7.7% 90.2% 3 0.9902 7.4 0.4% 97.5% 18 0.9820 9.6 1.6% 88.4% 13
0.9780 26.6 19.6% 85.6% 14 0.9474 23.1 19.7% 102.0% 37 0.9871 20.7
12.8% 88.6% 36 0.9904 11.9 3.0% 89.6% 39 0.9917 15.4 6.4% 89.7% 38
0.9968 6.8 0.0% 93.0% Testosterone 0.9992 3.3 0.0% 83.6% Diclofenac
0.9887 39.2 32.6% 88.1% Propafenone 0.9891 2.0 0.0% 91.7% Liver wt:
88 g/kg relative liver weight for mouse
TABLE-US-00009 TABLE 8 Dog liver microsome Remaining Remaining
T.sub.1/2 (T = 60 (NCF = 60 Cmpd R.sup.2 (min) min) min) 2 0.9911
30.3 24.6% 100.6% 3 0.9977 21.3 14.0% 100.1% 18 0.9932 24.1 18.7%
92.9% 13 0.9935 8.4 0.7% 98.4% 14 0.9932 40.8 36.2% 97.1% 37 0.9855
9.1 1.0% 94.1% 36 0.9908 35.0 29.9% 93.1% 39 0.9857 2.4 0.1% 96.3%
38 0.9961 33.5 28.3% 97.6% Testosterone 0.9900 19.8 12.5% 91.0%
Diclofenac 0.7395 >145 75.5% 89.6% Propafenone 0.9765 5.2 0.1%
89.6% Liver wt: 32 g/kg relative liver weight for dog
Metabolic Stability of Certain Compounds
[0150] Half-life and clearance rates for certain compounds were
assayed with human liver microsomes and human hepatocytes.
Compounds were incubated in the presence of human liver microsomes
(or human hepatocytes) in a 95% humidified incubator at 5% CO.sub.2
to start the reactions. At each time point (0, 5, 15, 30, 60, 90
min) the reactions were stopped, vortexed and centrifuged.
Supernatents were frozen until LC/MS/MS analysis. The results for
human liver microsomes and human hepactocytes are summarized in
TABLE 9, and TABLE 10, respectively.
TABLE-US-00010 TABLE 9 Cmpd % remaining Half-life (min) CL.sub.hep
(mL/min/kg)* 1 5 10.3 17.28 12 18 18 15.69 7 49 >45 ~11.54
*hepatic clearance where blood flow = 20 mL/min/kg
TABLE-US-00011 TABLE 10 Cmpd % remaining Half-life (min) CL.sub.hep
(mL/min/kg)* 1 48 226 8.7 12 56 >240 <8.7 7 100 >240
<8.7 *hepatic clearance where blood flow = 20 mL/min/kg
[0151] In another study, metabolism of compounds in hepatocytes
from various species was examined. Hepatocytes were contacted with
a compound, and the half-life of the compound was determined. The
results are summarized in TABLE 11.
TABLE-US-00012 TABLE 11 Half life of cmpds (min) Cmpd 1 18 14
7-Ethoxycoumarin 7-Hydroxycoumarin Mouse 30.9 60.0 62.79 37.86
15.82 Rat 14.2 18.8 27.41 38.24 12.41 Dog 16.7 30.6 20.26 9.14 9.38
Human 45.5 63.6 75.87 45.29 20.78
[0152] Certain compounds were assayed with human liver microsomes
(HLM) and human hepatocytes, and metabolites were detected.
Briefly, metabolic stability was tested at one TA concentration
(e.g., 1 .mu.M) in duplicate. Loss of test article over time was
evaluated in HLM (0.5 mg/mL) with and without NADPH at 0, 5, 10,
20, 30 and 45 min and hepatocytes (0.5.times.10.sup.6 cells/mL) at
0, 15, 30, 60, 120 and 240 min. Positive control (diclofenac) and
negative control (boiled HLM or heat inactivated hepatocytes) were
included. Diclofenac was monitored at incubation times similar to
test article while the negative control was measured at 0 and 45
min in HLM and 0 and 240 min in hepatocytes. Incubations were saved
and used for metabolite identification using LC/MS/MS. The parent
analyte/internal standard peak area ratios was converted to
percentage drug remaining, using the time=0 peak area ratio values
as 100%. The slope of the linear regression from log percentage
remaining versus incubation time relationships (-k) was determined
by linear regression fitting. From the individual log percentage
remaining time profiles, the mean half-life and intrinsic clearance
were reported. UHPLC-HRMS or UHPLC-MS/MS experiment was performed
on the authentic test articles to check for possible common
fragment ions. Selected microsomal samples from the 0, 10, 20, and
45 min aliquots, and the 0, 60, 120, and 240 min hepatocyte
incubations aliquots were used for preliminary metabolite
identification. The metabolites were summarized based on their mass
spectrometry peak areas. TABLE 12, TABLE 13 and TABLE 14 summarise
results for cmpds: 1, 12, and 7, respectively.
TABLE-US-00013 TABLE 12 [M + H] Retention Human liver Human
Description (m/z) time (min) Structure microsomes hepatocytes
Parent 366.0292 2.52 ##STR00077## d d Demethylation +
Glucuronidation 528.0482 1.97 ##STR00078## nd d Oxidation 382.0249
2.24 ##STR00079## d nd Demethylation 352.0141 2.27 ##STR00080## d d
d: detected, nd: not detected
TABLE-US-00014 TABLE 13 [M + H] Retention Human liver Human
Description (m/z) time (min) Structure microsomes hepatocytes
Parent 379.063 2.55 ##STR00081## d d Demethylation + Oxidation
381.0406 2.13 ##STR00082## d nd Didemethylation 351.0305 2.25
##STR00083## d d Oxidation 395.0552 2.28 ##STR00084## d nd d:
detected, nd: not detected
TABLE-US-00015 TABLE 14 [M + H] Retention time Human liver Human
Description (m/z) (min) Structure microsomes hepatocytes Parent
365.0458 2.40 ##STR00085## d d Oxidation 381.0402 2.21 ##STR00086##
d nd Demethylation 351.0301 2.25 ##STR00087## d d d: detected, nd:
not detected
Pharmacokinetics of Certain Compounds
[0153] Certain pharmacokinetics parameters were determined for
certain compounds in vivo for intravenous and oral administration.
Briefly, compounds were formulated and administered using IV bolus,
continuous IV infusion or oral administration. Blood was collected
at multiple time points over 24 hours and processed into plasma.
Plasma was analyses for parent using LC/MS/MS. Certain parameters
for certain compounds were obtained in duplicate. TABLE 15
summarises results with Cmp 14 for a continuous infusion study in
rat or dog. TABLE 16 summarises results for studies in BALB/c mice
or Sprague Dawley rats.
TABLE-US-00016 TABLE 15 Dose (mg/ T.sub.1/2 k.sub.elim Vd CL
MRT.sub.24-inf Css.sub.(3-24 h) kg/day) (h) (1/h) (mL/kg) (mL/h/kg)
(h) (ng/mL) .mu.M 24# 0.824 0.846 3457 2922 1.11 344 0.91 96# 1.00
0.703 4231 2976 1.73 1376 3.7 10{circumflex over ( )} 1.9 0.4 5643
2016 2.5 210 0.56 25{circumflex over ( )} 1.7 0.4 5663 2238 2.2 466
1.24 #rat; {circumflex over ( )}dog
TABLE-US-00017 TABLE 16 Dose (mg/kg), Cmax AUC T.sub.1/2 Tmax Cmpd
route Vehicle (ng/mL) h * (ng/mL) % F (hr) (hr) 2 5, IV captisol
9381 1991 0.033 2 20, po labrasol/tetraglycol# 2243 3890 49 1 2 20,
po labrasol/tetraglycol/water* 2835 4574 57 1 2 5, IV captisol 9381
1991 nd 0.033 2 20, po labrasol/tetraglycol.sup.# 2243 3896 40 1.6
1 2 20, po labrasol/tetraglycol/water* 2835 4574 47 1.5 1 11 5, IV
captisol 12362 2102 0.033 11 20, po labrasol/tetraglycol# 1673 3783
45 1 11 20, po labrasol/tetraglycol/water* 1168 3828 46 0.5 11 5,
IV captisol 12362 2871 0.8 0.033 11 20, po
labrasol/tetraglycol.sup.# 1673 3787 34 1 1 11 20, po
labrasol/tetraglycol/water* 1168 3832 34 0.8 0.5 5 5, IV captisol
12993 2324 0.033 5 20, po labrasol/tetraglycol# 2179 5192 56 1 5
20, po labrasol/tetraglycol/water* 1569 4354 47 1 5 5, IV captisol
12993 3207 1.7 0.033 5 20, po labrasol/tetraglycol.sup.# 2179 4939
47 3.1 0.5 5 20, po labrasol/tetraglycol/water* 1569 4081 39 2.8
0.5 14 5, IV captisol 10891 4131 0.8 0.03 14 20, po
labrasol/tetraglycol.sup.# nd nd nd nd nd 14 25, po
labrasol/tetraglycol/water* 2772 13152 64 3.1 1 14 10, IV captisol
39800 6059 1.43 0.03 14 100, po labrasol/tetraglycol 32999 146167
100 7.71 2 14 100, po PEG-400/tween 25967 92219 100 3.83 0 18 5, IV
captisol 14343 3738 0.8 0.033 18 20, po labrasol/tetraglycol.sup.#
3217 7497 50 1.4 1 18 20, po labrasol/tetraglycol/water* 6422 11029
74 0.9 1 .sup.#labrasol/tetraglycol: 9:1
*labrasol:tetraglycol:water is 72:8:20 nd: not determined
Parameters determined in BALB/c mice or Sprague Dawley rats
[0154] While the disclosure has been illustrated and described in
detail in the drawings and foregoing description, such illustration
and description are to be considered illustrative or exemplary and
not restrictive. The disclosure is not limited to the disclosed
embodiments. Variations to the disclosed embodiments can be
understood and effected by those skilled in the art in practicing
the claimed disclosure, from a study of the drawings, the
disclosure and the appended claims.
[0155] All references cited herein are incorporated herein by
reference in their entirety. To the extent publications and patents
or patent applications incorporated by reference contradict the
disclosure contained in the specification, the specification is
intended to supersede and/or take precedence over any such
contradictory material.
[0156] Unless otherwise defined, all terms (including technical and
scientific terms) are to be given their ordinary and customary
meaning to a person of ordinary skill in the art, and are not to be
limited to a special or customized meaning unless expressly so
defined herein. It should be noted that the use of particular
terminology when describing certain features or aspects of the
disclosure should not be taken to imply that the terminology is
being re-defined herein to be restricted to include any specific
characteristics of the features or aspects of the disclosure with
which that terminology is associated.
[0157] Where a range of values is provided, it is understood that
the upper and lower limit, and each intervening value between the
upper and lower limit of the range is encompassed within the
embodiments.
[0158] Terms and phrases used in this application, and variations
thereof, especially in the appended claims, unless otherwise
expressly stated, should be construed as open ended as opposed to
limiting. As examples of the foregoing, the term `including` should
be read to mean `including, without limitation,` `including but not
limited to,` or the like; the term `comprising` as used herein is
synonymous with `including,` `containing,` or `characterized by,`
and is inclusive or open-ended and does not exclude additional,
unrecited elements or method steps; the term `having` should be
interpreted as `having at least;` the term `includes` should be
interpreted as `includes but is not limited to;` the term `example`
is used to provide exemplary instances of the item in discussion,
not an exhaustive or limiting list thereof; adjectives such as
`known`, `normal`, `standard`, and terms of similar meaning should
not be construed as limiting the item described to a given time
period or to an item available as of a given time, but instead
should be read to encompass known, normal, or standard technologies
that may be available or known now or at any time in the future;
and use of terms like `preferably,` `preferred,` `desired,` or
`desirable,` and words of similar meaning should not be understood
as implying that certain features are critical, essential, or even
important to the structure or function of the invention, but
instead as merely intended to highlight alternative or additional
features that may or may not be utilized in a particular embodiment
of the invention. Likewise, a group of items linked with the
conjunction `and` should not be read as requiring that each and
every one of those items be present in the grouping, but rather
should be read as `and/or` unless expressly stated otherwise.
Similarly, a group of items linked with the conjunction `or` should
not be read as requiring mutual exclusivity among that group, but
rather should be read as `and/or` unless expressly stated
otherwise.
[0159] With respect to the use of substantially any plural and/or
singular terms herein, those having skill in the art can translate
from the plural to the singular and/or from the singular to the
plural as is appropriate to the context and/or application. The
various singular/plural permutations may be expressly set forth
herein for sake of clarity. The indefinite article "a" or "an" does
not exclude a plurality. The mere fact that certain measures are
recited in mutually different dependent claims does not indicate
that a combination of these measures cannot be used to advantage.
Any reference signs in the claims should not be construed as
limiting the scope.
[0160] It will be further understood by those within the art that
if a specific number of an introduced claim recitation is intended,
such an intent will be explicitly recited in the claim, and in the
absence of such recitation no such intent is present. For example,
as an aid to understanding, the following appended claims may
contain usage of the introductory phrases "at least one" and "one
or more" to introduce claim recitations. However, the use of such
phrases should not be construed to imply that the introduction of a
claim recitation by the indefinite articles "a" or "an" limits any
particular claim containing such introduced claim recitation to
embodiments containing only one such recitation, even when the same
claim includes the introductory phrases "one or more" or "at least
one" and indefinite articles such as "a" or "an" (e.g., "a" and/or
"an" should typically be interpreted to mean "at least one" or "one
or more"); the same holds true for the use of definite articles
used to introduce claim recitations. In addition, even if a
specific number of an introduced claim recitation is explicitly
recited, those skilled in the art will recognize that such
recitation should typically be interpreted to mean at least the
recited number (e.g., the bare recitation of "two recitations,"
without other modifiers, typically means at least two recitations,
or two or more recitations). Furthermore, in those instances where
a convention analogous to "at least one of A, B, and C, etc." is
used, in general such a construction is intended in the sense one
having skill in the art would understand the convention (e.g., "a
system having at least one of A, B, and C" would include but not be
limited to systems that have A alone, B alone, C alone, A and B
together, A and C together, B and C together, and/or A, B, and C
together, etc.). In those instances where a convention analogous to
"at least one of A, B, or C, etc." is used, in general such a
construction is intended in the sense one having skill in the art
would understand the convention (e.g., "a system having at least
one of A, B, or C" would include but not be limited to systems that
have A alone, B alone, C alone, A and B together, A and C together,
B and C together, and/or A, B, and C together, etc.). It will be
further understood by those within the art that virtually any
disjunctive word and/or phrase presenting two or more alternative
terms, whether in the description, claims, or drawings, should be
understood to contemplate the possibilities of including one of the
terms, either of the terms, or both terms. For example, the phrase
"A or B" will be understood to include the possibilities of "A" or
"B" or "A and B."
[0161] All numbers expressing quantities of ingredients, reaction
conditions, and so forth used in the specification 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 herein are approximations that may vary
depending upon the desired properties sought to be obtained. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of any claims in any
application claiming priority to the present application, each
numerical parameter should be construed in light of the number of
significant digits and ordinary rounding approaches.
[0162] Furthermore, although the foregoing has been described in
some detail by way of illustrations and examples for purposes of
clarity and understanding, it is apparent to those skilled in the
art that certain changes and modifications may be practiced.
Therefore, the description and examples should not be construed as
limiting the scope of the invention to the specific embodiments and
examples described herein, but rather to also cover all
modification and alternatives coming with the true scope and spirit
of the invention.
* * * * *