U.S. patent application number 10/899495 was filed with the patent office on 2005-01-06 for kinesin inhibitors.
This patent application is currently assigned to President and Fellows of Harvard College. Invention is credited to Feng, Yan, Kapoor, Tarun M., Maliga, Zoltan, Mayer, Thomas, Mitchison, Timothy J., Yarrow, Justin.
Application Number | 20050004156 10/899495 |
Document ID | / |
Family ID | 33554760 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050004156 |
Kind Code |
A1 |
Feng, Yan ; et al. |
January 6, 2005 |
Kinesin inhibitors
Abstract
The present invention provides for compounds, compositions,
methods and systems for inhibiting cell growth. More specifically,
the present invention provides for methods, compounds and
compositions which are capable of inhibiting mitosis in
metabolically active cells. Compounds, compositions and methods of
the present invention inhibit the activity of a protein involved in
the assembly and maintenance of the mitotic spindle. One class of
proteins which acts on the mitotic spindle is the family of mitotic
kinesins, a subset of the kinesin superfamily.
Inventors: |
Feng, Yan; (Brookline,
MA) ; Kapoor, Tarun M.; (New York, NY) ;
Mayer, Thomas; (Munchen, DE) ; Maliga, Zoltan;
(East Brunswick, NJ) ; Mitchison, Timothy J.;
(Brooklin, MA) ; Yarrow, Justin; (Boston,
MA) |
Correspondence
Address: |
Choate, Hall & Stewart
Exchange Place
53 State Street
Boston
MA
02109
US
|
Assignee: |
President and Fellows of Harvard
College
|
Family ID: |
33554760 |
Appl. No.: |
10/899495 |
Filed: |
July 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10899495 |
Jul 26, 2004 |
|
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09791339 |
Feb 23, 2001 |
|
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60184540 |
Feb 24, 2000 |
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Current U.S.
Class: |
514/287 ;
546/64 |
Current CPC
Class: |
C07D 471/14
20130101 |
Class at
Publication: |
514/287 ;
546/064 |
International
Class: |
C07D 471/14; A61K
031/4745 |
Goverment Interests
[0002] The work described in the present application was supported
by a grant from the National Institutes of Health (CA78048).
Claims
We claim:
1. A compound having the structure: 16wherein R.sub.1-4, 8-10, as
valency and stability permit are each independently selected from
the group consisting of substituted or unsubstituted, branched or
unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heterocycle, heteroalkyl, OH,
OR.sub.A, C(.dbd.O)R.sub.A, CO.sub.2H, CO.sub.2R.sub.A, CN,
halogen, SH, SR.sub.A, SOR.sub.A, SO.sub.2R.sub.A, NO.sub.2,
NH.sub.2, NHR.sub.A, N(R.sub.A).sub.2, hydrogen and NHC(O)R.sub.A,
wherein each occurrence of R.sub.A as valency and stability permit
is independently selected from the group consisting of substituted
or unsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,
heteroaryloxy, heteroarylthio, and hydrogen, and wherein R.sub.5-7,
11 as valency and stability permit are each independently selected
from the group consisting of substituted or unsubstituted, branched
or unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heterocycle, heteroalkyl, hydrogen,
--(C.dbd.O)R.sub.B, and --(SO.sub.2)R.sub.B, wherein each
occurrence of R.sub.B as valency and stability permit is
independently selected from the group consisting of substituted or
unsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,
heteroaryloxy, heteroarylthio, hydrogen, and --(C.dbd.O)R.sub.C,
wherein R.sub.C as valency and stability permit is selected from
the group consisting of substituted or unsubstituted, branched or
unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle, alkoxy,
aryloxy, alkylthio, arylthio, heteroaryloxy, heteroarylthio, and
hydrogen.
2. The compound of claim 1 having the structure: 17wherein X is H,
O, halogen, CF.sub.3, or carbon; and wherein R and R' as valency
and stability permit are each independently selected from the group
consisting of substituted or unsubstituted, branched or unbranched
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,
aryl, heteroaryl, heterocycle, heteroalkyl, hydrogen,
--(C.dbd.O)R.sub.D, and --(SO.sub.2)R.sub.D, wherein each
occurrence of R.sub.C as valency and stability permit is
independently selected from the group consisting of substituted or
unsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,
heteroaryloxy, heteroarylthio, hydrogen, and --(C.dbd.O)R.sub.E,
wherein R.sub.E as valency and stability permit is selected from
the group consisting of substituted or unsubstituted, branched or
unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle, alkoxy,
aryloxy, alkylthio, arylthio, heteroaryloxy, heteroarylthio, and
hydrogen.
3. (Canceled)
4. The compound of claim 1 having the structure: 18wherein Ar is
phenyl, p-hydroxyphenyl, m-hydroxyphenyl, m-methoxyphenyl or
m-fluorophenyl and R is as valency and stability permit are each
independently selected from the group consisting of substituted or
unsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heterocycle, heteroalkyl, hydrogen, --(C.dbd.O)R.sub.H, and
--(SO.sub.2)R.sub.H, wherein each occurrence of R.sub.H as valency
and stability permit is independently selected from the group
consisting of substituted or unsubstituted, branched or unbranched
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,
aryl, heteroaryl, heteroalkyl, heterocycle, alkoxy, aryloxy,
alkylthio, arylthio, heteroaryloxy, heteroarylthio, hydrogen, and
--(C.dbd.O)R.sub.I, wherein R.sub.I as valency and stability permit
is selected from the group consisting of substituted or
unsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,
heteroaryloxy, heteroarylthio, and hydrogen.
5. A pharmaceutical composition comprising at least one of the
compounds of claims 1, 2 or 4 and further comprising a
pharmaceutically acceptable carrier.
6. The composition of claim 5 wherein the compound has the
structure: 19
7. (Canceled)
8. (Canceled)
9. The composition of claim 6 wherein the compound has the
stereochemistry: 20
10. A method of treating cancer in a subject comprising
administering to a subject in need thereof a therapeutically
effective amount of at least one of any of the compounds of claims
1, 2 or 4 and a suitable pharmaceutically acceptable carrier.
11. The method of claim 10 wherein the compound has the structure:
21
12. The method of claim 11 wherein the compound has the
stereochemistry: 22
Description
PRIORITY INFORMATION
[0001] The present application claims priority under 35 U.S.C.
.sctn. 119(e) to the U.S. provisional patent application Ser. No.
60/184,540 by Mitchison et al. filed on Feb. 24, 2000. U.S. Ser.
No. 60/184,540 is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Cell-permeable small molecules can rapidly perturb the
function of their targets and are therefore powerful tools to
dissect dynamic cellular processes. However, such modulators are
not available for most of the proteins involved in essential
processes, and many of the ones that are available are nonspecific.
The only known small molecules that specifically affect the mitotic
machinery target tubulin (E. Hamel, Med. Res. Rev. 16, 207 (1996)),
a subunit of the microtubules in the mitotic spindle.
[0004] One class of proteins involved in the assembly and
maintenance of the mitotic spindle is the family of mitotic
kinesins, a subset of the kinesin superfamily. This superfamily
contains over 100 proteins, whose other functions include organelle
transport and membrane organization (R. D. Vale and R. J.
Fletterick, Annu. Rev. Cell Dev. Biol. 13, 745 (1997)). The first
evidence that mitotic kinesins are important in establishing
spindle bipolarity came from genetic studies: temperature-sensitive
mutants in the BimC family of kinesins do not form bipolar spindles
at the restrictive temperature (A. P. Enos and N. R. Morris, Cell
60, 1019 (1990); I. Hagan and M. Yanagida, Nature 356, 74 (1992);
M. A. Hoyt et al., J. Cell Biol. 118, 109 (1992)). Inhibition of
the BimC kinesin Eg5 with Eg5-specific antibodies also induced
monoasters similar to those observed after treatment with monastrol
(A. Blangy et al., Cell 83, 1159 (1995); K. E. Sawin et al., Nature
359, 540 (1992)). Like other kinesins, Eg5 can drive the movement
of microtubules in vitro (T. M. Kapoor and T. J. Mitchison, Proc.
Natl. Acad. Sci. U.S.A. 96, 9106 (1999)).
[0005] Enzymes in the kinesin superfamily use the free energy of
ATP hydrolysis to drive intracellular movement and influence
cytoskeleton organization (R. D. Vale and R. J. Fletterick, Annu.
Rev. Cell. Dev. Biol. 13, 745-777 (1997)). More than 90 members of
this family are known. Historically, kinesins have been proposed to
move cellular cargo along polar microtubule tracks. More recently
it has been shown that these ATPases can modulate dynamics of the
underlying microtubule network (A. Desai et al., Cell 96, 69-78
(1999)), couple movement of cargo to the microtubule polymerization
or depolymerization (K. W. Wood et al., Cell 91, 357-366 (1997)),
and crosslink microtubules in dynamic structures (D. J. Sharp et
al., J. Cell Biol. 144, 125-138 (1999)). Kinesins thus play central
roles in mitotic and meiotic spindle formation, chromosome
alignment and separation, axonal transport, endocytosis, secretion,
and membrane trafficking. The cargo associated with these motor
proteins includes intracellular vesicles, organelles, chromosomes,
kinetochores, intermediate filaments, microtubules, and even other
motors (reviewed in C. E. Walczak and T. J. Mitchison, Cell 85,
943-946 (1996); and N. Hirokawa, Science 279, 519-526 (1998)).
[0006] For many of these processes, more than one kinesin is
implicated, and the specific cargo associated with a given motor
protein has been difficult to establish. For example, conventional
kinesin (R. D. Vale et al., Cell 42, 39-50 (1985)) (the founding
member of the family) is one of a subset of kinesins involved in
organelle transport in mammalian cells. This group includes KIF1,
KIF2, KIFC2/C3, and KIF4; and more recently, 18 new murine KIFs
have been reported, many of which may functionally overlap with the
transport kinesins (reviewed in N. Hirokawa, Science 279, 519-526
(1998)). It thus has been difficult to tie down the in vivo
function(s) of conventional kinesin. Experiments using antisense
techniques and microinjection of inhibitory antibodies have been
further complicated by recent observations of efficient endoplasmic
reticulum to Golgi transport in the absence of microtubules, albeit
under restricted conditions (reviewed in G. S. Bloom and L. S.
Goldstein, J. Cell Biol. 140, 1277-1280 (1998)). Similar problems
have been encountered in dissecting the function of kinesins in
mitosis. Extensive genetic analysis of motors in Saccharomyces
cerevisiae has linked all but one of the six kinesins to spindle
function. None of these five motors are individually required for
the viability of yeast, implying that more than one motor is
associated with essential aspects of spindle movement (W. S.
Saunders and M. A. Hoyt, Cell 70, 451-458 (1992); M. A. Hoyt et
al., Proc. Natl. Acad Sci USA 94, 12747-12748 (1997)).
Immunodepletion and add-back approaches in Xenopus extract spindle
assembly assays have provided similarly ambiguous data (C. E.
Walczak et al., Curr. Biol. 8, 903-913 (1998)).
[0007] Small molecules that conditionally activate or inactivate a
protein are valuable tools for analyzing cellular functions of
proteins (D. T. Hung et al., Chem. Biol. 3, 623-639 (1996)). Their
use provides an alternative to conventional biochemical and genetic
approaches. However, to date there have been few reports of small
molecules that can reversibly alter the function of motor proteins.
Butanedione monoxime has been used to probe the role of myosin in
cell movement (L. P. Cramer and T. J. Mitchison, J. Cell Biol. 131,
179-189 (1995)), but its specificity has been questioned (G.
Steinberg and J. R. McIntosh, Eur. J. Cell Biol. 77, 284-293
(1998)). A natural product inhibitor of kinesin has been reported
(R. Sakowicz et al., Science 280, 292-295 (1998)), but is thought
not to be selective for different kinesins and thus is not useful
for probing the role of one specific kinesin in a complex process.
Hyman et al. (A. A. Hyman et al., Nature (London) 359, 533-536
(1992)) have used ATP analogs to distinguish between microtubule
motility at kinetochores driven by a kinesin and a dynein, but
again, this approach is unlikely to distinguish between different
kinesins. Thus currently there is a lack of small molecule
activators or inhibitors that are specific for one member of the
kinesin family. Such an inhibitory molecule with specificity for a
particular member of a kinesin class would be useful as an
anti-mitotic and also as an anti-cancer, anti-tumorigenic
compound.
SUMMARY OF THE INVENTION
[0008] The present invention provides compounds, compositions,
methods and systems for inhibiting cell growth. More specifically,
the present invention provides methods, compounds and compositions
that are capable of inhibiting mitosis in metabolically active
cells. Compounds, and compositions of the present invention inhibit
the activity of a protein involved in the assembly and maintenance
of the mitotic spindle. One class of proteins which acts on the
mitotic spindle is the family of mitotic kinesins, a subset of the
kinesin superfamily.
[0009] In one aspect, the present invention provides a compound
having the formula (I): 1
[0010] wherein R.sub.1-4, 8-10, as valency and stability permit are
each independently selected from the group consisting of
substituted or-unsubstituted, branched or unbranched alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heterocycle, heteroalkyl, OH, OR.sub.A,
C(.dbd.O)R.sub.A, CO.sub.2H, CO.sub.2R.sub.A, CN, halogen, SH,
SR.sub.A, SOR.sub.A, SO.sub.2R.sub.A, NO.sub.2, NH.sub.2,
NHR.sub.A, N(R.sub.A).sub.2, hydrogen and NHC(O)R.sub.A, wherein
each occurrence of R.sub.A as valency and stability permit is
independently selected from the group consisting of substituted or
unsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,
heteroaryloxy, heteroarylthio, and hydrogen, and
[0011] wherein R.sub.5-7, 11 as valency and stability permit are
each independently selected from the group consisting of
substituted or unsubstituted, branched or unbranched alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heterocycle, heteroalkyl, hydrogen, --(C.dbd.O)R.sub.B,
and --(SO.sub.2)R.sub.B, wherein each occurrence of R.sub.B as
valency and stability permit is independently selected from the
group consisting of substituted or unsubstituted, branched or
unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle, alkoxy,
aryloxy, alkylthio, arylthio, heteroaryloxy, heteroarylthio,
hydrogen, and --(C.dbd.O)R.sub.C, wherein R.sub.C as valency and
stability permit is selected from the group consisting of
substituted or unsubstituted, branched or unbranched alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio,
arylthio, heteroaryloxy, heteroarylthio, and hydrogen.
[0012] In another aspect, the present invention provides a compound
having the formula (II): 2
[0013] wherein X is H, O, halogen, CF.sub.3, or carbon; and wherein
R and R' as valency and stability permit are each independently
selected from the group consisting of substituted or unsubstituted,
branched or unbranched alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocycle,
heteroalkyl, hydrogen, --(C.dbd.O)R.sub.D, and --(SO.sub.2)R.sub.D,
wherein each occurrence of R.sub.D as valency and stability permit
is independently selected from the group consisting of substituted
or unsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,
heteroaryloxy, heteroarylthio, hydrogen, and --(C.dbd.O)R.sub.E,
wherein R.sub.E as valency and stability permit is selected from
the group consisting of substituted or unsubstituted, branched or
unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle, alkoxy,
aryloxy, alkylthio, arylthio, heteroaryloxy, heteroarylthio, and
hydrogen.
[0014] In yet another aspect, the present invention provides a
compound having the formula (III): 3
[0015] wherein R, R' and R" as as valency and stability permit are
each independently selected from the group consisting of
substituted or unsubstituted, branched or unbranched alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heterocycle, heteroalkyl, hydrogen, - (C.dbd.O)R.sub.F,
and --(SO.sub.2)R.sub.F, wherein each occurrence of R.sub.F as
valency and stability permit is independently selected from the
group consisting of substituted or unsubstituted, branched or
unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle, alkoxy,
aryloxy, alkylthio, arylthio, heteroaryloxy, heteroarylthio,
hydrogen, and --(C.dbd.O)R.sub.G, wherein R.sub.G as valency and
stability permit is selected from the group consisting of
substituted or unsubstituted, branched or unbranched alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio,
arylthio, heteroaryloxy, heteroarylthio, and hydrogen.
[0016] In yet another aspect, the present invention provides a
compound having the formula (IV) 4
[0017] wherein Ar is phenyl, p-hydroxyphenyl, m-hydroxyphenyl,
m-methoxyphenyl, m-halogenphenyl or m-fluorophenyl and R is as
valency and stability permit are each independently selected from
the group consisting of substituted or unsubstituted, branched or
unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heterocycle, heteroalkyl, hydrogen,
--(C.dbd.O)R.sub.H, and --(SO.sub.2)R.sub.H, wherein each
occurrence of R.sub.H as valency and stability permit is
independently selected from the group consisting of substituted or
unsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,
heteroaryloxy, heteroarylthio, hydrogen, and --(C.dbd.O)R.sub.I,
wherein R.sub.I as valency and stability permit is selected from
the group consisting of substituted or unsubstituted, branched or
unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle, alkoxy,
aryloxy, alkylthio, arylthio, heteroaryloxy, heteroarylthio, and
hydrogen.
[0018] In still yet another aspect, the present invention provides
a pharmaceutical composition comprising one or more of the
compounds (I-IV) as described by the present invention and further
comprising a pharmaceutically acceptable carrier.
[0019] In yet another aspect, the present invention provides a
pharmaceutical composition comprising a compound having the formula
(V): 5
[0020] and further comprising a pharmaceutically acceptable
carrier.
[0021] In yet another aspect, the present invention provides a
method of treating an individual with a cancerous growth comprising
administering to the individual a therapeutically effective and
non-toxic dose of a composition comprising a compound having the
formula (V): 6
[0022] In yet another aspect, the present invention provides a
method of treating an individual with a cancerous growth comprising
administering to the individual a therapeutically effective and
non-toxic dose of a composition comprising one or more of the
compounds I-IV as described by the present invention.
DESCRIPTION OF DRAWINGS
[0023] FIG. 1 depicts the fragmentation of the Golgi and the
formation of mono-astral spindles by cells treated with 22C16.
[0024] FIG. 2 depicts the results of experiments examining the
motility activity of full length Xenopus Eg5 with and without the
beta-carboline, 22C16. Also shown is the washout experiment
demonstrating that 22C16 acts reversibly to inhibit the motility of
Eg5.
[0025] FIG. 3 is a photograph of an automated screening microscope,
with a schematic of the steps in the screening process.
[0026] FIG. 4 is a schematic depiction of the protocol of the
synchronous screen for exocytosis. The BSC1 cells were mixed with
media containing the adenovirus with the VSVG-GFP construct and
plated at 3,000 cells/well (.about.80% confluency) in a 384-well
tray. After transduction, the cells were kept for 18 hours at
40.degree. C. (the non-permissive temperature) leading to
accumulation of VSVG-GFP in the ER. 50-100 nL of each chemical (in
DMSO) was diluted with 40 .mu.l of media and transferred to assay
wells, followed by incubation for another hour at 40.degree. C. The
cells were then either (a) retained at 40.degree. C. for 2 hours or
incubated for 2 hours at: (b) 20.degree. C.; or (c) 32.degree. C.
With no inhibitors present, VSVG-GFP behaves as follows: (a)
retained in ER; (b) exits the ER but is retained in the trans-Golgi
network; (c) exits the ER, traffics to the Golgi and continues to
the plasma membrane. Traffic was ended by transferring the trays to
4.degree. C. and fixing with 4% paraformaldehyde.
[0027] FIG. 5 shows the intracellular localization of VSVG-GFP
along the secretory pathway. These images demonstrate (a) the
retention of VSVG-GFP in the ER in cells kept at 40.degree. C. The
ER appears as a reticulum throughout the cell. (b) Retention of
VSVG-GFP in the perinuclear regions corresponding to the Golgi
complex in cells incubated at 20.degree. C.; and (c) Accumulation
of VSVG-GFP at the plasma membrane, following exit from the ER and
traffic through the Golgi in cells incubated at 32.degree. C.
[0028] FIG. 6 shows images of cells with disruption in membrane
traffic of VSVG-GFP due to various hits. These images are typical
examples of the type of disruptions observed during the primary
screen induced by the chemicals following the protocol summarized
in FIG. 4. To facilitate the presentation of the data, we only show
1/4 of the complete field.
[0029] FIG. 7 depicts a description of the NADH enzyme coupled
ATPase assay used to study the inhibition of the ability of
purified human recombinant Eg5 to hydrolyze ATP in the presence and
absence of 22C16 and monastrol.
[0030] FIG. 8 depicts data from an experiment examining the
inhibition of Eg5 ATPase activity by the beta-carboline, 22C1 6. As
indicated, the IC50 for this experiment is 1.1 uM.
[0031] FIG. 9 depicts data from an experiment examining the
inhibition of Eg5 ATPase activity by monastrol. As indicated, the
IC50 for this experiment is 5.3 uM.
[0032] FIG. 10 depicts the synthesis of monastroline.
[0033] FIG. 11 depicts the synthesis of derivatives of monastroline
(Ar=phenyl, p-hydroxyphenyl, m-hydroxyphenyl, m-methoxyphenyl and
m-fluorophenyl and R=hydrogen, methyl, ethyl, n-butyl, or
.alpha.-benzyl). For conjugation of monastroline and derivatives to
solid supports R can be C.sub.nCOOR, where n=1-20, preferably 1, 3,
5, 7, 9, 11 or 13, and wherein R is methyl, ethyl, n-butyl or
.alpha.-benzyl.
[0034] FIG. 12 depicts the synthesis of derivatives the
beta-carboline core of monastroline starting with derivatives of
tryptophan that are readily available commercially.
[0035] FIG. 13 depicts the derivatives of monastroline where X is
H, O Cl, Br, CF3, I or C.
[0036] FIG. 14 depicts the structure of a trans isomer of
monastroline which has an IC50 approximately 8 times lower than for
monastrol as assay by the ATPase assay described in Example 2.
DEFINITIONS
[0037] As discussed above, the present invention provides a novel
class of compounds useful for the treatment of cancer and other
uncontrolled cell proliferative conditions related thereto.
Compounds of this invention comprise those, as set forth above and
described herein, and are illustrated in part by the various
classes, subgenera and species disclosed elsewhere herein.
[0038] "Therapeutically effective": As used herein, the term
"therapeutically effective" is defined as an amount of a compound
or composition comprising the compound which is administered to an
individual in need thereof to slow or cease uncontrolled or
abnormal growth of cells in the individual without toxicity.
[0039] "Cancer or cancerous growth": As used herein, the term
"cancer" or "cancerous growth" means the uncontrolled, abnormal
growth of cells and includes within its scope all the well known
diseases that are caused by the uncontrolled and abnormal growth of
cells. Non-limiting examples of common cancers include bladder
cancer, breast cancer, colon cancer, endometrial cancer, head and
neck cancer, lung cancer, melanoma, non-hodgkin's lymphoma,
prostate cancer, and rectal cancer. A complete list of cancers is
available from the National Cancer Institute (Bethesda, Md.).
[0040] It will be appreciated by one of ordinary skill in the art
that numerous asymmetric centers exist in the compounds of the
present invention. Thus, inventive compounds and pharmaceutical
compositions thereof may be in the form of an individual
enantiomer, diastereomer or geometric isomer, or may be in the form
of a mixture of stereoisomers. Additionally, in certain preferred
embodiments, as detailed herein, the method of the present
invention provides for the stereoselective synthesis of alkaloids
and analogues thereof. Thus, in certain embodiments, the compounds
of the invention are enantiopure.
[0041] Additionally, the present invention provides
pharmaceutically acceptable derivatives of the foregoing compounds,
and methods of treating a subject using these compounds,
pharmaceutical compositions thereof, or either of these in
combination with one or more additional therapeutic agents. The
phrase, "pharmaceutically acceptable derivative", as used herein,
denotes any pharmaceutically acceptable salt, ester, or salt of
such ester, of such compound, or any other adduct or derivative
which, upon administration to a patient, is capable of providing
(directly or indirectly) a compound as otherwise described herein,
or a metabolite or residue thereof. Pharmaceutically acceptable
derivatives thus include among others pro-drugs. A pro-drug is a
derivative of a compound, usually with significantly reduced
pharmacological activity, which contains an additional moiety which
is susceptible to removal in vivo yielding the parent molecule as
the pharmacologically active species. An example of a pro-drug is
an ester which is cleaved in vivo to yield a compound of interest.
Pro-drugs of a variety of compounds, and materials and methods for
derivatizing the parent compounds to create the pro-drugs, are
known and may be adapted to the present invention. Certain
exemplary pharmaceutical compositions and pharmaceutically
acceptable derivatives will be discussed in more detail herein
below.
[0042] Certain compounds of the present invention, and definitions
of specific functional groups are also described in more detail
below. For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 75.sup.th Ed.,
inside cover, and specific functional groups are defined as
described therein. Additionally, general principles of organic
chemistry, as well as specific functional moieties and reactivity,
are described in "Organic Chemistry", Thomas Sorrell, University
Science Books, Sausalito: 1999, the entire contents of which are
incorporated herein by reference.
[0043] Furthermore, it will be appreciated by one of ordinary skill
in the art that the synthetic methods, as described herein, utilize
a variety of protecting groups. By the term "protecting group", has
used herein, it is meant that a particular functional moiety, e.g.,
O, S, or N, is temporarily blocked so that a reaction can be
carried out selectively at another reactive site in a
multifunctional compound. In preferred embodiments, a protecting
group reacts selectively in good yield to give a protected
substrate that is stable to the projected reactions; the protecting
group must be selectively removed in good yield by readily
available, preferably nontoxic reagents that do not attack the
other functional groups; the protecting group forms an easily
separable derivative (more preferably without the generation of new
stereogenic centers); and the protecting group has a minimum of
additional functionality to avoid further sites of reaction. As
detailed herein, oxygen, sulfur, nitrogen and carbon protecting
groups may be utilized. Exemplary protecting groups are detailed
herein, however, it will be appreciated that the present invention
is not intended to be limited to these protecting groups; rather, a
variety of additional equivalent protecting groups can be readily
identified using the above criteria and utilized in the method of
the present invention. Additionally, a variety of protecting groups
are described in "Protective Groups in Organic Synthesis" Third Ed.
Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New
York: 1999, the entire contents of which are hereby incorporated by
reference. Furthermore, a variety of carbon protecting groups are
described in Myers, A.; Kung, D. W.; Zhong, B.; Movassaghi, M.;
Kwon, S. J. Am. Chem. Soc. 1999,121, 8401-8402, the entire contents
of which are hereby incorporated by reference.
[0044] It will be appreciated that the compounds, as described
herein, may be substituted with any number of substituents or
functional moieties. In general, the term "substituted" whether
preceded by the term "optionally" or not, and substituents
contained in formulas of this invention, refer to the replacement
of hydrogen radicals in a given structure with the radical of a
specified substituent. When more than one position in any given
structure may be substituted with more than one substituent
selected from a specified group, the substituent may be either the
same or different at every position. As used herein, the term
"substituted" is contemplated to include all permissible
substituents of organic compounds. In a broad aspect, the
permissible substituents include acyclic and cyclic, branched and
unbranched, carbocyclic. and heterocyclic, aromatic and nonaromatic
substituents of organic compounds. For purposes of this invention,
heteroatoms such as nitrogen may have hydrogen substituents and/or
any permissible substituents of organic compounds described herein
which satisfy the valencies of the heteroatoms. Furthermore, this
invention is not intended to be limited in any manner by the
permissible substituents of organic compounds. Combinations of
substituents and variables envisioned by this invention are
preferably those that result in the formation of stable compounds
useful in the treatment of cancer and/or the inhibition of the
growth of or the killing of cancer cells. The term "stable", as
used herein, preferably refers to compounds which possess stability
sufficient to allow manufacture and which maintain the integrity of
the compound for a sufficient period of time to be useful for the
purposes detailed herein.
[0045] The term "aliphatic", as used herein, includes both
saturated and unsaturated, straight chain (i.e., unbranched),
branched, cyclic, or polycyclic aliphatic hydrocarbons, which are
optionally substituted with one or more functional groups. As will
be appreciated by one of ordinary skill in the art, "aliphatic" is
intended herein to include, but is not limited to, alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus,
as used herein, the term "alkyl" includes both straight, branched
and cyclic alkyl groups. An analogous convention applies to other
generic terms such as "alkenyl", "alkynyl" and the like.
Furthermore, as used herein, the terms "alkyl", "alkenyl",
"alkynyl" and the like encompass both substituted and unsubstituted
groups.
[0046] Unless otherwise specified, alkyl and other aliphatic groups
preferably contain 1-6, or 1- 3, contiguous aliphatic carbon atoms.
Illustrative aliphatic groups thus include, but are not limited to,
for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,
--CH.sub.2-cyclopropyl, allyl, n-butyl, sec-butyl, isobutyl,
tert-butyl, cyclobutyl, --CH.sub.2-cyclobutyl, n-pentyl,
sec-pentyl, isopentyl, tert-pentyl, cyclopentyl,
--CH.sub.2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl,
--CH.sub.2-cyclohexyl moieties and the like, which again, may bear
one or more substituents. Alkenyl groups include, but are not
limited to, for example, ethenyl, propenyl, butenyl,
1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups
include, but are not limited to, ethynyl, 2- propynyl (propargyl),
1-propynyl and the like.
[0047] The term "alkoxy", or "thioalkyl" as used herein refers to
an alkyl group, as previously defined, attached to the parent
molecular moiety through an oxygen atom or through a sulfur atom.
Examples of alkoxy, include but are not limited to, methoxy,
ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy and
n-hexoxy. Examples of thioalkyl include, but are not limited to
methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and
the like.
[0048] The term "alkylamino" refers to a group having the structure
--NHR' wherein R' is alkyl, as defined herein. Examples of
alkylamino include, but are not limited to, methylamino,
ethylamino, iso-propylamino and the like. In certain embodiments,
C.sub.1-C.sub.3 alkylamino groups are utilized in the present
invention.
[0049] Some examples of substituents of the above-described
aliphatic (and other) moieties of compounds of the invention
include, but are not limited to: F, Cl, Br, I, OH, NO.sub.2, CN,
C(O)-alkyl, C(O)-aryl, C(O)-heteroaryl, CO.sub.2-alkyl,
CO.sub.2-aryl, CO.sub.2-heteroaryl, CONH.sub.2, CONH-alkyl,
CONH-aryl, CONH-heteroaryl, OC(O)-alkyl, OC(O)-aryl,
OC(O)-heteroaryl, OCO.sub.2-alkyl, OCO.sub.2-aryl,
OCO.sub.2-heteroaryl, OCONH.sub.2, OCONH-alkyl, OCONH-aryl,
OCONH-heteroaryl, NHC(O)-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl,
NHCO.sub.2-alkyl, NHCO.sub.2-aryl, NHCONH-heteroaryl,
SO.sub.2-alkyl, SO.sub.2-aryl, C.sub.3-C.sub.6-cycloalkyl,
CF.sub.3, CH.sub.2CF.sub.3, CHCl.sub.2, CH.sub.2OH,
CH.sub.2CH.sub.2OH, CH.sub.2NH.sub.2, CH.sub.2SO.sub.2CH.sub.3,
aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy,
alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino,
arylamino, heteroarylamino, -alkyl-amino, thio, aryl-thio,
heteroarylthio, benzyl-thio, alkyl-thio, or methylthiomethyl.
Additional examples of generally applicable substituents are
illustrated by the specific embodiments shown in the Examples which
are described herein.
[0050] In general, the terms "aryl" and "heteroaryl", as used
herein, refer to stable mono- or polycyclic, heterocyclic,
polycyclic, and polyheterocyclic unsaturated moieties having
preferably 3-14 carbon atoms, each of which may be substituted or
unsubstituted. Substituents include, but are not limited to, any of
the previously mentioned substitutents, i.e., the substituents
recited for aliphatic moieties, or for other moieties as disclosed
herein, resulting in the formation of a stable compound. In certain
embodiments of the present invention, "aryl" refers to a mono- or
bicyclic carbocyclic ring system having one or two aromatic rings
including, but not limited to, phenyl, naphthyl,
tetrahydronaphthyl, indanyl, indenyl and the like. In certain
embodiments of the present invention, the term "heteroaryl", as
used herein, refers to a cyclic aromatic radical having from five
to ten ring atoms of which one ring atom is selected from S, O and
N; zero, one or two ring atoms are additional heteroatoms
independently selected from S, O and N; and the remaining ring
atoms are carbon, the radical being joined to the rest of the
molecule via any of the ring atoms, such as, for example, pyridyl,
pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl,
oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl,
furanyl, -quinolinyl, isoquinolinyl, and the like.
[0051] It will be appreciated that aryl and heteroaryl groups
(including bicyclic aryl groups) can be unsubstituted or
substituted, wherein substitution includes replacement of one, two
or three of the hydrogen atoms thereon independently with any one
or more of the following moieties including, but not limited to: F,
Cl, Br, I, OH, NO.sub.2, CN, C(O)-alkyl, C(O)-aryl,
C(O)-heteroaryl, CO.sub.2-alkyl, CO.sub.2-aryl,
CO.sub.2-heteroaryl, CONH.sub.2, CONH-alkyl, CONH-aryl,
CONH-heteroaryl, OC(O)-alkyl, OC(O)-aryl, OC(O)-heteroaryl,
OCO.sub.2-alkyl, OCO.sub.2-aryl, OCO.sub.2-heteroaryl, OCONH.sub.2,
OCONH-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC(O)-alkyl,
NHC(O)-aryl, NHC(O)-heteroaryl, NHCO.sub.2-alkyl, NHCO.sub.2-aryl,
NHCONH-heteroaryl, SO.sub.2-alkyl, SO.sub.2-aryl,
C.sub.3-C.sub.6-cycloalkyl, CF.sub.3, CH.sub.2CF.sub.3, CHCl.sub.2,
CH.sub.2OH, CH.sub.2CH.sub.2OH, CH.sub.2NH.sub.2,
CH.sub.2SO.sub.2CH .sub.3, aryl, heteroaryl, benzyl, benzyloxy,
aryloxy, heteroaryloxy, alkoxy, methoxymethoxy, methoxyethoxy,
amino, benzylamino, arylamino, heteroarylamino, alkyl-amino, thio,
aryl-thio, heteroarylthio, benzyl-thio, alkyl-thio, or
methylthiomethyl. Additional examples of generally applicable
substitutents are illustrated by the specific embodiments which are
described herein.
[0052] The term "cycloalkyl", as used herein, refers specifically
to groups having three to seven, preferably three to ten carbon
atoms. Suitable cycloalkyls include, but are not limited to
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and
the like, which, as in the case of other aliphatic, heteroaliphatic
or hetercyclic moieties, may optionally be substituted. F, Cl, Br,
I, OH, NO.sub.2, CN, C(O)-alkyl, C(O)-aryl, C(O)-heteroaryl,
CO.sub.2-alkyl, CO.sub.2-aryl, CO.sub.2-heteroaryl, CONH.sub.2,
CONH-alkyl, CONH-aryl, CONH-heteroaryl, OC(O)-alkyl, OC(O)-aryl,
OC(O)-heteroaryl, OCO.sub.2-alkyl, OCO.sub.2-aryl,
OCO.sub.2-heteroaryl, OCONH.sub.2, OCONH-alkyl, OCONH-aryl,
OCONH-heteroaryl, NHC(O)-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl,
NHCO.sub.2-alkyl, NHCO.sub.2-aryl, NHCONH-heteroaryl,
SO.sub.2-alkyl, SO.sub.2-aryl, C.sub.3-C.sub.6-cycloalkyl,
CF.sub.3, CH.sub.2CF.sub.3, CHCl.sub.2, CH.sub.2OH,
CH.sub.2CH.sub.2OH, CH.sub.2NH.sub.2, CH.sub.2SO.sub.2CH.sub.3,
aryl, heteroaryl, benzyl, benzyoxy, aryloxy, heteroaryloxy, alkoxy,
methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino,
heteroarylamino, alkyl-amino, thio, aryl-thio, heteroarylthio,
benzyl-thio, alkyl-thio, or methylthiomethyl. Additional examples
of generally applicable substitutents are illustrated by the
specific embodiments shown in the Examples which are described
herein.
[0053] The term "heteroaliphatic", as used herein, refers to
aliphatic moieties which contain one or more oxygen, sulfur,
nitrogen, phosphorous or silicon atoms, e.g., in place of carbon
atoms. Heteroaliphatic moieties may be branched, unbranched or
cyclic and include saturated and unsaturated heterocycles such as
morpholino, pyrrolidinyl, etc. In certain embodiments,
heteroaliphatic moieties are substituted by independent replacement
of one or more of the hydrogen atoms thereon with one or more
moieties including, but not limited to: F, Cl, Br, I, OH, NO.sub.2,
CN, C(O)-alkyl, C(O)-aryl, C(O)-heteroaryl, CO.sub.2-alkyl,
CO.sub.2-aryl, CO.sub.2-heteroaryl, CONH.sub.2, CONH-alkyl,
CONH-aryl, CONH-heteroaryl, OC(O)-alkyl, OC(O)-aryl,
OC(O)-heteroaryl, OCO.sub.2-alkyl, OCO.sub.2-aryl,
OCO.sub.2-heteroaryl, OCONH.sub.2, OCONH-alkyl, OCONH-aryl,
OCONH-heteroaryl, NHC(O)-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl,
NHCO.sub.2-alkyl, NHCO.sub.2-aryl, NHCONH-heteroaryl,
SO.sub.2-alkyl, SO.sub.2-aryl, C.sub.3-C.sub.6-cycloalkyl,
CF.sub.3, CH.sub.2CF.sub.3, CHCl.sub.2, CH.sub.2OH,
CH.sub.2CH.sub.2OH, CH.sub.2NH.sub.2, CH.sub.2SO.sub.2CH.sub.3,
aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy,
alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino,
arylamino, heteroarylamino, alkyl-amino, thio, aryl-thio,
heteroarylthio, benzyl-thio, alkyl-thio, or methylthiomethyl.
Additional examples of generally applicable substitutents are
illustrated by the specific embodiments shown in the Examples which
are described herein.
[0054] The terms "halo" and "halogen" as used herein refer to an
atom selected from fluorine, chlorine, bromine and iodine.
[0055] The term "haloalkyl" denotes an alkyl group, as defined
above, having one, two, or three halogen atoms attached thereto and
is exemplified by such groups as chloromethyl, bromoethyl,
trifluoromethyl, and the like.
[0056] The term "heterocycloalkyl" or "heterocycle", as used
herein, refers to a non-aromatic 5-, 6- or 7-membered ring or a bi-
or tri-cyclic group comprising fused six-membered rings having
between one and three heteroatoms independently selected from
oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0
to 1 double bonds and each 6-membered ring has 0 to 2 double bonds,
(ii) the nitrogen and sulfur heteroatoms may be optionally be
oxidized, (iii) the nitrogen heteroatom may optionally be
quaternized, and (iv) any of the above heterocyclic rings may be
fused to a benzene ring. Representative heterocycles include, but
are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,
imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,
oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,
isothiazolidinyl, and tetrahydrofuryl. In certain embodiments, a
"substituted heterocycloalkyl or heterocycle" group is utilized and
as used herein, refers to a heterocycloalkyl or heterocycle group,
as defined above, substituted by the independent replacement of
one, two or three of the hydrogen atoms thereon with but are not
limited to: F, Cl, Br, I, OH, NO.sub.2, CN, C(O)-alkyl, C(O)-aryl,
C(O)-heteroaryl, CO.sub.2-alkyl, CO.sub.2-aryl,
CO.sub.2-heteroaryl, CONH.sub.2, CONH-alkyl, CONH-aryl,
CONH-heteroaryl, OC(O)-alkyl, OC(O)-aryl, OC(O)-heteroaryl,
OCO.sub.2-alkyl, OCO.sub.2-aryl, OCO.sub.2-heteroaryl, OCONH.sub.2,
OCONH-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC(O)-alkyl,
NHC(O)-aryl, NHC(O)-heteroaryl, NHCO.sub.2-alkyl, NHCO.sub.2-aryl,
NHCONH-heteroaryl, SO.sub.2-alkyl, SO.sub.2-aryl,
C.sub.3-C.sub.6-cycloal- kyl, CF.sub.3, CH.sub.2CF.sub.3,
CHCl.sub.2, CH.sub.2OH, CH.sub.2CH.sub.2OH, CH.sub.2NH.sub.2,
CH.sub.2SO.sub.2CH.sub.3, aryl, heter benzyloxy, aryloxy,
heteroaryloxy, alkoxy, methoxymethoxy, methoxyethoxy, amino,
benzylamino, arylamino, heteroarylamino, alkyl-amino, thio,
aryl-thio, heteroarylthio, benzyl-thio, alkyl-thio, or
methylthiomethyl. Additional examples of generally applicable
substitutents are illustrated by the specific embodiments shown in
the Examples which are described herein.
DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0057] The present invention provides compounds, compositions,
methods and systems for inhibiting cell growth. More specifically,
the present invention provides for methods, compounds and
compositions which are capable of inhibiting mitosis in
metabolically active cells. Compounds, compositions and methods of
the present invention inhibit the activity of a protein involved in
the assembly and maintenance of the mitotic spindle. One class of
proteins which acts on the mitotic spindle is the family of mitotic
kinesins, a subset of the kinesin superfamily.
[0058] Monastrol
[0059] Mitchison and coworkers have demonstrated that the
dihydropyrimidine-based compound monastrol is capable of arresting
mammalian cells in mitosis with monopolar spindles (Mayer et al.
Science 286:971-974, 1999; incorporated herein by reference). In
vitro, monastrol specifically inhibited the motility of the mitotic
kinesin Eg5, a motor protein required for spindle bipolarity.
Monastrol was identified as causing monoastral spindles in mitotic
cells in a multistep screen. The initial screen utilized a
whole-cell immunodetection assay (Stockwell et al. Chem Biol
February 1999;6(2):71-83; WO 00/07017) to identify compounds that
increased the phosphorylation of nucleolin. Nucleolin is a
nucleolar protein that is specifically phosphorylated in cells
entering mitosis, and compounds that cause mitotic arrest would be
expected to have increases levels of phosphonucleolin. This initial
screen identified 139 compounds from a library of 16,320 small
molecules (Diverset E, Chembridge Corporation. San Diego, Calif.)
as having anti-mitotic activity.
[0060] A secondary screen, utilizing an in vitro tubulin
polymerization assay, ruled out molecules that target tubulin as
its mode of action. Of the 139 compounds selected in the first
screen, 86 compounds did not arrest mitosis by targeting tubulin
and were therefore deemed interesting for further study. These 86
compounds were then tested for their effects on microtubules, actin
and chromosomes. Twenty-seven of the 86 compounds had no observable
effect on the microtubule and actin cytoskeleton or on chromosome
distribution. Twelve of the 86 compounds had pleiotropic effects
and were not evaluated further. Forty-two of the 86 compounds
affected cells in interphase as well as in mitosis and thus were
not specifically affecting mitosis. Cells treated with these small
molecules had disorganized or partially depolymerized interphase
microtubules in addition to abnormal mitotic spindle structures and
misaligned chromosomes.
[0061] Five of the 86 compounds altered the mitotic spindle
specifically and were thus studied further. Monastrol was
identified from one of these five compounds. It was observed that
monkey epithelial kidney cells (BS-C.sub.71) treated with monastrol
had abnormal mitotic spindle. The normally bipolar mitotic spindle
was replaced by a monoastral microtubule array surrounded by a ring
of chromosomes. Interphase cells were not affected.
[0062] In addition, by studying the effects of monastrol and a
related compound DHP2 on microtubule motility, Mayer et al.
(Science 286:971-974, 1999) determined that the inhibition of
monastrol on the Eg5 kinesin is specific to monastrol. Furthermore,
monastrol's inhibiting effect on motility is specific for the Eg5
kinesin. Monastrol did not inhibit microtubule motility driven by
conventional kinesin (Mayer et al. Science 286:971-974, 1999.)
[0063] Monastroline
[0064] It is an aspect of the present invention that a compound
(and pharmaceutical compositions comprising the compound) having
the formula (I) arrests cells in mitosis and therefore inhibits
cell growth. The compound, a .beta.-carboline class compound, is
designated as 22C16 from the Diverset E library of small molecules
(Chembridge Corporation) and is also referred to herein as
monastroline.
[0065] It is readily appreciated by one skilled in the art that the
compound designated as 22C16 structure (depicted by the structural
formula (V)) contains two chiral centers and that the four
stereoisomers of 22C16 are encompassed by the description of the
present invention. Experiments on an optically purified
stereoisomer showed that a trans isomer of 22C16 shown in FIG. 14
has an IC50 which is approximately 8 times lower than monastrol.
7
[0066] The present invention teaches that monastroline affects the
mitotic machinery of mammalian cells by inhibiting the mitotic
activity of a kinesin. More specifically, 22C16 is capable of
inhibiting the ATPase activity of the human mitotic kinesin Eg5. As
previously described, enzymes in the kinesin family use the free
energy of ATP hydrolysis to drive intracellular movement and effect
cytoskeleton structure. Example 2 describes experiments
demonstrating that 22C16 inhibits the ATPase activity of a mitotic
kinesin. These experiments utilized a purified recombinant human
kinesin, Eg5.
[0067] In addition, 22C16 is capable of arresting cells in mitosis.
It was observed that 22C16 affected the spindles of mitotic
mammalian cells in a manner similar to that observed for monastrol
(Mayer et al. Science 286:971-974, 1999). Monastrol causes the
spindles in mitotic mammalian cells to form a mono-astral
microtubule array surrounded by a ring of chromosomes. This
phenotype was also observed when 22C16 was added to monkey-derived
BSC1 cells (see Example 1 and FIG. 1).
[0068] In experiments studying the secretion of a viral G protein,
approximately 140 compounds from the Diverset E library of small
molecules (Chembridge Corp.) were found to affect the transport of
a temperature sensitive mutant of a viral G protein fused to a
green fluorescent protein (GFP). More importantly, 42 of the 140
compounds were found to affect the Golgi of the cells resulting in
a fragmented Golgi phenotype. During the analysis of these 42
compounds for their effects on Golgi fragmentation and microtubule
structure, it was observed that one compound, 22C16, fragmented the
Golgi without affecting interphase microtubules. In addition, 22C16
caused a spindle defect similar to that observed for monostral
(FIG. 1). 22C16 causes spindles in mitotic mammalian cells to form
a mono-astral microtubule array surrounded by a ring of
chromosomes.
[0069] When the effects of 22C16 and monastrol on microtubule
structure were directly compared, it was shown by examining
formation of mono-astral structures that 22C16 has an IC50 (median
inhibitory concentration) of approximately 2 uM as compared to the
IC50 of approximately 20 uM for monastrol (Mayer and Mitchison,
unpublished observations). For experimental details on the cell
assay to determine mono-astral structures, see Mayer et al. Science
286:971-974, 1999. Images of live BS-C-l cells were taken as
described (Cramer et al. Curr. Opin. Cell. Biol. 67:82, 1994).
Briefly, for immunofluorescence, BS-C-1 cells were stained with
DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich) to visualize
DNA and anti-.alpha.-tubulin (DM1 A; Sigma-Aldrich, St. Louis) to
visualize spindle structure.
[0070] Since the effect of 22C16 on mono-astral formation in
mammalian cells was similar to that of cells treated with
monastrol, the ability of 22C16 to inhibit kinesins was examined.
Example 2 describes experiments demonstrating that 22C16
specifically affects the ability of the Eg5 kinesin to hydrolyze
ATP. It was shown that 22C16 inhibits the ability of Eg5, but not
conventional human kinesin, to hydrolyze ATP in the presence of
NADH and microtubules.
[0071] In addition, the ability of 22C16 to inhibit microtubule
motility driven by Eg5 was examined. For experimental protocols and
details, see Mayer et al. Science 286:971-974, 1999. Full length
Xenopus Eg5 was expressed in baculovirus according to standard
protocols (see Coligan et al. Current Protocols in Protein Science
and Ausubel et al. Current Protocols in Molecular Biology. John
Wiley & Sons. Incorporated herein by reference.) FIG. 2 shows
that 22C16 reversibly inhibits microtubule motility driven by
full-length Eg5. For the washout, Eg5-driven microtubule motility
in the presence of 22C16 was measured. The assay chamber was then
depleted of 22C16 and motility was immediately measured again.
Therefore, the results of the experiment depicted in FIG. 2 provide
direct evidence that 22C16 inhibits the microtubule motility driven
by Eg5.
[0072] Uses
[0073] Any system where the control of cellular growth and cell
division is desired may utilize compounds in the beta-carboline
class to regulate mitosis. More specifically, in a preferred
embodiment, the compound previously designated and described as
22C16 may be used to inhibit cell growth. One non-limiting example
of an application of 22C16 to a cellular system is the use of 22C16
as an anti-mitotic anti-cancer drug. Other examples include
controlling cell division and the immune system in diseases such as
rheumatoid arthritis.
[0074] In a preferred embodiment, a method of treating an
individual with uncontrolled or abnormal cell growth is provided.
Compositions comprising monastroline or derivatives with similar
biological activity are useful for treating individuals with cells
that having become cancerous tumors. As discussed monstroline and
derivatives with similar structure and biological activity are
provided, preferably in as pharmaceutical composition containing a
pharmaceutically acceptable carrier. The compositions containing
monstroline and/or derivatives can be administered to an individual
in need thereof at therapeutically effective amounts to slow or
cease the abnormal cell growth. Generally, abnormal cell growth is
associated with cancerous cells. However, other diseases resulting
from uncontrolled cell growth (e.g. cardiovascular diseases,
rheumatoid arthritis etc.) may be treated with compositions and
methods of the present invention.
[0075] Pharmaceutical Compositions
[0076] As discussed above, unexpectedly, the present invention
provides novel compounds having antitumor and anti-cell
proliferative activity, and thus the inventive compounds are useful
for the treatment of cancer. Accordingly, in another aspect of the
present invention, pharmaceutical compositions are provided,
wherein these compositions comprise any one of the compounds as
described herein, and optionally comprise a pharmaceutically
acceptable carrier.
[0077] In certain preferred embodiments, these compositions
optionally further comprise one or more additional therapeutic
agents. In certain other embodiments, the additional therapeutic
agent is an anticancer agent, as discussed in more detail herein.
It will also be appreciated that certain of the compounds of
present invention can exist in free form for treatment, or where
appropriate, as a pharmaceutically acceptable derivative thereof.
According to the present invention, a pharmaceutically acceptable
derivative includes, but is not limited to, pharmaceutically
acceptable salts, esters, salts of such esters, or any other adduct
or derivative which upon administration to a patient in need is
capable of providing, directly or indirectly, a compound as
otherwise described herein, or a metabolite or residue thereof,
e.g., a prodrug.
[0078] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts which are, within the scope of sound medical
judgement, suitable for use in contact with the tissues of humans
and lower animals without undue toxicity, irritation, allergic
response and the like, and are commensurate with a reasonable
benefit/risk ratio. Pharmaceutically acceptable salts are well
known in the art. For example, S. M. Berge, et al. describe
pharmaceutically acceptable salts in detail in J. Pharmaceutical
Sciences, 66: 1-19 (1977), incorporated herein by reference. The
salts can be prepared in situ during the final isolation and
purification of the compounds of the invention, or separately by
reacting the free base function with a suitable organic acid.
Examples of pharmaceutically acceptable, nontoxic acid addition
salts are salts of an amino group formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric
acid and perchloric acid or with organic acids such as acetic acid,
oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid
or malonic acid or by using other methods used in the art such as
ion exchange. Other pharmaceutically acceptable salts include
adipate, alginate, ascorbate, aspartate, benzenesulfonate,
benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hernisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium,. potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
[0079] Additionally, as used herein, the term "pharmaceutically
acceptable ester" refers to esters which hydrolyze in vivo and
include those that break down readily in the human body to leave
the parent compound or a salt thereof. Suitable ester groups
include, for example, those derived from pharmaceutically
acceptable aliphatic carboxylic acids, particularly alkanoic,
alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl
or alkenyl moiety advantageously has not more than 6 carbon atoms.
Examples of particular esters includes formates, acetates,
propionates, butyrates, acrylates and ethylsuccinates.
[0080] Furthermore, the term "pharmaceutically acceptable prodrugs"
as used herein refers to those prodrugs of the compounds of the
present invention which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and lower animals with undue toxicity, irritation, allergic
response, and the like, commensurate with a reasonable benefit/risk
ratio, and effective for their intended use, as well as the
zwitterionic forms, where possible, of the compounds of the
invention. The term "prodrug" refers to compounds that are rapidly
transformed in vivo to yield the parent compound of the above
formula, for example by hydrolysis in blood. A thorough discussion
is provided in T. Higuchi and V. Stella, Pro-drugs as Novel
Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and in
Edward B. Roche, ed., Bioreversible Carriers in Drug Design,
American Pharmaceutical Association and Pergamon Press, 1987, both
of which are incorporated herein by reference.
[0081] As described above, the pharmaceutical compositions of the
present invention additionally comprise a pharmaceutically
acceptable carrier, which, as used herein, includes any and all
solvents, diluents, or other liquid vehicle, dispersion or
suspension aids, surface active agents, isotonic agents, thickening
or emulsifying agents, preservatives, solid binders, lubricants and
the like, as suited to the particular dosage form desired.
Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W.
Martin (Mack Publishing Co., Easton, Pa., 1975) discloses various
carriers used in formulating pharmaceutical compositions and known
techniques for the preparation thereof. Except insofar as any
conventional carrier medium is incompatible with the anti-viral
compounds of the invention, such as by producing any undesirable
biological effect or otherwise interacting in a deleterious manner
with any other component(s) of the pharmaceutical composition, its
use is contemplated to be within the scope of this invention. Some
examples of materials which can serve as pharmaceutically
acceptable carriers include, but are not limited to, sugars such as
lactose, glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil; safflower oil; sesame oil; olive oil; corn oil and soybean
oil; glycols; such a propylene glycol; esters such as ethyl oleate
and ethyl laurate; agar; buffering agents such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol, and phosphate
buffer solutions, as well as other non-toxic compatible lubricants
such as sodium lauryl sulfate and magnesium stearate, as well as
coloring agents, releasing agents, coating agents, sweetening,
flavoring and perfuming agents, preservatives and antioxidants can
also be present in the composition, according to the judgment of
the formulator.
[0082] In yet another aspect, according to the methods of treatment
of the present invention, tumor cells are killed, or their growth
is inhibited by contacting said tumor cells with an inventive
compound or composition, as described herein. Thus, in still
another aspect of the invention, a method for the treatment of
cancer is provided comprising administering a therapeutically
effective amount of an inventive compound, or a pharmaceutical
composition comprising an inventive compound to a subject in need
thereof, in such amounts and for such time as is necessary to
achieve the desired result.
[0083] In certain embodiments of the present invention a
"therapeutically effective amount" of the inventive compound or
pharmaceutical composition is that amount effective for killing or
inhibiting the growth of tumor cells. The compounds and
compositions, according to the method of the present invention, may
be administered using any amount and any route of administration
effective for killing or inhibiting the growth of tumor cells.
Thus, the expression "amount effective to kill or inhibit the
growth of tumor cells", as used herein, refers to a sufficient
amount of agent to kill or inhibit the growth of tumor cells. The
exact amount required will vary from subject to subject, depending
on the species, age, and general condition of the subject, the
severity of the infection, the particular anticancer agent, its
mode of administration, and the like.
[0084] The anticancer compounds of the invention are preferably
formulated in dosage unit form for ease of administration and
uniformity of dosage. The expression "dosage unit form" as used
herein refers to a physically discrete unit of anticancer agent
appropriate for the patient to be treated. It will be understood,
however, that the total daily usage of the compounds and
compositions of the present invention will be decided by the
attending physician within the scope of sound medical judgment. The
specific therapeutically effective dose level for any particular
patient or organism will depend upon a variety of factors including
the disorder being treated and the severity of the disorder; the
activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidental with the specific compound employed; and like
factors well known in the medical arts.
[0085] Furthermore, after formulation with an appropriate
pharmaceutically acceptable carrier in a desired dosage, the
pharmaceutical compositions of this invention can be administered
to humans and other animals orally, rectally, parenterally,
intracisternally, intravaginally, intraperitoneally, topically (as
by powders, ointments, or drops), bucally, as an oral or nasal
spray, or the like, depending on the severity of the infection
being treated. In certain embodiments, the compounds of the
invention may be administered orally or parenterally at dosage
levels of about 0.01 mg/kg to about 50 mg/kg and preferably from
about 1 mg/kg to about 25 mg/kg, of subject body weight per day,
one or more times a day, to obtain the desired therapeutic
effect.
[0086] Liquid dosage forms for oral administration include, but are
not limited to, pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active compounds, the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0087] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0088] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0089] In order to prolong the effect of a drug, it is often
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This may be accomplished by the use of a
liquid suspension of crystalline or amorphous material with poor
water solubility. The rate of absorption of the drug then depends
upon its rate of dissolution which, in turn, may depend upon
crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle. Injectable
depot forms are made by forming microencapsule matrices of the drug
in biodegradable polymers such as polylactide-polyglycolide.
Depending upon the ratio of drug to polymer and the nature of the
particular polymer employed, the rate of drug release can be
controlled. Examples of other biodegradable polymers include
poly(orthoesters) and poly(anhydrides). Depot injectable
formulations are also prepared by entrapping the drug in liposomes
or microemulsions which are compatible with body tissues.
[0090] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
compounds of this invention with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active compound.
[0091] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar--agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin
and-bentonite clay, and i) lubricants such as talc, calcium
stearate, magnesium stearate, solid polyethylene glycols, sodium
lauryl sulfate, and mixtures thereof. In the case of capsules,
tablets and pills, the dosage form may also comprise buffering
agents.
[0092] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may
optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
which can be used include polymeric substances and waxes. Solid
compositions of a similar type may also be employed as fillers in
soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polethylene
glycols and the like.
[0093] The active compounds can also be in micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active compound may be admixed with at least one inert diluent such
as sucrose, lactose or starch. Such dosage forms may also comprise,
as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such
a magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions which can be used include polymeric
substances and waxes.
[0094] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, powders, solutions, sprays, inhalants or patches.
The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, ear drops, and
eye drops are also contemplated as being within the scope of this
invention. Additionally, the present invention contemplates the use
of transdermal patches, which have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux
of the compound across the skin. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the
compound in a polymer matrix or gel.
[0095] As discussed above, the compounds of the present invention
are useful as anticancer agents, and thus may be useful in the
treatment of cancer, by effecting tumor cell death or inhibiting
the growth of tumor cells. In general, the inventive anticancer
agents are useful in the treatment of cancers and other
proliferative disorders, including, but not limited to breast
cancer, cervical cancer, colon and rectal cancer, leukemia, lung
cancer, melanoma, multiple myeloma, non-Hodgkin's lymphoma, ovarian
cancer, pancreatic cancer, prostate cancer, and gastric cancer, to
name a few. In certain embodiments, the inventive anticancer agents
are active against leukemia cells and melanoma cells, and thus are
useful for the treatment of leukemias (e.g., myeloid, lymphocytic,
myelocytic and lymphoblastic leukemias) and malignant melanomas. In
still other embodiments, the inventive anticancer agents are active
against solid tumors and also kill and/or inhibit the growth of
multidrug resistant cells (MDR cells).
[0096] It will also be appreciated that the compounds and
pharmaceutical compositions of the present invention can be
employed in combination therapies, that is, the compounds and
pharmaceutical compositions can be administered concurrently with,
prior to, or subsequent to, one or more other desired therapeutics
or medical procedures. The particular combination of therapies
(therapeutics or procedures) to employ in a combination regimen
will take into account compatibility of the desired therapeutics
and/or procedures and the desired therapeutic effect to be
achieved. It will also be appreciated that the therapies employed
may achieve a desired effect for the same disorder (for example, an
inventive compound may be administered concurrently with another
anticancer-agent), or they may achieve different effects (e.g.,
control of any adverse effects).
[0097] For example, other therapies or anticancer agents that may
be used in combination with the inventive anticancer agents of the
present invention include surgery, radiotherapy (in but a few
examples, y-radiation, neutron beam radiotherapy, electron beam
radiotherapy, proton therapy, brachytherapy, and systemic
radioactive isotopes, to name a few), endocrine therapy, biologic
response modifiers (interferons, interleukins, and tumor necrosis
factor (TNF) to name a few), hyperthermia and cryotherapy, agents
to attenuate any adverse effects (e.g., antiemetics), and other
approved chemotherapeutic drugs, including, but not limited to,
alkylating drugs (mechlorethamine, chlorambucil, Cyclophosphamide,
Melphalan, Ifosfamide), antimetabolites (Methotrexate), purine
antagonists and pyrimidine antagonists (6-Mercaptopurine,
5-Fluorouracil, Cytarabile, Gemcitabine), spindle poisons
(Vinblastine, Vincristine, Vinorelbine, Paclitaxel),
podophyllotoxins (Etoposide, Irinotecan, Topotecan), antibiotics
(Doxorubicin, Bleomycin, Mitomycin), nitrosoureas (Carmustine,
Lomustine), inorganic ions (Cisplatin, Carboplatin), enzymes
(Asparaginase), and hormones (Tamoxifen, Leuprolide, Flutamide, and
Megestrol), to name a few. For a more comprehensive discussion of
updated cancer therapies see, http://www.nci.nih.gov/, a list of
the FDA approved oncology drugs at
http://www.fda.gov/cder/cancer/druglistframe.htm, and The Merck
Manual, Seventeenth Ed. 1999, the entire contents of which are
hereby incorporated by reference.
[0098] In still another aspect, the present invention also provides
a pharmaceutical pack or kit comprising one or more containers
filled with one or more of the ingredients of the pharmaceutical
compositions of the invention, and in certain embodiments, includes
an additional approved therapeutic agent for use as a combination
therapy. Optionally associated with such container(s) can be a
notice in the form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceutical products, which
notice reflects approval by the agency of manufacture, use or sale
for human administration.
Equivalents
[0099] The representative examples which follow are intended to
help illustrate the invention, and are not intended to, nor should
they be construed to, limit the scope of the-invention. Indeed,
various modifications of the invention and many further embodiments
thereof, in addition to those shown and described herein, will
become apparent to those skilled in the art from the full contents
of this document, including the examples which follow and the
references to the scientific and patent literature cited herein. It
should further be appreciated that the contents of those cited
references are incorporated herein by reference to help illustrate
the state of the art. The following examples contain important
additional information, exemplification and guidance which can be
adapted to the practice of this invention in its various
embodiments and the equivalents thereof.
EXAMPLE 1
Screen to Identify Small Molecule Inhibitors of Exocytosis
Automated Image Capture
[0100] Automated screening microscope was developed to allow
medium-throughput imaging-based screening. A Nikon inverted
fluorescence microscope equipped with a 20.times.dry lens (F 0.45)
was equipped with a cooled charged coupled device (CCD) camera and
the Metamorph software suite (Universal Imaging Corp.) for data
acquisition. Changes in the focal plane were obtained by
controlling the focal length of the optical path with a
piezo-electric collar attached to the 20.times. dry lens with steps
of 5 .mu.m on a total range of 300 .mu.m. Best focus was achieved
by using an algorithm that searches for maximum contrast on the
acquired image. The microscope set-up is diagrammed in FIG. 3.
[0101] The Metamorph software performs the following tasks:
[0102] (1) automatically center the lens in each well of a tissue
culture plate (in our case, 384-well plates);
[0103] (2) automatically focus the image, by collecting 25 images
in different focal planes and identifying the image with maximum
contrast; and
[0104] (3) transfer the final in-focus image (642 kilobytes) to the
hard drive of the attached computer (Pentium III, 1 Gigabyte RAM,
28 Gigabyte hard drive).
[0105] On average, data were collected from 20-40 cells per field.
Complete data acquisition for a 384-well tray was accomplished in
approximately one hour. The typical memory requirement for a screen
of 10,000 compounds is approximately 6 Gigabytes.
[0106] Implementation of the Synchronous Screen for Exocytosis
[0107] VSVG-ts045 (Gallione and Rose, J. Virology 54:374-82. 1985)
is a temperature sensitive mutant of the G protein of VSV
(vesicular stomatitis virus) that is retained in the ER if the
cells are grown at the non-permissive temperature of 40.degree. C.
When cells are transferred to the permissive temperature of
32.degree. C., VSVG-ts045 exits the ER and continues its traffic
through the Golgi complex, finally reaching the plasma membrane.
Others have shown that it is possible to add the green fluorescence
protein (GFP) to the cytoplasmic tail of VSVG-ts045 (abbreviated
here as VSVG-GFP), and that this chimera traffics in a similar way
(Hirschberg et al., J. Cell Biol. 143:1485-1503. 1998).
[0108] We generated a construct of VSVG-GFP and inserted it into an
adenovirus-based expression vector (He et al., Proc. Natl. Acad.
Sci. USA 95:2509-2514. 1998). The adenovirus containing the
construct was amplified in COS cells and aliquots of the media were
used for transduction in monkey-derived BSC1 cells. Almost 100% of
the cells express high amounts of VSVG-GFP that can be detected by
fluorescence microscopy 18 hours after infection. The protocol used
in the screen for exocytosis is shown in FIG. 4; an example of the
type of images that are acquired in the absence of added compounds
is shown in FIG. 5.
[0109] The images were retrieved using Metamorph, and scored for
variations in the pattern of VSVG-GFP traffic by direct visual
inspection. Images were classified into one of several phenotypes,
as shown in FIG. 6.
[0110] Summary of the Exocytic Screen
[0111] We tested the effect of .about.10,000 compounds (out of a
total of 16,320) from the Chembridge collection (Diverset E;
Chembridge Corporation) in the traffic of VSVG-GFP following the
protocol described above. About 140 hits were founds with obvious
effects on single steps in the traffic properties of VSVG-GFP (FIG.
6). Twenty-six hits that act at nominal concentrations of 100 .mu.M
or lower (based on dilutions of stock solutions) were selected for
further study. A summary of this stage of the screen is given in
Table 1.
[0112] We next used a suite of secondary screens to explore how
extensive the effects of these chemicals are on treated cells. This
secondary screen consisted of tests for (a) perturbations in the
intracellular organization of the tubulin-based cytoskeleton; (b)
variations in the intracellular distribution of ER, Golgi and
lysosomal markers; and (c) changes in the rate of uptake and
intracellular targeting to endosomes of Texas Red transferrin
internalized by receptor-mediated endocytosis.
[0113] It was observed in the primary screen described that one
compound 22C16 fragmented the Golgi of treated mammalian cells.
However, 22C16 did not depolymerize interphase microtubules and in
fact, caused the formation of monopolar spindles in mitotic cells.
FIG. 1 shows the effects of 22C16 on Golgi fragmentation and
spindle structure. As viewed in the lower right panel of FIG. 1,
22C16 causes the formation of monospindles with a mono-astral
microtubule array surrounded by a ring of chromosomes.
[0114] The observation of a mono-astral phenotype in mitotic cells
treated with 22C16 led to experiments to study the effects of 22C16
on Eg5 as was previously performed with monastrol (Mayer et al.
Science 286:971-974, 1999). The motility assay of 22C16 on Eg5 was
described in the preferred embodiments of this application and the
data are depicted in FIG. 2. The direct comparison of 22C16 and
monastrol on the monopolar spindle mono-astral formation on
mammalians was also described in the preferred embodiments.
EXAMPLE 2
ATPase Assay with Purified Human Eg5 Kinesin
[0115] Monastrol and 22C16 were both determined to block the ATPase
activity of human Eg5 (N-terminal 405 amino acids and 6-His tagged
at C terminus). Monastrol did not inhibit the activity of human
kinesin (N-terminal 560 amino acids of full length protein followed
by 6-His tag at C terminus).
[0116] Methods
[0117] Preparation of Recombinant Human Eg5 Kinesin (Eg5-405)
[0118] DNA encoding full length human Eg5 kinesin was amplified by
the polymerase chain reaction (PCR) using Vent DNA polymerase (NE
Biolabs, Beverly, Mass.) and subcloned into an expression plasmid
(pRSETa). For the PCR reaction, the template used was a pBluescript
vector containing the full length coding sequence for human Eg5 (a
gift from Anne Blangy). The 5' primer (5'-GCA ACG ATT AAT ATG GCG
TCG CAG CCA AAT TCG TCT GCG AAG) contained an Ase I cleavage site
upstream of the Eg5 start codon. The 3' primer (5'-GCA ACG CTC GAG
TCA GTG ATG ATG GTG GTG ATG CAT GAC TCT AAA ATT TTC TTC AGA AAT )
was complimentary to amino acid 405 and added a downstream six
histidine tag (6-HIS) followed by a UGA stop codon, and Xho I
cleavage site.
[0119] The resulting PCR DNA amplification product and also the
target plasmid to be used as the expression plasmid (pRSETa) were
double digested with Ase I/Xho I and Nde I/Xho I respectively (New
England Biolabs). Both products of the two restriction enzyme
double digests were resolved and purified by agarose gel
electrophoresis. The bands on the agarose gel corresponding to the
desired DNA fragments, more than 2 kb for pRSETa and 1.2 kb for
Eg5-405 were excised and purified (Qiagen Gel Purification Kit).
The cleaved and purified DNA fragments were ligated together using
T4 DNA ligase (New England Biolabs). The ligation products were
transformed into E. coli DH5.alpha. chemically competent cells
(Life Technologies), and selected by overnight growth on LB
ampicillin plates. Transformants were amplified by growth of E.
coli in LB ampicillin. Plasmids were purified (Qiagen Midiprep),
and sequenced (Harvard Medical School Biopolymer Facilities).
[0120] Purification of Eg5-405 and K560 (560 Amino Acid Kinesin
Construct):
[0121] BL21 pLysS (DE3) bacteria were transfected with the
expression plasmid described in the preceding section and grown
overnight at 37.degree. C. on LB plates containing 100 ug/ml
ampicillin (LB-amp). Several colonies were picked and grown at
37.degree. C. in 1 ml LB-amp, pooled and used to inoculate each of
six 1.5 L of LB-amp. These I L cultures were incubated at
37.degree. C. on a shaker (200 RPM) until the optical density
(O.D.) of the culture reached an absorbance of approximately
A.sub.600 nM=0.5 O.D. The cultures were cooled to 20.degree. C.,
induced with 24 mg/ml of isopropyl beta-D-thiogalactopyran- oside
(IPTG; Boehringer Mannheim) and incubated at room temperature for
approximately 3 hours. Cells were pelleted by centrifugation
(4000.times.g), rinsed in phosphate buffered saline (PBS) and
repelleted at 10,000.times.g). The bacterial pellets were flash
frozen in liquid nitrogen and stored at -80.degree. C.
[0122] The bacterial pellets were thawed on ice and resuspended in
a solution containing 50 mM potassium phosphate (pH 8.0), 250 mM
KCl, 0.1% Tween-20, 10 mM imidazole, 0.5 mM magnesium adenosine
triphosphate (Mg-ATP), 1 mM phenylmethanesulfonyl fluoride (PMSF),
and 2 mM benzimidine-HCl. To lyse the bacteria, lysozyme (1 mg/ml)
and 2-mercaptoethanol (5 mM) were added to the solution to result
in the indicated final concentration, incubated and sonicated (3
times 20 seconds, repeated 3 times incubating for 1 minute on ice
between each triple sonication) to break up the DNA and guarantee
bacterial lysis.
[0123] The lysate was spun at 40,000.times.g for approximately 35
minutes at 4.degree. C. with the resulting supernatant separated
from the pellet by decanting and then incubated with a
nickel-nitrilotriacetic acid resin (Ni-NTA; QIAGEN). The remaining
pellet was washed three times with a wash buffer (lysis buffer
supplemented with 10 mM 2-mercaptoethanol, no PMSF, and 0.1 mM
Mg-ATP) to extract any remaining protein. These washes were then
followed by a final wash using a low pH buffer (pH 6.0). The wash
solutions were then also added to the Ni-NTA resin. The resin
containing the desired tagged protein was poured into a column
(Biorad, 0.8.times.4 cm PolyPrep Chromatography Column) and allowed
to settle. The HIS-tagged proteins were eluted with a solution
containing 250 mM imidazole and 150 mM KCl (pH 7.0).
Protein-containing fractions of the eluate were loaded onto a
Superose 6 size-exclusion column (Pharmacia) and equilibrated with
a solution containing 80 mM potassium HEPES (pH 6.8), 200 mM KCl,
10 uM Mg-ATP, 1 mM dithiothreitol (DTT). Fractions containing
homogeneous proteins as determined by molecular weight and mobility
on SDS-PAGE were used for further enzymology experiments.
[0124] Polymerization of Microtubules:
[0125] A solution containing 1 mg/ml tubulin, 1 mM DTT, 1 mM
guanosine triphosphate (GTP), 1 mM MgCl.sub.2, 80 mM potassium
HEPES (pH 6.8), and 1 mM ethylene glycol-bis(beta-aminoethyl
ether)-N,N,NN'-tetraacetic acid (EGTA) was spun at 90,000.times.g
for 5 minutes. The solution was then warmed to 37.degree. C. for 2
minutes. Taxol was added in stepwise as 0.01, 0.1, and 1
equivalents. The polymerization solution was placed onto a solution
containing 40% glycerol, 80 mM potassium HEPES (pH 6.8), 1 mM
MgCl.sub.2, and 1 mM EGTA, and centrifuged at 90,000.times.g for
approximately 50 minutes. The resulting microtubule pellet was
washed extensively with and resuspended in resuspension buffer
containing 80 mM potassium HEPES (pH 6.8), 1 mM MgCl.sub.2, and 1
mM EGTA.
[0126] In Vitro NADH Enzyme Coupled ATPase: (Variation on Methods
Described in Crevel, Lockhart, and Cross. J. Mol. Biol. (1997) 273,
160-170.)
[0127] For a brief description as the ATPase assay used in the
present example, see FIG. 7.
[0128] A solution containing the microtubule resuspension buffer
was supplemented with 1 mM Mg-ATP, 100 uM nicotinamide adenine
dinucleotide (NADH), 1 mM phosphoenol pyruvate, 5 ug/ml pyruvate
kinase, 7.5 ug/ml lactate dehydrogenase, 0.7 uM resuspended
microtubules, and 0.5% dimethylsulfoxide (DMSO).
[0129] Next, serial dilutions of the compound 22C16 were prepared
in the supplemented microtubule resuspension buffer. Purified
recombinant human Eg5 protein was added resulting in a final
concentration of 50 uM. The subsequent fluorescent reaction was
measured in NUNC black walled 384 well plates at 340 nm using a
Wallac plate reader.
[0130] FIGS. 8 and 9 show the inhibition of Eg5 ATPase activity by
the beta-carboline 22C16 described by the present invention and by
monastrol respectively. Based on these assays, the IC50 for 22C16
equals 1.1 uM. For monastrol, the IC50=5.3 uM. Therefore, based on
the ATPase assay described 22C16 is approximately 5 times more
potent than monastrol in the inhibition of the ATPase activity of
the human kinesin Eg5.
EXAMPLE 3
Synthesis of Monastroline
[0131] The synthesis of monastroline (22C16) was performed via the
Pictet-Spengler reaction (Ber. 44:2030, 1911) Whaley and
Govindachari. Org. Reaction. 6:74, 1951; Ungemach et al. J. Amer.
Chem. Soc. 102:6976-6984, 1980). Recent examples of the use of the
Pictet-Spengler reaction are provided by Rousseau and Dodd (J. Org.
Chem. 63:2731-2737, 1998) and by Leonard et al. (Tet. Lett.
38:3071-3074, 1997).
[0132] FIG. 10 depicts the synthetic reaction scheme for
monastroline using the Pictet-Spengler reaction. D,L-tryptophan
(Sigma-Aldrich) was used as the starting material. D,L-tryptophan
was refluxed with 3-hydroxylbenzaldehyde (Sigma-Aldrich) in
methanol for approximately 6 hours. The racemic beta-carboline was
purified and refluxed with n-Bu-ICN in acetone for approximately 48
hours. Cis and trans isomers of monastroline were separated and
purified by standard silica gel chromatography.
EXAMPLE 4
Synthesis of Monastroline Derivatives
[0133] FIG. 11 depicts the synthesis of phenyl derivatives of
monastroline which have a structure similar to monastroline and
likely have similar biological activity. In FIG. 11, monastroline
and derivatives of monastroline may be conjugated to a solid
support through the R group wherein R is methyl, ethyl, n-butyl, or
alpha-benzyl. Alternatively or additionally, for conjugation to a
resin or other solid support, R is C.sub.nCOOR' where n is 1-20,
and preferably equal to 1, 3, 5, 7, 9, 1 1, or 13, and where R' is
methyl, ethyl, n-butyl, or alpha-benzyl.
[0134] The synthesis of beta-carboline derivatives and further
modifications of the beta-carboline core of monastroline is
depicted in FIG. 12. The synthesis of derivatives having
modifications, additions, and combinations thereof to heteroatom
functionality is depicted in FIG. 13.
[0135] Additionally syntheses of derivatives of monastroline are
described herein. The general synthetic scheme is depicted by the
following: 8
[0136] Synthetic reactions involving Step A are readily appreciated
by those skilled in the art. A representative reaction is shown
below.
[0137] Reaction of Tryptophan derivatives (4) with aldehyde buiding
blocks (5). 9
[0138] Syntheses of derivatives of monastroline starting with other
derivatives of tryptophan are readily appreciated by those skilled
in the art. Non-limiting examples are provided below. 10
[0139] Non-limiting examples of derivatives of monastroline
resulting from the reaction depicted by Step A are provided below.
The free acids can be further reacted with an isocynanate. The
carboxylic acids can be converted to the amine (Tetrahedron. Lett.
39(11):1291-1294, 1998). 1112
[0140] Syntheses involving the reaction depicted in Step B using
isocyanates are readily envisioned and appreciated by those skilled
in the art. A non-limiting example is provided below to further
illustrate reactions involving isocyanate in Step B.
[0141] Reaction of beta-carboline 4 with substituted isocyanates
(2). 13
[0142] Non-limiting examples of derivatives resulting from
reactions involving Step B are provided below. 1415
* * * * *
References