U.S. patent application number 10/452423 was filed with the patent office on 2003-12-25 for inhibitors of animal cell motility and growth.
Invention is credited to Anjum, Sarosh, Ankala, Sudha, Fenteany, Gabriel, Ghosh, Arun, McHenry, Kevin, Zhu, Shoutian.
Application Number | 20030236290 10/452423 |
Document ID | / |
Family ID | 29736433 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030236290 |
Kind Code |
A1 |
Fenteany, Gabriel ; et
al. |
December 25, 2003 |
Inhibitors of animal cell motility and growth
Abstract
Cell motility and growth inhibitors, including compounds of the
general structural formula 1 and use of the cell motility and cell
growth inhibitors, and pharmaceutically acceptable salts, prodrugs,
and solvates thereof, as therapeutic agents, are disclosed.
Inventors: |
Fenteany, Gabriel; (Oak
Park, IL) ; Ghosh, Arun; (River Forest, IL) ;
McHenry, Kevin; (Chicago, IL) ; Ankala, Sudha;
(Chicago, IL) ; Anjum, Sarosh; (Naperville,
IL) ; Zhu, Shoutian; (Chicago, IL) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Family ID: |
29736433 |
Appl. No.: |
10/452423 |
Filed: |
June 2, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60388141 |
Jun 12, 2002 |
|
|
|
Current U.S.
Class: |
514/376 ;
548/230 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 413/06 20130101; C07D 413/10 20130101; C07D 263/26
20130101 |
Class at
Publication: |
514/376 ;
548/230 |
International
Class: |
C07D 263/16; C07D
413/02; A61K 031/421; A61K 031/422 |
Goverment Interests
[0002] This invention was made with government support under CA
095177 by the National Institutes of Health. The government has
certain rights in this invention.
Claims
What is claimed is:
1. A compound having a formula 63wherein R.sup.1 is selected from
the group consisting of C(.dbd.O)C.sub.3-8cycloalkenyl,
C(.dbd.O)C.ident.C--R.sup.b, 64 C.sub.2-6alkenyl, and
C.sub.2-6alkynyl; R.sup.2 is selected from the group consisting of
hydro, C.sub.1-3alkyl, aryl, heteroaryl, C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneheteroaryl, C.sub.3-8cycloalkyl, and
C.sub.3-8heterocycloalkyl; R.sup.3 is selected from the group
consisting of hydro, C.sub.1-3alkyl, aryl, and heteroaryl; R.sup.a
is selected from the group consisting of hydro and C.sub.1-3alkyl;
R.sup.b is selected from the group consisting of hydro,
C.sub.1-6alkyl, CF.sub.3, C.sub.3-8cycloalkyl,
C.sub.3-8heterocycloalkyl, aryl, heteroaryl, S-aryl, O-aryl,
C.sub.1-3alkylenearyl, and C.sub.1-3alkyleneheteroaryl; R.sup.c is
selected from the group consisting of hydro, C.sub.1-3alkyl, and
fluoroC.sub.1-3alkyl, or R.sup.b and R.sup.c can be taken together
with the carbons to which they attached to form a five- or
six-membered aliphatic ring, optionally containing one or two
heteroatoms selected from oxygen, nitrogen, and sulfur; R.sup.d,
independently, is selected from the group consisting of hydro,
C.sub.1-3alkyl, and fluoroC.sub.1-4alkyl; and n is a number 0, 1,
or 2, and pharmaceutically acceptable salts, prodrugs, or hydrates
thereof.
2. The compound of claim 1 having a structure 65
3. The compound of claim 1 having a structure 66
4. The compound of claim 1 having a structure 67
5. The compound of claim 1 wherein R.sup.3 is hydro,
C.sub.1-3alkyl, and aryl; R.sup.2 is selected from the group
consisting of hydro, C.sub.1-3alkyl, aryl, C.sub.1-3alkylenearyl,
or heteroaryl; R.sup.1 is selected from the group consisting of
C(.dbd.O)C.sub.3-8cycloalkenyl, C(.dbd.O)C.ident.C--R.sup.b, and
68R.sup.a is hydro or C.sub.1-3alkyl; R.sup.b is selected from the
group consisting of hydro, C.sub.1-6alkyl, C.sub.3-8cycloalkyl,
CF.sub.3, aryl, heteroaryl, and S-aryl; R.sup.c is H or
C.sub.1-3alkyl; and n is 0 or 1.
6. The compound of claim 1 wherein R.sup.3 is selected from the
group consisting of hydro and phenyl; R.sup.2 is selected from the
group consisting of benzyl, hydro, isopropyl, methyl, and phenyl;
R.sup.1 is selected from the group consisting of
C(.dbd.O)C.ident.C--CH.sub.3 and 69R.sup.a is hydro and methyl;
R.sup.b is selected from the group consisting of hydro, methyl,
ethyl, trifluoromethyl, phenyl, pyridyl, naphthyl, thiophenyl,
furyl, thienyl, cyclopentyl, and pentyl; R.sup.c is hydro and
trifluoromethyl; or R.sup.a and R.sup.c are taken together with the
carbons to which they are attached to form a cyclohexenyl ring;
R.sup.d is selected from the group consisting of hydro, methyl, and
CH.sub.2CF.sub.3; and n is 0 or 1.
7. The compound of claim 1 wherein R.sup.3 is hydro or phenyl;
R.sup.2 is selected from the group consisting of benzyl, hydro,
isopropyl, phenyl, and methyl; and R.sup.1 is selected from the
group consisting of --C(.dbd.O)CH.dbd.CHCH.sub.3, 70717273
8. The compound of claim 1 having a structure 74
9. A compound of claim 1 having a biological IC.sub.50 value of
about 50 .mu.M or less.
10. A compound identified as compound 1, compounds 4-7, compounds 9
and 10, and compounds 12-73, as disclosed herein, and
pharmaceutically acceptable salts, prodrugs, or solvates
thereof.
11. A pharmaceutical composition comprising a compound of claim 1
together with a pharmaceutically acceptable diluent or carrier.
12. A method of treating a male or female animal for a condition
where inhibition of animal cell motility and cell growth is of a
therapeutic benefit comprising administering to said animal an
effective amount of a pharmaceutical composition comprising a
compound of claim 1, and a pharmaceutically acceptable diluent or
carrier.
13. The method of claim 12 wherein the inhibition is
reversible.
14. The method of claim 12 wherein the condition is a cancer.
15. A method of treating a condition where inhibition of cell
motility and cell growth is of therapeutic benefit, in a human or a
nonhuman animal body, comprising administering to said body a
therapeutically effective amount of a compound of claim 1.
16. A method of treating a male or female animal suffering from a
condition where inhibition of cell motility and cell growth is of a
therapeutic benefit comprising administering a therapeutically
effective amount of (a) a compound of claim 1, and (b) a second
therapeutically active ingredient used in a treatment of the
condition.
17. The method of claim 16 wherein (a) and (b) are administered
simultaneously or sequentially.
18. The method of claim 16 wherein the condition is a cancer.
19. The method of claim 18 wherein the second therapeutically
active ingredient is a chemotherapeutic agent or radiation.
20. A kit for the treatment of cancer comprising a compound of
claim 1 or a composition containing the same, packaged with
instructions for administration of the compound, or the
composition, to a mammal to treat the cancer.
21. The kit of claim 19 wherein the mammal is a human subject.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional
Patent Application Serial No. 60/388,141, filed on Jun. 12,
2002.
FIELD OF THE INVENTION
[0003] The present invention relates to a series of compounds, to
pharmaceutical compositions containing the compounds, and to their
use as therapeutic agents. In particular, the present invention
relates to compounds that are inhibitors of animal cell motility
and growth, and have utility in a variety of therapeutic areas
wherein such inhibition is considered beneficial, including the
treatment of various cancers.
BACKGROUND OF THE INVENTION
[0004] Cell shape change and motility are critical components in a
wide range of biological processes in mammals, including embryonic
development, tissue repair, angiogenesis, and immune system
function. Cell shape change and motility also are involved in
pathological events, such as cancer metastasis. While progress has
been made in identifying components of signal transduction pathways
leading to cell motility, a complete model of the mechanism is
still lacking. The precise roles of many proteins implicated in
these pathways are not yet elucidated, and a number of mechanisms
that cells use for movement may exist.
[0005] Cell motility is dependent on regulated actin filament
assembly, rearrangement, and disassembly. A large and growing
number of proteins are known to regulate and modulate the state of
the actin cytoskeleton, and some appear to have partly overlapping
functions. For example, actin polymerization and filament assembly
can be accomplished through de novo nucleation of new filaments by
the Arp2/3 complex or through elongation of existing filaments at
free barbed or fast-growing ends, generated by filament severing
and/or regulated dissociation of bound barbed-end capping proteins
(uncapping). Similarly, multiple routes exist for actin filament
bundling, crosslinking, and disassembly (depolymerization).
[0006] In addition to these end-point mechanisms of actin dynamics,
a number of different upstream signaling pathways leading to
changes in the actin cytoskeleton and cell morphology and behavior
have become apparent. The small Ras-related GTPases, e.g., Rac,
Rho, and Cdc42, in particular, have been implicated in the
regulation of the actin cytoskeleton and cell shape, and each plays
a distinct and specialized role. Rho proteins are associated
generally with formation of contractile actin/myosin bundles,
stress fibers, and focal adhesions. Rac proteins particularly are
associated with formation of lamellipodia (broad, sheet-like
membrane protrusions at the leading edge in the direction of
movement). Lamellipodial cell crawling resulting from activation of
Rac proteins is considered to be the most prevalent form of animal
cell motility. Cdc42 particularly is most associated with formation
of filpodia (finger-like membrane protrusions) and the control of
cell polarity. The Rho family small GTPases also have roles in
other cellular processes, such as control of cell growth and
cell-cell adhesion. In addition to these small GTPases,
phosphoinositides and calcium are known to regulate actin dynamics
and cell migration. However, a comprehensive understanding of the
signaling cascades leading to cell motility and the relationship
between these regulators remains elusive.
[0007] Progress in the art would be facilitated by the availability
of effective inhibitors of cell motility. A number of compounds
that target actin directly exist. The best known compounds are the
cytochalasins, which are cell-permeable destabilizers of actin
filaments, and phalloidin, which is a cell-impermeable stabilizer
of actin filaments (J. A. Cooper, J. Cell Biol, 105 (1987)). In
addition, latrunculins are cell-permeable disrupters of actin
filaments (I. Spector, Science, 219, 493 (1983)). Jasplakinolide is
a cell-permeable stabilizer of actin filaments (M. R. Bubb et al.,
Chem., 269, 14869 (1994)). A few compounds that target proteins
upstream of the actin cytoskeleton are known, such as the
Rho-kinase inhibitor Y-27632 (M. Uehata et al., Nature, 389, 990
(1997), and myosin light chain kinase inhibitors, such as ML-g (M.
Saitoh et al., Biochem. Biophys, Res. Commun., 140, 280 (1986)).
Recently, a cyclic peptide dimer was discovered that inhibits the
activity of N-WASP, a protein involved in Cdc42-mediated actin
nucleation by the Arp2/3 complex (J. R. Peterson et al., Proc.
Natl. Acad. Sci. USA, 98, 10624 (2001)). Nevertheless, there is a
dearth of available cell-permeable compounds that affect actin
dynamics and cell motility by inhibiting specific components of
signaling pathways to the actin cytoskeleton.
[0008] Presently, very few specific inhibitors of cell motility are
available, even though a great potential exists for such drugs as a
complement to existing anticancer therapies. Cell shape change and
motility are involved at two rate-limiting steps in cancer
progression: angiogenesis (i.e., blood vessel recruitment) and
metastasis (i.e., spreading of a tumor from one location in the
body to other locations). In combination with cell growth
inhibitors, treatment with specific cell motility inhibitors has
the potential to provide a more efficacious treatment of a cancer,
analogous to the multiple drug approach for treatment of HIV
infection and AIDS.
[0009] In particular, cell motility inhibitors have potential uses
such as, but not limited to, (a) an anticancer drug targeting
angiogenesis, (b) an anticancer drug targeting metastasis, and (c)
an anticancer drug targeting cell growth.
SUMMARY OF THE INVENTION
[0010] The present invention provides compounds of general
structural formula (I) 2
[0011] wherein R.sup.1 is selected from the group consisting of
C(.dbd.O)C.sub.3-8cycloalkenyl, C(.dbd.O)C.ident.C--R.sup.b, 3
[0012] C.sub.2-6alkenyl, and C.sub.2-6alkynyl;
[0013] R.sup.2 is selected from the group consisting of hydro,
C.sub.1-3alkyl, aryl, heteroaryl, C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneheteroaryl, C.sub.3-8cycloalkyl, and
C.sub.3-8heterocycloalkyl;
[0014] R.sup.3 is selected from the group consisting of hydro,
C.sub.1-3alkyl, aryl, and heteroaryl;
[0015] R.sup.a is selected from the group consisting of hydro and
C.sub.1-3alkyl;
[0016] R.sup.b is selected from the group consisting of hydro,
C.sub.1-6alkyl, CF.sub.3, C.sub.3-8cycloalkyl,
C.sub.3-8heterocycloalkyl, aryl, heteroaryl, S-aryl, O-aryl,
C.sub.1-3alkylenearyl, and C.sub.1-3alkyleneheteroaryl;
[0017] R.sup.c is selected from the group consisting of hydro,
C.sub.1-3alkyl, and fluoroC.sub.1-3alkyl, or
[0018] R.sup.b and R.sup.c can be taken together with the carbons
to which they are attached to form a five- or six-membered
aliphatic ring, optionally containing one or two heteroatoms
selected from oxygen, nitrogen, and sulfur;
[0019] R.sup.d, independently, is selected from the group
consisting of hydro, C.sub.1-3alkyl, and fluoroC.sub.1-4alkyl;
and
[0020] n is a number 0, 1, or 2, and
[0021] pharmaceutically acceptable salts, prodrugs, or solvates
(e.g., hydrates) thereof.
[0022] Another aspect of the present invention is to provide a cell
motility and growth inhibitor having a general structural formula
(II): 4
[0023] and pharmaceutically acceptable salts, prodrugs, or solvates
thereof.
[0024] Still another aspect of the present invention is to provide
a cell motility and growth inhibitor having a general structural
formula (III): 5
[0025] and pharmaceutically acceptable salts, prodrugs, or solvates
thereof.
[0026] Yet another aspect of the present invention is to provide a
cell motility and growth inhibitor having a general structural
formula (IV): 6
[0027] and pharmaceutically acceptable salts, prodrugs, or solvates
thereof.
[0028] As used herein, the term "a compound of the present
invention" is defined as a compound encompassed by general
structural formulae (I), (II), (III), and (IV), including, but not
limited to, the Examples and specific compounds disclosed
herein.
[0029] Another aspect of the present invention is to provide a cell
motility and growth inhibitor having a biological IC.sub.50 value
of about 50 .mu.M or less, preferably about 25 .mu.M or less, more
preferably about 15 .mu.M or less, and most preferably about 1
.mu.M or less, down to about 700 picomolar.
[0030] Yet another aspect of the present invention is to provide a
composition comprising a compound of the present invention and a
physiologically acceptable diluent or carrier.
[0031] Another aspect of the present invention is to provide a
method of treating an individual suffering from a disease or
condition wherein inhibition of cell motility and cell growth
provides a benefit, said method comprising administering a
therapeutically effective amount of a compound of the present
invention, or a composition containing the same, to the individual.
The method preferably provides a reversible inhibition of cell
motility and growth.
[0032] Still another aspect of the present invention is to provide
a method of treating an individual suffering from a cancer, said
method comprising administration of a therapeutically effective
amount of a compound of the present invention, or a composition
containing the same, to the individual, either alone or in
combination with other cancer treatment agents.
[0033] Another aspect of the present invention is to provide a
combination therapy comprising administration of therapeutically
effective amounts of (a) a compound of the present invention, or a
pharmaceutically acceptable salt, prodrug, or solvate thereof, and
(b) a second therapeutically active agent, to an individual,
simultaneously or sequentially, in the treatment of a disease or
condition wherein inhibition of cell growth and cell motility
provides a benefit. The disease or condition can be a cancer, and
the second therapeutically active agent can be a chemotherapeutic
agent or radiation, for example.
[0034] In another aspect, the present invention provides a kit for
the treatment of cancer comprising a compound of the present
invention, or a composition containing the same, packaged with
instructions for administration of the compound, or composition, to
a mammal, including a human, to treat the cancer. A compound of the
present invention and a second therapeutically active ingredient
for the treatment of cancer can be packaged together in a single
vial, packaged in separate vials, packaged as separate dosage
forms, and the like.
[0035] These and other aspects of the present invention will become
apparent from the following detailed description of the preferred
embodiments, taken in conjunction with the figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1A contains plots of % wound closure in MDCK cell
monolayers vs. time after application of different amounts of
compound 1;
[0037] FIG. 1B contains plots of % wound closure in MDCK cell
monolayers vs. time after application of a control compound (DMSO),
compound 1, and compound 11;
[0038] FIG. 2A contains bar graphs showing the % increase in number
of MDCK cells after application of a control compound (DMSO),
compound 1, and compound 11;
[0039] FIG. 2B contains bar graphs showing the % increase in number
of MDCK cells after administration of control compound (DMSO) or
compound 1, followed by rinsing of the control compound or compound
1 from the test system;
[0040] FIGS. 3A and 3B contain bar graphs showing developmental
defects in embryos treated with a control compound (DMSO) or
compound 1 over a 48- to 96-hour time period; and
[0041] FIGS. 4A and 4B contain bar graphs showing a delay in
blastopore closure arising from treatment with a control compound
(DMSO) and with various concentrations of compound 1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0042] Inhibitor compounds of the present invention have a general
structural formula (I): 7
[0043] wherein R.sup.1 is selected from the group consisting of
C(.dbd.O)C.sub.3-8cycloalkenyl, C(.dbd.O)C.ident.C--R.sup.b, 8
[0044] C.sub.1-6alkyl, C.sub.2-6alkenyl, and C.sub.2-6alkynyl;
[0045] R.sup.2 is selected from the group consisting of hydro,
C.sub.1-3alkyl, aryl, heteroaryl, C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneheteroaryl, C.sub.3-8cycloalkyl, and
C.sub.3-8heterocycloalkyl;
[0046] R.sup.3 is selected from the group consisting of hydro,
C.sub.1-3alkyl, aryl, and heteroaryl;
[0047] R.sup.a is selected from the group consisting of hydro and
C.sub.1-3alkyl;
[0048] R.sup.b is selected from the group consisting of hydro,
C.sub.1-6alkyl, CF.sub.3, C.sub.3-8cycloalkyl,
C.sub.3-8heterocycloalkyl, aryl, heteroaryl, S-aryl, O-aryl,
C.sub.1-3alkylenearyl, and C.sub.1-3alkyleneheteroaryl;
[0049] R.sup.c is selected from the group consisting of hydro,
C.sub.1-3alkyl, and fluoroC.sub.1-3alkyl, or
[0050] R.sup.b and R.sup.c can be taken together with the carbons
to which they are attached to form a five- or six-membered
aliphatic ring, optionally containing one or two heteroatoms
selected from oxygen, nitrogen, and sulfur;
[0051] R.sup.d, independently, is selected from the group
consisting of hydro, C.sub.1-3alkyl, and fluoroC.sub.1-4alkyl;
and
[0052] n is a number 0, 1, or 2, and pharmaceutically acceptable
salts, prodrugs, or solvates thereof.
[0053] In some preferred embodiments, the inhibitor compounds have
a general structural formula (II), (III), or (IV): 9
[0054] and pharmaceutically acceptable salts, prodrugs, or solvates
thereof.
[0055] As used herein, the term "alkyl" includes straight chained
and branched hydrocarbon groups containing the indicated number of
carbon atoms, typically methyl, ethyl, and straight chain and
branched propyl and butyl groups. The hydrocarbon group can contain
up to 16 carbon atoms. The term "alkyl" includes "bridged alkyl,"
i.e., a C.sub.6-C.sub.16 bicyclic or polycyclic hydrocarbon group,
for example, norbornyl, adamantyl, bicyclo[2.2.2]octyl,
bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, or decahydronaphthyl.
Alkyl groups can be substituted, for example, with fluoro (F),
hydroxy (OH), or amino (NH.sub.2) groups.
[0056] The term "cycloalkyl" is defined as a cyclic C.sub.3-C.sub.8
hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and
cyclopentyl. "Heterocycloalkyl" is defined similarly as cycloalkyl
except the ring contains one to three heteroatoms selected from the
group consisting of oxygen, nitrogen, and sulfur. Cycloalkyl and
heterocycloalkyl groups can be substituted, for example, with one
to three groups, independently selected from groups such as
C.sub.1-4alkyl, C.sub.1-3alkyleneOH, C(.dbd.O)NH.sub.2, NH.sub.2,
and OH.
[0057] The terms "alkenyl" and "alkynyl" are defined identically as
"alkyl," except for containing a carbon-carbon double bond or a
carbon-carbon triple bond, respectively. "Cycloalkenyl" is defined
similarly to, and is encompassed by, the term cycloalkyl, except a
carbon-carbon double bond is present in the ring.
[0058] The term "alkylene" refers to an alkyl group having a
substituent. For example, the term "C.sub.1-3alkylenearyl" refers
to an alkyl group containing one to three carbon atoms, and
substituted with an aryl group.
[0059] The term "halo" is defined herein as fluoro, bromo, chloro,
and iodo.
[0060] The term "haloalkyl" is defined herein as an alkyl group
substituted with one or more halo substituents, independently
selected from fluoro, chloro, bromo, and iodo. "Halocycloalkyl" is
encompassed by the term "haloalkyl," and is defined as a cycloalkyl
group having one or more halo substituents.
[0061] The term "aryl," alone or in combination, is defined herein
as a monocyclic or polycyclic aromatic group, preferably a
monocyclic or bicyclic aromatic group, e.g., phenyl or naphthyl.
Unless otherwise indicated, an "aryl" group can be unsubstituted or
substituted, for example, with one or more, and in particular one
to five, halo, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl,
heteroaryl, trifluoromethyl, OR.sup.b, OSi(C.sub.1-4alkyl).sub.3,
OC(.dbd.O)--C.sub.1-4alkyl, trifluoromethoxy,
SO.sub.2N(R.sup.b).sub.2, NHSO.sub.2aryl, N(R.sup.b).sub.2,
alkoxyalkyl, haloalkyl, nitro, cyano, acylamino, alkylthio,
arylthio, C(.dbd.O)N(R.sup.b).sub.2, C(.dbd.O)R.sup.a,
OC(.dbd.O)R.sup.a, C(.dbd.O)OR.sup.a, NR.sup.bC(.dbd.O)R.sup.a, and
OC.sub.1-3alkyleneNR.sup.aR.sup.b. Exemplary aryl groups include
phenyl, naphthyl, tetrahydronaphthyl, indenyl, indanyl, biphenyl,
chlorophenyl, nitrophenyl, fluorophenyl, methylphenyl,
methoxyphenyl, trifluoromethylphenyl, hydroxyphenyl, and the like.
The terms "arylC.sub.1-3alkyl" and "heteroarylC.sub.1-3alkyl" are
defined as an aryl or heteroaryl group having a C.sub.1-3alkyl
substituent.
[0062] The term "heteroaryl" is defined herein as a monocyclic or
bicyclic ring system containing one or two aromatic rings and
containing at least one nitrogen, oxygen, or sulfur atom in an
aromatic ring, and which can be unsubstituted or substituted, for
example, with one or more, and in particular one to three,
substituents, for example, the substituents listed above for aryl
groups. Examples of heteroaryl groups include thienyl, furyl,
pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazolyl,
isothiazolyl, isoxazolyl, imidizolyl, benzothiazolyl, pyrazinyl,
pyrimidinyl, thiazolyl, and thiadiazolyl.
[0063] The term "hydro" is defined as --H.
[0064] The term "hydroxy" is defined as --OH.
[0065] The term "alkoxy" is defined as --OR, wherein R is alkyl,
including cycloalkyl.
[0066] The term "alkoxyalkyl" is defined as an alkyl group wherein
a hydrogen has been replaced by an alkoxy group. The term
"(alkylthio)alkyl" is defined similarly as alkoxyalkyl, except a
sulfur atom, rather than an oxygen atom, is present.
[0067] The term "hydroxyalkyl" is defined as a hydroxy group
appended to an alkyl group.
[0068] The term "amino" is defined as --NH.sub.2, and the term
"alkylamino" is defined as --NR.sub.2, wherein at least one R is
alkyl and the second R is alkyl or hydrogen.
[0069] The term "5- or 6-membered aryl or heteroaryl group" as used
herein refers to carbocyclic and heterocyclic aromatic groups,
including, but not limited to, phenyl, thiophenyl, furyl, pyrrolyl,
imidazolyl, pyrimidinyl, and pyridinyl.
[0070] The term "acylamino" is defined as RC(.dbd.O)N--, wherein R
is alkyl or aryl.
[0071] The term "alkylthio" and "arylthio" are defined as --SR,
wherein R is alkyl or aryl, respectively.
[0072] The term "alkylsulfinyl" is defined as R--SO.sub.2--,
wherein R is alkyl.
[0073] The term "alkylsulfonyl" is defined as R--SO.sub.3--,
wherein R is alkyl.
[0074] The term "nitro" is defined as --NO.sub.2.
[0075] The term "trifluoromethyl" is defined as --CF.sub.3.
[0076] The term "trifluoromethoxy" is defined as --OCF.sub.3.
[0077] The term "cyano" is defined as --CN.
[0078] The carbon atom content of hydrocarbon-containing moieties
is indicated by a subscript designating the minimum and maximum
number of carbon atoms in the moiety, e.g., "C.sub.1-6alkyl" refers
to an alkyl group having one to six carbon atoms, inclusive.
[0079] In the structures herein, for a bond lacking a substituent,
the substituent is methyl, for example, 10
[0080] When no substituent is indicated as attached to a carbon
atom on a ring, it is understood that the carbon atom contains the
appropriate number of hydrogen atoms. In addition, when no
substituent is indicated as attached to a carbonyl group or a
nitrogen atom, for example, the substituent is understood to be
hydrogen, e.g., 11
[0081] and R--N is R--NH.sub.2.
[0082] The numbering system for the oxazolidinone ring system is
12
[0083] A compound is considered to be a inhibitor of cell motility
and cell growth if the compound effectively inhibits cell motility
and cell growth at a physiologically compatible concentration. To
be useful as a therapeutic compound, the compound also must be
nontoxic at such a concentration. Effective inhibition typically is
defined as a compound that inhibits cell motility and cell growth
by at least 50%, preferably at least 80%, and more preferably at
least 90%, at a physiologically compatible concentration.
[0084] As discussed in more detail hereafter, cell motility and
growth inhibition typically is measured using a dose-response assay
in which a sensitive assay system is contacted with a compound of
interest over a range of concentrations at which no or minimal
effect is observed, through higher concentrations at which partial
effect is observed, to saturating concentrations at which a maximum
effect is observed. Theoretically, such assays of the dose-response
effect of inhibitor compounds can be described as a sigmoidal
curve, expressing a degree of inhibition as a function of
concentration. The curve also theoretically passes through a point
at which the concentration is sufficient to inhibit activity to a
level that is 50% that of the difference between minimal and
maximal activity in the assay. This concentration is defined as the
Inhibitory Concentration (50%) or IC.sub.50. Comparisons between
the efficacy of inhibitors often are provided with reference to
comparative IC.sub.50 concentrations, wherein a higher IC.sub.50
indicates that the test compound is less potent, and a lower
IC.sub.50 indicates that the compound is more potent, than a
reference compound.
[0085] An "IC.sub.50 value" of a compound, therefore, is defined as
the concentration of the compound required to produce 50%
inhibition of biological activity. Inhibitors of cell motility and
cell growth disclosed herein preferably have an IC.sub.50 value of
less than about 50 .mu.M, more preferably less than about 25 .mu.M,
and still more preferably less than about 15 .mu.M. Most
preferably, a cell motility and cell growth inhibitor of the
present invention has an IC.sub.50 value of less than about 1
.mu.M, down to about 700 picomolar.
[0086] Similarly, the potency of inhibitor compounds can be related
in terms of the Effective Concentration (50%) or EC.sub.50, which
is a measure of dose-response activity in a cell-based or
animal-based model. EC.sub.50 measurements are useful to relate
properties of the compound that can influence its clinical utility,
such as compound solubility, ability to penetrate cell membranes,
partition coefficient, bioavailability, and the like. Two compounds
can exhibit a divergence in comparative IC.sub.50 and EC.sub.50
values, i.e., one compound can be more potent in a biochemical
assay and the second compound more potent in a cell-based assay
simply due to different properties of the compounds.
[0087] The term "pharmaceutically acceptable carrier" as used
herein refers to compounds suitable for use with recipient animals,
preferably mammals, and more preferably humans, and having a
toxicity, irritation, or allergic response commensurate with a
reasonable benefit/risk ratio, and effective for their intended
use.
[0088] The term "prodrug" as used herein refers to compounds that
transform rapidly in vivo to a compound of the invention, for
example, by hydrolysis. Prodrugs of the invention also can be
active in the prodrug form. A thorough discussion is provided in
Higuchi et al., Prodrugs as Novel Delivery Systems, Vol. 14, of the
A.C.S.D. Symposium Series, and in Roche (ed), Bioreversible
Carriers in Drug Design, American Pharmaceutical Association and
Pergamon Press, 1987.
[0089] In preferred embodiments of the present invention, R.sup.3
is hydro, C.sub.1-3alkyl, or aryl; R.sup.2 is hydro,
C.sub.1-3alkyl, aryl, C.sub.1-3alkylenearyl, or heteroaryl; R.sup.1
is C(.dbd.O)C.sub.3-8cycloa- lkenyl, C(.dbd.O)C.ident.C--R.sup.b,
or 13
[0090] R.sup.a is hydro or C.sub.1-3alkyl, R.sup.b is hydro,
C.sub.1-6alkyl, C.sub.3-8cycloalkyl, CF.sub.3, aryl, heteroaryl, or
S-aryl; R.sup.c is H or C.sub.1-3alkyl; and n is 0 or 1.
[0091] In other preferred embodiments, R.sup.3 is hydro or phenyl;
R.sup.2 is benzyl, hydro, isopropyl, methyl, or phenyl; R.sup.1 is
C(.dbd.O)C.ident.C--CH.sub.3 or 14
[0092] R.sup.a is hydro or methyl; R.sup.b is hydro, methyl, ethyl,
trifluoromethyl, phenyl, pyridyl, naphthyl, thiophenyl, furyl,
thienyl, cyclopentyl, or pentyl; R.sup.c is hydro or
trifluoromethyl; or R.sup.a and R.sup.c are taken together with the
carbons to which they are attached to form a cyclohexenyl ring;
R.sup.d is hydro, methyl, or CH.sub.2CF.sub.3; and n is 0 or 1.
[0093] In these embodiments, the aryl or heteroaryl rings
optionally are substituted with one or more of nitro, amino,
methoxy, trifluoromethyl, fluoro, chloro, methyl, phenyl, hydroxy,
NHSO.sub.2aryl, OSi(C.sub.1-4alkyl).sub.3, or OC(.dbd.O)tbutyl.
[0094] In most preferred embodiments, R.sup.3 is hydro or phenyl;
R.sup.2 is benzyl, hydro, isopropyl, phenyl, or methyl; and R.sup.1
is --C(.dbd.O)CH.dbd.CHCH.sub.3, 15161718
[0095] The aryl and heteroaryl substituents present on compounds of
the present invention can be unsubstituted or substituted groups
selected form the group consisting of 1920
[0096] Compounds of the present invention can contain one or more
asymmetric center, and, therefore, can exist as stereoisomers. The
present invention includes both racemic mixtures and individual
stereoisomers of the compounds of the present invention.
[0097] Pharmaceutically acceptable salts of compounds of the
present invention can be acid addition salts formed with
pharmaceutically acceptable acids. Examples of suitable salts
include, but are not limited to, the hydrochloride, hydrobromide,
sulfate, bisulfate, phosphate, hydrogen phosphate, acetate,
benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate,
gluconate, methanesulfonate, benzenesulfonate, and
p-toluenesulfonate salts. The compounds of the present invention
also can provide pharmaceutically acceptable metal salts, in
particular alkali metal salts and alkaline earth metal salts, with
bases. Examples include the sodium, potassium, magnesium, and
calcium salts.
[0098] Compounds of the present invention are potent inhibitors of
cell motility and cell growth. Thus, compounds of the present
invention are of interest for use in therapy, specifically for the
treatment of a variety of conditions where inhibition of cell
motility and growth is considered to be beneficial.
[0099] Cell motility and cell growth are particularly attractive
targets for inhibition because an inhibitor of cell motility and
growth provides effects which are beneficial in the treatment of
various disease states. The biochemical, physiological, and
clinical effects of cell motility and cell growth inhibitors
therefore suggest their utility in a variety of cancers by
targeting various control points in cancer progression, including
angiogenesis and metastasis. The compounds of the present
invention, therefore, are useful in treating various cancers, while
minimizing or eliminating adverse side effects due to the
reversible nature of the inhibitory effect.
[0100] An especially important use of the compounds of the present
invention is the treatment of a cancer. Thus, the present invention
is directed to the use of compounds of the present invention, a
pharmaceutically acceptable salt, prodrug, or solvate thereof, or a
pharmaceutical composition containing any of these entities, for
the manufacture of a medicament for the curative or prophylactic
treatment of a cancer in a mammal, including humans.
[0101] As used above and hereafter, the term "treatment" includes
preventing, lowering, stopping, or reversing the progression or
severity of the condition or symptoms being treated. As such, the
term "treatment" includes both medical therapeutic and/or
prophylactic administration, as appropriate, including, but not
limited to, the diseases and conditions discussed above.
[0102] It also is understood that "a compound of the present
invention," or a physiologically acceptable salt, prodrug, or
solvate thereof, can be administered as the neat compound, or as a
pharmaceutical composition containing either entity.
[0103] The present invention also is directed to a method of
treating conditions and disorders wherein inhibition of cell
motility and cell growth provides a benefit, in a human or nonhuman
animal body, comprising administering to said body a
therapeutically effective amount of a compound of the present
invention. According to another aspect of the present invention,
there is provided the use of a compound of formula (I) for the
manufacture of a medicament for the treatment of conditions and
disorders wherein inhibition of cell motility provides a
benefit.
[0104] In vivo methods of treatment are specifically contemplated.
Thus, for example, the present invention includes a method of
treating cancer in a mammal comprising the steps of administering
to the mammal (a) a compound of the present invention to inhibit
cell motility and cell growth and (b) an optional second active
compound or agent for effecting cancer treatment, e.g., a
chemotherapeutic agent or radiation, wherein the compound or
compounds are administered at concentrations effective to effect
inhibition of cell growth and cell motility in the mammal.
Administration to humans is specifically contemplated, but
administration to other animals, including pets, livestock, zoo
specimens, wildlife, and the like, also is contemplated.
[0105] Compounds of the present invention can be administered by
any suitable route, for example by oral, buccal, inhalation,
sublingual, rectal, vaginal, transurethral, nasal, topical,
percutaneous, i.e., transdermal, or parenteral (including
intravenous, intramuscular, subcutaneous, and intracoronary)
administration. Parenteral administration can be accomplished using
a needle and syringe, or using a high pressure technique, like
POWDERJECT.TM..
[0106] Oral administration of a compound of the present invention
is a preferred route. Oral administration is the most convenient
and avoids the disadvantages associated with other routes of
administration. For patients suffering from a swallowing disorder
or from impairment of drug absorption after oral administration,
the drug can be administered parenterally, e.g., sublingually or
buccally.
[0107] Compounds and pharmaceutical compositions suitable for use
in the present invention include those wherein the active
ingredient is administered in a sufficient amount to achieve its
intended purpose. More specifically, a "therapeutically effective
amount" means an amount effective to prevent development of a
disease in, or to alleviate the existing symptoms of a disease in,
the subject being treated. Therefore, a "therapeutically effective
amount" refers to that amount of the compound that results in
achieving the desired effect. Determination of a therapeutically
effective amount is well within the capability of those skilled in
the art, especially in light of the detailed disclosure provided
herein.
[0108] The toxicity and therapeutic efficacy of compounds of the
present invention can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, which is expressed as
the ratio between LD.sub.50 and ED.sub.50. Compounds that exhibit
high therapeutic indices are preferred. The data obtained from such
data can be used in formulating a dosage range for use in humans.
The dosage of such compounds preferably lies within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage can vary within this range depending
upon the dosage form employed, and the route of administration
utilized.
[0109] The exact formulation, route of administration, and dosage
is selected by the individual physician in view of the patient's
condition. Dosage amount and interval can be adjusted individually
to provide plasma levels of a compound of the present invention
which are sufficient to maintain therapeutic effects. Therefore,
the amount of a compound of the present invention administered is
dependent on the subject being treated, including the subject's
weight, the severity of the affliction, the manner of
administration, and the judgment of the prescribing physician.
[0110] Specifically, for administration to a human in the curative
or prophylactic treatment of the conditions and disorders
identified above, dosages of a compound of the present invention
generally are about 0.1 to about 1000 mg daily for an average adult
patient (70 kg). Thus, for a typical adult patient, individual
doses, for example, tablets or capsules, contain 0.1 to 500 mg of
active compound, in a suitable pharmaceutically acceptable vehicle
or carrier, for administration in single or multiple doses, once or
several times per day. Dosages for intravenous, buccal, or
sublingual administration typically also are 0.1 to 500 mg per
single dose as required. In practice, the physician determines the
actual dosing regimen which is most suitable for an individual
patient, and the dosage varies with the age, weight, and response
of the particular patient. The above dosages are exemplary of the
average case, but there can be individual instances in which higher
or lower dosages are merited, and such are within the scope of this
invention.
[0111] For human use, a compound of the present invention can be
administered alone, but generally is administered in admixture with
a pharmaceutical carrier selected with regard to the intended route
of administration and standard pharmaceutical practice.
Pharmaceutical compositions for use in accordance with the present
invention thus can be formulated in a conventional manner using one
or more physiologically acceptable carriers comprising excipients
and auxiliaries that facilitate processing of compounds of the
present invention into compositions that can be used
pharmaceutically.
[0112] Such pharmaceutical compositions can be manufactured in a
conventional manner, e.g., by conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping, or lyophilizing processes. Proper formulation is
related to the route of administration chosen. When a
therapeutically effective amount of a compound of the present
invention is administered orally, the composition typically is in
the form of a tablet, capsule, powder, solution, or elixir. When
administered in tablet form, the composition additionally can
contain a solid carrier, such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain about 5% to about 95% compound
of the present invention, and preferably from about 25% to about
90% compound of the present invention. When administered in liquid
form, a liquid carrier such as water, petroleum, or oils of animal
or plant origin can be added. The liquid form of the composition
can further contain physiological saline solution, dextrose or
other saccharide solutions, or glycols. When administered in liquid
form, the composition contains about 0.5% to about 90% by weight of
a compound of the present invention, and preferably about 1% to
about 50% of a compound of the present invention.
[0113] When a therapeutically effective amount of a compound of the
present invention is administered by intravenous, cutaneous, or
subcutaneous injecttion, the composition is in the form of a
pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable solutions, having due
regard to pH, isotonicity, stability, and the like, is within the
skill in the art. A preferred composition for intravenous,
cutaneous, or subcutaneous injection typically contains, in
addition to a compound of the present invention, an isotonic
vehicle.
[0114] For oral administration, the compounds can be formulated
readily by combining a compound of the present invention with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the present compounds to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for oral ingestion by a patient to be
treated. Pharmaceutical preparations for oral use can be obtained
by adding a compound of the present invention with a solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
include, for example, fillers and cellulose preparations. If
desired, disintegrating agents can be added.
[0115] For administration by inhalation, compounds of the present
invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebulizer, with the
use of a suitable propellant. In the case of a pressurized aerosol,
the dosage unit can be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of, e.g., gelatin, for use
in an inhaler or insufflator can be formulated containing a powder
mix of the compound and a suitable powder base such as lactose or
starch.
[0116] The compounds can be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampules or in multidose containers, with an
added preservative. The compositions can take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing,
and/or dispersing agents.
[0117] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds can be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils or synthetic
fatty acid esters. Aqueous injection suspensions can contain
substances which increase the viscosity of the suspension.
Optionally, the suspension also can contain suitable stabilizers or
agents that increase the solubility of the compounds and allow for
the preparation of highly concentrated solutions. Alternatively, a
present composition can be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0118] Compounds of the present invention also can be formulated in
rectal compositions, such as suppositories or retention enemas,
e.g., containing conventional suppository bases. In addition to the
formulations described previously, the compounds also can be
formulated as a depot preparation. Such long-acting formulations
can be administered by implantation (for example, subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the compounds can be formulated with suitable polymeric or
hydrophobic materials (for example, as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0119] For veterinary use, a compound of the present invention or a
nontoxic salt thereof, is administered as a suitably acceptable
formulation in accordance with normal veterinary practice. The
veterinarian readily can determine the dosing regimen and route of
administration that is most appropriate for a particular
animal.
[0120] Thus, the present invention provides a pharmaceutical
composition comprising a compound of the present invention,
together with a pharmaceutically acceptable diluent or carrier
therefor. The present invention also provides a process of
preparing a pharmaceutical composition comprising a compound of the
present invention, which process comprises mixing a compound of the
present invention, together with a pharmaceutically acceptable
diluent or carrier therefor.
[0121] In a particular embodiment, the invention includes a
pharmaceutical composition for the curative or prophylactic
treatment of cancer and other diseases and conditions wherein
inhibition of cell motility and cell growth provides a benefit in a
mammal, including humans, comprising a compound of the present
invention or a pharmaceutically acceptable salt thereof, together
with a pharmaceutically acceptable diluent or carrier.
[0122] A composition of the present invention can be administered
to an individual alone, or in concert with a second therapeutically
active agent. The second therapeutically active agent is a compound
useful in treating the disease or condition afflicting the
individual, and for which the individual is receiving treatment
with a compound of the present invention. For example, if an
individual is being treated for cancer, the individual can be
administered a therapeutically effective amount of a compound of
the present invention and a second therapeutically active agent
useful in the treatment of cancer, for example, a chemotherapeutic
drug or radiation. The cell motility and cell growth inhibitor of
the present invention and second therapeutically active agent can
be administered either simultaneously or sequentially. If
administered sequentially, either the cell motility inhibitor or
second therapeutically active agent can be administered first.
[0123] Compounds of the present invention can enhance the
therapeutic benefit of radiation and chemotherapy treatment,
including induction chemotherapy, primary (neoadjuvant)
chemotherapy, and both adjuvant radiation therapy and adjuvant
chemotherapy. In addition, radiation and chemotherapy are
frequently indicated as adjuvants to surgery in the treatment of
cancer. The goal of radiation and chemotherapy in the adjuvant
setting is to reduce the risk of recurrence and enhance
disease-free survival when the primary tumor has been controlled.
Chemotherapy is utilized as a treatment adjuvant for colon, lung,
and breast cancer, frequently when the disease is metastatic.
Adjuvant radiation therapy is indicated in several diseases
including colon, lung, and breast cancers as described above. For
example, radiation frequently is used both pre- and post-surgery as
components of the treatment strategy for rectal carcinoma.
Compounds of the present invention are particularly useful
following surgery in the treatment of cancer in combination with
radio- and/or chemotherapy.
[0124] Electromagnetic radiation treatment of other diseases not
listed herein also is contemplated by the present invention. The
terms "electromagnetic radiation" and "radiation" as used herein
include, but are not limited to, radiation having the wavelength of
10.sup.-20 to 100 meters. Preferred embodiments of the present
invention employ the electromagnetic radiation of: gamma-radiation
(10.sup.-20 to 10.sup.-13 m), X-ray radiation (10.sup.-12 to
10.sup.-9 m), ultraviolet light (10 nm to 400 nm), visible light
(400 nm to 700 nm), infrared radiation (700 nm to 1 mm), and
microwave radiation (1 mm to 30 cm).
[0125] Many cancer treatment protocols currently employ
radiosensitizers activated by electromagnetic radiation, e.g.,
X-rays. Examples of X-ray-activated radiosensitizers include, but
are not limited to, the following: metronidazole, misonidazole,
desmethylmisonidazole, pimonidazole, etanidazole, nimorazole,
mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide,
5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),
bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea,
cisplatin, and therapeutically effective analogs and derivatives of
the same.
[0126] Photodynamic therapy (PDT) of cancers employs visible light
as the radiation activator of the sensitizing agent. Examples of
photodynamic radiosensitizers include the following, but are not
limited to: hematoporphyrin derivatives, PHOTOFRIN.RTM.,
benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2),
pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines,
phthalocyanines, zinc phthalocyanine, and therapeutically effective
analogs and derivatives of the same.
[0127] Chemotherapeutic agents that can be used include, but are
not limited to, alkylating agents, antimetabolites, hormones and
antagonists, radio-isotopes, antibodies, as well as natural
products, and combinations thereof. For example, an inhibitor
compound of the present invention can be administered with
antibiotics, such as doxorubicin and other anthracycline analogs,
nitrogen mustards, such as cyclophosphamide, pyrimidine analogs,
such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its
natural and synthetic derivatives, and the like. As another
example, in the case of mixed tumors, such as adenocarcinoma of the
breast, where the tumors include gonadotropin-dependent and
gonadotropin-independent cells, the compound can be administered in
conjunction with leuprolide or goserelin (synthetic peptide analogs
of LH-RH). Other antineoplastic protocols include the use of an
inhibitor compound with another treatment modalite, e.g., surgery
or radiation, also referred to herein as "adjunt anti-neoplastic
modalities." Examples of chemotherapeutic agents useful for the
method of the present invention are listed in the following
table.
1 Alkylating agents Epipodophylotoxins Hormones and antagonists
Nitrogen mustards etoposide Adrenocorticosteroids/ mechlorethamine
teniposide antagonists cyclophosphamide Antibiotics prednisone and
equivalents ifosfamide actimomycin D dexamethasone melphalan
daunomycin ainoglutethimide chlorambucil (rubidomycin) Progestins
Nitrosoureas doxorubicin hydroxyprogesterone carmustine (BCNU)
(adriamycin) caproate lomustine (CCNU) mitoxantroneidarubicin
medroxyprogesterone acetate semustine (methyl-CCNU)
bleomycinsplicamycin megestrol acetate Ethylenimine/Methylmelamine
(mithramycin) Estrogens thriethylenemelamine (TEM) mitomycinC
diethylstilbestrol triethylene dactinomycin ethynyl estradiol/
thiophosphoramide Enzymes equivalents (thiotepa) L-asparaginase
Antiestrogen hexamethylmelamine Biological response tamoxifen (HMM,
altretamine) modifiers Androgens Alkyl sulfonates interferon-alpha
testosterone propionate busulfan IL-2 fluoxymesterone/equivalents
Triazines G-CSF Antiandrogens dacarbazine (DTIC) GM-CSF flutamide
Antimetabolites Differentiation Agents gonadotropin-releasing Folic
Acid analogs retinoic acid hormone analogs methotrexate derivatives
leuprolide trimetrexate Radiosensitizers Nonsteroidal antiandrogens
Pyrimidine analogs metronidazole flutamide 5-fluorouracil
misonidazole Photosensitizers fluorodeoxyuridine
desmethylmisonidazole hematoporphyrin derivatives gemcitabine
pimonidazole Photofrin .RTM. cytosine arabinoside etanidazole
benzoporphyrin derivatives (AraC, cytarabine) nimorazole Npe6
5-azacytidine RSU 1069 tin etioporphyrin (SnET2)
2,2'-difluorodeoxycytidine EO9 pheoboride-a Purine analogs RB 6145
bacteriochlorophyll-a 6-mercaptopurine SR4233 naphthalocyanines
6-thioguanine nicotinamide phthalocyanines azathioprine
5-bromodeozyuridine zinc phthalocyanines 2'-deoxycoformycin
5-iododeoxyuridine (pentostatin) bromodeoxycytidine
erythrohydroxynonyladenine Miscellaneous agents (EHNA) Platinum
coordination fludarabine phosphate complexes 2-chlorodeoxyadenosine
cisplatin (cladribine, 2-CdA) carboplatin Type I Topoisomerase
Anthracenedione Inhibitors mitoxantrone camptothecin Substituted
urea topotecan hydroxyurea irinotecan Methylhydrazine Natural
products derivatives Antimitotic drugs N-methylhydrazine paclitaxel
(MIH) Vinca alkaloids procarbazine vinblastine (VLB) Adrenocortical
vincristine suppressant vinorelbine mitotane (o,p'-DDD) Taxotere
.RTM. (docetaxel) ainoglutethimide estramustine Cytokines
estramustine phosphate interferon (.alpha., .beta., .gamma.)
interleukin-2
[0128] Examples of chemotherapeutic agents that are particularly
useful in conjunction with radiosensitizers include, for example,
adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin,
doxorubicin, interferon (alpha, beta, gamma), interleukin 2,
irinotecan, docetaxel, paclitaxel, topotecan, and therapeutically
effective analogs and derivatives of the same.
[0129] As appreciated by persons skilled in the art, reference
herein to treatment extends to prophylaxis, as well as to treatment
of established diseases or symptoms. It is further appreciated that
the amount of a compound of the invention required for use in
treatment varies with the nature of the condition being treated,
and with the age and the condition of the patient, and is
ultimately determined by the attendant physician or
veterinarian.
[0130] Compounds of the present invention can be prepared by any
suitable method known in the art, or by the following processes
which form part of the present invention. In the methods below,
R.sup.1, R.sup.2, and R.sup.3, as well as R.sup.a, R.sup.b,
R.sup.c, and R.sup.d, are defined above. For example, compounds of
the present invention can be prepared according to the following
synthetic schemes comprising converting a compound (V) to a
compound (VI) by Method A or Method B.
[0131] It should be understood that protecting groups can be
utilized in accordance with general principles of synthetic organic
chemistry to provide compounds of the present invention. Protecting
group-forming reagents, like benzyl chloroformate and
trichloroethyl chloroformate, are well known to persons skilled in
the art, for example, see T. W. Greene et al., "Protective Groups
in Organic Synthesis, Third Edition," John Wiley and Sons, Inc.,
NY, N.Y. (1999). These protecting groups are removed when necessary
by appropriate basic, acidic, or hydrogenolytic conditions known to
persons skilled in the art. Accordingly, compounds of the present
invention not specifically exemplified herein can be prepared by
persons skilled in the art.
[0132] In addition, a compound of the present invention can be
converted to another compound of the present invention. Thus, for
example, a particular R substituent can be interconverted to
prepare another suitably substituted compound of the present
invention. Examples of appropriate inter-conversions include, but
are not limited to, OR to hydroxy by suitable means (e.g., using an
agent such as BBr3 or a palladium catalyst, like
palladium-on-carbon, with hydrogen), or amino to substituted amino,
such as acylamino or sulphonylamino, using standard acylating or
sulfonylating conditions.
[0133] Compounds of the present invention can be prepared by the
methods above as individual stereoisomers or as a racemic mixture.
Individual stereoisomers of the compounds of the invention can be
prepared from racemates by resolution using methods known in the
art for the separation of racemic mixtures into their constituent
stereoisomers, for example, using HPLC on a chiral column, such as
Hypersil naphthyl urea, or using separation of salts of
stereoisomers. Compounds of the invention can be isolated in
association with solvent molecules by crystallization from, or
evaporation of, an appropriate solvent.
[0134] The pharmaceutically acceptable acid addition salts of the
compounds of the present invention that contain a basic center can
be prepared in a conventional manner. For example, a solution of
the free base can be treated with a suitable acid, either neat or
in a suitable solution, and the resulting salt isolated either by
filtration or by evaporation under vacuum of the reaction solvent.
Pharmaceutically acceptable base addition salts can be obtained in
an analogous manner by treating a solution of a compound of the
present invention with a suitable base. Both types of salt can be
formed or interconverted using ion-exchange resin techniques. Thus,
according to a further aspect of the invention, a method of
preparing a compound of the present invention, or prodrug thereof,
is provided, followed by (i) salt formation, or (ii) solvate (e.g.,
hydrate) formation. 21
[0135] Method A: 22
[0136] Reagents and Conditions: i) thionyl chloride (CO(Cl).sub.2),
benzene, dimethylformamide (DMF), catalyst (cat.), room temperature
(rt); ii) n-butyl lithium (n-BuLi), tetrahydrofuran (THF), acid
chloride (VIII), -78.degree. C. to rt.
[0137] Method B: 23
[0138] Reagents and Conditions: i) triethylamine (Et.sub.3N), THF,
pivaloyl chloride, -78.degree. C. to 0.degree. C.; ii) n-BuLi, THF,
acid anhydride (IX), -78.degree. C. to rt.
[0139] General Procedure for Method B:
[0140] To a cold (-78.degree. C.) solution of the unsaturated acid
(VII) (1.1 equiv) in anhydrous THF were added pivaloyl chloride
(1.1 equiv), followed by (1.2 equiv) of triethylamine. The reaction
mixture was stirred at -78.degree. C. for 30 minutes, at 0.degree.
C. for 1 hour, then recooled to -78.degree. C. In a separate flask,
n-BuLi (1.0 equiv) was added to a solution of a desired
oxazolidinone (V) (1.0 equiv) in anhydrous THF at -78.degree. C.
After stirring the reaction mixture at -78.degree. C. for 30
minutes, the mixed anhydride (IX) was added. The reaction mixture
was stirred at -78.degree. C. for 1 hour, then slowly allowed to
warm to room temperature. The reaction mixture was quenched with 4
ml of saturated aqueous ammonium chloride and extracted with ethyl
acetate. The combined organic phases were washed with brine and
dried over anhydrous sodium sulfate (Na.sub.2SO.sub.4). After
filtration, the solvent was removed under reduced pressure and the
residue was purified by column chromatography.
[0141] The following illustrates the synthesis of a compound of the
present invention, using Method A: 24
[0142] A solution of (4S)-4-benzyl-2-oxazolidinone (1.0 g, 5.6
mmol) in anhydrous THF (20 ml) was cooled to -78.degree. C. n-BuLi
(1.6M solution in hexanes, 3.5 ml, 5.6 mmol) was added dropwise to
the cooled solution. After 30 minutes, crotonyl chloride (0.59 ml,
6.2 mmol) was added to the mixture. The reaction was stirred for 30
minutes at -78.degree. C., then warmed slowly to room temperature.
The reaction mixture was quenched by addition of 4 ml saturated
aqueous ammonium chloride. The resultant reaction mixture was
diluted with ether, and washed with water and brine. The organic
layer was dried using anhydrous Na.sub.2SO.sub.4 and evaporated
under reduced pressure. Flash chromatography on a silica gel column
using ethyl acetate/hexane (1/3) as eluent provided compound 1,
i.e., (4S)-3-((E)-2-butenoyl)-4-benzyl-2-oxazolidinone, (1.50 g,
87%) as a white solid. m.p.: 83.degree. C. IR (KBr): 3027, 2922,
2539, 1772, 1682, 1351, 1209 cm.sup.-1. .sup.1H NMR (400 MHz,
CDCl.sub.3): 7.36-7.15 (m, 7H, aromatic H's, CH.dbd.CH); 4.75-4.70
(m, 1H, CHN); 4.23-4.15 (m, 2H, CH.sub.2O); 3.33 (dd, 1H, J=3.3,
13.4 Hz, CHHPh); 2.79 (dd, 1H, J=9.5, 13.4 Hz, CHHPh); 1.98 (dd,
3H, J=1.0, 6.2 Hz, CH.sub.3). .sup.13C NMR (100 MHz, CDCl.sub.3):
.delta. 164.69, 153.51, 146.95, 135.28, 129.37, 128.86, 127.23,
121.74, 66.01, 55.20, 37.79, 18.50.
[.alpha.].sub.D.sup.25=+7.77.degree. (c 2.00, CHCl.sub.3). HRMS:
Calcd for C.sub.14H.sub.16O.sub.3N (M+H).sup.+: 246.1125. Found:
246.1127.
[0143] Another compound, i.e., compound 11, having a structure:
25
[0144] was prepared from compound 1 as follows.
[0145] 10% Pd/C (0.10 g) was added to a solution of compound 1
(1.00 g, 4.08 mmol) in ethyl acetate (20.0 mL). The reaction
mixture was stirred in a hydrogen atmosphere (1 atm) for 1 hour,
then filtered through celite. The filtrate then was concentrated.
Flash chromatography over silica gel with hexane:ethyl acetate
(4:1) as eluent provided purified compound 11 (0.98 g, 97%) as a
colorless oil. IR (film): 2963, 1779, 1699, 1387, 1215, 771
cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 7.44-7.19 (m,
5H, Ph), 4.70-4.64 (m, 1H, CHN), 4.22-4.14 (m, 2H, CH.sub.2O), 3.29
(dd, 1H, J=3.2, 13.2 Hz, CHHPh), 3.01-2.83 (m, 2H, CH.sub.2CO),
2.77 (dd, 1H, J=9.6, 13.2 Hz, CHHPh), 1.77-1.68 (m, 2H,
CH.sub.2CH.sub.3), 1.03 (t, 3H, J=7.4 Hz, CH.sub.3); 13C NMR (100
MHz, CDCl.sub.3) .delta.: 173.12, 153.38, 135.24, 129.42, 128.85,
127.23, 66.06, 55.04, 37.84, 37.29, 17.61, 13.60;
[.alpha.].sub.D.sup.25=+71.0 (c 0.20, CHCl.sub.3); HRMS calcd for
C.sub.14H.sub.17O.sub.3N (M.sup.+): 247.1203; found: 247.1202.
[0146] Other compounds of the present invention, and some
comparative compounds, were synthesized using these same procedures
and appropriate starting materials. Additional examples of the
present invention, and comparative examples, include, but are not
limited to: 26272829303132333435363738394041424344
[0147] Compounds of the present invention were tested for an
ability to inhibit cell motility. The ability of a compound to
inhibit cell motility is related to the IC.sub.50 value for the
compound, i.e., the concentration of inhibitor required for 50%
inhibition of cell motility. The IC.sub.50 value for compounds of
the present invention were determined using the procedure set forth
hereafter.
[0148] The in vitro cell motility inhibitory IC.sub.50 values of
compounds set forth herein were determined by measuring the
inhibition as a function of the concentration of the test compound
over a range of 0 to 1 mM. The IC.sub.50 values of the compounds of
the present invention tested in the aforementioned assay ranged
from about 0.01 .mu.M to about 10 .mu.M.
[0149] The compounds of the present invention typically exhibit a
biological IC.sub.50 value of less than about 50 .mu.M, and
preferably less than about 40 .mu.M, and more preferably less than
about 30 .mu.M. The compounds of the present invention more
preferably exhibit a biological IC.sub.50 value of less than about
25 nM, and often less than about 20 nM. To achieve the full
advantage of the present invention, a present cell motility
inhibitor has an IC.sub.50 of about 700 pM (picomolar) to about 15
.mu.M.
[0150] A method of determining the ability of a compound to inhibit
or accelerate cell motility is a scrape-wound closure assay using
cultured mammalian epithelial cell monolayers. Mechanical scraping
of the epithelial monolayer induces cell sheet migration into the
resulting gap, a phenomenon characteristic of wounded epithelia and
mechanistically related to normal epithelial movements during
embryonic morphogenesis (A. Jacinto et al., Nat. Cell Biol, 3, E117
(2001)). Epithelial cell sheet migration in this method proceeds by
a Rac- and phosphoinositide-dependen- t cell crawling mechanism
resembling the motility of other animal cells that move as
individuals rather than as cell sheets (G. Fenteany et al., Curr.
Biol., 10, 831 (2000)).
[0151] Wound closure in MDCK cell monolayers primarily involves
cell spreading and motility rather than proliferation based on
observations and treatments that block cell growth without
inhibiting closure. However, after closure is complete and the gap
is covered, cell division is observed in the former wound area,
presumably as part of a homeo-static mechanism to regain the
original cell density (M. F. Olson et al., Science, 269; 1270
(1995)). Wound closure involves Rac- and phosphoinositide-dependent
actin polymerization and protrusion-driven cell crawling behavior,
where force generation for movement is distributed from the wound
margin to several rows of cells behind the margin. Accordingly,
this method is excellent for determining whether a compound affects
cell motility.
[0152] This test method showed that compound 1 is a potent
inhibitor of cell motility. In a separate assay, it also was
demonstrated that compound 1 inhibits cell proliferation. In
addition, compound 1 is biologically active at concentrations at
which there is no general toxicity, as determined by Trypan Blue
exclusion, morphological observations, and formation of new actin
bundles at the wound margin. Reversibility of the inhibitory
effects of compound 1 was demonstrated by washing compound 1 from
the culture medium and replacing it with un-treated medium.
[0153] Compound 1,
(4S)-3-((E)-2-butenoyl)-4-benzyl-2-oxazolidinone, is a
3,4-disubstituted oxazolidinone having an electrophilic
.alpha.,.beta.-unsaturated N-acyl group, which is theorized as
being related to inhibitory activity. Unlike the chemically
distinct 3,5-disubstituted oxazolidinones, such as linezolid,
compound 1, and other compounds of the present invention, do not
exhibit anti-bacterial activity against either Gram-positive or
Gram-negative bacteria. Compound 1 and other compounds of the
present invention, however, are novel and unexpected inhibitors of
eukaryotic cell motility and growth.
[0154] Compound 1 inhibits epithelial cell sheet migration during
wound closure in Madin-Darby Canine Kidney (MDCK) cells in a
statistically significant manner (p<0.05) (see FIG. 3). Based on
dose-response data, the calculated IC.sub.50 for inhibition of
wound closure at 12 hours by compound 1 is 14.0 .mu.M. This
inhibitory effect is reversible upon changing the medium to a
medium free of compound 1. It was found that compound 1 does not
act simply by inhibiting a serum component in the cell culture
medium because compound 1 also inhibited wound closure to a similar
degree relative to the control in serum-free conditions.
[0155] FIGS. 1A and 1B show percent wound closure in MDCK cell
monolayers in the presence or absence of compound 1 or compound 11.
Values are the mean with standard error of the mean (SEM) for
percent wound closure at the times indicated. FIG. 1A shows a
time-course of wound closure for different concentrations of
compound 1: 0.1% DMSO (control) (n=25) and 1 .mu.M (n=9), 5 .mu.M
(n=9), 10 .mu.M (n=15), 20 .mu.M (n=8), 50 .mu.M (n=19) of compound
1. The calculated IC.sub.50 for inhibition of wound closure at 12
hours by compound 1 from the dose-response experiments was 14.0
.mu.M (log IC.sub.50.+-.standard error of log
IC.sub.50=-4.853.+-.0.09815). FIG. 1B shows a time-course of wound
closure for the following treatments: 0.1% DMSO (control, n=25
wounded cell monolayers), 50 .mu.M compound 1 (n=19) or 50 .mu.M
compound 11 (n=10). The data presented in FIG. 1B shows
statistically significant differences for percent wound closure
between compound 1 (50 .mu.M) and control (0.1% DMSO) at all time
points.
[0156] It also was found that compound 1 inhibits cell motility and
wound closure in Madin-Darby canine kidney (MDCK) cell monolayers,
as shown by phase-contrast micrographs of cells under the following
treatment conditions: (A) 0.1% (v/v) DMSO control (solvent carrier
control with the same final solvent concentration as in the
experimental treatments) at 0, 12, and 36 hours after wounding; and
(B) 50 .mu.M compound 1 at 0, 12, and 36 hours after wounding.
[0157] Lamellipodial protrusion is known to drive most forms of
animal cell crawling. At any given time in the migrating cell
sheet, some cells are extending lamellipodia at the margin while
others are not. Although treatment with compound 1 does not
completely abolish formation of lamellipodia, treatment does result
in significantly fewer cells extending lamellipodia at the margin
see (Table 2), as determined by counting the number of
lamellipodial protrusions at the margin and dividing by the margin
perimeter length. Therefore, inhibition of cell sheet migration by
compound 1 at least in part is attributed to reduced formation of
lamellipodia at the would margin.
[0158] Table 1 shows that treatment with compound 1 leads to
decreased formation of lamellipodial protrusions at the wound
margin. Lamellipodial density at the margin of MDCK cell wounds was
determined under the following treatment conditions: 0.1% DMSO
(control, n=25 wounded monolayers), 50 .mu.M compound 1 (n=19), or
50 .mu.M compound 11 (n=10). Lamellipodial density is the number of
lamellipodial protrusions detected at the wound margin until
closure for the times indicated divided by margin perimeter length.
Values are mean.+-.SEM. Statistically significant differences
(p<0.05) from the control value are indicated with
asterisks.
2TABLE 1 Lamellipodial Density vs. Time Time (hours) Control
Compound 1 Compound 11 2 0.912 .+-. 0.205 0.341 .+-. 0.119 0.563
.+-. 0.148 4 1.425 .+-. 0.199 0.573 .+-. 0.194* 1.139 .+-. 0.270 6
1.592 .+-. 0.313 0.563 .+-. 0.142* 1.289 .+-. 0.276 8 1.697 .+-.
0.244 0.938 .+-. 0.184* 1.196 .+-. 0.312 10 1.389 .+-. 0.226 0.803
.+-. 0.203 1.609 .+-. 0.357 12 1.272 .+-. 0.215 0.520 .+-. 0.124*
1.368 .+-. 0.309
[0159] The course of wound closure frequently appears biphasic. In
the first few hours, there is often further gapping open of the
wound as damaged, but still attached, cells at the wound margin die
and perimarginal contractile actin/myosin bundles form and help
establish a stable wound margin. This is followed by increasing
protrusive activity at the margin and rapid closure of the wound, a
process inhibited by compound 1. This inhibition is reversible upon
washing compound 1 from the medium, indicating that inhibition is
not secondary to an irreversible cellular process, such as
apoptosis.
[0160] There is no evidence of toxicity or metabolic distress at
concentrations at which compound 1 inhibits cell migration as
determined by the Trypan blue exclusion cell viability assay. Cells
also remain normal in morphology with both cell-substratum and
cell-cell adhesion intact after treatment with compound 1.
Furthermore, treatment with compound 1 does not affect formation of
filamentous actin bundles at the wound margin, demonstrating that
cells are still metabolically capable of forming new actin
filaments, a process that requires adenosine triphosphate.
[0161] Compound 1 did not affect formation of filamentous actin
bundles at wound margin as shown in fluorescence micrographs of
rhodamine-phalloidin-stained MDCK cell monolayers 12 hours after
wounding under the following treatment conditions: (A) 0.1% DMSO
control and (B) 50 .mu.M compound 1. Although formation of
perimarginal actin bundles is not required for wound closure in
this system, these actin bundles may help to distribute force in
the first row of cells from actively protruding and moving cells to
less actively motile cells, thus making the closure process more
regular and uniform than it would be otherwise.
[0162] Compound 1 also inhibited cell growth in MDCK cells (FIG.
2A) at concentrations where no evidence of general toxicity exists,
and in a manner that was reversed by changing the medium to a
compound-free medium (FIG. 2B). FIGS. 2A and 2B show that compound
1, but not compound 11, reversibly inhibited MDCK cell
proliferation. In FIG. 2A, cells were plated at low cell density
(1.times.10.sup.4 cells/mL) and allowed to attach and begin to grow
for 48 hours prior to the start of experiment. The treatments were
0.1% DMSO (control, n=9 cultures), 50 .mu.M compound 1 (n=8), or 50
.mu.M compound 11 (n=7). Reported values are mean with SEM of the
percent increase in cell number from 0 to 48 hours after addition
of compound 1.
[0163] FIG. 2B shows that the inhibition of cell proliferation by
compound 1 was reversible by washing out the compound. The values
were mean with SEM of the percent increase in cell number another
48 hours after replacement of medium containing 50 .mu.M compound 1
(n=8 cultures) or 0.1% DMSO (n=9) with fresh medium containing 0.1%
DMSO.
[0164] As discussed in detail hereafter, it also was demonstrated
that compound 1 inhibited early development in frog embryos and
tissue dynamics in embryonic explants with equivalent potency. The
effects of compound 1 on frog embryos were most pronounced at
stages after most embryonic cell proliferation occurred, when
morphogenesis is mainly driven by cell motility and cell
rearrangement.
[0165] Compound 1 also did not affect the growth of either
Gram-positive or Gram-negative bacteria (Table 2). Furthermore,
although chiral 4-substituted oxazolidinones and
.alpha.,.beta.-unsaturated N-acyloxazolidinones have become widely
used in asymmetric organic synthesis, biological activity for these
oxazolidinones has not been reported (H. T. Sponsel et al., Am. J.
Physiol., 267, F257 (1994)). Compounds of the present invention,
therefore, represents a novel class of bioactive oxazolidinone.
[0166] Table 2 shows that compound 1 does not inhibit it the growth
of either Staphylococcus aureus (i.e., a Gram-postive bacterial
species) or Escherischia coli (i.e., a Gram-negative bacterial
species) in the presence of 0.1% DMSO (carrier solvent control) or
compound 1 (50 .mu.M), measured as absorbance at 600 nm. Reported
values are the mean.+-.SEM for triplicate samples.
3TABLE 2 Staphylococcus Staphylococcus Escherischia Escherischia
Time aureus aureus coli coli (hours) Control Compound 1 Control
Compound 1 0 0.091 .+-. 0.002 0.117 .+-. 0.037 0.128 .+-. 0.119
.+-. 0.001 0.004 1 0.109 .+-. 0.012 0.160 .+-. 0.056 0.190 .+-.
0.215 .+-. 0.005 0.004 2 0.259 .+-. 0.024 0.332 .+-. 0.130 0.572
.+-. 0.628 .+-. 0.024 0.009 3 0.753 .+-. 0.038 0.615 .+-. 0.162
1.137 .+-. 1.127 .+-. 0.019 0.018 4 1.394 .+-. 0.060 0.937 .+-.
0.181 1.614 .+-. 1.613 .+-. 0.035 0.017 17 2.725 .+-. 2.595 .+-.
0.024 0.008 27 3.695 .+-. 0.410 3.265 .+-. 0.196 31 4.067 .+-.
0.467 3.608 .+-. 0.271 38 2.854 .+-. 2.795 .+-. 0.050 0.023
[0167] It is theorized, but not relied upon herein, that in some
embodiments, the .alpha.,.beta.-unsaturated acyl moiety on nitrogen
atom of the oxazolidinone ring of compound 1 is a reactive
electrophile. Compound 1, therefore, has the potential to form a
covalent complex with a nucleophilic cellular target by
1,4-addition of the nucleophile to the .alpha.,.beta.-unsaturated
acyl group. Consistent with this theory, the closest saturated
analog to compound 1, i.e., compound 11, displays no statistically
significant bioactivity in either wound closure or cell
proliferaion assays, even at the highest concenraion tested (500
.mu.M). Compound 11 is the product of reductive hydrogenation of
compound 1, and is identical in structure to compound 1 except that
compound 11 lacks the C--C double bond, therefore making it
chemically unreactive to a nucleohile.
[0168] However, an interaction based on addition of a nucleophilic
target to the compound would be covalent and probably stable, but
the effects of compound 1 in both wound closure and cell
proliferation assays is reversible. An alternative theory,
therefore, is that the interaction can be labile and reversible.
The immediate target can be hypothesized as an endogenous
nucleophilic molecule, such as glutathione, or an intracellular
metabolite as opposed to a macromolecule, resulting in a conjugate,
which then mediates the biological effects of treatment, presumably
by interaction with a protein or other biological macromolecule.
Another hypothesis is that the .alpha.,.beta.-unsaturated N-acyl
group of compound 1 may be important for a reason unrelated to its
potential reactivity, such as reduced conformational
flexibility.
[0169] Additional tests illustrating inhibition of cell motility
and cell growth provided by compounds of the present invention,
were performed on frog embryos. In particular, cell behavior and
movements that underlie many aspects of gastrulation, neurulation,
and body axis formation have been extensively characterized in the
frog embryo (R. Keller et al., Philos. Trans. R. Soc. Lond. B Biol.
Sci., 355, 397-922 (2000)). Xenopus laevis (African clawed frog) is
an established model organism in developmental biology having
unique advantages in studies of vertebrate morphogenesis, such as
extremely well-characterized embryology, availability of a wide
range of explant and transplant techniques and manipulations, an
extensive fate mapping of the embryo, and a relative ease with
which direct observations of cell behavior can be made.
[0170] Xenopus laevis has large eggs (e.g., 1.2 mm diameter) and
early embryos that develop externally in an aqueous environment,
making the species useful for both embryological analysis and
delivery of pharmacological agents with precise temporal control.
Furthermore, Xenopus laevis oocytes, extracts, and embryos are
well-suited for biochemical analysis and have been used extensively
to isolate proteins, characterize protein function, and study the
biochemistry of the cell cycle and other cellular processes.
[0171] In order to identify compounds that specifically perturb
morphogenesis in the frog embryo, a suitable method for scoring
embryos by characteristic defects was developed. A useful starting
point was the "dorsoanterior index" (DAI). The DAI originally was
constructed by adding new morphological characteristics to the
older "index of axis deficiency" (S. R. Scharf et al., Dev. Biol.,
99, 75-87 (1983)). The DAI is a scale of dorsoanterior development
wherein embryos are assigned a score based on degree of
dorsoanterior phenotype (K. R. Kao et al., Dev. Biol., 127, 64-77
(1988)). The scale encompasses a score of 0 (for the most
axis-deficient or ventralized embryos) to 10 (for embryos with the
most enhanced dorsoanterior structures), with a score of 5
signifying a normal embryo.
[0172] Because the DAI focuses only on dorsoanterior phenotypes,
other categories were added to describe possible defects more
comprehensively, with an emphasis on characteristics that are
easily and unambiguously recognized in intact embryos, and that are
diagnostic of possible effects on morphogenesis during gastrulation
and neurulation. The most useful and readily scored categories that
covered the most frequent deficiencies were reduced anterior
structures and microcephaly, delayed appearance or absence of eye
pigmentation, overall shortness along the anteroposterior axis,
bent axis, bent tail only, reduced posterior structures/tail,
malformed or reduced fins, delayed or failed neural tube closure
(neural fold fusion), abnormal pigmentation pattern, ventral
swelling (edema), and delayed or abnormal visceral organogenesis
and yolk resorption. In addition, blastopore closure, an earlier
process that marks the completion of gastrulation, was
observed.
[0173] In these tests, test compounds were added individually to
wells each containing 4-8 embryos in 12-well plates. Embryos at
stage 8.5-9, according to the classification of Nieuwkoop and Faber
(P. I. Nieuwkoop et al., Normal Table of Xenopus laevis (Daudin).
Amsterdam: North-Holland Publ.) (1967)), were used because these
are late blastula stages before gastrulation begins at stage 10,
which allowed focusing primarily on compounds that affect
morphogenetic events in gastrulation and neurulation. These
embryos, therefore, have undergone a midblastula transition at
stage 8 (cleavage division 12) when zygotic transcription begins
(J. Newport et al., Cell, 30, 675-686 (1982); J. Newport et al.,
Cell, 30, 687-696 (1982)) but have not yet started the
morphogenetic movements of gastrulation. In separate experiments,
as indicated in the figures, a test compound was added to embryos
at the 2-cell stage (after the first cleavage division, Nieuwkoop
and Faber stage 2) in order to evaluate whether additional effects
were observed with earlier treatment, for example, defects due to
inhibition of inductive events at the earlier stages.
[0174] This test led to the discovery that compounds of the present
invention are inhibitors of morphogenesis in the frog embryo. The
structures of the compounds used in the study are summarized in
Table 3.
4TABLE 3 Structures of N-Acyloxazolidinones Bn = benzyl, i-Pr =
isopropyl, Me = methyl, Ph = phenyl Com- pound R.sup.1 R.sup.2
R.sup.5 R.sup.4 R.sup.3 1 45 H Bn H H 6 46 H H H H 11 47 H Bn H H
13 48 H Bn H H 15 49 H i-Pr H H 17 50 H Bn H H 23 51 H Bn H H 25 52
H Me H Ph 26 53 Me H Ph H 30 54 H Ph H H 31 55 H Bn H H 33 56 H Bn
H H 35 57 H Bn H H 41 58 H Bn H H 45 59 Bn H H H 52 60 H Bn H H 54
61 H Bn H H 55 62 H Bn H H
[0175] Of the characteristics evaluated, anteroposterior length,
head development, eye pigmentation, visceral organogenesis, and
yolk resorption were significantly different by Student's t-test in
embryos treated with compound 1 at stage 8.5-9 from the control
embryos, as observed later in post-neurula embryos at 48, 72, or 96
hours after treatment (FIG. 3A). FIG. 3 shows that treatment with
compound 1 results in specific developmental defects.
[0176] In FIGS. 3A and 3B, significance by Student's t-tests
(individual experimental vs. control) was indicated as follows: *
0.01.ltoreq.p<0.05; ** 0.001.ltoreq.p<0.01; *** p<0.001.
Each experiment was conducted in triplicate on at least three
separate occasions in 12-well plates for a total of 9 or more wells
per treatment (with 4-8 embryos for each well). Reported values
were expressed as the mean and standard error of the mean (SEM).
FIG. 3A illustrates the percentage of embryos with specific defects
48 hours (white bar), 72 hours (black bar) and 96 hours (striped
bar) after addition of compound 1 to embryos at stage 8.5-9 for
n=101 embryos (0.1% DMSO control) and n=129 embryos (15 .mu.M
compound 1).
[0177] The EC.sub.50 of compound 1 with respect to the defective
phenotype at 72 hours after treatment is 13.9 .mu.M (log
EC.sub.50+standard error of log EC.sub.50=--4.858+0.01959, Table
2), based on dose-response data using five different concentrations
over the range of the response. This value is equivalent to the
compound's IC.sub.50 for inhibition of cell migration in a
mammalian epithelial cell culture model. The compound 1-treated
defective embryos were alive over the course of the experiment with
a functioning muscularture and nervous system based on apparently
normal movement of the embryos. However, compound 1 became lethal
to the embryos at concentrations above 2 to 3 times the
EC.sub.50.
[0178] Compound 1-treated embryos were markedly shorter from head
to tail than control embryos at every time point, indicating an
inhibitory effect on anteroposterior elongation. Development of the
head was delayed, with significant frequencies of a reduced
forehead (slightly microcephalic) phenotype observed in
compound-treated embryos. The eye anlage formed later with compound
1 treatment, and eyes were subsequently less pigmented at every
stage over the course of their development. Visceral organogenesis
and accompanying yolk resorption were inhibited in treated embryos
compared to control embryos. Most easily observed was the delay in
formation of the gastrointestinal tract. The anal canal was
significantly under-developed in compound 1-treated embryos but
clearly defined in the control embryos at 96 hours.
[0179] Treatment of embryos at the 2-cell stage with compound 1
also had significant effects on anteroposterior elongation, head
and eye development, visceral organogenesis, and yolk resorption
(FIG. 3B). FIG. 3B shows percentage of embryos with specific
defects at 48 hours after addition of compound 1 to the embryos at
the 2-cell stage (after the first cleavage division, Nieuwkoop and
Faber stage 2) for n=171 (0.1% DMSO control) and 98 (15 .mu.M
compound 1). In addition, reduction in the length of the tail was
evident with treatment at the 2-cell stage (FIG. 3B), but not when
compound 1 was added to stage 8.5-9 embryos. Therefore, the effects
of compound 1 on anteroposterior elongation, head and eye
development, visceral organogenesis, and yolk resorption are
mediated primarily by a post-blastula activity, whereas reduction
in the tail is due exclusively to a pre-gastrula activity.
[0180] In addition to the defects observed in post-neurula embryos,
blastopore closure was delayed in gastrulating embryos following
compound 1 treatment at either stage 8.5-9 (FIG. 4A) or the 2-cell
stage (FIG. 4B). FIG. 4 shows that treatment with compound 1
results in delayed blastopore closure.
[0181] FIG. 4A shows the percentage of embryos (mean and SEM) with
open blastopore 12 hours after addition of compound 1 to embryos at
stage 8.5-9 for n=101 (0.1% DMSO control), 123 (10 .mu.M compound
1), 129 (15 .mu.M compound 1), 119 (20 .mu.M compound 1), 45 (50
.mu.M compound 1), 46 (75 .mu.M compound 1), and 50 (100 .mu.M
compound 1).
[0182] FIG. 4B shows the percentage of embryos (mean and SEM) with
an open blastopore 24 hours after addition of compound to embryos
at the 2-cell stage for n=171 (0.1% DMSO control), 87 (10 .mu.M
compound 1), 84 (15 .mu.M compound 1), and 97 (20 .mu.M compound
1).
[0183] Blastopore closure marks the completion of gastrulation at
the end of stage 12, and is the result of the tissue movements of
convergent extension (R. Keller et al., Development, 103, 193-209
(1988)), which involves tissue narrowing (convergence) coupled with
tissue elongation (extension).
[0184] Morphogenesis during gastrulation and neurulation occurs
largely by cell movements and rearrangements. There is reduced cell
division and increased cell motility after the midblastula
transition at stage 8 in the frog embryo (J. Newport et al., Cell,
30, 675-686 (1982); L. D. Etkin, Dev. Biol., 5, 509-225 (1988)). At
gastrulation in Xenopus laevis, which begins at stage 10, cells at
the dorsal lip of the presumptive blastopore begin to invaginate by
an apical actin/myosin-dependent contraction, becoming bottle
shaped (J. Hardin et al., Development, 103, 211-230 (1988)). The
subsequent involution of tissue rolling over the blastopore lip to
form the primary germ layers is mainly the result of convergent
extension, a process driven by protrusive cell motility and
intercalation in prospective mesodermal and neural tissues (R.
Keller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci., 355,
897-922 (2000)). Convergent extension continues through
neurulation, underlying the elongation of the anteroposterior axis
that turns a spherical embryo into a cigar-shaped post-neurula
embryo. These movements, therefore, are responsible for
establishing much of the body plan of the organism, establishing
its three-dimensional organization and positioning tissues such
that they are properly oriented relative to one another, allowing
appropriate inductive, and physical interactions required for later
organ development to take place.
[0185] It is hypothesized, but not relied upon herein, that the
effects of compound 1 are mediated by an activity or activities
after stage 9 during gastrulation and neurulation, and its effects
on blastopore closure and anteroposterior elongation are consistent
with inhibition of convergent extension. In fact, compound 1
inhibits convergent extension in Keller sandwich explants. It is
noteworthy that compound 1 also inhibits migration of mammalian
epithelial cells in culture, and a similar activity in the frog
embryo can account for many effects of compound 1 on development.
Therefore, compound 1 affects cell motility, cell polarity, and/or
cell adhesion in the embryo.
[0186] In order to determine the time following exposure to
compound 1 before which its activity becomes irreversible (at which
time the embryo would be committed to a defective phenotype), stage
8.5-9 embryos were treated with compound 1, then compound 1 was
completely washed out at intervals of 3 hours from different
parallel samples by replacing the medium with a compound-free
medium. Defects were scored and tested results by Student's t-test
against those for parallel control embryos, wherein DMSO carrier
solvent was initially added to a final concentration of 0.1% (v/v)
and then washed out at the same times as the experimental embryos.
Exposure of stage 8.5-9 embryos to 15 .mu.M compound 1 for up to 9
hours before washing out compound 1 had no significant effect on
later development. However, a 12-hour or more exposure to compound
1 resulted in significant defects later in the postneurula
analyses.
[0187] In an analogous experiment, when compound 1 was added to
two-cell stage embryos and then washed out every hour from
different parallel samples, the effects became irreversible between
only 2 and 3 hours of treatment. These results indicate that
between 9 and 12 hours following treatment of late blastula embryos
and between 2 and 3 hours following treatment of two-cell-stage
embryos with compound 1, the embryos become irreversibly committed
to the defective phenotype.
[0188] Dose-response experiments were conducted to calculate the
EC.sub.50 values of the different compounds of the present
invention with respect to the defective phenotype (Table 4).
Although defective, the treated embryos were alive with a
functioning musculature and nervous system. However, at
concentrations greater than 2-3 times the EC.sub.50, the compounds
became lethal to the embryos.
5TABLE 4 EC.sub.50 Values of Various N-Acyloxazolidinones EC.sub.50
values of each compound with respect to the defective phenotype at
72 hr following treatment, calculated from dose- response data
using 5 different concentrations (with n .gtoreq. 36 embryos for
each concentration). NA = no statistically significant biological
activity at any concentration. Compound EC.sub.50 (.mu.M) 1 13.9 6
39.1 11 NA 13 54.6 15 25.9 17 19.1 23 58.1 25 13.9 26 12.7 30 10.1
31 5.9 33 47.6 35 NA 41 27.1 45 8.9 52 6.1 54 30.4 55 NA
[0189] Compound 17, the cis N-crotonyl isomer of compound 1, was
essentially as active as compound 1 in the embryo. However,
compounds of the present invention in which the N-acyl moeity was
larger than the crotonyl displayed a reduced activity (compound 13,
compound 15, compound 23, compound 33, compound 35, compound 41,
compound 54, and compound 55). Some of these compounds also contain
a less electrophilic .alpha.,.beta.-unsaturated bond.
[0190] In addition to an unsaturated N-acyl group of moderate size,
hydrophobic substitutions on other positions of the oxazolidinone
ring may affect activity. Compound 6, which possesses an N-crotonyl
group, but no substituents on ring C4 or C5 positions, displayed
reduced activity. The 4-methyl, 5-phenyl enantiomers, i.e.,
compound 25 and compound 26, both were highly potent, as was
compound 45, the enantiomer of compound 1. These results suggest a
tolerance for different stereochemistries at these positions.
Treatment with each of the biologically active compounds resulted
in defects in the embryos that were qualitatively similar to those
caused by treatment with compound 1, suggesting intervention along
common or overlapping biological pathways.
[0191] In summary, compounds of structural formula (I) exhibit
specific inhibitory effects on gastrulation and neurulation in the
frog embryo has been found. These compounds of structural formula
(I) inhibit blastopore closure, anteroposterior elongation,
development of the head and eyes, visceral organogenesis and yolk
resorption. Between 9 and 12 hours after treatment of late blastula
embryos with a present compound, the embryos become irreversibly
committed to the defective phenotype.
[0192] Experimental Procedures
[0193] Cell culture conditions:
[0194] Madin-Darby canine kidney (MDCK) cells (American Type
Culture Collection cell line CCL-34) were cultured in Minimum
Essential Medium (with Earle's balanced salts, non-essential amino
acids, L-glutamine and sodium pyruvate) supplemented with 10%
newborn calf serum at 37.degree. C. and 5% carbon dioxide
(CO.sub.2). Main cultures were grown in either 25 cm.sup.3 or 75
cm.sup.3 tissue culture flasks with medium changes every two days.
When cultures were about 75% confluent, cells were passaged by
rinsing twice with single strength phosphate buffered saline (PBS)
and treating with a solution of 0.05% (w/v) trypsin/0.02% (w/v)
ethylenediaminetetraacetic acid (EDTA) in PBS to detach cells from
the flasks. After cells were detached, an equal volume of
serum-containing medium was added to inhibit the trypsin and cell
density was determined using a hemacytometer. Cells were replated
following dilution in fresh medium on new tissue culture flasks for
continued culture and multiwell tissue culture plates for
experiments. No culture used exceeded 15 passages. Experimental
cultures were grown in 12-, 24-, 48-, or 96-well tissue culture
plates with medium changes every two days.
[0195] Wound closure assay and compound screening:
[0196] MDCK cells were plated on multiwell tissue culture plates
and cultured at 37.degree. C. and 5% CO.sub.2 with medium changes
every two days until confluent. When the cultures reached
confluence, medium was changed again and all experimental
treatments were begun a day later. Compounds were solubilized in
dimethyl sulfoxide (DMSO) and added with fresh medium to cell
cultures at initial screening concentrations of 10 and 100 .mu.M.
Controls consisted of parallel wells to which DMSO solvent carrier
alone was added at the same concentration as that added with
experimental treatments (not exceeding 0.1% (v/v)). DMSO alone at
this concentration had no detectable effect on the cells.
Monolayers were scraped 30 min later with a micropipette and
ultramicro tips, allowing small wounds of consistent size to be
generated. Progress of wound closure was examined at set time
intervals following wounding by microscope, and inhibitory or
acceleratory effects on wound closure relative to parallel controls
noted. Compounds that exhibited biological activity then were
tested further at a range of concentrations with greater sample
size. Digital images of the wounded monolayers were taken every 2
hours for 18 hours, and then at 30, 36, 54, 60, and 72 hours
following wounding. The number of lamellipodia at the wound margin
was also counted at these times.
[0197] Imaging and analysis:
[0198] Wound closure assays were performed using either a Zeiss
Axiovert 200 inverted microscope with a Zeiss AxioCam CCD camera
and Improvision OpenLab image software running on an Apple Power
Mac G4 computer or a Zeiss Axiovert 25 inverted microscope with a
Roper Scientific/Photometrics CoolSNAP-Pro CCD camera and Roper
Scientific/Photometrics RS Image software on an Apple Power Mac G4
computer. Subsequent morpho-metric analyses using the digital
images were performed using the public domain NIH Image program
(developed at the U.S. National Institutes of Health and available
on the Internet at http://rsb.info.nih.gov/nih-image/). These
analyses entailed tracing the wound margin in each of the digital
images to determine the length of the margin perimeter and the
remaining open area. Microsoft Excel and GraphPad Prism software
were used for routine tabulation, analysis and graphing of the
data. The dose-response data was used to determine the IC.sub.50
for inhibition of wound closure by compound 1 using GraphPad Prism
software.
[0199] Filamentous actin staining:
[0200] MDCK cell monolayers on glass coverslips were fixed with
3.7% formaldehyde in PBS 12 hours after wounding. Cells were
permeabilized with 0.1% (w/v) TRITON.RTM. X-100 in PBS, stained
with 50 nM tetramethylrhodamine-conjugated phalloidin in PBS, and
washed twice with PBS. Coverslips were mounted in 90% glycerol/10%
ten strength PBS with 2 mg/mL p-phenylenediamine on glass slides
and then examined by fluorescence microscopy.
[0201] MDCK cell proliferation:
[0202] MDCK cells were plated on multiwell tissue culture plates at
low density (1.times.10.sup.4 cells/mL). Cells were allowed to
attach and begin to grow for 48 hours before the start of the
experiment. Fresh medium with or without compound was then added,
and cells were incubated for 48 hours. At both 0 and 48 hours, some
of the parallel wells for control and compound treatments were
washed twice with PBS and treated with trypsin/EDTA solution to
detach the cells. An equal volume of medium was added, cells were
collected and counted using a hemacytometer. For reversibility,
some of the parallel cultures for both control and compound
treatments were grown another 48 hours after washing twice with PBS
and replacing medium with fresh compound-free medium. At both the
time of washing out and 48 hours later, some of the parallel wells
for control and compound treatments were washed twice with PBS,
detached from the plates with trypsin/EDTA solution, and the number
of cells was counted.
[0203] Bacterial growth:
[0204] Staphylococcus aureus or Escherischia coli (DH5a strain)
were grown in Luria-Bertani (LB) medium for 12 hours at 37.degree.
C. in a shaking incubator. Dilutions into fresh LB medium with 0.1%
DMSO (solvent carrier control) or 50 .mu.M compound 1 were made to
an absorbance at 600 nm (A.sub.600) of about 0.1. Bacteria were
incubated at 37.degree. C. with shaking during the experiment, and
A.sub.600 measurements were made at set time intervals during log
growth phase until the plateau phase was reached.
[0205] Xenopus laevis Manipulation and Embryo Preparation
[0206] Standard procedures were used in the care of animals, gamete
preparation, fertilization of oocytes, and culture of embryos (B.
K. Kay et al., Methods in Cell Biology, Volumn 36 (San Diego:
Academic Press, Inc.) (1991)). In order to stimulate ovulation,
female frogs were injected with 500 units of human chorionic
gonadotropin (HCG). Eggs were squeezed from female frogs 12 hours
later. Testes were removed from euthanized male frogs that had been
primed with 50 units of HCG 4-6 hours earlier. The testes were
stored at 4.degree. C. in single strength Marc's Modified Ringer's
solution with 50 .mu.g/ml gentamycin and used for no more than 10
days. Small pieces of testes were macerated and spread over eggs
for in vitro fertilization in Petri dishes. Fertilized eggs were
placed in one-tenth strength Modified Barth's Saline (MBS) with 50
.mu.g/ml gentamycin at constant temperature (19.degree. C.) in a
temperature-controlled incubator and development allowed to proceed
under these conditions. Embryos were dejellied by briefly swirling
them in a solution of 2% (w/v) L-cysteine, pH 8.0. The embryos then
were rinsed extensively with one-tenth strength MBS to wash out the
cysteine. Experiments were started within an hour of dejellying the
embryos.
[0207] Assay Protocol
[0208] Experiments were carried out in 12-well flat-bottom
polystyrene plates with 4-8 embryos per well in 1.0 ml of one-tenth
strength MBS with 50 .mu.g/ml gentamycin at 19.degree. C. Staging
of embryos was done according to the classification of Nieuwkoop
and Faber. Embryos at stage 8.5-9 (late blastula stages prior to
the debut of gastrulation at stage 10) or at the 2-cell stage
(after the first cleavage division, Nieuwkoop and Faber stage 2),
as indicated in the figures, were treated with a test compound or
dimethylsulfoxide (DMSO) carrier solvent and maintained at
19.degree. C. DMSO concentration did not exceed 0.1% (v/v) in any
experiment. DMSO alone at this concentration had no detectable
effect on the embryos. For compound 22 and compound 41,
dimethylformamide (DMF) was used as solvent because these compounds
were more soluble in DMF, with final concentration of solvent again
not exceeding 0.1% (v/v) in any experiment. As with 0.1% DMSO, this
concentration of DMF had no effect on development of the frog
embryos. Each experiment was conducted in triplicate on at least
three separate occasions for a total of 9 or more wells per
treatment. Embryos were observed using a stereomicroscope and
scored for defects or delayed development at set times (0, 3, 6, 9,
12, 24, 48, 72, and 96 hours) following treatment.
[0209] Scoring Method
[0210] A range of morphological criteria were used to score for
defects in embryogenesis as a function of time after addition of
small molecules to live intact embryos. These criteria included
delayed blastopore closure, reduced anterior structures and
microcephaly, delayed appearance or absence of eye pigmentation,
overall shortness along the anteroposterior axis, bent axis, bent
tail only, reduced posterior structures/tail, malformed or reduced
fins, delayed or failed neural tube closure (neural fold fusion),
abnormal pigmentation pattern, ventral swelling (edema), and
delayed or abnormal visceral organogenesis and yolk resorption.
Scoring was done for all embryos in each well at the time of
observation. Any dead embryos in a well were noted as such and not
used for scoring defects. A separate record of lethality was
maintained. Measurements of anteroposterior length from head to
tail were made using an eyepiece reticle at the time of
observation. If an embryo was not positioned well for accurate
scoring, the plate was shaken gently or the embryo was carefully
moved into a better position for observation using a hair loop.
Digital images were taken for documentation purposes for each well
using a CCD camera attached to the stereomicroscope. GraphPad Prism
software was used to graph data, perform statistical analyses and
calculate EC.sub.50 values.
[0211] Obviously, many modifications and variations of the
invention as hereinbefore set forth can be made without departing
from the spirit and scope thereof, and, therefore, only such
limitations should be imposed as are indicated by the appended
claims.
* * * * *
References