U.S. patent application number 17/233668 was filed with the patent office on 2021-10-14 for indolinone derivatives as inhibitors of maternal embryonic leucine zipper kinase.
The applicant listed for this patent is BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM. Invention is credited to Kevin N. Dalby, Ramakrishna Edupuganti, Ju Hyeon Lee, Juliana Taliaferro.
Application Number | 20210317107 17/233668 |
Document ID | / |
Family ID | 1000005655079 |
Filed Date | 2021-10-14 |
United States Patent
Application |
20210317107 |
Kind Code |
A1 |
Dalby; Kevin N. ; et
al. |
October 14, 2021 |
INDOLINONE DERIVATIVES AS INHIBITORS OF MATERNAL EMBRYONIC LEUCINE
ZIPPER KINASE
Abstract
The present disclosure relates to indolinone compounds,
compositions, and methods for the inhibition of maternal embryonic
leucine zipper kinase (MELK). The present disclosure further
relates to indolinone compounds, compositions, and methods for the
treatment or prevention of a cancer (for example, triple negative
breast cancer).
Inventors: |
Dalby; Kevin N.; (Leander,
TX) ; Edupuganti; Ramakrishna; (Austin, TX) ;
Taliaferro; Juliana; (Austin, TX) ; Lee; Ju
Hyeon; (Austin, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM |
Austin |
TX |
US |
|
|
Family ID: |
1000005655079 |
Appl. No.: |
17/233668 |
Filed: |
April 19, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16489803 |
Aug 29, 2019 |
10981896 |
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PCT/US2018/020668 |
Mar 2, 2018 |
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17233668 |
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62465992 |
Mar 2, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
C07D 405/14 20130101; C07D 209/34 20130101; C07D 403/12 20130101;
C07D 413/14 20130101; C07D 401/14 20130101 |
International
Class: |
C07D 403/12 20060101
C07D403/12; C07D 209/34 20060101 C07D209/34; C07D 401/14 20060101
C07D401/14; C07D 405/14 20060101 C07D405/14; C07D 413/14 20060101
C07D413/14 |
Claims
1.-11. (canceled)
12. A method for the treatment of a cancer, comprising:
administering an effective amount of a compound of Formula I to a
host in need thereof: ##STR00154## wherein: R.sup.1 is hydrogen;
R.sup.2 is selected from carboxylic acid, ester, amide, acyl,
alkoxy, sulfonamide, alkylamino, aminoacyl, or amino; and R.sup.3
is selected from aryl or heteroaryl; or a pharmaceutically
acceptable salt thereof.
13. The method of claim 12, wherein the compound comprises the
formula: ##STR00155## wherein: R.sup.1 is hydrogen; and R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, or amino; or a pharmaceutically
acceptable salt thereof.
14. The method of claim 12, wherein R.sup.2 is ester.
15. The method of claim 12, wherein R.sup.3 is selected from
phenyl, benzyl, pyridine, or furan.
16. The method of claim 12, wherein R.sup.3 is unsubstituted
aryl.
17. The method of claim 12, wherein R.sup.3 is phenyl.
18. The method of claim 12, wherein R.sup.3 is substituted
aryl.
19. The method of claim 12, wherein R.sup.3 is pyridine.
20. The method of claim 12, wherein R.sup.3 is furan.
21. The method of claim 12, wherein the compound is
##STR00156##
22. The method of claim 12, wherein the compound is
##STR00157##
23. (canceled)
24. The method of claim 12, wherein the cancer is triple negative
breast cancer.
25. The method of claim 12, wherein the cancer is glioblastoma
multiforme.
26. The method of claim 12, wherein the compound is administered in
combination with an additional chemotherapeutic agent.
27. A method for the treatment of a cancer, comprising:
administering an effective amount of a compound of Formula III to a
host in need thereof: ##STR00158## wherein: R.sup.1 is hydrogen;
R.sup.2 is selected from carboxylic acid, ester, amide, acyl,
alkoxy, sulfonamide, alkylamino, aminoacyl, amino, nitro, aryl, or
heteroaryl; each R.sup.4 is independently selected from hydrogen,
alkyl, carboxylic acid, ester, amide, acyl, alkoxy, hydroxyl,
hydroxyalkyl, sulfonamide, alkylamino, aminoacyl, amino, nitro,
heterocycloalkyl, heterocycloalkylalkyl, or NR.sup.5R.sup.6; or two
R.sup.4 come together to form a carbocyclic ring or a heterocyclic
ring; R.sup.5 and R.sup.6 are independently selected from hydrogen,
alkyl, alkylamino, heterocycloalkyl, acyl, or ester; and x is
selected from 1 or 2; or a pharmaceutically acceptable salt
thereof.
28. The method of claim 27, wherein R.sup.2 is ester.
29. The method of claim 27, wherein R.sup.4 is selected from
hydrogen or alkyl.
30. The method of claim 27, wherein x is 1.
31. The method of claim 27, wherein the cancer is triple negative
breast cancer.
32. The method of claim 27, wherein the cancer is glioblastoma
multiforme.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 62/465,992 filed Mar. 2, 2017, which is
expressly incorporated herein by reference in its entirety.
FIELD
[0002] The present disclosure relates to indolinone compounds,
compositions, and methods for the inhibition of maternal embryonic
leucine zipper kinase (MELK). The present disclosure further
relates to indolinone compounds, compositions, and methods for the
treatment or prevention of a cancer (for example, triple negative
breast cancer).
BACKGROUND
[0003] Over the last two decades, protein kinases have represented
a major field for drug discovery and development. However, one of
these protein kinases, maternal embryonic leucine zipper kinase
(MELK), has yet to be targeted by an FDA approved therapeutic.
[0004] Maternal embryonic leucine zipper kinase (MELK) is
evolutionarily conserved in eukaryotes from nematodes to humans. In
contrast to most members of the AMPK-RK family, which mediate cell
survival under stressful metabolic conditions, MELK has been
implicated in multiple cellular processes, including cell cycle
checkpoint regulation, proliferation, apoptosis, and RNA
processing. MELK is expressed in the early stages of murine
embryonic development, but MELK knockout mice develop normally with
no obvious pathologic phenotype, suggesting that MELK's
developmentally-related functions may be redundant. Yet despite its
apparent dispensable nature in differentiated adult cells, evidence
has implicated MELK's importance in proliferating progenitor
populations, including multipotent neural progenitors, myoblasts,
and mammary progenitors. Interestingly, MELK inhibition does not
affect survival in normal neural stem cells, but siRNA-mediated
MELK knockdown induces apoptosis selectively in glioma stem cells.
Such data reinforces the redundancy of MELK function in
noncancerous cells, but also implicates the existence of an
exploitable target in certain cancer stem cell populations.
[0005] In addition to its putative role in cancer stem cells,
upregulated MELK mRNA and protein levels have been observed in a
wide array of cancer cell types and clinical tumor samples. Of
particular note is the fact that MELK expression correlates with
poor prognosis in the most aggressive subsets of disease, including
glioblastoma multiforme (GBM) and triple negative breast cancer
(TNBC). Factors contributing to poor outlook for TNBC patients in
part stems from the cancer's ability not only to proliferate
quickly, but also its propensity to spread and recur in distant
organs. From a molecular biology perspective, mounting evidence
continues to implicate MELK in direct and transcriptional
regulation of cell division in the context of malignancy.
Furthermore, MELK has also been preliminarily linked to metastasis
through its involvement with TGF- driven epithelial-to-mesenchymal
transition (EMT). The cancer-specific expression pattern, combined
with the clinical and biological data have therefore justifiably
fostered strong interest in MELK as a clinical target.
[0006] The compounds, compositions, and methods disclosed herein
address these and other needs.
SUMMARY
[0007] Disclosed herein are indolinone compounds, compositions, and
methods for the potent and selective inhibition of maternal
embryonic leucine zipper (MELK) kinase. In some embodiments, the
present disclosure further relates to indolinone compounds,
compositions, and methods for the treatment or prevention of a
cancer (for example, triple negative breast cancer).
[0008] In one aspect, disclosed herein is a compound of Formula
I:
##STR00001##
[0009] wherein: [0010] R.sup.1 is hydrogen; [0011] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, or amino; and [0012] R.sup.3 is
selected from aryl or heteroaryl; [0013] or a pharmaceutically
acceptable salt thereof.
[0014] In one aspect, disclosed herein is a compound of Formula
II:
##STR00002##
[0015] wherein: [0016] R.sup.1 is hydrogen; and [0017] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, or amino; [0018] or a
pharmaceutically acceptable salt thereof.
[0019] In one aspect, disclosed herein is a compound of Formula
III:
##STR00003##
[0020] wherein: [0021] R.sup.1 is hydrogen; [0022] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, amino, nitro, aryl, or
heteroaryl; [0023] each R.sup.4 is independently selected from
hydrogen, alkyl, carboxylic acid, ester, amide, acyl, alkoxy,
hydroxyl, hydroxyalkyl, sulfonamide, alkylamino, aminoacyl, amino,
nitro, heterocycloalkyl, heterocycloalkylalkyl, or NR.sup.5R.sup.6;
or [0024] two R.sup.4 come together to form a carbocyclic ring or a
heterocyclic ring; [0025] R.sup.5 and R.sup.6 are independently
selected from hydrogen, alkyl, alkylamino, heterocycloalkyl, acyl,
or ester; and [0026] x is selected from 1 or 2; [0027] or a
pharmaceutically acceptable salt thereof.
[0028] In one aspect, disclosed herein is a compound of Formula
IV:
##STR00004##
[0029] wherein: [0030] R.sup.1 is hydrogen; [0031] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, amino, nitro, aryl, or
heteroaryl; [0032] each R.sup.4 is independently selected from
hydrogen, alkyl, carboxylic acid, ester, amide, acyl, alkoxy,
hydroxyl, hydroxyalkyl, sulfonamide, alkylamino, aminoacyl, amino,
nitro, heterocycloalkyl, heterocycloalkylalkyl, or NR.sup.5R.sup.6;
or [0033] two R.sup.4 come together to form a carbocyclic ring or a
heterocyclic ring; [0034] R.sup.5 and R.sup.6 are independently
selected from hydrogen, alkyl, alkylamino, heterocycloalkyl, acyl,
or ester; [0035] R.sup.7 is selected from NR.sup.8R.sup.9, alkyl,
alkylamino, heterocycloalkyl, or heterocycloalkylalkyl; [0036]
R.sup.8 and R.sup.9 are independently selected from hydrogen,
alkyl, alkylamino, heterocycloalkyl, heterocycloalkylalkyl, or
acyl; and [0037] x is selected from 1 or 2; [0038] or a
pharmaceutically acceptable salt thereof.
[0039] In one embodiment, the compound is
##STR00005##
[0040] In another embodiment, the compound is
##STR00006##
[0041] In one aspect, provided herein is a method for the treatment
of a cancer, comprising: administering an effective amount of a
compound of Formula I to a host in need thereof:
##STR00007##
[0042] wherein: [0043] R.sup.1 is hydrogen; [0044] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, or amino; and [0045] R.sup.3 is
selected from aryl or heteroaryl; [0046] or a pharmaceutically
acceptable salt thereof.
[0047] In another aspect, provided herein is a method for the
treatment of a cancer, comprising: administering an effective
amount of a compound of Formula II to a host in need thereof:
##STR00008##
[0048] wherein: [0049] R.sup.1 is hydrogen; and [0050] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, or amino; [0051] or a
pharmaceutically acceptable salt thereof.
[0052] In one aspect, provided herein is a method for the treatment
of a cancer, comprising: administering an effective amount of a
compound of Formula III to a host in need thereof:
##STR00009##
[0053] wherein: [0054] R.sup.1 is hydrogen; [0055] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, amino, nitro, aryl, or
heteroaryl; [0056] each R.sup.4 is independently selected from
hydrogen, alkyl, carboxylic acid, ester, amide, acyl, alkoxy,
hydroxyl, hydroxyalkyl, sulfonamide, alkylamino, aminoacyl, amino,
nitro, heterocycloalkyl, heterocycloalkylalkyl, or NR.sup.5R.sup.6;
or [0057] two R.sup.4 come together to form a carbocyclic ring or a
heterocyclic ring; [0058] R.sup.5 and R.sup.6 are independently
selected from hydrogen, alkyl, alkylamino, heterocycloalkyl, acyl,
or ester; and [0059] x is selected from 1 or 2; [0060] or a
pharmaceutically acceptable salt thereof.
[0061] In one aspect, provided herein is a method for the treatment
of a cancer, comprising: administering an effective amount of a
compound of Formula IV to a host in need thereof:
##STR00010##
[0062] wherein: [0063] R.sup.1 is hydrogen; [0064] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, amino, nitro, aryl, or
heteroaryl; [0065] each R.sup.4 is independently selected from
hydrogen, alkyl, carboxylic acid, ester, amide, acyl, alkoxy,
hydroxyl, hydroxyalkyl, sulfonamide, alkylamino, aminoacyl, amino,
nitro, heterocycloalkyl, heterocycloalkylalkyl, or NR.sup.5R.sup.6;
or [0066] two R.sup.4 come together to form a carbocyclic ring or a
heterocyclic ring; [0067] R.sup.5 and R.sup.6 are independently
selected from hydrogen, alkyl, alkylamino, heterocycloalkyl, acyl,
or ester; [0068] R.sup.7 is selected from NR.sup.8R.sup.9, alkyl,
alkylamino, heterocycloalkyl, or heterocycloalkylalkyl; [0069]
R.sup.8 and R.sup.9 are independently selected from hydrogen,
alkyl, alkylamino, heterocycloalkyl, heterocycloalkylalkyl, or
acyl; and [0070] x is selected from 1 or 2; [0071] or a
pharmaceutically acceptable salt thereof.
[0072] In one embodiment, the cancer is selected from triple
negative breast cancer or glioblastoma multiforme. In one
embodiment, the cancer is triple negative breast cancer. In one
embodiment, the cancer is glioblastoma multiforme.
[0073] In one embodiment, a compound of Formula I, II, III, or IV
is administered in combination with an additional chemotherapeutic
agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several aspects
described below.
[0075] FIG. 1 shows the top 3 screening hits for inhibition of
maternal embryonic leucine zipper kinase (MELK), with key predicted
hydrogen-bonding interactions with MELK binding site and their
IC.sub.50's: A) Nintedanib; B) S1529 (Hesperadin); and C)
CC-401.
[0076] FIG. 2 shows the molecular interactions between nintedanib
and Cpd2 with MELK. Molecular modeling studies were performed using
Gold 5.1 (Cambridge Crystallographic Data Center). A) Docking of
nintedanib (IC.sub.50.about.43 nM) into the ATP-binding site of the
MELK conformation PDB 4BKY. B) Crystal structure of a
benzodipyrazole inhibitor (Cpd2) in complex with the MELK catalytic
domain (PDB: 4BKZ).
[0077] FIG. 3 shows the top-down view of the ATP binding pocket of
MELK (PDB: 4BKY) and predicted binding poses of compounds 15
(6-CO.sub.2Me, A) and 17 (5-CO.sub.2Me, B). A) Compound 15 forms
hydrogen bonds with C89, E87, and K40 of the ATP binding pocket.
6th position substitution sterically prevents 15 from achieving
optimal complementarity with the curvature of the binding site. B)
5th position methyl ester substitution in 17 forms the same
hydrogen bonds, but allows deeper penetration of the compound into
the binding site, shortening hydrogen bond lengths between MELK and
the compound by up to an angstrom in some cases. Measurements were
generated using Pymol software. H-bonds are shown as dashed black
lines. ATP binding pocket is shaded in light purple. Ribbon
backbones of residues 13-19 and 23-27 are excluded for clarity.
[0078] FIG. 4 shows inhibitor 17 suppresses cell proliferation of
triple negative breast cancer (TNBC) cells in vitro. Inhibitor 17
was more effective than inhibitor 19 at suppressing cell
proliferation, and cells expressing high levels of MELK were more
sensitive to inhibitor 17 than cells expressing low levels of MELK.
Cells were seeded in 96-well plates and the next day treated with
inhibitors at the indicated concentrations. Seventy-two hours
later, cell viability was determined using the CellTiter-Blue Cell
Viability Assay for the following cell lines: A) HCC70; B) BT549;
C) SUM159; and D) MCF-10A cell lines. E) MELK expression levels are
shown for HCC70, BT549, and SUM159 cell lines. Tubulin is shown as
the loading control.
[0079] FIG. 5 shows time course and IC.sub.50 plots for the three
most potent inhibitors, compounds 16, 17, and 21. (a) Time course
for inhibition at different concentrations for compound 16. (b)
IC.sub.50 plot showing fractional activity vs. inhibitor
concentration for compound 16. (c) Time course for inhibition at
different concentrations for compound 17. (d) IC.sub.50 plot
showing fractional activity vs. inhibitor concentration for
compound 17. (e) Time course for inhibition at different
concentrations for compound 21. (f) IC.sub.50 plot showing
fractional activity vs. inhibitor concentration for compound 21.
Rates derived for compounds 16 and 21 represent the average for
three independent experiments, whereas those for compound 17 are
the average for 4 separate experiments. K values were fit directly
in Prism.RTM. using experimentally determined K.sub.M.sup.ATP of
6.+-.1.5 .mu.M (two independent replicates) and an ATP
concentration of 40 .mu.M.
[0080] FIG. 6 shows the X-ray structure for the 5-CO.sub.2Me analog
(compound 17).
DETAILED DESCRIPTION
[0081] Disclosed herein are indolinone compounds, compositions, and
methods for the potent and selective inhibition of maternal
embryonic leucine zipper (MELK) kinase. In some embodiments, the
present disclosure further relates to indolinone compounds,
compositions, and methods for the treatment or prevention of a
cancer (for example, triple negative breast cancer).
[0082] Reference will now be made in detail to the embodiments of
the invention, examples of which are illustrated in the drawings
and the examples. This invention may, however, be embodied in many
different forms and should not be construed as limited to the
embodiments set forth herein.
[0083] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. The
following definitions are provided for the full understanding of
terms used in this specification.
Terminology
[0084] As used in the specification and claims, the singular form
"a," "an," and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0085] As used herein, the terms "may," "optionally," and "may
optionally" are used interchangeably and are meant to include cases
in which the condition occurs as well as cases in which the
condition does not occur. Thus, for example, the statement that a
formulation "may include an excipient" is meant to include cases in
which the formulation includes an excipient as well as cases in
which the formulation does not include an excipient.
[0086] As used here, the terms "beneficial agent" and "active
agent" are used interchangeably herein to refer to a chemical
compound or composition that has a beneficial biological effect.
Beneficial biological effects include both therapeutic effects,
i.e., treatment of a disorder or other undesirable physiological
condition, and prophylactic effects, i.e., prevention of a disorder
or other undesirable physiological condition. The terms also
encompass pharmaceutically acceptable, pharmacologically active
derivatives of beneficial agents specifically mentioned herein,
including, but not limited to, salts, esters, amides, prodrugs,
active metabolites, isomers, fragments, analogs, and the like. When
the terms "beneficial agent" or "active agent" are used, then, or
when a particular agent is specifically identified, it is to be
understood that the term includes the agent per se as well as
pharmaceutically acceptable, pharmacologically active salts,
esters, amides, prodrugs, conjugates, active metabolites, isomers,
fragments, analogs, etc.
[0087] As used herein, the terms "treating" or "treatment" of a
subject includes the administration of a drug to a subject with the
purpose of preventing, curing, healing, alleviating, relieving,
altering, remedying, ameliorating, improving, stabilizing or
affecting a disease or disorder, or a symptom of a disease or
disorder. The terms "treating" and "treatment" can also refer to
reduction in severity and/or frequency of symptoms, elimination of
symptoms and/or underlying cause, prevention of the occurrence of
symptoms and/or their underlying cause, and improvement or
remediation of damage.
[0088] As used herein, the term "preventing" a disorder or unwanted
physiological event in a subject refers specifically to the
prevention of the occurrence of symptoms and/or their underlying
cause, wherein the subject may or may not exhibit heightened
susceptibility to the disorder or event.
[0089] By the term "effective amount" of a therapeutic agent is
meant a nontoxic but sufficient amount of a beneficial agent to
provide the desired effect. The amount of beneficial agent that is
"effective" will vary from subject to subject, depending on the age
and general condition of the subject, the particular beneficial
agent or agents, and the like. Thus, it is not always possible to
specify an exact "effective amount." However, an appropriate
"effective" amount in any subject case may be determined by one of
ordinary skill in the art using routine experimentation. Also, as
used herein, and unless specifically stated otherwise, an
"effective amount" of a beneficial can also refer to an amount
covering both therapeutically effective amounts and
prophylactically effective amounts.
[0090] An "effective amount" of a drug necessary to achieve a
therapeutic effect may vary according to factors such as the age,
sex, and weight of the subject. Dosage regimens can be adjusted to
provide the optimum therapeutic response. For example, several
divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation.
[0091] As used herein, a "therapeutically effective amount" of a
therapeutic agent refers to an amount that is effective to achieve
a desired therapeutic result, and a "prophylactically effective
amount" of a therapeutic agent refers to an amount that is
effective to prevent an unwanted physiological condition.
Therapeutically effective and prophylactically effective amounts of
a given therapeutic agent will typically vary with respect to
factors such as the type and severity of the disorder or disease
being treated and the age, gender, and weight of the subject.
[0092] The term "therapeutically effective amount" can also refer
to an amount of a therapeutic agent, or a rate of delivery of a
therapeutic agent (e.g., amount over time), effective to facilitate
a desired therapeutic effect. The precise desired therapeutic
effect will vary according to the condition to be treated, the
tolerance of the subject, the drug and/or drug formulation to be
administered (e.g., the potency of the therapeutic agent (drug),
the concentration of drug in the formulation, and the like), and a
variety of other factors that are appreciated by those of ordinary
skill in the art.
[0093] As used herein, the term "pharmaceutically acceptable"
component can refer to a component that is not biologically or
otherwise undesirable, i.e., the component may be incorporated into
a pharmaceutical formulation of the invention and administered to a
subject as described herein without causing any significant
undesirable biological effects or interacting in a deleterious
manner with any of the other components of the formulation in which
it is contained.
[0094] When the term "pharmaceutically acceptable" is used to refer
to an excipient, it is generally implied that the component has met
the required standards of toxicological and manufacturing testing
or that it is included on the Inactive Ingredient Guide prepared by
the U.S. Food and Drug Administration.
[0095] Also, as used herein, the term "pharmacologically active"
(or simply "active"), as in a "pharmacologically active" derivative
or analog, can refer to a derivative or analog (e.g., a salt,
ester, amide, conjugate, metabolite, isomer, fragment, etc.) having
the same type of pharmacological activity as the parent compound
and approximately equivalent in degree.
[0096] As used herein, the term "subject" or "host" can refer to
living organisms such as mammals, including, but not limited to
humans, livestock, dogs, cats, and other mammals. Administration of
the therapeutic agents can be carried out at dosages and for
periods of time effective for treatment of a subject. In some
embodiments, the subject is a human.
Chemical Terminology
[0097] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic, and
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
below. The permissible substituents can be one or more and the same
or different for appropriate organic compounds. For purposes of
this disclosure, the heteroatoms, such as nitrogen, can have
hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valences of
the heteroatoms. This disclosure is not intended to be limited in
any manner by the permissible substituents of organic compounds.
Also, the terms "substitution" or "substituted with" include the
implicit proviso that such substitution is in accordance with
permitted valence of the substituted atom and the substituent, and
that the substitution results in a stable compound, e.g., a
compound that does not spontaneously undergo transformation such as
by rearrangement, cyclization, elimination, etc.
[0098] "Z.sup.1," "Z.sup.2," "Z.sup.3," and "Z.sup.4" are used
herein as generic symbols to represent various specific
substituents. These symbols can be any substituent, not limited to
those disclosed herein, and when they are defined to be certain
substituents in one instance, they can, in another instance, be
defined as some other substituents.
[0099] The term "aliphatic" as used herein refers to a non-aromatic
hydrocarbon group and includes branched and unbranched, alkyl,
alkenyl, or alkynyl groups.
[0100] The term "alkyl" as used herein is a branched or unbranched
saturated hydrocarbon group. In some embodiments, the alkyl
comprises 1 to 10 carbon atoms, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,
octyl, nonyl, decyl, and the like. The alkyl group can also be
substituted or unsubstituted. The alkyl group can be substituted
with one or more groups including, but not limited to, alkyl,
halogenated alkyl, alkoxy, alkenyl, alkynyl, acyl, aryl,
heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,
hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone,
sulfoxide, or thiol, as described below.
[0101] Throughout the specification "alkyl" is generally used to
refer to both unsubstituted alkyl groups and substituted alkyl
groups; however, substituted alkyl groups are also specifically
referred to herein by identifying the specific substituent(s) on
the alkyl group. For example, the term "halogenated alkyl"
specifically refers to an alkyl group that is substituted with one
or more halide, e.g., fluorine, chlorine, bromine, or iodine. The
term "alkoxyalkyl" specifically refers to an alkyl group that is
substituted with one or more alkoxy groups, as described below. The
term "alkylamino" specifically refers to an alkyl group that is
substituted with one or more amino groups, as described below, and
the like. When "alkyl" is used in one instance and a specific term
such as "alkylalcohol" is used in another, it is not meant to imply
that the term "alkyl" does not also refer to specific terms such as
"alkylalcohol" and the like. This practice is also used for other
groups described herein. That is, while a term such as "cycloalkyl"
refers to both unsubstituted and substituted cycloalkyl moieties,
the substituted moieties can, in addition, be specifically
identified herein; for example, a particular substituted cycloalkyl
can be referred to as, e.g., an "alkylcycloalkyl." Similarly, a
substituted alkoxy can be specifically referred to as, e.g., a
"halogenated alkoxy," a particular substituted alkenyl can be,
e.g., an "alkenylalcohol," and the like. Again, the practice of
using a general term, such as "cycloalkyl," and a specific term,
such as "alkylcycloalkyl," is not meant to imply that the general
term does not also include the specific term.
[0102] The term "alkoxy" as used herein is an alkyl group bound
through a single, terminal ether linkage; that is, an "alkoxy"
group can be defined as --OZ.sup.1 where Z.sup.1 is alkyl as
defined above.
[0103] The term "alkenyl" as used herein is a hydrocarbon group of
from 2 to 24 carbon atoms with a structural formula containing at
least one carbon-carbon double bond. Asymmetric structures such as
(Z.sup.1Z.sup.2)C.dbd.C(Z.sup.3Z.sup.4) are intended to include
both the E and Z isomers. This can be presumed in structural
formulae herein wherein an asymmetric alkene is present, or it can
be explicitly indicated by the bond symbol C.dbd.C. The alkenyl
group can be substituted with one or more groups including, but not
limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl,
acyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol, as described below.
[0104] The term "alkynyl" as used herein is a hydrocarbon group of
2 to 24 carbon atoms with a structural formula containing at least
one carbon-carbon triple bond. The alkynyl group can be substituted
with one or more groups including, but not limited to, alkyl,
halogenated alkyl, alkoxy, alkenyl, alkynyl, acyl, aryl,
heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,
hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone,
sulfoxide, or thiol, as described below.
[0105] The term "aryl" as used herein is a group that contains any
carbon-based aromatic group including, but not limited to, benzene,
naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The
term "heteroaryl" is defined as a group that contains an aromatic
group that has at least one heteroatom incorporated within the ring
of the aromatic group. Examples of heteroatoms include, but are not
limited to, nitrogen, oxygen, sulfur, and phosphorus. The term
"non-heteroaryl," which is included in the term "aryl," defines a
group that contains an aromatic group that does not contain a
heteroatom. The aryl or heteroaryl group can be substituted or
unsubstituted. The aryl or heteroaryl group can be substituted with
one or more groups including, but not limited to, alkyl,
halogenated alkyl, alkoxy, alkenyl, alkynyl, acyl, aryl,
heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,
hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone,
sulfoxide, or thiol as described herein. The term "biaryl" is a
specific type of aryl group and is included in the definition of
aryl. Biaryl refers to two aryl groups that are bound together via
a fused ring structure, as in naphthalene, or are attached via one
or more carbon-carbon bonds, as in biphenyl.
[0106] The term "cycloalkyl" as used herein is a non-aromatic
carbon-based ring composed of at least three carbon atoms. Examples
of cycloalkyl groups include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, etc. The term
"heterocycloalkyl" is a cycloalkyl group as defined above where at
least one of the carbon atoms of the ring is substituted with a
heteroatom such as, but not limited to, nitrogen, oxygen, sulfur,
or phosphorus. The cycloalkyl group and heterocycloalkyl group can
be substituted or unsubstituted. The cycloalkyl group and
heterocycloalkyl group can be substituted with one or more groups
including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl,
acyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,
ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl,
sulfone, sulfoxide, or thiol as described herein.
[0107] The term "cycloalkenyl" as used herein is a non-aromatic
carbon-based ring composed of at least three carbon atoms and
containing at least one double bound, i.e., C.dbd.C. Examples of
cycloalkenyl groups include, but are not limited to, cyclopropenyl,
cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl,
cyclohexadienyl, and the like. The term "heterocycloalkenyl" is a
type of cycloalkenyl group as defined above, and is included within
the meaning of the term "cycloalkenyl," where at least one of the
carbon atoms of the ring is substituted with a heteroatom such as,
but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The
cycloalkenyl group and heterocycloalkenyl group can be substituted
or unsubstituted. The cycloalkenyl group and heterocycloalkenyl
group can be substituted with one or more groups including, but not
limited to, alkyl, alkoxy, alkenyl, alkynyl, acyl, aryl,
heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,
hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone,
sulfoxide, or thiol as described herein.
[0108] The term "cyclic group" is used herein to refer to either
aryl groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl,
cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic
groups have one or more ring systems that can be substituted or
unsubstituted. A cyclic group can contain one or more aryl groups,
one or more non-aryl groups, or one or more aryl groups and one or
more non-aryl groups.
[0109] The term "aldehyde" as used herein is represented by the
formula --C(O)H. Throughout this specification "C(O)" or "CO" is a
short hand notation for C.dbd.O.
[0110] The terms "amine" or "amino" as used herein are represented
by the formula --NZ.sup.1Z.sup.2, where Z.sup.1 and Z.sup.2 can
each be substitution group as described herein, such as hydrogen,
an alkyl, halogenated alkyl, alkenyl, alkynyl, acyl, aryl,
heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or
heterocycloalkenyl group described above. The term "aminoacyl"
specifically refers to an amino group that is substituted with one
or more acyl groups, as described below.
[0111] The term "carboxylic acid" as used herein is represented by
the formula --C(O)OH. A "carboxylate" or "carboxyl" group as used
herein is represented by the formula --C(O)O.sup.-.
[0112] The term "ester" as used herein is represented by the
formula --OC(O)Z.sup.1 or --C(O)OZ.sup.1, where Z.sup.1 can be an
alkyl, halogenated alkyl, alkenyl, alkynyl, acyl, aryl, heteroaryl,
cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl
group described above. The term "alkylester" indicates an alkyl
group as defined herein covalently bound to the group it
substitutes by an ester linkage. The ester linkage may be in either
orientation, e.g., a group of the formula --O(C.dbd.O)alkyl or a
group of the formula --(C+O)Oalkyl.
[0113] The term "ether" as used herein is represented by the
formula Z.sup.1OZ.sup.2, where Z.sup.1 and Z.sup.2 can be,
independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, acyl,
aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or
heterocycloalkenyl group described above.
[0114] The term "ketone" as used herein is represented by the
formula Z.sup.1C(O)Z.sup.2, where Z.sup.1 and Z.sup.2 can be,
independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, acyl,
aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or
heterocycloalkenyl group described above.
[0115] The term "acyl" refers to a group of the formula
--C(O)Z.sup.1, wherein Z.sup.1 can be an alkyl, halogenated alkyl,
alkenyl, alkynyl, acyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,
heterocycloalkyl, or heterocycloalkenyl group described above. For
example, the term "C.sub.2-7 acyl" as used herein, either alone or
in combination with another substituent, means a C.sub.1-6 alkyl
group linked through a carbonyl group such as --C(O)--C.sub.1-6
alkyl.
[0116] The term "amide" or "carboxamide" refers to a group of the
formula --C(O)NZ.sup.1Z.sup.2 where Z.sup.1 and Z.sup.2 can be,
independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, acyl,
aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or
heterocycloalkenyl group described above; or together with the
nitrogen to which they are bonded, Z.sup.1 and Z.sup.2 can form a
heterocyclic ring.
[0117] The term "halide" or "halogen" or "halo" as used herein
refers to the fluorine, chlorine, bromine, and iodine.
[0118] The term "hydroxyl" or "hydroxy" as used herein is
represented by the formula --OH.
[0119] The term "nitro" as used herein is represented by the
formula --NO.sub.2.
[0120] The term "silyl" as used herein is represented by the
formula --SiZ.sup.1Z.sup.2Z.sup.3, where Z.sup.1, Z.sup.2, and
Z.sup.3 can be, independently, hydrogen, alkyl, halogenated alkyl,
alkoxy, alkenyl, alkynyl, acyl, aryl, heteroaryl, cycloalkyl,
cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group
described above.
[0121] The term "sulfonyl" is used herein to refer to the sulfo-oxo
group represented by the formula --S(O).sub.2Z.sup.1, where Z.sup.1
can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl,
acyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl,
or heterocycloalkenyl group described above.
[0122] The term "sulfonylamino" or "sulfonamide" as used herein is
represented by the formula --S(O).sub.2NH--.
[0123] The term "thiol" as used herein is represented by the
formula --SH.
[0124] The term "thio" as used herein is represented by the formula
--S--.
[0125] "R.sup.1," "R.sup.2," "R.sup.3," "R.sup.n," etc., where n is
some integer, as used herein can, independently, possess one or
more of the groups listed above. For example, if R.sup.1 is a
straight chain alkyl group, one of the hydrogen atoms of the alkyl
group can optionally be substituted with a hydroxyl group, an
alkoxyl group, an amine group, an alkyl group, a halide, and the
like. Depending upon the groups that are selected, a first group
can be incorporated within second group or, alternatively, the
first group can be pendant (i.e., attached) to the second group.
For example, with the phrase "an alkyl group comprising an amino
group," the amino group can be incorporated within the backbone of
the alkyl group. Alternatively, the amino group can be attached to
the backbone of the alkyl group. The nature of the group(s) that is
(are) selected will determine if the first group is embedded or
attached to the second group.
[0126] Unless stated to the contrary, a formula with chemical bonds
shown only as solid lines and not as wedges or dashed lines
contemplates each possible isomer, e.g., each enantiomer,
diastereomer, and meso compound, and a mixture of isomers, such as
a racemic or scalemic mixture.
[0127] Reference will now be made in detail to specific aspects of
the disclosed materials, compounds, compositions, and methods,
examples of which are illustrated in the accompanying Examples and
Figures.
Compounds
[0128] In one aspect, disclosed herein is a compound of Formula
I:
##STR00011##
[0129] wherein: [0130] R.sup.1 is hydrogen; [0131] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, or amino; and [0132] R.sup.3 is
selected from aryl or heteroaryl; [0133] or a pharmaceutically
acceptable salt thereof.
[0134] In another aspect, disclosed herein is a compound of Formula
II:
##STR00012##
[0135] wherein: [0136] R.sup.1 is hydrogen; and [0137] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, or amino; [0138] or a
pharmaceutically acceptable salt thereof.
[0139] In one aspect, disclosed herein is a compound of Formula
III:
##STR00013##
[0140] wherein: [0141] R.sup.1 is hydrogen; [0142] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, amino, nitro, aryl, or
heteroaryl; [0143] each R.sup.4 is independently selected from
hydrogen, alkyl, carboxylic acid, ester, amide, acyl, alkoxy,
hydroxyl, hydroxyalkyl, sulfonamide, alkylamino, aminoacyl, amino,
nitro, heterocycloalkyl, heterocycloalkylalkyl, or NR.sup.5R.sup.6;
or [0144] two R.sup.4 come together to form a carbocyclic ring or a
heterocyclic ring; [0145] R.sup.5 and R.sup.6 are independently
selected from hydrogen, alkyl, alkylamino, heterocycloalkyl, acyl,
or ester; and [0146] x is selected from 1 or 2; [0147] or a
pharmaceutically acceptable salt thereof.
[0148] In one aspect, disclosed herein is a compound of Formula
IV:
##STR00014##
[0149] wherein: [0150] R.sup.1 is hydrogen; [0151] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, amino, nitro, aryl, or
heteroaryl; [0152] each R.sup.4 is independently selected from
hydrogen, alkyl, carboxylic acid, ester, amide, acyl, alkoxy,
hydroxyl, hydroxyalkyl, sulfonamide, alkylamino, aminoacyl, amino,
nitro, heterocycloalkyl, heterocycloalkylalkyl, or NR.sup.5R.sup.6;
or [0153] two R.sup.4 come together to form a carbocyclic ring or a
heterocyclic ring; [0154] R.sup.5 and R.sup.6 are independently
selected from hydrogen, alkyl, alkylamino, heterocycloalkyl, acyl,
or ester; [0155] R.sup.7 is selected from NR.sup.8R.sup.9, alkyl,
alkylamino, heterocycloalkyl, or heterocycloalkylalkyl; [0156]
R.sup.8 and R.sup.9 are independently selected from hydrogen,
alkyl, alkylamino, heterocycloalkyl, heterocycloalkylalkyl, or
acyl; and [0157] x is selected from 1 or 2; [0158] or a
pharmaceutically acceptable salt thereof.
[0159] In one embodiment, R.sup.2 is unsubstituted. In one
embodiment, R.sup.2 is substituted. In one embodiment, R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, or amino. In one embodiment,
R.sup.2 is selected from carboxylic acid, ester, amide, acyl,
alkoxy, sulfonamide, alkylamino, aminoacyl, amino, nitro, aryl, or
heteroaryl. In one embodiment, R.sup.2 is ester. In one embodiment,
R.sup.2 is alkylester. In one embodiment, R.sup.2 is methyl ester.
In one embodiment, R.sup.2 is carboxylic acid. In one embodiment,
R.sup.2 is ester. In one embodiment, R.sup.2 is amide. In one
embodiment, R.sup.2 is acyl. In one embodiment, R.sup.2 is
sulfonamide. In one embodiment, R.sup.2 is alkylamino. In one
embodiment, R.sup.2 is aminoacyl. In one embodiment, R.sup.2 is
amino. In one embodiment, R.sup.2 is nitro. In one embodiment,
R.sup.2 is aryl. In one embodiment, R.sup.2 is heteroaryl.
[0160] In one embodiment, R.sup.2 is alkoxy. In one embodiment,
R.sup.2 is substituted alkoxy. In one embodiment, R.sup.2 is
alkoxy, wherein the alkoxy is substituted with alkyl, aryl,
alkylaryl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, or
heterocycloalkylalkyl.
[0161] In one embodiment, R.sup.3 is unsubstituted. In one
embodiment, R.sup.3 is substituted. In one embodiment, R.sup.3 is
selected from aryl or heteroaryl. In one embodiment, R.sup.3 is
selected from phenyl, benzyl, pyridine, or furan. In one
embodiment, R.sup.3 is unsubstituted aryl. In one embodiment,
R.sup.3 is phenyl. In one embodiment, R.sup.3 is substituted aryl.
In one embodiment, R.sup.3 is substituted aryl, wherein the
substituted aryl is an aryl group substituted with alkyl, amino,
alkylamino, amide, aminoacyl, hydroxy, alkoxy, aryloxy, carboxylic
acid, or ester, or a combination thereof. In one embodiment,
R.sup.3 is pyridine. In one embodiment, R.sup.3 is a pyridine
derivative. In one embodiment, R.sup.3 is a substituted pyridine.
In one embodiment, R.sup.3 is furan.
[0162] In one embodiment, R.sup.3 is unsubstituted heteroaryl. In
one embodiment, R.sup.3 is substituted heteroaryl. In one
embodiment, R.sup.3 is substituted heteroaryl, wherein the
substituted heteroaryl is a heteroaryl group substituted with
alkyl, amino, alkylamino, amide, aminoacyl, hydroxy, alkoxy,
aryloxy, carboxylic acid, or ester, or a combination thereof.
[0163] In some embodiments, each R.sup.4 is independently selected
from hydrogen, alkyl, carboxylic acid, ester, amide, acyl, alkoxy,
hydroxyl, hydroxyalkyl, sulfonamide, alkylamino, aminoacyl, amino,
nitro, heterocycloalkyl, heterocycloalkylalkyl, or NR.sup.5R.sup.6.
In some embodiments, two R.sup.4 come together to form a
carbocyclic ring or a heterocyclic ring.
[0164] In some embodiments, two R.sup.4 come together to form an
unsubstituted or substituted carbocyclic ring; or an unsubstituted
or substituted heterocyclic ring.
[0165] In some embodiments, R.sup.4 is hydrogen. In some
embodiments, R.sup.4 is alkyl. In some embodiments, R.sup.4 is
carboxylic acid. In some embodiments, R.sup.4 is ester. In some
embodiments, R.sup.4 is amide. In some embodiments, R.sup.4 is
acyl. In some embodiments, R.sup.4 is alkoxy. In some embodiments,
R.sup.4 is hydroxyl. In some embodiments, R.sup.4 is hydroxyalkyl.
In some embodiments, R.sup.4 is sulfonamide. In some embodiments,
R.sup.4 is alkylamino. In some embodiments, R.sup.4 is aminoacyl.
In some embodiments, R.sup.4 is amino. In some embodiments, R.sup.4
is nitro. In some embodiments, R.sup.4 is heterocycloalkyl. In some
embodiments, R.sup.4 is heterocycloalkylalkyl. In some embodiments,
R.sup.4 is NR.sup.5R.sup.6.
[0166] In some embodiments, R.sup.5 and R.sup.6 are independently
selected from hydrogen, alkyl, alkylamino, heterocycloalkyl, acyl,
or ester. In some embodiments, R.sup.5 is hydrogen. In some
embodiments, R.sup.5 is alkyl. In some embodiments, R.sup.5 is
alkylamino. In some embodiments, R.sup.5 is heterocycloalkyl. In
some embodiments, R.sup.5 is acyl. In some embodiments, R.sup.5 is
ester. In some embodiments, R.sup.6 is hydrogen. In some
embodiments, R.sup.6 is alkyl. In some embodiments, R.sup.6 is
alkylamino. In some embodiments, R.sup.6 is heterocycloalkyl. In
some embodiments, R.sup.6 is acyl. In some embodiments, R.sup.6 is
ester.
[0167] In some embodiments, R.sup.7 is selected from
NR.sup.8R.sup.9, alkyl, alkylamino, or heterocycloalkyl. In some
embodiments, R.sup.7 is NR.sup.8R.sup.9. In some embodiments,
R.sup.7 is alkyl. In some embodiments, R.sup.7 is alkylamino. In
some embodiments, R.sup.7 is heterocycloalkyl.
[0168] In some embodiments, R.sup.8 and R.sup.9 are independently
selected from hydrogen, alkyl, alkylamino, heterocycloalkyl,
heterocycloalkylalkyl, or acyl. In some embodiments, R.sup.8 is
hydrogen. In some embodiments, R.sup.8 is alkyl. In some
embodiments, R.sup.8 is alkylamino. In some embodiments, R.sup.8 is
heterocycloalkyl. In some embodiments, R.sup.8 is
heterocycloalkylalkyl. In some embodiments, R.sup.8 is acyl. In
some embodiments, R.sup.8 is substituted acyl. In some embodiments,
R.sup.8 is acyl, wherein the acyl is substituted with hydrogen,
alkyl, alkylamino, heterocycloalkyl, or heterocycloalkylalkyl. In
some embodiments, R.sup.9 is hydrogen. In some embodiments, R.sup.9
is alkyl. In some embodiments, R.sup.9 is alkylamino. In some
embodiments, R.sup.9 is heterocycloalkyl. In some embodiments,
R.sup.9 is heterocycloalkylalkyl. In some embodiments, R.sup.9 is
acyl. In some embodiments, R.sup.9 is substituted acyl. In some
embodiments, R.sup.9 is acyl, wherein the acyl is substituted with
hydrogen, alkyl, alkylamino, heterocycloalkyl, or
heterocycloalkylalkyl.
[0169] In some embodiments, x is selected from 1 or 2. In some
embodiments, x is 2. In some embodiments, x is 1.
[0170] In one embodiment, the compound is
##STR00015##
[0171] In another embodiment, the compound is
##STR00016##
[0172] In some embodiments, the compound of Formula I, II, III, or
IV is selected from the compounds listed in Table 1.
TABLE-US-00001 TABLE 1 Non-limiting examples of compounds of
Formula I, II, III, or IV Compound Structure 17 ##STR00017## 18
##STR00018## 19 ##STR00019## 21 ##STR00020## 24 ##STR00021## 25
##STR00022##
[0173] In some embodiments, the compound of Formula I, II, III, or
IV is selected from the compounds listed in Table 2.
TABLE-US-00002 TABLE 2 Additional non-limiting examples of
compounds of Formula I, II, III, or IV Table 2 Structure No.
Structure 91 ##STR00023## 92 ##STR00024## 93 ##STR00025## 94
##STR00026## 95 ##STR00027## 96 ##STR00028## 97 ##STR00029## 98
##STR00030## 100 ##STR00031## 102 ##STR00032## 103 ##STR00033## 104
##STR00034## 105 ##STR00035## 106 ##STR00036## 107 ##STR00037## 108
##STR00038##
[0174] In some embodiments, the compound of Formula I, II, III, or
IV is selected from the compounds listed in Table 3.
TABLE-US-00003 TABLE 3 Additional non-limiting examples of
compounds of Formula I, II, III, or IV Table 3 Structure No.
Structure 109 ##STR00039## 110 ##STR00040## 111 ##STR00041## 112
##STR00042## 113 ##STR00043## 114 ##STR00044## 115 ##STR00045## 116
##STR00046## 117 ##STR00047## 118 ##STR00048## 119 ##STR00049## 120
##STR00050## 121 ##STR00051## 122 ##STR00052## 123 ##STR00053## 124
##STR00054## 125 ##STR00055## 126 ##STR00056## 127 ##STR00057## 128
##STR00058## 129 ##STR00059## 130 ##STR00060## 131 ##STR00061## 132
##STR00062##
[0175] In some embodiments, the compound is:
##STR00063##
Methods
[0176] In one aspect, provided herein is a method for the treatment
of a cancer, comprising: administering an effective amount of a
compound of Formula I to a host in need thereof:
##STR00064##
[0177] wherein: [0178] R.sup.1 is hydrogen; [0179] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, or amino; and [0180] R.sup.3 is
selected from aryl or heteroaryl; [0181] or a pharmaceutically
acceptable salt thereof.
[0182] In another aspect, provided herein is a method for the
treatment of a cancer, comprising: administering an effective
amount of a compound of Formula II to a host in need thereof:
##STR00065##
[0183] wherein: [0184] R.sup.1 is hydrogen; and [0185] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, or amino; [0186] or a
pharmaceutically acceptable salt thereof.
[0187] In one aspect, provided herein is a method for the treatment
of a cancer, comprising: administering an effective amount of a
compound of Formula III to a host in need thereof:
##STR00066##
[0188] wherein: [0189] R.sup.1 is hydrogen; [0190] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, amino, nitro, aryl, or
heteroaryl; [0191] each R.sup.4 is independently selected from
hydrogen, alkyl, carboxylic acid, ester, amide, acyl, alkoxy,
hydroxyl, hydroxyalkyl, sulfonamide, alkylamino, aminoacyl, amino,
nitro, heterocycloalkyl, heterocycloalkylalkyl, or NR.sup.5R.sup.6;
or [0192] two R.sup.4 come together to form a carbocyclic ring or a
heterocyclic ring; [0193] R.sup.5 and R.sup.6 are independently
selected from hydrogen, alkyl, alkylamino, heterocycloalkyl, acyl,
or ester; and [0194] x is selected from 1 or 2; [0195] or a
pharmaceutically acceptable salt thereof.
[0196] In one aspect, provided herein is a method for the treatment
of a cancer, comprising: administering an effective amount of a
compound of Formula IV to a host in need thereof:
##STR00067##
[0197] wherein: [0198] R.sup.1 is hydrogen; [0199] R.sup.2 is
selected from carboxylic acid, ester, amide, acyl, alkoxy,
sulfonamide, alkylamino, aminoacyl, amino, nitro, aryl, or
heteroaryl; [0200] each R.sup.4 is independently selected from
hydrogen, alkyl, carboxylic acid, ester, amide, acyl, alkoxy,
hydroxyl, hydroxyalkyl, sulfonamide, alkylamino, aminoacyl, amino,
nitro, heterocycloalkyl, heterocycloalkylalkyl, or NR.sup.5R.sup.6;
or [0201] two R.sup.4 come together to form a carbocyclic ring or a
heterocyclic ring; [0202] R.sup.5 and R.sup.6 are independently
selected from hydrogen, alkyl, alkylamino, heterocycloalkyl, acyl,
or ester; [0203] R.sup.7 is selected from NR.sup.8R.sup.9, alkyl,
alkylamino, heterocycloalkyl, or heterocycloalkylalkyl; [0204]
R.sup.8 and R.sup.9 are independently selected from hydrogen,
alkyl, alkylamino, heterocycloalkyl, heterocycloalkylalkyl, or
acyl; and [0205] x is selected from 1 or 2; [0206] or a
pharmaceutically acceptable salt thereof.
[0207] In one embodiment, the cancer is selected from triple
negative breast cancer or glioblastoma multiforme. In one
embodiment, the cancer is triple negative breast cancer. In one
embodiment, the cancer is glioblastoma multiforme.
[0208] In some embodiments, the methods described herein are used
for the treatment of the prevention of a cancer, for example,
melanoma, lung cancer (including lung adenocarcinoma, basal cell
carcinoma, squamous cell carcinoma, large cell carcinoma,
bronchioloalveolar carcinoma, bronchogenic carcinoma,
non-small-cell carcinoma, small cell carcinoma, mesothelioma);
breast cancer (including triple negative breast cancer (TNBC),
ductal carcinoma, lobular carcinoma, inflammatory breast cancer,
clear cell carcinoma, mucinous carcinoma, serosal cavities breast
carcinoma); colorectal cancer (colon cancer, rectal cancer,
colorectal adenocarcinoma); anal cancer; pancreatic cancer
(including pancreatic adenocarcinoma, islet cell carcinoma,
neuroendocrine tumors); prostate cancer; prostate adenocarcinoma;
ovarian carcinoma (ovarian epithelial carcinoma or surface
epithelial-stromal tumor including serous tumor, endometrioid tumor
and mucinous cystadenocarcinoma, sex-cord-stromal tumor); liver and
bile duct carcinoma (including hepatocellular carcinoma,
cholangiocarcinoma, hemangioma); esophageal carcinoma (including
esophageal adenocarcinoma and squamous cell carcinoma); oral and
oropharyngeal squamous cell carcinoma; salivary gland adenoid
cystic carcinoma; bladder cancer; bladder carcinoma; carcinoma of
the uterus (including endometrial adenocarcinoma, ocular, uterine
papillary serous carcinoma, uterine clear-cell carcinoma, uterine
sarcomas, leiomyosarcomas, mixed mullerian tumors); glioma,
glioblastoma, medulloblastoma, and other tumors of the brain;
kidney cancers (including renal cell carcinoma, clear cell
carcinoma, Wilm's tumor); cancer of the head and neck (including
squamous cell carcinomas); cancer of the stomach (gastric cancers,
stomach adenocarcinoma, gastrointestinal stromal tumor); testicular
cancer; germ cell tumor; neuroendocrine tumor; cervical cancer;
carcinoids of the gastrointestinal tract, breast, and other organs;
signet ring cell carcinoma; mesenchymal tumors including sarcomas,
fibrosarcomas, haemangioma, angiomatosis, haemangiopericytoma,
pseudoangiomatous stromal hyperplasia, myofibroblastoma,
fibromatosis, inflammatory myofibroblastic tumor, lipoma,
angiolipoma, granular cell tumor, neurofibroma, schwannoma,
angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma,
leiomyoma, leiomysarcoma, skin, including melanoma, cervical,
retinoblastoma, head and neck cancer, pancreatic, brain, thyroid,
testicular, renal, bladder, soft tissue, adenal gland, urethra,
cancers of the penis, myxosarcoma, chondrosarcoma, osteosarcoma,
chordoma, malignant fibrous histiocytoma, lymphangiosarcoma,
mesothelioma, squamous cell carcinoma; epidermoid carcinoma,
malignant skin adnexal tumors, adenocarcinoma, hepatoma,
hepatocellular carcinoma, renal cell carcinoma, hypernephroma,
cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma,
seminoma, embryonal cell carcinoma, glioma anaplastic; glioblastoma
multiforme, neuroblastoma, medulloblastoma, malignant meningioma,
malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma,
medullary carcinoma of thyroid, bronchial carcinoid,
pheochromocytoma, Islet cell carcinoma, malignant carcinoid,
malignant paraganglioma, melanoma, Merkel cell neoplasm,
cystosarcoma phylloide, salivary cancers, thymic carcinomas, and
cancers of the vagina among others.
[0209] In one embodiment of the above methods, the compound
administered is:
##STR00068##
[0210] In another embodiment of the above methods, the compound
administered is:
##STR00069##
[0211] In another aspect, provided herein is a method for the
treatment of a cancer, comprising: administering an effective
amount of a compound to a host in need thereof, wherein the
compound is:
##STR00070##
Combinations Therapies--Additional Chemotherapeutic Agents
[0212] In one embodiment, a compound of Formula I, II, III, or IV
is administered in combination with an additional chemotherapeutic
agent. In one embodiment, disclosed herein is a composition
comprising a compound of Formula I and an additional
chemotherapeutic agent. In one embodiment, disclosed herein is a
composition comprising a compound of Formula II and an additional
chemotherapeutic agent. In one embodiment, disclosed herein is a
composition comprising a compound of Formula III and an additional
chemotherapeutic agent. In one embodiment, disclosed herein is a
composition comprising a compound of Formula IV and an additional
chemotherapeutic agent.
[0213] Additional chemotherapeutic agents include, but are not
limited to, radioactive molecules, toxins, also referred to as
cytotoxins or cytotoxic agents, which includes any agent that is
detrimental to the viability of cells, agents, and liposomes or
other vesicles containing chemotherapeutic compounds. Examples of
suitable chemotherapeutic agents include but are not limited to
1-dehydrotestosterone, 5-fluorouracil decarbazine,
6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin,
aldesleukin, alkylating agents, allopurinol sodium, altretamine,
amifostine, anastrozole, anthramycin (AMC)), anti-mitotic agents,
cis-dichlorodiamine platinum (II) (DDP) cisplatin), diamino
dichloro platinum, anthracyclines, antibiotics, antis,
asparaginase, BCG live (intravesical), betamethasone sodium
phosphate and betamethasone acetate, bicalutamide, bleomycin
sulfate, busulfan, calcium leucouorin, calicheamicin, capecitabine,
carboplatin, lomustine (CCNU), carmustine (BSNU), Chlorambucil,
Cisplatin, Cladribine, Colchicin, conjugated estrogens,
Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine,
cytochalasin B, Cytoxan, Dacarbazine, Dactinomycin, dactinomycin
(formerly actinomycin), daunirubicin HCL, daunorucbicin citrate,
denileukin diftitox, Dexrazoxane, Dibromomannitol, dihydroxy
anthracin dione, Docetaxel, dolasetron mesylate, doxorubicin HCL,
dronabinol, E. coli L-asparaginase, emetine, epoetin-.alpha.,
Erwinia L-asparaginase, esterified estrogens, estradiol,
estramustine phosphate sodium, ethidium bromide, ethinyl estradiol,
etidronate, etoposide citrororum factor, etoposide phosphate,
filgrastim, floxuridine, fluconazole, fludarabine phosphate,
fluorouracil, flutamide, folinic acid, gemcitabine HCL,
glucocorticoids, goserelin acetate, gramicidin D, granisetron HCL,
hydroxyurea, idarubicin HCL, ifosfamide, interferon .alpha.-2b,
irinotecan HCL, letrozole, leucovorin calcium, leuprolide acetate,
levamisole HCL, lidocaine, lomustine, maytansinoid, mechlorethamine
HCL, medroxyprogesterone acetate, megestrol acetate, melphalan HCL,
mercaptipurine, mesna, methotrexate, methyltestosterone,
mithramycin, mitomycin C, mitotane, mitoxantrone, nilutamide,
octreotide acetate, ondansetron HCL, paclitaxel, pamidronate
disodium, pentostatin, pilocarpine HCL, plimycin, polifeprosan 20
with carmustine implant, porfimer sodium, procaine, procarbazine
HCL, propranolol, rituximab, sargramostim, streptozotocin,
tamoxifen, taxol, teniposide, tenoposide, testolactone, tetracaine,
thioepa chlorambucil, thioguanine, thiotepa, topotecan HCL,
toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastine
sulfate, vincristine sulfate, and vinorelbine tartrate.
[0214] Additional chemotherapeutic agents or therapeutic agents
that can be administered in combination with the compounds
disclosed herein can include bevacizumab, sutinib, sorafenib,
2-methoxyestradiol, finasunate, vatalanib, vandetanib, aflibercept,
volociximab, etaracizumab, cilengitide, erlotinib, cetuximab,
panitumumab, gefitinib, trastuzumab, atacicept, rituximab,
alemtuzumab, aldesleukine, atlizumab, tocilizumab, temsirolimus,
everolimus, lucatumumab, dacetuzumab, atiprimod, natalizumab,
bortezomib, carfilzomib, marizomib, tanespimycin, saquinavir
mesylate, ritonavir, nelfinavir mesylate, indinavir sulfate,
belinostat, panobinostat, mapatumumab, lexatumumab, oblimersen,
plitidepsin, talmapimod, enzastaurin, tipifarnib, perifosine,
imatinib, dasatinib, lenalidomide, thalidomide, simvastatin, and
celecoxib.
Compositions
[0215] Compositions, as described herein, comprising an active
compound and an excipient of some sort may be useful in a variety
of applications. For example, pharmaceutical compositions
comprising an active compound and an excipient may be useful for
the treatment or prevention of a cancer, for example, triple
negative breast cancer.
[0216] "Excipients" include any and all solvents, diluents or other
liquid vehicles, dispersion or suspension aids, surface active
agents, isotonic agents, thickening or emulsifying agents,
preservatives, solid binders, lubricants and the like, as suited to
the particular dosage form desired. General considerations in
formulation and/or manufacture can be found, for example, in
Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.
Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The
Science and Practice of Pharmacy, 21st Edition (Lippincott Williams
& Wilkins, 2005).
[0217] Exemplary excipients include, but are not limited to, any
non-toxic, inert solid, semi-solid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. Some
examples of materials which can serve as excipients include, but
are not limited to, sugars such as lactose, glucose, and sucrose;
starches such as corn starch and potato starch; cellulose and its
derivatives such as sodium carboxymethyl cellulose, ethyl
cellulose, and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients such as cocoa butter and suppository
waxes; oils such as peanut oil, cottonseed oil; safflower oil;
sesame oil; olive oil; corn oil and soybean oil; glycols such as
propylene glycol; esters such as ethyl oleate and ethyl laurate;
agar; detergents such as Tween 80; buffering agents such as
magnesium hydroxide and aluminum hydroxide; alginic acid;
pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol; and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator. As would be appreciated by one
of skill in this art, the excipients may be chosen based on what
the composition is useful for. For example, with a pharmaceutical
composition or cosmetic composition, the choice of the excipient
will depend on the route of administration, the agent being
delivered, time course of delivery of the agent, etc., and can be
administered to humans and/or to animals, orally, rectally,
parenterally, intracisternally, intravaginally, intranasally,
intraperitoneally, topically (as by powders, creams, ointments, or
drops), bucally, or as an oral or nasal spray.
[0218] Exemplary diluents include calcium carbonate, sodium
carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate,
calcium hydrogen phosphate, sodium phosphate lactose, sucrose,
cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,
inositol, sodium chloride, dry starch, cornstarch, powdered sugar,
etc., and combinations thereof.
[0219] Exemplary granulating and/or dispersing agents include
potato starch, corn starch, tapioca starch, sodium starch
glycolate, clays, alginic acid, guar gum, citrus pulp, agar,
bentonite, cellulose and wood products, natural sponge,
cation-exchange resins, calcium carbonate, silicates, sodium
carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone),
sodium carboxymethyl starch (sodium starch glycolate),
carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose (croscarmellose), methylcellulose, pregelatinized starch
(starch 1500), microcrystalline starch, water insoluble starch,
calcium carboxymethyl cellulose, magnesium aluminum silicate
(Veegum), sodium lauryl sulfate, quaternary ammonium compounds,
etc., and combinations thereof.
[0220] Exemplary surface active agents and/or emulsifiers include
natural emulsifiers (e.g. acacia, agar, alginic acid, sodium
alginate, tragacanth, chondrux, cholesterol, xanthan, pectin,
gelatin, egg yolk, casein, wool fat, cholesterol, wax, and
lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and
Veegum [magnesium aluminum silicate]), long chain amino acid
derivatives, high molecular weight alcohols (e.g. stearyl alcohol,
cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene
glycol distearate, glyceryl monostearate, and propylene glycol
monostearate, polyvinyl alcohol), carbomers (e.g. carboxy
polymethylene, polyacrylic acid, acrylic acid polymer, and
carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.
carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene
sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween
60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan
monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan
tristearate [Span 65], glyceryl monooleate, sorbitan monooleate
[Span 80]), polyoxyethylene esters (e.g. polyoxyethylene
monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil,
polyethoxylated castor oil, polyoxymethylene stearate, and
Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid
esters (e.g. Cremophor), polyoxyethylene ethers, (e.g.
polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone),
diethylene glycol monolaurate, triethanolamine oleate, sodium
oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate,
sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium
bromide, cetylpyridinium chloride, benzalkonium chloride, docusate
sodium, etc. and/or combinations thereof.
[0221] Exemplary binding agents include starch (e.g. cornstarch and
starch paste), gelatin, sugars (e.g. sucrose, glucose, dextrose,
dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and
synthetic gums (e.g. acacia, sodium alginate, extract of Irish
moss, panwar gum, ghatti gum, mucilage of isapol husks,
carboxymethylcellulose, methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, microcrystalline cellulose, cellulose acetate,
poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and
larch arabogalactan), alginates, polyethylene oxide, polyethylene
glycol, inorganic calcium salts, silicic acid, polymethacrylates,
waxes, water, alcohol, etc., and/or combinations thereof.
[0222] Exemplary preservatives include antioxidants, chelating
agents, antimicrobial preservatives, antifungal preservatives,
alcohol preservatives, acidic preservatives, and other
preservatives.
[0223] Exemplary antioxidants include alpha tocopherol, ascorbic
acid, acorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, monothioglycerol, potassium metabisulfite,
propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite,
sodium metabisulfite, and sodium sulfite.
[0224] Exemplary chelating agents include
ethylenediaminetetraacetic acid (EDTA) and salts and hydrates
thereof (e.g., sodium edetate, disodium edetate, trisodium edetate,
calcium disodium edetate, dipotassium edetate, and the like),
citric acid and salts and hydrates thereof (e.g., citric acid
monohydrate), fumaric acid and salts and hydrates thereof, malic
acid and salts and hydrates thereof, phosphoric acid and salts and
hydrates thereof, and tartaric acid and salts and hydrates thereof.
Exemplary antimicrobial preservatives include benzalkonium
chloride, benzethonium chloride, benzyl alcohol, bronopol,
cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol,
chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin,
hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol,
phenylmercuric nitrate, propylene glycol, and thimerosal.
[0225] Exemplary antifungal preservatives include butyl paraben,
methyl paraben, ethyl paraben, propyl paraben, benzoic acid,
hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium
benzoate, sodium propionate, and sorbic acid.
[0226] Exemplary alcohol preservatives include ethanol,
polyethylene glycol, phenol, phenolic compounds, bisphenol,
chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
[0227] Exemplary acidic preservatives include vitamin A, vitamin C,
vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic
acid, ascorbic acid, sorbic acid, and phytic acid.
[0228] Other preservatives include tocopherol, tocopherol acetate,
deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),
butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl
sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium
bisulfite, sodium metabisulfite, potassium sulfite, potassium
metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115,
Germaben II, Neolone, Kathon, and Euxyl. In certain embodiments,
the preservative is an anti-oxidant. In other embodiments, the
preservative is a chelating agent.
[0229] Exemplary buffering agents include citrate buffer solutions,
acetate buffer solutions, phosphate buffer solutions, ammonium
chloride, calcium carbonate, calcium chloride, calcium citrate,
calcium glubionate, calcium gluceptate, calcium gluconate,
D-gluconic acid, calcium glycerophosphate, calcium lactate,
propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium
phosphate, phosphoric acid, tribasic calcium phosphate, calcium
hydroxide phosphate, potassium acetate, potassium chloride,
potassium gluconate, potassium mixtures, dibasic potassium
phosphate, monobasic potassium phosphate, potassium phosphate
mixtures, sodium acetate, sodium bicarbonate, sodium chloride,
sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic
sodium phosphate, sodium phosphate mixtures, tromethamine,
magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free
water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and
combinations thereof.
[0230] Exemplary lubricating agents include magnesium stearate,
calcium stearate, stearic acid, silica, talc, malt, glyceryl
behanate, hydrogenated vegetable oils, polyethylene glycol, sodium
benzoate, sodium acetate, sodium chloride, leucine, magnesium
lauryl sulfate, sodium lauryl sulfate, etc., and combinations
thereof.
[0231] Exemplary natural oils include almond, apricot kernel,
avocado, babassu, bergamot, black current seed, borage, cade,
chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa
butter, coconut, cod liver, coffee, corn, cotton seed, emu,
Eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd,
grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui
nut, lavandin, lavender, lemon, Litsea cubeba, macademia nut,
mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,
orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,
pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,
sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,
soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut,
and wheat germ oils. Exemplary synthetic oils include, but are not
limited to, butyl stearate, caprylic triglyceride, capric
triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,
isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,
silicone oil, and combinations thereof.
[0232] Additionally, the composition may further comprise a
polymer. Exemplary polymers contemplated herein include, but are
not limited to, cellulosic polymers and copolymers, for example,
cellulose ethers such as methylcellulose (MC),
hydroxyethylcellulose (HEC), hydroxypropyl cellulose (HPC),
hydroxypropyl methyl cellulose (HPMC), methylhydroxyethylcellulose
(MHEC), methylhydroxypropylcellulose (MHPC), carboxymethyl
cellulose (CMC) and its various salts, including, e.g., the sodium
salt, hydroxyethylcarboxymethylcellulose (HECMC) and its various
salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various
salts, other polysaccharides and polysaccharide derivatives such as
starch, dextran, dextran derivatives, chitosan, and alginic acid
and its various salts, carageenan, various gums, including xanthan
gum, guar gum, gum arabic, gum karaya, gum ghatti, konjac and gum
tragacanth, glycosaminoglycans and proteoglycans such as hyaluronic
acid and its salts, proteins such as gelatin, collagen, albumin,
and fibrin, other polymers, for example, polyhydroxyacids such as
polylactide, polyglycolide, polyl(lactide-co-glycolide) and
poly(.epsilon.-caprolactone-co-glycolide)-, carboxyvinyl polymers
and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP),
polyacrylic acid and its salts, polyacrylamide, polyacilic
acid/acrylamide copolymer, polyalkylene oxides such as polyethylene
oxide, polypropylene oxide, poly(ethylene oxide-propylene oxide),
and a Pluronic polymer, polyoxyethylene (polyethylene glycol),
polyanhydrides, polyvinylalchol, polyethyleneamine and
polypyrridine, polyethylene glycol (PEG) polymers, such as
PEGylated lipids (e.g., PEG-stearate,
1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene
glycol)-1000],
1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene
glycol)-2000], and
1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene
glycol)-5000]), copolymers and salts thereof.
[0233] Additionally, the composition may further comprise an
emulsifying agent. Exemplary emulsifying agents include, but are
not limited to, a polyethylene glycol (PEG), a polypropylene
glycol, a polyvinyl alcohol, a poly-N-vinyl pyrrolidone and
copolymers thereof, poloxamer nonionic surfactants, neutral
water-soluble polysaccharides (e.g., dextran, Ficoll, celluloses),
non-cationic poly(meth)acrylates, non-cationic polyacrylates, such
as poly(meth)acrylic acid, and esters amide and hydroxyalkyl amides
thereof, natural emulsifiers (e.g. acacia, agar, alginic acid,
sodium alginate, tragacanth, chondrux, cholesterol, xanthan,
pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and
lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and
Veegum [magnesium aluminum silicate]), long chain amino acid
derivatives, high molecular weight alcohols (e.g. stearyl alcohol,
cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene
glycol distearate, glyceryl monostearate, and propylene glycol
monostearate, polyvinyl alcohol), carbomers (e.g. carboxy
polymethylene, polyacrylic acid, acrylic acid polymer, and
carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.
carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene
sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween
60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan
monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan
tristearate [Span 65], glyceryl monooleate, sorbitan monooleate
[Span 80]), polyoxyethylene esters (e.g. polyoxyethylene
monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil,
polyethoxylated castor oil, polyoxymethylene stearate, and
Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid
esters (e.g. Cremophor), polyoxyethylene ethers, (e.g.
polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone),
diethylene glycol monolaurate, triethanolamine oleate, sodium
oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate,
sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium
bromide, cetylpyridinium chloride, benzalkonium chloride, docusate
sodium, etc. and/or combinations thereof. In certain embodiments,
the emulsifying agent is cholesterol.
[0234] Liquid compositions include emulsions, microemulsions,
solutions, suspensions, syrups, and elixirs. In addition to the
active compound, the liquid composition may contain inert diluents
commonly used in the art such as, for example, water or other
solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. Besides inert diluents,
the oral compositions can also include adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening, flavoring,
and perfuming agents.
[0235] Injectable compositions, for example, injectable aqueous or
oleaginous suspensions may be formulated according to the known art
using suitable dispersing or wetting agents and suspending agents.
The sterile injectable preparation may also be a injectable
solution, suspension, or emulsion in a nontoxic parenterally
acceptable diluent or solvent, for example, as a solution in
1,3-butanediol. Among the acceptable vehicles and solvents for
pharmaceutical or cosmetic compositions that may be employed are
water, Ringer's solution, U.S.P. and isotonic sodium chloride
solution. In addition, sterile, fixed oils are conventionally
employed as a solvent or suspending medium. Any bland fixed oil can
be employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid are used in the preparation of
injectables. In certain embodiments, the particles are suspended in
a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose
and 0.1% (v/v) Tween 80. The injectable composition can be
sterilized, for example, by filtration through a bacteria-retaining
filter, or by incorporating sterilizing agents in the form of
sterile solid compositions which can be dissolved or dispersed in
sterile water or other sterile injectable medium prior to use.
[0236] Compositions for rectal or vaginal administration may be in
the form of suppositories which can be prepared by mixing the
particles with suitable non-irritating excipients or carriers such
as cocoa butter, polyethylene glycol, or a suppository wax which
are solid at ambient temperature but liquid at body temperature and
therefore melt in the rectum or vaginal cavity and release the
particles.
[0237] Solid compositions include capsules, tablets, pills,
powders, and granules. In such solid compositions, the particles
are mixed with at least one excipient and/or a) fillers or
extenders such as starches, lactose, sucrose, glucose, mannitol,
and silicic acid, b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,
sucrose, and acacia, c) humectants such as glycerol, d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, e) solution retarding agents such as paraffin, 0
absorption accelerators such as quaternary ammonium compounds, g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate, h) absorbents such as kaolin and bentonite clay, and
i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets, and pills, the dosage
form may also comprise buffering agents. Solid compositions of a
similar type may also be employed as fillers in soft and
hard-filled gelatin capsules using such excipients as lactose or
milk sugar as well as high molecular weight polyethylene glycols
and the like.
[0238] Tablets, capsules, pills, and granules can be prepared with
coatings and shells such as enteric coatings and other coatings
well known in the pharmaceutical formulating art. They may
optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
which can be used include polymeric substances and waxes.
[0239] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0240] Compositions for topical or transdermal administration
include ointments, pastes, creams, lotions, gels, powders,
solutions, sprays, inhalants, or patches. The active compound is
admixed with an excipient and any needed preservatives or buffers
as may be required.
[0241] The ointments, pastes, creams, and gels may contain, in
addition to the active compound, excipients such as animal and
vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc, and zinc oxide, or mixtures thereof.
[0242] Powders and sprays can contain, in addition to the active
compound, excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates, and polyamide powder, or mixtures of
these substances. Sprays can additionally contain customary
propellants such as chlorofluorohydrocarbons.
[0243] Transdermal patches have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the nanoparticles in a
proper medium. Absorption enhancers can also be used to increase
the flux of the compound across the skin. The rate can be
controlled by either providing a rate controlling membrane or by
dispersing the particles in a polymer matrix or gel.
[0244] The active ingredient may be administered in such amounts,
time, and route deemed necessary in order to achieve the desired
result. The exact amount of the active ingredient will vary from
subject to subject, depending on the species, age, and general
condition of the subject, the severity of the infection, the
particular active ingredient, its mode of administration, its mode
of activity, and the like. The active ingredient, whether the
active compound itself, or the active compound in combination with
an agent, is preferably formulated in dosage unit form for ease of
administration and uniformity of dosage. It will be understood,
however, that the total daily usage of the active ingredient will
be decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically effective dose level
for any particular subject will depend upon a variety of factors
including the disorder being treated and the severity of the
disorder; the activity of the active ingredient employed; the
specific composition employed; the age, body weight, general
health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific
active ingredient employed; the duration of the treatment; drugs
used in combination or coincidental with the specific active
ingredient employed; and like factors well known in the medical
arts.
[0245] The active ingredient may be administered by any route. In
some embodiments, the active ingredient is administered via a
variety of routes, including oral, intravenous, intramuscular,
intra-arterial, intramedullary, intrathecal, subcutaneous,
intraventricular, transdermal, interdermal, rectal, intravaginal,
intraperitoneal, topical (as by powders, ointments, creams, and/or
drops), mucosal, nasal, bucal, enteral, sublingual; by
intratracheal instillation, bronchial instillation, and/or
inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
In general the most appropriate route of administration will depend
upon a variety of factors including the nature of the active
ingredient (e.g., its stability in the environment of the
gastrointestinal tract), the condition of the subject (e.g.,
whether the subject is able to tolerate oral administration),
etc.
[0246] The exact amount of an active ingredient required to achieve
a therapeutically or prophylactically effective amount will vary
from subject to subject, depending on species, age, and general
condition of a subject, severity of the side effects or disorder,
identity of the particular compound(s), mode of administration, and
the like. The amount to be administered to, for example, a child or
an adolescent can be determined by a medical practitioner or person
skilled in the art and can be lower or the same as that
administered to an adult.
EXAMPLES
[0247] The following examples are set forth below to illustrate the
compounds, compositions, methods, and results according to the
disclosed subject matter. These examples are not intended to be
inclusive of all aspects of the subject matter disclosed herein,
but rather to illustrate representative methods and results. These
examples are not intended to exclude equivalents and variations of
the present invention which are apparent to one skilled in the
art.
Example 1. Potent and Selective Indolinone Derivatives that Inhibit
Maternal Embryonic Leucine Zipper Kinase (MELK) and Inhibit Triple
Negative Breast Cancer (TNBC) Cell Growth
[0248] Despite recent advances in molecularly directed therapy,
triple negative breast cancer (TNBC) remains one of the most
aggressive forms of breast cancer still without a suitable target
for specific inhibitors. Maternal embryonic leucine zipper kinase
(MELK) is highly expressed in TNBC where the level of
overexpression correlates with poor prognosis and an aggressive
disease course. This example describes the identification of a
series of ATP-competitive indolinone derivatives with subnanomolar
inhibition constants towards MELK. The most potent compound
inhibits the proliferation of TNBC cells and exhibits a selectivity
for cells expressing high levels of MELK.
[0249] In contrast to most members of the AMPK-RK family, which
mediate cell survival under stressful metabolic conditions (Gil M,
et al. Gene. 1997; 195(2):295-301; Beullens M, et al. The Journal
of biological chemistry. 2005; 280(48):40003-40011). MELK has been
implicated in multiple cellular processes, including cell cycle
checkpoint regulation (Blot J, et al. Dev Biol. 2002;
241(2):327-338; Davezac N, et al. Oncogene. 2002;
21(50):7630-7641), proliferation (Joshi K, et al. Stem cells
(Dayton, Ohio). 2013; Saito R, et al. Cancer science. 2012;
103(1):42-49), apoptosis (Lin M L, et al. Breast cancer research:
BCR. 2007; 9(1):R17; Pickard M R, et al. Biochimica et biophysica
acta. 2011; 1812(9):1146-1153), and RNA processing (Vulsteke V, et
al. The Journal of biological chemistry. 2004; 279(10):8642-8647).
MELK is expressed in the early stages of murine embryonic
development (Heyer B S, et al. Molecular reproduction and
development. 1997; 47(2):148-156), but MELK knockout mice develop
normally with no obvious pathologic phenotype, suggesting that
MELK's developmentally-related functions may be redundant (Wang Y,
et al. eLife. 2014:e01763). Yet despite its apparent dispensable
nature in differentiated adult cells, evidence has implicated
MELK's importance in proliferating progenitor populations,
including multipotent neural progenitors (Nakano I, et al. The
Journal of cell biology. 2005; 170(3):413-427), myoblasts (Niesler
C U, et al. Experimental physiology. 2007; 92(1):207-217), and
mammary progenitors (Hebbard L W, et al. Cancer research. 2010;
70(21):8863-8873). Interestingly, MELK inhibition does not affect
survival in normal neural stem cells, but siRNA-mediated MELK
knockdown induces apoptosis selectively in glioma stem cells
(Nakano I, et al. Neuro-oncology. 2011; 13(6):622-634; Nakano I, et
al. Journal of neuroscience research. 2008; 86(1):48-60). Such data
reinforces the redundancy of MELK function in noncancerous cells,
but also implicates the existence of an exploitable target in
certain cancer stem cell populations.
[0250] In addition to its putative role in cancer stem cells,
upregulated MELK mRNA and protein levels have been observed in a
wide array of cancer cell types and clinical tumor samples (Gray D,
et al. Cancer research. 2005; 65(21):9751-9761; Ryu B, et al. PloS
one. 2007; 2(7):e594; Marie S K, et al. International journal of
cancer. Journal international du cancer. 2008; 122(4):807-815; Li
Y, et al. Lung cancer (Amsterdam, Netherlands). 2013; Li Y, et al.
Lung cancer (Amsterdam, Netherlands). 2013; Rajkumar T, et al. BMC
cancer. 2011; 11:80; Risinger J I, et al. Frontiers in oncology.
2013; 3:139). Of particular note is the fact that MELK expression
correlates with poor prognosis in the most aggressive subsets of
disease, including glioblastoma multiforme (GBM) (Nakano I, et al.
Journal of neuroscience research. 2008; 86(1):48-60; Marie S K, et
al. International journal of cancer. Journal international du
cancer. 2008; 122(4):807-815; Kappadakunnel M, et al. Journal of
neuro-oncology. 2010; 96(3):359-367) and triple negative breast
cancer (TNBC) (Wang Y, et al. eLife. 2014:e01763; Komatsu M, et al.
International journal of oncology. 2013; 42(2):478-506; Al-Ejeh F,
et al. Oncogenesis. 2014; 3:e100). Factors contributing to poor
outlook for TNBC patients in part stems from the cancer's ability
not only to proliferate quickly, but also its propensity to spread
and recur in distant organs. From a molecular biology perspective,
mounting evidence continues to implicate MELK in direct and
transcriptional regulation of cell division in the context of
malignancy (Joshi K, et al. Stem cells (Dayton, Ohio). 2013; Wang
Y, et al. eLife. 2014:e01763; Marie S K, et al. Proteome Sci. 2016;
14:6). Furthermore, MELK has also been preliminarily linked to
metastasis through its involvement with TGF-.beta. driven
epithelial-to-mesenchymal transition (EMT) (Seong H A, et al. The
Journal of biological chemistry. 2010; 285(40):30959-30970). The
cancer-specific expression pattern, combined with the clinical and
biological data have therefore justifiably fostered strong interest
in MELK as a clinical target.
[0251] The present example discloses novel chemical probes to
inhibit MELK and small molecule therapeutics that can target a
cancer (for example, TNBC). To that end, the design and development
of a new structural class of highly potent indolinone MELK
inhibitors is described in this example, including compounds 16,
17, and 21, with subnanomolar inhibition constants (K.sub.i). In
addition to potency, the new class of inhibitors disclosed herein
show selectivity for MELK relative to other functionally and
evolutionarily related kinases. Finally, these inhibitors were
applied to relevant TNBC cell lines. It was observed that the
inhibitors impact TNBC cell viability and proliferation, with
little effect on an immortalized breast epithelial cell line which
does not express high levels of MELK.
[0252] Over the last two decades, protein kinases have represented
a major field for drug development. As such, diverse methods of
kinase inhibitor discovery have emerged and have been extensively
employed in both the industrial and academic settings.
Target-centric strategies focus on screening small molecule
libraries against a single kinase of interest, whereas
compound-centric approaches profile activities of a single compound
against the entire kinome (Miduturu C V, et al. Chemistry &
biology. 2011; 18(7):868-879). Within the target-centric method,
screening libraries may take on the form of fragment-based,
directed, or diverse high-throughput compound collections.
[0253] In addition to addressing selectivity, cross-screening has
become increasingly utilized as a means of drug discovery.
Cross-screening unearths off-target effects in cases where
inhibitory properties of the small molecules are characterized
(e.g. single known kinase inhibitor or directed kinase inhibitor
libraries) (Uitdehaag J C, et al. Br J Pharmacol. 2012;
166(3):858-876). Such off-target activity then serves as an initial
starting point for development of untargeted or under targeted
kinase inhibitors (Mathea S, et al. ACS Chem Biol. 2016;
11(6):1595-1602; Elkins J M, et al. Nat Biotechnol. 2016;
34(1):95-103). Alternatively, compound-centric approaches may also
be used to improve selectivity of the primary compound, engineering
out inhibition of particular undesired targets (Mathea S, et al.
ACS Chem Biol. 2016). In each of the above strategies, structural
guidance is typically essential at every stage to provide critical
insight for scaffold modification.
[0254] To date, no inhibitors developed with MELK as a primary
target are FDA approved. Thus, as an initial experiment, a curated
library of approximately 800 known kinase inhibitors in duplicate
plate format was examined for ability to inhibit MELK as an
off-target. Compounds were ranked according to average percent
inhibition at both 10 and 1 .mu.M, revealing 18 compounds with
.gtoreq.50% inhibition at 1 .mu.M. The ten most potent compounds
were further characterized by determining each compound's
respective IC.sub.50 (Table 4). The indolinone scaffold or a
similar bicyclic core structure populated the top hits. Indeed,
indolinone motifs represent a common pharmacophore in
ATP-competitive inhibitors (Prakash C R, et al. Mini reviews in
medicinal chemistry. 2012; 12(2):98-119; Aronov A M, et al. Journal
of medicinal chemistry. 2008; 51(5):1214-1222).
TABLE-US-00004 TABLE 4 Cross-inhibition of MELK by known kinase
inhibitors Compound Structure Core IC.sub.50 .+-. SE (nM)
Nintedanib ##STR00071## Indolinone 43 .+-. 3.4 Hesperadin
##STR00072## Indolinone 225 .+-. 50 420099 ##STR00073##
Benzimidazo- isoquinolinone 330 .+-. 37 CC401 ##STR00074## Indazole
340 .+-. 70 PIK75 ##STR00075## Imidazopyridine 400 .+-. 72 HA-1004
##STR00076## Isoquinoline 440 .+-. 210 AT9283 ##STR00077##
Benzimidazole 685 .+-. 125 527450 ##STR00078## Indolinone 720 .+-.
100 572660 ##STR00079## Indolinone 760 .+-. 190 AZD776 ##STR00080##
Thiofuran 900 .+-. 91
[0255] The most potent of the top inhibitor candidates was
nintedanib (BIBF-1120, Vargatef.RTM./Ofev.RTM.), which displayed an
IC.sub.50 of 43 nM. Nintedanib is already FDA-approved for the
treatment of idiopathic pulmonary fibrosis and is currently
undergoing clinical trials for treatment of non-small cell lung
cancer, metastatic colorectal cancer, and ovarian cancer. However,
it is a multi-kinase inhibitor whose primary described mechanism of
action is inhibition of the growth factor receptors VEGFR, PDGFR,
and FGFR. To improve upon nintedanib's potency and selectivity
towards MELK, a medicinal chemistry program was designed around its
structural features, using the indolinone core as a primary
scaffold.
[0256] While nintedanib was clearly the most potent molecule,
(IC.sub.50=43 nM), additional observations from the screen also
directed the medicinal chemistry design strategy. The top three
most potent inhibitors all exhibit a common alternating
donor/acceptor hydrogen-bonding pattern that has also been observed
with previously published inhibitors (FIG. 1). Heteroatoms and
substituents of the bicyclic core are spatially oriented to
interact with both the hinge region and the conserved catalytic
lysine, respectively. In addition to nintedanib, an indolinone
scaffold forms the core of three other candidate compounds (Table
4, entries 2, 8, and 9), while others contained similar
heterocyclic motifs. Notably, several of the indolinones contain
substituents at the 5- or 6-position, which are predicted through
modeling studies to interact via hydrogen bonding with Lys 40 of
the binding pocket.
[0257] Molecular modeling studies using Gold 5.1 (Cambridge
Crystallographic Data Center) reinforce the similarities in binding
pose and electrostatic contacts between nintedanib and other
published inhibitors (FIG. 2). In the hinge region, the backbone of
C89 interacts with the enamine nitrogen and the carbonyl of the
indolinone ring of nintedanib (FIG. 2A), mirroring the hydrogen
bonding pattern seen in the crystal structure of MELK with Cpd2
(FIG. 2B, PDB 4BKY) (Canevari G, et al. Biochemistry. 2013;
52(37):6380-6387). With both nintedanib and Cpd2, K40 is positioned
to interact with bicyclic core substituents, either directly or via
a water molecule. Interaction with K40 has been previously
described as an "activity cliff" with other MELK inhibitors, in
that loss of this interaction results in loss of compound activity
towards MELK (Toure B B, et al. J Med Chem. 2016; 59(10):4711-4723;
Furtmann N, et al. J Med Chem. 2015; 58(1):252-264). Taken
together, the screening and modeling data suggest that MELK not
only accommodates various substituents at the 5th and 6th positions
of the indolinone ring, but also that it may be a critical element
for maintaining inhibitor potency. Thus, a derivative library
incorporating 5- and 6-substituted indolinone inhibitors was
synthesized.
Synthesis of 5, or 6-Substituted Indolinone Derivatives 15-25
[0258] After identifying the MELK inhibitor compounds in Table 4, a
number of 5, or 6-substituted indolinone derivatives were then
synthesized in order to improve potency and selectivity (See
Compounds 15-25). The general synthetic routes are outlined in
Schemes 1 and 2. The key intermediates 6-10 were prepared by
acylation of indolinones 1-5 and subsequent condensation with
ortho-benzoic acid triethylester. Aromatic amine intermediate 14
was prepared by literature procedures as illustrated in Scheme 1
(Roth G J, et al. J Med Chem. 2009; 52(14):4466-4480). Acylation of
N-methyl-4-nitroaniline 11 gave chloroactyl amide 12, which was
then treated with N-methylpiperazine to displace chloride; followed
by catalytic reduction of the nitro group which gave the key
aromatic amine intermediate 14.
##STR00081## ##STR00082##
[0259] Final indolinone analogs 15-19 were prepared by addition of
14 to substituted indolinones 6-10 and subsequent elimination of
ethanol, followed by acetyl cleavage using piperidine in one pot
(Scheme 2). Additional 5 or 6-substituted amide analogs 22-25 were
synthesized from the corresponding 5 or 6-substituted methyl ester
derivatives by hydrolysis using aqueous 1 N NaOH and subsequent
standard amide coupling reactions using N-methyl amine and N,
N-dimehtylamine after TBTU or HBTU activation (Scheme 2). The 5, or
6-substituted indolinone derivatives 15-25 in Table 5 were
evaluated for their ability to inhibit MELK.
##STR00083## ##STR00084##
5-Substituted Indolinone Derivatives Improve Potency Towards
MELK
[0260] First, efforts were focused on 6-substituted indolinone
derivatives. After synthesizing nintedanib (15), the methyl ester
was hydrolyzed to the carboxylic acid 20. Longer amines were also
synthesized containing amides 22 and 23 (similar to the amine motif
in the known MELK inhibitor Cpd1 (Canevari G, et al. Biochemistry.
2013; 52(37):6380-6387). Unfortunately, all the 6-substituted
indolinone derivatives were found to be less potent compared to the
lead compound 15 (Table 5). Next, attention was shifted to the 5th
position of the indolinone ring. Initial compounds synthesized
included 5-substituted methyl ester, methyl ketone and fluoro
indolinones 17-19. Surprisingly, both 5-CO.sub.2Me 17 (K.sub.i=0.39
nM) and 5-F derivative 19 (K.sub.i=3.1 nM) were found to be more
potent compared to the lead compound 15 (K.sub.i=5.6 nM). Further,
the unsubstituted indolinone 16 was also found to be active
(K.sub.i=0.47 nM), challenging the notion that the 5 or
6-substituents are critical for binding MELK. Hydrolysis of the
methyl ester produced inhibitor 19 which retained MELK activity
(K.sub.i=0.68 nM). 5-substituted amide analogs 24 (K=2.3 nM) and 25
(K.sub.i=8.8 nM) showed lower activities compared to the 5-methyl
ester analog 17 (K.sub.i=0.39 nM). Also, complete loss of MELK
activity (K.sub.i>5000 nM) was observed when the key
intermediates 6, 8 and 14 were tested. This suggests the importance
of the two key fragments in combination.
##STR00085##
TABLE-US-00005 TABLE 5 Characterization of indolinone derivatives.
Com- pound R.sub.1 R.sub.2 IC.sub.50 (nM) K.sub.i .sup.a SE (nM) 15
CO.sub.2Me H .sup. 43 .+-. 3.4 5.6 .+-. 0.4 16 H H 3.6 .+-. 0.4
0.47 .+-. 0.06 17 H CO.sub.2Me 3 .+-. 0.8 0.39 .+-. 0.09 18 H COMe
354 .+-. 35 46 .+-. 4.6 19 H F .sup. 24 .+-. 4.7 3.1 .+-. 0.6 20
CO.sub.2H H 1145 .+-. 168 149 .+-. 22 21 H CO.sub.2H 5.2 .+-. 0.5
0.68 .+-. 0.07 22 CONH(CH.sub.2).sub.3NMe.sub.2 H 2700 .+-. 540 358
.+-. 71 23 CONH(CH.sub.2).sub.3NH.sub.2 H >5000 >650 24 H
CONHMe .sup. 18 .+-. 3.8 2.3 .+-. 0.5 25 H CONMe.sub.2 67 .+-. 11
8.8 .+-. 1.4 .sup.a K.sub.i calculated using Equation 4, where
[ATP]= 40 .mu.M, K.sub.M.sup.app (ATP) = 6 .mu.M
TABLE-US-00006 ##STR00086## ##STR00087## ##STR00088## R.sub.1
R.sub.2 IC.sub.50 14 15 intermediate 6 CO.sub.2Me H >5000 nM
IC50 => 5000 nM IC.sub.50 = 706 .+-. 113 8 H CO.sub.2Me >5000
nM K.sub.i = 92 .+-. 14
TABLE-US-00007 TABLE 6 Fragment and intermediate potency (K.sub.i's
of synthetic intermediates) Compound K.sub.i .+-. SD (nM) 6 >650
8 >650 14 >650 15 84 .+-. 13
[0261] To further understand the substantial increase in potency by
shifting the methyl ester from the 6 to the 5 position, both
compound 15 (6-CO.sub.2Me) and compound 17 (5-CO.sub.2Me) were
docked with MELK (PDB: 4BKY) and analyzed using Gold5.2 with the
ChemPLP scoring function. The methyl ester substituents of both
compounds are within direct or water-bridged hydrogen bonding
distance of K40 (FIG. 3). A methyl ester in the 5th position,
however is predicted to force 17 deeper into the narrow groove
leading to K40. This would likely shorten the H-bond distance, and
strengthen the interaction. Moreover, this modification enables 17
to adopt a conformation more complementary to the shape of the
binding pocket, facilitating the interaction of the indolinone core
with E87 through an additional hydrogen bond. Concordantly,
retained potency of 16 (unsubstituted indolinone) suggests that
suboptimal substituent position significantly affects inhibitor
binding. Alternatively, the potency of 16 may also be explained by
the ability of an unsubstituted indolinone to adopt a greater
number of binding modes.
MELK Derivatives Show Differential Selectivity Towards Functionally
Related Kinases
[0262] In light of the same chemical reaction they all perform,
kinases by definition have a characteristic binding site for ATP.
While critical residues must be present for the phosphoryl transfer
reaction (e.g., those to coordinate magnesium ions and the
phosphoester chain), there is considerable diversity in residues
that additionally influence the volume and shape of the ATP binding
pocket, giving rise to the possibility of kinase inhibitor
selectivity. Such parameters not only determine how tightly an
enzyme can bind ATP, but also dictate what small molecules will
effectively compete with ATP and take advantage of unique surface
complementarities. Therefore, these new inhibitors were
investigated in the context of inhibiting MELK's most closely
related family members AMPK and NUAK1. It was also determined
whether the most potent MELK inhibitor derivatives affected other
kinases involved in putative MELK-related pathways (the cell cycle
and CHK1), or those driving certain AMPK-RK signaling pathways
(CAMKKII). In contrast, ERK2 served as an unrelated control.
[0263] The top three most potent MELK inhibitor derivatives,
compounds 16, 17, and 21, were analyzed in dose-response inhibition
assays and used apparent K.sub.M.sup.ATP values determined under
our experimental conditions to calculate K (Table 8). Compound 16,
which contains no substituent at either the 5- or 6-position of the
indolinone, was the least selective inhibitor of the three (Table
7). Compound 16 inhibited both AMPK and NUAK1 quite well, resulting
in only a 5-10 fold difference in K compared to MELK. Addition of a
methyl ester at the 5-position (compound 17) resulted in comparable
potency towards MELK, but increased selectivity towards other
kinases. Hydrolysis of the methyl ester to the carboxylate
(compound 21) yielded the best selectivity profile of the three
inhibitors tested, displaying >100-fold difference in relative K
compared to MELK for CHK1, CAMKK2, and ERK2. All three compounds
remained fairly tight binding inhibitors of closely related kinases
AMPK and NUAK1.
##STR00089##
TABLE-US-00008 TABLE 7 Selectivity of MELK inhibitor derivatives
against selected kinases. Compound 16 Compound 17 Compound 21
Kinase K.sub.i .phi..sup.a K.sub.i .phi..sup.a K.sub.i .phi..sup.a
MELK 0.47 .+-. 0.06 1.00 0.39 .+-. 0.09 1.00 0.68 .+-. 0.07 1.00
AMPK 4.2 .+-. 0.3 8.9 .+-. 0.2 9.2 .+-. 1.7 24 .+-. 0.3 4.9 .+-.
0.6 7.2 .+-. 0.2 NUAK1 1.7 .+-. 0.17 3.6 .+-. 0.2 .sup. 11 .+-.
0.86 28 .+-. 0.2 8.6 .+-. 1.6 13 .+-. 0.2 CHK1 10 .+-. 0.5 18 .+-.
0.2 7 .+-. 0.17 18 .+-. 0.2 81 .+-. 5.6 120 .+-. 0.1 CAMKK2 15 .+-.
1.8 32 .+-. 0.2 31 .+-. 11 80 .+-. 0.4 85 .+-. 9.2 125 .+-. 0.2
ERK2 1160 .+-. 320 2500 .+-. 0.3.sup. 3000 .+-. 1300 7700 .+-. 0.5
19000 .+-. 3800 28000 .+-. 0.2 .sup.a.phi. =
K.sub.i.sup.Enzyme/K.sub.i.sup.MELK
[0264] While not appreciably affecting potency towards MELK,
additional negative charge at the 5-position enhances compound
selectivity. Such a trend is particularly apparent in the case of
CHK1. MELK has been implicated in regulation of the cell cycle
though inhibitory phosphorylation of the phosphatase CDC25B, whose
main function is to promote mitotic entry through dephosphorylation
and activation of CDK1/cyclin B (Davezac N, et al. Oncogene. 2002;
21(50):7630-7641). It has additionally been reported that MELK
overexpression may increase DNA damage and replication stress
tolerance in cancer cells, as MELK inhibition resulted in a
prolonged ATM-CHK2 response (Beke L, et al. Biosci Rep. 2015). The
reported roles of MELK in cancer cells therefore suggest potential
intimate relation with those functions of CHK1. Thus, a MELK
inhibitor that affords additional selectivity against CHK1, like
compound 21, is a valuable tool for further delineation of MELK's
role in the context of cell division and DNA damage and repair.
Finally, it should be noted that while Compound 17 is the most
potent in vitro, it is possible that its methyl ester is hydrolyzed
in vivo due to the prevalence of esterases at the cellular and
whole organism level. Thus, not only is compound 21 the most
selective of the three, it is also a likely potent species present
to inhibit MELK in a living system.
Inhibitor 17 Decreases Cell Proliferation of TNBC Cells
[0265] As MELK was found to be essential in basal-like breast
cancer cells (Wang Y, et al. eLife. 2014:e01763), the
CellTiter-Blue assay was used to test the ability of two compounds
(17 and 19, FIG. 4A) to inhibit the proliferation of various TNBC
subtypes (Lehmann B D, et al. The Journal of clinical
investigation. 2011; 121(7):2750-2767), HCC70 (BL2 subtype), BT-549
(mesenchymal) and SUM-159 (mesenchymal stem-like), as well as the
immortalized mammary epithelial cell line MCF10A (classified as
Basal B by molecular profiling) (Kao J, et al. PloS one. 2009;
4(7):e6146). Proliferation of the HCC70, BT-549, and SUM-159 cells
were clearly impacted by treatment with 17, whereas the MCF10A
cells remained largely unaffected at 10 .mu.M. In contrast, 19,
inhibited all cell lines. Such data is congruent with our previous
results (Table 5), as 17 is around 10 fold more potent than the
5-fluoro derivative 19 in vitro, and 17 is a more effective
inhibitor of proliferation in cells. The ability of 19 to inhibit
proliferation in MCF10A cells may be explained by the fact that
5-fluoro derivative may be less selective, much like the
unsubstituted indolinone 16. Furthermore, cell lines expressing
high levels of MELK protein (HCC70 and BT549) were more sensitive
to 17 than those cells with low levels of MELK expression (SUM159).
Collectively, these data show that 17 selectively inhibits
proliferation of TNBC cells expressing high levels of MELK.
SUMMARY
[0266] In conclusion, 5-substituted indolinones were identified as
a novel MELK inhibitor scaffold though targeted kinase library
inhibitor screening. Derivatization of the most potent MELK
inhibitor identified in the initial screen (nintedanib) led to
three tightly binding MELK inhibitors with subnanomolar inhibition
constants (compounds 16,17, and 21). The unsubstituted indolinone
was the least selective against a small subset of evolutionarily
and functionally related kinases. However, compound 21 afforded
selectivity against 3 out of 5 kinases tested, particularly against
the mitotic kinase CHK1. The most potent MELK inhibitor, compound
17, decreases viability and proliferation of multiple TNBC cell
lines with high MELK expression. Conversely, compound 17 shows
little effect on low MELK-expressing MCF-10A cells, an immortalized
breast epithelial cell line.
Methods
Chemistry
[0267] General Information. Reagents and starting materials
including indolinones 1-3, and 5, and N-Methyl-4-nitroaniline 11
were purchased from various commercial sources including
Sigma-Aldrich or Matrix Scientific and used without further
purification unless otherwise stated. 5-Acetylindolinone 4 and
aromatic amine intermediate 14 were prepared by literature
procedures (Roth G J, et al. J Med Chem. 2009; 52(14):4466-4480;
Heckel A B, et al. Inventor; Boehringer Ingelheim International
GmbH (Ingelheim, DE), assignee. Cycloalkyl-containing
5-acylindolinones, the preparation thereof and their use as
medicaments. 2007). All reactions were carried out in oven- or
flame-dried glassware under argon. Thin-layer chromatography (TLC)
was performed using pre-coated TLC plates with silica gel 60 F254
(EMD) or with aluminum oxide 60 F254 neutral. Flash column
chromatography was performed using 40-63 .mu.m (230-400 mesh ASTM)
silica gel (EMD). Melting points were recorded on a Thomas Hoover
capillary melting point apparatus. NMR spectra were recorded on a
Varian MR spectrometer. High-resolution mass and liquid
chromatography mass spectral data were obtained at the University
of Texas at Austin. Compounds were characterized by NMR and HRMS or
LCMS.
[0268] 5-Acetylindolin-2-one (4). By following acylation procedure
reported in a patent (Heckel A B, et al. Inventor; Boehringer
Ingelheim International GmbH (Ingelheim, DE), assignee.
Cycloalkyl-containing 5-acylindolinones, the preparation thereof
and their use as medicaments. 2007). 5-acetylindolin-2-one was
prepared from indolin-2-one and AlCl.sub.3. The crude was
recrystallized from ethyl acetate to obtain 72% of the title
compound. .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 10.76 (br s,
1H, NH), 7.85 (m, 1H, Ar--H), 7.80 (m, 1H, Ar--H), 6.90 (d, J=8.0
Hz, 1H, Ar--H), 3.55 (s, 2H), 2.50 (s, 3H, Ac).
[0269] General procedure for the synthesis of
N-acetyl-3-(ethoxy(phenyl)methylene)-2-oxoindolines (Compounds
6-10). Methyl
(Z)-1-acetyl-3-(ethoxy(phenyl)methylene)-2-oxoindoline-6-carboxyla-
te (6) (Roth G J, et al. J Med Chem. 2009; 52(14):4466-4480).
Indolinone 1 (1000 mg, 52.3 mmol) was suspended in acetic anhydride
(10 mL) and refluxed at 130.degree. C. for 8 h. The reaction
mixture was allowed to cool to 50.degree. C. and
(triethoxymethyl)benzene (2930 mg, 131 mmol) was added. The
resulting reaction mixture was stirred at 120.degree. C. for 6 h.
Then, volatiles were removed in vacuo and petroleum ether was added
to the obtained residue. After triturating for 15 minutes, the
separated solids were filtered and washed with petroleum ether and
then dried under vacuum to afford 974 mg (51%) of title compound.
.sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 8.75 (s, 1H), 8.10 (d,
J=8.0 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.49-7.58 (m, 5H), 4.01 (q,
J=7.2 Hz, 2H), 3.87 (s, 3H), 2.44 (s, 3H), 1.35 (t, J=8.0 Hz, 3H).
HRMS m/z found 365.1260, calcd for C.sub.21H.sub.19NO.sub.5
[M].sup.+365.1263.
[0270] (Z)-1-acetyl-3-(ethoxy(phenyl)methylene)indolin-2-one (7).
The title compound was synthesized in 46% yield using similar
procedure as described for the synthesis of compound 6 by swapping
indolinone 2 for inodolinone 1. Major conformer .sup.1H NMR (400
MHz, DMSO-d.sub.6): 8.17-8.13 (m, 1H), 8.02-7.99 (m, 1H), 7.56-7.46
(m, 5H), 7.30-7.22 (m, 2H), 3.94 (q, J=7.2 Hz, 2H), 2.43 (s, 3H),
1.33 (t, J=7.4 Hz, 3H). LCMS m/z found 308.1, calcd for
C.sub.19H.sub.18NO.sub.3 [M+H].sup.+308.1.
[0271] Methyl
(Z)-1-acetyl-3-(ethoxy(phenyl)methylene)-2-oxoindoline-5-carboxylate
(8). The title compound was synthesized in 48% yield using similar
procedure as described for the synthesis of compound 6 by swapping
indolinone 3 for inodolinone 1. .sup.1H NMR (400 MHz,
DMSO-d.sub.6): .delta. 8.61 (d, J=1.6 Hz, 1H), 8.23 (d, J=8.4 Hz,
1H), 7.92 (dd, J=8.4 Hz, J=1.6 Hz, 1H), 7.58-7.49 (m, 5H), 3.99 (q,
J=7.0 Hz, 2H), 3.87 (s, 3H), 2.45 (s, 3H), 1.37 (t, J=7.0 Hz, 3H).
HRMS m/z found 346.1058, calcd for C.sub.19H.sub.17NNaO.sub.4
[M-Ac+Na].sup.+346.1055.
[0272]
(Z)-1,1'-(3-(Ethoxy(phenyl)methylene)-2-oxoindoline-1,5-diyl)bis(et-
han-1-one) (9). The title compound was synthesized in 47% yield
using similar procedure as described for the synthesis of compound
6 by swapping indolinone 4 for inodolinone 1. .sup.1H NMR (400 MHz,
DMSO-d.sub.6): .delta. 8.58 (d, J=2.0 Hz, 1H), 8.23 (d, J=8.4 Hz,
1H), 7.94 (dd, J=8.4 Hz, J=2.0 Hz, 1H), 7.58-7.49 (m, 5H), 3.99 (q,
J=7.0 Hz, 2H), 2.61 (s, 3H), 2.45 (s, 3H), 1.39 (t, J=8.0 Hz, 3H).
LCMS m/z found 350.1, calcd for C.sub.21H.sub.20NO.sub.4
[M+H].sup.+350.1.
[0273]
(Z)-1-acetyl-3-(Ethoxy(phenyl)methylene)-5-fluoroindolin-2-one
(10). The title compound was synthesized in 48% yield using similar
procedure as described for the synthesis of compound 6 by swapping
indolinone 5 for inodolinone 1. Major conformer .sup.1H NMR (400
MHz, DMSO-d.sub.6): .delta. 8.16 (m, 1H), 7.71 (m, 1H), 7.57-7.47
(m, 5H), 7.12 (m, 1H), 3.98 (q, J=7.0 Hz, 2H), 2.41 (s, 3H), 1.33
(t, J=8.0 Hz, 3H). .sup.19F NMR (376 MHz, DMSO-d.sub.6): .delta.
-117.6 (m). LCMS m/z found 326.1, calcd for
C.sub.19H.sub.17FNO.sub.3 [M+H].sup.+326.1.
[0274]
N-(4-Aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (14)
The title acetamide compound was prepared from
N-methyl-4-nitroaniline 11 using similar procedure as described in
literature (Roth G J, et al. J Med Chem. 2009; 52(14):4466-4480).
.sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 2.10 (s, 3H),
2.14-2.43 (m, 8H), 2.79 (s, 2H), 3.03 (s, 3H), 5.23 (s, 2H),
6.52-6.57 (m, 2H), 6.88-6.92 (m, 2H). HRMS m/z found 263.1866,
calcd for C.sub.14H.sub.23N.sub.4O [M+H].sup.+263.1872.
[0275] General procedure for the synthesis of final compounds
15-19. Methyl
(Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)am-
ino)(phenyl)methylene)-2-oxoindoline-6-carboxylate (15). To a
suspension of methyl
(Z)-1-acetyl-3-(ethoxy(phenyl)methylene)-2-oxoindoline-6-carbox-
ylate (6) (500 mg, 1.368 mmol) in DMF (3.5 mL) was added
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (14)
(395 mg, 1.505 mmol, 1.1 equiv.) at room temperature. After heating
the reaction mixture at 80.degree. C. for 1 h, it was allowed to
cool to RT. Piperidine (297 .mu.L, 3.010 mmol, 2.2 equiv.) was then
added and stirred for 2 h. Volatiles were removed in vacuo and
water was added to the obtained residue and stirred for 15 min.
Precipitate was then filtered under suction and cake was washed
with water, then with minimum amount of cold methanol, and then
ether. The obtained product was purified by column chromatography
(neutral Al.sub.2O.sub.3, 0-10% methanol in CH.sub.2Cl.sub.2) to
afford 532 mg (72%) of target molecule 15. Major conformer .sup.1H
NMR (400 MHz, DMSO-d.sub.6): .delta. 12.22 (s, 1H), 10.98 (s, 1H),
7.66-7.47 (m, 5H), 7.42 (s, 1H), 7.24-7.09 (m, 3H), 6.89 (d, J=8.0
Hz, 2H), 5.83 (d, J=8.0 Hz, 1H), 3.77 (s, 3H), 3.06 (s, 3H), 2.69
(s, 2H), 2.34-2.06 (brs, 8H), 2.10 (s, 3H). HRMS m/z found
540.2606, calcd for C.sub.31H.sub.34N.sub.5O.sub.4
[M+H].sup.+540.2605.
[0276]
(Z)--N-Methyl-2-(4-methylpiperazin-1-yl)-N-(4-(((2-oxoindolin-3-yli-
dene)(phenyl)methyl)amino)phenyl)acetamide (16) The title compound
was synthesized in 64% yield using similar procedure as described
for the synthesis of compound 15 by swapping
(Z)-1-acetyl-3-(ethoxy(phenyl)methylene)indolin-2-one (7) for
inodolinone derivative 6. A 58:42 mixture of conformers .sup.1H NMR
(400 MHz, DMSO-d.sub.6): .delta.12.19 and 12.02 (s, 1H), 10.74 and
10.59 (s, 1H), 7.61-5.75 (m, 13H), 3.17-2.64 (m, 5H), 2.36-2.07 (m,
11H). HRMS m/z found 482.2408, calcd for
C.sub.29H.sub.32N.sub.5O.sub.2 [M+H].sup.+482.2400.
[0277]
Methyl(Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phen-
yl)amino)(phenyl)methylene)-2-oxoindoline-5-carboxylate (17). The
title compound was synthesized in 73% yield using similar procedure
as described for the synthesis of compound 15 by swapping Methyl
(Z)-1-acetyl-3-(ethoxy(phenyl)methylene)-2-oxoindoline-5-carboxylate
(8) for inodolinone derivative 6. Major conformer .sup.1H NMR (400
MHz, DMSO-d.sub.6): .delta. 11.97 (s, 1H), 11.13 (s, 1H), 7.63-7.47
(m, 6H), 7.13 (d, J=8.4 Hz, 2H), 6.94 (d, J=8.4 Hz, 1H), 6.88 (d,
J=8.4 Hz, 2H), 6.51 (s, 1H), 3.63 (s, 3H), 3.06 (s, 3H), 2.69 (s,
2H), 2.19 (brs, 8H), 2.10 (s, 3H). HRMS m/z found 540.2613, calcd
for C.sub.31H.sub.34N.sub.5O.sub.4 [M+H].sup.+540.2605.
[0278]
(Z)--N-(4-(((5-Acetyl-2-oxoindolin-3-ylidene)(phenyl)methyl)amino)p-
henyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (18). The title
compound was synthesized in 63% yield using similar procedure as
described for the synthesis of compound 15 by swapping
(Z)-1,1'-(3-(Ethoxy(phenyl)methylene)-2-oxoindoline-1,5-diyl)bis(ethan-1--
one) (9) for inodolinone derivative 6. Major conformer .sup.1H NMR
(400 MHz, DMSO-d.sub.6): .delta. 11.94 (s, 1H), 11.14 (s, 1H),
7.63-7.50 (m, 6H), 7.13 (d, J=8.4 Hz, 2H), 6.94 (d, J=8.4 Hz, 1H),
6.90 (d, J=8.4 Hz, 2H), 6.39 (s, 1H), 3.06 (s, 3H), 2.71 (s, 2H),
2.32-2.16 (brs, 8H), 2.14 (s, 6H). HRMS m/z found 523.2585, calcd
for C.sub.31H.sub.33N.sub.5O.sub.3 [M].sup.+523.2583.
[0279]
(Z)--N-(4-(((5-Fluoro-2-oxoindolin-3-ylidene)(phenyl)methyl)amino)p-
henyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (19). The title
compound was synthesized in 69% yield using similar procedure as
described for the synthesis of compound 15 by swapping
(2)-1-acetyl-3-(Ethoxy(phenyl)methylene)-5-fluoroindolin-2-one (10)
for inodolinone derivative 6. .sup.1H NMR (400 MHz, DMSO-d.sub.6):
.delta. 12.09 (s, 1H), 10.76 (s, 1H), 7.63-7.48 (m, 5H), 7.12 (d,
J=8.4 Hz, 2H), 6.90-6.79 (m, 3H), 6.72 (m, 1H), 5.37 (dd, J=10.4
Hz, 2.4 Hz, 1H), 3.05 (s, 3H), 2.67 (s, 2H), 2.18 (brs, 8H), 2.10
(s, 3H). .sup.19F NMR (376 MHz, DMSO-d.sub.6): .delta. -123.3 (m).
HRMS m/z found 500.2455, calcd for C.sub.29H.sub.31FN.sub.5O.sub.2
[M+H].sup.+500.2456.
[0280] General procedure for the synthesis of final compounds
22-25.
(Z)--N-(3-(Dimethylamino)propyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1--
yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide
(22). 6-carboxylic acid methyl ester 15 (250 mg, 0.46 mmol) was
added to 1:1 mixture of methanol and dioxane (8 mL). The resulting
suspension was heated to 50.degree. C. and then 1N aqueous NaOH
solution (2.5 mL) was added. The solution was stirred for 5 h at
80.degree. C. and then allowed to cool to room temperature.
Volatiles were removed in vacuo and water was added to the obtained
residue. After stirred for 10 minutes, the separated solids were
filtered and washed with water and diethyl ether, and then dried
under vacuum to afford crude
(Z)-3-(((4-(N-Methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(ph-
enyl)methylene)-2-oxoindoline-6-carboxylic acid (20). Major
conformer .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 12.20 (s,
1H), 10.96 (s, 1H), 7.63-7.48 (m, 5H), 7.42 (s, 1H), 7.17 (dd,
J=8.0 Hz, 1.2 Hz, 1H), 7.13 (d, J=8.4 Hz, 2H), 6.88 (d, J=8.0 Hz,
2H), 5.81 (d, J=8.4 Hz, 1H), 3.06 (s, 3H), 2.67 (s, 2H), 2.20 (brs,
8H), 2.12 (s, 3H). HRMS m/z found 525.2371, calcd for
C.sub.30H.sub.31N.sub.5O.sub.4 [M].sup.+525.2376.
[0281] To a suspension of crude 6-carboxylic acid 20 (1.0 equiv.),
HBTU or TBTU (1.2 equiv.), HOBt (1.2 equiv.) in dimethylformamide
was added DIPEA (16 equiv.) at room temperature.
N,N-dimethylpropane-1,3-diamine (1.5 equiv.) was then added and
continued stirred for 2 h. Volatiles were removed in vacuo and the
obtained product was purified by column chromatography (neutral
Al.sub.2O.sub.3, 0-10% methanol in CH.sub.2Cl.sub.2) or HPLC to
afford the target molecule 2-oxoindoline-6-carboxamide 22. Major
conformer .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.12.10 (s,
1H), 10.93 (s, 1H), 8.24 (t, J=5.2 Hz, 1H), 7.64-7.49 (m, 5H), 7.33
(d, J=1.6 Hz, 1H), 7.12 (d, J=8.4 Hz, 2H), 7.05 (dd, J=8.4 Hz, 1.6
Hz, 1H), 6.87 (d, J=8.4 Hz, 2H), 5.74 (d, J=8.0 Hz, 1H), 3.20 (q,
J=8.0 Hz, 2H), 3.05 (s, 3H), 2.69 (s, 2H), 2.33-2.05 (m, 19H), 1.59
(m, 2H). HRMS m/z found 610.3494, calcd for
C.sub.35H.sub.44N.sub.7O.sub.3 [M+H].sup.+ 610.3500.
[0282]
(Z)--N-(3-Aminopropyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)a-
cetamido)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide
(23). The title compound was synthesized using similar procedure as
described for the synthesis of compound 22 by substituting
tert-butyl (3-aminopropyl)carbamate for dimethylpropane-1,3-diamine
followed by removal of Boc protecting with treatment of 20% TFA in
dichloromethane at rt for 3 hr. Major conformer .sup.1H NMR (400
MHz, DMSO-d.sub.6): .delta.12.15 (s, 1H), 10.98 (s, 1H), 8.37 (t,
J=5.2 Hz, 1H), 7.72-7.50 (m, 7H), 7.35 (s, 1H), 7.16 (d, J=8.4 Hz,
2H), 7.08 (dd, J=8.0 Hz, 1.6 Hz, 1H), 6.89 (d, J=8.4 Hz, 2H), 5.74
(d, J=8.4 Hz, 1H), 3.48-2.69 (m, 20H), 1.74 (m, 2H). HRMS m/z found
582.3186, calcd for C.sub.33H.sub.40N.sub.7O.sub.3 [M+H].sup.+
582.3187.
[0283]
(Z)--N-Methyl-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)-
phenyl)amino)(phenyl)methylene)-2-oxoindoline-5-carboxamide (24).
(Z)-3-(((4-(N-Methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(ph-
enyl)methylene)-2-oxoindoline-5-carboxylic acid (21) was
synthesized using similar procedure as described for the synthesis
of compound 20 by swapping indolinone 17 for inodolinone derivative
15. Major conformer .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.
12.00 (s, 1H), 11.09 (s, 1H), 7.60-7.47 (m, 6H), 7.12 (d, J=8.4 Hz,
2H), 6.92 (d, J=8.0 Hz, 1H), 6.85 (d, J=8.8 Hz, 2H), 6.59 (s, 1H),
3.05 (s, 3H), 2.69 (s, 2H), 2.19 (brs, 8H), 2.11 (s, 3H). HRMS m/z
found 526.2453, calcd for C.sub.30H.sub.32N.sub.5O.sub.4
[M+H].sup.+ 526.2449.
[0284] The title compound 24 was synthesized using similar
procedure as described for the synthesis of compound 22 by
substituting methylamine hydrochloride for
dimethylpropane-1,3-diamine. Major conformer .sup.1H NMR (400 MHz,
DMSO-d.sub.6): .delta. 12.03 (s, 1H), 10.97 (s, 1H), 7.93 (m, 1H),
7.62-7.46 (m, 5H), 7.39 (dd, J=8.4 Hz, 1.6 Hz, 1H), 7.11 (d, J=8.4
Hz, 2H), 6.85 (d, J=8.4 Hz, 1H), 6.83 (d, J=8.8 Hz, 2H), 6.53 (s,
1H), 3.05 (s, 3H), 2.69 (s, 2H), 2.63 (d, J=4.8 Hz, 3H), 2.19 (brs,
8H), 2.11 (s, 3H). HRMS m/z found 539.2782, calcd for
C.sub.31H.sub.35N.sub.6O.sub.3 [M+H].sup.+ 539.2765.
[0285]
(Z)--N,N-Dimethyl-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetam-
ido)phenyl)amino)(phenyl)methylene)-2-oxoindoline-5-carboxamide
(25). The title compound was synthesized using similar procedure as
described for the synthesis of compound 22 by substituting
dimethylamine hydrochloride for dimethylpropane-1,3-diamine. Major
conformer .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 12.03 (s,
1H), 10.94 (s, 1H), 7.62-7.48 (m, 5H), 7.11 (d, J=8.4 Hz, 2H), 7.03
(dd, J=8.0 Hz, 1.6 Hz, 1H), 6.88 (d, J=8.0 Hz, 1H), 6.84 (d, J=8.0
Hz, 2H), 5.86 (s, 1H), 3.05 (s, 3H), 2.76 (s, 6H), 2.69 (s, 2H),
2.18 (brs, 8H), 2.10 (s, 3H). HRMS m/z found 553.2930, calcd for
C.sub.32H.sub.37N.sub.6O.sub.3 [M+H].sup.+ 553.2922.
MELK Assays and Selectivity Screening
Inhibitor Library Screening
[0286] MELK and its substrate, Bcl-G were both recombinantly
expressed and purified for use in screening assays (See Methods).
752 compounds from an in-house curated inhibitor library were
subjected to a p81-based kinase assay. 10 nM MELK 340 and 10 .mu.M
Bcl-GL in kinase assay buffer (50 mM HEPES pH 7.5, 100 mM KCl, 0.1
mM EDTA, 0.1 mM EGTA, 10 mM MgCl.sub.2, 10 .mu.g/mL BSA) with 10 mM
DTT were added to either 10 .mu.M or 1 .mu.M inhibitor aliquoted
into 96 well plates (final 1% DMSO). The mixture was incubated at
room temperature for 30 minutes prior to initiation of the assay
with 40 .mu.M .gamma.-.sup.32P-ATP (100-1000 CPM/pmol). 40 .mu.L
aliquots were spotted onto a p81 96 well filter plate (Unifilter,
Whatman), quenched, and washed with 75 mM 0-phosphoric acid 8
times, followed by a final wash with acetone for drying. Wells were
then filled with scintillation fluid, sealed, and quantified using
a MicroBeta TriLux liquid scintillation counter (PerkinElmer). Each
inhibitor plate was assayed in duplicate, with at least 4 wells
without MELK to establish background and at least 4 wells without
inhibitor as a negative control. All readings were corrected for
background signal based on the average counts from the wells
without enzyme. Percent inhibition, defined as
[100-(CPM+inhibitor/average CPM of positive controls)*100], was
determined first at 10 .mu.M inhibitor. The top 50 inhibitors were
then re-screened in duplicate at 1 .mu.M.
Characterization of Hits and Compound 15 (MELK-in-1)
Derivatives
[0287] The top 10 hits from the 1 .mu.M screening and derivatives
of MELK-In-1 were subjected to the same assay conditions with
varied inhibitor concentrations (0.0005-50 .mu.M) to generate a
dose-response curve and IC.sub.50. At set time points (0.25, 0.5,
1, 1.5, and 2 min for assays containing 10 nM MELK and 1, 2, 4, 6,
and 10 min for assays containing 1 nM MELK), 30 .mu.L aliquots were
taken from each reaction and spotted onto 2.times.2 cm squares of
p81 paper. Papers were washed 4 times in 75 mM 0-phosphoric acid
and once in acetone. Labeled protein was quantified by its
associated CPM determined on a Packard 1500 scintillation counter
at a sigma value of 2. CPM were translated into nmol .sup.32P
incorporated using Equation 1.
nmolP = ( C .times. P .times. M / 3 ) .times. ( 1 .times. 10 5 ) (
1 .times. 10 - 9 ) .times. Specific .times. .times. Activity
Equation .times. .times. 1 ##EQU00001##
[0288] Rates in the presence of each concentration of inhibitor
were determined by plotting nmol .sup.32P incorporated vs. time.
These rates were normalized to rates obtained without inhibitor,
and data were plotted in terms of fractional activity. In most
cases, data were fit to Equation 2. If the IC.sub.50 approached the
amount of enzyme used in the assay, data were re-fit to the
equation for tight-binding inhibitors (the Morrison equation,
Equation 2). To validate IC.sub.50 values for MELK-In-7, 8, 9, and
11, assays were repeated in the presence of 1 nM enzyme instead of
10 nM. Data were transformed as discussed and fit to Equation
3.
v i v 0 = 1 - [ I ] ( [ I ] + IC 5 .times. 0 ) , Equation .times.
.times. 2 ##EQU00002##
where v.sub.i is the observed rate with inhibitor, v.sub.0 is the
rate in the absence of inhibitor, [I] is inhibitor concentration in
nM, and IC.sub.50 is the concentration of inhibitor at which half
maximal change in v.sub.0 is observed.
v i v 0 = 1 - ( E T + [ I ] + IC 5 .times. 0 ) - E T + [ I ] + IC 5
.times. 0 - 4 .times. E T .function. [ I ] 2 .times. E T , Equation
.times. .times. 3 ##EQU00003##
where E.sub.T was constrained to the total enzyme in the assay.
[0289] IC.sub.50 values were used to estimate each inhibitor's K
using the Cheng-Prusoff relationship for competitive inhibitors
(Equation 4). Apparent K.sub.M.sup.ATP for MELK was experimentally
determined for screening and derivative conditions by performing
two independent dose-response curves with varied ATP (0-1.28 mM).
The value obtained, K.sub.M.sup.ATP=6.+-.1.5 .mu.M, was used as a
parameter in Equation 4 in the course of fitting the K.sub.i.
K i = IC 5 .times. 0 / ( 1 + [ A .times. T .times. P ] K M A
.times. T .times. P ) Equation .times. .times. 4 ##EQU00004##
Selectivity Screening
[0290] Candidates for inhibitor selectivity characterization were
chosen based on an initial single-timepoint commercial kinome
profiling screen performed with inhibitor 17 (MELK-In-7)
(KinomeScan, DiscoveRx, San Diego, Calif.), primary sequence
relation to MELK, and laboratory availability. CHK1 and NUAK1 were
purchased from SignalChem (Vancouver, BC). The NUAK2, CHK, and SAMS
peptides were purchased from BioSyn (Lewisville, Tex.). The
sequence of CHK and NUAK2 peptides are described elsewhere (Sanchez
Y, et al. Science (New York, N.Y.). 1997; 277(5331):1497-1501;
Scott J W, et al. Sci Rep. 2015; 5:14436). ERK2, AMPK, CAMKK2, and
Ets1 were produced in house as previously described (Waas W F,
Dalby K N. The Journal of biological chemistry. 2002;
277(15):12532-12540; Neumann D, et al. Protein Expr Purif. 2003;
30(2):230-237; Waas W F, Dalby K N. Protein Expr Purif. 2001;
23(1):191-197). Apparent K.sub.M values for ATP under specific
assay conditions were determined using respective experimental
conditions in Table 8 with varied ATP (0-1.28 mM). All selectivity
dose-response assays were performed in kinase assay buffer (see
Inhibitor Library Screen) with 2 mM DTT and 100 .mu.M
.gamma.-.sup.32P-ATP with additional conditions listed in Table 8.
IC.sub.50 and K.sub.M.sup.ATP values were subsequently used to
calculate K (Equation 4). Relative selectivity was determined by
comparing K.sub.i.sup.Enzyme/K.sub.i.sup.MELK (termed .phi. in
Table 7). All IC.sub.50 and/or K.sub.i data were fit using
Prism.RTM. (GraphPad) using equations 2, 3, and 4, as appropriate.
Standard error from linear regression data was propagated
internally in Prism and taken into account in nonlinear regression
to determine IC.sub.50 or K.sub.i.
Cell Culture and Reagents
[0291] HCC70, BT-549, and SUM159 human TNBC cell lines and MCF10A
human breast epithelial cell line were purchased from American Type
Culture Collection. The murine 4T1-Luc TNBC cells were purchased
from PerkinElmer. HCC70, BT-549, and 4T1-Luc cells were maintained
in RPMI 1640 medium (Life Technologies Inc., Grand Island, N.Y.,
USA) and MDA-MB-231-LM2 cells in Dulbecco's modified Eagle's
medium/F12 medium (Life Technologies Inc., Grand Island, N.Y.,
USA), both types of medium supplemented with FBS (10%) and
antibiotic/antimycotic (1%). SUM159 cells were maintained in Ham's
F-12 medium (Life Technologies Inc., Grand Island, N.Y., USA)
supplemented with FBS (5%), antibiotic/antimycotic (1%), insulin (5
.mu.g/mL), and hydrocortisone (1 .mu.g/mL). MCF10A cells were
maintained in Dulbecco's modified Eagle's medium/F12 medium
supplemented with horse serum (10%), antibiotic/antimycotic (1%),
insulin (10 .mu.g/mL), EGF (20 ng/mL), cholera toxin (100 ng/mL),
and hydrocortisone (500 .mu.g/mL). All cell lines used in this
study were validated by the Characterized Cell Line Core Facility
at MD Anderson Cancer Center by using a short tandem repeat method
based on primer extension to detect single base deviations.
Western Blotting
[0292] Western blotting was done as described previously
(Bartholomeusz C, et al. Clinical cancer research: an official
journal of the American Association for Cancer Research. 2010;
16(6):1802-1811). Proteins of interest were probed using the
following primary antibodies (1:1000 dilution) purchased from Cell
Signaling Technology (Danvers, Mass., USA) or other suppliers as
indicated: anti-fibronectin (1:500 dilution; BD Transduction),
anti-vimentin, anti-E-cadherin (1:1000 dilution; BD Transduction),
anti-.beta.-catenin, anti-snail (1:1000 dilution; Santa Cruz), and
anti-.alpha.-tubulin (clone DM1A, T9026, Sigma-Aldrich, St. Louis,
Mo., USA). Secondary antibodies were horseradish
peroxidase-conjugated IgG (1:10,000 dilution; Invitrogen) for
chemiluminescent signal detection and the corresponding Alexa
Fluor-conjugated IgG (1:5000 dilution; Invitrogen) for fluorescence
signal detection.
Cell Proliferation Assay
[0293] Cell proliferation was determined using the CellTiter-Blue
viability assay as described previously (Gloeckner H, et al.
Journal of immunological methods. 2001; 252(1-2):131-138). Cells
were seeded in 96-well plates and treated the next day with MELK
inhibitors (0-20 .mu.M). At 72 h after MELK inhibitor treatment,
optical density at 595 nm was determined.
Cloning and Recombinant Expression of MELK and Bcl-G
[0294] The plasmid containing full length MELK (GenBank Accession
number NM_014791) was a generous gift of Dr. Garth Powis (Sanford
Burnham Prebys). The catalytic domain of MELK (amino acids 1-340)
was subcloned into pET28a using the NdeI/XhoI restriction sites.
Insertion and sequence were verified by DNA sequencing. Plasmids
were transformed into BL21(DE3) E. coli, grown in TB media to an
OD.sub.595 of 0.6, and induced with 0.5 mM IPTG. Cultures were
grown for 5 hours at 25.degree. C., harvested by centrifugation at
15,000 rpm at 4.degree. C. for 30 minutes, and flash frozen for
storage at -80.degree. C. Pellets were resuspended in Ni-NTA lysis
buffer (20 mM Tris pH 8, 0.5 M NaCl, 0.03% Brij-35, 1% Triton
X-100, 5 mM imidazole, 1 mM benzamidine, 0.1 mM TPCK, 0.1 mM PMSF,
and 0.1% B-mercaptoethanol) and sonicated. Lysates were cleared by
centrifugation and supernatants incubated with nickel beads
(Qiagen) at 4.degree. C. for 1 hour. Beads were washed (20 mM Tris
pH 8, 0.03% Brij-35, 10 mM imidazole, 1 mM benzamidine, 0.1 mM
TPCK, 0.1 mM PMSF, and 0.1% B-mercaptoethanol) and
hexahistadine-tagged MELK was subsequently eluted (20 mM Tris pH 8,
0.03% Brij-35, 250 mM imidazole, 1 mM benzamidine, 0.1 mM TPCK, 0.1
mM PMSF, and 0.1% B-mercaptoethanol). Nickel eluates were
subsequently dialyzed into equilibration buffer (20 mM Tris pH 8,
0.1 mM EDTA, 0.1 mM EGTA, 0.1% B-mercaptoethanol), loaded onto an
anion exchange column (MonoQ 10/100, GE Healthcare), and eluted
over a 17 column volume linear gradient of 0-0.5 M NaCl. Fractions
were analyzed by SDS-PAGE. Protein was dialyzed into storage buffer
(25 mM HEPES pH 7.5, 50 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM DTT,
10% glycerol) and stored at -80.degree. C.
[0295] Full-length cDNA for Bcl-GL (GenBank Accession number
BC025778) was purchased from Open Biosystems and subcloned into
pET28a using LIC. After insertion and sequence verification,
plasmids were transformed into BL21(DE3) E. coli, cultured in TB
media to an OD.sub.595 of 0.6, and induced with 50 .mu.M IPTG.
Flasks were incubated overnight (18 hours) at 18.degree. C. and
harvested and purified using nickel affinity and anion exchange
columns as described for MELK.
TABLE-US-00009 TABLE 8 Conditions used in selectivity screening.
Apparent Time K.sub.M.sup.ATP .+-. SE Enzyme Conditions Course
(.mu.M) AMPK 10 nM AMPK, 100 .mu.M 0.25-2 min 98 .+-. 8.4 SAMS, 50
.mu.M AMP CAMKK2 50 nM CAMKK2, 200 .mu.M 0.5-6 min 265 .+-. 25.sup.
NUAK2 peptide, 150 .mu.M total Ca2+, 1 .mu.M calmodulin CHK1 5 nM
CHK1, 100 .mu.M CHK 0.25-4 min 125 .+-. 2.5 peptide ERK2 1 nM ERK2,
20 .mu.M Ets-1 0.25-4 min 98 .+-. 14 NUAK1 10 nM NUAK1, 100 .mu.M
0.25-4 min 60 .+-. 3.6 CHK peptide
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252(1-2):131-138.
Example 2. Synthesis of Indolinone Derivatives 91-108
[0346] A number of indolinone derivatives were synthesized in order
to improve potency and selectivity. (See Compounds 91-108). The
general synthetic routes are outlined in Schemes 3 and 4. The key
intermediates 79-90 were prepared via modified Bischler-napieralski
reactions of 2-alkynylaryl isocyanates 67-78 with iron trichloride
(Cantagrel, G., et al. J. Org. Lett., 2009, 11, 4262-4265).
##STR00090## ##STR00091## ##STR00092## ##STR00093##
##STR00094## ##STR00095##
[0347] Stereoselectively converted final indolinone
(Z)-3-(aminoarylmethylene)oxindoles analogs were synthesized by
addition of compound 14 to 3-(arylchloromethylene)oxindoles 79-90.
which can be subsequently stereoselectively converted to
(Z)-3-(aminoarylmethylene)oxindoles (scheme 4). Additional amino
analogs 111 and 112 were prepared from the corresponding nitro
derivatives 99 and 101 through reduction by tin (II) chloride.
Alcohol analogs 113 and 114 were also obtained via TBS-deprotection
using TBAF. 5-acid derivatives 107-108 were synthesized through
hydrolysis using aqueous 1N NaOH (Scheme 4).
Methyl 4-amino-3-((trimethylsilyl)ethynyl)benzoate (27)
##STR00096##
[0349] A combined suspension of methyl
4-amino-3-((trimethylsilyl)ethynyl)benzoate (1.50 g, 5.41 mmol),
copper (I) iodide (21 mg, 0.11 mmol),
bis(triphenylphosphine)palladium(II) dichloride (76 mg, 0.11 mmol),
and ethynyltrimethylsilane (0.90 mL, 6.50 mmol) were stirred in
toluene and TEA (1/1, 46 mL) at ambient temperature for overnight.
The reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with hexane/ethyl
acetate (5/1, v/v) to obtain the compound 27 as a white solid (1.33
g, 99% yield):
[0350] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.00 (d, J=2.0 Hz,
1H), 7.77 (dd, J=8.6, 2.0 Hz, 1H), 6.65 (d, J=8.5 Hz, 1H), 4.69
(bs, 2H), 3.83 (s, 3H), 0.26 (s, 9H);
[0351] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 166.6, 152.0,
134.7, 131.7, 119.2, 113.2, 107.0, 100.6, 100.6, 51.8, 0.2.
Methyl 4-amino-3-ethynylbenzoate (28)
##STR00097##
[0353] To a solution of methyl
4-amino-3-((trimethylsilyl)ethynyl)benzoate (1.34 g, 5.42 mmol) in
THF (11 mL) was added tetrabutylammonium fluoride solution (5.96
ml, 1M in THF). After addition, the reaction was stirred at ambient
temperature for 2 h. All the volatile solvent was removed under
reduced pressure, and the residue was dissolved in DCM (20 ml).
Water (15 mL) was added, and the aqueous layer was extracted with
DCM (15 mL.times.2). The combined organic layer was washed with
brine (30 mL), dried over Na.sub.2SO.sub.4, and filtered. The
organic layer was concentrated under reduced pressure and the
residue was purified by silica gel chromatography, by column
chromatography with hexane/ethyl acetate (2/1, v/v) to obtain the
compound 28 as a white solid (949 mg, quant. yield):
[0354] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.01 (d, J=2.0 Hz,
1H), 7.79 (dd, J=8.6, 2.0 Hz, 1H), 6.65 (d, J=8.6 Hz, 1H), 4.74
(bs, 2H), 3.83 (s, 3H), 3.39 (s, 1H);
[0355] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 166.6, 152.3,
134.9, 131.9, 119.1, 113.3, 105.8, 83.1, 76.8, 51.8, 29.8.
Methyl 4-amino-3-((4-nitrophenyl)ethynyl)benzoate (31)
##STR00098##
[0357] A suspension of methyl 4-amino-3-ethynylbenzoate (1.01 g,
5.78 mmol), 1-bromo-4-nitrobenzene (1.40 g, 6.93 mmol), copper (I)
iodide (55 mg, 0.29 mmol), bis(triphenylphosphine)palladium(II)
dichloride (203 mg, 0.29 mmol), TEA (2.42 ml, 1.73 mmol) were
stirred in THF (10 mL) at ambient temperature for overnight. The
reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with hexane/ethyl
acetate (1/1, v/v) to obtain the compound 31 as a yellow solid
(1.71 g, quant. yield):
[0358] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.27 (d, J=8.3
Hz, 2H), 7.95 (d, J=8.3 Hz, 2H), 7.89 (d, J=2.1 Hz, 1H), 7.70 (dd,
J=8.7, 2.1 Hz, 1H), 6.78 (d, J=8.3 Hz, 1H), 6.60 (bs, 2H), 3.77 (s,
3H).
Methyl 4-amino-3-((4-(methoxycarbonyl)phenyl)ethynyl)benzoate
(32)
##STR00099##
[0360] A suspension of methyl 4-amino-3-ethynylbenzoate (2.04 g,
11.65 mmol), methyl 4-bromobenzoate (3.01 g, 13.97 mmol), copper
(I) iodide (111 mg, 0.58 mmol),
bis(triphenylphosphine)palladium(II) dichloride (409 mg, 0.58
mmol), TEA (4.87 ml, 34.93 mmol) were stirred in THF (19 mL) at
ambient temperature for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with hexane/ethyl acetate (1/1, v/v) to
obtain the compound 32 as a brown solid (3.05 g, 85% yield):
[0361] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 87.98 (d, J=8.1
Hz, 2H), 7.86 (d, J=2.1 Hz, 1H), 7.81 (d, J=8.7 Hz, 2H), 7.68 (dd,
J=8.7, 2.1 Hz, 1H), 6.77 (d, J=8.7 Hz, 1H), 6.50 (s, 2H), 3.87 (s,
3H), 3.77 (s, 3H);
[0362] .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. 165.7, 165.6,
153.8, 134.3, 131.6, 131.5, 129.2, 128.8, 127.6, 116.3, 113.4,
104.1, 93.4, 88.9, 52.3, 51.5.
Trimethyl((3-nitrophenyl)ethynyl)silane (39)
##STR00100##
[0364] A suspension of 1-iodo-3-nitrobenzene (3.00 g, 12.05 mmol),
ethynyltrimethylsilane (2.00 mL, 14.46 mmol), copper (I) iodide
(115 mg, 0.60 mmol), bis(triphenylphosphine)palladium(II)
dichloride (423 mg, 0.60 mmol), TEA (5.04 ml, 36.14 mmol) were
stirred in THF (20 mL) at ambient temperature for overnight. The
reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with hexane/ethyl
acetate (10/1, v/v) to obtain the compound 39 (2.64 g, quant.
yield):
[0365] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.31 (t, J=2.0 Hz,
1H), 8.16 (dd, J=8.3, 1.1 Hz, 1H), 7.75 (dt, J=7.7, 1.3 Hz, 1H),
7.49 (t, J=8.0 Hz, 1H), 0.27 (s, 9H);
[0366] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 137.7, 129.4,
126.9, 125.1, 123.3, 102.3, 97.8, -0.1.
(Benzo [d][1,3]dioxol-5-ylethynyl)trimethylsilane (40)
##STR00101##
[0368] A suspension of 5-iodobenzo[d][1,3]dioxole (3.00 g, 12.10
mmol), ethynyltrimethylsilane (2.01 mL, 14.52 mmol), copper (I)
iodide (115 mg, 0.61 mmol), bis(triphenylphosphine)palladium(II)
dichloride (424 mg, 0.61 mmol), TEA (5.06 ml, 36.29 mmol) were
stirred in THF (20 mL) at ambient temperature for overnight. The
reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with hexane/ethyl
acetate (10/1, v/v) to obtain the compound 40 (2.64 g, quant.
yield):
[0369] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 6.98 (dd, J=8.1,
1.6 Hz, 1H), 6.89 (d, J=1.6 Hz, 1H), 6.70 (d, J=8.0 Hz, 1H), 5.92
(s, 2H), 0.23 (s, 9H);
[0370] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 148.1, 147.4,
126.8, 116.5, 111.9, 108.4, 105.1, 101.4, 92.3, 0.1.
1-Ethynyl-3-nitrobenzene (41)
##STR00102##
[0372] To a solution of trimethyl((3-nitrophenyl)ethynyl)silane
(4.24 g, 19.34 mmol) in THF (39 mL) was added tetrabutylammonium
fluoride solution (21.27 ml, 1M in THF). After addition, the
reaction was stirred at ambient temperature for 2 h. All the
volatile solvent was removed under reduced pressure, and the
residue was dissolved in DCM (20 ml). Water (15 mL) was added, and
the aqueous layer was extracted with DCM (15 mL.times.2). The
combined organic layer was washed with brine (30 mL), dried over
Na.sub.2SO.sub.4, and filtered. The organic layer was concentrated
under reduced pressure and the residue was purified by silica gel
chromatography, by column chromatography with hexane/ethyl acetate
(20/1, v/v) to obtain the compound 41 as a yellow solid (1.77 mg,
quant. yield):
[0373] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.31 (t, J=1.9 Hz,
1H), 8.20 (dd, J=8.3, 1.1 Hz, 1H), 7.79 (dt, J=7.7, 1.3 Hz, 1H),
7.53 (t, J=8.0 Hz, 1H), 3.24 (s, 1H);
[0374] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 137.9, 129.5,
127.0, 124.0, 123.6, 81.2, 80.0.
5-Ethynylbenzo[d][1,3]dioxole (42)
##STR00103##
[0376] To a solution of (benzo
[d][1,3]dioxol-5-ylethynyl)trimethylsilane (3.77 g, 17.27 mmol) in
THF (35 mL) was added tetrabutylammonium fluoride solution (19.00
ml, 1M in THF). After addition, the reaction was stirred at ambient
temperature for 2 h. All the volatile solvent was removed under
reduced pressure, and the residue was dissolved in DCM (20 ml).
Water (15 mL) was added, and the aqueous layer was extracted with
DCM (15 mL.times.2). The combined organic layer was washed with
brine (30 mL), dried over Na.sub.2SO.sub.4, and filtered. The
organic layer was concentrated under reduced pressure and the
residue was purified by silica gel chromatography, by column
chromatography with hexane/ethyl acetate (20/1, v/v) to obtain the
compound 42 as a brown solid (1.74 mg, 98% yield):
[0377] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.00 (dd, J=8.0,
1.6 Hz, 1H), 6.91 (d, J=1.6 Hz, 1H), 6.71 (d, J=8.0 Hz, 1H), 5.92
(s, 2H), 2.98 (s, 1H);
[0378] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 148.3, 147.4,
126.8, 115.2, 112.0, 108.4, 101.4, 83.6, 75.7.
Methyl 4-amino-3-((3-nitrophenyl)ethynyl)benzoate (43)
##STR00104##
[0380] A combined suspension of methyl 4-amino-3-iodobenzoate (2.78
g, 10.04 mmol), copper (I) iodide (38 mg, 0.20 mmol),
bis(triphenylphosphine)palladium(II) dichloride (141 mg, 0.20
mmol), and 1-ethynyl-3-nitrobenzene (1.77 g, 12.05 mmol) were
stirred in toluene and TEA (1/1, 54 mL) at ambient temperature for
overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with hexane/ethyl acetate (3/1, v/v) to obtain the compound 43 as a
yellow solid (2.33 g, 78% yield):
[0381] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.62 (t, J=1.9
Hz, 1H), 8.22 (dd, J=8.3, 1.1 Hz, 1H), 8.08 (dt, J=7.7, 1.3 Hz,
1H), 7.90 (d, J=2.3 Hz, 1H), 7.70 (q, J=8.0 Hz, 1H), 7.69 (d, J=8.7
Hz, 1H), 6.77 (d, J=8.7 Hz, 1H), 6.60 (s, 2H), 3.77 (s, 3H);
[0382] .sup.13C NMR (125 MHz, DMSO-d.sub.6) .delta. 165.6, 153.7,
147.9, 137.5, 134.4, 131.6, 130.1, 125.9, 124.4, 123.0, 116.2,
113.4, 103.8, 91.9, 88.1, 51.5.
Methyl 4-amino-3-(p-tolylethynyl)benzoate (44)
##STR00105##
[0384] A combined suspension of methyl 4-amino-3-iodobenzoate (2.50
g, 9.02 mmol), copper (I) iodide (35 mg, 0.18 mmol),
bis(triphenylphosphine)palladium(II) dichloride (127 mg, 0.18
mmol), and 1-ethynyl-4-methylbenzene (1.37 mL, 10.83 mmol) were
stirred in toluene and TEA (1/1, 76 mL) at ambient temperature for
overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with hexane/ethyl acetate (5/1, v/v) to obtain the compound 44 as a
white solid (2.39 g, quant. yield):
[0385] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.81 (d, J=2.0
Hz, 1H), 7.65 (dd, J=8.7, 2.1 Hz, 1H), 7.56-53 (m, 2H), 7.24-7.21
(m, 2H), 6.76 (d, J=8.7 Hz, 1H), 6.36 (s, 2H), 3.75 (s, 3H), 2.33
(s, 3H);
[0386] .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. 165.7, 153.4,
138.2, 133.8, 131.4, 131.0, 129.2, 119.6, 116.2, 113.2, 105.1,
94.4, 84.9, 51.5, 21.1.
Methyl 4-amino-3-((4-ethylphenyl)ethynyl)benzoate (45)
##STR00106##
[0388] A combined suspension of methyl 4-amino-3-iodobenzoate (2.0
g, 7.22 mmol), copper (I) iodide (28 mg, 0.14 mmol),
bis(triphenylphosphine)palladium(II) dichloride (101 mg, 0.14
mmol), and 1-ethyl-4-ethynylbenzene (1.22 mL, 8.66 mmol) were
stirred in toluene and TEA (1/1, 60 mL) at ambient temperature for
overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with hexane/ethyl acetate (5/1, v/v) to obtain the compound 45 as a
white solid (2.02 g, quant. yield):
[0389] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.07 (d, J=2.0 Hz,
1H), 7.78 (dd, J=8.6, 2.1 Hz, 1H), 7.41 (d, J=8.2 Hz, 2H), 7.12 (d,
J=8.2 Hz, 2H), 6.64 (d, J=8.6 Hz, 1H), 4.86 (s, 2H), 3.81 (s, 3H),
2.6 (q, J=7.6 Hz, 2H), 1.19 (t, J=7.6 Hz, 3H);
[0390] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 166.6, 151.7,
144.8, 134.1, 131.3, 131.1, 127.9, 119.8, 118.8, 113.1, 107.1,
95.2, 84.1, 51.6, 28.7, 15.2.
Methyl 4-amino-3-((4-methoxyphenyl)ethynyl)benzoate (46)
##STR00107##
[0392] A combined suspension of methyl 4-amino-3-iodobenzoate (3.0
g, 10.83 mmol), copper (I) iodide (41 mg, 0.22 mmol),
bis(triphenylphosphine)palladium(II) dichloride (152 mg, 0.22
mmol), and 1-ethynyl-4-methoxybenzene (1.69 mL, 12.99 mmol) were
stirred in toluene and TEA (1/1, 90 mL) at ambient temperature for
overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with hexane/ethyl acetate (3/1, v/v) to obtain the compound 46 as a
white solid (2.96 g, 97% yield):
[0393] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.06 (d, J=2.1 Hz,
1H), 7.77 (dd, J=8.6, 2.1 Hz, 1H), 7.41 (d, J=8.4 Hz, 2H), 6.82 (d,
J=8.4 Hz, 2H), 6.65 (d, J=8.6 Hz, 1H), 4.88 (s, 2H), 3.82 (s, 3H),
3.73 (s, 3H);
[0394] .sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 166.6, 159.6,
151.6, 134.0, 132.8, 131.0, 118.8, 114.7, 113.9, 113.1, 107.2,
95.0, 83.4, 55.1, 51.6.
Methyl 4-amino-3-((3-methoxyphenyl)ethynyl)benzoate (47)
##STR00108##
[0396] A combined suspension of methyl 4-amino-3-iodobenzoate (3.0
g, 10.83 mmol), copper (I) iodide (41 mg, 0.22 mmol),
bis(triphenylphosphine)palladium(II) dichloride (152 mg, 0.22
mmol), and 1-ethynyl-3-methoxybenzene (1.69 mL, 12.99 mmol) were
stirred in toluene and TEA (1/1, 90 mL) at ambient temperature for
overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with hexane/ethyl acetate (3/1, v/v) to obtain the compound 47 as a
white solid (3.00 g, 98% yield):
[0397] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.07 (d, J=2.1 Hz,
1H), 7.78 (dd, J=8.2, 2.0 Hz, 1H), 7.21 (t, J=7.9 Hz, 1H), 7.08
(dt, J=7.7, 1.2 Hz, 2H), 7.03-7.02 (m, 2H), 6.87-6.86 (m, 1H), 6.66
(d, J=8.6 Hz, 1H), 4.90 (s, 2H), 3.82 (s, 3H), 3.74 (s, 3H);
[0398] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 166.5, 159.2,
151.7, 134.3, 131.3, 129.4, 123.8, 123.7, 118.8, 116.2, 114.8,
113.2, 106.7, 94.9, 84.6, 55.1, 51.6.
Methyl 4-amino-3-(benzo[d][1,3]dioxol-5-ylethynyl)benzoate (48)
##STR00109##
[0400] A combined suspension of methyl 4-amino-3-iodobenzoate (2.74
g, 9.90 mmol), copper (I) iodide (38 mg, 1.98 mmol),
bis(triphenylphosphine)palladium(II) dichloride (139 mg, 1.98
mmol), and 5-ethynylbenzo[d][1,3]dioxole (1.74 g, 11.89 mmol) were
stirred in toluene and TEA (1/1, 82 mL) at ambient temperature for
overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with hexane/ethyl acetate (3/1, v/v) to obtain the compound 48 as a
white solid (2.92 g, quant. yield):
[0401] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.04 (d, J=2.0 Hz,
1H), 7.78 (dd, J=8.6, 2.0 Hz, 1H), 7.03 (dd, J=8.0, 1.6 Hz, 1H),
6.94 (d, J=1.6 Hz, 1H), 6.77 (d, J=8.0 Hz, 1H), 6.67 (d, J=8.6 Hz,
1H), 5.97 (s, 2H), 4.74 (bs, 2H), 3.85 (s, 3H);
[0402] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 166.7, 151.5,
148.1, 147.5, 134.3, 131.3, 126.2, 119.3, 116.1, 113.3, 111.4,
108.6, 107.2, 101.5, 95.1, 83.2, 51.8.
4-((Trimethylsilyl)ethynyl)phenol (52)
##STR00110##
[0404] A suspension of 4-iodophenol (3.00 g, 13.64 mmol),
ethynyltrimethylsilane (2.27 mL, 16.36 mmol), copper (I) iodide
(130 mg, 0.68 mmol), bis(triphenylphosphine)palladium(II)
dichloride (479 mg, 0.68 mmol), TEA (5.70 ml, 40.91 mmol) were
stirred in THF (23 mL) at ambient temperature for overnight. The
reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with hexane/ethyl
acetate (2/1, v/v) to obtain the compound 52 (1.76 g, 68%
yield):
[0405] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.33 (d, J=8.7 Hz,
2H), 6.72 (d, J=8.7 Hz, 2H), 5.82 (bs, 1H), 0.23 (s, 9H);
[0406] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 155.8, 133.8,
115.6, 115.4, 105.6, 92.9, 0.1.
(4-((Trimethylsilyl)ethynyl)phenyl)methanol (53)
##STR00111##
[0408] A suspension of (4-iodophenyl)methanol (3.00 g, 13.64 mmol),
ethynyltrimethylsilane (2.13 mL, 15.38 mmol), copper (I) iodide
(122 mg, 0.64 mmol), bis(triphenylphosphine)palladium(II)
dichloride (450 mg, 0.64 mmol), TEA (5.36 ml, 38.46 mmol) were
stirred in THF (22 mL) at ambient temperature for overnight. The
reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with hexane/ethyl
acetate (20/1, v/v) to obtain the compound 53 (2.66 g, quant.
yield):
[0409] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.44 (d, J=8.4 Hz,
2H), 7.26 (d, J=8.5 Hz, 2H), 4.63 (s, 2H), 2.17 (bs, 1H), 0.25 (s,
9H);
[0410] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 141.3, 132.2,
126.7, 122.4, 105.0, 94.3, 64.9, 0.1.
2-(4-((Trimethylsilyl)ethynyl)phenyl)ethan-1-ol (54)
##STR00112##
[0412] A suspension of (4-iodophenyl)ethanol (3.00 g, 12.09 mmol),
ethynyltrimethylsilane (2.01 mL, 14.51 mmol), copper (I) iodide
(115 mg, 0.60 mmol), bis(triphenylphosphine)palladium(II)
dichloride (424 mg, 0.60 mmol), TEA (5.06 ml, 36.28 mmol) were
stirred in THF (20 mL) at ambient temperature for overnight. The
reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with hexane/ethyl
acetate (5/1, v/v) to obtain the compound 54 (2.64 g, quant.
yield):
[0413] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.38 (d, J=8.3 Hz,
2H), 7.08 (d, J=8.2 Hz, 2H), 3.66 (td, J=7.0, 3.9 Hz, 2H), 2.72 (t,
J=6.8 Hz, 2H), 0.25 (s, 9H);
[0414] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 139.4, 132.0,
128.9, 121.0, 105.1, 93.7, 63.0, 38.9, 0.01.
4-Ethynylphenol (55)
##STR00113##
[0416] To a solution of 4-((trimethylsilyl)ethynyl)phenol (1.76 g,
9.25 mmol) in THF (19 mL) was added tetrabutylammonium fluoride
solution (10.17 ml, 1M in THF). After addition, the reaction was
stirred at ambient temperature for 2 h. All the volatile solvent
was removed under reduced pressure, and the residue was dissolved
in DCM (20 ml). Water (15 mL) was added, and the aqueous layer was
extracted with DCM (15 mL.times.2). The combined organic layer was
washed with brine (30 mL), dried over Na.sub.2SO.sub.4, and
filtered. The organic layer was concentrated under reduced pressure
and the residue was purified by silica gel chromatography, by
column chromatography with hexane/ethyl acetate (5/1, v/v) to
obtain the compound 55 as a white solid (511 mg, 47% yield):
[0417] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.39 (d, J=8.7 Hz,
2H), 6.78 (d, J=8.7 Hz, 2H), 5.21 (bs, 1H), 3.00 (s, 1H);
[0418] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 156.1, 134.0,
115.6, 114.5, 83.7, 76.0.
(4-Ethynylphenyl)methanol (56)
##STR00114##
[0420] To a solution of (4-((trimethylsilyl)ethynyl)phenyl)methanol
(2.96 g, 14.47 mmol) in THF (29 mL) was added tetrabutylammonium
fluoride solution (15.92 ml, 1M in THF). After addition, the
reaction was stirred at ambient temperature for 2 h. All the
volatile solvent was removed under reduced pressure, and the
residue was dissolved in DCM (20 ml). Water (15 mL) was added, and
the aqueous layer was extracted with DCM (15 mL.times.2). The
combined organic layer was washed with brine (30 mL), dried over
Na.sub.2SO.sub.4, and filtered. The organic layer was concentrated
under reduced pressure and the residue was purified by silica gel
chromatography, by column chromatography with hexane/ethyl acetate
(3/1, v/v) to obtain the compound 56 as a white solid (1.66 mg, 87%
yield):
[0421] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.41 (d, J=8.3 Hz,
2H), 7.17 (d, J=8.6 Hz, 2H), 4.48 (s, 2H), 3.59 (s, 1H), 3.09 (s,
1H);
[0422] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 141.5, 132.2,
126.7, 121.0, 83.5, 77.4, 64.3.
2-(4-Ethynylphenyl)ethan-1-ol (57)
##STR00115##
[0424] To a solution of
2-(4-((trimethylsilyl)ethynyl)phenyl)ethan-1-ol (2.85 g, 13.07
mmol) in THF (26 mL) was added tetrabutylammonium fluoride solution
(14.37 ml, 1M in THF). After addition, the reaction was stirred at
ambient temperature for 2 h. All the volatile solvent was removed
under reduced pressure, and the residue was dissolved in DCM (10
ml). Water (10 mL) was added, and the aqueous layer was extracted
with DCM (15 mL.times.2). The combined organic layer was washed
with brine (30 mL), dried over Na.sub.2SO.sub.4, and filtered. The
organic layer was concentrated under reduced pressure and the
residue was purified by silica gel chromatography, by column
chromatography with hexane/ethyl acetate (1/1, v/v) to obtain the
compound 57 as a white solid (1.67 mg, 94% yield):
[0425] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.39 (d, J=8.3 Hz,
2H), 7.10 (d, J=8.3 Hz, 2H), 3.66 (td, J=6.9, 2.7 Hz, 2H), 3.28
(bs, 1H), 3.09 (s, 1H), 2.72 (t, J=6.8 Hz, 2H);
[0426] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 139.6, 132.0,
128.9, 119.8, 83.6, 7.2, 62.8, 38.7.
tert-Butyl(4-ethynylphenoxy)dimethylsilane (58)
##STR00116##
[0428] To a solution of 4-ethynylphenol (511 mg, 4.33 mmol) and
imidazole (295 mg, 4.33 mmol) in DMF (13 mL) was added TBDMSCl (652
mg, 4.33 mmol). This reaction mixture was stirred at ambient
temperature for overnight. All the volatile solvent was removed
under reduced pressure, and the residue was dissolved in DCM (10
ml). Water (10 mL) was added, and the aqueous layer was extracted
with DCM (10 mL.times.2). The combined organic layer was washed
with brine (20 mL), dried over Na.sub.2SO.sub.4, and filtered. The
organic layer was concentrated under reduced pressure and the
residue was purified by silica gel chromatography, by column
chromatography with hexane/ethyl acetate (10/1, v/v) to obtain the
compound 58 as a brown solid (675 mg, 67% yield):
[0429] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.41 (d, J=8.6 Hz,
2H), 6.81 (d, J=8.6 Hz, 2H), 3.02 (s, 1H), 1.01 (s, 9H), 0.23 (s,
6H);
[0430] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 156.4, 133.7,
120.3, 115.0, 83.8, 76.1, 25.8, 18.3, -4.3.
tert-Butyl((4-ethynylbenzyl)oxy)dimethylsilane (59)
##STR00117##
[0432] To a solution of (4-ethynylphenyl)methanol (1.66 g, 12.54
mmol) and imidazole (1.71 g, 25.08 mmol) in DMF (37 mL) was added
TBDMSCl (2.84 g, 18.81 mmol). This reaction mixture was stirred at
ambient temperature for overnight. All the volatile solvent was
removed under reduced pressure, and the residue was dissolved in
DCM (10 ml). Water (10 mL) was added, and the aqueous layer was
extracted with DCM (10 mL.times.2). The combined organic layer was
washed with brine (20 mL), dried over Na.sub.2SO.sub.4, and
filtered. The organic layer was concentrated under reduced pressure
and the residue was purified by silica gel chromatography, by
column chromatography with hexane/ethyl acetate (10/1, v/v) to
obtain the compound 59 as a brown solid (2.91 mg, 94% yield):
[0433] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.46 (d, J=8.2 Hz,
2H), 7.28 (d, J=8.8 Hz, 2H), 4.73 (s, 2H), 3.04 (s, 1H), 0.94 (s,
9H), 0.10 (s, 6H);
[0434] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 142.5, 132.2,
126.0, 120.6, 83.9, 64.7, 26.1, 18.5, -5.1.
tert-Butyl(4-ethynylphenethoxy)dimethylsilane (60)
##STR00118##
[0436] To a solution of 2-(4-ethynylphenyl)ethan-1-ol (1.67 g,
11.40 mmol) and imidazole (1.55 g, 22.79 mmol) in DMF (34 mL) was
added TBDMSCl (2.58 g, 17.09 mmol). This reaction mixture was
stirred at ambient temperature for overnight. All the volatile
solvent was removed under reduced pressure, and the residue was
dissolved in DMF (34 ml). Water (10 mL) was added, and the aqueous
layer was extracted with DCM (10 mL.times.2). The combined organic
layer was washed with brine (20 mL), dried over Na.sub.2SO.sub.4,
and filtered. The organic layer was concentrated under reduced
pressure and the residue was purified by silica gel chromatography,
by column chromatography with hexane/ethyl acetate (1/1, v/v) to
obtain the compound 60 as a brown solid (2.67 g, 90% yield):
[0437] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.45 (d, J=8.2 Hz,
2H), 7.20 (d, J=8.2 Hz, 2H), 3.84 (t, J=6.8, 2H), 3.08 (s, 1H),
2.84 (t, J=6.8 Hz, 2H), 0.93 (s, 9H), 0.03 (s, 6H);
[0438] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 140.3, 132.0,
129.2, 120.0, 83.9, 76.9, 64.1, 39.5, 26.0, 18.3, -5.4.
Methyl
4-amino-3-((4-((tert-butyldimethylsilyl)oxy)phenyl)ethynyl)benzoate
(61)
##STR00119##
[0440] A combined suspension of methyl 4-amino-3-iodobenzoate (1.21
g, 4.36 mmol), copper (I) iodide (17 mg, 0.87 mmol),
bis(triphenylphosphine)palladium(II) dichloride (61 mg, 0.87 mmol),
and tert-butyl(4-ethynylphenoxy)dimethylsilane (1.22 g, 5.23 mmol)
were stirred in toluene and TEA (1/1, 36 mL) at ambient temperature
for overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with hexane/ethyl acetate (5/1, v/v) to obtain the compound 61 as a
brown solid (1.66 g, quant. yield):
[0441] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.06 (d, J=2.1 Hz,
1H), 7.80 (dd, J=8.6, 2.0 Hz, 1H), 7.41 (d, J=8.6 Hz, 2H), 6.82 (d,
J=8.6 Hz, 2H), 6.68 (d, J=8.5 Hz, 1H), 4.73 (s, 2H), 3.86 (s, 3H),
0.99 (s, 9H), 0.22 (s, 6H);
[0442] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 166.8, 156.3,
151.5, 134.3, 133.1, 131.2, 120.4, 119.4, 115.7, 113.3, 107.6,
95.2, 83.6, 51.8, 25.7, 18.3, -4.3.
Methyl 4-amino-3-((4-(hydroxymethyl)phenyl)ethynyl)benzoate
(62)
##STR00120##
[0444] A combined suspension of methyl 4-amino-3-iodobenzoate (2.72
g, 9.83 mmol), copper (I) iodide (37 mg, 0.20 mmol),
bis(triphenylphosphine)palladium(II) dichloride (138 mg, 0.20
mmol), and tert-butyl((4-ethynylbenzyl)oxy)dimethylsilane (2.91 g,
11.79 mmol) were stirred in toluene and TEA (1/1, 82 mL) at ambient
temperature for overnight. The reaction solvent was evaporated
under reduced pressure, and the residue was purified by column
chromatography with hexane/ethyl acetate (7/1, v/v) to obtain the
compound 62 as a brown solid (3.89 g, quant. yield):
Methyl
4-amino-3-((4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)ethynyl-
)benzoate (63)
##STR00121##
[0446] A combined suspension of methyl 4-amino-3-iodobenzoate (2.36
g, 8.53 mmol), copper (I) iodide (33 mg, 0.17 mmol),
bis(triphenylphosphine)palladium(II) dichloride (120 mg, 0.17
mmol), and tert-butyl(4-ethynylphenethoxy)dimethylsilane (2.67 g,
10.23 mmol) were stirred in toluene and TEA (1/1, 72 mL) at ambient
temperature for overnight. The reaction solvent was evaporated
under reduced pressure, and the residue was purified by column
chromatography with hexane/ethyl acetate (3/1, v/v) to obtain the
compound 63 as a brown solid (3.66 g, quant. yield):
[0447] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.04 (d, J=2.1 Hz,
1H), 7.75 (dd, J=8.6, 2.0 Hz, 1H), 7.39 (d, J=8.2 Hz, 2H), 7.12 (d,
J=8.3 Hz, 2H), 6.62 (d, J=8.6 Hz, 1H), 4.94 (s, 2H), 3.78 (s, 3H),
3.75 (t, J=6.7 Hz, 2H), 2.75 (t, J=6.7 Hz, 2H), 0.85 (s, 9H), -0.05
(s, 6H);
[0448] .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 166.4, 151.7,
139.9, 134.1, 131.1, 129.1, 120.3, 118.6, 113.0, 106.8, 95.0, 84.3,
63.8, 51.4, 39.2, 25.7, 18.0, -5.7.
N-(4-Ethynylphenyl)acetamide (65)
##STR00122##
[0450] To a solution of 4-ethynylaniline (3.00 g, 25.61 mmol) in
DCM (75 mL) was added acetic anhydride (2.71 ml, 28.68 mmol). After
addition, the reaction was stirred at ambient temperature for 6 h.
And the, water (75 mL) was added, and the aqueous layer was
extracted with DCM (15 mL.times.2). The combined organic layer was
washed with brine (30 mL), dried over Na.sub.2SO.sub.4, and
filtered. The organic layer was concentrated under reduced pressure
and the residue was purified by silica gel chromatography, by
column chromatography with hexane/ethyl acetate (2/1, v/v) to
obtain the compound 65 as a white solid (3.41 g, 84% yield):
[0451] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.10.10 (s, 1H),
7.59 (d, J=8.7 Hz, 2H), 7.39 (d, J=8.7 Hz, 2H), 4.06 (s, 1H), 2.05
(s, 3H);
[0452] .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. 168.5, 139.8,
132.3, 118.7, 115.8, 83.6, 79.7, 24.1.
Methyl 3-((4-acetamidophenyl)ethynyl)-4-aminobenzoate (66)
##STR00123##
[0454] A combined suspension of methyl 4-amino-3-iodobenzoate (3.00
g, 10.83 mmol), copper (I) iodide (41 mg, 0.22 mmol),
bis(triphenylphosphine)palladium(II) dichloride (152 mg, 0.22
mmol), and N-(4-ethynylphenyl)acetamide (2.07 g, 12.99 mmol) were
stirred in toluene and TEA (1/1, 90 mL) at ambient temperature for
overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with hexane/ethyl acetate (1/1, v/v) to obtain the compound 66 as a
white solid (2.18 g, 65% yield):
[0455] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 710.11 (s, 1H),
7.80 (d, J=2.0 Hz, 1H), 7.65-7.56 (m, 5H), 6.75 (d, J=8.7 Hz, 1H),
6.33 (s, 2H), 3.76 (s, 3H), 2.06 (s, 3H);
[0456] .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. 168.5, 165.7,
153.4, 139.5, 133.6, 132.1, 130.8, 118.6, 116.8, 116.2, 113.2,
105.2, 94.4, 84.6, 51.5, 24.1.
Methyl
(Z)-3-(chloro(4-nitrophenyl)methylene)-2-oxoindoline-5-carboxylate
(79)
##STR00124##
[0458] To a solution of triphosgene (371 mg, 1.25 mmol) in toluene
(169 mL) were added methyl
4-amino-3-((4-nitrophenyl)ethynyl)benzoate (1.00 g, 3.38 mmol) and
TEA (1.04 mL, 7.43 mmol). After addition, the reaction was stirred
at ambient temperature for 3 h. All the volatile solvent was
removed under reduced pressure, the crude methyl
4-isocyanato-3-((4-nitrophenyl)ethynyl)benzoate was obtained as a
solid (2.14 g), and then directly was used to next step. This solid
compound (2.14 g, 6.85 mmol) was dissolved in DCM (69 mL).
FeCl.sub.3 (1.67 g, 10.27 mmol) was added to the solution. The
reaction mixture was stirred at ambient temperature for overnight.
The reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with ethyl
acetate/ethanol (50/1, v/v) to obtain the major compound 79 as a
yellow solid (624 mg, 52% yield):
[0459] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 11.15 (s, 1H),
8.69 (d, J=1.6 Hz, 1H), 8.30 (d, J=8.9 Hz, 2H), 8.01 (dd, J=8.2,
1.7 Hz, 1H), 7.86 (d, J=8.9 Hz, 2H), 7.00 (d, J=8.2 Hz, 1H), 3.86
(s, 3H).
Methyl
(Z)-3-(chloro(4-(methoxycarbonyl)phenyl)methylene)-2-oxoindoline-5--
carboxylate (80)
##STR00125##
[0461] To a solution of triphosgene (355 mg, 1.20 mmol) in toluene
(162 mL) were added methyl
4-amino-3-((4-(methoxycarbonyl)phenyl)ethynyl)benzoate (1.00 g,
3.23 mmol) and TEA (0.99 mL, 7.11 mmol). After addition, the
reaction was stirred at ambient temperature for 3 h. All the
volatile solvent was removed under reduced pressure, the crude
methyl 4-isocyanato-3-((4-(methoxycarbonyl)phenyl)eth-ynyl)benzoate
was obtained as a solid (2.08 g), and then directly was used to
next step. This solid compound (2.08 g, 6.19 mmol) was dissolved in
DCM (62 mL). FeCl.sub.3 (1.51 g, 9.29 mmol) was added to the
solution. The reaction mixture was stirred at ambient temperature
for overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with ethyl acetate/ethanol (50/1, v/v) to obtain the major compound
80 as a yellow solid (304 mg, 25% yield):
[0462] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.88 (s, 1H), 8.11
(d, J=8.7 Hz, 2H), 8.05 (dd, J=8.2, 1.6 Hz, 2H), 7.61 (d, J=8.8 Hz,
2H), 6.85 (d, J=8.2 Hz, 1H), 3.94 (s, 6H).
Methyl
(Z)-3-(chloro(3-nitrophenyl)methylene)-2-oxoindoline-5-carboxylate
(81)
##STR00126##
[0464] To a solution of triphosgene (371 mg, 1.25 mmol) in toluene
(169 mL) were added methyl
4-amino-3-((3-nitrophenyl)ethynyl)benzoate (1.00 g, 3.36 mmol) and
TEA (1.04 mL, 7.43 mmol). After addition, the reaction was stirred
at ambient temperature for 3 h. All the volatile solvent was
removed under reduced pressure, the crude methyl
4-isocyanato-3-((3-nitrophenyl)ethynyl)benzoate was obtained as a
solid (4.45 g), and then directly was used to next step. This solid
compound (4.45 g, 13.81 mmol) was dissolved in DCM (138 mL).
FeCl.sub.3 (3.36 g, 20.72 mmol) was added to the solution. The
reaction mixture was stirred at ambient temperature for overnight.
The reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with ethyl
acetate/ethanol (50/1, v/v) to obtain the major compound 81 as a
yellow solid (820 mg, 68% yield):
[0465] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.11.27 (s, 1H),
8.50 (d, J=1.5 Hz, 2H), 8.11 (d, J=8.0 Hz, 1H), 7.94 (t, J=8.3 Hz,
1H), 7.81 (dd, J=8.3, 1.7 Hz, 1H), 6.73 (d, J=1.6 Hz, 1H), 3.63 (s,
3H).
Methyl (Z)-3-(chloro(p-tolyl)methylene)-2-oxoindoline-5-carboxylate
(82)
##STR00127##
[0467] To a solution of triphosgene (414 mg, 1.40 mmol) in toluene
(189 mL) were added methyl 4-amino-3-(p-tolylethynyl)benzoate (1.00
g, 3.77 mmol) and TEA (1.16 mL, 8.29 mmol). After addition, the
reaction was stirred at ambient temperature for 3 h. All the
volatile solvent was removed under reduced pressure, the crude
methyl 4-isocyanato-3-(p-tolylethynyl)benzoate was obtained as a
solid (2.70 g), and then directly was used to next step. This solid
compound (2.70 g, 9.27 mmol) was dissolved in DCM (93 mL).
FeCl.sub.3 (2.26 g, 13.91 mmol) was added to the solution. The
reaction mixture was stirred at ambient temperature for overnight.
The reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with ethyl
acetate/ethanol (50/1, v/v) to obtain the major compound 82 as a
yellow solid (1.24 g, quant. yield):
Methyl
(Z)-3-(chloro(4-ethylphenyl)methylene)-2-oxoindoline-5-carboxylate
(83)
##STR00128##
[0469] To a solution of triphosgene (393 mg, 1.32 mmol) in toluene
(180 mL) were added methyl
4-amino-3-((4-ethylphenyl)ethynyl)benzoate (1.00 g, 3.58 mmol) and
TEA (1.10 mL, 7.88 mmol). After addition, the reaction was stirred
at ambient temperature for 3 h. All the volatile solvent was
removed under reduced pressure, the crude methyl
3-((4-ethylphenyl)ethynyl)-4-isocyanatobenzoate was obtained as a
solid (2.60 g), and then directly was used to next step. This solid
compound (2.60 g, 8.51 mmol) was dissolved in DCM (85 mL).
FeCl.sub.3 (2.07 g, 12.76 mmol) was added to the solution. The
reaction mixture was stirred at ambient temperature for overnight.
The reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with ethyl
acetate/ethanol (50/1, v/v) to obtain the major compound 83 as a
yellow solid (1.22 g, quant. yield):
[0470] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.05 (s, 1H),
8.68 (d, J=1.6 Hz, 1H), 7.95 (dd, J=8.2, 1.7 Hz, 1H), 7.50 (d,
J=8.2 Hz, 2H), 7.28 (d, J=8.3 Hz, 2H), 6.95 (d, J=8.2 Hz, 1H), 3.84
(s, 3H), 2.67 (q, J=7.6 Hz, 2H), 1.22 (t, J=7.6 Hz, 3H);
[0471] .sup.13C NMR (125 MHz, DMSO-d.sub.6) .delta. 166.0, 165.3,
146.5, 146.2, 146.1, 134.3, 132.1, 129.5, 127.3, 125.5, 123.7,
122.5, 122.4, 109.6, 52.1, 28.1, 15.3.
Methyl
(Z)-3-(chloro(4-methoxyphenyl)methylene)-2-oxoindoline-5-carboxylat-
e (84)
##STR00129##
[0473] To a solution of triphosgene (390 mg, 1.32 mmol) in toluene
(178 mL) were added methyl
4-amino-3-((4-methoxyphenyl)ethynyl)benzoate (1.00 g, 3.56 mmol)
and TEA (1.09 mL, 7.82 mmol). After addition, the reaction was
stirred at ambient temperature for 3 h. All the volatile solvent
was removed under reduced pressure, the crude methyl
4-isocyanato-3-((4-methoxyphenyl)ethynyl)benzoate was obtained as a
solid (2.30 g), and then directly was used to next step. This solid
compound (2.30 g, 7.48 mmol) was dissolved in DCM (75 mL).
FeCl.sub.3 (1.82 g, 11.23 mmol) was added to the solution. The
reaction mixture was stirred at ambient temperature for overnight.
The reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with ethyl
acetate/ethanol (50/1, v/v) to obtain the major compound 84 as a
yellow solid (932 mg, 76% yield):
[0474] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 11.01 (s, 1H),
8.68 (d, J=1.7 Hz, 1H), 7.93 (dd, J=8.2, 1.7 Hz, 1H), 7.56 (d,
J=8.9 Hz, 2H), 6.97 (d, J=8.9 Hz, 2H), 6.94 (dd, J=8.2, 0.6 Hz,
1H), 3.83 (s, 3H), 3.82 (s, 3H).
Methyl
(Z)-3-(chloro(3-methoxyphenyl)methylene)-2-oxoindoline-5-carboxylat-
e (85)
##STR00130##
[0476] To a solution of triphosgene (390 mg, 1.32 mmol) in toluene
(178 mL) were added methyl
4-amino-3-((4-methoxyphenyl)ethynyl)benzoate (1.00 g, 3.56 mmol)
and TEA (1.09 mL, 7.82 mmol). After addition, the reaction was
stirred at ambient temperature for 3 h. All the volatile solvent
was removed under reduced pressure, the crude methyl
4-isocyanato-3-((4-methoxyphenyl)ethynyl)benzoate was obtained as a
solid (2.27 g), and then directly was used to next step. This solid
compound (2.27 g, 7.38 mmol) was dissolved in DCM (74 mL).
FeCl.sub.3 (1.80 g, 11.08 mmol) was added to the solution. The
reaction mixture was stirred at ambient temperature for overnight.
The reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with ethyl
acetate/ethanol (50/1, v/v) to obtain the major compound 85 as a
yellow solid (887 mg, 73% yield):
Methyl
(Z)-3-(benzo[d][1,3]dioxol-5-ylchloromethylene)-2-oxoindoline-5-car-
boxylate (86)
##STR00131##
[0478] To a solution of triphosgene (372 mg, 1.25 mmol) in toluene
(169 mL) were added methyl
4-amino-3-(benzo[d][1,3]dioxol-5-ylethynyl)benzoate (1.00 g, 3.39
mmol) and TEA (1.04 mL, 7.45 mmol). After addition, the reaction
was stirred at ambient temperature for 3 h. All the volatile
solvent was removed under reduced pressure, the crude methyl
3-(benzo[d][1,3]dioxol-5-ylethynyl)-4-isocyanatobenzoate was
obtained as a solid (2.18 g), and then directly was used to next
step. This solid compound (2.18 g, 6.77 mmol) was dissolved in DCM
(68 mL). FeCl.sub.3 (1.65 g, 10.15 mmol) was added to the solution.
The reaction mixture was stirred at ambient temperature for
overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with ethyl acetate/ethanol (50/1, v/v) to obtain the major compound
86 as a yellow solid (1.21 g, quant. yield):
[0479] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.02 (s, 1H),
8.67 (s, 1H), 7.95 (d, J=8.1 Hz, 1H), 7.18 (s, 1H), 7.13 (d, J=8.2
Hz, 1H), 7.00-6.95 (m, 2H), 6.12 (s, 2H), 3.85 (s, 3H);
[0480] .sup.13C NMR (125 MHz, DMSO-d.sub.6) .delta. 166.0, 165.3,
149.1, 146.7, 146.0, 145.3, 132.0, 130.4, 125.4, 124.5, 123.7,
122.5, 122.4, 109.9, 109.5, 107.9, 101.7, 52.1.
Methyl
(Z)-3-((4-((tert-butyldimethylsilyl)oxy)phenyl)chloromethylene)-2-o-
xoindoline-5-carboxylate (87)
##STR00132##
[0482] To a solution of triphosgene (288 mg, 0.97 mmol) in toluene
(131 mL) were added methyl
4-amino-3-((3-nitrophenyl)ethynyl)benzoate (1.00 g, 2.62 mmol) and
TEA (0.80 mL, 5.77 mmol). After addition, the reaction was stirred
at ambient temperature for 3 h. All the volatile solvent was
removed under reduced pressure, the crude methyl
3-((4-((tert-butyldimethylsilyl)oxy)phenyl)ethynyl)-4-isocyanatobenzoate
was obtained as a solid (2.12 g), and then directly was used to
next step. This solid compound (2.12 g, 5.19 mmol) was dissolved in
DCM (52 mL). FeCl.sub.3 (1.26 g, 7.79 mmol) was added to the
solution. The reaction mixture was stirred at ambient temperature
for overnight. The reaction solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with ethyl acetate/ethanol (50/1, v/v) to obtain the major compound
87 as a yellow solid (218 mg, 19% yield):
[0483] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.08 (s, 1H),
7.86 (d, J=8.2 Hz, 1H), 7.44 (d, J=8.7 Hz, 2H), 7.33 (s, 1H), 6.96
(dd, J=8.5, 2.1 Hz, 3H), 3.74 (s, 3H), 1.03 (s, 9H), 0.29 (s, 6H);
NMR (100 MHz, CDCl.sub.3) .delta. 168.2, 166.7, 158.4, 145.4,
143.2, 131.5, 130.7, 130.5, 124.1, 123.5, 123.2, 122.3, 120.7,
109.9, 52.0, 25.7, 25.7, 18.3, -4.28, -4.30.
Methyl
(Z)-3-(chloro(4-(hydroxymethyl)phenyl)methylene)-2-oxoindoline-5-ca-
rboxylate (88)
##STR00133##
[0485] To a solution of triphosgene (278 mg, 0.94 mmol) in toluene
(126 mL) were added methyl
4-amino-3-((4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)ethynyl)benzoa-
te (1.00 g, 2.53 mmol) and TEA (0.78 mL, 5.56 mmol). After
addition, the reaction was stirred at ambient temperature for 3 h.
All the volatile solvent was removed under reduced pressure, the
crude methyl
3-((4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)ethynyl)-4-isocyanatob-
enzoate was obtained as a solid (2.04 g), and then directly was
used to next step. This solid compound (2.04 g, 4.85 mmol) was
dissolved in DCM (49 mL). FeCl.sub.3 (1.18 g, 7.27 mmol) was added
to the solution. The reaction mixture was stirred at ambient
temperature for overnight. The reaction solvent was evaporated
under reduced pressure, and the residue was purified by column
chromatography with ethyl acetate/ethanol (50/1, v/v) to obtain the
major compound 88 as a yellow solid (869 mg, 34% yield):
Methyl
(Z)-3-((4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)chloromethy-
lene)-2-oxoindoline-5-carboxylate (89)
##STR00134##
[0487] To a solution of triphosgene (268 mg, 0.90 mmol) in toluene
(122 mL) were added methyl
4-amino-3-((4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)ethynyl)benzo-
ate (1.00 g, 2.44 mmol) and TEA (0.75 mL, 5.37 mmol). After
addition, the reaction was stirred at ambient temperature for 3 h.
All the volatile solvent was removed under reduced pressure, the
crude methyl
3-((4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)ethynyl)-4-isocyanato-
benzoate was obtained as a solid (2.00 g), and then directly was
used to next step. This solid compound (2.00 g, 4.60 mmol) was
dissolved in DCM (46 mL). FeCl.sub.3 (1.12 g, 6.90 mmol) was added
to the solution. The reaction mixture was stirred at ambient
temperature for overnight. The reaction solvent was evaporated
under reduced pressure, and the residue was purified by column
chromatography with ethyl acetate/ethanol (50/1, v/v) to obtain the
major compound 89 as a yellow solid (247 mg, 21% yield):
[0488] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 9.10 (s, 1H), 8.28
(s, 1H), 8.22 (dd, J=8.2, 1.7 Hz, 1H), 7.72 (d, J=8.2 Hz, 2H), 7.50
(d, J=8.2 Hz, 2H), 7.47 (s, 1H). 7.02 (d, J=8.2 Hz, 1H), 4.15 (s,
3H), 4.06 (t, J=7.1 Hz, 2H), 3.10 (t, J=7.1 Hz, 2H), 1.10 (s, 9H),
0.23 (s, 6H);
[0489] .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 167.0, 166.2,
149.0, 144.2, 142.4, 135.0, 132.2, 129.4, 129.0, 126.9, 124.3,
123.4, 123.2, 109.2, 64.3, 52.3, 39.7, 26.1, 18.5, 0.2, -5.2.
Methyl
(Z)-3-((4-acetamidophenyl)chloromethylene)-2-oxoindoline-5-carboxyl-
ate (90)
##STR00135##
[0491] To a solution of triphosgene (107 mg, 0.36 mmol) in toluene
(49 mL) were added methyl
3-((4-acetamidophenyl)ethynyl)-4-aminobenzoate (300 mg, 0.97 mmol)
and TEA (0.30 mL, 2.14 mmol). After addition, the reaction was
stirred at ambient temperature for 3 h. All the volatile solvent
was removed under reduced pressure, the crude methyl
3-((4-acetamidophenyl)ethynyl)-4-isocyanatobenzoate was obtained as
a solid (628 mg), and then directly was used to next step. This
solid compound (628 mg, 1.88 mmol) was dissolved in DCM (19 mL).
FeCl.sub.3 (455 mg, 2.82 mmol) was added to the solution. The
reaction mixture was stirred at ambient temperature for overnight.
The reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with ethyl
acetate/ethanol (50/1, v/v) to obtain the major compound 90 as a
yellow solid (186 mg, 52% yield):
[0492] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 12.24 (s, 1H),
8.03 (d, J=2.0 Hz, 1H), 8.00 (s, 1H), 7.78 (dd, J=8.5, 2.1 Hz, 2H),
7.52, (d, J=8.7 Hz, 2H), 7.42 (d, J=8.6 Hz, 2H), 6.67 (d, J=8.6 Hz,
1H), 3.85 (s, 3H), 2.15 (s, 3H), 2.05 (s, 3H).
Methyl
(Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)ami-
no)(4-nitrophenyl)methylene)-2-oxoindoline-5-carboxylate (91)
##STR00136##
[0494] A solution of methyl
(Z)-3-(chloro(4-nitrophenyl)methylene)-2-oxoindoline-5-carboxylate
(188 mg, 0.52 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (158
mg, 0.60 mmol) and TEA (0.15 mL, 1.05 mmol) in EtOH (1.5 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 91 as a yellow solid (267 mg, 87%
yield):
[0495] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.85 (s, 1H),
11.20 (s, 1H), 8.44 (d, J=8.4 Hz, 2H), 7.88 (d, J=8.4 Hz, 2H), 7.59
(dd, J=8.2, 1.7 Hz, 1H), 7.16 (d, J=8.1 Hz, 2H), 6.99 (d, J=8.2 Hz,
2H), 6.96 (d, J=8.2 Hz, 1H), 6.27 (s, 1H), 3.59 (s, 3H), 3.06 (bs,
2H), 2.66 (bs, 2H), 2.14 (bs, 6H), 2.08 (s, 3H);
[0496] .sup.1H NMR (125 MHz, DMSO-d.sub.6) .delta. 170.2, 168.6,
166.2, 154.7, 148.2, 140.7, 140.5, 138.9, 136.9, 130.8, 127.9,
125.8, 124.5, 123.4, 121.3, 119.1, 109.1, 97.6, 59.2, 54.5, 52.4,
51.6, 45.7, 36.7; HRMS (ESI-TOF) m/z calcd for
C.sub.31H.sub.32N.sub.6O.sub.6 [M+Na.sup.+] 607.2276 found
607.2278.
Methyl
(Z)-3-((4-(methoxycarbonyl)phenyl)((4-(N-methyl-2-(4-methylpiperazi-
n-1-yl)acetamido)
phenyl)amino)methylene)-2-oxoindoline-5-carboxylate (92)
##STR00137##
[0498] A solution of methyl
(Z)-3-(chloro(4-(methoxycarbonyl)phenyl)methylene)-2-oxoindoline-5-carbox-
ylate (10 mg, 0.03 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (81
mg, 0.04 mmol) and TEA (0.5 .mu.L, 0.04 mmol) in EtOH (0.3 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 92 as a yellow solid (9.4 mg, 59%
yield):
[0499] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 11.95 (s, 1H),
8.24 (d, J=8.5 Hz, 2H), 7.93 (s, 1H), 7.74 (dd, J=8.2, 1.7 Hz, 1H),
7.57 (d, J=8.5 Hz, 2H), 6.99 (d, J=8.4 Hz, 2H), 6.94 (dd, J=8.2,
0.6 Hz, 1H), 6.79 (d, J=8.6 Hz, 2H), 6.67 (s, 1H), 3.99 (s, 3H),
3.71 (s, 3H), 3.17 (bs, 2H), 2.78 (bs, 2H), 2.38 (bs, 4H), 2.25 (s,
3H), 1.25 (s, 6H).
Methyl
(Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)ami-
no)(3-nitrophenyl)methylene)-2-oxoindoline-5-carboxylate (93)
##STR00138##
[0501] A solution of methyl
(Z)-3-(chloro(3-nitrophenyl)methylene)-2-oxoindoline-5-carboxylate
(58 mg, 0.16 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (48
mg, 0.18 mmol) and TEA (0.05 mL, 0.32 mmol) in EtOH (0.5 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 93 as a yellow solid (84 mg, 90%
yield):
[0502] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.86 (s, 1H),
11.21 (s, 1H), 8.48 (dd, J=7.6, 1.4 Hz, 2H), 8.00 (d, J=7.8 Hz,
1H), 7.87 (t, J=8.1 Hz, 1H), 7.60 (dd, J=8.2, 1.7 Hz, 1H), 7.16 (d,
J=8.2 Hz, 2H), 7.00 (d, J=8.2 Hz, 2H), 6.96 (d, J=8.2 Hz, 1H), 6.42
(s, 1H), 3.62 (s, 3H), 3.05 (bs, 2H), 2.14 (bs, 5H), 2.09 (s, 3H);
HRMS (ESI-TOF) m/z calcd for C.sub.31H.sub.32N.sub.6O.sub.6
[M+H.sup.+] 584.2383 found 585.2459.
Methyl
(Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)ami-
no)(p-tolyl)methylene)-2-oxoindoline-5-carboxylate (94)
##STR00139##
[0504] A solution of methyl
(Z)-3-(chloro(p-tolyl)methylene)-2-oxoindoline-5-carboxylate (100
mg, 0.31 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (92
mg, 0.35 mmol) and TEA (0.09 mL, 0.61 mmol) in EtOH (1.0 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 94 as a yellow solid (169 mg, quant.
yield):
[0505] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.94 (s, 1H),
11.12 (s, 1H), 7.57 (dd, J=8.2, 1.7 Hz, 1H), 7.40-7.36 (m, 4H),
7.14 (d, J=8.1 Hz, 2H), 6.93 (d, J=8.2 Hz, 1H), 6.90 (d, J=8.3 Hz,
2H), 6.50 (s, 1H), 3.64 (s, 3H), 3.05 (bs, 2H), 2.69 (bs, 1H), 2.43
(s, 3H), 2.19 (bs, 6H), 2.10 (s, 3H);
[0506] .sup.13C NMR (125 MHz, DMSO-d.sub.6) .delta. 170.4, 168.6,
166.4, 157.3, 140.4, 140.0, 139.8, 137.5, 130.0, 129.3, 128.4,
127.8, 125.4, 124.1, 123.6, 121.3, 120.0, 108.8, 97.5, 59.2, 54.6,
52.4, 51.6, 45.8, 36.7, 21.1;
[0507] HRMS (ESI-TOF) m/z calcd for C.sub.31H.sub.32N.sub.6O.sub.6
[M+H.sup.+] 553.2689 found 554.2772.
Methyl
(Z)-3-((4-ethylphenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)aceta-
mido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate (95)
##STR00140##
[0509] A solution of methyl
(Z)-3-(chloro(4-ethylphenyl)methylene)-2-oxoindoline-5-carboxylate
(100 mg, 0.29 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (88
mg, 0.34 mmol) and TEA (0.08 mL, 0.59 mmol) in EtOH (1.0 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 95 as a yellow solid (166 mg, quant.
yield):
[0510] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.95 (s, 1H),
11.13 (s, 1H), 7.58 (dd, J=8.2, 1.7 Hz, 1H), 7.42-7.38 (m, 5H),
7.13 (d, J=8.2 Hz, 2H), 6.93 (d, J=8.2 Hz, 1H), 6.89 (d, J=8.2 Hz,
2H), 6.51 (s, 1H), 3.62 (s, 3H), 3.05 (bs, 2H), 2.72 (q, J=7.6 Hz,
2H), 2.68 (bs, 2H), 2.18 (bs, 2H), 2.10 (s, 3H), 1.27 (t, J=7.6 Hz,
3H);
[0511] .sup.13C NMR (125 MHz, DMSO-d.sub.6) .delta. 170.4, 168.6,
166.4, 157.3, 146.4, 140.4, 139.8, 137.5, 129.5, 128.9, 128.5,
127.7, 125.6, 124.0, 123.6, 121.3, 119.5, 108.9, 97.5, 59.2, 54.6,
52.4, 51.5, 45.8, 36.7, 28.3, 15.8; HRMS (ESI-TOF) m/z calcd for
C.sub.31H.sub.32N.sub.6O.sub.6 [M+H.sup.+] 567.2846 found
568.2941.
Methyl
(Z)-3-((4-methoxyphenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)ace-
tamido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate (96)
##STR00141##
[0513] A solution of methyl
(Z)-3-(chloro(4-methoxyphenyl)methylene)-2-oxoindoline-5-carboxylate
(100 mg, 0.29 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (88
mg, 0.34 mmol) and TEA (0.08 mL, 0.59 mmol) in EtOH (1.0 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 96 as a yellow solid (164 mg, 99%
yield):
[0514] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.91 (s, 1H),
11.12 (s, 1H), 7.59 (dd, J=8.2, 1.7 Hz, 1H), 7.43 (d, J=8.6 Hz,
2H), 7.15 (d, J=8.8 Hz, 3H), 6.94 (d, J=8.2 Hz, 1H), 6.90 (d, J=8.4
Hz, 2H), 6.65 (d, J=1.5 Hz, 1H), 3.87 (s, 3H), 3.65 (s, 3H), 3.07
(bs, 3H), 2.87 (bs, 2H), 2.65 (bs. 4H);
[0515] .sup.13C NMR (125 MHz, DMSO-d.sub.6) .delta. 170.4, 166.4,
160.7, 156.8, 140.4, 139.1, 130.2, 127.8, 125.4, 124.15, 124.09,
123.4, 121.2, 119.6, 115.0, 108.8, 97.8, 57.7, 55.5, 52.6, 51.5,
49.1; HRMS (ESI-TOF) m/z calcd for C.sub.32H.sub.35N.sub.5O.sub.5
[M+Na.sup.+] 592.2530, found 592.2531.
Methyl
(Z)-3-((3-methoxyphenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)ace-
tamido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate (97)
##STR00142##
[0517] A solution of methyl
(Z)-3-(chloro(3-methoxyphenyl)methylene)-2-oxoindoline-5-carboxylate
(150 mg, 0.44 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (132
mg, 0.50 mmol) and TEA (0.12 mL, 0.87 mmol) in EtOH (1.2 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 97 as a yellow solid (172 mg, 69%
yield):
[0518] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.98 (s, 1H),
11.12 (s, 1H), 7.59 (d, J=8.3 Hz, 1H), 7.49 (t, J=8.0 Hz, 1H),
7.19-7.14 (m, 3H), 7.10 (s, 1H), 7.05 (d, J=7.6 Hz, 1H), 6.93 (t,
J=8.4 Hz, 3H), 6.65 (s, 1H), 3.74 (s, 3H), 3.66 (s, 3H), 3.06 (bs,
2H), 2.70 (bs, 2H), 2.19 (bs, 2H), 2.10 (s, 3H);
[0519] .sup.13C NMR (125 MHz, DMSO-d.sub.6) .delta. 170.4, 168.6,
166.4, 160.0, 156.7, 140.4, 139.9, 133.3, 130.8, 127.7, 125.5,
123.8, 123.5, 121.3, 120.5, 119.7, 115.9, 114.0, 108.8, 97.3, 59.1,
55.5, 54.6, 52.4, 51.5, 45.8;
[0520] HRMS (ESI-TOF) m/z calcd for C.sub.32H.sub.35N.sub.5O.sub.5
[M+H.sup.+] 570.2711, found 570.2714.
Methyl
(Z)-3-(benzo[d][1,3]dioxol-5-yl-((4-(N-methyl-2-(4-methylpiperazin--
1-yl)acetamido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate
(98)
##STR00143##
[0522] A solution of methyl
(Z)-3-(benzo[d][1,3]dioxol-5-ylchloromethylene)-2-oxoindoline-5-carboxyla-
te (150 mg, 0.42 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (127
mg, 0.48 mmol) and TEA (0.12 mL, 0.84 mmol) in EtOH (1.2 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 98 as a yellow solid (179 mg, 73%
yield):
[0523] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.85 (s, 1H),
11.10 (s, 1H), 7.60 (dd, J=8.2, 1.7 Hz, 1H), 7.18 (d, J=8.3 Hz,
2H), 7.11 (d, J=8.1 Hz, 1H), 7.10 (s, 1H), 6.98 (dd, J=8.0, 1.7 Hz,
1H), 6.95-6.93 (m, 3H), 6.72 (s, 1H), 6.16 (s, 1H), 6.12 (s, 1H),
3.69 (s, 3H), 3.08 (bs, 2H), 2.73 (bs, 2H), 2.21 (bs, 3H), 2.11 (s,
3H);
[0524] .sup.1H NMR (125 MHz, DMSO-d.sub.6) .delta. 170.4, 168.6,
166.4, 156.6, 148.7, 148.1, 140.4, 139.9, 127.8, 125.6, 125.4,
124.0, 123.5, 122.8, 121.3, 119.6, 109.4, 109.0, 108.8, 101.7,
97.6, 59.2, 54.6, 52.3, 51.6, 45.7, 36.7;
[0525] HRMS (ESI-TOF) m/z calcd for C.sub.32H.sub.33N.sub.5O.sub.6
[M+H.sup.+] 584.2504, found 584.2509.
Methyl
(Z)-3-((4-((tert-butyldimethylsilyl)oxy)phenyl)((4-(N-methyl-2-(4-m-
ethylpiperazin-1-yl)
acetamido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate
(99)
##STR00144##
[0527] A solution of methyl
(Z)-3-((4-((tert-butyldimethylsilyl)oxy)phenyl)chloromethylene)-2-oxoindo-
line-5-carboxylate (200 mg, 0.45 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (136
mg, 0.52 mmol) and TEA (0.13 mL, 0.90 mmol) in EtOH (1.3 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 99 (166 mg, 54% yield):
[0528] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 11.93 (s, 1H),
10.08 (s, 1H), 7.74 (dd, J=8.2, 1.6 Hz, 1H), 7.31 (d, J=8.5 Hz,
2H), 7.05 (s, 1H), 7.00 (dd, J=8.3, 1.7 Hz, 3H), 6.97 (d, J=8.4 Hz,
2H), 6.75 (d, J=8.8 Hz, 2H), 3.75 (s, 3H), 3.19 (s, 3H), 2.84 (s,
2H), 2.44 (bs, 6H), 2.27 (s, 3H), 1.04 (s, 9H), 0.91 (s, 2H), 0.29
(s, 6H);
[0529] .sup.1H NMR (125 MHz, CDCl.sub.3) .delta. 171.5, 169.6,
167.5, 157.9, 157.4, 139.9, 139.4, 138.5, 130.4, 127.8, 126.2,
124.9, 124.4, 123.6, 122.7, 121.4, 120.5, 109.1, 98.3, 59.7, 54.9,
53.3, 51.8, 46.1, 37.5, 25.7, 18.4, -3.4, -4.3.
Methyl
(Z)-3-((4-(hydroxymethyl)phenyl)((4-(N-methyl-2-(4-methylpiperazin--
1-yl)acetamido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate
(100)
##STR00145##
[0531] A solution of methyl
(Z)-3-(chloro(4-(hydroxymethyl)phenyl)methylene)-2-oxoindoline-5-carboxyl-
ate (17 mg, 0.05 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (15
mg, 0.06 mmol) and TEA (0.1 .mu.L, 0.10 mmol) in EtOH (0.1 .mu.L)
was stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 100 (15 mg, 52% yield):
[0532] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.11.97 (s, 1H),
11.13 (s, 1H), 7.57 (d, J=8.2 Hz, 1H), 7.51 (d, J=7.8 Hz, 2H), 7.44
(d, J=7.8 Hz, 2H), 7.13 (d, J=8.2 Hz, 2H), 6.93 (d, J=8.3 Hz, 1H),
6.90 (d, J=8.2 Hz, 2H), 6.51 (s, 1H), 5.49 (bs, 1H), 4.64 (s, 2H),
3.63 (s, 3H), 3.05 (bs, 2H), 2.69 (bs, 2H), 2.18 (bs, 6H), 2.10 (s,
3H);
[0533] .sup.1H NMR (125 MHz, DMSO-d.sub.6) .delta. 170.4, 168.6,
166.4, 157.2, 145.0, 140.4, 139.8, 130.3, 128.3, 127.8, 127.7,
127.0, 125.5, 124.0, 123.6, 121.3, 119.6, 108.8, 97.6, 62.4, 59.2,
54.6, 52.5, 52.4, 51.6, 51.5, 45.8, 36.7; HRMS (ESI-TOF) m/z calcd
for C.sub.31H.sub.32N.sub.6O.sub.6 [M+H.sup.+] 569.2638 found
570.2713.
Methyl
(Z)-3-((4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)((4-(N-meth-
yl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)methylene)-2-oxoindoli-
ne-5-carboxylate (101)
##STR00146##
[0535] A solution of methyl
(Z)-3-((4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)
chloromethylene)-2-oxoindoline-5-carboxylate (138 mg, 0.29 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (88
mg, 0.34 mmol) and TEA (0.8 .mu.L, 0.10 mmol) in EtOH (0.8 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 101 (154 mg, 76% yield):
[0536] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 12.03 (s, 1H),
10.04 (s, 1H), 7.73 (dd, J=8.2, 1.6 Hz, 1H), 7.45 (d, J=7.9 Hz,
2H), 7.39 (d, J=8.2 Hz, 2H), 6.99 (d, J=8.2 Hz, 3H), 6.84 (d, J=8.7
Hz, 2H), 6.69 (d, J=1.7 Hz, 1H), 3.95 (t, J=7.0 Hz, 2H), 3.75 (s,
3H), 3.20 (s, 2H), 3.00 (t, J=7.0, 2H), 2.45 (bs, 6H), 2.28 (s,
3H), 0.92 (s, 9H), 0.07 (s, 6H);
[0537] .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 171.4, 169.5,
167.4, 157.3, 142.3, 139.9, 139.3, 138.5, 130.5, 130.1, 128.4,
128.3, 127.9, 126.2, 124.3, 123.2, 122.6, 120.7, 115.7, 109.0,
98.6, 64.3, 59.6, 54.9, 53.3, 51.7.
Methyl
(Z)-3-((4-acetamidophenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)a-
cetamido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate
(102)
##STR00147##
[0539] A solution of methyl
(Z)-3-((4-acetamidophenyl)chloromethylene)-2-oxoindoline-5-carboxylate
(10 mg, 0.03 mmol),
N-(4-aminophenyl)-N-methyl-2-(4-methylpiperazin-1-yl)acetamide (8.1
mg, 0.03 mmol) and TEA (0.01 mL, 0.06 mmol) in EtOH (0.1 mL) was
stirred under refluxed for overnight. The reaction solvent was
evaporated under reduced pressure, and the residue was purified by
column chromatography with dichloromethane/ethanol (50/1, v/v) to
obtain the final compound 102 as a yellow solid (14 mg, 88%
yield):
[0540] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 11.89 (s, 1H),
11.12 (s, 1H), 10.26 (s, 1H), 7.77 (d, J=8.6 Hz, 2H), 7.58 (dd,
J=8.2, 1.7 Hz, 1H), 7.41 (d, J=8.6 Hz, 2H), 7.15 (d, J=8.2 Hz, 2H),
6.93 (d, J=8.2 Hz, 1H), 6.90 (d, J=5.0 Hz, 2H), 6.69 (s, 1H), 3.64
(s, 3H), 3.06 (bs, 2H), 2.67 (bs, 2H), 2.18 (bs, 4H), 2.11 (s, 3H),
2.10 (bs, 2H); HRMS (ESI-TOF) m/z calcd for
C.sub.33H.sub.36N.sub.6O.sub.5 [M+H+] 596.2747 found 597.2818.
Methyl
(Z)-3-((4-aminophenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)aceta-
mido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate (103)
##STR00148##
[0542] To a solution of methyl
(Z)-3-((4-aminophenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)p-
henyl)amino)methylene)-2-oxoindoline-5-carboxylate (70 mg, 0.12
mmol) in EtOH (12 mL) was added SnCl.sub.2 (91 mg, 0.48 mmol). This
reaction mixture was stirred at 70.degree. C. for overnight. The
reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with
dichloromethane/ethanol (50/1, v/v) to obtain the final compound
103 as a yellow solid (66 mg, quant. % yield):
[0543] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.11.79 (s, 1H),
11.03 (s, 1H), 7.58 (dd, J=8.2, 1.8 Hz, 1H), 7.18 (d, J=8.3 Hz,
2H), 7.10 (d, J=8.3 Hz, 3H), 6.92 (d, J=8.2 Hz, 1H), 6.88 (d, J=8.3
Hz, 2H), 6.68 (d, J=8.3 Hz, 2H), 5.76 (bs, 2H), 3.69 (s, 3H), 3.08
(bs, 2H), 2.89 (bs, 2H), 2.71 (bs, 4H), 2.50 (s, 3H);
[0544] .sup.1H NMR (125 MHz, DMSO-d.sub.6) .delta. 170.5, 168.2,
166.6, 158.1, 151.0, 140.2, 138.7, 129.9, 127.8, 125.1, 124.5,
123.1, 121.2, 119.9, 117.9, 113.9, 108.6, 97.3, 57.7, 56.0, 52.6,
51.6, 48.9, 48.6, 36.7; HRMS (ESI-TOF) m/z calcd for
C.sub.31H.sub.34N.sub.604 [M+Na.sup.+] 577.2534, found
577.2537.
Methyl
(Z)-3-((3-aminophenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)aceta-
mido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate (104)
##STR00149##
[0546] To a solution of methyl
(Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(3--
nitrophenyl)methylene)-2-oxoindoline-5-carboxylate (13 mg, 0.12
mmol) in EtOH (2.1 mL) was added SnCl.sub.2 (17 mg, 0.09 mmol).
This reaction mixture was stirred at 70.degree. C. for overnight.
The reaction solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with
dichloromethane/ethanol (50/1, v/v) to obtain the final compound
104 as a yellow solid (9.2 mg, 76% yield):
[0547] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 12.03 (s, 1H),
11.10 (s, 1H), 7.59 (dd, J=8.2, 1.7 Hz, 2H), 7.26 (t, J=7.8 Hz,
2H), 7.18 (d, J=8.2 Hz, 2H), 6.93 (d, J=8.2 Hz, 1H), 6.82-6.78 (m,
2H), 6.63 (d, J=7.5 Hz, 1H), 6.59 (s, 1H), 5.44 (bs, 2H), 3.67 (s,
3H), 3.08 (bs, 2H), 2.91 (bs, 2H), 2.67 (bs, 2H), 2.50 (s, 3H);
HRMS (ESI-TOF) m/z calcd for C.sub.31H.sub.32N.sub.6O.sub.6
[M+H.sup.+] 554.2642 found 555.2706.
Methyl
(Z)-3-((4-hydroxyphenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)ace-
tamido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate
(105)
##STR00150##
[0549] To a solution of methyl
(Z)-3-((4-((tert-butyldimethylsilyl)oxy)phenyl)((4-(N-methyl-2-(4-methylp-
iperazin-1-yl)acetamido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylat-
e (132 mg, 0.20 mmol) in THF (1.0 mL) was added tetrabutylammonium
fluoride solution (0.22 ml, 1M in THF). After addition, the
reaction was stirred at ambient temperature for 2 h. All the
volatile solvent was removed under reduced pressure, and the
residue was purified by silica gel chromatography, by column
chromatography with dichloromethane/ethanol (50/1, v/v) to obtain
the final compound 105 (95 mg, 87% yield):
[0550] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.11.87 (s, 1H),
11.07 (s, 1H), 10.07 (bs, 1H), 7.58 (dd, J=8.2, 1.7 Hz, 1H), 7.27
(d, J=8.6 Hz, 2H), 7.15 (d, J=8.2 Hz, 2H), 6.94-6.90 (m, 3H), 6.86
(d, J=8.4 Hz, 2H), 6.81 (d, J=1.6 Hz, 1H), 3.67 (s, 3H), 3.07 (bs,
2H), 2.72 (bs, 2H), 2.21 (bs, 6H), 2.10 (s, 3H);
[0551] HRMS (ESI-TOF) m/z calcd for C.sub.31H.sub.32N.sub.6O.sub.6
[M+H.sup.+] 555.2482 found 556.2560.
Methyl
(Z)-3-((4-(2-hydroxyethyl)phenyl)((4-(N-methyl-2-(4-methylpiperazin-
-1-yl)acetamido)phenyl)amino)methylene)-2-oxoindoline-5-carboxylate
(106)
##STR00151##
[0553] To a solution of methyl
(Z)-3-((4-(2-((tert-butyldimethylsilyl)oxy)ethyl)phenyl)((4-(N-methyl-2-(-
4-methylpiperazin-1-yl)acetamido)phenyl)amino)methylene)-2-oxoindoline-5-c-
arboxylate (154 mg, 0.22 mmol) in THF (1.0 mL) was added
tetrabutylammonium fluoride solution (0.24 ml, 1M in THF). After
addition, the reaction was stirred at ambient temperature for 2 h.
All the volatile solvent was removed under reduced pressure, and
the residue was purified by silica gel chromatography, by column
chromatography with dichloromethane/ethanol (50/1, v/v) to obtain
the final compound 106 (89 mg, 69% yield):
[0554] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.11.95 (s, 1H),
11.13 (s, 1H), 7.58 (dd, J=8.1, 1.7 Hz, 1H), 7.45-7.36 (m, 5H),
7.13 (d, J=8.2 Hz, 2H), 6.93 (d, J=8.2 Hz, 1H), 6.88 (d, J=8.2 Hz,
2H), 6.51 (s, 1H), 4.79 (t, J=5.2 Hz, 1H), 3.70-3.67 (m, 2H), 3.35
(s, 3H), 3.05 (bs, 2H), 2.85 (t, J=7.3 Hz, 2H), 2.69 (bs, 1H),
2.53-2.47 (m, 5H), 2.19 (bs, 2H), 2.10 (s, 3H);
[0555] .sup.13C NMR (125 MHz, DMSO-d.sub.6) .delta. 170.4, 168.6,
166.4, 157.3, 141.9, 140.4, 139.8, 137.5, 129.9, 129.8, 128.3,
127.7, 125.6, 124.0, 123.6, 121.3, 119.5, 108.9, 97.5, 62.4, 59.2,
54.6, 52.4, 51.6, 45.8, 36.7; HRMS (ESI-TOF) m/z calcd for
C.sub.33H.sub.37N.sub.5O.sub.5 [M+H+] 583.2795 found 584.2872.
(Z)-3-((4-Aminophenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)ph-
enyl)amino)methylene)-2-oxoindoline-5-carboxylic acid (107)
##STR00152##
[0557] To a solution of methyl
(Z)-3-((4-aminophenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)
phenyl)amino)methylene)-2-oxoindoline-5-carboxylate (300 mg, 0.54
mmol) in MeOH/1,4-dioxane (1/1, 9 mL) was added aqueous IN NaOH
(3.0 mL) at 50.degree. C. This reaction mixture was stirred at
80.degree. C. for 6 h. The reaction solvent was evaporated under
reduced pressure, and the residue was filtered by acetonitrile (5.0
mL) and diethyl ether (5.0 mL) to obtain the final compound 107 as
a yellow solid (280 mg, 96% yield):
[0558] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.84 (s, 1H),
10.65 (s, 1H), 7.57 (dd, J=7.9, 1.5 Hz, 1H), 7.35 (s, 1H), 7.10
(bs, 1H), 7.08 (d, J=8.4 Hz, 3H), 6.73 (d, J=8.1 Hz, 3H), 6.60 (d,
J=8.2 Hz, 2H), 5.66 (s, 2H), 3.06 (bs, 2H), 2.73 (bs, 2H), 2.24
(bs, 6H), 2.10 (s, 3H);
[0559] .sup.1H NMR (125 MHz, DMSO-d.sub.6) .delta. 171.3, 170.9,
168.7, 156.3, 150.6, 137.1, 132.5, 130.1, 127.5, 125.4, 123.0,
122.4, 120.5, 118.3, 113.7, 107.3, 101.3, 58.9, 54.6, 52.4, 45.8,
36.8;
[0560] HRMS (ESI-TOF) m/z calcd for C.sub.30H.sub.32N.sub.6O.sub.4
[M+H.sup.+] 541.2558, found 541.2571.
(Z)-3-((3-Aminophenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)ph-
enyl)amino)methylene)-2-oxoindoline-5-carboxylic acid (108)
##STR00153##
[0562] To a solution of methyl
(Z)-3-((3-aminophenyl)((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)p-
henyl)amino)methylene)-2-oxoindoline-5-carboxylate (23.8 mg, 0.14
mmol) in MeOH/1,4-dioxane (1/1, 3 mL) was added aqueous 1N NaOH
(0.8 mL) at 50.degree. C. This reaction mixture was stirred at
80.degree. C. for 6 h. The reaction solvent was evaporated under
reduced pressure, and the residue was filtered by acetonitrile (5
mL) and diethyl ether (5 mL) to obtain the final compound 108 as a
yellow solid (18.6 mg, 80% yield): HRMS (ESI-TOF) m/z calcd for
C.sub.31H.sub.32N.sub.6O.sub.6 [M+H.sup.+] 540.2485 found
541.2549.
[0563] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
[0564] Those skilled in the art will appreciate that numerous
changes and modifications can be made to the preferred embodiments
of the invention and that such changes and modifications can be
made without departing from the spirit of the invention. It is,
therefore, intended that the appended claims cover all such
equivalent variations as fall within the true spirit and scope of
the invention.
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