U.S. patent application number 11/615183 was filed with the patent office on 2007-09-13 for inhibitors of phosphatidyl myo-inositol cycle.
Invention is credited to Alan P. Kozikowski, Garth Powis, Lixin Qiao.
Application Number | 20070213398 11/615183 |
Document ID | / |
Family ID | 29741186 |
Filed Date | 2007-09-13 |
United States Patent
Application |
20070213398 |
Kind Code |
A1 |
Kozikowski; Alan P. ; et
al. |
September 13, 2007 |
INHIBITORS OF PHOSPHATIDYL MYO-INOSITOL CYCLE
Abstract
The present invention relates to the preparation and biological
activity of 3-deoxy-Dmyo-inositol ether lipid analogs as inhibitors
of phosphatidylinositol-3-kinase signaling and cancer cell growth.
The compounds of the present invention are useful as anti-tumor 5
agents which effectively inhibit the growth of mammalian cells.
Inventors: |
Kozikowski; Alan P.;
(Princeton, NJ) ; Qiao; Lixin; (Arlington, VA)
; Powis; Garth; (Tucson, AZ) |
Correspondence
Address: |
PEPPER HAMILTON LLP
ONE MELLON CENTER, 50TH FLOOR
500 GRANT STREET
PITTSBURGH
PA
15219
US
|
Family ID: |
29741186 |
Appl. No.: |
11/615183 |
Filed: |
December 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10733115 |
Dec 11, 2003 |
7153843 |
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11615183 |
Dec 22, 2006 |
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09879765 |
Jun 12, 2001 |
6667340 |
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10733115 |
Dec 11, 2003 |
|
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|
09339948 |
Jun 25, 1999 |
6245754 |
|
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09879765 |
Jun 12, 2001 |
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60090877 |
Jun 26, 1998 |
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60223421 |
Aug 7, 2000 |
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60223724 |
Aug 8, 2000 |
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60235269 |
Sep 26, 2000 |
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60235270 |
Sep 26, 2000 |
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Current U.S.
Class: |
514/520 ;
558/271 |
Current CPC
Class: |
C07F 9/4081 20130101;
C07C 69/96 20130101; C07C 2601/14 20170501; C07F 9/117 20130101;
C07C 69/013 20130101 |
Class at
Publication: |
514/520 ;
558/271 |
International
Class: |
A61K 31/265 20060101
A61K031/265; C07C 69/96 20060101 C07C069/96 |
Claims
1. A 3-deoxy-D-myo-inositol analog having the formula: ##STR11##
wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are individually,
(C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl,
(C.sub.9-C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is
(C.sub.1-C.sub.25) alkyl (C.sub.6-C.sub.102) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl or
(C.sub.9-C.sub.32)) alkenylaryl; R.sup.4 ad R.sup.5 are
individually hydrogen or a phosphate group; or when R.sup.4 or
R.sup.5 is not hydrogen, a pharmaceutically acceptable salt
thereof.
2. A method of treating cancer comprising administering a
therapeutically effective amount of a compound to a subject
according to claim 1.
3. The method of claim 2, wherein said cancer is one that is
characterized by cells that over-express the EGF and/or the PDGF
receptor, cells that express a mutant ras, or cells that comprise a
Bcr/Abl transfection.
4. The method of claim 3, wherein said cancer is selected from the
group consisting of colon, pancreatic, prostate, head and neck,
breast, gastric, renal, brain and CML.
Description
RELATED APPLICATIONS
[0001] This application claims priority to and is a continuation of
the co-pending U.S. patent application Ser. No. 10/733,115 filed
Dec. 12, 2003, which is a continuation of U.S. application Ser. No.
09/879,765 filed Jun. 12, 2001, which is a continuation-in-part of
application U.S. Ser. No. 09/339,948 filed Jun. 25, 1999, now U.S.
Pat. No. 6,245,754, which claimed the benefit of priority from U.S.
Provisional Patent Application Ser. No. 60/090,877 filed on Jun.
26, 1998 and also claims the benefit of U.S. Provisional
Application Serial No. 60/223,421 filed on Aug. 7, 2000, and U.S.
Provisional Application No. 60,223,724 filed on Aug. 8, 2000, and
U.S. Provisional Application No. 60/235,269 filed on Sep. 26, 2000,
and U.S. Provisional Application No. 60/35,270 filed on Sep. 26,
2000.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to specific compounds designed
to inhibit cell growth signaling. In particular, PtdIns-3-kinase
anti-metabolites are rationally designed to provide compounds that
inhibit cell differentiation and/or cell proliferation, and/or
which promote apoptosis by antagonizing myo-inositol cell growth
signaling. The present invention also relates to therapeutic
methods, e.g., treatment of cancer, including the administration of
the compounds according to the invention.
[0004] 2. Background of the Invention
[0005] For mammalian cells to survive, they must be able to respond
rapidly to changes in their environment. Furthermore, for cells to
reproduce and carry out other cooperative functions, they must be
able to communicate efficiently with each other. Cells most
frequently adapt to their environment and communicate with one
another by means of chemical signals. An important feature of these
signaling mechanisms is that in almost all cases a cell is able to
detect a chemical signal without it being necessary for the
chemical messenger itself to enter the cell. This permits the cell
to maintain the homeostasis of its internal environment, thereby
permitting the cell to respond to its external environment without
being adversely affected by it.
[0006] These sensing functions are carried out by a variety of
receptors, which are dispersed on the outer surface of the cell and
function as "molecular antennae". These receptors detect an
incoming messenger and activate a signal pathway that ultimately
regulates a cellular process such as secretion, contraction,
metabolism or growth. In the cell's cellular plasma membrane,
transduction mechanisms translate external signals into internal
signals, which are then carried throughout the interior of the cell
by chemicals known as "second messengers."
[0007] In molecular terms, the process depends on a series of
proteins within the cellular plasma membrane, each of which
transmits information by inducing a conformational change in the
protein next in line. At some point, the information is assigned to
small molecules or even to ions within the cell's cytoplasm, which
serve as the above-mentioned second messengers. The diffusion of
the second messengers enables a signal to propagate rapidly
throughout the cell.
[0008] Several major signal pathways are now known, but two seem to
be of primary importance. One employs cyclic nucleotides as second
messengers. These cyclic nucleotides activate a number of proteins
inside the cell, which then cause a specific cellular response. The
other major pathway employs a combination of second messengers that
includes calcium ions as well as two substances whose origin is
remarkable: myo-inositol-1,4,5-trisphosphate (IP3) and
diacylglycerol (DAG). These compounds are cannibalized from the
plasma membrane itself, by enzymes which are activated by specific
cellular membrane receptors. However, this pathway requires that
myo-inositol, in its non-phosphorylated form, be initially
synthesized by the cell from glucose or obtained from the
extracellular environment. Recently, another phosphatidylinositol
signaling pathway has been identified and linked to the action of
some growth factors and oncogenes. Phosphatidylinositol-3'-kinase
(also designated type 1 phosphatidylinositol kinase) is found
associated with a number of protein tyrosine kinases including the
ligand-activated receptors for insulin, platelet derived growth
factor (PDGF), epidermal growth factor (EGF), and
colony-stimulating factor-1 (CSF-1) as well as proto-oncogene and
oncogene tyrosine kinases (Y. Fukui et al., Oncogene Res., 4, 283
(1989)). This enzyme phosphorylates the D-3 position of the
myo-inositol ring of phosphatidylinositols to give a class of
phosphafdylinositol-3'-phosphates that are not substrates for
hydrolysis by phosphatidylinositol phospholipase C. Accordingly,
these compounds apparently exert their signaling action
independently of the inositol phosphate pathway.
[0009] Biased on the potential effects thereof on cell
proliferation, differentiation and apoptosis, it would be
beneficial if compounds could be obtained which selectively block
phosphatidylinositol signaling pathways. More specifically, it
would be beneficial if compounds could be obtained which antagonize
myo-inositol metabolites produced by PtdIns-3-Kinase. Such
compounds have significant therapeutic potential, in particular for
treatment of cancer and other conditions involving abnormal cell
differentiation and proliferation. Compounds having improved
selectivity, solubility and stability and particularly
desirable.
SUMMARY AND OBJECTS OF THE INVENTION
[0010] It is an object of the invention to provide novel compounds
which inhibit the phosphatidylinositol signaling pathway.
[0011] It is a more specific object of the invention to provide
novel compounds which are antagonistic of myo-inositol metabolites
provided by PtdIns-3-Kinase.
[0012] It is an even more specific object of the invention to
provide novel analogs of 3-deoxy-D-myo-inositol which inhibit the
phosphatidylinisitol signaling pathway.
[0013] It is still a more specific object of the invention to
provide compounds having the formulae (I) and (II) set forth below:
##STR1## wherein X is O or CH.sub.2, R.sup.1 and R.sup.2 are
individually, (C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl,
(C.sub.9-C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is
(C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl or
(C.sub.9-C.sub.32) alkenylaryl, with the proviso that when X is O,
R.sup.3 is not (C.sub.15) alkyl; R.sup.4 and R.sup.5 are
individually hydrogen or a phosphate group; or when R.sup.4 or
R.sup.5 is not hydrogen, a pharmaceutically acceptable salt
thereof, and ##STR2## wherein X is O or CH.sub.2; R.sup.1 and
R.sup.2 are individually, (C.sub.2-C.sub.25) alkyl,
(C.sub.6-C.sub.10) aryl, (C.sub.3-C.sub.8) cycloalkyl,
(C.sub.2-C.sub.22) alkenyl, (C.sub.5-C.sub.8) cycloalkenyl,
(C.sub.7-C.sub.32) aralkyl, (C.sub.7-C.sub.32) alkylaryl,
(C.sub.9-C.sub.32) aralkenyl, (C.sub.9-C.sub.32) alkenylaryl or
C(O)R.sup.3; and R.sup.3 is (C.sub.1-C.sub.25) alkyl,
(C.sub.6-C.sub.10) aryl, (C.sub.3-C.sub.8) cycloalkyl,
(C.sub.2-C.sub.22) alkenyl, (C.sub.5-C.sub.8) cycloalkenyl,
(C.sub.7-C.sub.32) aralkyl, (C.sub.7-C.sub.32) alkylaryl,
(C.sub.9-C.sub.32) aralkenyl or (C.sub.9-C.sub.12) alkenylaryl;
R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group;
or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically
acceptable salt thereof.
[0014] It is a more specific object of the invention to treat
cancer by the administration of at least one compound of the
formulae (I) or (II), ##STR3## wherein X is O or CH.sub.2; R.sup.1
and R.sup.2 are individually. (C.sub.1-C.sub.25) alkyl,
(C.sub.6-C.sub.10) aryl, (C.sub.3-C.sub.8) cycloalkyl,
(C.sub.2-C.sub.22) alkenyl, (C.sub.5-C.sub.8) cycloalkenyl,
(C.sub.7-C.sub.32) aralkyl, (C.sub.7-C.sub.32) alkylaryl
(C.sub.9-C.sub.32) aralkenyl, (C.sub.9-C.sub.32) alkenylaryl or
C(O)R.sup.3; and R.sup.3 is (C.sub.1-C.sub.25) alkyl,
(C.sub.6-C.sub.10) aryl, (C.sub.3-C.sub.8) cycloalkyl,
(C.sub.2-C.sub.22) alkenyl, (C.sub.5-C.sub.8) cycloalkenyl,
(C.sub.7-C.sub.32) aralkyl (C.sub.7-C.sub.32) alkylaryl
(C.sub.9-C.sub.32) aralkenyl or (C.sub.9-C.sub.32) alklenylaryl,
with the proviso that when X is O, R.sup.3 is not (C.sub.16) alkyl;
R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group,
or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically
acceptable salt thereof; and ##STR4## wherein X is O or CH.sub.2;
R.sup.1 and R.sup.2 are individually, (C.sub.1-C.sub.25) alkyl,
(C.sub.6-C.sub.10) aryl, (C.sub.3-C.sub.8) cycloalkyl,
(C.sub.2-C.sub.22) alkenyl, (C.sub.5-C.sub.8) cycloalkenyl,
(C.sub.7-C.sub.32) aralkyl, (C.sub.7-C.sub.32) alkylaryl,
(C.sub.9-C.sub.32) aralkenyl, (C.sub.9-C.sub.32) alkenylaryl or
C(O)R.sup.3; and R.sup.3 is (C.sub.1-C.sub.25) alkyl,
(C.sub.6-C.sub.10) aryl, (C.sub.3-C.sub.8) cycloalkyl,
(C.sub.2-C.sub.22) alkenyl, (C.sub.5-C.sub.8) cycloalkenyl,
(C.sub.7-C.sub.32) aralkyl, (C.sub.7-C.sub.32) alkylaryl,
(C.sub.9-C.sub.32) aralkenyl or (C.sub.9-C.sub.32) alkenylaryl;
R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group;
or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically
acceptable salt thereof.
[0015] It is another object of the invention to provide
pharmaceutical compositions comprising at least one novel compound
that inhibits the phosphatidylinositol signaling pathway, and more
preferably a compound that antagonizes myo-inositol metabolites
produced by PtdIns-3-Kinase.
[0016] It is a more specific object of the invention to provide
pharmaceutical compositions that comprise at least one compound
having the formulae (I) or (II): ##STR5##
[0017] wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are
individually, (C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl,
(C.sub.9-C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is
(C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl or
(C.sub.9-C.sub.32) alkenylaryl, with the proviso that when X is O,
R.sup.3 is not (C.sub.15) alkyl; R.sup.4 and R.sup.5 are
individually hydrogen or a phosphate group; or when R.sup.4 or
R.sup.5 is not hydrogen, a pharmaceutically acceptable salt
thereof; or
[0018] wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are
individually, (C19-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3 C.sub.8) ##STR6## cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl,
(C.sub.9-C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is
(C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl or
(C.sub.9-C.sub.32) alkenylaryl; R.sup.4 and R.sup.5 are
individually hydrogen or a phosphate group; or when R.sup.4 or
R.sup.5 is not hydrogen, a pharmaceutically acceptable salt
thereof; which inhibit the phosphatidylinositol signaling pathway
and thereby inhibit cell proliferation and/or differentiation
and/or promote apoptosis.
[0019] It is another object of the invention to provide novel
therapies based on inhibiting in vivo the phosphatidylinositol
signaling pathway.
[0020] It is a more specific object of the invention to provide
novel therapies that result in the inhibition of cell proliferation
and/or differentiation and/or the promotion of cell apoptosis
comprising the administration of a compound that antagonizes
myo-inositol cell growth signaling.
[0021] It is an even more specific object of the invention to
provide novel therapies that result in the inhibition of cell
proliferation and/or differentiation and/or promotion of cell
apoptosis by the administration of a compound having formulae (I)
or (II): ##STR7## wherein X is O or CH.sub.2; R.sup.1 and R.sup.2
are individually, (C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10)
aryl, (C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl,
(C.sub.9-C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is
(C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.328) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) ##STR8## aralkenyl
or (C.sub.9-C.sub.32) alkenylaryl, with the proviso that when X is
O, R.sup.3 is not (C.sub.15) alkyl; R.sup.4 and R.sup.5 are
individually hydrogen or a phosphate group; or when R.sup.4 or
R.sup.5 is not hydrogen, a pharmaceutically acceptable salt
thereof; or wherein X is O or CH.sub.2; R.sup.1and R.sup.2 are
individually, (C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl,
(C.sub.9-C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is
(C.sub.1-C.sub.25) alkyl, (C.sub.6 -C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl or
(C.sub.9-C.sub.32) alkenylaryl; R.sup.4 and R.sup.5 are
individually hydrogen or a phosphate group, or when R.sup.4 or
R.sup.5 is not hydrogen, a pharmaceutically acceptable salt
thereof.
[0022] In a preferred embodiment, such therapies will comprise
treatment of cancer and other neoplastic conditions and/or will
comprise treatment of arthritis, inflamation or modulation of
platelet aggregation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a schematic diagram of the signaling by
PtdIns-3-kinase leading to cancer cell proliferation;
[0024] FIG. 2 is a schematic diagram of phosphatidylinositol
(PtdIns),
[0025] FIG. 3 shows the structure of
3-deoxy-phosphatidyl-myo-inositol (30) (DPI);
[0026] FIG. 4 shows the generic structure of phosphonate analogs of
DPI (30);
[0027] FIG. 5 is a schematic diagram of the synthesis of the
3-deoxy-phosphatidyl-myo-inositol phosphonate analog (50);
[0028] FIG. 6 shows a generic structure of D-3-deoxyPtdIns ether
lipid analogs designed according to the invention;
[0029] FIG. 7 is a schematic diagram of the synthesis of OMDPI
(60);
[0030] FIG. 8 illustrates the synthesis of
1-O-octadecyl-2-O-Me-sn-glycerol (84);
[0031] FIG. 9 is a schematic outline of the synthesis of the
phosphonate analog (90) of OMDPI;
[0032] FIG. 10 shows the results for PH Domain inhibition; and
[0033] FIGS. 11 and 12 show the cell growth inhibiting ability of
DPI (30) and OMDPI (60), respectively.
[0034] DETAILED DESCRIPTION OF THE INVENTION
[0035] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
[0036] The present invention includes novel compounds which are
rationally designed to inhibit cell growth. Rational design of the
novel compounds of the present invention includes identifying a
mechanism associated with cell growth. Information relating to the
mechanism is then analyzed such that compound structures having
possible activity in interfering with such mechanism are
formulated. In particular, novel structures are synthesized based
on "building blocks", wherein each building block has a feature
potentially capable of interfering with a particular mechanism
associated with cell growth. Compounds having different building
block combinations are then synthesized and their activity in
relation to the identified mechanism tested. Such tests are
conducted in vitro and/or in vivo. The information obtained through
such tests is then incorporated in a new cycle of rational drug
design. The design-synthesis-testing cycle is repeated until a lead
compound having the desired properties is identified. The lead
compound is then clinically tested.
Identification of a Mechanism Associated with Cell Growth
[0037] Growth factors and certain oncogenes activate a range of
phospholipid-mediated signal transduction pathways associated with
cell proliferation. Phosphatidyl myoinositol (PI) occupies a unique
position in that it can undergo reversible phosphorylation at
multiple sites to generate five different phosphoinositides. PI
metabolites regulate two pathways important for cell proliferation,
the inositol phosphate/diacylglycerol signaling pathway and the
phosphate/diacylglycerol 3-phosphate (PI-3-kinase) pathway.
[0038] In the first pathway, PI specific phospholipase C (PI-PLC)
hydrolyses a minor membrane phospholipid, PI(4,5)P.sub.2 to give
the water soluble Ins(1,4,5)P.sub.3, and a lipophilic
diacylglycerol (DAG). Ins(1,4,5)P.sub.3 interacts specifically with
membrane receptors to release Ca.sup.2+, a key event in cellular
signal transduction. DAG is an endogenous activator of protein
kinase C (PKC). Ins(1,4,5)P.sub.3 is metabolized either by
hydrolysis of the phosphate at position 5, giving Ins(1,4)P.sub.2
or phosphorylation at position 3 giving Ins(1,3,4,5)P.sub.4.
Ins(1,4)P.sub.2 is not active as a Ca.sup.2+ mobilizing agent and
is subsequently degraded by other phosphatases. However, it has
been suggested that Ins(1,3,4,5)P.sub.4 may play a role in
refilling the intracellular Ca.sup.2+ stores with extracellular
Ca.sup.2+. Together, the increase in Ca.sup.2+ concentration and
the increased activity of PKC lead to a sequence of events that
culminate in DNA synthesis and cell proliferation.
[0039] In the second pathway, PI-3-kinase has been found to be
associated with almost every growth factor receptor or oncogene
transformation. PI-3-kinase phosphorylates PI at position 3 of the
myo-inositol ring to give a class of PIs that are poor substrates
for hydrolyses by PI-PLC, e.g., PI(3,4)P.sub.2 and
PI(3,4,5)P.sub.3.
[0040] PtdIns-3-kinases are a family of enzymes that phosphorylate
the D-3-OH position of the myo-inositol ring of the minor cell
membrane phospholipid phosphatidylinositol (PtdIns). The most
studied member of the PtdIns-3-kinase family is a heterodimer
consisting of an 85 kDa regulatory subunit (p85) and a 110 kDa
catalytic subunit (p110). All of the known isoforms of p110 are
capable of phosphorylating both PtdIns and PtdIns(4)P in vitro,
however, PtdIns (4,5)P.sub.2 is the preferred substrate in vivo.
PtdIns-3-kinase is activated by a wide range of growth factor
receptor and oncogene proteintyrosine kinases as well as by
p21.sup.Ras.
[0041] The exact mechanism by which 3-phosphorylated PIs modulate
cell growth is not known but they appear to be important modulators
of protein interaction and enzyme activity through binding to
specific sites on proteins. For example, binding of PI(3,4)P.sub.2
PI(4,5)P.sub.2 or PI(3,4,5)P.sub.3 to pleckstrin-homology (PH)
domains on enzymes such as Akt (protein kinase B) leads to enzyme
activation, whereas the Src-homology-2 (SH2) domain that mediates
protein tyrosine phosphate binding binds specifically
PI(3,4,5)P.sub.3. PtdIns-3-kinase is activated by binding of the
src-homology 2 (SH2) domain of the p85 regulatory subunit of
PtdIns-3-kinase to phosphorylated tyrosine residues on activated
growth factor receptors and oncogenic protein tyrosine kinases
which cause a conformational change at the active site of the p110
catalytic subunit and brings PtdIns-3-kinase from the cytoplasm to
the inner surface of the plasma membrane where PtdIns substrates
are located. Also, PtdIns-3-kinase itself becomes tyrosine
phosphorylated, however, this phosphorylation apparently does not
result in any increased activity of the enzyme.
[0042] FIG. 1 depicts schematically the means by which
PtdIns-3-kinase signaling is theorized to enhance cancer cell
proliferation. Essentially, activation of PtdIns-3-kinase (PI-3-K)
leads to the formation of PtdIns-3-phosphates which bind to the PH
domains of enzymes such as Akt, PtdIns PLC-.gamma. and activation
of PKC-.zeta..
[0043] Several lines of evidence suggest an essential role for
PtdIns-3-kinase in the modulation of cancer cell growth and the
cancer phenotype. For example, cells transfected with a mutant PDGF
receptor that retains protein tyrosine kinase activity, but which
do not associate with or activate PtdIns-3-kinase, fail to show a
mitogenic response to PDGF, unlike cells transfected with the
wild-type PDGF receptor ("Role of phosphatidylinositol kinase in
PDGF receptor signal transduction"; Coughlin et al.; Science,
243:1191-1194 (1989)). Also it has been reported that a mutant
CSF-1 receptor which contains a kinase-insert deletion results in
significantly reduced association with PtdIns-3-kinase. Moreover,
this mutant receptor is only capable of conferring CSF-1-dependent
transformation to some cells and has lost the ability to transform
other cells ("Phosphatidylinositol-3-kinase is necessary for
12-O-tetradecanoylphorbol-13-acetate-induced cell transformation
and activated protein 1 activation"; Huang et al.; J. Biol. Chem.,
272:4187-4194 (1997)).
[0044] Further, it has been reported that active PtdIns-3-kinase is
necessary for phorbol ester mediated transformation of cells. In
particular, it has been reported that polyoma middle T mutants
which associate with and activate pp60.sup.c-src tyrosine kinase,
but which fail to activate PtdIns-3-kinase are nontransforming
("Common elements in growth factor stimulation and oncogenic
transformation: 85 kd phosphoprotein and phosphatidylinositol
kinase activity"; Kaplan et al.; Cell, 50:1021-1029 (1987)) It is
further known that the levels of cellular PtdIns-3-phosphates are
elevated in transforming mutants of middle T but not by
transformation of defective mutants, suggesting that these
compounds play a significant role in transformation.
[0045] It is also known that PtdIns-3-kinase prevents apoptosis and
is necessary or the inhibition of apoptosis caused by nerve growth
factor in PC 12 phemochromocytoma cells ("Requirement for
phosphatidylinositol-3-kinase in the prevention of apoptosis by
nerve growth factor"; Yao et al.; Science, 267:2003-2006 (1995))
and by IL-3 and IL-4 in 5 hematopoietic cells ("Signaling through
the lipid products of phosphoinositide-3-OH kinase"; Toker et al.;
Nature, 387:673-676 (1997)).
[0046] Based on the foregoing, PtdIns-3-kinase has generated
considerable interest as a target for the development of anticancer
drugs to block the activity of increased growth factor signaling or
oncogene expression. More particularly, based on what has been
reported about this enzyme disease conditions that potentially
would be susceptible to growth inhibition by PtdIns-3-kinase
inhibitors include cancers that over-express PDGF receptors such as
colon, pancreatic, prostate and head and neck tumors, and tumors
overexpressing EGE receptor such as breast, gastric and prostate
tumors. Also, tumors expressing mutant ras such as colon and
pancreatic cancer and CML which is characterized by a Bcr/Abl
(Philadelphia chromosome) translocation (where Bcr/Abl has been
shown to require PtdIns-3-kinase for its effects) may also be
amenable to treatment by compounds that affect PtdIns-3-kinase
activity.
[0047] Essentially, because of the important role PtdIns-3-kinase
apparently plays in effecting cell growth, it provides an exciting
avenue for designing therapeutic protocols based on controlling
PtdIns-3-kinase activity. More specifically, compounds which
mediate PtdIns-3-kinase activity potentially may be used to control
(inhibit) tumor cell growth.
[0048] A direct approach for modulating PtdIns-3-kinase and the
biological pathways it affects is to design therapeutic protocols
based on compounds having PtdIns-3-kinase inhibitory activity.
Supplying such compounds to target cells potentially should reduce
or block cell proliferation attributable to the inhibition of
PtdIns-3-kinase.
[0049] An example thereof is the fungal metabolite wortmannin,
which is an irreversible inhibitor of p110 PtdIns-3 -kinase (having
an IC.sub.50 of 4 nM) ("Wortmannin inactivates phosphoinositide
3-kinase by covalent modification of Lys-802, a residue involved in
the phosphate transfer reaction"; Wymann et al.; Mol. Cell Biol,
16:1722-1733 (1996)). Because of this activity, wortmannin has been
used extensively as a pharmacological probe of the functions of
PtdIns-3-kinase. Based on such inhibitory activity, it was
initially hoped that wortmannin might be a useful anticancer drug
against tumors with activated PtdIns-3-kinase signaling ("In vitro
and in vivo activity of the phosphatidylinositol-3-kinase
inhibitor, wortmannin"; Schultz et al.; Anticancer Res.,
15:1135-1140 (1995).
[0050] Unfortunately, while although wortmannin has shown
anti-tumor activity against a variety of tumors, it lacks target
selectivity and is toxic to normal tissues, particularly the liver
and hematopoietic system. This has precluded its further
therapeutic development. Such lack of selectivity is apparently
attributable to the fact that wortmannin inhibits other
serine/threonine kinases of the PtdIns-3-kinase family, e.g., mTOR
and DNA-dependent protein kinase, with IC.sub.50s of 2 to 4 nM. The
unrelated enzyme phospholipase A2 is also inhibited by wortmannin,
with an IC.sub.50 of 2 nM ("Wortmannin and its structural analogue
demethoxyviridin inhibit stimulated phospholipase A.sub.2 activity
in Swiss 3T3 cells"; Cross et al.; J. Biol. Chem., 270:25352-25355
(1995)).
[0051] The poor selectivity of PtdIns3-kinase inhibition by
wortmannin suggests that the binding site of PtdIns-3-kinase does
not have unique structural features recognizable by this inhibitor.
This, in turn suggests that designing PtdIns-3-kinase inhibitors
having acceptable selectivity requires detailed structural analysis
of the active sites of PtdIns-3kinase and related enzymes whose
activity is indiscriminately inhibited by known inhibitors such as
wortmannin.
[0052] Another potential approach for controlling PtdIns-3-kinase
activity and thereby cell growth which is the focus of the present
invention, is directed to PtdIns-3-kinase metabolites. More
specifically, the subject approach is based on rationally designing
compounds which are antagonists of myo-inositol second messengers
produced by PtdIns-3-kinase which reduce or block cell growth by
antagonizing myo-inositol cell growth signaling. Preferably
antagonists arc designed which reduce or block cell proliferation
while leaving other aspects of myo-inositol signaling unaffected.
The designed antagonists should provide a novel basis for
therapeutic protocols based on the selective control of cancer cell
growth signaling which do not disrupt the function of normal
cells.
Metabolic Products of, Phosphatidylinositol-3-kinase
[0053] The products of PtdIns-3-kinase, i.e., PtdIns-3-phosphates,
are responsible for the effects of PtdIns-3-kinase on tumor growth
and apoptosis. Only recently has their mechanism of action begun to
be understood. PtdIns-3-phosphates are found in the cell as small
amounts of PtdIns-3-phosphate and larger amounts of
PtdIns(3,4)P.sub.2 and PtdIns(3,4,5)P.sub.3 ("Phosphoinositide
3-kinase is activated by phosphopeptides that bind to the SH2
domains of 84-kDa subunit"; Carpenter et al.; J. Biol. Chem.
268:9478-9483 (1993)). PtdIns-3-phosphates have the unique ability
to bind to specific protein domains, a property not shared by
non-3-phosphorylated PtdIns, resulting in the activation of key
signaling proteins involved in cell growth and death. The
pleckstrin homology (PH) domain is a protein module of
approximately 120 amino acids found in a number of signaling
proteins activated by PtdIns-3-phosphate binding, The PH domain of
these proteins hinds specifically to PtdIns-3-phosphates present in
the inner plasma membrane resulting in the translocation of the
signaling proteins from the cytosol to the plasma membrane where
their substrates are located. Binding of PtdIns-3-phosphates to PH
domains may also result in a direct increase in the catalytic
activity of the enzyme ("PH domains: diverse sequences with a
common fold recruit signaling molecules to the cell surfaces";
Lemmon et al.; Cell, 85:621-624 (1996)).
[0054] The most extensively studied examples of PH domain-regulated
signaling are the PH domain dependent activation by
PtdIns(3,4)P.sub.2 and PtdIns(3,4,5)P.sub.3 of the serine/threonine
kinase Akt (PKB/Rac) and of PtdIns-PLC.gamma.. Binding of the PH
domain to membrane PtdIns-3-phosphates causes the translocation of
Akt to the plasma membrane bringing it into contact with membrane
bound Akt kinase, which is itself activated by
PtdIns(3.4,5)P.sub.3, which then phosphorylates and activates Akt
("Characterization of a 3-phosphoinositide-dependent protein kinase
which phosphorylates and activates protein kinase Ba"; Alessi et
al., Curr. BioL, 7:261-269 (1997)), ("Dual role of
Phosphatidylinositol 3,4,5-triphosphate in the activation of
protein kinase B"; Stokoe et al., Science, 277:567-570 (1997)). Akt
is a proto-oncogene that inhibits apoptosis by phosphorylating Bad,
thus, promoting its binding to, and blocking the activity of the
cell survival factor Bcl-x ("A bad kinase makes good"; Franke et
al.; Nature, 390:116-124 (1997)).
[0055] Accordingly, the inhibition of Akt activation potentiates
cancer cell apoptosis. Translocation of PtdIns-PLC.gamma. to the
plasma membrane brings it into contact with its substrate
PtdIns(4,5)P.sub.2 resulting in more efficient hydrolysis to
Ins(1,4,5)P.sub.3 and diacylglycerol. The binding of
PtdIns(3,4,5)P.sub.3 to the SH2 domain of PtdIns-PLC.gamma., as
well as the better recognized SH2 binding to tyrosine phosphate
residues on autophosphorylated growth factor receptors, provides
additional mechanisms for translocating PtdIns-PLC.gamma. to the
plasma membrane (Id.). An increase in intracellular free Ca.sup.2+
caused by the release of intracellular stores of Ca.sup.2+ by
Ins(1,4,5)P.sub.3 together with the activation of protein kinase C
by diacylglycerol leads to a series of events that culminate in
increased cell proliferation. PKC-.zeta. is also directly activated
by PtdIns(3,4,5)P.sub.3 (Activation of zeta isozyme of protein
kinase C by phosphatidylinositol 3,4,5-triphosphate; Nakanishi et
al.: J. Biol. Chem., 268:13-16 (1993)). Thus, an increase in
PtdIns-3-phosphates in the cell membrane results in the activation
of two different pathways, one leading to increased cell
proliferation, the other to inhibition of cell death. These
separate pathways explain the growth stimulating and transformation
related effects of PtdIns-3-kinase.
Design of PtdIns-3-Kinase Anti-Metabolites
[0056] As disclosed supra, the focus of the subject invention is to
produce by rational methods antagonists of PtdIns-3-kinase
metabolites. In order do so, the present inventors have elected to
rationally designs antagonists of PtdIns-3-kinase metabolites such
as PtdIns-3-phosphates, by utilizing the structure of the
PtdIns-3-kinase substrate as a starting structure for modification.
In particular, different modifications to this starting structure
are judiciously selected and the effects thereof on activity
evaluated, so that ideally an effective antagonist is produced. One
method for obtaining effective antagonists is to maintain a high
structural similarity between the antagonist and the substrate.
That is, the modification is based on a balance between the new
features providing the desired antagonistic effect and maintaining
sufficient structural similarity such that metabolites are not
produced. These antagonists will advantageously be sufficiently
similar in structure to the metabolites such that they effectively
interfere with the processing of the metabolites in the signaling
cycle, down stream of the PtdIns-3-kinase step. Effective
antagonists should have sufficient structural similarity to these
metabolites so that they effectively compete with the metabolites
for interaction with sites available for the step following
phosphorylation by PtdIns-3-kinase, while at the same time being
unaffected by this interaction. This should block the signaling
cycle both upstream and downstream of the PtdIns-3-kinase mediated
phosphorylation.
[0057] The starting structure of the modified metabolite
phosphatidylinositol (PtdIns) is contained in FIG. 2. As explained,
PtdIns 20 is utilized as the starting structure for designing
PtdIns-3-kinase cell growth signaling antagonists. More
specifically, the present inventors elected to focus on three
specific sites in the PtdIns structure as candidates to be modified
in order to obtain analogs of PtdIns-3-kinase metabolites which
function as effective antagonists. Ideally, such antagonists will
exhibit the desired pharmaceutical properties in vivo and will
selectively antagonize myo-inositol metabolites produced by
PtdIns-3-kinase while not disrupting other cell signaling pathways,
in particular of normal cells. The rationale for selecting these
specific modifications is based on the present inventors' analysis
and understanding of the chemistry associated with cell growth
signaling.
A) The 3 Position of the myo Inositol Ring
[0058] The first position selected for modification was the
3position of the myo-inositol ring. This was selected because
analogs of myo-inositol in which the 3-hydroxyl group is removed or
replaced can not be phosphorylated by PtdIns-3-kinase and appear to
act as inhibitors of PtdIns-3-kinase signaling.
D-3-deoxy-substituted myo-inositols are taken up by the
myo-inositol transporter of cells and incorporated into cellular
PtdIns by PtdIns synthetase leading to the selective growth
inhibition of (some) transformed relative to normal cells. However,
the affinity of the D-3-deoxy-substituted myo-inositols for uptake
and PtdIns synthesis is less than that of myo-inositol itself and
at physiological concentrations myo-inositol inhibits their growth
inhibitor activity. D-3-deoxysubstituted PtdIns inhibits the growth
of cancer cells in the presence of myo-inositol. In fact,
D-3-deoxy-PtdIns and a more active analog have been reported to
exhibit anti-tumor activity against human tumor xenografts in SCID
mice ("Synthesis and Biology of 1D-3-Deoxyphosphatidylinisitol: A
Putative Anti-metabolite of phosphatidylinositol-3-phosphate and an
Inhibitor of Cancer Cell Colony Formation", Kozikowski, A. P. et
al., J. of Medicinal Chem. Vol. 38, 7:1053-1056 (1995)), the
contents of which are hereby incorporated by reference. Also,
treatment of NIH 3T3 cells with 1D-3-deoxy-PtdIns blocks the
activation of Akt due to inhibition of PH domain binding. Moreover,
D-3deoxy-myo-inositols kill cells by inducing apoptosis which is
consistent with the role of PtdIns-3-kinase and Akt in preventing
apoptosis (Id.).
[0059] The position for a first modification, site 21, corresponds
to the 3 position on the inositol ring. As discussed above,
PtdIns-3-kinase phosphorylates the D-3-OH position of the myo
inositol ring. Modifying the 3 position of the inositol ring to
preclude phosphorylation should interrupt the PtdIns-3-kinase
signaling cycle. Precluding phosphorylation by PtdIns-3-kinase is
achieved by removing the oxy group at the 3 position of the
inositol ring.
[0060] The resultant modified structures
3-deoxy-phosphatidyl-myo-inositol 30 (DPI) is contained in FIG. 3.
In fact, DPI is recalcitrant to phosphorylation by PtdIns-3-kinase
and therefore possesses cell growth inhibiting activity. Assays of
the biological activity of DPI show that the compound inhibits
colony formation by HT-29 human colon carcinoma cells. DPI exhibits
all IC.sub.50 of 35 .mu.M (Id.).
[0061] Also, it has been demonstrated by the inventors that the 3
position of the myo-inositol ring can be modified to include
non-phosphorylable substituents. In particular, the 3 position
hydrogen atom in DPI can be substituted by a halogen, such as
fluorine or chlorine. The synthesis and biological activity of such
substituted DPI analogs is the subject of U.S. Pat. No. 5,227,508,
the contents of which are hereby incorporated in their entirety.
For example, it has been shown that the PtdIns analog bearing a
fluorine atom in place of the 3-hydroxy group inhibits colony
formation by HT-29 human colon carcinoma cells with an IC.sub.50 of
37 .mu.M.
B) The DAG sn-3 Oxygen Position
[0062] A second site selected by the inventors for the rational
drug design of PtdIns antagonists is the sn-3 oxygen of the DAG.
This was chosen because in the PtdIns signaling cycle,
PtdIns-3-kinase metabolites are hydrolyzed by PI-PLC at the sn-3
oxo position. Therefore, precluding hydrolysis by substituting the
sn-3 oxygen by a non hydrolyzable group should allow the
concentration of PtdIns-3-kinase anti-metabolites to remain at a
high level, thereby inhibiting PtdIns-3-kinase activity.
[0063] More specifically, the present inventors elected to modify
the 3-sn oxygen position of the PtdIns analog, preferably to
preclude hydrolysis by PI-PLC, by replacing the oxygen with a
methylene group (CH.sub.2). This modification was made because it
is hypothesized that maintaining a high concentration of
PtdIns-3-kinase anti-metabolites requires that such metabolites be
present in the environment of PtdIns-3-kinase. Moreover, it is
believed that the low potency of these compounds may be due to
their hydrolysis by phospholipases including PI-PLC. Also, DAG
produced by hydrolysis can activate PKC, which may lead to tumor
cell proliferation. By contrast, the present inventors seek to
obtain novel antagonists which act as PtdIns-3-kinase
anti-metabolites which are not hydrolyzable at the 3-sn oxygen by
PI-PLC. These antagonists are designed based on a double
modification of the starting structure. In particular, both the 3
position 21 of the myo-inositol ring and the sn-3 oxygen position
23 of the DAG were modified.
[0064] FIG. 4 shows the generic structure of phosphonate analogs of
DPI. Also, the synthesis of the 3 -deoxy-phosphatidyl-myo-inositol
phosphonate analog
1-O-[(3S)-3,4-bis(palmitoyloxy)butylphosphonyl]-1D-3-deoxy-myo-ino-
sitol (50) is schematically outlined in FIG. 5. For the synthesis
of the phosphonate analog (50), the dichloride (48) is prepared
from (S)-3,4-bis(palmitolyloxy)butyl-hosphonic acid (47) with
oxalyl chloride in the presence of a catalytic amount of DMF at
room temperature. The inositol component,
ID-2,4,5,6-tetra-O-benzyl-3-deoxy-myo-inositol (49) is obtained as
reported before. Phosphorylation of (49) with (48) in the presence
of a base provides monoesterchloride intermediate which is
transformed into (40) by hydrolysis, a reaction which proceeds in a
surprisingly sluggish manner, After purification by preparative
TLC, catalytic hydrogenation of (40) using Pd(OH).sub.2/C in
tert-butanol provides the target
phosphonatel-O-[(3S)-3,4-bis(palmitoyloxy)butyl-phosphonyl]-1D-3-deoxy-my-
o-inositol (50) in good yield.
C) The Diacvlglycerol Position
[0065] A third site of interest for rational drug design of PtdIns
antagonists selected by the inventors was the diacylglycerol at
position 25 (the lipid ester moiety in the DAG). The diacylglycerol
position was selected for modification in order to potentially
enhance the PtdIns-3-kinase anti-metabolite properties of the
compounds designed according to the invention.
[0066] Specifically, rational modification of the diacylglycerol
ester lipid at position 25 was effected by substituting the
diacylglycerol group with a lipid moiety of a compound having known
PtdIns-3-kinase inhibition properties and/or anti-tumor properties.
It is noted that diacylglycerol, which is an endogenous activator
of PKC and tumor cell growth, and which is liberated upon PtdIns
analog hydrolysis, has antagonizing effects against the inhibition
of PtdIns-3-kinase signaling. By contrast, the lipid moiety which
was incorporated in the designed compounds lacks the antagonist
effects of diacylglycerol against PtdIns-3-kinase signaling
inhibition.
[0067] Thus, novel compounds were designed potentially to increase
the potency of D-3 deoxyPtdIns and to reduce the possibility of
unwanted side effects stemming from the metabolic production of
diacylglycerol.
[0068] FIG. 6 shows a generic structure of D-3-deoxyPtdIns ether
lipid analogs designed according to the invention. Design of
3-deoxy-PI ether lipid analogs is of especial interest because of
the potential enhanced stability of these compounds to
phospholipases. This will potentially occur because the 3-deoxy-PI
ether lipid should not function as a substrate for PI-PLC. A
further advantage of the ether lipids is that they have previously
been shown to possess intrinsic anti-tumor activity against a
variety of tumor types. In fact, some ether lipid analogs which
have undergone clinical testing as anti-tumor agents are inhibitors
of PI-3-kinase. They affect several aspects of lipid intracellular
signaling, and their anti-tumor activity may arise, from a
combination of effects on the signaling pathway. In this regard,
1-O-octadecyl-2-O-methylglycero-phosphocholine (edelfosfine) and a
number of related compounds are known inhibitors of PI-PLC with
IC.sub.50s in the low .mu.M range.
[0069] In particular, the compound
1-O-(2-O-methyl-1-O-octadecyl-sn-glycero-3-phospho)1D-3-deoxy-myo-inosito-
l (OMDPI) was synthesized by modifying DPI to replace the
dipalmitoylglycerol group with
1-O-octadecyl-2-O-methyl-sn-glycerol. The synthesis of OMDPI (60)
is schematically outlined in FIG. 7.
[0070] The starting material for the D-3-deoxy-PtdIns ether lipid
analog is the regioisomeric mixture of viburnitol (i.e.,
3-deoxy-myo-inositol) 1,2:4,5- and 1,2:5,6-diacetonides (62), (63),
obtained from L-quebrachitol. Controlled acidic hydrolysis of the
more labile trans acetonide moieties in this mixture provides
monoacetonide (64) in 79% yield. All of the three required O-benzyl
groups are then introduced simultaneously with benzyl bromide and
NaH in DMF (74% yield), and the remaining cis-acetonide gas removed
by acidic hydrolysis (96% yield). The resulting diol (66) is
protected selectively at the equatorial 1-hydroxyl by reacting its
cyclic dibutylstannylene derivative with chloromethyl methyl ether.
Following benzylation of the 2-hydroxyl (73% yield) and acidic
hydrolysis of the MOM ether (77% yield) resulted in the formation
of the key intermediate, 2,4,S,6-tetra-O-benzylburnitol (69), in
crystalline form.
[0071] Another component for the synthesis of
3-deoxy-phosphatidylinositol ether lipid analog is
1,2disubstituted-sn-glycerol, which is obtained in high yield and
in high enantiomeric excess by carrying out the asymmetric
dihydroxylation of allyl 4-methoxyphenyl ether (80).
1-O-octadecyl-2-O-Me-sn-glycerol (84) is illustrated in FIG. 8 as
an example. Selective monoalkylation with 1 bromooctadecane is
achieved via the 1-2-O-stannylene intermediate, and the resulting
secondary alcohol (82) is then methylated to provide (83). This
particular strategy allows facile manipulation of the size of the
alkyl side chains, a feature which was found to significantly
affect the solubility of the resultant PtdIns analogs under the
assay conditions. Final removal of the 3-O-(4-methoxyphenyl) group
with ceric ammonium nitrate (CAN) provides the desired glycerol
(84).
[0072] Subsequently phosphitylation of intermediates (69) with
O-benzyl N,N,N'N'-tetraisopropyl-phosphorodiamidite catalyzed by
diisopropylammonium tetrazolide provided the phosphoramidite (75)
in quantitative yield which was then coupled with ether lipid (84)
in the presence of tetrazole. The resulting phosphates were
oxidized to the phosphates (76) with tert-butyl hydroperoxide (74%
yield for 3 steps). Final hydrogenolysis then provides the desired
ether lipid analog
1-O-(2-O-methyl-1-O-octadecyl-sn-glycero-3-phospho)-1D-3-deoxy-myo-inosit-
ol (60) in 96% yield.
[0073] She synthesis of the phosphonate analog
1-O-[(3S)-methoxy-4-(octadecyloxy)butylphosphonyl]-1D-3-deoxy-myo-inosito-
l (90) of OMDPI is schematically outlined in FIG. 9. Methyl
phosphonate (94) underwent an S.sub.N2 reaction with glyceryl
triflate to yield the phosphonate (95). Lastly, hydrogenation of
(95) delivered (90).
Biological Activity
[0074] To confirm the efficacy of the subject analogs in inhibiting
PtdIns-3-Kinase, and in particular the inhibition of cell
proliferation and/or differentiation and/or induction of apoptosis
of cancer cells, the following experiments were controlled. These
experiments were effected in particular to assess the anti-tumor
activity and the binding properties of compounds according to the
invention.
A) Anti-Tumor Activity
[0075] Specifically, anti-tumor activity was assessed by growing
HT-29 colon cancer cells colonies in soft agarose which were then
exposed to compounds according to the invention for 7 days and the
colonies then counted. Values were expressed as the IC.sub.50 for
inhibition of colony formation and are the mean of 3 determinations
.+-.S.E. The results of these experiments are contained in Table 1
below. TABLE-US-00001 TABLE 1 In Vitro Anti-Tumor Activity Compound
Soft Agarose IC.sub.50 (.mu.M) 3-deoxy-PtdIns (30) 35 .+-. 9
3-flouro-PtdIns (35) 37 .+-. 3 2,3-dideoxy-PtdIns 50 .+-. 7
3-deoxy-PtdIns phosphonate (50) 10 .+-. 2 OMDPI (60) 2.1 .+-. 0.1
OMDPI phosphonate (90) 45 .+-. 7
[0076] As shown in Table 1,3-deoxy-PtdIns (30) and
3-Fluoro-3-deoxy-PtdIns (35) inhibited colony formation of HT-29
human colon carcinoma cells with IC.sub.50 values of 35 and 37
.mu.M, respectively, while 3-chloro-3-deoxy-PtdIns (not shown) was
virtually inactive (<20% growth inhibition at maximum tested
concentration). The ether lipid analog OMDPI (60) was found to be
15-fold more active in its growth inhibitory activity (2 .mu.M)
compared to DPI. Replacement of the phosphate group of DPI by a
phosphonate was found to increase the growth inhibition by over a
3-fold (IC.sub.50 for (50) is 10 .mu.M). However, the same
modification decreased the activity of OMDPI (IC.sub.50 for (90) is
45 .mu.M).
[0077] These results indicate that replacement of the
diacylglycerol moiety with an ether 20 lipid group resulted in an
over 15-fold increase in growth inhibition activity (compare
compounds (30) and (60)). Replacement of phosphate by phosphonate
increased the growth inhibiting activity of 3-deoxy-PI by almost
3-fold (compare compounds (30) and (50)). However, it decreased the
growth inhibiting activity of the 3-deoxy ether lipid analog
(compare (60) and (90)).
[0078] Based on the observation that replacement of the
diacylglycerol moiety of D-3-deoxy-PtdIns with ether lipid provided
over 15-fold increase in in vitro growth inhibitory potency against
HT-29 tumor cells, the activity of
1-O-octadecyl-2-O-methyl-sn-glycero3-phospho-myo-inositol (that is
the ether lipid analog of PtdIns with a myo-inositol group instead
of a D-3-deoxymyo-inositol group) was then tested.
1-O-octadecyl-2-O-methyl-sn-glycero-3-phospho-myo-inositol was
found to be a poor inhibitor of PtdIns-PLC and only a weak
inhibitor of cancer cell growth. Based on these results, it appears
that the increase in anti-tumor activity of OMDPI is not solely
attributable to the incorporation of the ether lipid moiety.
Rather, the enhanced anti-tumor activity of OMDPI apparently is the
synergistic result of both the modification of the 3 position of
the myo-inositol ring 21 and the diacylglycero position 25.
B) PH Domain Inhibition
[0079] PtdIns-3-phosphates bind to and activate PH domain
containing enzymes. Accordingly, the ability of the
D-3-deoxy-PtdIns analogs to inhibit the activation of the PH domain
dependent enzyme Akt was investigated. NIH 3T3 cells were
transiently transfected with human Akt with a hemagglutinin (HA)
epitope tag. The cells were exposed to the D-3-deoxy-PtdIns analogs
for 6 hours and stimulated with PDGF to activate Akt. The Akt was
immunoprecipitated with anti-HA antibody and its ability to
phosphorylate histone-H2B measured using [.gamma..sup.32P]-ATP.
Histone H2B was separated by SDS PAGE and bands on the gel
quantitated using a phosphorimager. The results are shown in FIG.
10. PDGF resulted in a marked increase in Akt activity in the cells
and both D-3-deoxy-PtdIns and D-3-deoxy-PtdIns ether lipid blocked
Akt activation. Wortmannin, an inhibitor of PtdIns-3-kinase,
blocked Akt activation as expected.
[0080] Also, the in vitro inhibition of bovine PI-PLC and of bovine
brain p110/p85 PI-3-kinase were measured as previously described
("In vitro and in vivo activity of the
phosphatidylinositol-3-kinase inhibitor, wortmannin"; Schultz et
al.; Anticancer Res., 15:1135-1140 (1995)). The results of this
binding experiment are summarized in Table 2: TABLE-US-00002 TABLE
2 PH Domain Inhibition PtdIns-PLC PI-3-K IC50 W) IC50 (gM) (30) N/A
>250 (35) 8 30 (50) N/A N/A (60) 19.9 2.5 (90) 10 5.3 N/A = not
active, with <20% inhibition at 100 gM.
[0081] The results show that all the compounds are only weak
inhibitors of PI-PLC compared to 1-O-octadecyl-2-O-methyl
glycerophosphocholine which has an IC.sub.50 under the same assay
conditions of around 1 .mu.M. However, the 3-deoxy ether lipid PIs
are relatively potent inhibitors of PI-3-kinase with IC.sub.50
values of 2 to 5 .mu.M.
1-O-octadecyl-2-O-methylglycerophosphocholine has previously been
found to be an inhibitor of PI-3kinase with an IC.sub.50 of 35
.mu.M, while the myo-inositol containing analog is a much weaker
inhibitor with an IC.sub.50 of 90 .mu.M. Thus, the presence of a
3-deoxy-myo-inositol moiety appears to impart PI-3-kinase
inhibiting activity to the compounds.
[0082] Based on the unexpectedly low anti-tumor and PI-3-kinase
inhibition ID.sub.50 values obtained with the lead compound OMDPI,
in vivo and human clinical tests are being designed to establish
anticancer protocols based on OMDPI.
[0083] Example 1 illustrates the synthesis of compounds (50), (60)
and (90). Example 2 shows in vivo activity and toxicity studies
comparing OMDPI (60) and DPI (30) as anti-tumor agents.
EXAMPLE 1
Preparation of Compound 50:
[0084] To a suspension of 110 mg (170 gmol) of (47) in 2 mL of
CH.sub.2Cl.sub.2, and 5 .mu.L of DMF under N.sub.2, was added 119
.mu.L (1.36 mmol) of oxalyl chloride. The white solid disappeared
in five minutes, the resulting solution is then stirred at room
temperature for another 4 hours. After removal of the solvent in
vacuum, the residue (compound (48)) is dried and used directly in
the next step without further purification.
[0085] To a solution of (48) and 89 mg (170 .mu.mol) of (49) in 2
mL of CH.sub.2Cl.sub.2, was added 89 .mu.L (0.51 mmol) of
i-pr.sub.2NEt and 5 mg of DMAP. The resulting mixture was allowed
to stir at room temperature overnight followed by hydrolysis with
water. The product was then extracted with CDCl.sub.3 and dried
over MgS0.sub.4. After concentration, the residue was purified by
preparative TLC developed by CH.sub.2Cl.sub.2/MeOH (v/v 9/1),
giving 105 mg (54%) of (40) as a yellow syrup.
[0086] 99.1 mg of (40) in 11 mL of tert-butanol is hydrogenated
under 5 bar H.sub.2 over 56 mg of 20% Pd(OH).sub.2/C for 12 hours.
After filtration, the filtrate is concentrated and dried in vacuum,
leaving 64.9 mg (95%) of
1-O-[(3S)-3,4-bis(palmitoyloxy)butylphosphonyl]-1D-3-deoxy-myo-inositol
(50) as a white solid.
Preparation of Compound 60:
[0087] A solution containing 1.80 g (1.74 mmol) of (69) in 50 ML of
tert-butanol was hydrogenated in a Parr shaker under 70 psi of
H.sub.2 for 36 h, using 1.0 g of 20% Pd(OH).sub.2/C (Aldrich,
.ltoreq.50% H.sub.2O) as catalyst. The catalyst was filtered out
and the filter cake was washed with 100 nI of MeOH/CHCl.sub.3
(v/v=1/1). The filtrate was concentrated and dried in vacuo leaving
0.98 g (96%) of (60) as a white powder.
Preparation of Compound 90:
[0088] To a solution of 262 mg (0.5 mmol) of (99), 113 mg (0.6
mmol) of ammonium O-benzyl-H-phosphonate, 0.2 mL of pyridine in 2
mL of CH.sub.2Cl.sub.2, was added 74 .mu.L, (0.6 mmol) of pivaloyl
chloride. The mixture was stirred at room temperature for 10
minutes, then diluted with 50 mL of EtOAc. The organic layer was
washed with 10 mL.times.2 saturated aqueous CUS04, dried over
MgS0.sub.4. After evaporation, the residue was purified by column
chromatography on silica gel with EtOAc/hexane 1:1, affording 335
mg (94%) of (93) as a colorless oil.
[0089] Under N.sub.2, to a solution of 170 mg (0.25 mmol) of (93)
and 20 mg (60%, 0.5 mmol) of NaH in 2 mL anhydrous THF, is added 31
.mu.L (0.5 mmol) of MeI. The resulting mixture was allowed to stir
at room temperature overnight and then partitioned in 50 mL of
EtOAc and 5 mL of H.sub.20. The organic layer was washed with brine
and dried over MgS0.sub.4. After evaporations the residue was
purified by column chromatography on silica gel with EtOAc/hexane
2:1, giving 112 mg (65%) of (94) as a colorless oil.
[0090] Under N.sub.2, to a solution of 77 mg (0.11 mmol) of (94) in
2 mL of anhydrous THF at -78.degree. C., is added 56 .mu.L (0.11
mmol, 2.0 N in Hexane) of n-BuLi. After stirring at -78.degree. C.
for 30 min, a solution of triflate in 1 mL of THF was dropped in.
The resulting reaction mixture was warmed to room temperature
slowly and stirred overnight. 1 mL of MeOH was added, and the
reaction mixture was then concentrated. After column chromatography
on silica gel with EtOAc/hexane 1/1, 57.9 mg (51%) of (95) is
obtained as a colorless oil.
[0091] A solution of 49 mg (47 .mu.mol) of (95) in 3 mL of EtOH was
hydrogenated over 25 mg of 20% Pd(OH).sub.2/C under atm. of H.sub.2
at room temperature for 20 hours. After filtration, the filter cake
was washed with 20 mL of CHCl.sub.3/MeOH (v/v=1/1). Evaporation and
drying in vacuo left 25.7 mg (93%) of
1-O-[(3S)-methoxy-4-(octadecyloxy)butylphosphonyl]-1D-3-deoxy-myo-inosito-
l (90) as a white powder.
In Vivo Activity of DPI and OMDPI
[0092] Preliminary studies of in vivo anti-tumor activity were
conducted in SCID (severe combined immunodeficient) mice implanted
subcutaneously wit 10 HT-29 human colon adenocarcinoma cells.
Injection of compounds (30) and (60) was begun 4 days after tumor
inoculation in groups of 8 mice as 4 daily intraperitoneal
injections of the compounds suspended in 3% EtOH, 3% Tween 20, 0.9%
NaCl. Tumor volume was measured with calipers on day 10. As shown
in Table 3, compound (30) was lethal at a 5 daily dose of 500
mg,/kg and exhibited no anti-tumor activity at half this dose.
Compound (60) was not toxic at the highest dose tested of 150 mg/kg
per day and inhibited tumor growth by 67%. However, at doses of 100
and 50 mg/kg per day, the compound did not elicit anti-tumor
activity.
[0093] 10.sup.7 tumor cells were injected s.c. on day 0. Palpable
tumor was detected on day 4 with a mean volume of 0.009 cm.sub.3.
Drugs were injected i.p. suspended in sterile 3% ethanol, 0.1%
Tween 20, and 0.9% NaCl. TABLE-US-00003 TABLE 3 Anti-Tumor Activity
Against Established HT-29 Human Colon Cancer in SCID Tumor
Coumpound volume.sup.b on Dose (mg/kg) Schedule.sup.a day 10
(cm.sup.3) T/C % P.sup.c Control 0.27 .+-. 0.04 (30) 500 i.p., qd
4-5 lethal 250 i.p., qd 4-7 0.30 .+-. 0.06 111.1 NS (60) 150 i.p.,
qd 4-7 0.09 .+-. 0.07 33.3 <0.05 100 i.p., qd 4-7 0.32 .+-. 0.09
118.0 NS 50 i.p., qd 4-7 0.258 .+-. 0.05 103.7 NS .sup.ae.g. 250
mg.kg i.p., qd 4-7 means that the 250 mg/kg does is given as an
intraperitoneal injection each day from days 4 to 7 (four daily
injections) after the tumors are implanted. .sup.bTumor volume
values are the means for 8 mice per group with S.E. .sup.cThe P
column is the significance value for Student's test comparing the
tumor volumes in the treated group tot he tumor volumes in the
control group. 0.05 is usually the maximum value for significance.
NS is `not significant`, meaning that these studies are not
repeated.
[0094] Therefore, based on these results, OMDPI exhibited
significant in vivo anti-tumor 20 activity against both established
human MCF-7 breast cancer and established HT-29 colon tumor
xenografts implanted in SCID mice. OMDPI administered by a 4 or 5
day daily i.p. schedule resulted in a 60% inhibition of the growth
of MCF-7 breast cancer and a 67% inhibition of the growth of HT-29
colon cancer xenografts. The activity of OMDPI administered by a 10
day schedule provides 80% inhibition of the growth of MCF-7 breast
cancer xenografts.
[0095] Also, the relative ability of D-3-deoxy-PtdIns and OMDPI,
when administered i.p. as a daily micellar suspension to inhibit
the growth of MCF-7 human breast cancer xenografts in SCID mice,
was further evaluated. These results are contained in FIGS. 11 and
12. Based on these results, it can be seen that D-3-deoxy-PtdIns
exhibits only moderate anti-tumor activity (T/C 62%, p<0.05).
More specifically, OMDPI exhibited good anti-tumor activity when
administered for 10 days at a dose of 75 mg/kg (T/C 20%, p<0.05)
and slightly less activity when administered for 5 days at 100
mg/kg (T/C 39%, p<0.05). These were the maximum tolerated doses
that could be given by these schedules. Not shown are the effects
of cyclophosphamide 265 mg/kg which was administered on day 5 as a
positive control and which resulted in a 43% inhibition of the
growth of MCF-7 xenografts. The results of these experiments
demonstrate OMDPI has significant antitumor activity (T/C 20%)
against a relatively chemotherapy resistant human tumor xenograft
(MCF-7 breast cancer) when administered over 10 days.
Preliminary Toxicology Tests Comparing DPI (30) and OMDPI (60)
[0096] In order to evaluate clinical safety, toxicity tests were
conducted by administering DPI or OMDPI to groups of 4 male BALB/C
mice daily by i.p. injection for 5 days at the maximum tolerated
dose. These results are contained in Table 4. As shown therein,
OMDPI had no effect on body weight or platelet count and did not
result in elevation of serum liver or kidney enzymes. Moreover,
there was a 33% decrease in total white blood cell count. It should
be noted, however, that this is not sufficient for a cytotoxic drug
to be considered myelosuppresive. Thus, while the cause of death by
OMDPI remains undetermined it is not liver or renal toxic and is
only weakly myelosuppresive.
[0097] As discussed, Table 4 contains the toxicity results obtained
with groups of 4 male BALB/C mice, which were administered the
compounds daily by i.p. injection for 5 days at the doses shown and
killed 24 hr after the last dose. Values are mean .+-.S.E. The p
values are shown only where there was a significant difference to
control. TABLE-US-00004 TABLE 4 Preliminary ToxicoLQU Studies of
DPI and OMDPI in Mice Weight White Total Compound Change % blood
cells Platelets .times. 10.sup.6 BUN Protein ALT Control +6.6 .+-.
1.9 8.7 .+-. 0.7 1.1 .+-. 0.1 20.5 .+-. 1.5 5.4 .+-. 0.2 34.5 .+-.
2.7 deoxy-PtdIns -4.3 .+-. 1.9 7.9 .+-. 0.5 0.9 .+-. 0.1 15.7 .+-.
0.5 4.6 .+-. 0.1 30.5 .+-. 4.6 300 mg/kg p < 0.05 deoxy-PtdIns
+2.3 .+-. 1.5 5.8 .+-. 0.3 0.9 .+-. 0.1 18.7 .+-. 1.0 4.9 .+-. 0.1
32.7 .+-. 2.4 Ether Lipid p < 0.05 p < 0.05 100 mg/kg
[0098] Unlike D-3-deoxy-PtdIns, OMDPI exhibits good aqueous
solubility, i.e., sufficient for a 10 mg/ml solution control in
0.1% Tween 20 to be used for intraperitoneal injection as an
opalescent solution. Therefore, OMDPI should be readily formulated
for intravenous use, should this be determined to be the optimal
route for clinical administration. Also, it is believed that OMDPI
should exhibit good anti-tumor activity and exhibit good
bioavailability if orally administered. In this regard, it was
noted that the ether lipids
1-O-octadecyl-2-O-methylglycero-phosphocholine (edelfosine) and
hexadecylphosphocholine (mitelfosine) have shown clinical
anti-tumor activity when administered orally as well as
parenterally.
[0099] Based on this result,
1-O-(2-O-methyl-1-O-octadecyl-sn-glycero-3-phospho)-1D-3-deoxy-myo-inosit-
ol (OMDPI) is presently the lead compound of the invention for
further development, essentially because of its superior activity
relative to D-3-deoxy-PtdIns and also because of its increased
aqueous solubility.
[0100] However, while OMDPI is currently the lead compound, the
present invention is broadly directed to the identification of
compounds that inhibit PtdIns-3-Kinase signaling. In the preferred
embodiment, such compounds will be synthesized by modification of a
PtdIns-3-Kinase substrate, most preferably
phosphatidylinositol.
[0101] The design of such compounds is discussed above. In an
especially preferred embodiment, these compounds will comprise
3-deoxy-D-myo-inositol analogs having the ##STR9## formula (I):
[0102] wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are
individually, (C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl,
(C.sub.9-C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is
(C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl or
(C.sub.9-C.sub.32) alkenylaryl, with the proviso that when X is O,
R.sup.3 is not (C.sub.15) alkyl; R.sup.4 and R.sup.5 are
individually hydrogen or a phosphate group; or when R.sup.4 or
R.sup.5 is not hydrogen, a pharmaceutically acceptable salt
thereof; or will comprise 3-deoxy-D-myo-inositol analogs having the
formula (II): ##STR10##
[0103] wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are
individually, (C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl,
(C.sub.9-C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is
(C.sub.1-C.sub.25) alkyl, (C.sub.6-C.sub.10) aryl,
(C.sub.3-C.sub.8) cycloalkyl, (C.sub.2-C.sub.22) alkenyl,
(C.sub.5-C.sub.8) cycloalkenyl, (C.sub.7-C.sub.32) aralkyl,
(C.sub.7-C.sub.32) alkylaryl, (C.sub.9-C.sub.32) aralkenyl or
(C.sub.9-C.sub.32) alkenylaryl; R.sup.4 and R.sup.5 are
individually hydrogen or a phosphate group; or when R.sup.4 or
R.sup.5 is not hydrogen, a pharmaceutically acceptable salt
thereof.
[0104] Based on the results obtained with the compounds synthesized
to date, it is anticipated that these compounds will comprise
significant therapeutic application. In particular, these compounds
should inhibit PtdIns-3-Kinase signaling and the biological effects
associated therewith. More specifically, these compounds should
selectively inhibit PtdIns-3-Kinase cell Proliferation and/or
differentiation and/or promote apoptosis especially of cancer and
other neoplastic cells.
[0105] Thus, the compounds produced according to the invention will
be used to treat conditions wherein inhibition of PtdIns-3-Kinase
signaling is therapeutically beneficial. This will include
conditions that involve abnormal cell growth and/or differentiation
such as cancers and other neoplastic conditions. Also, the subject
compounds may be used to treat other conditions involving abnormal
cell proliferation and/or differentiation such as dermatological
conditions and disorders. Also, the subject compounds may be useful
in treating inflammatory conditions such as arthritis, psoriasis,
autoimmune disorders such as myasthenia gravis, lupus, multiple
sclerosis, and others, and conditions involving abnormal platelet
aggregation. The preferred indication is cancer, especially cancers
involving over-expression of EGF and/or the PDGF receptor, cancers
that express mutant ras, or cancers which comprise a Bcr/Abl
translocation. Examples of cancers which may be treated according
to the invention include colon, pancreatic, prostate, head and neck
gastric, renal, brain and CML.
[0106] The subject therapies will comprise administration of at
least one compound according to the invention in an amount
sufficient to elicit a therapeutic response, e.g., inhibition of
tumor cell proliferation and/or differentiation and/or promotion of
apoptosis.
[0107] The compound may be administered by any pharmaceutically
acceptable means, by either systemic or local administration.
Suitable modes of administration include oral, dermal, e.g., via
transdermal patch, inhalation, via infusion, intranasal, rectal,
vaginal, topical parenteral (e.g., via intraperitoneal,
intravenous, intramuscular, subcutaneous, injection).
[0108] Typically, oral administration or administration via
injection is preferred. The subject compounds may be administered
in a single dosage or chronically dependent upon the particular
disease, condition of patient, toxicity of compound, and whether
this compound is being utilized alone or in combination with other
therapies. Chronic or repeated administration will likely be
preferred based on other chemotherapies.
[0109] The subject compounds will be administered in a
pharmaceutically acceptable formulation or composition. Examples of
such formulations include injectable solutions, tablets, milk, or
suspensions, creams, oil-in-water and water-in-oil emulsions,
microcapsules and microvesicles.
[0110] These compositions will comprise conventional pharmaceutical
excipients and carriers typically used in drug formulations, e.g.,
water, saline solutions, such as phosphate buffered saline,
buffers, surfactants.
[0111] The subject compounds may be free or entrapped in
microcapsules, in colloidal drug delivery systems such as
liposomes, microemulsions, and macroemulsions. Suitable materials
and methods for preparing pharmaceutical formulations are disclosed
in Remington's Pharmaceutical Chemistry, 16' Edition, (1980). Also,
solid formulations containing the subject compounds, such as
tablets, and capsule formulations may be prepared.
[0112] Suitable examples thereof include semipermeable materials of
solid hydrophobic polymers containing the subject compound which
may be in the form of shaped articles, e.g., films or
microcapsules, as well as various other polymers and copolymers
known in the art.
[0113] The dosage effective amount of compounds according to the
invention will vary depending upon factors including the particular
compound, toxicity, and inhibitory activity, the condition treated,
and whether the compound is administered alone or with other
therapies. Typically a dosage effective amount will range from
about 0.0001 mg/kg to 1500 mg/kg, more preferably 1 to 1000 mg/kg,
more preferably from about 1 to 150 mg/kg of body weight, and most
preferably about 50 to 100 mg/kg of body weight.
[0114] The subjects treated will typically comprise mammals and
most preferably will be human subjects, e.g., human cancer
subjects.
[0115] The compounds of the invention may be used alone or in
combination. Additionally, the treated compounds may be utilized
with other types of treatments, e.g., cancer treatments. For
example, the subject compounds may be used with other
chemotherapies, e.g., tamoxifen, taxol, methothrexate, biologicals,
such as antibodies, growth factors, lymphokines, or radiation, etc.
Combination therapies may result in synergistic results.
[0116] The preferred indication is cancer, especially the cancers
identified previously.
[0117] While the invention has been described in terms of preferred
embodiments, the skilled artisan will appreciate that various
modifications, substitutions, omissions and changes may be made
without departing from the spirit thereof. Accordingly, it is
intended that the scope of the present invention be limited solely
by the scope of the following claims, including equivalents
thereof.
* * * * *