U.S. patent application number 11/359095 was filed with the patent office on 2006-06-29 for tubulin binding agents and corresponding prodrug constructs.
Invention is credited to David J. Chaplin, Zhi Chen, James M. Dorsey, Klaus Edvardson, Charles M. Garner, Anjan Ghatak, Usha R. Ghatak, Mallinath Hadimani, Jimmy Kessler, Vani P. Mocharla, Kevin G. Pinney, Joseph Prezioso.
Application Number | 20060142252 11/359095 |
Document ID | / |
Family ID | 33130475 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060142252 |
Kind Code |
A1 |
Pinney; Kevin G. ; et
al. |
June 29, 2006 |
Tubulin binding agents and corresponding prodrug constructs
Abstract
A diverse set of tubulin binding agents have been discovered
which are structurally characterized, in a general sense, by a
semi-rigid molecular framework capable of maintaining aryl-aryl,
pseudo pi stacking distances appropriate for molecular recognition
of tubulin. In phenolic or amino form, these ligands may be further
functionalized to prepare phosphate esters, phosphate salts,
phosphoramidates, and other prodrugs capable of demonstrating
selective targeting and destruction of tumor cell vasculature.
Inventors: |
Pinney; Kevin G.; (Woodway,
TX) ; Mocharla; Vani P.; (San Diego, CA) ;
Chen; Zhi; (Hamden, CT) ; Garner; Charles M.;
(Woodway, TX) ; Ghatak; Anjan; (Salt Lake City,
UT) ; Ghatak; Usha R.; (West Bengal, IN) ;
Hadimani; Mallinath; (Waco, TX) ; Kessler; Jimmy;
(Sugarland, TX) ; Dorsey; James M.; (Durham,
NC) ; Edvardson; Klaus; (Klampenborg, DK) ;
Chaplin; David J.; (Oxford, GB) ; Prezioso;
Joseph; (Boston, MA) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY;AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
33130475 |
Appl. No.: |
11/359095 |
Filed: |
February 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10404525 |
Apr 1, 2003 |
7001926 |
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11359095 |
Feb 21, 2006 |
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09804280 |
Mar 12, 2001 |
6593374 |
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10404525 |
Apr 1, 2003 |
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10218833 |
Aug 14, 2002 |
6956054 |
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11359095 |
Feb 21, 2006 |
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09505402 |
Feb 16, 2000 |
6538038 |
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10218833 |
Aug 14, 2002 |
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60188295 |
Mar 10, 2000 |
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60120478 |
Feb 18, 1999 |
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Current U.S.
Class: |
514/125 ;
514/129; 514/567; 514/718 |
Current CPC
Class: |
C07C 217/94 20130101;
C07C 43/23 20130101; C07C 49/755 20130101; C07C 205/37 20130101;
C07C 205/45 20130101; C07D 333/56 20130101; C07C 45/673 20130101;
C07C 45/64 20130101; C07C 225/22 20130101; C07D 333/64 20130101;
A61K 31/075 20130101; C07C 45/673 20130101; C07C 49/84 20130101;
C07C 217/84 20130101; C07C 45/512 20130101; C07F 9/12 20130101;
C07C 45/673 20130101; C07F 9/65517 20130101; C07F 9/655354
20130101; C07C 223/06 20130101; C07C 45/64 20130101; C07D 307/81
20130101; C07D 307/80 20130101; C07C 49/755 20130101; C07C 49/84
20130101; C07C 49/755 20130101; C07C 43/215 20130101; C07C 49/84
20130101; A61K 31/66 20130101; C07C 237/08 20130101; C07C 45/512
20130101; C07F 9/247 20130101; C07C 2602/10 20170501; C07F 9/18
20130101; C07C 2602/08 20170501 |
Class at
Publication: |
514/125 ;
514/129; 514/567; 514/718 |
International
Class: |
A61K 31/66 20060101
A61K031/66; A61K 31/075 20060101 A61K031/075 |
Claims
1. A method for treating a vascular proliferative disorder in an
animal comprising administering to an animal an effective amount of
a compound of formula I: ##STR6## wherein R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are independently
selected from the group consisting of H, OH, amine, lower alkoxy,
phosphate, phosphoramidate, or amino acid acyl group; is optionally
a single covalent bond or a double covalent bond; X is a single
covalent bond or a carbonyl group, and Y is optionally H or OH.
2. The method of claim 1, wherein the vascular proliferative
disorder is characterized by the presence of nonmalignant
proliferating vasculature.
3. The method of claim 2, wherein the malignant proliferating
vasculature is associated with a tumor or other neoplastic
disease.
4. The method of claim 2, wherein the vascular proliferative
disorder is characterized by the presence of nonmalignant
proliferating vasculature.
5. The method of claim 4, wherein the nonmalignant proliferating
vasculature is associated with an ocular disease selected from the
group comprising wet or age-related macular degeneration, diabetic
retinopathy, retinopathy of prematurity, diabetic macular edema,
uveitis, or corneal neovascularization.
6. The method of claim 4, wherein the nonmalignant proliferating
vasculature is associated with a nonocular disease state such as
psoriasis, rheumatoid arthritis, atheroma, restenosis, Kaposi's
sarcoma, haemangioma, and in general, inflammatory diseases
characterized by vascular proliferation.
7. The method of claim 1, wherein at least one of R.sub.2, R.sub.3,
R4, R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is lower alkoxy.
8. The method of claim 1, wherein R.sub.4 is OMe.
9. The method of claim 1, wherein X is a single bond.
10. The method of claim 1, wherein X is a carbonyl.
11. The method of claim 1, wherein at least one of R.sub.2,
R.sub.3, R4, R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is
phosphate.
12. The method of claim 1, wherein the compound is selected from:
##STR7## ##STR8## ##STR9## ##STR10##
13. A method for selectively reducing the flow of blood to at least
a portion of a neoplastic region thereby causing substantial
necrosis of tissue in the neoplastic region without substantial
necrosis of tissue in adjoining regions by administering a compound
of formula I: ##STR11## wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, R.sub.7, and R.sub.8 are independently selected from the
group consisting of H, OH, amine, lower alkoxy, phosphate,
phosphoramidate, or amino acid acyl group; is optionally a single
covalent bond or a double covalent bond; X is a single covalent
bond or a carbonyl, and Y is optionally H or OH.
14. The method of claim 13, wherein the reduction in tumor blood
flow is reversible such that normal tumor blood flow is restored
following cessation of treatment.
15. The method of claim 13, wherein at least one of R.sub.2,
R.sub.3, R4, R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is alkoxy.
16. The method of claim 13, wherein R.sub.4 is OMe.
17. The method of claim 13, wherein X is a single bond.
18. The method of claim 13, wherein X is a carbonyl.
19. The method of claim 13, wherein at least one of R.sub.2,
R.sub.3, R4, R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is
phosphate.
20. The method of claim 13, wherein the compound is selected from:
##STR12## ##STR13## ##STR14## ##STR15##
21. A method for treating neoplastic disease in an animal
comprising administering to an animal an antiproliferative amount
of a compound of formula I: ##STR16## wherein R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are independently
selected from the group consisting of H, OH, amine, lower alkoxy,
phosphate, phosphoramidate, or amino acid acyl group; is optionally
a single covalent bond or a double covalent bond; X is a single
covalent bond or a carbonyl, and Y is optionally H or OH.
22. The method of claim 21, wherein the reduction in tumor blood
flow is reversible such that normal tumor blood flow is restored
following cessation of treatment.
23. The method of claim 21, wherein at least one of R.sub.2,
R.sub.3, R4, R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is lower
alkoxy.
24. The method of claim 21, wherein R.sub.4 is OMe.
25. The method of claim 21, wherein X is a single bond.
26. The method of claim 21, wherein X is a carbonyl.
27. The method of claim 21, wherein at least one of R.sub.2,
R.sub.3, R4, R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is
phosphate.
28. The method of claim 21, wherein the compound is selected from:
##STR17## ##STR18## ##STR19## ##STR20##
29. A method for inhibiting tubulin polymerization by contacting a
tubulin-containing system with a compound of formula I: ##STR21##
wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and
R.sub.8 are independently selected from the group consisting of H,
OH, amine, lower alkoxy, phosphate, phosphoramidate, or amino acid
acyl group; is optionally a single covalent bond or a double
covalent bond; X is single covalent bond or a carbonyl group, and Y
is optionally H or OH.
30. The method of claim 29, wherein said system is a tumor
cell.
31. The method of claim 29, wherein at least one of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is lower
alkoxy.
32. The method of claim 29, wherein R.sub.4 is OMe.
33. The method of claim 29, wherein X is a single bond.
34. The method of claim 29, wherein X is a carbonyl.
35. The method of claim 29, wherein at least one of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is
phosphate.
36. The method of claim 29, wherein the compound is selected from:
##STR22## ##STR23## ##STR24## ##STR25##
37. A pharmaceutical formulation containing a compound of formula I
in a pharmaceutically suitable carrier: ##STR26## wherein R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are
independently selected from the group consisting of H, OH, amine,
lower alkoxy, phosphate, phosphoramidate, or amino acid acyl group;
is optionally a single covalent bond or a double covalent bond; X
is a single covalent bond or a carbonyl, and Y is optionally H or
OH.
38. The method of claim 37, wherein at least one of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is lower
alkoxy.
39. The method of claim 37, wherein R.sub.4 is OMe.
40. The method of claim 37, wherein X is a single bond.
41. The method of claim 37, wherein X is a carbonyl.
42. The method of claim 37, wherein at least one of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, or R.sub.8 is
phosphate.
43. The method of claim 37, wherein the compound is selected from:
##STR27## ##STR28## ##STR29## ##STR30##
Description
RELATED APPLICATIONS
[0001] This is a Continuation of U.S. patent application Ser. No.
10/404,525, filed Apr. 1, 2003, which itself is a
Continuation-In-Part of co-pending U.S. patent application Ser. No.
09/804,280, filed Mar. 12, 2001, which itself claims priority to
U.S. provisional patent application Ser. No. 60/188,295, filed on
Mar. 10, 2000. This application also claims the priority benefit of
co-pending U.S. patent application Er. No. 10/218,833, filed Aug.
14, 2002, which itself claims priority to both U.S. patent
application Ser. No. 09/505,402, filed Feb. 16, 2000 and U.S.
provisional patent application Ser. No. 60/120,478, filed Feb. 18,
1999. Attention is called to U.S. Pat. No. 6,162,930 issued to
Pinney et al. on Dec. 19, 2000, which is incorporated in pertinent
part by reference herein for the reasons cited.
BACKGROUND OF THE INVENTION
[0002] The cytoskeletal protein tubulin is among the most
attractive therapeutic drug targets for the treatment of solid
tumors. A particularly successful class of chemotherapeutics
mediates its anti-tumor effect through a direct binding interaction
with tubulin. This clinically promising class of therapeutics,
called Tubulin Binding Agents, exhibit potent tumor cell
cytotoxicity by efficiently inhibiting the polymerization of
.alpha..beta.-tubulin heterodimers into the microtubule structures
that are required to facilitate mitosis or cell division (Hamel,
Medicinal Research Reviews, 1996).
[0003] Currently, the most widely recognized and clinically useful
antitumor agents are Vinca Alkaloids, such as Vinblastine and
Vincristine (Owellen et al, Cancer Res., 1976; Lavielle et al, J.
Med. Chem., 1991) along with Taxanes such Taxol (Kingston, J. Nat.
Prod., 1990; Schiff et al, Nature, 1979; Swindell et al, J. Cell
Biol., 1981). Additionally, natural products such as Rhizoxin
(Nakada et al, Tetrahedron Lett., 1993; Boger et al, J. Org. Chem.,
1992; Rao, et al, Tetrahedron Lett., 1992; Kobayashi et al, Pure
Appl. Chem., 1992; Kobayashi et al, Indian J. Chem., 1993; Rao et
al, Tetrahedron Lett., 1993), the Combretastatins (Lin et al,
Biochemistry, 1989; Pettit et al, J. Nat. Prod., 1987; Pettit et
al, J. Org. Chem., 1985; Pettit et al, Can. J. Chem., 1982; Dorr et
al, Invest. New Drugs, 1996), Curacin A (Gerwick et al, J. Org.
Chem., 59:1243, 1994), Podophyllotoxin (Hammonds et al, J. Med.
Microbiol, 1996; Coretese et al, J. Biol. Chem., 1977), Epothilones
A and B (Nicolau et al., Nature, 1997), Dolastatin-10 (Pettit et
al, J. Am. Chem. Soc., 1987; Pettit et al, Anti-Cancer Drug Des.,
1998), and Welwistatin (Zhang et al, Molecular Pharmacology, 1996),
as well as certain synthetic analogues including Phenstatin (Pettit
G R et al., J. Med. Chem., 1998), 2-styrylquinazolin-4(3H)-ones
("SQOs", Jiang et al, J. Med. Chem., 1990), and highly oxygenated
derivatives of cis- and trans-stilbene and dihydrostilbene (Cushman
et al, J. Med. Chem., 1991) are all known to mediate tumor
cytotoxic activity through a mode of action that includes tubulin
binding and subsequent inhibition of mitosis.
[0004] Normally, during the metaphase of cell mitosis, the nuclear
membrane has broken down and tubulin is able to form centrosomes
(also called microtubule organizing centers) that facilitate the
formation of the microtubule spindle apparatus to which the
dividing chromosomes become attached. Subsequent polymerization and
depolymerization of the spindle apparatus mitigates the separation
of the daughter chromosomes during anaphase such that each daughter
cell contains a full complement of chromosomes. As
antiproliferatives or antimitotic agents, Tubulin Binding Agents
exploit the relatively rapid mitosis that occurs in proliferating
tumor cells. By binding to tubulin and inhibiting the formation of
the spindle apparatus in a tumor cell, the Tubulin Binding Agent
can cause significant tumor cell cytotoxicity with relatively minor
effects on the slowly dividing normal cells of the patient.
[0005] The exact nature of tubulin binding site interactions remain
largely unknown, and they definitely vary between each class of
Tubulin Binding Agent. Photoaffinity labeling and other binding
site elucidation techniques have identified three key binding sites
on tubulin: 1) the Colchicine site (Floyd et al, Biochemistry,
1989; Staretz et al, J. Org. Chem., 1993; Williams et al, J. Biol.
Chem., 1985; Wolff et al, Proc. Natl. Acad. Sci. U.S.A., 1991), 2)
the Vinca Alkaloid site (Safa et al, Biochemistry, 1987), and 3) a
site on the polymerized microtubule to which taxol binds (Rao et
al, J. Natl. Cancer Inst., 1992; Lin et al, Biochemistry, 1989;
Sawada et al, Bioconjugate Chem, 1993; Sawada et al, Biochem.
Biophys. Res. Commun., 1991; Sawada et al, Biochem. Pharmacol.,
1993). An important aspect of this work requires a detailed
understanding, at the molecular level, of the "small molecule"
binding domain of both the .alpha. and .beta. subunits of tubulin.
The tertiary structure of the .alpha.,.beta. tubulin heterodimer
was reported in 1998 by Downing and co-workers at a resolution of
3.7 .ANG. using a technique known as electron crystallography
(Nogales et al, Nature, 1998). This brilliant accomplishment
culminates decades of work directed toward the elucidation of this
structure and should facilitate the identification of small
molecule binding sites, such as the colchicine site, using
techniques such as photoaffinity and chemical affinity labeling
(Chavan et al, Bioconjugate Chem., 1993; Hahn et al, Photochem.
Photobiol., 1992).
[0006] An aggressive chemotherapeutic strategy for the treatment
and maintenance of solid tumor cancers continues to rely on the
development of architecturally new and biologically more potent
Tubulin Binding Agents which mediate their effect through a direct
binding interaction with tubulin. The present invention addresses
this urgent need by providing a structurally novel class of Tubulin
Binding Agent compositions with potent antiproliferative activity
and tumor cell cytotoxicity. In addition, the present invention
provides the important discovery that corresponding prodrug
constructs of these agents have selective effects on the tumor
vasculature that are independent of any antimitotic effect on the
cells of the tumor. These agents are capable of selectively
shutting down the flow of blood to a tumor and causing secondary
tumor cell death. Thus the present compositions have expanded
clinical utility over known tubulin binding agents.
SUMMARY OF THE INVENTION
[0007] The present invention relates to a discovery of
dihydronaphthalene compounds that result from the judicious
combination of a non-tubulin binding molecular template which, when
suitably modified with structural features such as hydroxyl
moieties and arylalkoxy groups, are found to function as Tubulin
Binding Agents capable of inhibiting tubulin polymerization and
tumor cell proliferation.
[0008] One important aspect of the present invention provides a
compound of the following general formula I: ##STR1## wherein:
[0009] R.sub.1 is optionally H, halogen, or lower alkoxy, [0010]
R.sub.2 through R.sub.8 are independently selected from the group
consisting of H, OH, amine, lower alkoxy, phosphate,
phosphoramidate, or amino acid acyl group, [0011] is optionally a
single covalent bond or double covalent bond, [0012] X is
optionally a single covalent bond or a carbonyl group, and [0013] Y
is optionally H or OH.
[0014] In a more specific embodiment, the present invention focuses
on dihydronaphthalene derivatives, particularly compounds of the
following general Formula Ia: ##STR2## wherein: [0015] R.sub.1a,
R.sub.1b, R.sub.1c, R.sub.1d, and R.sub.1e are independently
selected from the group consisting of H, halogen, or lower alkoxy,
[0016] R.sub.2 through R.sub.6 are independently selected from the
group consisting of H, OH, halogen, amine, lower alkoxy, phosphate,
phosphoramidate, or amino acid acyl; and [0017] X is a single
covalent bond or a carbonyl group.
[0018] Compounds of Formula Ia can be synthesized according to the
following general synthetic scheme: ##STR3##
[0019] A particularly preferred dihydronaphthalene derivative is
the compound of the following structure (1): ##STR4##
[0020] In a further specific embodiment, the compound of the
following general Formula Ib: ##STR5## wherein: [0021] R.sub.1a,
R.sub.1b, R.sub.1c, R.sub.1d, and R.sub.1e are independently
selected from the group consisting of H, halogen, or lower alkoxy,
[0022] R.sub.2 through R.sub.6 are independently selected from the
group consisting of H. OH, halogen, amine, lower alkoxy, phosphate,
phosphoramidate, or amino acid acyl; and [0023] X is a single
covalent bond or a carbonyl group.
[0024] In a second aspect, the invention contemplates methods of
contacting a tubulin-containing system with an effective amount of
a compound of Formula I. Methods are also provided for treating a
warm-blooded animal afflicted with a neoplastic disease comprising
administering an effective amount of compound capable of inhibiting
the proliferation of the neoplastic disease. In a preferred
embodiment, the antiproliferative effect has the direct result of
causing tumor cell cytotoxicity due to inhibition of mitosis.
[0025] In a third aspect, the invention broadly contemplates the
provision of a method for treating a warm-blooded animal having a
vascular proliferative disorder comprising administering an
effective amount of a compound of the present invention to achieve
targeted vascular toxicity at a locality of proliferating
vasculature, wherein in the proliferating vasculature is malignant
or nonmalignant.
[0026] In yet another aspect, the invention broadly contemplates
the provision of a method for administering an effective amount of
a compound of the present invention to selectively reduce the flow
of blood to at least a portion of a neoplastic region, thereby
causing substantial necrosis of tissue in the neoplastic region
without substantial necrosis of tissue in adjoining regions. In a
preferred embodiment, the effect of reduced tumor blood flow is
reversible so that normal tumor blood flow is restored following
cessation of treatment.
[0027] The details of one or more embodiments of the invention are
set forth in the accompanying description below. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
Other features, objects, and advantages of the invention will be
apparent from the description. In the specification and the
appended claims, the singular forms also include the plural unless
the context clearly dictates otherwise. Unless defined otherwise,
all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 illustrates a synthetic route for large-scale
preparation of starting material required for synthesis of tubulin
binding agents.
[0029] FIG. 2 illustrates a synthetic route for the preparation of
an exemplary hydroxyl-substituted tetralone.
[0030] FIG. 3 illustrates a synthetic route for the preparation of
an exemplary dihydroxy-substituted tetralone.
[0031] FIG. 4 illustrates a synthetic route for the preparation of
exemplary amine-substituted tetralones.
[0032] FIG. 5 A) depicts exemplary aryl-substituted
dihydronaphthalene tubulin binding agents and; and B) amine,
aryl-substituted dihydronaphthalene and tetrahydronaphthalene
tubulin binding agents and corresponding prodrug constructs.
[0033] FIG. 6 A) depicts exemplary hydroxyl, aryl-substituted
dihydronaphthalene and tetrahydronaphthalene tubulin binding
agents; and B) corresponding prodrug constructs.
[0034] FIG. 7 A) depicts exemplary aroyl-substituted
dihydronaphthalene tubulin binding agents and corresponding prodrug
constructs; B) an exemplary aroyl-substituted naphthalene prodrug
construct.
[0035] FIG. 8 illustrates a general synthetic route for the
preparation of aryl-substituted dihydronaphthalene and
tetrahydronaphthalene tubulin binding agents.
[0036] FIG. 9 illustrates a synthetic route for the preparation of
an exemplary amine, aryl-substituted dihydronaphthalene and its
corresponding phosphoramidate prodrug.
[0037] FIG. 10 illustrates a synthetic route for the preparation of
an exemplary hydroxyl, aryl-substituted dihydronaphthalene tubulin
binding agent.
[0038] FIG. 11 illustrates a synthetic route for the preparation of
an exemplary hydroxyl, aryl-substituted dihydronaphthalene tubulin
binding agent and its corresponding phosphate prodrug.
[0039] FIG. 12 illustrates a synthetic route for the preparation of
an exemplary aroyl-substituted dihydronaphthalene, its
corresponding phosphate prodrug, and an exemplary aroyl-substituted
naphthalene phosphate prodrug.
[0040] FIG. 13 illustrates a synthetic route for the preparation of
an exemplary dihydroxy, aryl-substituted dihydronaphthalene tubulin
binding agent and its corresponding diphosphate prodrug.
[0041] FIG. 14 illustrate a synthetic route for the preparation of
exemplary diaryl-substituted dihydronaphthalene compositions.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The compounds of the present invention demonstrate
remarkable cytotoxicity against a variety of human cancer cell
lines. The ability of an agent to inhibit tubulin polymerization
and microtubule formation is an important property of many
anticancer agents. Disruption of microtubules that comprise the
cytoskeleton and mitotic spindle apparatus can interfere
dramatically with the ability of a cell to successfully complete
cell division. The compounds of the present invention are highly
cytotoxic to actively proliferating cells, inhibiting their mitoic
division and often causing their selective apoptosis while leaving
normal quiescent cells relatively unaffected.
[0043] Further significance is given to new drugs that bind to the
colchicine site since it has recently been shown that many tubulin
binding agents which bind to this site also demonstrate activity
against malignant proliferating vasculature. Antivascular
chemotherapy is an emerging area of cancer chemotherapy which
centers on the development of drugs, called Vascular Targeting
Agents ("VTAs") or vascular damaging agents, that selectively
target the vasculature of tumor cells rather than the tumor cells
themselves. Much of the research in anti-vascular cancer therapy
has focused on understanding the process of new blood vessel
formation, known as angiogenesis, and identifying anti-angiogenic
agents which inhibit the formation of new blood vessels.
Angiogenesis is characterized by the proliferation of tumor
endothelial cells and generation of new vasculature to support the
growth of a tumor. This growth is stimulated by certain growth
factors produced by the tumor itself. One of these growth factors,
Vascular Endothelial Growth Factor ("VEGF"), is relatively specific
towards endothelial cells, by virtue of the restricted and
up-regulated expression of its cognate receptor. Various
anti-angiogenic strategies have been developed to inhibit this
signaling process at one or more steps in the biochemical pathway
in order to prevent the growth and establishment of the tumor
vasculature. However, anti-angiogenic therapies act slowly and must
be chronically administered over a period of months to years in
order to produce a desired effect.
[0044] In contrast to anti-angiogenic agents, VTAs attack solid
tumors by selectively targeting the established tumor vasculature
and causing extensive shutdown of tumor blood flow. A single dose
of a VTA can cause a rapid and selective shutdown of the tumor
neovasculature within a period of minutes to hours, leading
eventually to tumor necrosis by induction of hypoxia and nutrient
depletion. This vascular-mediated cytotoxic mechanism of VTA action
is quite divorced from that of anti-angiogenic agents that inhibit
the formation of new tumor vascularization, rather than interfering
with the existing tumor vasculature. Other agents have been known
to disrupt tumor vasculature but differ in that they also manifest
substantial normal tissue toxicity at their maximum tolerated dose.
In contrast, genuine VTAs retain their vascular shutdown activity
at a fraction of their maximum tolerated dose. Combretastatin A-4
Disodium Phosphate Prodrug ("CA4DP") is the lead drug of a group of
VTAs currently in clinical trials (U.S. Pat. No.5,561,122; Chaplin
et al, Anticancer Res., 1999; Tozer et al, Cancer Res., 1999;
Pettit and Rhodes, Anti-Cancer Drug Des., 1998; Iyer et al, Cancer
Res., 1998; Dark et al, Cancer Res., 1997;; ). Other Tubulin
binding VTAs that have been discovered include the Colchicinoid
ZD6126 (Davis et al., Cancer Research, 2002 ) and the
Combretastatin analog AVE8032 (Lejeune et al, Proceedings of the
AACR., 2002). It is thought that tubulin-binding VTAs selectively
destabilize the microtubule cytoskeleton of tumor endothelial
cells, causing a profound alteration in the shape of the cell which
ultimately leads to occlusion of the tumor blood vessel and
shutdown of blood flow to the tumor (Kanthou, Blood, 2002). Thus
the invention provides the discovery that the compounds of the
invention as well as analogs thereof, are vascular targeting agents
(VTAs), and thus are useful for the treatment of malignant vascular
proliferative disorders, such as solid tumor cancers, as well as
other nonmalignant vascular proliferative disorders, such as
retinal neovascularization and restenosis.
[0045] In one embodiment, the present invention is directed to the
administration of a vascular targeting agent ("VTA"), particularly
a tubulin binding VTA, for the treatment of malignant or
non-malignant vascular proliferative disorders in ocular
tissue.
[0046] Neovascularization of ocular tissue is a pathogenic
condition characterized by vascular proliferation and occurs in a
variety of ocular diseases with varying degrees of vision failure.
The administration of a VTA for the pharmacological control of the
neovascularization associated with non-malignant vascular
proliferative disorders such as wet macular degeneration,
proliferative diabetic retinopathy or retinopathy of prematurity
would potentially benefit patients for which few therapeutic
options are available. In another embodiment, the invention
provides the administration of a VTA for the pharmacological
control of neovascularization associated with malignant vascular
proliferative disorders such as ocular tumors.
[0047] The blood-retinal barrier (BRB) is composed of specialized
nonfenestrated tightly-joined endothelial cells that form a
transport barrier for certain substances between the retinal
capillaries and the retinal tissue. The nascent vessels of the
cornea and retina associated with the retinopathies are aberrant,
much like the vessels associated with solid tumors. Tubulin binding
agents, inhibitors of tubulin polymerization and vascular targeting
agents, may be able to attack the aberrant vessels because these
vessels do not share architectural similarities with the blood
retinal barrier. Tubulin binding agents may halt the progression of
the disease much like they do with a tumor-vasculature.
[0048] The compounds of the present invention are also contemplated
for use in the treatment of vascular disease, particularly
atherosclerosis and restenosis. Atherosclerosis is the most common
form of vascular disease and leads to insufficient blood supply to
critical body organs, resulting in heart attack, stroke, and kidney
failure. Additionally, atherosclerosis causes major complications
in those suffering from hypertension and diabetes, as well as
tobacco smokers. Atherosclerosis is a form of chronic vascular
injury in which some of the normal vascular smooth muscle cells
("VSMC") in the artery wall, which ordinarily control vascular tone
regulating blood flow, change their nature and develop
"cancer-like" behavior. These VSMC become abnormally proliferative,
secreting substances (growth factors, tissue-degradation enzymes
and other proteins) which enable them to invade and spread into the
inner vessel lining, blocking blood flow and making that vessel
abnormally susceptible to being completely blocked by local blood
clotting, resulting in the death of the tissue served by that
artery.
[0049] Restenosis, the recurrence of stenosis or artery stricture
after corrective surgery, is an accelerated form of
atherosclerosis. Recent evidence has supported a unifying
hypothesis of vascular injury in which coronary artery restenosis
along with coronary vein graft and cardiac allograft
atherosclerosis can be considered to represent a much-accelerated
form of the same pathogenic process that results in spontaneous
atherosclerosis. Restenosis is due to a complex series of
fibroproliferative responses to vascular injury involving potent
growth-regulatory molecules, including platelet-derived growth
factor (PDGF) and basic fibroblast growth factor (bFGF), also
common to the later stages in atherosclerotic lesions, resulting in
vascular smooth muscle cell proliferation, migration and neointimal
accumulation.
[0050] Restenosis occurs after coronary artery bypass surgery
(CAB), endarterectomy, and heart transplantation, and particularly
after heart balloon angioplasty, atherectomy, laser ablation or
endovascular stenting (in each of which one-third of patients
redevelop artery-blockage (restenosis) by 6 months), and is
responsible for recurrence of symptoms (or death), often requiring
repeat revascularization surgery. Despite over a decade of research
and significant improvements in the primary success rate of the
various medical and surgical treatments of atherosclerotic disease,
including angioplasty, bypass grafting and endarterectomy,
secondary failure due to late restenosis continues to occur in
30-50% of patients.
[0051] The most effective way to prevent this disease is at the
cellular level, as opposed to repeated revascularization surgery
which can carry a significant risk of complications or death,
consumes time and money, and is inconvenient to the patient.
[0052] As used herein, the following terms in quotations shall have
the indicated meanings, whether in plural or singular form:
[0053] "Amino acid acyl group" in the amino acid acylamino group is
an acyl group derived from the amino acid. The amino acids may be
enumerated by .alpha.-amino acids, .beta.-amino acids and
.gamma.-amino acids. Examples of preferred amino acids include
glycine, alanine, leucine, serine, lysine, glutamic acid, asparatic
acid, threonine, valine, isoleucine, ornithine, glutamine,
asparagines, tyrosine, phenylalanine, cysteine, methionine,
arginine, .beta.-alanine, tryptophan, proline, histidine, etc. The
preferred amino acid is serine and the preferred amino acid acyl
group is a serinamide.
[0054] "Amine" refers to a free amine NH.sub.2 or a lower
alkylamino.
[0055] "Animal" refers to any warm-blooded mammal, preferably a
human.
[0056] "Alkyl" refers to a group containing from 1 to 8 carbon
atoms and may be straight chained or branched. An alkyl group is an
optionally substituted straight, branched or cyclic saturated
hydrocarbon group. When substituted, alkyl groups may be
substituted with up to four substituent groups, R as defined, at
any available point of attachment. When the alkyl group is said to
be substituted with an alkyl group, this is used interchangeably
with "branched alkyl group". Exemplary unsubstituted such groups
include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl,
isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl,
octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and
the like. Exemplary substituents may include but are not limited to
one or more of the following groups: halo (such as F, Cl, Br, I),
haloalkyl (such as CCl.sub.3 or CF.sub.3), alkoxy, alkylthio,
hydroxy, carboxy (--COOH), alkyloxycarbonyl (--C(O)R),
alkylcarbonyloxy (--OCOR), amino (--NH.sub.2), carbamoyl
(--NHCOOR-- or --OCONHR--), urea (--NHCONHR--) or thiol (--SH).
Alkyl groups as defined may also comprise one or more carbon to
carbon double bonds or one or more carbon to carbon triple
bonds.
[0057] "Aryl" refers to groups with aromaticity, including 5- and
6-membered single-ring aromatic groups that may include from zero
to four heteroatoms, as well as multicyclic systems with at least
one aromatic ring. Examples of aryl groups include benzene, phenyl,
pyrrole, furan, thiophene, thiazole, isothiazole, imidazole,
triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine,
pyrazine, pyridazine, and pyrimidine, and the like. The aromatic
ring can be substituted at one or more ring positions with such
substituents as described above, as for example, halogen, hydroxyl,
alkoxy, etc. The preferred aryl group of the present invention is a
benzene ring.
[0058] "Aroyl" refers to the --(C.dbd.O)-aryl groups, wherein aryl
is defined as hereinabove. The aryl group is bonded to the core
compound through a carbonyl bridge.
[0059] "Cycloalkyl" is a species of alkyl containing from 3 to 15
carbon atoms, without alternating or resonating double bonds
between carbon atoms. It may contain from 1 to 4 rings. Exemplary
unsubstituted such groups include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, adamantyl, etc. Exemplary substituents
include one or more of the following groups: halogen, alkyl,
alkoxy, alkyl hydroxy, amino, nitro, cyano, thiol and/or
alkylthio.
[0060] "Halogen" or "Halo" refers to chlorine, bromine, fluorine or
iodine.
[0061] "Lower alkoxy" refers to --O-alkyl groups, wherein alkyl is
as defined hereinabove. The alkoxy group is bonded to the core
compound through the oxygen bridge. The alkoxy group may be
straight chained or branched; although the straight-chain is
preferred. Examples include methoxy, ethyloxy, propoxy, butyloxy,
t-butyloxy, i-propoxy, and the like. Preferred alkoxy groups
contain 1-4 carbon atoms, especially preferred alkoxy groups
contain 1-3 carbon atoms. The most preferred alkoxy group is
methoxy.
[0062] "Lower alkylamino" refers to a group wherein one or two
alkyl groups is bonded to an amino nitrogen, i.e., NH(alkyl). The
nitrogen is the bridge connecting the alkyl group the core
compound. Examples include NHMe, NHEt, NHPr, and the like.
[0063] "Prodrug" refers to a precursor form of the drug which is
metabolically converted in vivo to produce the active drug.
Preferred prodrugs of the present invention include the phosphate,
phosphoramidate, or amino acid acyl groups as defined herein. The
phosphate ester salt moiety may also include
(--OP(O)(O-alkyl).sub.2 or (--OP(O)(O--NH.sub.4.sup.+).sub.2).
[0064] "Phenolic moiety" means herein a hydroxy group when it
refers to an R group on an aryl ring.
[0065] "Phosphate", "Phosphate moiety", or "Phosphate prodrug salt"
refers to phosphate ester salt moiety
(--OP(O)(O.sup.-M.sup.+).sub.2), a phosphate triester moiety
(--OP(O)(OR).sub.2) or a phosphate diester moiety
(--OP(O)(OR)(O.sup.-M.sup.+), where M is a salt and R is chosen to
be any appropriate alkyl or branched alkyl substituent (the two R
groups may be the same alkyl group or may be mixed), or benzyl, or
aryl groups. The salt M is advantageously Na, K and Li, but the
invention is not limited in this respect.
[0066] "Phosphoramidate" refers to a phosphoamidate ester salt
moiety (--NP(O)(O.sup.-M.sup.+).sub.2), a phosphoramidate diester
moiety (--NP(O)(OR).sub.2), or a phosphamidate disalt moiety
(--NP(O)(OR)(O.sup.-M.sup.+), where M is a salt and R is chosen to
be any appropriate alkyl or branched alkyl substituent (the two R
groups may be the same alkyl group or may be mixed), or benzyl, or
aryl groups. The salt M is advantageously Na, K and Li, but the
invention is not limited in this respect.
[0067] "Salt" is a pharmaceutically acceptable salt and can include
acid addition salts such as the hydrochlorides, hydrobromides,
phosphates, sulphates, hydrogen sulphates, alkylsulphonates,
arylsulphonates, acetates, benzoates, citrates, maleates,
fumarates, succinates, lactates, and tartrates; alkali metal
cations such as Na, K, Li, alkali earth metal salts such as Mg or
Ca or organic amine salts such as those disclosed in PCT
International Application Nos. WO02/22626 or WO00/48606.
[0068] "Tubulin Binding Agent" shall refer to a ligand of tubulin
or a compound capable of binding to either .alpha..beta.-tubulin
heterodimers or microtubules and interfering with the
polymerization or depolymerization of microtubules.
[0069] "Tumors", "Cancers", or "Neoplastic Disease" shall be used
interchangeably and include (but are not limited to) the following:
[0070] 1) carcinomas, including that of the bladder, breast, colon,
kidney, liver, lung, including small cell lung cancer, esophagus,
gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate,
and skin, including squamous cell carcinoma; [0071] 2)
hematopoietic tumors of lymphoid lineage, including leukemia, acute
lymphocytic leukemia, acute lymphoblastic leukemia, B-cell
lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins
lymphoma, hairy cell lymphoma and Burkett's lymphoma; [0072] 3)
hematopoietic tumors of myeloid lineage, including acute and
chronic myelogenous leukemias, myelodysplastic syndrome and
promyelocytic leukemia; [0073] 4) tumors of mesenchymal origin,
including fibrosarcoma and rhabdomyosarcoma; [0074] 5) tumors of
the central and peripheral nervous system, including astrocytoma,
neuroblastoma, glioma and schwannomas; and [0075] 6) other tumors,
including melanoma, seminoma, teratocarcinoma, osteosarcoma,
xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer,
anaplastic thyroid cancer and Kaposi's sarcoma.
[0076] "Vascular toxicity" refers to the selective destruction,
damage, or occlusion, whether reversible or irreversible, partial
or complete, of proliferating vasculature.
[0077] "Malignant proliferating vasculature" refers to the
endothelium, artery, blood vessel, or neovasculature formed by a
malignant disease state, such as a tumor.
[0078] "Nonmalignant proliferating vasculature" refers to the
endothelium, artery, blood vessel, or neovasculature formed by
undesirable or pathological angiogenesis and that is associated
with disease states other than a malignant disease state, including
without limitation ocular diseases such wet or age-related macular
degeneration, diabetic retinopathy, retinopathy of prematurity,
diabetic molecular edema, uveitis, and corneal neovascularization,
or other nonocular disease states such as psoriasis, rheumatoid
arthritis, atheroma, restenosis, Kaposi's sarcoma, haemangioma, and
in general, inflammatory diseases characterized by vascular
proliferation.
[0079] "Antiproliferative" or "antimitotic" refer to the ability of
the compounds of the present invention to directly inhibit the
proliferation of tumor cells and impart direct cytotoxicity towards
tumor cells.
[0080] "Treating" (or "treat") as used herein includes its
generally accepted meaning which encompasses prohibiting,
preventing, restraining, and slowing, stopping, or reversing
progression, severity, of a resultant symptom. As such, the methods
of this invention encompass both therapeutic and prophylactic
administration.
[0081] "Effective amount" refers to the amount or dose of the
compound, upon single or multiple dose administration to the
patient, which provides the desired effect in the patient under
diagnosis or treatment.
[0082] An effective amount can be readily determined by the
attending diagnostician, as one skilled in the art, by the use of
known techniques and by observing results obtained under analogous
circumstances. In determining the effective amount or dose of
compound administered, a number of factors are considered by the
attending diagnostician, including, but not limited to: the species
of mammal; its size, age, and general health; the specific disease
involved; the degree of or involvement or the severity of the
disease; the response of the individual patient; the particular
compound administered; the mode of administration; the
bioavailability characteristics of the preparation administered;
the dose regimen selected; the use of concomitant medication; and
other relevant circumstances.
[0083] A typical daily dose will contain from about 0.1 mg/kg to
about 1000 mg/kg of the active compound of this invention.
Preferably, daily doses will be about 10 mg/kg to about 100 mg/kg,
and most preferably about 10 mg.
[0084] In effecting treatment of a patient afflicted with a
condition, disease or disorder described herein, a compound of the
present invention can be administered systemically in any form or
mode which makes the compound bioavailable in effective amounts.
Systemic administration may be accomplished by administration of a
compound of the present invention into the bloodstream at a site
which is separated by a measurable distance from the diseased or
affected organ or tissue. For example, compounds of the present
invention can be administered orally, parenterally, subcutaneously,
intramuscularly, intravenously, transdermally, intranasally,
rectally, buccally, and the like. Oral or intravenous
administration is generally preferred for treating neoplastic
disease or cancer. Alternatively, the compound may be administered
non-systemically by local administration of the compound of the
present invention directly at the diseased or affected organ or
tissue. Treatment of ocular diseases characterized by the presence
of non-malignant proliferative vasculature or neovascularization,
can be achieved using non-systemic administration methods such as
intravitreal injection, sub-Tenon's injection, ophthalmic drops,
iontophoresis, topical formulation and implants and/or inserts. One
skilled in the art of preparing formulations can readily select the
proper form and mode of administration depending upon the
particular characteristics of the compound selected, the disease
state to be treated, the stage of the disease, and other relevant
circumstances.
[0085] It will be understood by the skilled reader that all of the
compounds used in the present invention are capable of forming
salts, and that the salt forms of pharmaceuticals are commonly
used, often because they are more readily crystallized and purified
than are the free bases. In all cases, the use of the
pharmaceuticals described above as salts is contemplated in the
description herein, and often is preferred, and the
pharmaceutically acceptable salts of all of the compounds are
includes in the names of them.
[0086] According to another aspect, the present invention provides
a pharmaceutical composition, which comprises a compound of the
present invention or a pharmaceutically acceptable salt thereof as
defined hereinabove and a pharmaceutically acceptable diluent or
carrier.
[0087] The pharmaceutical compositions are prepared by known
procedures using well-known and readily available ingredients. In
making the compositions of the present invention, the active
ingredient will usually be mixed with a carrier, or diluted by a
carrier, or enclosed within a carrier, and may be in the form of a
capsule, sachet, paper, or other container. When the carrier serves
as a diluent, it may be a solid, semi-solid, or liquid material
which acts as a vehicle, excipient, or medium for the active
ingredient. The compositions can be in the form of tablets, pills,
powders, lozenges, sachets, cachets, elixirs, suspensions,
emulsions, solutions, syrups, aerosols, ointments containing, for
example, up to 10% by weight of active compound, soft and hard
gelatin capsules, suppositories, sterile injectable solutions, and
sterile packaged powders.
[0088] Some examples of suitable carriers, excipients, and diluents
include lactose, dextrose, sucrose, sorbitol, mannitol, starches,
gum, acacia, calcium phosphate, alginates, tragcanth, gelatin,
calcium silicate, micro-crystalline cellulose,
polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose,
methyl and propyl hydroxybenzoates, talc, magnesium stearate, and
mineral oil. The formulations can additionally include lubricating
agents, wetting agents, emulsifying and suspending agents,
preserving agents, sweetening agents, or flavoring agents.
Compositions of the invention may be formulated so as to provide
quick, sustained, or delayed release of the active ingredient after
administration to the patient by employing procedures well know in
the art.
[0089] The compositions are preferably formulated in a unite dosage
form, each dosage containing from about 1 mg to about 500 mg, more
preferably about 5 mg to about 300 mg (for example 25 mg) of the
active ingredient. The term "unit dosage form" refers to a
physically discrete unit suitable as unitary dosages for human
subjects and other mammals, each unit containing a predetermined
quantity of active material calculated to produce the desired
therapeutic effect, in association with a suitable pharmaceutical
carrier, diluent, or excipient. The following formulation examples
are illustrative only and are not intended to limit the scope of
the invention in any way.
[0090] The compositions of the present invention may be formulated
in a conventional manner using one or more pharmaceutically
acceptable carriers. Thus, the active compounds of the invention
may be formulated for oral, buccal, transdermal (e.g., patch),
intranasal, parenteral (e.g., intravenous, intramuscular or
subcutaneous) or rectal administration or in a form suitable for
administration by inhalation or insufflation.
[0091] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose of calcium phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycollate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form, of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, methyl cellulose or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters or ethyl alcohol); and
preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic
acid).
[0092] For buccal administration the composition may take the form
of tablets or lozenges formulated in conventional manner.
[0093] The active compounds of the invention may be formulated for
parenteral administration by injection, including using
conventional catheterization techniques or infusion. Formulations
for injection may be presented in unit dosage form, e.g., in
ampules or in multi-dose containers, with an added preservative.
The compositions may take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain formulating
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in a powder form for
reconstitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use.
[0094] The active compounds of the invention may also be formulated
in rectal compositions such as suppositories or retention enemas,
e.g., containing conventional suppository bases such as cocoa
butter or other glycerides.
[0095] For intranasal administration or administration by
inhalation, the active compounds of the invention are conveniently
delivered in the form of a solution or suspension form a pump spray
container that is squeezed or pumped by the patient or as an
aerosol spray presentation from a pressurized container or a
nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount. The
pressurized container or nebulizer may contain a solution or
suspension of the active compound. Capsules and cartridges (made,
for example, from gelatin) for use in an inhaler or insufflator may
be formulated containing a powder mix of a compound of the
invention and a suitable powder base such as lactose or starch.
[0096] Tablets or capsules of the compounds may be administered
singly or two or more at a time as appropriate. It is also possible
to administer the compounds in sustained release formulations.
[0097] The physician will determine the actual dosage which will be
most suitable for an individual patient and it will vary with the
age, weight and response of the particular patient. The above
dosages are exemplary of the average case. There can of course, be
individual instances where higher or lower dosage ranges are
merited, and such are within the scope of this invention.
[0098] The compounds of the present invention can be administered
by inhalation or in the form of a suppository or pessary, or they
may be applied topically in the form of a lotion, solution, cream,
ointment or dusting powder. An alternative means of transdermal
administration is by use of a skin patch. For example, they can be
incorporated into a cream consisting of an aqueous emulsion of
polyethylene glycols or liquid paraffin. They can also be
incorporated, at a concentration of between 1 and 10% by weight,
into an ointment consisting of a white wax or white soft paraffin
base together with such stabilizers an preservatives as may be
required.
[0099] "Administering" means any of the standard methods of
administering a compound to a subject, known to those skilled in
the art. Examples include, but are not limited to intravenous,
intramuscular or intraperitoneal administration.
[0100] The clonogenic toxicity may be increased by imbalancing
Ca.sup.2+ cytosolic levels or nucleotide pools or in combination
thereof.
[0101] For the purposes of this invention "pharmaceutically
acceptable carriers" means any of the standard pharmaceutical
carriers. Examples of suitable carriers are well known in the art
and may-include, but are not limited to, any of the standard
pharmaceutical carriers such as a phosphate buffered saline
solutions phosphate buffered saline containing POLYSORB 80, water,
emulsions such as oilwater emulsion, and various type of wetting
agents. Other carrier may also include sterile solutions, tablets,
coated tablets, and capsules.
[0102] Typically such carriers contain excipients such as starch,
milk, sugar, certain types of clay gelatin, stearic acid or salts
thereof, magnesium or calcium stearate, talc, vegetable fats or
oils, gums glycols, or other known excipients. Such carriers may
also include flavor and color additives or other ingredients.
Compositions comprising such carriers are formulated by well known
convention methods.
[0103] Methods of determining an "effective amount" are well known
to those skilled in the art and depend upon factors including, but
not limited to: the size of the patient and the carrier used.
[0104] The invention is further defined by reference to the
following examples and preparations which describe the manner and
process of making and using the invention and are illustrative
rather than limiting. It will be apparent to those skilled in the
art that many modifications, both to the materials and methods, may
be practiced without departing from the purpose and interest of the
invention.
EXAMPLES
Example 1
Large-Scale Preparations of tert-butyldimethylsiloxy Protected
Mono-Bromide as a Starting Material for Synthesis of Tubulin
Binding Agents
[0105] The following reactions are illustrated in FIG. 1:
3-tert-butyldimethylsiloxy (TBSO)-4-methoxybenzaldehyde (1)
[0106] To a 1-L round-bottom flask was added Isovanillin (80 g, 526
mmol) and DMAP (1 g) under inert atmosphere. Dry dichloromethane
(.about.450 mL) was added, followed by triethylamine (81 mL, 580
mol), at which point the solid entirely dissolved. The mixture was
cooled to 0.degree. C. and tert-butyldimethylsilyl ("TBS") chloride
("TBSCI", 89 g, 590 mmol) was added in one portion. The mixture
began almost immediately to precipitate solid. The mixture was
allowed to stir for 1.5 h at 0.degree. C., at which point TLC (30%
EtOAc/hexanes) showed an almost complete absence of isovanillin.
The mixture was allowed to stir overnight, then the precipitate was
filtered off through Celite. The filtrate was washed with water
(200 mL) followed by saturated NaCl solution (200 mL) and dried
over MgSO4. This was filtered into a tared 1-L flask and
concentrated by distillation on a rotary evaporator, followed by
aspirator vacuum to approximately constant weight, yielding a deep
red-brown liquid (149.4 g; theoretical=140 g). This material was
taken into the next reaction without further characterization.
1-(3-tert-butyldimethylsiloxy(TBSO)-4-methoxyphenyl)ethanol (2)
[0107] The entire crude product from the preceding reaction (-526
mol) was-transferred as a solution in dry ether (200 mL) to a 2-L
round bottom flask equipped with a very large magnetic stirring
bar. An additional 500 mL of dry ether were added and the mixture
was cooled to 0.degree. C. Then methyllithium (500 mL of a 1.4 M
solution, 700 mmol) was added over .about.40 minutes by cannula,
and the mixture was allowed to stir overnight. The deep red mixture
was re-cooled to 0.degree. C. and treated with water (200 mL) very
cautiously at first. The mixture became a heterogeneous yellow. In
a separatory funnel, the aqueous phase was separated and the
organic phase was washed once with saturated NaCl solution and
dried over MgSO.sub.4. After filtration and concentration by
distillation on a rotary evaporator followed by aspirator vacuum, a
deep red liquid was obtained (136.8 g), and was found to be free of
starting material by TLC. This material was taken into the next
reaction without further characterization.
3-tert-butyldimethylsiloxy(TBSO)-4-methoxyacetophenone (3)
[0108] The entire amount of the crude alcohol from the preceding
reaction was transferred to a 3-L round bottom flask as a solution
in .about.1.5 L of dry dichloromethane. Celite (62 g, oven dried),
K.sub.2CO.sub.3 (16 g) and a very large magnetic stirring bar were
added. PCC (115 g) was then added in portions over a 2-hr period,
during which time the heterogeneous yellow mixture became dark
brown. At the end of the addition, large amounts of the starting
alcohol were still present by TLC (25% EtOAc/hexanes) so the
mixture was allowed to stir overnight. At this point, the starting
alcohol was absent (or nearly so) by TLC, and the mixture was
filtered through a 3-cm pad of silica gel, rinsing well with
dichloromethane. The mud-brown solution was concentrated by
distillation on a rotary evaporator followed by aspirator vacuum to
yield an opaque brown liquid. This was purified in 30 mL portions
by Kugelrohr distillation (.about.0.5 Torr, 140.degree. C.) to
yield 104.4 g of a brown liquid which crystallized on brief
standing. This was dissolved in hot hexanes (104 mL) and filtered
hot through Celite to yield a clear yellow solution. This was
seeded and left in a refrigerator (.about.5.degree. C.) overnight.
The crystalline product was filtered cold, washed quickly with a
small amount of cold hexanes and dried under pump vacuum to give
84.8 g (303 mol, 58% yield from isovanillin) recrystallized light
yellow solid, pure by .sup.1H and .sup.13C NMR. A second crop of
crystals (6.3 g) were obtained by dissolving the concentrated
filtrate in hot hexanes (20 mL) followed by seeding and standing
overnight.
[0109] 3. .sup.1H NMR (CDCl.sub.3): 0.15 (s, 6H); 0.98 (s, 9H);
2.52 (s, 3H); 3.85 (s, 3H); 6.85 (d, 1H, J=8.4); 7.45 (s, 1H); 7.56
(dd, 1H, J =8.4, 2.2).
[0110] .sup.13C NMR (CDCl.sub.3): -4.8, 18.4, 25.6, 26.3, 55.4,
110.7, 120.2, 123.5, 130.5, 144.7, 155.3, 196.8.
.alpha.-halo-3-tert-butyldimethylsiloxy(TBSO)-4-methoxyacetophenone
(5)
[0111] An important part of the present invention is a new
efficient method of converting 3-TBSO-4-methoxyacetophenone (3) to
.alpha.-halo-3-TBSO-4-methoxyacetophenone (5) by treatment of the
trimethylsilyl enol ether (4)
[1-(3-TBSO-4-methoxyphenyl)-1-trimethylsiloxy(TMSO)-ethylene] with
elemental halogen. Bromine is the preferred halogen. It is
understood that chlorine and iodine may be utilized in place of
bromine should iodo or chloro analogs be desired.
Example 2
Synthesis of Substituted Tetralones
[0112] A fundamental intermediate in the synthesis of the compounds
of the present invention is a substituted tetralone structure (see
FIG. 8).
a. Synthesis of Mono-Hydroxy Substituted Tetralone
[0113] A procedure for the synthesis of mono-hydroxy substituted
tetralone from tetrahydronaphthalene is illustrated with an
exemplary synthetic route in FIG. 2. A mixture of
6-methoxy-1,2,3,4-tetrahydronaphthalene (6) and TMEDA was treated
with sec-butyllithium at room temperature followed by addition of
trimethyl borate. The resultant mixture was subsequently treated
with acetic acid and 35% hydrogen peroxide to form the
hydroxytetrahydronaphthalenes 7 and 8 which are easily separated by
column chromatography.
[0114] The 5-hydroxy isomer 7 was converted to
5-acetoxy-6-methoxy-1,2,3,4-tetrahydronaphthalene (9) in 95% yield.
Reaction of the 9 with DDQ in dioxane-water led to the formation of
tetralone 10 as a single isomer in 96 % yield. The correct position
of the keto group was confirmed by single-crystal X-ray
crystallography. The acetate was removed by treating tetralone 10
with sodium bicarbonate in methanol to form
5-hydroxy-6-methoxy-1-tetralone (11).
[0115] 5-Hydroxy-6-methoxy-1-tetralone (11): .sup.1H-NMR (300 MHz,
CDCl.sub.3) .delta. 2.11 (m, 2H), 2.60 (t, 2H, J=6.2 Hz), 2.93 (t,
2H, J=6.1 Hz), 3.95 (s, 3H), 5.75 (bs, 1H), 6.83 (d, 1H, J=8.6 Hz),
7.68 (d, 1H, J=8.6 Hz).
b. Synthesis of Dihydroxy-Substituted Tetralones
[0116] In order to synthesize dihydroxy-substituted
dihydronaphthalenes and their corresponding prodrugs, the invention
provides the synthesis di-hydroxytetralones as intermediates. The
synthesis of an exemplary dihydroxy tetralone is provided in FIG.
3. 5,7-dihydroxy-6-methoxy-1-tetralone (19) is synthesized by
protection of the 7-hydroxy group in the tetrahydronaphthalene 17
as its corresponding TBS ether (13) proceeded in high yield. In
regard to regioselective introduction of the hydroxy group, we
anticipated that due to different ortho-directing effects of
methoxy and -OTBS groups, it should prove possible to obtain one of
the isomers in much higher amount over the other. Accordingly, when
tetrahydronaphthalene 13 was treated sequentially with
sec-butyllithium-TMEDA, trimethylborate, acetic acid, and 35%
hydrogen peroxide, and
5-hydroxy-6-methoxy-7-(tert-butyldimethylsiloxy)-1,2,3,4-tetrahydronaphth-
alene (14) was obtained as a single product. The TBS protecting
group was removed by treating tetrahydronaphthalene 14 with 1M TBAF
in THF and the two hydroxy groups of the resultant
tetrahydronaphthalene 15 were converted to the corresponding
acetates to give
5,7-diacetoxy-6-methoxy-1,2,3,4-tetrahydronaphthalene (16).
Unfortunately, all of our efforts to convert the
tetrahydronaphthalene 16 to the corresponding tetralone failed
using DDQ under various conditions. In order to overcome the
problem of oxidation, we considered different oxidizing agents.
After various trials, chromium (VI) oxide in acetic acid-water was
found to be the reagent of choice. Thus, tetrahydronaphthalene 16
was converted to 5,7-diacetoxy-6-methoxy-1-tetralone (17) in 54%
yield. Approximately 5% of isomeric tetralone 18 was also formed.
Again in this case, the para-directing effect of the methoxy group
led to the formation of the tetralone 17 predominantly. The
acetates in tetralone 17 were removed by treatment with potassium
carbonate in methanol to produce
5,7-dihydroxy-6-methoxy-1-tetralone (19).
[0117] 5,7-Diacetoxy-6-methoxy-1-tetralone (19): .sup.1H-NMR (300
MHz, CDCl.sub.3) .delta. 2.08-2.14 (m, 2H), 2.32 (s, 3H), 2.36 (s,
3H), 2.61 (t, 2H, J=6.6 Hz), 2.74 (t, 2H, J=6.0 Hz), 7.69 (s,
1H).
c. Synthesis of Amino-Functionalized Tetralones
6-Methoxy-5-Nitro-1-Tetralone and 6-Methoxy-7-Nitro-l-Tetralone
[0118] A procedure for the synthesis of mono-amino substituted
tetralone from tetrahydronaphthalene is illustrated with an
exemplary synthetic route in FIG. 4. To an ice-cold, stirred
solution of 6-methoxy-1-tetralone (20, 17.6 g, 0.10 mol) in acetone
(30 mL) was added dropwise a mixture of sulfuric acid (18 mL,
96.0%) and nitric acid (15 mL, 68.0-70.0%). After the addition was
complete, the reaction was stirred at 0.degree. C. for 6 hours, and
TLC was employed to monitor the reaction progress. The reaction
mixture was poured into ice-water, and the mixture was partitioned
between CH.sub.2Cl.sub.2 (3.times.200 mL) and water (200 mL). The
organic layer was washed by saturated NaHCO.sub.3 solution and
water (200 mL each), dried over anhydrous sodium sulfate, and,
after filtration, the organic layer was concentrated in vacuo to
provide a yellow oil. 6-Methoxy-5-nitro-1-tetralone (21, 7.74 g,
0.035 mol) and 6-methoxy-7-nitro-1-tetralone (22, 6.63 g, 0.030
mol) were obtained after purification by column chromatography:
[0119] 6-Methoxy-5-nitro-1-tetralone (21): .sup.1H-NMR (CDCl3, 300
MHz) .delta. 2.15 (m, 2H), 2.65 (t, J=6.2 Hz, 2H), 2.87 (t, J=6.0
Hz, 2H), 7.02 (d, J=8.7 Hz, 1H), 8.19 (d, J=8.9 Hz, 1H).
[0120] 6-Methoxy-7-nitro-1-tetralone (22): .sup.1H-NMR (CDCl.sub.3,
300MHz) .delta. 2.15 (m, 2H), 2.67 (t, J=6.2 Hz, 2H), 3.01 (t,
J=6.1 Hz, 2H), 6.90 (s, 1H), 8.52 (s, 1H).
[0121] To a solution of acetic acid (20 mL) in H.sub.2O (100 mL),
7-nitro-6-methoxy-1-tetralone (21) (2.21 g, 10.0 mol) was added.
The solution was heated to reflux for 1 hour, and then cooled down
to RT. NaHCO.sub.3 (60 mL, saturated solution) was added, and the
mixture was partitioned between CH.sub.2Cl.sub.2 and water. The
organic layer was dried over anhydrous sodium sulfate. After
filtration, the organic layer was concentrated in vacuo to provide
(23) as a red oil.
[0122] 7-Amino-6-methoxy-1-tetralone (23): (1.74 g, 9.1 mol, 91%)
.sup.1H-NMR (CDCl.sub.3, 300 MHz) 2.07 (m, 2H), 2.57 (t, J=6.5 Hz,
2H), 2.86 (t, J=6.0 Hz, 2H), 3.73 (b, 2H), 2.91 (s, 3H), 6.59 (s,
1H), 7.36 (s, 1H).
[0123] Hydroxy-functionalized tetralones were also obtained from
amino-functionalized counterpart. For example, 23 (0.96 g, 5.0 mol)
was dissolved in a mixture of sulfuric acid (96%, 2.1 mL) and
H.sub.2O (3.9 mL). The solution was cooled to 0 C, and ice (5.0 g)
was added resulting in the crystallization of a solid. A solution
of NaNO.sub.2 (0.48 g, 7 mol) in H.sub.2O (5 mL) was added dropwise
at 0.degree. C., After the solution had been stirred for an
additional 10 min, a few crystals of urea were added to decompose
any excess sodium nitrite. To the cold solution of benzenediazonium
bisulfate was added a solution of cupric nitrate trihydrate (19.0
g, 78.6 mol) in H.sub.2O (150 mL) at 0.degree. C. With vigorous
stirring, cuprous oxide (0.72 g, 5.0 mol) was added to the
solution. The solution was stirred for 10 more min, and TLC was
employed to monitor the reaction. The mixture was partitioned
between ethyl ether and water. The organic layer was dried over
anhydrous sodium sulfate, and after filtration, the organic layer
was concentrated in vacuo to provide (12) as a yellow oil.
[0124] 7-Hydroxy-6-methoxy-1-tetralone (12): (0.22 g, 1.15 mol,
23%) .sup.1H-NMR (CDCl.sub.3, 300 MHz) 2.09 (m, 2H), 2.59 (t, J=6.5
Hz, 2H), 2.88 (t, J=6.0 Hz, 2H), 3.95 (s, 1H), 6.66 (s, 1H), 7.56
(s, 1H). C-NMR (CDCl.sub.3, 75 MHz) 23.6, 29.5, 38.6, 56.0, 109.6,
112.2, 126.5, 138.4, 144.4, 151.0, 197.2.
Example 3
Aryl Substituted Dihydronaphthalene-Based Tubulin Binding
Agents
[0125] Our interest in preparing the following aryl-substituted
dihydronaphthalene ligands was based on our molecular recognition
studies which suggest an optimal aryl-aryl distance (centroid to
centroid) for enhanced tubulin binding. Exemplary aryl-substituted
dihydronaphthalene ligands are depicted in FIG. 5A.
[0126] Each aryl-substituted compound was synthesized according the
generalized synthetic scheme illustrated in FIG. 8.
a. Synthesis of Aryl-substituted Tetrahydronaphthalen-1-ol
[0127] To a stirred solution of n-butyllithium (3.7 mL, 1.6 M in
hexane solution, 6.0 mol) in dry ether (40 mL), a solution of
3,4,5-trimethoxyphenylbromide (0.74 g, 3.0 mol) in ether (20 mL)
was added under dry nitrogen at -78.degree. C. The solution was
stirred for 1 h in order to form 3,4,5-trimethoxyphenyllithum
("TPL"). Substituted tetralone reagent (3.0 mol) was added at
-20.degree. C., and the stirring was continued for 2 h (-20.degree.
C.--RT). The mixture was partitioned between CH.sub.2Cl.sub.2 and
water, the organic layer was dried over anhydrous sodium sulfate,
and, after filtration, the organic layer was concentrated in vacuo
to provide a tetrahydronaphthalen-1-ol as a yellow oil. Each
compound was purified by column chromatography.
[0128] Oxi-com 146 (24) (61% yield) .sup.1H-NMR (CDCl.sub.3,300MHz)
.delta. 1.82 (m, 1H), 1.96 (m, 1H), 2.10 (t, J=5.7 Hz, 2H), 2.82
(t, J=5.9 Hz, 2H), 3.68 (s, 3H), 3.78 (s, 3H), 6.55 (s, 2H), 6.22
(d, J=2.6 Hz, 1H), 6.80 (dd, J=2.7, 8.4 Hz, 1H), 7.08 (d, J=8.4 Hz,
1H). .sup.13C-NMR (CDCl.sub.3, 75 MHz) .delta. 14.1, 19.8, 28.9,
41.3, 55.3, 56.1, 60.8, 75.7, 103.8, 113.0, 114.3, 129.7, 129.9,
136.5, 142.6, 144.4, 152.5, 157.9. HRMS (EI) M.sup.+, calcd for
C.sub.20H.sub.24O.sub.5 344.1624, found 344.1622.
[0129] Oxi-com 150 (25) (41% yield) .sup.1H-NMR (CDCl.sub.3, 300
MHz) .delta. 1.84 (m, 1H), 2.00 (m, 1H), 2.12 (m, 2H), 2.22 (s,
1H), 2.67 (m, 1H), 2.94 (m, 1H), 3.78 (s, 6H), 3.85 (s, 3H), 3.87
(s, 3H), 6.55 (s, 2H), 6.70 (d, J=8.0 Hz, 1H), 6.77 (d, J=8.1 Hz,
1H), 7.13 (t, J=7.9 Hz, 1H). .sup.13C-NMR (CDCl.sub.3, 75MHz)
.delta. 19.2, 23.6, 41.0, 55.7, 56.4, 61.1, 75.8, 104.1, 108.8,
120.9, 126.9, 127.1, 136.7, 143.0, 145.0, 152.8, 157.0. HRMS (EI)
M.sup.+, calcd for C.sub.20H.sub.24O.sub.5 344.1624, found
344.1622.
[0130] Oxi-com 156 (26) (58% yield) .sup.1H-NMR (CDCl.sub.3, 300
MHz) 6 1.82 (m, 1H), 2.00 (m, 4H), 2.88 (t, J=6.2, 2H), 3.79 (s,
6H), 3.80 (s, 3H), 3.85 (s, 3H), 6.57 (s, 2H), 6.69 (m, 2H), 6.99
(d, J=7.5 Hz, 1H). .sup.13C-NMR (CDCl.sub.3, 75MHz) .delta. 19.7,
30.2, 41.4, 55.2, 56.1, 60.8, 75.2, 103.7, 112.8, 112.9, 130.2,
134.1, 136.4, 139.1, 144.9, 152.5, 158.7. HRMS (EI) M.sup.+, calcd
for C.sub.20H.sub.240.sub.5 344.1624, found 344.1626.
b. Synthesis of Aryl-Substituted Dihydronaphthalenes.
[0131] To a solution of acetic acid (10 mL) in H.sub.2O (60 mL),
compounds 24, 25, 26, (1 mol) were added respectively. The solution
was heated to reflux for 1 hour, and then cooled down to RT.
NaHCO.sub.3 (20 mL, saturated solution) was added, and the mixture
was partitioned between CH.sub.2Cl.sub.2 and water. The organic
layer was dried over anhydrous sodium sulfate, after filtration,
the organic layer was concentrated in vacuo to provide the
following compounds as a yellow oil. Each compound was purified by
column chromatography.
[0132] Oxi-com 148 (27) (92% yield) .sup.1H-NMR (CDCl.sub.3,
300MHz) .delta. 2.83 (m, 2H), 2.79 (t, J=7.7 Hz, 2H), 3.71 (s, 3H),
3.85 (s, 6H), 3.89 (s, 3H), 6.11 (t, J=4.6 Hz, 1H), 6.57 (s, 2H),
6.65 (d, J=2.6 Hz, 1H), 6.73 (dd, J=2.6, 8.2 Hz, 1H), 7.13 (d,
J=8.2 Hz, 1H). .sup.13C-NMR (CDCl.sub.3, 75 MHz) .delta. 24.1,
27.6, 55.7, 56.4, 61.3, 106.0, 112.0, 112.3, 128.4, 128.5, 129.2,
136.3, 136.7, 137.3, 140.1, 153.3, 158.4. HRMS (EI) M.sup.+, calcd
for C.sub.20H.sub.22O.sub.4 326.1518, found 326.1507. Anal. Calcd
for C.sub.20H.sub.22O.sub.4: C, 73.60; H, 6.79. Found: C, 73.77; H,
6.93.
[0133] Oxi-com 153 (28) (90% yield) .sup.1H-NMR (CDCl.sub.3, 300
MHz) 6 2.39 (m, 2H), 2.86 (t, J=8.3 Hz, 2H), 3.84 (s, 6H), 3.87 (s,
3H), 3.89 (s, 3H), 6.10 (t, J=4.6 Hz, 1H), 6.55 (s, 2H), 6.71 (d,
J=7.7 Hz, 1H), 6.82 (d, J=8.2 Hz, 1H), 7.11 (t, J=8.1 Hz, 1H).
13C-NMR (CDCl.sub.3, 75 MHz) .delta. 19.8, 22.8, 55.6, 56.1, 60.9,
105.7, 109.7, 118.5, 124.5, 126.2, 127.7, 136.0, 136.8, 136.9,
139.7, 152.8, 156.0. HRMS (EI) M.sup.+, calcd for
C.sub.20H.sub.22O.sub.4 326.1518, found 326.1518. Anal. Calcd for
C.sub.20H.sub.22O.sub.4: C, 73.60; H, 6.79. Found: C, 73.77; H,
6.84.
[0134] Oxi-com 141 (29) (90% yield) .sup.1H-NMR (CDCl.sub.3, 300
MHz) .delta. 2.83 (m, 2H), 2.83 (t, J=7.6 Hz, 2H), 3.82 (s, 3H),
3.85 (s, 6H), 3.89 (s, 3H), 5.95 (t, J=4.5Hz, 1H), 6.56 (s, 2H),
6.66 (dd, J=2.6, 8.4 Hz, 1H), 6.73 (d, J=2.6 Hz, 1H), 7.00 (d,
J=8.4 Hz, 1H). .sup.13C-NMR (CDCl.sub.3, 75 MHz) .delta. 23.8,
29.2, 55.7, 56.5, 61.4, 106.1, 111.2, 114.2, 125.2, 127.1, 128.5,
137.1, 137.4, 139.0, 139.9, 153.4, 159.0. HRMS (EI) M.sup.+, calcd
for C.sub.20H.sub.22O.sub.4 326.1518, found 326.1515.
Example 4
Synthesis of a Functionalized Aryl and Aroyl-Substituted
Dihydronaphthalene-Based Tubulin Binding Agents and Corresponding
Prodrugs
a) Synthesis of Amino, Aryl-Substituted Dihydronaphthalene
Analogs
[0135] Amine, Aryl-substituted dihydronaphthelene compounds and
their corresponding prodrugs are contemplated as part of the
invention. Exemplary compounds are depicted in FIG. 5. The C-5
amine dihydronaphthalene derivative, Oxi-com 142 (31), and its
phosphoramidate prodrug Oxi-com 143 (32) were synthesized as
illustrated in FIG. 9.
[0136] To a stirred solution of n-butyllithium (15.0 mL, 1.6 M in
hexane solution, 24.0 mol) in dry ether (160 mL), a solution of
3,4,5-trimethoxyphenylbromide (2.97 g, 12.0 mol) in ether (40 mL)
was added under dry nitrogen at -78.degree. C. The solution was
stirred for 1 h in order to form 3,4,5-trimethoxyphenyllithum.
[0137] 6-Methoxy-5-nitro-1-tetralone (21) (2.65 g, 12.0 mol) was
added at -20 .degree. C. to the trimethoxyphenyllithium and
stirring was continued for 2 h (-20.degree. C.--RT). The mixture
was partitioned between CH.sub.2Cl.sub.2 and water, the organic
layer was dried over anhydrous sodium sulfate, and, after
filtration, the organic layer was concentrated in vacuo, to afford
1-Hydroxy-6-methoxy-5-nitro-1-(3',4',5'-trimethoxyphenyl)tetralin
(30) as a crude yellow oil. (GC-MS shows the yield is about 55%).
This compound, without purification, was added to a refluxing
mixture of acetic acid (10 mL) and water (80 mL), and iron (0.5 g)
was added. After heating at reflux for 1 h, the mixture was
partitioned between CH.sub.2Cl.sub.2 (3.times.100 mL) and water
(100 mL). The organic layer was washed with NaHCO.sub.3 (sat.) and
water (100 mL each), dried over anhydrous sodium sulfate, and,
after filtration, the organic layer was concentrated in vacuo to
provide a yellow oil. Purification by column chromatography
afforded
5-Amino-6-methoxy-1-(3',4',5'-trimethoxyphenyl)-3,4-dihydronapht-
halene (31):
[0138] Oxi-com 142 (31) (2.03 g, 5.95 mol). .sup.1H-NMR
(CDCl.sub.3, 300 MHz) .delta. 2.43 (m, 2H), 2.68 (t, J=7.8 Hz, 2H),
3.84 (s, 6H), 3.86 (s, 3H), 3.89 (s, 3H), 5.93 (t, J=4.7 Hz, 1H),
6.51 (d, J=8.4 Hz, 1H), 6.56 (s, 2H), 6.60 (d, J=8.4 Hz, 1H).
.sup.13C-NMR (CDCl.sub.3, 75 MHz) .delta. 22.0, 23,3, 56.0, 56.5,
61.3, 106.4, 107.5, 117.2, 121.6, 124.5, 128.6, 132.8, 137.4,
137.6, 140.4, 147.5, 153.2. HRMS (EI) M.sup.+, calcd for
C.sub.20H.sub.23NO.sub.4 341.1627, found 341.1635. Anal. Calcd for
C.sub.20H.sub.23NO.sub.4: C, 70.36; H, 6.79; N: 4.10. Found: C,
70.24; H, 6.74; N, 4.08.
[0139] To a stirred solution of C1P(OC.sub.2H.sub.5).sub.2 (0.157
g, 1.0 mol) in dry ethyl ether (20 mL) was added slowly a solution
of 31 (0.341 g, 1 mol) in dry ethyl ether (30 mL) at -78.degree. C.
Once this addition was complete, a solution of N,
N-diisopropylethylamine (0.28 g, 2.2 mol) in dry ethyl ether (2 mL)
was added. The solution was stirred at -78.degree. C. for 2 hours,
followed by stirring at RT for 10 hours. The solution was filtered
and concentrated in vacuo to provide a yellow oil. The yellow oil
was dissolved in dry CH.sub.2Cl.sub.2 (10 mL), then cooled to
-40.degree. C. A solution of MCPBA (0.28 g) in dry CH.sub.2Cl.sub.2
(10 mL) was added. After stirring at RT for 1 h, the organic layer
was washed with saturated Na.sub.2SO.sub.4 solution and water (10
mL each), dried over anhydrous sodium sulfate, and, after
filtration, the organic layer was concentrated in vacuo to provide
a yellow oil. Purification by column chromatography afforded
6-methoxy-1-(3',4',5'-trimethoxyphenyl)-5-diethylphosphoramidate-3,4-dihy-
dronaphthalene (32):
[0140] Oxi-com 143 (32): (0.286 g, 0.60 mol). .sup.1H-NMR
(CDCl.sup.3, 300 MHz) .delta. 1.33 (t, J=7.0 Hz, 6H), 2.75 (t,
J=8.5 Hz, 2H), 3.21 (t, J=8.6 Hz, 2H), 3.82 (s, 3H), 3.84 (s, 6H),
3.92 (s, 3H), 4.11 (m, 4H), 6.46 (s, 2H), 6.50 (d, J=8.4 Hz, 1H),
6.57 (d, J=8.6 Hz, 1H). .sup.13C-NMR (CDC.sub.13, 75MHz)
.delta.16.2, 16.3, 24.8, 32.4, 55.6, 56.2, 61.0, 62.9, 63.0, 107.0,
107.6, 123.7, 124.7, 129.1, 131.4, 133.4, 134.8, 152.8, 153.1.
.sup.31P-NMR (CDCl.sub.3, 300MHz) .delta. 6.51. HRMS (EI) M.sup.+,
calcd for C.sub.24H.sub.32NPO.sub.7477.1916, found 477.1928.
[0141] Acyl amino acid ester prodrugs of Amino, Aryl-Substituted
Dihydronaphthalene Analogs were also synthesized. The L-serinamide
prodrug derivative of 31 was obtained by dissolving
3,4-dihydro-5-FMOC-L-serinamide-6-methoxy-1-(3',4',5'-trimethoxyphenyl)
naphthalene (0.0435 g, 0.0679 mmol) in CH.sub.2Cl.sub.2 (1.2 mL)
and CH.sub.3OH (1.2 mL). While stirring, NaOH (0.003 g, 0.000075
mmol) was added and allowed to react at room temperature for 16 h.
After completion, extraction of the organic layer was accomplished
with EtOAc, water, and saturated NaCl (100 mL each). The organic
layer was dried with anhydrous sodium sulfate. Purification by
preparative TLC and flash column chromatography (silica gel, 90:10
methylene chloride:methanol) afforded the desired serinamide
product (33)
[0142] Oxi-com 229 (33): (0.010 g, 0.024 mmol, 36%) .sup.1H-NMR
(CDCl.sub.3, 300 MHz) .delta. 2.30 (m, 2H) 2.70 (t, J=7.70 Hz, 2
H), 3.77 (s, 3 H), 3.82 (s, 6H), 3.89 (s, 3 H), 4.00 (m, 3H), 5.98
(t, J=4.45 Hz, 1H), 6.57 (s, 2H), 6.67 (d, J=8.55 Hz, 1H), 6.98 (d,
J=8.55 Hz, 1H), .sup.13C-NMR (CDCl.sub.3, 300 MHz) .delta. 23.0,
24.0, 55.8, 56.1, 60.9, 105.8, 107.7, 125.1, 128.8, 135.3, 136.7,
139.5, 152.8, 152.9
b) Synthesis of Hydroxy-Dihydronaphthalene Analogs
[0143] Dihydronaphthalene compounds functionalized with hydroxy
groups are contemplated in the present invention. Exemplary
compounds are depicted in FIG. 6A. The exemplary C-7
hydroxy-dihydronaphthalene analog Oxi-com 158 (36) was synthesized
based on the synthetic pathway illustrated in FIG. 10.
[0144] To a well-stirred solution of
7-Hydroxy-6-methoxy-1-tetralone (12, 80.0 mg, 0.42 mol) in
CH.sub.2Cl.sub.2, was added Et.sub.3N (47 mg, 0.46 mol), followed
by DMAP (5.1 mg, 0.042 mol) and TBSCI (69 mg, 0.46 mol) at
0.degree. C. under dry nitrogen. After 2 h (at rt), the mixture was
partitioned between CH.sub.2Cl.sub.2 and water. The organic layer
was dried over anhydrous sodium sulfate, and after filtration, the
organic layer was concentrated in vacuo to provide (34) as a yellow
oil.
[0145] 7-TBSO-6-methoxy-1-tetralone(34): (122 mg, 0.40 mol, 95%)
.sup.1H-NMR (CDCl.sub.3, 300 MHz) 0.15 (s, 6H), 0.99 (s, 9H), 2.11
(m, 2H), 2.58 (t, J=6.1 Hz, 2H), 2.88 (t, J=6.0 Hz, 2H), 3.86 (s,
3H), 6.63 (s, 1H), 7.56 (s, 1H).
[0146] To a stirred solution of n-butyllithium (0.5 mL, 1.6 M in
hexane solution, 0.80 mol) in dry ether (20 mL), a solution of
3,4,5-trimethoxyphenylbromide (98.8 mg, 0.40 mol) in ether (10 mL)
was added under dry nitrogen at -78.degree. C. The solution was
stirred for 1 h in order to form 3,4,5-trimethoxyphenyllithum.
7-[(tertButyldimethylsilyl)oxy]-6-methoxy-1-tetralone (122 mg, 0.40
mol) was added at -20.degree. C., and stirring was continued for 2
h (-20.degree. C.--rt). The mixture was partitioned between
CH.sub.2Cl.sub.2 and water, and the organic layer was dried over
anhydrous sodium sulfate. After filtration, the organic layer was
concentrated in vacuo to provide (35) as a yellow oil.
[0147]
7-TBSO-1-hydroxy-6-methoxy-1-(3',4',5'-trimethoxyphenyl)-tetralone
(35): (137 mg, 0.29 mol, 73%) .sup.1H-NMR (CDCl.sub.3, 300MHz) 0.04
(s, 6H), 0.89 (s, 9H), 1.80 (b, 2H), 2.11 (m, 4H), 2.81 (t, J=6.5,
2H), 3.79 (s, 6H), 3.81 (s, 3H), 3.84 (s, 3H), 6.49 (s, 1H), 6.56
(s, 2H), 6.59 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 75MHz) -4.3, 18.8,
20.2, 26.1, 29.9, 41.5, 55.8, 56.5, 61.2, 75.6, 104.2, 111.9,
121.2, 131.4, 134.2, 136.8, 143.8, 145.3, 150.9, 152.9.
[0148] To a solution of acetic acid (10 mL) in H.sub.2O (60 mL),
was added
7-TBSO-1-hydroxy-6-methoxy-1-(3',4',5'-trimethoxyphenyl)-tetralone
(137 mg, 0.29 mol). The solution was heated to reflux for 8 hour,
and then cooled down to RT. NaHCO.sub.3 (20 mL, saturated solution)
was added, and the mixture was partitioned between CH.sub.2Cl.sub.2
and water. The organic layer was dried over anhydrous sodium
sulfate, and after filtration, the organic layer was concentrated
in vacuo to provide 36 as a yellow oil.
[0149] Oxi-com 158 (36) (84.3 mg, 0.25 mol, 86%) .sup.1H-NMR
(CDCl.sub.3, 300MHz) 2.37 (m, 2H), 2.78 (t, J=7.7 Hz, 2H), 3.84 (s,
6H), 3.88 (s, 3H), 3.91 (s, 3H), 5.41 (s, 1H), 5.98 (t, J=4.6 Hz,
1H), 6.54 (s, 2H), 6.68 (s, 1H), 6.81 (s, 1H).
[0150] Similar dihydronaphthalenes functionalized in an analogous
fashion at any other carbon position can readily be prepared. An
exemplary detailed synthesis for the preparation of Oxi-com 196 is
provided in FIG. 11.
[0151] Oxi-com 196 (37):.sub.--.sup.1H-NMR (300 MHz): .delta.
2.34-2.41 (2H, m), 2.88 (2H, t, J=7.6 Hz), 3.84 (6H, s), 3.88 (6H,
s), 5.72 (1H, s), 5.97 (1H, t, J=4.6 Hz), 6.55 (2H), 6.59 (1H, d,
J=8.4 Hz), 6.63 (1H, d, J=8.4 Hz). .sup.3C-NMR (75 MHz): .delta.
20.04, 22.62, 55.7, 55.87, 60.72, 105.63, 107.05, 117.21, 122.13,
125.18, 128.73, 136.,72, 136.75, 139.32, 141.8, 145.68, 152.67.
[0152] Oxi-com 238 (38): .sup.1H NMR (CDCl.sub.3, 360 MHz): .delta.
7.08 (d, J=8.9 Hz, 1H), .delta. 6.53 (m, 3H), .delta. 6.26 (d,
J=8.3 Hz, 1H), .delta. 5.86 (t, J=4.6 Hz, 1H), .delta. 5.67 (s,
1H), .delta. 3.84 (s, 6H), .delta. 3.68 (s, 3H), 6 2.91 (m, 2H),
2.4 (m, 2H).
[0153] Oxi-com 240 (39) .sup.1H NMR (CDCl.sub.3, 360 MHz): .delta.
6.81 (m, 3H), .delta. 6.55 (d, J=8.6 Hz, 1H), .delta. 6.27 (d,
J=7.9 Hz, 1H), .delta. 5.9 (t, J=4.6 Hz, 1H), .delta. 5.67 (s, 1H),
.delta. 3.85 (s, 3H), .delta. 3.77 (s, 3H), .delta. 3.65 (s, 3H),
.delta. 2.92 (m, 2H), 6 2.41 (m, 2H).
[0154] Oxi-com 242 (40) .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.
6.88 (m, 3H), .delta. 6.62 (d, J=8.4 Hz, 1H), .delta. 6.57 (d,
J=8.4 Hz, 1H), .delta. 5.95 (t, J=4.7 Hz, 1H), .delta. 5.72 (s,
1H), .delta. 3.92 (s, 3H), .delta. 3.88 (s, 3H), .delta. 3.86 (s,
3H), .delta. 2.88 (t, J=7.7 Hz, 2H), .delta. 2.4 (m, 2H).
d) Synthesis of Dihydronaphthalene Phosphate Prodrugs
[0155] Phosphate Prodrugs in the form of phosphate salts and esters
of hydroxyl functionalized dihydronaphthalene derivatives (and
related phenolic analogs) are envisioned, for example phosphate
ester disodium salts of C-5 (Oxi-com 197, 43) and C-7
Dihydronaphthalene (42) as illustrated in FIG. 6B. We have
developed a synthetic route which will readily yield the C-5
disodium phosphate salt of dihydronaphthalene by phosphorylation of
37 illustrated in FIG. 11.
[0156] The phenol Oxi-com 196 (37) was converted to its disodium
phosphate prodrug salt using by sequential treatment of CCl4,
iPr2Net, DMAP, and dibenzylphosphite in acetonitrole to produce the
dibenzyl phosphate derivative (46) in 86% yield. The dibenzyl
phosphate derivative was then stirred with NaI and TMS1 in
acetonitrile at RT for 30 min to debenzylate the compound. After
removal of acetonitrile and drying, the residue was stirred
overnight with NaOMe in methanol to form a salt. Crystallization in
acetone-water produced the prodrug Oxi-com 197 as a white
powder.
[0157] Oxi-com 197 (43): .sup.1H-NMR (300 MHz) .delta. 2.31-2.35
(2H, m), 2.94 (2H, t, J=7.7 Hz), 3.80 (6H, s), 3.82 (6H, s), 6.07
(1H, t, J=4.3 Hz), 6.72-6.74 (4H, overlapping singlet and two
doublets). .sup.13C-NMR (90.55 MHz) .delta. 24.64, 25.32, 58.35,
58.81, 63.83, 109.08, 111.84, 123.63, 129.55, 131.00, 134.39,
138.61, 140.63, 141.40, 142.85, 154.49, 155.06. .sup.31P-NMR
(121.48 MHz) .delta. 4.33
[0158] Related phosphate prodrug derivatives of the
1-trimethoxyphenyl-dihydronaphthalene system (FIG. 3B) were
synthesized in a similar fashion.
[0159] Oxi-com 239 (44) .sup.1H NMR (D.sub.2O, 360 MHz): .delta.
7.22(d, J=8.1 Hz, 1H), .delta. 6.72 (m, 3H), .delta. 6.46 (d, J=8.7
Hz, 1H), .delta. 5.99 (t, J=4.6 Hz, 1H), .delta. 3.91 (s, 3H),
.delta. 3.82 (s, 3H), .delta. 3.71 (s, 3H), .delta. 3.05 (m, 2H),
.delta. 2.39 (m, 2H). .sup.31P NMR (D.sub.2O, 360 MHz): .delta. 1.9
(s, 1P)
[0160] Oxi-com 241 (45) .sup.1H NMR (D.sub.2O, 360 MHz): .delta.
7.1 (d, J=9.0 Hz, 1H), 6 7.04 (dd, J=8.9 Hz, J=3.1 Hz, 3H), .delta.
6.93 (d, J=3.1 Hz, 1H), .delta. 6.74 (d, J=8.7, 1H), .delta. 6.45
(d, J=8.4 Hz, 1H), .delta. 6.0 (t, J=4.5 Hz, 1H), .delta. 3.83 (s,
3H), .delta. 3.81 (s, 3H), .delta. 3.67 (s, 3H), .delta. 3.07 (m,
2H), .delta. 2.4 (m, 2H).
[0161] .sup.31P NMR (D.sub.2O, 360 MHz): .delta. 1.98 (s, 1P)
[0162] Oxi-com 243 (46) .sup.1H NMR (D.sub.2O, 300 MHz): .delta.
7.1 (d, J=8.0 Hz, 1H), .delta. 7.02 (m, 2H), .delta. 6.79 (d, J=9.6
Hz, 1H), .delta. 6.76 (d, J=9.2 Hz, 1H), .delta. 6.08 (t, J=3.8 Hz,
1H), .delta. 3.9 (s, 3H), .delta. 3.84 (s, 3H), .delta. 3.81 (s,
3H), .delta. 3.07 (t, J=7.6 Hz, 2H), .delta. 2.32 (m, 2H).
[0163] .sup.31P NMR (D.sub.2O, 300 MHz): .delta. 4.32 (s, 1P)
e) Synthesis of Hydroxy, Aroyl-Substituted Dihydronaphthalene and
Naphthalene Prodrugs
[0164] Aryol-substituted dihydronaphthalene ligands have previously
described in U.S. Pat. No. 6,162,930. Hydroxyl functionalized
derivatives and corresponding phosphate prodrugs of this compound
are now provided (see FIG. 7A). A detailed synthetic route for this
compound is illustrated in FIG. 12.
[0165] Oxi-com 224 (47): .sup.1H NMR: in D.sub.2O .delta. (PPM)
7.07 (s, 2H, Ph-H), 6.80 (d, 1H, J=8.4 Hz, Ar--H), 6.72 (d, 1H,
J=8.5 Hz, Ar--H), 6.45 (t, 1H, J=4.4 vinylic-H), 3.75 (s, 3H,
--OCH.sub.3), 3.74 (s, 9H, --OCH.sub.3), 2.82 (t, 2H, J=7.7 Hz,
--CH.sub.2), 2.37(m, 2H, --CH.sub.2) .sup.31P NMR: in D.sub.2O
.delta. (PPM) --3.60 (not calibrated) Phosphate prodrugs of
hydroxyl functionalized naphthalene derivatives (and related
phenolic analogs) are also provided in the present invention. An
exemplary compound is depicted in FIG. 7B. This compound was
synthesized as illustrated in FIG. 12.
[0166] Oxi-com 225 (48):.sub.--.sup.1H NMR in D.sub.2O.delta. (PPM)
8.43 (d, 1H, J=8.6 Hz, Ar--H), 7.72 (d, 1H, J=9.2 Hz, Ar--H), 7.61
(t, 1H, J=6.9 Hz, Ar--H), 7.50 (d, 1H, J=6.7 Hz, Ar--H), 7.43 (d,
1H, J=9.3 Hz, Ar--H), 3.97 (s, 3H, --OCH.sub.3), 3.87 (s, 3H,
--OCH.sub.3), 3.76 (s, 6H, --OCH.sub.3)
[0167] .sup.31P NMR: in D.sub.2O.delta. (PPM) -1.39 (not
calibrated)
f) Synthesis of Diphosphate Prodrugs
[0168] It is now known that while CA4P is a potent vascular
targeting and destruction agent in vivo, it is likely that CA1P (a
diphosphate) may prove to be as active or even more active than
CA4P in vivo. Since CA4P is enzymatically converted to CA4, which
in turn interacts with tubulin to cause vascular disruption, it is
reasonable to expect that the new tubulin binding ligands described
herein may prove to be enhanced vascular targeting agents once
functionalized as diphosphates. The syntheses of these compounds
will parallel the methodology described in the various synthetic
schemes delineated within this application and will be readily
apparent to persons skilled in the art. A representative synthesis
is illustrated in FIG. 13.
Example 4
Inhibition of Tubulin Polymerization Assay
[0169] IC.sub.50 values for tubulin polymerization were determined
according to a previously described procedure (Bai et al, Cancer
Research, 1996). Purified tubulin is obtained from bovine brain
cells as described in Hamel and Lin (Hamel and Lin, Biochemistry,
1984). Various amounts of inhibitor were preincubated for 15
minutes at 37.degree. C. with purified tubulin. After the
incubation period, the reaction was cooled and GTP was added to
induce tubulin polymerization. Polymerization was then monitored in
a Gilford spectrophotometer at 350 nm. The final reaction mixtures
(0.25 ml) contained 1.5 mg/ml tubulin, 0.6 mg/ml
microtubule-associated proteins (MAPs), 0.5 mM GTP, 0.5 mM
MgCl.sub.2, 4% DMSO and 0.1M 4-morpholineethanesulfonate buffer
(MES, pH 6.4). IC.sub.50 is the amount of inhibitor needed to
inhibit tubulin polymerization 50% with respect to the amount of
inhibition that occurs in the absence of inhibitor. TABLE-US-00001
TABLE 1 In Vitro Inhibition of Tubulin Polymerization. Compound
IC.sub.50 (.mu.M) CA-4 1.2 (.+-.0.02) Oxi-com 141, 29 1-2 Oxi-com
142, 31 0.5-1.0 Oxi-com 143, 32 Inactive (>40) Oxi-com 148, 27 1
Oxi-com 156, 26 1-4 Oxi-com 196, 37 0.5-1 Oxi-com 199, 50 Inactive
(>40)
Example 5
In vitro Cytotoxicity Activity Against Cancer Cell Lines
a) Human Cancer Cell Lines
[0170] The activity of several compounds were tested against a
variety of cell lines derived from human tumors, using an assay
system similar to a procedure previously described (Monks et al, J.
Natl. Cancer Inst., 1991). Briefly, the cell suspensions, diluted
according to the particular cell type and the expected target cell
density (5,000-40,000 cells per well based on cell growth
characteristics), were added by pipet (100 .mu.l) to 96-well
microtiter plates. Inoculates were allowed a preincubation time of
24-28 hours at 37.degree. C. for stabilization. Incubation with the
inhibitor compounds lasted for 48 hours in 5% CO.sub.2 atmosphere
and 100% humidity. Determination of cell growth was performed by in
situ fixation of cells, followed by staining with a protein-binding
dye sulforhodamine B (SRB), which binds to the basic amino acids of
cellular macromolecules. The solubilized stain was measured
spectrophotometrically.
[0171] Several compounds were evaluated for cytotoxic activity
against human P388 leukemia cell lines. The effective dose or
ED.sub.50 value (defined as the effective dosage required to
inhibit 50% of cell growth) was measured. These and additional
compounds were evaluated in terms of growth inhibitory activity
against several other human cancer cell lines including: central
nervous system ("CNS", SF-295), pancreas (BXPC-3), non-small cell
lung cancer ("lung-NSC", NCI-H460), breast (MCF-7), colon (KM20L2),
and prostate (DU-145). The results are described in Table 2 below.
The growth inhibition GI.sub.50 (defined as the dosage required to
inhibit tumor cell growth by 50%) is listed for each cell line.
TABLE-US-00002 TABLE 2 In vitro Cytotoxicity against Human Cancer
Cell Lines ED50 (ug/ml) for P-388 GI.sub.50 (.mu.g/ml) for Cell
Line Compound Cell Line SF-295 BXPC-3 NCI-H460 MCF-7 KM20L2 DU-145
Oxi-com 156, 26 0.13 0.043 0.67 0.16 0.11 0.38 Oxi-com 141, 29
0.0033 0.0054 0.0038 0.0010 0.0032 0.0037 Oxi-com 142, 31 0.034
0.0029 0.0034 0.0026 <0.001 0.0027 0.0038 Oxi-com 143, 32 20.4
2.2 2.0 3.4 3.0 2.5 2.8 Oxi-com 158, 36 5.4 1.8 3.2 1.5 2.6 3.4
Oci-com 196, 37 0.00175 0.00012 0.10 0.0032 <0.0001 0.27 0.00040
Oxi-com 197, 43 0.00182 0.0033 0.28 0.011 0.0045 0.33 0.0055
b) Murine Cancer Cell Lines
[0172] The following compounds were tested for in vitro
antiproliferative activity against the murine hemangioendothelioma
MHEC-5T cell line using a standard MTT assay (see Mosman, J.
Immunol. Methods, 1983). In actively proliferating cells, an
increase in MTT conversion is spectrophotometrically quantified by
the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) to the insoluble formazan dye by enzymes
associated with metabolic activity. A compound with growth
inhibitory activity will cause a reduction in dye formation
relative to cells exposed to a vehicle control. The IC.sub.50 value
(defined as the amount of compound required to inhibit growth of
50% of cells with respect to a control treatment) for each compound
was determined at one hour and five days at (see Table 3 below).
TABLE-US-00003 TABLE 3 In vitro Cytotoxicity against Murine cancer
cell lines Compound IC.sub.50 (.mu.M) at 1 hour IC.sub.50 (.mu.M)
at 5 days Oxi-com 196, 37 0.11 0.004 Oxi-com 197, 43 0.10 0.003
Oxi-com 199, 50 >44.8 >44.8 Oxi-com 200, 49 >50 16.2
Oxi-com 224, 47 0.4 0.003 Oxi-com 225, 48 0.4 0.005 Oxi-com 229, 33
>44 >1.4 Oxi-com 238, 38 14.0 0.04 Oxi-com 239, 44 2.4 0.06
Oxi-com 240, 39 8.0 0.03 Oxi-com 241, 45 2.5 0.08 Oxi-com 243, 46
20. 0.30
Example 6
Inhibition of Tumor Blood Flow
[0173] The antivascular effects of the C-5 dihydronaphthalene
phosphate prodrug Oxi-com 197, 43 was assessed in tumor-bearing
mice using a Fluorescent Bead Assay. A MHEC-5T hemangioendothelioma
tumor model was established by subcutaneous injection of
0.5.times.106 cultured transformed cell murine myocardial vascular
endothelial cell line ("MHEC5-T") cells into the right flank of Fox
Chase CB-17 Severe Combined Immunodeficient ("SCID") mice. When
transplanted tumors reached a size of 500 mm.sup.3 (a size without
development of necrosis), the mice received a single
intraperitoneal (i.p.) injection of saline control or compound at
doses ranging from 3.2 to 25 mg/kg. At 24 hours post-treatment,
mice were injected intravenously with 0.25 ml of diluted FluoSphere
beads (1:6 in physiological saline) in the tail vein, sacrificed
after 3 minutes, and tumor was excised for cryosectioning. Tumor
cryosections at a thickness of 8 um were directly examined using
quantitative fluorescent microscopy. Blood vessels were indicated
by blue fluorescence from injected beads. For quantification, image
analysis of 3 sections from three tumors treated in each group were
examined and vascular shutdown was expressed as vessel area
(mm.sup.2) per tumor tissue area (mm.sup.2) as a percentage of the
control ("% VAPM") and as vessel number per tumor tissue area
(mm.sup.2) as a percentage of the control ("% VNPM"). The results
as shown in Table 11 indicate a clear dose-dependent effect of the
agent on tumor blood flow as indicated by the reduction in blood
vessel number and vessel area. Administration of a 25 mg/kg dose of
Oxi-com 197 was particularly effective, causing a 90% reduction in
tumor vessel number relative to the control. TABLE-US-00004 TABLE 4
Vascular Targeting Activity of Oxi-com 197 prodrug Dose (mg/kg) %
VAPM % VNPM 0 100 .+-. 13.5 100 .+-. 10.8 3.2 101.6 .+-. 36.6 89.1
.+-. 25.6 6.3 65.4 .+-. 4.9 66.5 .+-. 0.5 12.5 32.2 .+-. 6.8 55.5
.+-. 0.8 25 13.0 .+-. 5.0 10.4 .+-. 6.3
[0174] Additional compounds of the present invention were tested
for antivascular effects at two dosages (100 mg/kg and 10 mg/kg)
using the same Fluorescent Bead Assay as in the previous
experiment. The results are summarized in Table 5 below.
TABLE-US-00005 TABLE 5 Vascular Targeting Activity of Aroyl
dihydronaphthalene and Aroyl Napthalene phosphate prodrugs % VAPM
at 100 mg/kg % VAPM at 10 mg/kg Compound dose dose Oxi-com 224, 47
46 17 Oxi-com 225, 48 60 35
Example 7
Evaluation of Tumor Growth Control in vivo
[0175] The antitumor activity of C-5 dihydronaphthalene phosphate
prodrug, Oxi-com 197, was assessed in tumor-bearing mice by
measuring its effects on tumor volume in comparison with CA4P. A
human breast adenocarcinoma model was established by subcutaneous
injection of cultured MDA-MB-231 cells in Fox Chase CB-17 SCID
mice. When the average tumor size reached 50-100 mm.sup.3, mice
were randomly divided into several groups (n=10) with no
significant difference in body weight and tumor size. Mice were
administered CA4P or Oxi-com 197 in saline carrier at doses of 25,
50 or 100mg/kg by daily intraperitoneal (i.p.) injection for 5
consecutive days (Q1.times.5). Saline carrier only was used as the
control treatment. On Day 15 3, 7, 10, 13, 17 and 23, tumors were
excised from animals in each treatment group (n=2) and measured by
width and length. Tumor volume was calculated according to the
following formula: Length.times.Width.sup.2.times.0.4. The dosage
effects of Oxi-com 197 is illustrated in FIG. 14. Administration of
25, 50, and 100 mg/kg doses of the drug significantly inhibited
tumor growth relative to control treatment. C-5-DHN-P was also
observed to have enhanced antitumor activity relative to CA4P.
OTHER EMBODIMENTS
[0176] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. For instance, in addition to the
various metal salts described for the phosphates and
phosphoramidates, any appropriate metal or non-metal cation and, in
fact, any appropriately related salt construct can be employed
without departing from the spirit and scope of the invention. For
therapeutic and/or prophylactic anti-tumor purposes, the prodrugs
of the present invention would be administered at a dosage of from
about 5 mg/m.sup.2 to about 100 mg/m.sup.2 while intravascular
infusion of the prodrug is preferred other modes of parenteral
topical or enteral administration are usable under certain
conditions.
[0177] The present invention also involves uses of the novel
compounds described in manners relating to their useful effects on
tubulin polymerization and abnormal vasculature. Certainly a method
for inhibiting tubulin polymerization is a part of the present
invention. This involves contacting a tubulin containing system
with an effective amount of a compound described in the present
invention. This tubulin containing system may be in a tumor cell,
thereby inhibiting neoplastic disease by administering an effective
amount of a compound of the present invention. Patients may thus be
treated. In cases of cancer treatment, it is believed that many
neoplasias such as leukemia, lung cancer, colon cancer, thyroid
cancer, CNS, melanoma, ovarian cancer, renal cancer, prostate
cancer and breast cancers may be effectively treated by the
administration of an effective amounts of the compounds described
in the present invention. Pharmaceutical preparations may be
prepared by mixing the compounds of the present invention with a
pharmaceutically acceptable carrier. This may be in tablet or
intravascular form. In one important aspect, macular degeneration,
and related diseases of the eye where vascularization is involved,
may be treated by a method comprising administering an effective
amount of a compound described in the present invention. Psoriasis
may also be treated by administering an effective amount of the
compound of the present invention. Likewise, any disease or
condition caused or enhanced by undesired vascularization may be
treated by administering an effective amount of a compound of the
present invention.
[0178] In addition to their tumor-selective vascular targeting and
destruction capabilities, it is contemplated that all the compounds
of the present invention have potential application in the
treatment of other diseases where the issue of vascularization is
of great significance. Representative examples of these diseases
include: diseases associated with ocular neovascularization
(corneal and retinal), psoriasis and arthritis. All such similar
substitutes and modifications apparent to those skilled in the art
are deemed to be within the spirit, scope and concept of the
invention as defined by the appended claims.
REFERENCES
[0179] The following citations are incorporated in pertinent part
by reference herein for the reasons cited. [0180] 1. Bai, R.;
Schwartz, R. E.; Kepler, J. A.; Pettit, G. R.; Hamel, E.,
Characterization of the Interaction of Cryptophycin with Tubulin:
Binding in the Vinca Domain, Competitive Inhibition of Dolastatin
10 Binding, and an Unusual Aggregation Reaction, Cancer Res., 1996,
56, 4398-4406. [0181] 2. Boger, D. L.; Curran, T. T., Synthesis of
the Lower Subunit of Rhizoxin, J. Org. Chem. 1992, 57, 2235. [0182]
3. Chaplin D J, Pettit G R, Hill S A. Anti-vascular approaches to
solid tumor therapy: Evaluation of combretastatin A4 phophate.
Anticancer Res., 1999; 19:189-196. [0183] 4. Chavan, A. J.;
Richardson, S. K.; Kim, H.; Haley, B. E.; Watt, D. S., Forskolin
Photoaffinity Probes for the Evaluation of Tubulin Binding Sites,
Bioconjugate Chem. 1993, 4, 268. [0184] 5. Cortese, F.;
Bhattacharyya, B.; Wolff, J., Podophyllotoxin as a Probe for the
Colchicine Binding Site of Tubulin, J. Biol. Chem., 1977, 252,
1134. [0185] 6. Cushman, M.; Nagarathnam, D.; Gopal, D.;
Chakraborti, A. K.; Lin, C. M.; Hamel, E. Synthesis and Evaluation
of Stilbene and Dihydrostilbene Derivatives as Potential Anticancer
Agents That Inhibit Tubulin Polymerization, J. Med. Chem. 1991, 34,
2579. [0186] 7. Dark, G. G., Hill, S. A., Prise, V. G., Tozer, G.
M., Pettit, G. R., Chaplin, D. J., Combretastatin A-4, an Agent
That Displays Potent and Selective Toxicity Toward Tumor
Vasculature, Cancer Res., 1997, 57, 1829-1834. [0187] 8. Davis P D,
Dougherty G J, Blakey D C, Galbraith S M, Tozer G M, Holder A L,
Naylor M A, Nolan J, Stratford M R, Chaplin D J, Hill S A. ZD6126:
A Novel Vascular-targeting Agent that causes selective destruction
of tumor vasculature. Cancer Research. 2002. 62(24): 7247-53.
[0188] 9. Dorr, R. T.; Dvorakova, K.; Snead, K.; Alberts, D. S.;
Salmon, S. E.; Pettit, G. R., Antitumor Activity of Combretastatin
A4 Phosphate, a Natural Product Tubulin Inhibitor, Invest. New
Drugs, 1996, 14, 131. [0189] 10. Floyd. L. J.; Barnes, L. D.;
Williams, R. F., Photoaffinity Labeling of Tubulin with
(2-Nitro-4-azidophenyl)deacetylcolchicine: Direct Evidence for Two
Colchicine Binding Sites, Biochemistry, 1989, 28, 8515. [0190] 11.
Gerwick, W. H.; Proteau, P. J.; Nagle, D. G.; Hamel, E.; Blokhin,
A.; Slate, D. L., Structure of Curacin A, a Novel Antimitotic,
Antiproliferative, and Brine Shrimp Toxic Natural Product from the
Marine Cyanobacterium Lyngbya majuscula, J. Org. Chem. 1994, 59,
1243. [0191] 12. Hahn, K. M.; Hastie, S. B.; Sundberg, R. J.,
Synthesis and Evaluation of 2-Diazo-3,3,3-trifluoropropanoyl
Derivatives of Colchicine and Podophyllotoxin as Photoaffinity
Labels: Reactivity, Photochemistry, and Tubulin Binding, Photochem.
Photobiol. 1992, 55, 17. [0192] 13. Hamel, E., Antimitotic Natural
Products and Their Interactions with Tubulin, Medicinal Research
Reviews, 1996, 16, 207. [0193] 14. Hamel, E.; Lin, C. M.,
Separation of Active Tubulin and Microtubule-Associated Proteins by
Ultracentrifugation and Isolation of a Component Causing the
Formation of Microtubule Bundles, Biochemistry, 1984, 23,
4173-4184. [0194] 15. Hammonds, T. R.; Denyer, S. P.; Jackson, D.
E.; Irving, W. L., Studies To Show That With Podophyllotoxin the
Early Replicative Stages of Herpes Simplex Virus Type I Depend Upon
Functional Cytoplasmic Microtubules, J. Med. Microbiol., 1996, 45,
167. [0195] 16. Iyer S, Chaplin D J, Rosenthal D S, et al.
Induction of apoptosis in proliferating human endothelial cells by
the tumor-specific antiangiogenesis agent combretastatin A-4.
Cancer Res. 1998; 58:4510-4514. [0196] 17. Jiang, J. B.; Hesson, D.
P.; Dusak, B. A.; Dexter, D. L.; Kang, G. J.; Hamel, E., Synthesis
and Biological Evaluation of 2-Styrylquinazolin-4(3H)-ones, a New
Class of Antimitotic Anticancer Agents Which Inhibit Tubulin
Polymerization, J. Med. Chem. 1990, 33, 1721. [0197] 18. Kanthou C,
Tozer G M. The tumor vascular targeting agent CA4P induces
reorganization of the actin cytoskeleton and early membrane
blebbing in human endothelial cells. Blood. 2002. 99(6):2060-9.
[0198] 19. Kingston, D. G. I.; Samaranayake, G.; Ivey, C. A., The
Chemistry of Taxol, a Clinically Useful Anticancer Agent, J. Nat.
Prod. 1990, 53, 1. [0199] 20. Kobayashi, S.; Nakada, M.; Ohno, M.,
Synthetic Study on an Antitumor Antibiotic Rhizoxin by Using an
Enzymatic Process on Prochiral beta-Substituted Glutarates, Pure
Appl. Chem. 1992, 64, 1121. [0200] 21. Kobayashi, S.; Nakada, M.;
Ohno, M., Synthetic Study on an Antitumor Antibiotic Rhizoxin by
Using an Enzymatic Process on Prochiral beta-Substituted Glutarates
Indian J. Chem., Sect. B. 1993, 32B, 159. [0201] 22. Lavielle, G.;
Havtefaye, P.; Schaeffer, C.; Boutin, J. A.; Cudennec, C. A.;
Pierre, A., New .alpha.-Amino Phosphonic Acid Derivatives of
Vinblastine: Chemistry and Antitumor Activity, J. Med. Chem. 1991,
34, 1998. [0202] 23. Lejeune P, Hodge T G, Vrignaud, Bissery M-C.
In vivo antitumor activity and tumor necrosis induced by AVE8062A,
a tumor vasculature targeting agent. Proceedings of the AACR.
ABSTRACT#781. 2002, 43: 156. [0203] 24. Lin, C. M.; Ho, H. H.;
Pettit, G. R.; Hamel, E., Antimitotic Natural Products
Combretastatin A-4 and Combretastatin A-2: Studies on the Mechanism
of Their Inhibition of the Binding of Colchicine to Tubulin,
Biochemistry 1989, 28, 6984. [0204] 25. Monks, A.; Scudiero, D.;
Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.;
Langley, J.; Cronise, P.; Vaigro-Wolff, A.; Gray-Goodrich;
Campbell; Mayo; Boyd, M., Feasibility of a High-Flux Anticancer
Drug Screen Using a Diverse Panel of Cultured Human Tumor Cell
Lines, J. Natl. Cancer Inst., 1991, 83, 757-766. [0205] 26.
Mossman, T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assay. J.
Immunol. Methods, 1983, 16, 195-200. [0206] 27. Nakada, M.;
Kobayashi, S.; Iwasaki, S.; Ohno, M., The First Total Synthesis of
the Antitumor Macrolide Rhizoxin: Synthesis of the Key Building
Blocks, Tetrahedron Lett. 1993, 34, 1035. [0207] 28. Nicolaou, K.
C., Winssinger, N., Pastor, J., Ninkovic, S., Sarabia, F., He, Y.,
Vourloumis, D., Yang, Z., Oi, T., Giannakakou, P., Hamel, E.,
Synthesis of Epothilones A and B in Solid and Solution Phase,
Nature, 1997, 387, 268-272. [0208] 29. Nogales, E., Wolf, S. G.,
and Downing, K. H., Structure of the .alpha.,.beta. Tubulin Dimer
by Electron Crystallography, Nature, 1998, 391, 199-203. [0209] 30.
Owellen, R. J.; Hartke, C. A.; Kickerson, R. M.; Hains, F. O.,
Inhibition of Tubulin-Microtubule Polymerization by Drugs of the
Vinca Alkaloid Class, Cancer Res. 1976, 36, 1499. [0210] 31.
Pettit, G. R.; Rhodes, M. R., Antineoplastic agents 393. Synthesis
of the trans-isomer of CA4P. Anti-Cancer Drug Des., 1998, 13, 183.
[0211] 32. Pettit, G. R., Srirangam, J. K., Barkoczy, J., Williams,
M. D., Boyd, M. R., Hamel, E., Pettit, R. K., Hogan F., Bai, R.,
Chapuis, J. C., McAllister, S. C., Schmidt, J. M., Antineoplastic
Agents 365: Dolastatin 10 SAR Probes, Anti-Cancer Drug Des., 1998,
13, 243-277. [0212] 33. Pettit, G. R., Toki, B., Herald, D. L.,
Verdier-Pinard, P., Boyd, M. R., Hamel, E., Pettit, R. K.,
Antineoplastic Agents 379. Synthesis of Phenstatin Phosphate, J.
Med. Chem., 1998, 41, 1688-1695. [0213] 34. Pettit, G. R.; Cragg,
G. M.; Singh, S. B., Antineoplastic agents, 122. Constituents of
Combretum caffrum, J. Nat. Prod. 1987, 50, 386. [0214] 35. Pettit,
G. R., Kamano, Y., Herald, C. L., Tuinman, A. A., Boettner, F. E.,
Kizu, H., Schmidt, J. M., Baczynskyj, L., Tomer, K. B., Bontems, R.
J., The Isolation and Structure of a Remarkable Marine Animal
Antineoplastic Constituent: Dolastatin 10, J. Am. Chem. Soc., 1987,
109, 6883-6885. [0215] 36. Pettit, G. R.; Singh, S. B.; Cragg, G.
M., Synthesis of Natural (-)-Combretastatin, J. Org. Chem. 1985,
50, 3404. [0216] 37. Pettit, G. R.; Cragg, G. M.; Herald, D. L.;
Schmidt, J. M.; Lohavanijaya, P., Isolation and Structure of
combretastatin, Can, J. Chem. 1982, 60, 1374. [0217] 38. Rao, A. V.
R.; Bhanu, M. N.; Sharma, G. V. M., Studies Directed Towards the
Total Synthesis of Rhizoxin: Stereoselective Synthesis of C-12 to
C-18 Segment, Tetrahedron Lett. 1993, 34, 707. [0218] 39. Rao, S.;
Horwitz, S. B.; Ringel, I., Direct Photoaffinity Labeling of
Tubulin with Taxol, J. Natl. Cancer Inst., 1992, 84, 785. [0219]
40. Rao, A. V. R.; Sharma, G. V. M.; Bhanu, M. N., Radical Mediated
Enantioselective Construction of C-1 to C-9 Segment of Rhizoxin,
Tetrahedron Lett. 1992, 33, 3907. [0220] 41. Safa, A. R.; Hamel,
E.; Felsted, R. L., Photoaffinity Labeling of Tubulin Subunits with
a Photoactive Analog of Vinblastine, Biochemistry 1987, 26, 97.
[0221] 42. Sawada, T.; Kato, Y.; Kobayashi, H.; Hashimoto, Y.;
Watanabe, T.; Sugiyama, Y.; Iwasaki, S., A Fluorescent Probe and a
Photoaffinity Labeling Reagent to Study the Binding Site of
Maytansine and Rhizoxin on Tubulin, Bioconjugate Chem., 1993, 4,
284. 31. [0222] 43. Sawada, T.; Kobayashi, H.; Hashimoto, Y.;
Iwasaki, S., Identification of the Fragment Photoaffinity-labeled
with Azidodansyl-rhizoxin as Met-363-Lys-379 on beta-Tubulin,
Biochem. Pharmacol. 1993, 45, 1387. [0223] 44. Schiff, P. B.; Fant,
J.; Horwitz, S. B., Promotion of Microtubule Assembly In Vitro by
Taxol, Nature, 1979, 277, 665. [0224] 45. Staretz, M. E.; Hastie,
S. B., Synthesis, Photochemical Reactions, and Tubulin Binding of
Novel Photoaffinity Labeling Derivatives of Colchicine, J. Org.
Chem. 1993, 58, 1589. [0225] 46. Swindell, C. S.; Krauss, N. E.;
Horwitz, S. B.; Ringel, I., Biologically Active Taxol Analogs with
Deleted A-ring Side Chain Substituents and Variable C-2
C'Configurations, J. Med. Chem. 1991, 34, 1176. (d) Parness, J.;
Horwitz, S. B., Taxol Binds to Polymerized Tubulin In Vitro, J.
Cell Biol. 1981, 91, 479. [0226] 47. Tozer, G. M.; Prise, V. E.;
Wilson, J.; Locke, R. J.; Vojnovic, B.; Stratford, M. R. L.;
Dennis, M. F.; Chaplin, D. J., Combretastatin A-4 Phosphate as a
Tumor Vascular-Targeting Agent: Early Effects in Tumors and Normal
Tissues. Cancer Res., 1999, 59, 1626. [0227] 48. Williams, R. F.;
Mumford, C. L.; Williams, G. A.; Floyd, L. J.; Aivaliotis, M. J.;
Martinez, R. A.; Robinson, A. K.; Barnes, L. D., A Photoaffinity
Derivative of Colchicine:
6-(4'-Azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine:
Photolabeling and Location of the Colchicine-binding Site on the
alpha-subunit of Tubulin, J. Biol. Chem. 1985, 260, 13794. [0228]
49. Zhang, X.; Smith, C. D., Microtubule Effects of Welwistatin, a
Cyanobacterial Indolinone that Circumvents Multiple Drug
Resistance, Molecular Pharmacology, 1996, 49, 288.
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