U.S. patent application number 15/383413 was filed with the patent office on 2018-05-31 for novel ezrin inhibitors and methods of making and using.
The applicant listed for this patent is Georgetown University. Invention is credited to Milton L. Brown, Gullay Bulut, George Kosturko, Mikell Paige, Jeffrey A. Torestsky, Aykut Uren.
Application Number | 20180148437 15/383413 |
Document ID | / |
Family ID | 46051475 |
Filed Date | 2018-05-31 |
United States Patent
Application |
20180148437 |
Kind Code |
A1 |
Brown; Milton L. ; et
al. |
May 31, 2018 |
Novel Ezrin Inhibitors and Methods of Making and Using
Abstract
The invention encompasses compound and pharmaceutical
composition comprising the compound of the following Formula (I):
or pharmaceutically acceptable salts or prodrugs thereof, that are
useful for inhibiting ezrin protein in a cell or for inhibiting the
growh of a cancer cell.
Inventors: |
Brown; Milton L.;
(Brookeville, MD) ; Paige; Mikell; (Fairfax,
VA) ; Torestsky; Jeffrey A.; (Silver Spring, MD)
; Uren; Aykut; (Rockville, MD) ; Kosturko;
George; (Alexandria, VA) ; Bulut; Gullay;
(Washington, DC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Georgetown University |
Washington |
DC |
US |
|
|
Family ID: |
46051475 |
Appl. No.: |
15/383413 |
Filed: |
December 19, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14047362 |
Oct 7, 2013 |
9522908 |
|
|
15383413 |
|
|
|
|
13818223 |
|
|
|
|
PCT/US2011/048635 |
Aug 22, 2011 |
|
|
|
14047362 |
|
|
|
|
61375823 |
Aug 21, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 237/30 20130101;
C07D 471/04 20130101; C07D 413/12 20130101; C07D 215/38 20130101;
C07D 217/24 20130101; C07D 401/12 20130101; C07D 215/24 20130101;
C07D 401/06 20130101; C07D 209/48 20130101 |
International
Class: |
C07D 413/12 20060101
C07D413/12; C07D 401/12 20060101 C07D401/12; C07D 215/24 20060101
C07D215/24; C07D 471/04 20060101 C07D471/04; C07D 209/48 20060101
C07D209/48; C07D 215/38 20060101 C07D215/38; C07D 401/06 20060101
C07D401/06; C07D 237/30 20060101 C07D237/30; C07D 217/24 20060101
C07D217/24 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Part of the work performed during development of this
invention utilized U.S. Government funds under Department of
Defense, Grant No. W81XWH-10-1-0137. The U.S. Government has
certain rights in this invention.
Claims
1. A compound of ##STR00109## or pharmaceutically acceptable salts
or prodrugs thereof, wherein E, F and G are optionally
independently present; ##STR00110## wherein Y.sub.1, Y.sub.2,
Y.sub.3, and Y.sub.4 are each independently N or C--Z; wherein Z is
H or a C.sub.1 to C.sub.6 linear, branched or cyclic alkyl; M and D
are not both hydrogen; D is H, halogen, OH, aryl, aryloxy,
NO.sub.2, CN, carboxy, SH, CF.sub.3, C.sub.1 to C.sub.10 alkoxy,
NH.sub.2, or NR.sub.13R.sub.14; wherein R.sub.13 and R.sub.14 are
each independently H, C.sub.1 to C.sub.15 linear chain or branched
chain alkyl; when none of E, F and G are present, M is H, halogen,
OH, aryl, aryloxy, NO.sub.2, CN, carboxy, SH, CF.sub.3, C.sub.1 to
C.sub.10 alkoxy, NH.sub.2, or NR.sub.13R.sub.14; when at least one
of E, F and G are present, M is --NH--, --O--, or --S--; E is a
bond, linear, branched or cyclic C.sub.1 to C.sub.10 alkylene,
alkene, alkyne, or ether; F is a bond, O, S, ##STR00111## and G is
##STR00112## wherein R.sub.1-R.sub.12 and X.sub.1-X.sub.5 are each
independently selected from H, halogen, OH, aryl, aryloxy,
NO.sub.2, CN, carboxy, SH, CF.sub.3, C.sub.1 to C.sub.5 alkoxy,
C.sub.1 to C.sub.5 alkyl group, NHCONH.sub.2, NH--SO.sub.2CH.sub.3,
NH--NH--NH.sub.2, NH.sub.2, or NR.sub.13R.sub.14; with the proviso
that when R.sub.3 is OH and ##STR00113## R.sub.2 and R.sub.4 are
not both H, OH, or the same halogen.
2.-61. (canceled)
62. A pharmaceutical composition comprising a compound or a
pharmaceutically acceptable salt thereof of claim 1 and a
pharmaceutically acceptable carrier.
63. A method of inhibiting ezrin protein in a cell comprising
administering at least one compound of claim 1.
64. The method of claim 63, wherein the cell is an abnormal cancer
cell.
65. A method of inhibiting the growth of a cancer cell comprising
administering to the cell an amount of at least one compound of
claim 1.
66. The method of claim 65 wherein the cancer cell is selected from
the group consisting of a lung cancer cell, a breast cancer cell, a
colon cancer cell, a malignant melanoma cell, an ovarian carcinoma
cell, a brain tumor cell, a soft tissue sarcoma cell,
rhabdomyosarcoma, prostate cancer and pancreas cancerand an
osteosarcoma cell.
67. The method of claim 66 wherein the cancer cell is an
osteosarcoma cell.
68. The method of claim 67, wherein the osteosarcoma cell is in a
subject.
69. A method of reducing the likelihood of cancer metastasis
comprising administering to a subject in need of a therapeutically
effective amount of at least one compound of claim 1.
70. The method of claim 69 wherein the cancer is selected from the
group consisting of lung cancer, breast cancer, colon cancer,
malignant melanoma, ovarian carcinoma, brain tumors, soft tissue
sarcomas, rhabdomyosarcoma, prostate cancer and pancreas cancer and
osteosarcoma.
71. The method of claim 70 wherein the cancer is osteosarcoma.
Description
[0001] This application claims the benefit of U.S. provisional
patent application No. 61/375,823, filed Aug. 21, 2010, Which is
incorporated herein by reference.
SEQUENCE LISTING INFORMATION:
[0003] A computer readable text file, entitled "036681-5010-02US
SequenceListing.txt," with a file size of about 1 kb contains the
sequence listing for this application and is hereby incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0004] The invention encompasses novel compounds and
pharmaceutically acceptable salts thereof. The invention also
encompasses methods for inhibiting the function of ezrin protein in
a cell and methods for inhibiting the cancer cell or tumor growth,
which methods include administering at least one compound of the
invention or a pharmaceutically acceptable salt thereof.
BACKGROUND OF THE INVENTION
[0005] Ezrin is a multifunctional protein that connects the actin
cytoskeleton to extracellular matrix through transmembrane
proteins. High ezrin expression is associated with lung metastasis
and poor survival rates in cancer.
[0006] Osteosarcoma (OS) is the most common type of primary bone
cancer in children and adolescents. The pathogenesis underlying the
disease has been difficult to establish due to its heterogenous
histology and complex etiology. Treatment of the localized disease
has improved with introduction of neoadjuvant chemotherapy,
increasing the 5-year survival to 60-70%. However, 5-year survival
of patients with metastasis at diagnosis decreases to 30% (Zhang,
P., et at., Clin. Cancer Res., 14, 2962-2969 (2008); Rosen G., et
at., Cancer, 49, 1221-1230 (1982); Ferrari, S. & Palmerini, E.,
Curr. Opin. Oncol., 19, 341-346 (2007); Bacci, G., et al., Cancer,
106, 1154-1161 (2006)). In OS, the predominatnt site of recurrence
and the main cause of death are pulmonary metastases Dunn, D. &
Dehner, L. P., Cancer, 40, 3054-3064 (1977)). Targeting the
underlying molecular events that lead to metastasis could provide
dramatic benefits for the treatment of patients with poor
prognosis.
[0007] Ezrin is a member of the ERM (Ezrin/Radixin/Moesin) family
of proteins and. is conserved through evolution both structurally
and functionally (Fievet, B., et al., Biochim. Biophys. Acta, 1773,
653-660 (2007)). By regulating membrane-cytoskeleton complexes, it
plays key roles in normal cellular processes like maintenance of
membrane dynamics, survival, adhesion, motility, cytokinesis,
phagocytosis and integration of membrane transport with signaling
pathways (Bretscher, A., et al., Nat. Rev. Mol. Cell Biol., 3,
586-599 (2002)). Both in vivo and in vitro studies show ezrin
function is actively regulated by its conformational changes
(Fievet, B., et al., Biochim. Biophys. Acta, 1773, 653-660 (2007)).
Ezrin exists in an inactive conformation, in which the membrane and
actin binding sites are masked by intramolecular interaction of the
N-terminal and the last 100 amino acids of the long Carboxy
terminal domains (Gary, R., & Bretscher, Mol. Biol. Cell, 6,
1061-1075 (1995)). In its active-open confirmation, it functions as
a crosslinker between the plasma membrane and the cortical
cytoskeleton. Two factors are reported to be involved in this
conformational transition, binding of N-terminal domain to the
phosphotidylinositol 4,5 biphosphates (PIP.sub.2) and
phosphorylation of a conserved threonine at residue 567 (T567) in
the F-actin binding site (Fievet, B., et al., Biochim. Biophys.
Acta, 1773, 653-660 (2007)). Several serine/threonine kinases, Rho
kinase (ROCK), protein kinase C-alpha (PKC.alpha.) and MST4, are
reported to be important for T567 phosphorylation (Matsui, T., et
al. J. Cell Biol., 140, 647-657 (1998); Ren, L., et al. Oncogene,
28, 792-802 (2009); Ten Klooster, J. P., et al. Dev. Cell, 16,
551-562 (2009)). In its active form, ezin can interact with
membrane proteins either directly or through adaptor proteins. It
binds to adhesion related proteins with single transmembrane
domains such as CD43, CD44, CD95, ICAM-1, 2, -3 and PA.2.26 antigen
directly through their cytoplasmic tails (Louvet-Vallee, S., Biol.
Cell, 92, 305-316 (2000)), which modulates cell motility and
cellular morphology (Legg, J. W. & Isache, Curr, Biol., 8,
705-708 (1998)). Ezrin binding to adaptor proteins such as
EBP50/NHE-RF and E3KARP regulates the activity of ion transporters,
endocytosis of plasma membrane proteins and interaction of F-actin
to specific plasma membrane domains (Bre scher, A., et al., Nat.
Rev. Mol. Cell Biol., 3, 586-599 (2002)), In addition to plasma
membrane proteins, ezrin associates with cytoplasmic signaling
proteins and is involved in several signaling pathways, such as but
not limited to Rho and PI3K/Akt pathways (Gautreau, A., et al.,
Proc. Natl. Acad. Sci. USA, 96, 7300-7305 (1999); and Hirao, M., et
al. J. Cell Biol., 135, 37-51 (1996)). Ezrin can modulate these
pathways at both upstream and the downstream levels.
[0008] Accumulating evidence from experimental mouse models, as
well as canine and human patients validate that ezrin is a key
factor in metastases. In this study, we identified that the
compounds described herein directly interact with ezrin and inhibit
its biological function in multiple assays.
SUMMARY OF THE INVENTION
[0009] The invention encompasses a compound of Formula (I),
##STR00001##
and salts or prodrugs thereof, wherein: [0010] E, F and G are
optionally independently present;
##STR00002##
[0011] wherein [0012] Y.sub.1, Y.sub.2, Y.sub.3, and Y.sub.4 are
each independently N or C--Z; wherein Z is H or a C.sub.1 to
C.sub.6, linear, branched or cyclic alkyl group; [0013] M and D are
not both hydrogen; [0014] D is H, halogen, OH, aryl, aryloxy,
NO.sub.2, CN, carboxy, SH, CF.sub.3, C.sub.1 to C.sub.10alkoxy,
NH.sub.2, or NR.sub.13R.sub.14; wherein R.sub.13 and R.sub.14 are
each independently H, C.sub.1 to C.sub.15 straight chain or
branched chain alkyl; [0015] wherein when none of E, F and G are
present, M is H, halogen, OH, aryl, aryloxy, NO.sub.2, CN, carboxy,
SH, CF.sub.3, C.sub.1 to C.sub.10 alkoxy, NH.sub.2, or
NR.sub.13R.sub.14; [0016] wherein when at least one of E, F and G
are present, M is --NH--, --O--, or --S--; E is a bond, linear,
branched or cyclic C.sub.1 to C.sub.10 alkylene, alkene, alkyne, or
ether; F is a bond, O, S,
##STR00003##
[0016] and G is
##STR00004##
[0018] wherein
[0019] R.sub.1-R.sub.12 and X.sub.1-X.sub.5 are each independently
selected from H, halogen, OH, aryl, aryloxy, NO.sub.2, CN, carboxy,
SH, CF.sub.3, C.sub.1 to C.sub.5 alkoxy group, C.sub.1 to C.sub.5
alkyl group, NHCONH.sub.2, NH--SO.sub.2CH.sub.3,
NH--NH--NH.sub.2,NH.sub.2, or NR.sub.13R.sub.14;
##STR00005##
with the proviso that when R.sub.3 is OH and R.sub.2 and R.sub.4
are not both H, OH, or the same halogen.
[0020] According to one embodiment,
##STR00006##
and none of E, F and G are present. In a more specific embodiment,
D is hydrogen; and M is NR.sub.13R.sub.14 in the above-described
compound.
[0021] According to another embodiment,
##STR00007##
and none of E, F and G are present;
[0022] wherein at least one of M or D is NH.sub.2 or
NR.sub.13R.sub.14.
[0023] In other embodiments,
##STR00008##
and E, F and G are all present; [0024] wherein M is --NH--. In a
more specific embodiment, D is H, E is a C.sub.1 to C.sub.5 linear
alkylene, F is a bond, and G is
##STR00009##
[0025] In other embodiments,
##STR00010##
and E is --(CH.sub.2).sub.2--, [0026] F is bond, and G is
##STR00011##
[0027] wherein R.sub.3 is hydroxy or methoxy.
[0028] In other embodiments,
##STR00012##
and E, F and G are all present;
[0029] wherein F is a bond; and G is
##STR00013##
[0030] In other embodiments,
##STR00014##
and E, F and G are all present.
[0031] In other embodiments,
##STR00015##
none of E, F, and G are present. In a more specific embodiment, M
is a C.sub.1 to C.sub.10 alkoxy; and D is NH.sub.2 in the
above-described compound.
[0032] In other embodiments,
##STR00016##
none of F, F, G are present, and M is methoxy.
[0033] In one specific embodiment,
##STR00017##
E is a C.sub.1 to C.sub.5 linear, branched or substitute alkylene;
[0034] F is a bond; and [0035] G is
##STR00018##
[0036] In another specific embodiment,
##STR00019##
and [0037] E is --(CH.sub.2).sub.2--; [0038] F is a bond; [0039] G
is
##STR00020##
[0040] wherein X.sub.2 is H, hydroxy, fluorine, methoxy or
methyl.
[0041] In another embodiment,
##STR00021##
E is a C.sub.1 to C.sub.5 linear alkylene; [0042] F is
##STR00022##
[0042] G is
##STR00023##
[0044] wherein R.sub.9 is NH.sub.2, --N(CH.sub.3).sub.2, or
NR.sub.13R.sub.14.
[0045] In specific embodiments,
##STR00024##
E is a C.sub.1 to C.sub.5 linear alkylene; [0046] F is
##STR00025##
[0046] and [0047] G is
##STR00026##
[0048] wherein R.sub.9 is or NH.sub.2or NR.sub.13R.sub.14.
[0049] In other embodiments,
##STR00027##
E is a C.sub.1 to C.sub.5 linear alkylene; [0050] F is
##STR00028##
[0050] and [0051] G is
##STR00029##
[0052] wherein D is hydrogen and R.sub.9 is
--N(CH.sub.3).sub.2.
[0053] In other embodiments,
##STR00030##
E is --(CH.sub.2).sub.2--; [0054] F is
##STR00031##
[0054] and [0055] G is
##STR00032##
[0056] wherein R.sub.9 is NH.sub.2, --N(CH.sub.3).sub.2, or
NR.sub.13R.sub.14.
[0057] In other embodiments,
##STR00033##
E is --(CH.sub.2).sub.2--, or C.sub.1 to C.sub.5 linear alkylene;
[0058] F is a bond; and [0059] G is
##STR00034##
[0060] wherein R.sub.3 is hydroxy or methoxy.
[0061] In other embodiments,
##STR00035##
and none of E, F and G are present.
[0062] In other embodiments,
##STR00036##
E is --(CH.sub.2).sub.2--, or C.sub.1 to C.sub.6 linear, branched,
or cyclic alkylene; [0063] F is a bond; and [0064] G is
##STR00037##
[0065] wherein R.sup.3 is hydroxy or methoxy.
[0066] According to one embodiment,
##STR00038##
E is --(CH.sub.2).sub.2--, or C.sub.1 to C.sub.6 linear, branched,
or cyclic alkylene; [0067] F is a bond; and [0068] G is
##STR00039##
[0069] wherein D is hydrogen; M is --NH--; R.sub.1 and R.sub.5 are
both hydrogen; R.sub.3 is hydroxy or methoxy.
[0070] Specific embodiments of the invention encompass but are not
limited to:
##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044##
##STR00045##
[0071] The present invention includes, but is not limited to
compounds of Formula I and salts thereof.
[0072] In certain illustrative embodiment, the invention
encompasses a pharmaceutical composition comprising a compound of
formula I or a pharmaceutically acceptable salt thereof.
[0073] In one aspect, there is provided a method of inhibiting
ezrin protein function in a cell comprising administering at least
of one compound as described herein. In one embodiment, the cell is
an abnormal cancer cell.
[0074] In another aspect, there is provided a method of inhibiting
the growth of a cancer cell comprising administering to the cell at
least one compound described herein in an amount sufficient to
inhibit the growth of the cancer cell. In one embodiment, the cell
is selected from the group consisting of a lung cancer cell, a
breast cancer cell, a colon cancer cell, a malignant melanoma cell,
an ovarian carcinoma cell, a brain tumor cell, a soft tissue
sarcoma cell, a rhabdomyosarcoma cell, a pancreatic cancer cell, a
prostate cancer cell and an osteosarcoma cell. In one specific
embodiment, the cell is an osteosarcoma cell. In another
embodiment, the cell is in a subject, and the subject is in need of
treatment for cancer.
[0075] In a further aspect of the present invention, there is
provided a method reducing the likelihood of cancer metastasis in a
subject in need of treatment thereof, the method comprising
administering a therapeutically effective amount of at least one
compound described herein. In one embodiment, the cancer is
selected from the group consisting of lung cancer, breast cancer,
colon cancer, malignant melanoma, ovarian carcinoma, brain tumors,
soft tissue sarcomas, rhabdomyosarcoma, pancreatic cancer, prostate
cancer and osteosarcoma. In one specific embodiment, the cancer is
an osteosarcoma.
[0076] The present invention may be understood more fully by
reference to the figures, detailed description, and examples, which
are intended to exemplify non-limiting embodiments of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0077] FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, and FIG. 1E illustrate
that NSC305787 and NSC668394 directly interact with ezrin. (FIG.
1A) WT and T567D mutant forms of recombinant mouse ezrin proteins
were expressed in bacteria. The most prominent band in total
bacterial lysate following IPTG induction appeared just above 75 kD
molecular weight marker on a 10% PAGE gel stained with coomassie
blue (lanes 2 vs 3 and 5 vs 6). The best fractions from column
chromatography purifications for each protein are shown on lanes 4
and 7 (arrow). (FIG. 1B) K12 OS cell lysate was mixed with ezrin
proteins and subjected to immunoprecipitation (IP) with an ezrin
antibody. Cellular actin binding to these two proteins was detected
by immunoblotting (IB) with an actin antibody. Equal loading of
ezrin proteins was determined by blotting with an ezrin antibody.
T567D ezrin showed stronger binding to actin than the WT. (FIG. 1C)
Chemical structures of NSC305787 and NSC668394 (FIG. 1D) Direct
binding of NSC305787 to WT ezrin protein is analyzed by SPR.
Average affinity for NSC305787 binding to ezrin from 5 independent
experiments was calculated to be 5.85 .mu.M (.+-.s.d. 3.85 .mu.M).
A representative set of binding curves are presented with the
steady state affinity curve given in the inset. (FIG. 1E) NSC668394
bound to WT ezrin with an average KD of 12.59 .mu.M (data from 5
independent experiments with .+-.s.d. 6.35 .mu.M). Black lines show
actual data points, red lines show curve fits for 1:1 binding model
in Biacore T-100 evaluation software.
[0078] FIG. 2A, FIG. 2B, and FIG. 2C illustrate NSC305787 and
NSC668394 inhibit ezrin T567 phosphorylation. (FIG. 2A) K7M2
metastatic OS cell line was treated with NSC305787 (10 .mu.M) and
NSC668394 (10 .mu.M) for 6 hours. NSC305787 and NSC668394 inhibited
phosphorylation of endogenous ezrin protein and its interaction
with actin without altering cellular ezrin protein levels (FIG. 2B
and FIG. 2C). The effect of NSC305787 and NSC668394 on recombinant
ezrin phosporylation by recombinant PKC, was tested in an in vitro
kinase assay. Phoshorylation of ezrin was detected by a
phosphospecific antibody following PAGE and immunoblotting.
Experiments were repeated three times and densitometric analysis of
bands was used for calculation of % inhibition (graphs). Error bars
represents s.d. from three independent experiments. Kinase
activities of PKC,, PKC,.alpha. and PKC,.gamma. were also evaluated
with a nonspecific substrate in the presence of NSC305787 and
NSC668394 by a radioactive in vitro kinase assay. Data analysis was
done by using a sigmoidal dose-response equation where log
EC.sub.50 for each curve with the ezrin substrate and the
non-specific substrate were compared to each other (P=0.0118 for
NSC305787 and P=0.0084 for NSC668394).
[0079] FIG. 3A, FIG. 3B, and FIG. 3C illustrate NSC305787 and
NSC668394 inhibit ezrin mediated invasion of OS cells. (FIG. 3A)
Endogenous ezrin protein levels in K12 and K7M2 cells are shown.
Equal loading is determined by blotting with an actin antibody.
Both NSC305787 (FIG. 3B) and NSC668394 (FIG. 3C) at 1 .mu.M and 10
.mu.M concentrations inhibited invasion of K7M2 cells through a
HUVEC monolayer when compared to the control (1% DMSO). NSC305787
did not inhibit invasion of less metastatic K12 cells at both
concentrations. NSC668394 also did not inhibit invasion by these
cells at 1 .mu.M, but a slight inhibition was observed at 10 .mu.M
treatment. Error bars represent standard deviation from duplicate
data points. The experiments were performed in duplicates. Cell
index is a measurement parameter with no units. In this assay a
decrease in cell index represents invasion of HUVEC layer by OS
cells. It is measured according to the following formula: Cell
index=((Rt-R0)/F where, Rt is resistance at time point t, R0 is
background resistance (measured with media alone, no cells), and F
is frequency at which measurement is taken (10 kHz).
[0080] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, and FIG. 4E illustrate
NSC305787 and NSC668394 create reduced cell motility phenotypes in
zebrafish. (FIG. 4A) Beginning in the late blastula period,
blastodisc cells begin to spread over the yolk cell in the process
of epiboly. Reduction of ezrin protein levels by antisense
morpholino oligonucleotides (MO1) results in epiboly defects
characterized by cells not spreading over the yolk cell but instead
piling up at the animal pole. MO1 injected embryos were scored for
epiboly defects at the 90% epiboly stage: WT (wild-type, not
injected) 4/73 (5%); 3 ng MO1, 5/12 (42%); 6 ng MO1 33/48 (69%); 12
ng MO1 20/29 (69%). (FIG. 4B) When embryos were treated with 10
.mu.M NSC305787, 12 of 81 embryos (15%) had epiboly defects that
completely mimicked the MO1 injected embryos. At 20 .mu.M
NSC305787, 71 of 71 embryos (100%) had epiboly defects. (FIG. 4C)
Normal eye development follows lateral movement of progenitor cells
to form two eyes in untreated (WT) embryos. Treatment with 10 .mu.M
NSC668394 inhibited motility of eye progenitor cells resulting in
cycloptic embryos at 28 hours post fertilization (hpf) in 97 of the
99 embryos (98%). (FIG. 4D) NSC668394 treated embryos continued to
grow and formed a single functional eye at 6 days post
fertilization. (FIG. 4E) Percentile of animals with the observed
phenotypes upon MO1, NSC305787 and NSC668394 treatment are
given.
[0081] FIG. 5A and FIG. 5B illustrate NSC305787 and NSC668394
inhibit OS metastatic growth in lung organ culture. GFP expressing
K7M2 OS cells (2.times.10.sup.5) were injected to tail vein of
female BALB/c mice. Within 15 min of tumor cells injection, mice
were euthanized and lungs were injected with a mixed agarose/medium
solution and then were removed. Complete transverse sections (1-2
mm in thickness) were made from each lobe and 4-5 lung sections
were placed on a single 1.5.times.0.7 cm sterile Gelfoam section.
Lung sections were incubated at 37.degree. C. in humidified
conditions of 5% CO.sub.2. Fresh medium or small molecules (10
.mu.M) were replaced every other day. (FIG. 5A) Quantitation of the
fluorescence signal from NSC305787 (10 .mu.M) and NSC668394 (10
.mu.M) treated organ cultures over time. Metastatic burden was
quantified by measuring the fluorescent area of metastatic cells in
each lung section at each time point and was expressed as mean
fluorescent area (mean fluorescent area of each lung section over 4
lung sections). Mean fluorescent area was normalized to 100 pixels
for day 0 to allow quantitative evaluation of metastatic
progression over time. Data are represented as mean .s.d. from
three independent experiments. (FIG. 5B) Fluorescence pictures of a
representative culture treated with 10 .mu.M NSC305787 and 10 .mu.M
NSC668394 are given on the right panel.
[0082] FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D illustrate NSC305787
and NSC668394 inhibit in vivo OS metastatic growth in lungs. (FIG.
6A) K7M2 metastatic OS tumor cells)(1.times.10.sup.6) were injected
via tail vein. Vehicle (1% DMSO). NSC305787 (240 .mu.g/kg) and
NSC668394 (226 .mu.g/kg) were injected 5 days a week
intraperitoneally. Kaplan-Meier survival curves show percent
survival for NSC305787 and NSC668394 treatment over time. Median
survival of vehicle, NSC305787 and NSC668394 treated K7M2 cells
were 28.5, 50, and 49 days after tumor cell injection. Overall
survival of NSC305787 treated mice is significantly different than
the vehicle treated group (P value of NSC305787* is 0.0337 and
NSC668394** is 0.0524). (FIG. 6B) Fluorescence pictures of whole
lungs are given. There is a significant decrease in the number of
the GFP expressing metastatic foci in the lung tissues of the
NSC305787 and NSC668394 treated groups. (FIG. 6C) GFP expressing
ezrin independent MNNG, human OS cells were injected to mice and
treated with vehicle (1% DMSO), NSC305787 (240 .mu.g/kg) and
NSC668394 (226 .mu.g/kg). Survival of each group is not different
probably because MNNG is an ezrin independent cell line. Median
survival of vehicle, NSC305787 and NSC668394 treated MNNG cells
were 50.5, 49, and 48.5 days after tumor cell injection. (FIG. 6D)
Fluorescent images of the lung tissues of this group are given.
There is not a significant difference in the number of the GFP
expressing metastatic foci in the lung tissues of the NSC305787 and
NSC668394 treated groups.
[0083] FIG. 7 provides results illustrating the functional activity
of select compounds of the present invention on ezrin protein. The
chemical structure of the embodied compounds is detailed and the
corresponding binding and viability values are listed. The binding
potential of select compounds to ezrin was analyzed by using SPR.
Recombinant WT ezrin was immobilized onto a Biacore CMS sensorchip
and compounds were injected one-at-a-time at 6 different
concentrations. SPR sensogranis and K.sub.D values were determined
using Biacore software. An average affinity for compounds binding
to ezrin from 3 independent experiments was calculated. The
embodied compounds have comparable affinity to WT ezrin as NSC
668394. The viability and growth inhibition activity of select
compounds were determined by treatment of high ezrin expressed K7M2
and low ezrin expressed K12 osteosarcoma cells with a dose range of
the select compound for 24 hours. IC.sub.50 values, which
represents the concentration at which growth is inhibited for 50%
of total cell number, was measured by WST-1 (ROCHE). The embodied
compounds show parallel growth inhibition activity to that of NSC
668394.
[0084] FIG. 8 illustrates that select compounds inhibit ezrin
mediated migration. Anti-migration potential of the embodied
compounds were validated using an electric cell migration system.
High ezrin expressed K7M2 cells and low ezrin expressed K12 cells
were treated with non-toxic concentrations of a compound and their
corresponding anti-migration activity was measured. Data is
represented as the ratio of compound K7M2 anti-migration activity
divided by the K12 anti-migration activity, The X-axis details the
non-toxic concentration of the compound used in the study. Embodied
compounds GK2-057 has greater anti-migratory activity on the high
ezrin expressed, metastatic osteosarcoma cells compared to NSC
668394, while benign to the K12 cell line.
[0085] FIG. 9 and FIG. 10 provide data showing compounds reduced
T567 phosphorylation of ezrin and functional activity. K7M2 cells
that were treated with embodied compounds and resolved protein
lysates were immunoblotted for co-precipitated phosphorylated ezrin
(TOP), or actin (BOTTOM). 4.0.times.10.sup.6 K7M2 cells were plated
in 15 cm dishes. After 24 h, the plates are at least 70% confluent.
Media was removed and 10 .mu.M of compound in SF DMEM was added.
After the ezrin protein was incubated for 5 h with the indicated
compound, the plates were lysed with PLB containing Calyculin A. 2
.mu.L of Ezrin Ab and 10 .mu.M of the compound (prepared in PLB)
were added to the lysate and allowed to incubate overnight (14 h),
followed by tumbling with Agarose IgG beads. The mixture was run on
10% acrylamide gel and transferred overnight.
[0086] FIG. 10 provides data showing compounds reduced T567
phosphorylation of ezrin and functional activity. K7M2 cells that
were treated with embodied compounds and resolved protein lysates
were immunoblotted for co-precipitated phospholated ezrin (TOP), or
actin (BOTTOM). 4.0'10.sup.6 K7M2 cells were plated in 15 cm
dishes. After 24 h, the plates are at least 70% confluent. Media
was removed and either 7 .mu.M or 10 .mu.M of compound in SF DMEM
was added. After the ezrin protein was incubated for 5 h with the
indicated compound, the plates were lysed with PLB containing
Calyculin A. 2 .mu.L of Ezrin Ab and 10 .mu.M of the compound
(prepared in PLB) were added to the lysate and allowed to incubate
overnight (14 h), followed by tumbling with Agarose IgG beads. The
mixture was run on 10% acrylamide gel and transferred
overnight.
[0087] FIG. 11A, FIG. 11B, and FIG. 11C show the binding of ezrin
and poly-A-binding protein 1 (PABP1) by immunoprecipitation and
ELISA. (FIG. 11A) Total cell lysates from 7M2. osteosarcoma cells
were immunoprecipitated by using an anti-ezrin antibody. As a
control, non-specific total IgG was used. Immunoprecipitates were
run on SDS-PAGE and Western Blot was performed using anti-ezrin
(lower panel) and anti-PABP1 (upper panel) antibodies. PABP1
protein was detected in immunoprecipitates of ezrin, suggetsing
that these two proteins interact with each other in a protein
complex. (FIG. 11B) Ezrin-PABP1 interaction was also evaluated in
an ELISA experiment where recombinant ezrin protein was
immmobilized on surface and total cell lysates from osteosarcoma
cells were applied on top. Amount of PABP1 binding to ezrin on the
surface was detected by a anti-PABP1 primary antibody followed by
enzyme linked secondary antibody. (FIG. 11C) Immunoprecipitation
and western experiment was prepared as described in panel A. Cells
were treated with NSC305787 (compound 8) and NSC668394 (compound
16) for 60 min prior to immunoprecipitation. Both NSC305787 and
NSC668394 inhibited interaction of PABP1 with ezrin
DETAILED DESCRIPTION OF THE INVENTION
[0088] As used herein and unless otherwise indicated, the term
"alkyl" means a substituted or unsubstituted, saturated, monovalent
linear or branched hydrocarbon chain. Examples of alkyl groups
include, but are not limited to, C.sub.1 to C.sub.15 linear,
branched or cyclic alkyl, such as methyl, ethyl, propyl, isopropyl,
cyclopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl,
3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl,
2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl,
2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,
2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl,
isobutyl, sec-butyl, t-butyl, cyclobutyl, pentyl, isopentyl,
neopentyl, hexyl, and cyclohexyl and longer alkyl groups, such as
heptyl, octyl, nonyl and decyl. An alkyl can be unsubstituted or
substituted with one or two suitable substituents."
[0089] As used herein and unless otherwise indicated, the terms
"alkoxy" or "alkyloxy" means an --O-alkyl, wherein alkyl is as
defined herein. An alkoxy can be unsubstituted or substituted with
one or two suitable substituents. Preferably, the alkyl chain of an
alkyloxy is from 1 to 5 carbon atoms in length, referred to herein,
for example, as "(C.sub.1-C.sub.5)alkoxy." Preferably, the alkyl
chain of an alkyloxy is from 1 to 10 carbon atoms in length,
referred to herein, for example, as "(C.sub.1-C.sub.10)alkoxy."
[0090] As used herein and unless otherwise indicated, the term
"alkylene" means a linear or branched or cyclic saturated divalent
hydrocarbon radical. An alkylene can be unsubstituted or
substituted with one or two suitable substituents. Examples of
alkylene groups include, but are not limited to, C.sub.1 to
C.sub.10 linear chain or branched chain alkylene, such as,
methylene, ethylene, propylene, isopropylene, cyclopropylene,
2-methylpropylene, 2-methylpropylene, 2-methylbutylene,
3-methylbutylene, 2-methylbutylene, 2,2-dimethylpropylene,
2-methylpentylene, 3-methylpentylene, 4-methylpentylene,
2-methylpentylene, 3-methylpentylene, 4-methylpentylene,
2,2-dimethylbutylene, 3,3-dimethylbutylene, 2-ethylbutylene,
butylene, isobutylene, t-butylene, cyclobutylene, pentylene,
isopentylene, neopentylene, cyclopentylene, hexylene,
2-methylpentylene, 3-methylpentylene, and cyclohexylene.
[0091] As used herein and unless otherwise indicated, the terms
"alkene" or "alkenyl group" means a monovalent linear, branched or
cyclic hydrocarbon chain having one or more double bonds therein.
The double bond of an alkene can be unconjugated or conjugated to
another unsaturated group. An alkene can be unsubstituted or
substituted with one or two suitable substituents. Suitable alkenes
include, but are not limited to (C.sub.2-C.sub.8)alkenyl groups,
such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl,
pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl,
4-(2-methyl-3-butene)-pentenyl, An alkene can be unsubstituted or
substituted with one or two suitable substituents.
[0092] As used herein and unless otherwise indicated, the terms
"alkyne" or "alkynyl group" means monovalent linear, branched or
cyclic hydrocarbon chain having one or more triple bonds therein.
The triple bond of an alkynyl group can be unconjugated or
conjugated to another unsaturated group. An alkyne can be
unsubstituted or substituted with one or two suitable substituents.
Suitable alkynes include, but are not limited to,
(C.sub.2-C.sub.8)alkynyl groups, such as ethynyl, propynyl,
butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl,
4-propyl-2-pentynyl, and 4-butyl-2-hexynyl. An alkynyl group can be
unsubstituted or substituted with one or two suitable
substituents.
[0093] As used herein and unless otherwise indicated, the term
"aryl" means a monocyclic or polycyclic aromatic ring comprising
carbon and hydrogen atoms. Examples of suitable aryl groups
include, but are not limited to, phenyl, tolyl, anthacenyl,
fluorenyl, indenyl, azulenyl, and naphthyl. An aryl group can be
unsubstituted or substituted with one or two suitable substituents.
Preferably, the aryl group is a monocyclic ring, wherein the ring
comprises 6 carbon atoms, referred to herein as
"(C.sub.6)aryl."
[0094] As used herein and unless otherwise indicated, the term
"aryloxy" means an --O-aryl group, wherein aryl is as defined
herein. An aryloxy group can be unsubstituted or substituted with
one or two suitable substituents. Preferably, the aryl ring of an
aryloxy group is a monocyclic ring, wherein the ring comprises 6
carbon atoms, referred to herein as "(C.sub.6)aryloxy."
[0095] As used herein and unless otherwise indicated, the term
"ether" means a group of formula alkyl-O-alkyl, alkyl-O-alkynyl,
alkyl-O-aryl, alkenyl-O-alkenyl, alkenyl-O-alkynyl, alkenyl-O-aryl,
alkynyl-O-alkynyl, alkynyl-O-aryl, aryl-O-aryl, wherein "alkyl",
"alkenyl", "alkynyl" and "aryl" are defined herein.
[0096] As used herein and unless otherwise indicated, the term
"carboxy" means a radical of the formula: --COOH.
[0097] As used herein and unless otherwise indicated, the term
"halogen" means fluorine, chlorine, bromine, or iodine.
Correspondingly, the meaning of the terms "halo" and "Hal"
encompass fluoro, chloro, bromo, and iodo.
[0098] As used herein and unless otherwise indicated, the terms
"substituted" and "a suitable substituent" means a group that does
not nullify the synthetic or pharmaceutical utility of the
compounds of the invention or the intermediates useful for
preparing them. Examples of substituted groups or suitable
substituents include, but are not limited to:
(C.sub.1-C.sub.10)alkyl; (C.sub.1-C.sub.10)alkenyl;
(C.sub.1-C.sub.10)alkynyl; (C.sub.6)aryl;
(C.sub.3-C.sub.5)heteroaryl; (C.sub.3-C.sub.7)cycloalkyl;
(C.sub.1-C.sub.10)alkoxy; (C6)aryloxy; --CN; --OH; SH, oxo; halo,
--NO.sub.2, 1'CO.sub.2H; --NH.sub.2; --NHOH,
--NH((C.sub.1-C.sub.10)alkyl); --NH((C.sub.1-C.sub.10)alkyl).sub.2;
--NH((C.sub.6)aryl); --NHO((C.sub.1-C.sub.10)alkyl);
--N(O(C.sub.1-C.sub.10)alkyl).sub.2; --NH(O(C.sub.6)aryl);
--S((C.sub.1-C.sub.10)alkyl); --S((C.sub.1-C.sub.10)alkyl).sub.2;
--S((C.sub.6)aryl); (.dbd.O); C(S), --N((C.sub.6)aryl).sub.2;
--CHO; --C(O)((C.sub.1-C.sub.10)alkyl); --C(O)((C.sub.6)aryl);
--CO.sub.2((C.sub.1-C.sub.10)alkyl); and --CO.sub.2((C.sub.6)aryl),
--C(S)((C.sub.1-C.sub.10)alkyl); --C(S)((C.sub.6)aryl);
--SO.sub.2((C.sub.1-C.sub.10)alkyl); --SO.sub.2((C.sub.6)aryl), and
--SO.sub.3H, --C(S)O((C.sub.1-C.sub.10)alkyl);
--C(S)(O)((C.sub.6)aryl). In certain illustrative embodiments, the
substituents can be one or more than one suitable groups, such as,
but not limited to, --F, --Cl, --Br, --OH, azido, --SH, alkyl,
aryl, heteroalky, alkyoxyl, alkylthiol, amino, hydroxylamino,
N-alkylamino, --N,N-dialkylamino, --N,N-dimethylamino, acyl,
alkyloxycarbonyl, sulfonyl, urea, --NO.sub.2, triazolyl. One of
skill in art can readily choose a suitable substituent based on the
stability and pharmacological and synthetic activity of the
compound of the invention.
[0099] As used herein and unless otherwise indicated, the term
"compounds of the invention" means, collectively, the compounds of
formula I and pharmaceutically acceptable salts thereof as well as
compounds depicted herein. The compounds of the invention are
identified herein by their chemical structure and/or chemical name.
Where a compound is referred to by both a chemical structure and a
chemical name, and that chemical structure and chemical name
conflict, the chemical structure is determinative of the compound's
identity. The compounds of the invention may contain one or more
chiral centers and/or double bonds and, therefore, exist as
stereoisomers, such as double-bond isomers (i.e., geometric
isomers), enantiomers, or diastereomers. According to the
invention, the chemical structures depicted herein, and therefore
the compounds of the invention, encompass all of the corresponding
compound's enantiomers and stereoisomers, that is, both the
stereomerically pure form (e.g., geometrically pure,
enantiomerically pure, or diastereomerically pure) and enantiomeric
and stereoisomeric mixtures. Enantiomeric and stereoisomeric
mixtures can be resolved into their component enantiomers or
stereoisomers by well known methods, such as chiral-phase gas
chromatography, chiral-phase high performance liquid
chromatography, crystallizing the compound as a chiral salt
complex, or crystallizing the compound in a chiral solvent.
Enantiomers and stereoisomers can also be obtained from
stereomerically- or enantiomerically-pure intermediates, reagents,
and catalysts by well known asymmetric synthetic methods.
[0100] The phrase "pharmaceutically acceptable salt(s)," as used
herein includes but is not limited to salts of acidic or basic
groups that may be present in compounds used in the present
compositions. Compounds included in the present compositions that
are basic in nature are capable of forming a wide variety of salts
with various inorganic and organic acids. The acids that may be
used to prepare pharmaceutically acceptable acid salts of such
basic compounds are those that form non-toxic acid salts, i.e.,
salts containing pharmacologically acceptable anions including, but
not limited to, sulfuric, citric, maleic, acetic, oxalic,
hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate,
bisulfate, phosphate, acid phosphate, isonicotinate, acetate,
lactate. salicylate, citrate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,
benzoate, glutamate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds
included in the present compositions that include an amino moiety
may form pharmaceutically acceptable salts with various amino
acids, in addition to the acids mentioned above. Compounds included
in the present compositions that are acidic in nature are capable
of forming base salts with various pharmacologically acceptable
cations. Examples of such salts include alkali metal or alkaline
earth metal salts and, particularly, calcium, magnesium, sodium
lithium, zinc, potassium, and iron salts.
[0101] As used herein and unless otherwise indicated, the term
"prodrug" means a derivative of a compound that can hydrolyze,
oxidize, or otherwise react under biological conditions (in vitro
or in vivo) to provide the compound. Examples of prodrugs include,
but are not limited to, compounds that comprise biohydrolyzable
moieties such as biohydrolyzable amides, biohydrolyzable esters,
biohydrolyzable carbamates, biohydrolyzable carbonates,
biohydrolyzable ureides, and biohydrolyzable phosphate analogues.
Other examples of prodrugs include compounds that comprise
ofigonudeotides, peptides, lipids, aliphatic and aromatic groups,
or NO, NO.sub.2, ONO, and ONO.sub.2 moieties. Prodrugs can
typically be prepared using well known methods, such as those
described in Burger's Medicinal Chemistry and Drug Discovery, pp.
172, 178, 949, 982 (Manfred E. Wolff ed., 5th ed. 1995), and Design
of Prodrugs (H. Bundgaard ed., Elselvier, N.Y. 1985).
[0102] The term "ezrin" as used herein is used as it is in the art.
It is understood that ezrin is a protein known to be involved in
connections of cytoskeletal structures within the cell to the
plasma cell membrane. Ezrin is known to have domains in common with
talin protein. Ezrin exists in an inactive conformation, in which
the membrane and actin binding sites are masked by intramolecular
interaction of the N-terminal and the last 100 amino acids of the
long carboxy terminal domains. In its active-open confirmation,
ezrin functions as a crosslinker between the plasma membrane and
the cortical cytoskeleton. Two factors are reported to be involved
in this conformational transition, binding of N-terminal domain to
the phosphotidylinositol 4,5 biphosphates (PIP.sub.2) and
phosphorylation of a conserved threonine at residue 567 (T567) in
the F-actin binding site. Several serine/threonine kinases, Rho
kinase (ROCK), protein kinase C-alpha (PKC.alpha.) and MST4, are
also important for T567 phosphorylation. In its active form, ezin
can interact with membrane proteins either directly or through
adaptor proteins. Ezrin binds to adhesion related proteins with
single transmembrane domains such as CD43, CD44, CD95, ICAM-1, -2,
-3 and PA.2.26 antigen directly through their cytoplasmic tails,
which modulates cell motility and cellular morphology. Ezrin
binding to adaptor proteins such as EBP50/NHE-RF and E3KARP
regulates the activity of ion transporters, endocytosis of plasma
membrane proteins and interaction of F-actin to specific plasma
membrane domains. In addition to plasma membrane proteins, ezrin
associates with cytoplasmic signaling proteins and is involved in
several signaling pathways, such as but not limited to Rho and
PI3K/Akt pathways. In addition, ezrin may also be involved in the
modulation of nuclear processes such as transcription and RNA
processing. For example, ezrin may bind to one or more types of
Forkhead Box (Fox) proteins, which would suggest that ezrin may
also play a role in transcription. Ezrin can also hind to
poly-A-binding protein 1 (PABP1), see FIG. 11, which suggests that
ezrin may play a role in translation and/or possibly mRNA transport
from the nucleus to the cytoplasm, which would also affect
translation. Ezrin can modulate these pathways at both upstream and
the downstream levels.
[0103] As used herein, "inhibiting the function of ezrin" can be
related to any of these currently known functions of ezrin.
Moreover, the term "inhibiting," when used in connection with ezrin
function, means a reduction in any downstream effect in which ezrin
has a role. The inhibition could be due to any aspect of normal
ezrin function, such as but not limited to the reduced binding of
ezrin to any of its natural binding partners, the inhibition or
reduction of the phosphorylation of ezrin, a loss of association of
ezrin with any of its adaptor proteins and the like.
[0104] As used herein and unless otherwise indicated, the term
"subject" means mammals and non-mammals. Mammals means any member
of the mammalia class including, but not limited to, humans,
non-human primates such as chimpanzees and other ape and monkey
species; farm animals such as cattle, horses, sheep, goats, and
swine; domestic animals such as rabbits, dogs, and cats; laboratory
animals including rodents, such as rats, mice, and guinea pigs; and
the like. Examples of non-mammals include, but are not limited to,
birds, and the like. The term "subject" does not denote a
particular age or sex, and is used herein interchangeably with
"patient."
[0105] The terms "treating" or "inhibiting" when used in connection
with an abnormal condition, e.g., cancer, are intended to include
preventing, eradicating, reducing the likelihood or preventing the
resulting increase of undesired physiological activity associated
with a disorder, for example, in the context of the therapeutic or
prophylactic methods of the invention. In another embodiment, the
term treating or inhibiting includes antagonistic effects, e.g.,
diminishment of the activity or production of mediators of a
disorder, reducing the rate of proliferation of abnormal cells,
etc.
[0106] As used herein and unless otherwise indicated, the terms
"cancer" or "cancer cell" refer to abnormal cell growth or
proliferation that may or may not include spontaneous or induced
phenotypic changes. As used herein, "cancer" includes but is not
limited to such abnormal conditions as hypertrophy, neoplasia,
hyperplasia, benign and malignant cancer. As used herein, the term
"tumor" is a general term that includes hypertrophies, neoplasias,
hyperplasias, benign cancers and malignant cancers. Accordingly,
certain embodiments of the present invention include but are not
limited to treating a hypertrophy, a neoplasia, a hyperplasia, a
benign or a malignant cancer in a subject. In additional
embodiments, the present invention is directed to preventing or
reducing the likelihood of metastasis and/or recurrence of a
hypertrophy, a neoplasia, a hyperplasia, a benign or a malignant
cancer within a subject comprising administering at least one
compound of the present invention to the subject. For example, at
least one compound of the present invention may be administered
after tumor resection/removal/ablation, etc. to reduce the
likelihood of recurrence of the tumor in the subject. In another
example, at least one compound of the present invention may be
administered to reduce the likelihood of metastasis of the tumor in
the subject.
[0107] As used herein and unless otherwise indicated, the phrase
"therapeutically effective amount" of a composition of the
invention is measured by the therapeutic effectiveness of a
compound of the invention, wherein at least one adverse effect of a
disorder is ameliorated or alleviated.
[0108] When administered to a. cell, the compounds of the invention
can be optionally administered in isolated form. As used herein,
"isolated" means that the compounds of the invention are separated
from other components of either (a) a natural source, such as a
plant or cell, preferably bacterial culture, or (b) a synthetic
organic chemical reaction mixture. In one embodiment, the compounds
of the invention are purified. As used herein, "purified" means
that when isolated, the isolate contains at least about 80% of a
compound of the invention by weight of the isolate. In one
embodiment, the isolates contain at least about 90%, at least about
95% or at least about 99% of the compound of the invention by
weight.
[0109] The invention encompasses compound and pharmaceutical
composition comprising the compound of the following Formula
(1):
##STR00046##
or pharmaceutically acceptable salts or prodrugs thereof.
[0110] According to one aspect the present invention provides a
compound and pharmaceutical compositions comprising the compound of
the following Formula (I): or pharmaceutically acceptable salts and
prodrugs thereof,
wherein: [0111] E, F and G are optionally independently
present;
##STR00047##
[0112] wherein [0113] Y.sub.1, Y.sub.2, Y.sub.3, and Y.sub.4 are
each independently N or C--Z; wherein Z is H or a C.sub.1 to
C.sub.6 linear, branched or cyclic alkyl group; [0114] M and D are
not both hydrogen; [0115] D is H, halogen, OH, aryl, aryloxy,
NO.sub.2, CN, carboxy, SH, CF.sub.3, C.sub.1 to C.sub.10 alkoxy,
NH.sub.2, or NR.sub.13R.sub.14; wherein R.sub.13 and R.sub.14 are
each independently H, C.sub.1 to C.sub.15 straight chain or
branched chain alkyl; [0116] wherein when none of E, F and G are
present, M is H, halogen, OH, aryl, aryloxy, NO.sub.2, CN, carboxy,
SH, CF.sub.3, C.sub.1 to C.sub.10 alkoxy, NH.sub.2, or
NR.sub.13R.sub.14; [0117] wherein when at least one of E, F and G
are present. M is --NH--, --O--, or --S--; [0118] E is a bond,
linear, branched or cyclic C.sub.1 to C.sub.10 alkylene, alkene,
alkyne, or ether; [0119] F is a bond, O, S.
##STR00048##
[0119] and [0120] G is
##STR00049##
[0121] wherein [0122] R.sub.1-R.sub.12 and X.sub.1-X.sub.5 are each
independently selected from H, halogen, OH, aryl, aryloxy,
NO.sub.2, CN, carboxy, SH, CF.sub.3, C.sub.1 to C.sub.5 alkoxy
group, C.sub.1 to C.sub.5 alkyl group, NHCONH.sub.2,
NH--SO.sub.2CH.sub.3, NH--NH--NH.sub.2, NH.sub.2, or
NR.sub.13R.sub.14; [0123] with the proviso that when R.sub.3 is OH
and
[0123] ##STR00050## [0124] R.sub.2 and R.sub.4 are not both H, OH,
or the same halogen.
[0125] According to one embodiment,
##STR00051##
and none of E, F and G are present. In a more specific embodiment,
D is hydrogen; and M is NR.sub.13R.sub.14 in the above-described
compound.
[0126] According to another embodiment,
##STR00052##
and none of E, F and G are present;
[0127] wherein at least one of M or D is NH.sub.2 or
NR.sub.13R.sub.14.
[0128] In other embodiments,
##STR00053##
and E, F and G are all present;
[0129] wherein M is --NH--. In a more specific embodiment, D is H,
E is a C.sub.1 to C.sub.5 linear alkylene, F is a bond, and G
is
##STR00054##
[0130] In other embodiments,
##STR00055##
and E is --(CH.sub.2).sub.2--, [0131] F is bond, and [0132] G
is
##STR00056##
[0133] wherein R.sub.3 is hydroxy or methoxy.
[0134] In other embodiments,
##STR00057##
and E, F and G are all present;
[0135] wherein F is a bond; and G is
##STR00058##
[0136] In other embodiments,
##STR00059##
and E, F and G are all present.
[0137] In other embodiments,
##STR00060##
none of E, F, and G are present. In a more specific embodiment. M
is a C.sub.1 to C.sub.10 alkoxy; and D is NH.sub.2in the
above-described compound.
[0138] In other embodiments,
##STR00061##
none of E, F, G are present, and M is methoxy.
[0139] In one specific embodiment,
##STR00062##
E is a C.sub.1 to C linear, branched or substitute alkylene; [0140]
F is a bond; and [0141] G is
##STR00063##
[0142] In another specific embodiment,
##STR00064##
and [0143] E is --(CH.sub.2).sub.2--; [0144] F is a bond; [0145] G
is
##STR00065##
[0146] wherein X.sub.2 hydroxy, fluorine, methoxy or methyl.
[0147] In another embodiment,
##STR00066##
E is a C.sub.1 to C.sub.5 linear alkylene; [0148] F is
##STR00067##
[0148] G is
##STR00068##
[0150] wherein R.sub.9 is NH.sub.2, --N(CH.sub.3).sub.2, or
NR.sub.13R.sub.14.
[0151] In specific embodiments,
##STR00069##
E is a C.sub.1 to C.sub.5 linear alkylene; [0152] F is
##STR00070##
[0152] and [0153] G is
##STR00071##
[0154] wherein R.sub.9 is NH.sub.2 or NR.sub.13R.sub.14.
[0155] In other embodiments,
##STR00072##
E is a C.sub.1 to C.sub.5 linear alkylene; [0156] F is
##STR00073##
[0156] and [0157] G is
##STR00074##
[0158] wherein D is hydrogen and R.sub.9 is
--N(CH.sub.3).sub.2.
[0159] In other embodiments,
##STR00075##
E is --(CH.sub.2).sub.2--, [0160] F is
##STR00076##
[0160] and [0161] G is
##STR00077##
[0162] wherein R.sub.9 is NH.sub.2, --N(CH.sub.3).sub.2, or
NR.sub.13R.sub.14.
[0163] In other embodiments,
##STR00078##
E is --(CH.sub.2).sub.2--, or C.sub.1 to C.sub.5 linear alkylene;
[0164] F is a bond; and [0165] G is
##STR00079##
[0166] wherein R.sub.3 is hydroxy or methoxy.
[0167] In other embodiments,
##STR00080##
and none of E, F and G are present.
[0168] In other embodiments,
##STR00081##
E is --(CH.sub.2).sub.2--, or C.sub.1 to C.sub.6 linear, branched,
or cyclic alkylene; [0169] F is a bond; and [0170] G is
##STR00082##
[0171] wherein R.sup.3 is hydroxy or methoxy.
[0172] According to one embodiment,
##STR00083##
E is --(CH.sub.2).sub.2--, or C.sub.1 to C.sub.6 linear, branched,
or cyclic alkylene; [0173] F is a bond; and [0174] G is
##STR00084##
[0175] wherein D is hydrogen; M is --NH--; R.sub.1 and R.sub.5 are
both hydrogen; R.sub.3 is hydroxy or methoxy.
[0176] Specific embodiments of the invention encompass but are not
limited to:
##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089##
##STR00090##
[0177] It will be understood that above compounds are illustrative
only and not intended to limit the scope of the claims to only
those compounds.
[0178] The compounds of the invention can he synthesized by organic
chemistry techniques known to those of ordinary skill in the
art.
[0179] In accordance with the invention, a composition comprising a
compound of the invention or a pharmaceutically acceptable salt, is
administered to a cell, such as lung cell, a breast cancer cell, a
lung cancer cell, a malignant melanoma cell, an ovarian carcinoma
cell, a brain tumor cell, a soil tissue sarcoma cell, and an
osteosarcoma cell.
[0180] The invention also encompasses method of inhibiting the
growth of a cancer cell, such as lung cancer cell, a breast cancer
cell, a colon cancer cell, a malignant melanoma cell, an ovarian
carcinoma cell, a brain tumor cell, a soft tissue sarcoma cell, a
rhabdomyosarcoma cell, a pancreatic cancer cell, a prostate cancer
cell and an osteosarcoma cell, which comprises administering to the
cell a pharmaceutically effective amount of a composition
comprising a compound of Formula I, or a pharmaceutically
acceptable salt or prodrug thereof.
[0181] The invention also encompasses a method of reducing the
likelihood of cancer metastasis, such as lung cancer metastasis,
breast cancer metastasis, colon cancer metastasis, malignant
melanoma metastasis, ovarian carcinoma metastasis, brain tumor
metastasis, soft tissue sarcoma metastasis, rhabdomyosarcoma
metastasis, pancreatic cancer metastasis, prostate cancer
metastasis and osteosarcoma metastasis, which comprises
administering to a subject in need of such treatment a
therapeutically effective amount of a composition comprising a
compound of Formula I, or a pharamaceutically acceptable salt or
prodrug thereof.
[0182] In one embodiment, "treatment" or "treating" refers to an
amelioration of a. disease or disorder, or at least one detectable
symptom thereof. In another embodiment, "treatment" or "treating"
refers to an amelioration of at least one measurable physical
parameter, not necessarily discernible by the patient. In yet
another embodiment, "treatment" or "treating" refers to inhibiting
the progression of a disease or disorder, either physically, e.g.,
stabilization of a discernible symptom, physiologically, e.g.,
stabilization of a physical parameter, or both. In yet another
embodiment, "treatment" or "treating" refers to delaying the onset
of a disease or disorder.
[0183] In certain embodiments, the compositions of the invention
are administered to a patient, for example a human, as a
preventative measure against diseases, including preventing the
occurrence of a tumor or preventing the progression of a tumor.
[0184] As used herein, the term "prevent," as it relates to tumors
and/or abnormal cell growth, indicates that a compound of the
present invention is administered to a subject to at least
partially inhibit the or reduce the likelihood of growth, division,
spread, or proliferation of tumor cells. Of course, the term
"prevent" also encompasses prohibiting entirely the emergence of
new tumors or any of the associated symptoms from detectably
appearing. Thus a subject may be "pretreated," by administering the
one or more compounds of the present invention to prevent tumors
from arising. The phrase "preventing the progression," as it
relates to tumors, is used to mean a procedure designed to at least
partially inhibit the detectable appearance of one or more
additional tumors or aberrant cell growth in a patient already
exhibiting one or more symptoms of the presence of a tumor or
aberrant cell growth, and is also used to mean at least partially
prohibiting the already-present symptoms of cancer from worsening
in the subject.
[0185] As used herein, the term "administer" and "administering"
are used to mean introducing at least one compound or composition
into a subject. When administration is for the purpose of
treatment, the substance is provided at, or after the diagnosis of
an abnormal cell growth, such as a tumor. The therapeutic
administration of this substance serves to inhibit cell growth of
the tumor or abnormal cell growth.
[0186] As used herein, the term "coadminister" is used to mean that
each of at least two compounds are administered during a time frame
wherein the respective periods of biological activity overlap. Thus
the term includes sequential as well as coextensive administration
of the compositions of the present invention. If more than one
substance is coadministered, the routes of administration of the
two or more substances need not be the same. The scope of the
invention is not limited by the identity of the substance which may
be coadministered with the compositions of the present invention.
For example, one of the compounds of the present invention may be
co-administered with another compound of the present invention or
another other pharmaceutically active substances, such as vinca
alkaloids, nucleic acid inhibitors, platinum agents, interleukin-2,
interferons, alkylating agents, antimetabolites, corticosteroids,
DNA intercalating agents, anthracyclines, and ureas. Examples of
specific agents in addition to those exemplified herein, include
hydroxyurea, 5-fluorouracil, anthramycin, asparaginase, bleomycin,
dactinomycin, dacabazine, cytarabine, busulfan, thiotepa,
lomustine, mechlorehamine, cyclophosphamide, melphalan,
mechlorethamine, chlorambucil, carmustine, 6-thioguanine,
methotrexate, etc.
[0187] Due to the activity of the compounds of the invention, the
compounds are advantageously useful in yeterinary and human
medicine. As described above, the compounds of the invention are
useful for the treatment or prevention of conditions caused by
uncontrolled cell growth, hyperproliferation of cells, tumor
growth, and cancers, for example, lung cancer, pancreatic cancer,
leukemia, breast cancer, liver cancer, kidney cancer, human
glioblastoma and prostate cancer.
[0188] The invention provides methods of treatment and prophylaxis
by administration to a subject of a therapeutically effective
amount of a composition comprising a compound of the invention. The
subject can be a mammal, including, but is not limited to, an
animal such a cow, horse, sheep, pig, chicken, cat, dog, mouse,
rat, rabbit, guinea pig, non-human primate or human.
[0189] The present compositions, which comprise one or more
compounds of the invention can be administered intravenously,
intravenously intramuscularly intraperitonealy and orally.
[0190] Suitable dosage ranges of the compounds of the invention,
regardless of the route of administration, are generally about
0.0001 milligram to 2000 milligrams of a compound of the invention
per kilogram body weight. In one specific embodiment, the dose is
about 0.001 milligram to about 1500 milligrams per kilogram body
weight, more specifically about 0.01 milligram to about 1000
milligrams per kilogram body weight, more specifically about 0.1
milligram to about 500 milligrams per kilogram body weight, and yet
more specifically about 1 milligram to about 100 milligrams per
kilogram body weight.
[0191] The compounds and the compositions of the invention may also
be administered by any other route, for example, by infusion or
bolus injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.),
and they may be administered together with another biologically
active agent. Administration can be systemic or local. Various
delivery systems are known, e.g., encapsulation in liposomes,
microparticles, microcapsules, capsules, etc., and can be used to
administer a compound or composition of the invention. In certain
embodiments, more than one compound of the invention is
administered to a patient. Methods of administration include but
are not limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, oral, sublingual,
intranasal, intracerebral, intravaginal, transdermal, rectally, by
inhalation, or topically. The preferred mode of administration is
left to the discretion of the practitioner, and will depend in-part
upon the site of the medical condition.
[0192] In specific embodiments, it may be desirable to administer
one or more compounds or compositions of the invention locally to
the area in need of treatment. This may be achieved, for example,
and not by way of limitation, by local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as but not limited to silastic membranes, or fibers. In one
embodiment, administration can be by direct injection at the site
(or former site) of an atherosclerotic plaque tissue.
[0193] Pulmonary administration can also be employed, e.g., by use
of an inhaler or nebulizer, and formulation with an aerosolizing
agent, or via, perfusion in a fluorocarbon or synthetic pulmonary
surfactant. in certain embodiments, the compounds of the invention
can be formulated as a suppository, with traditional binders and
vehicles such as triglycerides.
[0194] The present compositions will contain a therapeutically
effective amount of a compound of the invention, optionally more
than one compound of the invention, preferably in purified form,
together with a suitable amount of a pharmaceutically acceptable
vehicle so as to provide the form for proper administration to the
patient.
[0195] In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Phartnacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "vehicle" refers to a diluent,
adjuvant, excipient, or carrier with which a compound of the
invention is administered. Such pharmaceutical vehicles can be
liquids, such as water and oils, including those of petroleum,
animal, vegetable or synthetic origin, such as peanut oil, soybean
oil, mineral oil, sesame oil and the like. The pharmaceutical
vehicles can be saline, gum acacia, gelatin, starch paste, talc,
keratin, colloidal silica, urea, and the like. In addition,
auxiliary, stabilizing, thickening, lubricating and coloring agents
may be used. When administered to a patient, the compounds of the
invention and pharmaceutically acceptable vehicles are preferably
sterile. Water is a preferred vehicle when the compound of the
invention is administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid vehicles, particularly for injectable solutions. Suitable
pharmaceutical vehicles also include excipients such as starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The present compositions, if desired, can
also contain minor amounts of wetting or emulsifying agents, or pH
buffering agents.
[0196] The present compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, pellets, capsules, capsules
containing liquids, powders, sustained-release formulations,
suppositories, emulsions, aerosols, sprays, suspensions, or any
other form suitable for use.
[0197] In another embodiment, the compounds and/or compositions of
the invention are formulated in accordance with routine procedures
as a pharmaceutical composition adapted for intravenous
administration to human beings. Typically, compounds and/or
compositions of the invention for intravenous administration are
solutions in sterile isotonic aqueous buffer. Where necessary, the
compositions may also include a solubilizing agent. Compositions
for intravenous administration may optionally include a local
anesthetic such as lignocaine to ease pain at the site of the
injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where the compound of the invention is to
be administered by infusion, it can be dispensed, for example, with
an infusion bottle containing sterile pharmaceutical grade water or
saline. Where the compound of the invention is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0198] In one specific embodiment, the compositions of the
invention can be administered orally. Formulations for oral
delivery may be in the form of tablets, lozenges, aqueous or oily
suspensions, granules, powders, emulsions, capsules, syrups, or
elixirs, for example. Orally administered compositions may contain
one or more optionally agents, for example, sweetening agents such
as fructose, aspartame or saccharin; flavoring agents such as
peppermint, oil of wintergreen, or cherry; coloring agents; and
preserving agents, to provide a pharmaceutically palatable
preparation. Moreover, where in tablet or pill form, the
compositions may be coated to delay disintegration and absorption
in the gastrointestinal tract thereby providing a sustained action
over an extended period of time. Selectively permeable membranes
surrounding an osmotically active driving compound are also
suitable for orally administered compounds of the invention. In one
particular platform, fluid from the environment surrounding the
capsule is imbibed by the driving compound, which swells to
displace the agent or agent composition through an aperture. These
delivery platforms can provide an essentially zero order delivery
profile as opposed to the spiked profiles of immediate release
formulations. A time delay material such as glycerol monostearate
or glycerol stearate may also be used. Oral compositions can
include standard vehicles such as mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Such vehicles are preferably of pharmaceutical
grade.
[0199] The amount of a compound of the invention that will be
effective in the treatment of a particular disorder or condition
disclosed herein will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques.
In addition, in vitro or in vivo assays may optionally be employed
to help identify optimal dosage ranges. The precise dose to be
employed in the compositions will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. In specific embodiments of the
invention, the oral dose of at least one compound of the present
invention is about 0.01 milligram to about 100 milligrams per
kilogram body weight, or from about 0.1 milligram to about 50
milligrams per kilogram body weight, or from about 0.5 milligram to
about 20 milligrams per kilogram body weight, or from about 1
milligram to about 10 milligrams per kilogram body weight.
[0200] Suitable dosage ranges for parenteral, for example,
intravenous (i.v.) administration are 0.01 milligram to 100
milligrams per kilogram body weight, 0.1 milligram to 35 milligrams
per kilogram body weight, and 1 milligram to 10 milligrams per
kilogram body weight. Suitable dosage ranges for intranasal
administration are generally about 0.01 pg/kg body weight to 1
mg/kg body weight. Suppositories generally contain 0.01 milligram
to 50 milligrams of a compound of the invention per kilogram body
weight and comprise active ingredient in the range of 0.5% to 10%
by weight. Recommended dosages for intradermal, intramuscular,
intraperitoneal, subcutaneous, epidural, sublingual, intracerebral,
intravaginal, transdermal administration or administration by
inhalation are in the range of 0.001 milligram to 200 milligrams
per kilogram of body weight. Suitable doses of the compounds of the
invention for topical administration are in the range of 0.001
milligram to 1 milligram, depending on the area to which the
compound is administered. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems. Such inial models and systems are well known in the
art.
[0201] In other embodiments, a composition of the invention for
parenteral, for example, intravenous administration includes about
0.001 milligram to about 2000 milligrams of a compound of the
invention, more preferably about 0.01 milligram to about 1000
milligrams of a compound of the invention, more preferably about
0.1 milligram to about 500 milligrams of a compound of the
invention, and yet more preferably about 1 milligram to about 200
milligrams of a compound of the invention.
[0202] The invention also provides pharmaceutical packs or kits
comprising one or more containers filled with one or more compounds
of the invention, Optionally associated with such container(s) can
be a notice in the form prescribed by a governmental agency
regulating the manufacture, use or sale of pharmaceuticals or
biological products, which notice reflects approval by the agency
of manufacture, use or sale for human administration. In a certain
embodiment, the kit contains more than one compound of the
invention.
[0203] The compounds of the invention can be assayed in vitro and
in vivo, for the desired therapeutic or prophylactic activity,
prior to use in humans. For example, in vitro assays can be used to
determine whether administration of a specific compound of the
invention or a combination of compounds of the invention can be
used for treating a particular disorder or condition disclosed
herein. The compounds of the invention may also be demonstrated to
be effective and safe using animal model systems.
[0204] Other methods will be known to the skilled artisan and are
within the scope of the invention.
EXAMPLES
[0205] The compounds of the invention showed pharmacological
efficacy in treating or preventing various disorders. Example 1
I. Synthetic Procedure for Novel 6- or 7- Substituted aza- or
diazanaphthalene-5.8-dione Scaffolds as Anti-Metastatic Agents
Targeting osteosarcoma.
[0206] The novel 6- or 7- substituted aza- or
diazanaphthalene-5.8-dione scaffolds were synthesized by the route
illustrated in scheme 1. Condensation of the primary amine with the
dione gives a mixture of 6- or 7-substituted adduct, which can be
separated by silica gel chromatography.
##STR00091##
[0207] The starting material in the reaction above can be
synthesized using the well-known "Henry Reaction," as disclosed in
PCT Application No. PCT/US2003/026300 (Publication No. WO
2004/026305), which is incorporated by reference, as shown in
Scheme 1a, below. Compounds 4 and 5 below were transformed to
compounds 6 and 7 using the Henry reaction, and compounds 6 and 7
were transformed to compounds 8 and 9 using the reactions as
disclosed in Kita, Y., et al., J. Org. Chem. 61:223-227 (1996),
which is incorporated by reference.
##STR00092##
[0208] The precursor 5.8-dione derivatives for the synthesis of the
claimed compounds were generated using methods described in the
literature as discussed herein.
[0209] (A) isoquinoline 5,8-dione precursor (Y1, Y3 and Y4=C, Y2=N)
used in the methods of the present invention were prepared
according to the following reaction scheme 2. The detailed
procedure is described in (i) Tett. Lett., 32, 4872-4873, and (ii)
Bioorganic and Medicinal chemistry Letter, 17, 6091.
##STR00093##
[0210] (B) Diazanaphthalene-5,8-diones precursor (Y2 and Y4=C, Y1
and Y3=N) used in the process of the present invention were
prepared according to the following reaction scheme 3.
##STR00094##
(C) Phthalazine-5,8-dione precursors (Y1 and Y4=C, Y2 and Y3=N)
used in the process of the present invention were prepared
according to the following reaction scheme 4. The detailed
procedure is described in (i) J. Med Chem., 48, 744-752, and (ii)
Bioorganic and Medical Chemistry Letters, 27, 2577-2580.
##STR00095##
II. Synthetic Procedure for Novel phthalimide Analogs as
Anti-Metastatic Agents Targeting osteosarcoma.
[0211] Syntheses of the phthalimide analogs were accomplished by
condensation of the primary amine with the requisite phthalic
anhydride in acetic acid under microwave conditions for 1 hour. The
isolated adduct was then purified by silica gel chromatography to
give the product.
##STR00096##
[0212] (A) The condensation of phthalic anhydride (Y.sub.1=N,
Y.sub.2, Y.sub.3 and Y.sub.4=C) and the primary amine (R.sub.1 and
R.sub.3=H, and R.sub.2=OH, and n=2) of the above-described reaction
is described in scheme 6.
##STR00097##
[0213] (B) The condensation of phthalic anhydride (Y.sub.2=N,
Y.sub.1, Y.sub.3 and Y.sub.4=C) and the primary amine (R.sub.1 and
R.sub.3=H, and R.sub.2=OH, and n=2) of the above-described reaction
is described in scheme 7.
##STR00098##
III. Synthetic Procedure of Novel Substituted Serotonin Scaffolds
Anti-Metastatic Agents Targeting osteosarcoma.
[0214] Synthesis of the analogs incorporating the serotonin core
can be accomplished by condensation of the requisite tryptamine
analog with the corresponding dione to give both the 6- and
7-substituted adducts. The mixture was separated by silica gel
chromatography to give each analog.
##STR00099##
[0215] Synthesis of the precursor tryptamine analogs were
accomplished according to the following reaction scheme 9. The
detailed procedure is described in (i) J. Med. Chem., 22, 63-69,
and (ii) J. Fluorine Chemistry, 97, 161-164.
##STR00100## ##STR00101##
IV. Synthetic Procedure of Novel 3,4,5-trisubstituted benzylamino
and benzamide Scaffolds.
[0216] 3,4,5-trisubstituted benzylamino and benzamido scaffolds
used in the process of the present invention were prepared
according to the following reaction scheme 10. The detailed
procedure is described in (i) U.S. Pat. No. 5,283,336 and (ii) J.
Med. Chem., 50, 3497.
##STR00102##
V. Synthetic Procedure of Chloro Derivatives
[0217] Chloro derivatices of the present invention were prepared
according to the following reaction scheme 11. The detailed
procedure is described in J. Organic Chemistry, 9, 359-372.
##STR00103##
VI. Synthetic Procedure of GK3-015
[0218] GK3-015 was prepared according to the following reaction
scheme 12.
##STR00104##
VII. Synthetic Procedures of GK2-197, GK2-229, GK2-303
[0219] GK2-197, GK2-229, GK2-303 were prepared according to the
following reaction in scheme 13.
##STR00105##
VIII. Synthetic Procedure of the 7-aminoquinoline
[0220] 7-aminoquinoline was prepared according to the following
reaction in scheme 14.
##STR00106##
IX. Synthetic Procedure of Halo-Serotonin Analogs
[0221] Halo-serotonin analogs were prepared according to the
following reaction in scheme 15.
##STR00107## ##STR00108##
Methods
Ezrin Purification
[0222] Expression of untagged WT and mutant (T567D) of ezrin
constructs in pQE16 expression vector were propagated in E.coli
strain M15 pREP4(Qiagen, Valencia, Calif.). T567D ezrin cDNA was
prepared from WT ezrin cDNA by site directed mutagenesis by using
QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene, Cedar
Creek, Tex.). Both ezrin proteins were purified using
hydroxyapatite (Bio-Rad, Hercules, Calif.) column on AKTA Explorer
machine and dialysed into 20 mM MES, 150 mM NaCl, pH6.7 as
described previously (Reczek, D., et al., J. Cell Biol. 139,
169-179 (1997)). Protein purity was estimated based on absence of
nonspecific bands on cootnassie stained gels.
Immunoprecipitation
[0223] For analysis of actin binding to FL and mutant ezrin, K12
cell lysate was incubated with recombinant ezrin protein, then
subjected to immunoprecipitation with ezrin antibody (Sigma, St.
Louis, Mo.) and immunoblotted with actin antibody (Santa Cruz,
Santa. Cruz, Calif.). For analysis of inhibition of small molecules
on ezrin phosphorylation and actin binding, K7M2 cell lysates were
treated with compounds at 10 .mu.M concentration for 6 hours,
followed by immunoprecipitation with the ezrin antibody and
immunoblotted with phospho-ezrin (Cell Signaling, Danvers, Mass.),
actin and ezrin antibodies.
In Vitro Kinase Assays
[0224] 500 ng of recombinant ezrin protein in kinase assay mix (200
.mu.M ATP) was incubated with the compounds at 1-100 .mu.M
concentrations for 15 minutes on ice, then 50 ng PKC, (Millipore,
Billerica, Mass.) was added. Reaction was performed at 30.degree.
C. for 30 min and stopped by adding 2.times. sample buffer.
NSC305787 and NSC668394 profiling on PKC, , .alpha. and .gamma.
were performed by utilizing the Kinase Inhibitor Compound Profiling
Service at Kinexus Bioinformatics Corporation (Vancouver,
Calif.).
Surface Plasmon Resonance (SPR)
[0225] Compounds for initial screening were acquired from 4
libraries (Challenge Set, Diversity Set, Mechanistic Set and
Natural Product Set) of the Developmental Therapeutics Program,
National Cancer Institute. Their direct ezrin binding potential was
analyzed by using Biacore T100 v2.0.3 instrument (GE Healthcare,
Piscataway N.J.). The technique requires immobilization of the
ligand on sensorchip and the analyte is then injected over the chip
surface through a fluidics system. Existing interactions are
measured based on total mass change on the chip surface.
Recombinant WT ezrin protein was used as the ligand and immobilized
on to a Biacore CMS sensorchip and compounds were injected one at a
time as the binding interactions were recorded. HBS-P, which
contained 10 mM Hepes (pH 7.4), 150 mM NaCl and 0.05% surfactant
P-20 was used as the standard running buffer. At initial screening,
each molecule was injected for 1 min at 10 or 100 .mu.M
concentrations. Any molecule giving more than 10 resonance units
(RU) binding signal with an acceptable curve shape was selected as
an initial hit. During detailed SPR analysis, NSC305787 was
injected at 1 .mu.M, 2 .mu.M, 4 .mu.M, 8 .mu.M, 32 .mu.M and 64
.mu.M concentrations in duplicates. NSC668394 was injected at 1,5
.mu.M, 3 .mu.M, 6 .mu.M, 12.5 .mu.M, 25 .sub..mu.M, 50 .mu.M and
100 .mu.M concentrations in triplicates. Results were analyzed by
using Biacore T-100 v2.0.3 analysis software.
Chemotaxis, Cell Viability Assays
[0226] Chemotaxis experiments were performed in 96 well Boyden
chamber as described previously (Chen. K., et al., Pediatr. Blood
Cancer, 51, 349-355 (2008)). To measure effect of compounds on
cellular toxicity, WST viability assay (Roche, Indianapolis, Ind.)
was performed in parallel to chemotaxis experiments.
Invasion Assays
[0227] The anti-invasive potential of NSC305787 and NSC668394 were
evaluated by using electric cell impedance sensing (ECIS) on a
Roche xCELLigence system (Roche, Indianapolis, Ind.). Briefly,
HUVEC cells (25,000/well) were seeded in a 96-well plate in EGM-2
media (Lonza, Basel, CH). Following formation of a confluent HUVEC
monolayer (app 32 hrs), EGM-2 media was aspirated and a layer of OS
cells (10,000 cells/well) was added in DMEM media containing the
compounds. This time point was accepted as 0 hr of treatment and
invasion was monitored during the following 6 hrs by measuring
changes in resistance at the cell-electrode interphase.
Zebrafish Embryo Development Assay
[0228] Animals. Zebrafish (Danio rerio) were raised, maintained and
crossed as described before (Westerfield, M. University of Oregon
Press, Eugene. Oreg., 1993), Development of embryos was studied at
28.degree. C. and staging was determined by morphological
characteristics (Kimmel, C. B., et al., Dev. Dyn. 203, 253-310
(1995)).
Morpholino Oligonucleotide Injections.
[0229] A translation blocking anti-ezrin morpholine oligonucleotide
(MO), described and validated by Link et al. (Link, V., et al., J.
Cell Sci., 119, 2073-2083 (2006)), MO1, 5'-CGCGAACATTTACTGGTTTAGG
(SEQ ID NO:1), was synthesized by Gene-Tools, LCC (Philomath,
Oreg.). MOs were microinjected into one to four cell stage embryos.
Chemical screening.
[0230] Zebrafish embryos were arrayed, three per well, in 96-well
plates. Compounds were added at 1-33 .mu.M concentrations. Embryos
were observed, and photographed, at 70% epiboly, 24-28 hours post
fertilization (hpf) and 48 hpf.
Pulmonary Metastasis Assay (PuMA)
[0231] The technique of isolated lung organ culture was performed
as reported by Mendoza et al (Mendoza, A., et al., J. Clin.
Invest., 120(8):2979-2988 (2010)).
In Vivo Experimental Metastasis Model
[0232] GFP expressing K7M2 or MNNG tumor cells (1.times.10.sup.6)
were delivered by tail vein to nude mice. One day after injection,
vehicle, NSC305787 (240 .mu.g/kg/inj) and NSC 608394 (226
.mu.g/kg/inj) were injected 5 days a week intraperitoneally.
Statistical Analysis
[0233] Statistical analyses were performed with Prism (GraphPad
Software, LaJolla, Calif.).
Results
[0234] Initial Screening of Small Molecules for Direct Binding to
ezrin
[0235] Small molecule libraries were screened to identify compounds
that directly bind to ezrin protein. To achieve this goal,
full-length recombinant ezrin protein was expressed in bacteria and
the protein was purified by column chromatography as described
earlier (Reczek, D., et al., J. Cell Biol., 139, 169-179 (1997)).
Recombinant full length ezrin was prepared to a concentration of
100 .mu.g/ml with >90% purity (FIG. 1A). To validate that
recombinant mouse ezrin protein purified from bacteria has the
appropriate tertiary structure, its actin binding ability was
evaluated. Phosphorylation of threonine at position 567 provides an
open conformation of ezrin and increases its actin binding
capacity, A threonine (T) to aspartic acid (D) substitution was
used to mimic phosphorylation in this residue (Gautreau, A., et
al., J. Cell Biol., 150, 193-203 (2000)). Wild type (WT) and
phosphomimicking mutant (T567D) of recombinant ezrin protein were
purified (FIG. 1A). Actin binding of these two constructs was
evaluated by immunoprecipitation. Cell lysates from low ezrin
expressing K12 osteosarcoma cells were incubated with recombinant
ezrin proteins. Cellular actin protein co-immunoprecipitated was
detected by immunoblotting. Significantly higher levels of actin
binding to the T567D mutant were detected compared to WT ezrin
(FIG. 1B). Increased binding of T567D ezrin to purified actin was
also demonstrated in an ELISA assay (data not shown). These results
suggested that at least a portion of recombinant ezrin made in
bacteria was folded properly to replicate expected mammalian
structure. p Surface plasmon resonance (SPR) allows measurement of
direct molecular interactions in real-time and in a label free
setting (Fivash, M., et al., Curr. Opin. Biotechnol., 9. 97-101
(1998) Malmqvist, M., et al., Biochem Soc Trans., 27, 335-340
(1999)). SDR to screen small molecule libraries for compounds that
directly bind to recombinant WT ezrin protein. Ezrin was
immobilized on Biacore T100 sensorchips and small molecules were
injected over the surface one at a time at a single concentration
(10 .mu.M or 100 .mu.M). Four libraries for screening (Challenge
Set, Diversity Set, Mechanistic Set and Natural Product Set) were
provided by Developmental Therapeutics Program of the National
Cancer Institute. At this initial screen any molecule that showed
meaningful binding to recombinant ezrin over the background values
was selected as a primary hit. The 65 primary hits were evaluated
with functional assays, which identified two lead compounds,
NSC305787 and NSC668394 (FIG. 1C). These two compounds were used to
develop and generate the novel compounds disclosed herein. Detailed
SPR analysis of the two lead compounds in 5 independent experiments
yielded an average K.sub.D of 5.85 .mu.M (.+-.s.d. 3.85 .mu.M) for
the affinity of NSC305787 (FIG. ID) and 12,59 .mu.M (.+-.s.d. 6.35
.mu.M) for the affinity of NSC668394 (FIG. 1E) binding to ezrin.
K.sub.D values of NSC305787 and NSC668394 binding to actin, which
was used as a negative control were 91.4 .mu.M and 603 .mu.M,
respectively (data not shown). None of the 65 primary hits showed
differential binding between WT and T567D mutant ezrin
proteins.
[0236] The secondary functional assays used for lead compound
selection included ezrin phosphorylation, actin binding,
chemotaxis, zebrafish embryonic development and mouse lung organ
culture. Furthermore, drugability based on their solubility,
potential in vivo toxicity, chemical stability and potential for
derivatization were also considered for elimination of some primary
hits.
NSC305787 and NSC668394 Inhibit Endogenous Ezrin 1567
phosphorylation and Actin Binding.
[0237] Ezrin phosphorylation at T567 is critical for its
activation, which then enables interaction of ezrin with other
cellular proteins including actin (Matsui, T., et al., J. Cell
Biol., 140, 647-657 (1998)). Both NSC305787 and NSC668394 inhibited
T567 phosphorylation and actin binding of ezrin at 10 .mu.M
concentration in K7M2 OS cells. Treatment with the compounds did
not alter cellular ezrin protein levels (FIG. 2A).
Kinase Independent Inhibition of Ezrin T567 phosporylation by PKC,
by Directly Targeting the Substrate.
[0238] Dynamic regulation of ezrin phosphorylation during
metastatic progression is linked to protein kinase C (PKC)
activation. Members of this family of serine/threonine kinases that
phosphorylate ezrin at T567 in OS cells include PKC, alpha
(.alpha.), iota () and gamma (.gamma.) (Ren, L., et al., Oncogene,
28, 792-802 (2009)). Phosphorylation of ezrin by PKC, was inhibited
by NSC305787 with an IC.sub.50 of 5.5 .mu.M and by NSC668394 with
an IC.sub.50 of 7.81 .mu.M (FIG. 2B, FIG. 2C). To determine whether
reduced phosphorylation of ezrin resulted from inhibition of the
kinase, the effect of lead compounds on three PKC isoforms (PKC,,
PKC,.alpha.. and PKC,.gamma.) with a nonspecific substrate was
tested. NSC305787 required 10-fold higher concentration (.about.50
.mu.M) to inhibit all three PKC isoforms than that required to
inhibit ezrin phosphorylation (FIG. 2B). NSC668394 did not show any
significant inhibition of PKC activity with the doses tested in
this experiment (maximum 100 .mu.M) (FIG. 2C).
[0239] Furthermore, direct interaction experiments with Biacore in
SPR analysis revealed that NSC668394 bound to PKC, with a K.sub.D
of 160.2 .mu.M and NSC305787 bound to PKC, with a K.sub.D of 172.4
.mu.M (data not shown). These results strongly suggest that
NSC305787 and NSC668394 inhibit ezrin T567 phosphorylation
primarily due to their binding to ezrin and not due to inhibition
of PKC, kinase activity.
OS Cell's Invasive Phenotype is Inhibited by NSC305787 and
NSC668394
[0240] Higher levels of ezrin protein in K7M2 cells compared to K12
cells leads to enhanced metastatic potential of K7M2 cells (FIG.
3A) (Khanna, C., et al., Cancer Res., 61, 3750-3759 (2001)). All 65
initial hits were tested for inhibiting cell motility of both cell
lines in a modified Boyden chamber chemotaxis assay. Any molecule
that inhibited chemotaxis without any cellular toxicity was given
higher priority. The anti-invasive potential of NSC305787 and
NSC668394 were further evaluated by using electric cell impedance
sensing (ECIS) on a Roche xCELLigence system. This technique
involves monitoring cell-cell interactions in real-time by
measuring changes in cell resistance as a monolayer of human
umbilical vein endothelial cells (HUVEC) is disrupted by invading
tumor cells. Both NSC305787 and NSC668394 inhibited invasion of
metastatic K7M2 cells on HUVEC monolayer (FIG. 3B, FIG. 3C).
NSC305787 did not have any effect on invasion of K12 cells at 1
.mu.M and 10 .mu.M concentrations, whereas NSC668394 inhibited
invasion by K12 cells slightly at 10 .mu.M and did not have any
effect at 1 .mu.M concentration (FIG. 3B, FIG. 3C). Both compounds
were not toxic to K7M2, K12 and HUVEC cells at these concentrations
(data not shown).
NSC305787 and NSC668394 Inhibit Cell Motility During Zebrafish
Embryonic Development
[0241] Inhibition of ezrin protein expression by morpholine
oligonucleotides (MO) results in a unique phenotype in zebrafish
embryos (Link, V., et al., J. Cell Sci., 119, 2073-2083 (2006)).
All 65 primary hit compounds were tested on early zebrafish embryo
development. Small molecules that killed the embryos prior to 70%
epiboly during gastrulation were eliminated based on toxicity. The
ezrin MO phenotype, characterized by reduced epiboly movements
resulting from defective germ layer morphogenesis, was confirmed by
microinjection of MO1 as described by Link et al. (FIG. 4A, FIG.
4E). Treatment with 10 .mu.M NSC305787 mimicked the ezrin MO1
phenotype (FIG. 4B, FIG. 4E). Embryos treated with 10 .mu.M
NSC668394 showed normal development at earlier stages, but had a
very distinctive cycloptic eye phenotype by 28 hpf (FIG. 4C, FIG.
4E). If NSC668394 was removed before 48 hpf, the animals survived
up to 7 days, they were able to swim, and the cycloptic eye
appeared to be otherwise functional as the single eye moved and
responded to light (FIG. 4D). In normal zebrafish development, the
eye field extends across the midline and as progenitor cells
divide, they move laterally to form two separate eyes. Therefore,
the observed cycloptic phenotype suggests stalling of eye precursor
cells in the midline. Since inhibition of hedgehog pathway in sheep
embryogenesis creates cycloptic lambs, we tested NSC668394 on Gli
reporter assays in mammalian cell lines, but did not observe any
significant inhibition (data not shown).
Prevention of Metastastatic Growth in a Lung Organ Culture
Assay
[0242] In OS, the predominant site of recurrence and main cause of
death is pulmonary metastasis (Dunn, D. &. Dehner, L. P.,
Cancer, 40, 3054-3064 (1977)). An ex vivo mouse lung organ culture
assay was performed to evaluate the inhibitory potential of the
lead compounds. In this method, tumor cells reaching the lung
following tail vein injection grew in the lung slices kept in organ
culture, resembling in vivo lung metastasis (Ren, L., et al.,
Oncogene, 28, 792-802 (2009)). When green fluorescent protein (GFP)
expressing metastatic K7M2 cells were injected through tail vein of
mice, metastatic foci were followed up in the lung organ cultures,
which was quantitated by GFP fluorescence. When mice were injected
with less metastatic and low ezrin expressing K12 cells, no
surviving tumor cells were observed in organ culture (data not
shown). NSC305787 and NSC668394 treatment at 10 .mu.M concentration
significantly inhibited the lung metastasis of K7M2 high ezrin
expressing OS cells in this organ culture assay (FIG. 5A, FIG.
5B).
NSC305787 and NSC668394 Inhibit Ezrin Dependent in vivo OS
Metastasis Growth in Mouse Lung.
[0243] After observing inhibition of lung metastatic growth in lung
organ culture, the effects of NSC305787 and NSC668394 on in vivo
lung metastasis model were tested. Following injection of GFP
expressing K7M2 cells through tail vein, vehicle treated animals
died due to progressive lung metastases in approximately 4 weeks
(median survival 28.5 days). NSC305787 and NSC668394 treated
animals survived up to 50 and 49 days, respectively (FIG. 6A).
Overall survival of NSC305787 treated mice was significantly
different than vehicle treated group (P=0.0337). NSC668394 treated
group showed a very strong correlation (P=0.0524), When the lung
tissues were harvested and analyzed, there was a significant
difference between the vehicle treated and the small molecule
treated groups (FIG. 6B).
[0244] MNNG is a human OS cell line and maintains its metastatic
phenotype even after inhibition of ezrin expression by siRNA (data
not shown). MNNG cells were used as a negative control for
ezrin-dependent specificity. Animals injected with GFP expressing
MNNG cells by tail vein and treated with NSC305787 and NSC668394
did not demonstrate any difference in survival between control and
treatment groups. Median survival of vehicle, NSC305787 and
NSC668394 treated MNNG cells were 50.5, 49, and 48.5 days,
respectively (FIG. 6C). We did not observe a difference between the
vehicle, NSC305787 and NSC668394 treated groups for their number of
GFP expressing metastatic foci in lung tissues (FIG. 6D).
[0245] The present invention is not to be limited in scope by the
specific embodiments disclosed in the examples which are intended
as illustrations of a few aspects of the invention and any
embodiments which are functionally equivalent are within the scope
of this invention. Indeed, various modifications of the invention
in addition to those shown and described herein will become
apparent to those skilled in the art and are intended to fall
within the appended claims.
[0246] A number of references have been cited, the entire
disclosures of which are incorporated herein by reference.
Sequence CWU 1
1
1122DNAArtificial SequenceTranslation blocking anti-ezrin
morpholino oligonecleotide (MO) 1cgcgaacatt tactggttta gg 22
* * * * *