U.S. patent application number 13/769024 was filed with the patent office on 2013-06-20 for biocompatible biodegradable fumagillin analog conjugates.
This patent application is currently assigned to Mersana Therapeutics, Inc. The applicant listed for this patent is Laura C. Akullian, Charles E. Hammond, John J. Kane, Russell C. Petter, Cheri A. Stevenson, Mao Yin, Aleksandr Yurkovetskiy. Invention is credited to Laura C. Akullian, Charles E. Hammond, John J. Kane, Russell C. Petter, Cheri A. Stevenson, Mao Yin, Aleksandr Yurkovetskiy.
Application Number | 20130158046 13/769024 |
Document ID | / |
Family ID | 40600044 |
Filed Date | 2013-06-20 |
United States Patent
Application |
20130158046 |
Kind Code |
A1 |
Akullian; Laura C. ; et
al. |
June 20, 2013 |
BIOCOMPATIBLE BIODEGRADABLE FUMAGILLIN ANALOG CONJUGATES
Abstract
Fumagillin analog polymer conjugates, methods of making
fumagillin analog polymer conjugates, compositions comprising a
polymer conjugate of a fumagillin analog, and methods for treating
cancer, or treating angiogenic diseases comprising administering to
a subject in need thereof an effective amount of a polymer
conjugate of a fumagillin analog, are described. Also described are
novel fumagillin analogs, methods of making fumagillin analogs,
compositions comprising at least one fumagillin analog, and methods
for treating cancer, or treating angiogenic diseases comprising
administering to a subject in need thereof an effective amount of a
fumagillin analog.
Inventors: |
Akullian; Laura C.;
(Lunenburg, MA) ; Petter; Russell C.; (Stow,
MA) ; Kane; John J.; (Queens Village, NY) ;
Hammond; Charles E.; (Billerica, MA) ; Yin; Mao;
(Needham, MA) ; Yurkovetskiy; Aleksandr;
(Littleton, MA) ; Stevenson; Cheri A.; (Haverhill,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Akullian; Laura C.
Petter; Russell C.
Kane; John J.
Hammond; Charles E.
Yin; Mao
Yurkovetskiy; Aleksandr
Stevenson; Cheri A. |
Lunenburg
Stow
Queens Village
Billerica
Needham
Littleton
Haverhill |
MA
MA
NY
MA
MA
MA
MA |
US
US
US
US
US
US
US |
|
|
Assignee: |
Mersana Therapeutics, Inc
Cambridge
MA
|
Family ID: |
40600044 |
Appl. No.: |
13/769024 |
Filed: |
February 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12276856 |
Nov 24, 2008 |
8399512 |
|
|
13769024 |
|
|
|
|
61004582 |
Nov 28, 2007 |
|
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Current U.S.
Class: |
514/254.1 ;
514/409; 514/475; 544/374; 548/407; 549/332 |
Current CPC
Class: |
C07D 405/14 20130101;
C07D 303/18 20130101; C07D 303/36 20130101; A61K 47/59 20170801;
C07D 487/08 20130101; A61P 9/00 20180101; C07D 303/40 20130101;
A61P 35/02 20180101; A61P 35/00 20180101; C07D 303/22 20130101 |
Class at
Publication: |
514/254.1 ;
549/332; 514/475; 544/374; 548/407; 514/409 |
International
Class: |
C07D 303/40 20060101
C07D303/40; C07D 303/18 20060101 C07D303/18 |
Claims
1.-24. (canceled)
25. A compound of the Formula V: ##STR00205## wherein, X is O,
S(.dbd.O), optionally substituted CH.sub.2, or optionally
substituted NH; q is 0, 1, or 2; R is
(2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane,
2-methylhepta-2,5-diene, (2R,3S)-2-isopentyl-3-methyloxirane,
6-methylhept-2-ene, (Z)-acetaldehyde O-benzyl oxime,
C.sub.2-heterocyclic-C.sub.1-C.sub.6 alkyl,
C.sub.2-heterocyclic-C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-heterocyclic-C.sub.1-C.sub.6-alkenyl-COO--C.sub.1-C.sub.6
alkyl, C.sub.2-heterocyclic-C.sub.1-C.sub.6
alkyl-COO--C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6
alkyl=N--O--C.sub.1-C.sub.6 alkyl-aryl, C(O)C.sub.1-C.sub.6 alkyl,
CN, or halogen; R.sub.8 is selected from the group consisting of
VI, VII, VIII, IX, X, XI, XII, and XIIA whose formulas are
represented below: ##STR00206## R'--CO.sub.2H, optionally
substituted --NH.sub.2--, or --N cyclic imide, NHC(O)
(C.sub.1-C.sub.6 alkyl)-C(O)R'', R' is meta or para in relation to
the --S-- atom; and R'' is --OH, --O--C.sub.1-C.sub.6 alkyl, or
--NH.sub.2 optionally acylated through the carboxyl group of an
amino acid; ##STR00207## wherein R.sub.9 is H or C(O)R.sub.11;
R.sub.10 is --NH.sub.2, --NHCH(C.sub.1-C.sub.6 alkyl)-, --NHC(O)
(C.sub.1-C.sub.6 alkyl), N-cyclized imide; --NH acylated through
the carboxyl group of an amino acid, wherein the nitrogen of the
amino group of the amino acid is optionally protected, and R.sub.11
is OH, OC.sub.1-C.sub.6 alkyl, or optionally substituted
--NH.sub.2; ##STR00208## wherein N.sub.12 is H, C.sub.1-C.sub.6
alkyl, --(C.sub.1-C.sub.6)--COOH,
--(C.sub.1-C.sub.6)--C(O)O--(C.sub.1-C.sub.6),
--CH.sub.2CH.sub.2O--R.sub.13, --C(O)(C.sub.1-C.sub.6 alkyl), an
amino acid attached through the carboxyl group of the amino acid;
R.sub.13 is --H or an amino acid attached through the carboxyl
group of the amino acid, wherein the nitrogen of the amino acid is
optionally protected, C(O) (C.sub.1-C.sub.6 alkyl)-COR''; R'' is
--OH, --OC.sub.1-C.sub.6 alkyl, or --NH.sub.2 optionally acylated
through the carboxyl group of an amino acid; [-------] represents
an optional methylene bridge (--CH.sub.2--) between carbons 2 and 5
of the piperazine moiety; and Z' is a bond, --C.sub.1-C.sub.6
alkyl, --NHC(O)--, or --NHSO.sub.2--; ##STR00209## wherein R.sub.14
is --H, CO.sub.2H, --CO.sub.2(C.sub.1-C.sub.6 alkyl),
--C(O)NH--C.sub.1-C.sub.6 alkyl-OH, wherein the 0 of
--C(O)NH--C.sub.1-C.sub.6 alkyl-OH is optionally acylated with the
carboxyl group of an amino acid; optionally substituted --NH,
C.sub.1-C.sub.6-alkyl-NH.sub.2, wherein the NH.sub.2 is optionally
substituted; and [-------] represents an optional ethylene bridge
(--CH.sub.2CH.sub.2--) between carbons 1 and 4 of the cyclohexane
moiety; ##STR00210## Z' is a bond, --CH.sub.2--, --CH.sub.2--S--,
CH.sub.2CH.sub.2--, --C(H) (Me)-, NHCH.sub.2----NHCH(CH.sub.3)--,
--NHCH.sub.2CH.sub.2--; R.sub.15 is H, optionally substituted --NH,
--NHC(O) (C.sub.1-C.sub.6-alkyl), --N cyclized imide optionally
containing a heteroatom within the cyclic structure,
--NHC(O)CH.sub.2OCH.sub.2C(O)OH, NHC(O)CH(C.sub.1-C.sub.6 alkyl)-N
cyclized imide, --NHC(O)CH(R'')NHC(O)--(C.sub.1-C.sub.6
alkyl)-C(O)OH, --NHC(O)--(C.sub.1-C.sub.6 alkyl)-C(O)OH,
--C(O)O(C.sub.1-C.sub.6 alkyl), --C(O)N(H)(C.sub.1-C.sub.6
alkyl)-OH, or NO.sub.2; and R'' or --C.sub.1-C.sub.6 alkyl;
##STR00211## wherein Y' is C.sub.1-C.sub.3 alkyl, or
NH--C.sub.1-C.sub.3 alkyl and is attached to positions 1, 2, or 3
of the indole; and R.sub.16 is H, C.sub.1-C.sub.6 alkyl,
--CH.sub.2COOH, or --CH.sub.2CH.sub.2OH, wherein the O of
--CH.sub.2CH.sub.2OH can be optionally acylated with an amino acid;
##STR00212## wherein R'' is --OH, --OC.sub.1-C.sub.6 alkyl, --NH
optionally acylated through the carboxyl group of an amino acid;
and ##STR00213## wherein Y'' is C(O)N(CH.sub.3)(OCH.sub.3),
C(O)OCH.sub.3, CH.sub.2Cl, or NHC(O)CH.sub.2Cl.
26.-31. (canceled)
32. A composition comprising the compound or pharmaceutically
acceptable salt of the compound of claim 25 and a pharmaceutically
acceptable carrier.
33.-35. (canceled)
36. A composition comprising a compound or pharmaceutically
acceptable salt of the compound of claim 25 and a pharmaceutically
acceptable carrier.
37. The composition of claim 36, wherein the pharmaceutically
acceptable carrier is suitable for injectable administration and
the composition comprises an injectable dosage form.
38. A method of treating cancer, comprising administering to a
subject in need thereof a compound or a pharmaceutically acceptable
salt of a compound of claim 25, in an amount effective to treat the
cancer.
39. The method of claim 38, wherein the cancer is selected from the
group consisting of anal, astrocytoma, leukemia, lymphoma, head and
neck, liver, testicular, cervical, sarcoma, hemangioma, esophageal,
eye, laryngeal, mouth, mesothelioma, skin, myeloma, oral, rectal,
throat, bladder, breast, uterus, ovary, prostate, lung, colon,
pancreas, renal, and gastric.
40. A method of treating an angiogenic disease, comprising
administering to a subject in need thereof a compound or a
pharmaceutically acceptable salt of a compound of claim 25, in an
amount effective to inhibit angiogenesis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119(e) to co-pending U.S. application No. 61/004,582,
filed on Nov. 28, 2007, entitled "Biocompatible Biodegradable
Fumagillin Analog Conjugates," which is incorporated in its
entirety by reference.
[0002] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art as
known to those skilled therein as of the date of the invention
described and claimed herein.
[0003] This patent disclosure contains material that is subject to
copyright protection. The copyright owner has no objection to the
facsimile reproduction by anyone of the patent document or the
patent disclosure, as it appears in the U.S. Patent and Trademark
Office patent file or records, but otherwise reserves any and all
copyright rights whatsoever.
FIELD OF THE INVENTION
[0004] Polymer conjugates of fumagillin analogs, compositions
comprising a fumagillin analog conjugate, and fumagillin analogs
are described herein. Methods for treating or preventing cancer, or
treating an angiogenic disease, comprising the administration of an
effective amount of a fumagillin analog conjugate, are also
described. Methods for treating or preventing cancer or treating an
angiogenic disease, comprising the administration of an effective
amount of a fumagillin analog, are also described.
BACKGROUND OF THE INVENTION
[0005] Angiogenesis is the fundamental process by which new blood
vessels are formed and is essential to a variety of normal body
activities (such as reproduction, development and wound repair).
Although the process is not completely understood, it is believed
to involve a complex interplay of molecules which both stimulate
and inhibit the growth of endothelial cells, the primary cells of
the capillary blood vessels. Under normal conditions, these
molecules appear to maintain the microvasculature in a quiescent
state (i.e., one of no capillary growth) for prolonged periods
which may last for as long as weeks or in some cases, decades. When
necessary, however, (such as during wound repair), these same cells
can undergo rapid proliferation and turnover within a five day
period (Folkman, J. and Shing, Y., (1992) Journal of Biological
Chemistry, 267(16): 10931-10934, and Folkman, J. and Klagsbrun, M.,
(1987) Science, 235: 442-447).
[0006] Although angiogenesis is a highly regulated process under
normal conditions, many diseases (characterized as "angiogenic
diseases") are driven by persistent unregulated angiogenesis.
Otherwise stated, unregulated angiogenesis may either cause a
particular disease directly or exacerbate an existing pathological
condition. For example, growth and metastasis of solid tumors are
angiogenesis-dependent (Folkman, J., (1986) Cancer Research, 46:
467-473 and Folkman, J., (1989) Journal of the National Cancer
Institute, 82: 4-6). It has been shown that tumors which enlarge to
greater than 2 mm must obtain their own blood supply and do so by
inducing the growth of new capillary blood vessels. Once these new
blood vessels become embedded in the tumor, they provide a means
for tumor cells to enter the circulation and metastasize to distant
sites, such as the liver, lung or bone (Weidner, N., et al., (1991)
The New England Journal of Medicine, 324(1):1-8). In another
example, ocular neovascularization has been implicated as the most
common cause of blindness and dominates approximately 20 eye
diseases. In certain existing conditions such as arthritis, newly
formed capillary blood vessels invade the joints and destroy
cartilage. In diabetes, new capillaries formed in the retina invade
the vitreous, bleed, and cause blindness.
[0007] Fumagillin is a known compound which has been used as an
antimicrobial and antiprotozoal. Its physicochemical properties and
method of production are well known (U.S. Pat. No. 2,803,586 and
Turner, J. and Tarbell, J., (1962) Proc. Nat. Acad. Sci. USA, 48:
733-735). Fumagillin and certain types of Fumagillin analogs have
also been reported to exhibit anti-angiogenic activity. However,
the use of such inhibitors (e.g., TNP-470) may be limited by their
rapid metabolic degradation, erratic blood levels, and by
dose-limiting central nervous system (CNS) side effects.
Additionally, these molecules have physical and chemical properties
that make them undesirable as therapeutics, for example, low water
solubility, very short half-life values, unacceptable neurotoxic
side-effects, and possible disruption of normal angiogenic
processes.
[0008] One objective in the field of drug delivery systems is to
deliver medications intact to specifically targeted areas of the
body through a system that can control the rate and time of
administration of the therapeutic agent by means of either a
physiological or chemical trigger. Over the past decade, materials
such as polymeric microspheres, polymer micelles, soluble polymers
and hydrogel-type materials have been shown to be effective in
enhancing drug targeting specificity, lowering systemic drug
toxicity, improving treatment absorption rates, and providing
protection for pharmaceuticals against biochemical degradation, and
thus have shown great potential for use in biomedical applications,
particularly as components of drug delivery devices.
[0009] Despite the known usefulness of fumagillin derivatives, they
have not been used successfully as treatments because of the
failure to overcome the problems of the low water solubility, short
half-life values, and neurotoxic side-effects of these compounds.
Accordingly, there is still a need for angiogenesis inhibitors
which are more potent, less neurotoxic, more stable, and/or have
longer serum half-lives than presently known angiogenesis
inhibitors. The combination of drug delivery platform technology
and fumagillin derivatives produces new agents useful in the
treatment of diseases and disease states associated with
angiogenesis.
DESCRIPTION OF THE FIGURES
[0010] FIG. 1 depicts the activity of fumagillin conjugates against
B16-melanoma cells in a mouse model.
[0011] FIG. 2 depicts the activity of fumagillin conjugates against
A2058 human melanoma cells in a mouse model.
[0012] FIG. 3 depicts the activity of fumagillin conjugates against
PC3 human prostate cancer cells in a mouse model.
[0013] FIG. 4 and FIG. 5 depict the effect of fumagillin conjugates
on behavior patterns in mice using an Open Field Behavioral Events
assay.
[0014] FIG. 6 depicts fumagillin conjugates (Fm-analog)
accumulation in rat CSF.
SUMMARY OF THE INVENTION
[0015] In one aspect, biocompatible biodegradable conjugates are
provided which comprise at least one fumagillin analog conjugated
directly or indirectly to a water soluble polyal, the conjugate
having the structure of Formula I:
[fumagillin analog].sub.m-polyal (I)
[0016] wherein fumagillin analog is any fumagillin core structure
which demonstrates MetAP-2 inhibition;
[0017] m is an integer from 1 to 40; and
[0018] polyal is any polymer having at least one acetal oxygen atom
or ketal oxygen atom in each monomer unit positioned within the
main chain of the polymer.
[0019] In some embodiments, the fumagillin analog is represented by
the Formula IIA:
##STR00001##
[0020] wherein
[0021] X is O, S(.dbd.O).sub.q, optionally substituted CH.sub.2, or
optionally substituted NH;
[0022] R is (2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane,
2-methylhepta-2,5-diene, (2R,3S)-2-isopentyl-3-methyloxirane,
6-methylhept-2-ene, (Z)-acetaldehyde O-benzyl oxime,
C.sub.2-heterocyclic-C.sub.1-C.sub.6 alkyl,
C.sub.2-heterocyclic-C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-heterocyclic-C.sub.1-C.sub.6-alkenyl-COO--C.sub.1-C.sub.6
alkyl, C.sub.2-heterocyclic-C.sub.1-C.sub.6
alkyl-COO--C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6
alkyl=N--O--C.sub.1-C.sub.6 alkyl-aryl, C(O)C.sub.1-C.sub.6 alkyl,
CN, or halogen;
[0023] M is O, or
##STR00002##
[0024] q is 0, 1, or and
[0025] the fumagillin core structure can be optionally substituted
at C-7 and C-8, independently, with C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, OH, ketone, or
alkoxy.
[0026] In some embodiments, the fumagillin analog of the conjugate
has the core structure A:
##STR00003##
[0027] wherein C-7 and C-8 are optionally substituted;
[0028] X is O, S(.dbd.O).sub.q, optionally substituted NH, or
optionally substituted CH.sub.2;
[0029] M is O, or
##STR00004##
[0030] q is 0, 1, or 2; and
[0031] the fumagillin analog is directly or indirectly attached to
the polyal through X.
[0032] In other embodiments, the fumagillin analog of the conjugate
has the core structure B:
##STR00005##
[0033] wherein C-7 and C-8 are optionally substituted;
[0034] X is O, S(.dbd.O).sub.4, optionally substituted NH, or
optionally substituted CH.sub.2;
[0035] M is O, or
##STR00006##
[0036] q is 0, 1, or 2; and
[0037] the fumagillin analog is directly or indirectly attached to
the polyal through X.
[0038] In still other embodiments, the fumagillin analog of the
conjugate has the core structure C:
##STR00007##
[0039] wherein C-7 and C-8 are optionally substituted;
[0040] X is O, S(.dbd.O).sub.q, optionally substituted NH, or
optionally substituted CH.sub.2;
[0041] M is O, or
##STR00008##
[0042] q is 0, 1, or 2; and
[0043] the fumagillin analog is directly or indirectly attached to
the polyal through X.
[0044] In some embodiments, the polyal has the structure
##STR00009## [0045] wherein for each occurrence of the n bracketed
structure, either
[0046] one of R.sub.1 and R.sub.2 is hydrogen, and the other is a
biocompatible group and includes a carbon atom covalently attached
to C.sub.1 or
[0047] each occurrence of R.sub.1 and R.sub.2 is a biocompatible
group and includes a carbon atom covalently attached to
C.sub.1;
[0048] R.sub.x includes a carbon atom covalently attached to
C.sub.2;
[0049] n is an integer;
[0050] each occurrence of R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is
a biocompatible group and is independently hydrogen or an organic
moiety; and
[0051] for each occurrence of the bracketed structure n, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6
comprises a functional group suitable for coupling directly or
indirectly with the fumagillin analog.
[0052] In another aspect, biocompatible biodegradable conjugates
are described which comprise at least one fumagillin analog of the
formula II conjugated to a water soluble polyal
##STR00010##
[0053] wherein
[0054] X is O, S(.dbd.O).sub.q, optionally substituted CH.sub.2, or
optionally substituted NH;
[0055] R is (2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane,
2-methylhepta-2,5-diene, (2R,3S)-2-isopentyl-3-methyloxirane,
6-methylhept-2-ene, (Z)-acetaldehyde O-benzyl oxime,
C.sub.2-heterocyclic-C.sub.1-C.sub.6 alkyl,
C.sub.2-heterocyclic-C.sub.2-C.sub.6alkenyl, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6alkenyl,
C.sub.2-heterocyclic-C.sub.1-C.sub.6-alkenyl-COO--C.sub.1-C.sub.6alkyl,
C.sub.2-heterocyclic-C.sub.1-C.sub.6alkyl-COO--C.sub.1-C.sub.6-alkyl,
C.sub.1-C.sub.6alkylN-O--C.sub.1-C.sub.6 alkyl-aryl,
C(O)C.sub.1-C.sub.6 alkyl, CN, or halogen;
[0056] M is O, or
##STR00011##
[0057] q is 0, 1, or 2;
[0058] wherein the fumagillin core structure can be optionally
substituted at C-7 and C-8, independently, with
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, OH, ketone, or alkoxy;
[0059] wherein the fumagillin analog is conjugated by covalent
attachment of X to a free hydroxyl of the polyal through a spacer
moiety of the Formula III
##STR00012##
[0060] wherein Tether is an organic moiety with a molecular weight
between about null and about 1000 covalently attached to both the X
of Formula II and to Linker;
[0061] wherein Linker is an organic moiety covalently attached to
both Tether and to a free hydroxyl of the polyal, having a
molecular weight between about null and about 1000; and
[0062] wherein the spacer moiety of Formula III comprises one or
more labile bonds capable of enzymatic or chemical cleavage, so as
to provide a fumagillin analog conjugate having either a higher
water solubility than, a longer biological half-life than, or less
neurotoxicity than, the unconjugated fumagillin analog.
[0063] In another aspect, biocompatible biodegradable polymer
conjugates of the Formula IV are described:
##STR00013##
[0064] wherein
[0065] X is O, S(.dbd.O).sub.q, optionally substituted CH.sub.2, or
optionally substituted NH;
[0066] R is (2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane,
2-methylhepta-2,5-diene, (2R,3S)-2-isopentyl-3-methyloxirane,
6-methylhept-2-ene, (Z)-acetaldehyde O-benzyl oxime,
C.sub.2-heterocyclic-C.sub.1-C.sub.6 alkyl,
C.sub.2-heterocyclic-C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-heterocyclic-C.sub.1-C.sub.6-alkenyl-COO--C.sub.1-C.sub.6
alkyl, C.sub.2-heterocyclic-C.sub.1-C.sub.6
alkyl-COO--C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6
alkyl-N--O--C.sub.1-C.sub.6 alkyl-aryl, C(O)C.sub.1-C.sub.6 alkyl,
CN, or halogen;
[0067] W is null, a bond, --C(O)--, --NH--, --C.sub.1-C.sub.6
alkyl-, --C.sub.2-C.sub.6alkenyl-, --C.sub.2-C.sub.6 alkynyl-,
--C.sub.1-C.sub.6 alkoxy-, -aryl-, -heteroaryl-, -cycloalkyl-,
--C(O)bicycloalkylC(O)--, --SO.sub.2-bicycloalkyl-,
-heterocycloalkyl-, -heterobicycloalkyl-, --C(O)C.sub.1-C.sub.6
alkyl heteroaryl-O--,
--C(O)--C.sub.1-C.sub.6-alkenyl-aryl-O--N(C.sub.1-C.sub.6
alkyl).sub.2-, --C(O)-heterocycloalkyl-C.sub.1-C.sub.6 alkyl-O--,
--C(O)-heterocycloalkyl-C.sub.1-C.sub.6 alkyl-COO--,
--C(O)-heterobicycloalkyl-C.sub.1-C.sub.6 alkyl-COO--,
--C(O)-heterobicycloalkyl-C.sub.1-C.sub.6alkyl-C(O)--,
--C(O)NH(C.sub.1-C.sub.6 alkyl) heteroaryl(O)--,
--C(O)NH(C.sub.1-C.sub.6 alkyl) aryl(O)--, --C(O)C.sub.1-C.sub.6
alkylaryl-, --NEC(O)C.sub.1-C.sub.6-alkylaryl-,
--C(O)C.sub.1-C.sub.6-alkenyl-aryl-O--NH(C.sub.1-C.sub.6-alkyl)-,
--C(O)NH(C.sub.1-C.sub.6 alkyl)cycloalkylCOO--, --C(O)cycloalkyl
C.sub.1-C.sub.6 alkyl NH--, --NHC(O)C.sub.1-C.sub.6 alkyl-,
--NHC(O)NHC.sub.1-C.sub.6 alkyl-, --SO.sub.2NH--,
--SO.sub.2NHC.sub.1-C.sub.6alkyl-, --SO.sub.2N(C.sub.1-C.sub.6
alkyl-, --NHSO.sub.2C.sub.1-C.sub.6 alkyl-,
--CO.sub.2C.sub.1-C.sub.6 alkyl-, --CONHC.sub.1-C.sub.6 alkyl-,
--CON(C.sub.1-C.sub.6 alkyl)-, --C.sub.1-C.sub.6
alkyl-O--C.sub.1-C.sub.6 alkyl N(C.sub.1-C.sub.6 alkyl).sub.2-,
--C(O)NHC(O)C.sub.1-C.sub.6 alkyl S C.sub.0-C.sub.6 alkyl
aryl-,
[0068] --C.sub.1-C.sub.6alkyl
NH--SO.sub.2-heterocyclo-C.sub.1-C.sub.6 alkyl-O--,
--C(O)NHC(O)C.sub.1-C.sub.6 alkyl SC.sub.0-C.sub.6alkylNH--,
--C(O)NH--C.sub.1-C.sub.6 alkyl-aryl-, --C(O)heterocycloalkyl-,
--C(O)--C.sub.1-C.sub.6-alkyl-S-aryl-, --C(O)heterobicycloalkyl-,
--C(O)--NH--C(O)--C.sub.1-C.sub.6 alkyl-, or
--C(O)--NH--C(O)--C.sub.0-C.sub.6 alkyl-S-aryl-, each optionally
substituted;
Q is null, --NH--, -amino acid-, --NH-amino acid-,
--(C.sub.1-C.sub.6 alkyl COO)--, --(OOCC.sub.1-C.sub.6 alkyl
COO)--, --(C.sub.1-C.sub.6 alkyl-O-amino acid)-, or
--(C.sub.1-C.sub.6 alkyl-O)--;
[0069] Y is null, an oxalic, malonic, succinic, glutaric, adipic,
pimelic, suberic, azelaic, sebacic, phthalic, isophthalic,
terephthalic, diglycolic acid, oxaglutaric, tartaric, glutamic,
fumaric, or aspartic moiety, including amide, imide, or
cyclic-imide derivatives of each thereof, and each optionally
substituted; and
[0070] Z is a polyal with the structure:
##STR00014## [0071] wherein for each occurrence of the n bracketed
structure, either
[0072] one of R.sub.1 and R.sub.2 is hydrogen, and the other is a
biocompatible group and includes a carbon atom covalently attached
to C.sub.1 or
[0073] each occurrence of R.sub.1 and R.sub.2 is a biocompatible
group and includes a carbon atom covalently attached to
C.sub.1;
[0074] R.sub.x includes a carbon atom covalently attached to
C.sub.2;
[0075] n is an integer;
[0076] each occurrence of R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is
a biocompatible group and is independently hydrogen or an organic
moiety; and
[0077] for each occurrence of the bracketed structure n, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6
comprises a functional group suitable for coupling with W, Y, or
Q.
[0078] In another aspect, compounds of the Formula V are
described:
##STR00015##
[0079] wherein,
[0080] X is O, S(.dbd.O).sub.q, optionally substituted CH.sub.2, or
optionally substituted NH;
[0081] q is 0, 1, or 2;
[0082] R is (2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane,
2-methylhepta-2,5-diene, (2R,3S)-2-isopentyl-3-methyloxirane,
6-methylhept-2-ene, (Z)-acetaldehyde O-benzyl oxime,
C.sub.2-heterocyclic-C.sub.1-C.sub.6 alkyl,
C.sub.2-heterocyclic-C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6 alkenyl,
C.sub.2-heterocyclic-C.sub.1-C.sub.6-alkenyl-COO--C.sub.1-C.sub.6alkyl,
C.sub.2-heterocyclic-C.sub.1-C.sub.6alkyl-COO--C.sub.1-C.sub.6-alkyl,
C.sub.1-C.sub.6alkyl=N--O--C.sub.1-C.sub.6 alkyl-aryl,
C(O)C.sub.1-C.sub.6 alkyl, CN, or halogen;
[0083] R.sub.8 is selected from the group consisting of VI, VII,
VIII, IX, X, XI, XII, and XIIA whose formulas are represented
below:
##STR00016##
[0084] R' is --CO.sub.2H, optionally substituted --NH.sub.2, or --N
cyclic imide, NHC(O)(C.sub.1-C.sub.6 alkyl)-C(O)R'', R' is meta or
para in relation to the S-- atom; and
[0085] R'' is --OH, --O--C.sub.1-C.sub.6 alkyl, or --NH.sub.2
optionally acylated through the carboxyl group of an amino
acid;
##STR00017##
[0086] wherein R.sub.9 is H or C(O)R.sub.11;
[0087] R.sub.10 is --NH.sub.2, --NHCH(C.sub.1-C.sub.6 alkyl)-,
--NHC(O)(C.sub.1-C.sub.6 alkyl), N-cyclized imide; --NH acylated
through the carboxyl group of an amino acid, wherein the nitrogen
of the amino group of the amino acid is optionally protected;
and
[0088] R.sub.11 is OH, OC.sub.1-C.sub.6 alkyl, or optionally
substituted --NH.sub.2;
##STR00018##
[0089] wherein R.sub.12 is H, C.sub.1-C.sub.6 alkyl,
--(C.sub.1-C.sub.6)--COOH,
--(C.sub.1-C.sub.6)--C(O)O--(C.sub.1-C.sub.6),
--CH.sub.2CH.sub.2O--R.sub.13, --C(O)(C.sub.1-C.sub.6-alkyl), or an
amino acid attached through the carboxyl group of the amino
acid;
[0090] R.sub.13 is --H or an amino acid attached through the
carboxyl group of the amino acid, wherein the nitrogen of the amino
acid is optionally protected, C(O)(C.sub.1-C.sub.6
alkyl)-COR'';
[0091] R'' is OH, --OC.sub.1-C.sub.6 alkyl, or --NH.sub.2
optionally acylated through the carboxyl group of an amino
acid;
[0092] [-------] represents an optional methylene bridge
(--C.sub.2--) between carbons 2 and 5 of the piperazine moiety;
and
[0093] Z' is a bond, --C.sub.1-C.sub.6 alkyl, --NHC(O)--, or
--NHSO.sub.2--;
##STR00019##
[0094] wherein R.sub.14 is --H, --CO.sub.2H,
--CO.sub.2(C.sub.1-C.sub.6 alkyl), --C(O)NH--C.sub.1-C.sub.6
alkyl-OH, wherein the O of --C(O)NH--C.sub.1-C.sub.6 alkyl-OH is
optionally acylated with the carboxyl group of an amino acid;
optionally substituted --NH.sub.2, C.sub.1-C.sub.6-alkyl-N.sub.2,
wherein the NH.sub.2 is optionally substituted; and
[0095] [-------] represents an optional ethylene bridge
(--CH.sub.2CH.sub.2--) between carbons 1 and 4 of the cyclohexane
moiety;
##STR00020##
[0096] Z' is a bond, --CH.sub.2--, --CH.sub.2--S--,
--CH.sub.2CH.sub.2--, --C(H)(Me)-, --NHCH.sub.2--,
--NHCH(CH.sub.3)--, or --NHCH.sub.2CH.sub.2--;
[0097] R.sub.15 is H, optionally substituted --NH.sub.2,
--NHC(O)(C.sub.1-C.sub.6-alkyl), --N cyclized imide optionally
containing a heteroatom within the cyclic structure,
--NHC(O)CH.sub.2OCH.sub.2C(O)OH, NHC(O)CH(C.sub.1-C.sub.6 alkyl)-N
cyclized imide, --NHC(O)CH(R'')NHC(O)--(C.sub.1-C.sub.6
alkyl)-C(O)OH, --NHC(O)--(C.sub.1-C.sub.6 alkyl)-C(O)OH,
--C(O)O(C.sub.1-C.sub.6 alkyl), --C(O)N(H)(C.sub.1-C.sub.6
alkyl)-OH, or NO.sub.2; and
[0098] R'' is --H, or --C.sub.1-C.sub.6 alkyl;
##STR00021##
[0099] wherein Y' is C.sub.1-C.sub.3 alkyl, or NH--C.sub.1-C.sub.3
alkyl and is attached to positions 1, 2, or 3 of indole; and
[0100] R.sub.16 is H, C.sub.1-C.sub.6 alkyl, --CH.sub.2COOH, or
--CH.sub.2CH.sub.2OH, wherein the O of --CH.sub.2CH.sub.2OH can be
optionally acylated with an amino acid;
##STR00022##
[0101] wherein R'' is --OH, --OC.sub.1-C.sub.6 alkyl, or --NH.sub.2
optionally acylated through the carboxyl group of an amino acid;
and
##STR00023##
[0102] wherein Y'' is C(O)N(CH.sub.3)(OCH.sub.3), C(O)OCH.sub.3,
CH.sub.2Cl, or NHC(O)CH.sub.2Cl.
[0103] Pharmaceutical compositions comprising an effective amount
of a fumagillin analog conjugate and a pharmaceutically acceptable
carrier are described.
[0104] Pharmaceutical compositions comprising an effective amount
of a fumagillin analog and a pharmaceutically acceptable carrier
are described. Also, fumagillin analogs are provided as a
pharmaceutically acceptable prodrug, hydrated salt, such as a
pharmaceutically acceptable salt, or mixtures thereof.
[0105] In another aspect, methods for treating cancer, or treating
an angiogenic disease, comprising the administration of an
effective amount of a fumagillin analog conjugate are
described.
[0106] In another aspect, methods for treating cancer, or treating
an angiogenic disease, comprising the administration of an
effective amount of a fumagillin analog are described,
DETAILED DESCRIPTION OF THE INVENTION
[0107] The following definitions are used in connection with the
fumagillin analog conjugates and the fumagillin analogs described
herein:
[0108] "Alkyl" refers to a hydrocarbon chain that may be a straight
chain or branched chain, containing the indicated number of carbon
atoms. For example, C.sub.1-C.sub.6 indicates that the group may
have from 1 to 6 (inclusive) carbon atoms in it.
[0109] "Aryl" refers to cyclic aromatic carbon ring systems
containing from 6 to 18 carbons. Examples of an aryl group include,
but are not limited to, phenyl, naphthyl, anthracenyl, tetracenyl,
and phenanthrenyl. An aryl group can be unsubstituted or
substituted with one or more of the following groups: H, halogen,
CN, OH, aryl, arylalkyl, heteroaryl, heteroaryl, heteroarylalkyl,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-3
fluorinated-alkyl, C.sub.3-6 cycloalkyl,
C.sub.3-6cycloalkyl-C.sub.1-3alkyl, NO.sub.2, NH.sub.2, NHC.sub.1-6
alkyl, N(C.sub.1-6 alkyl).sub.2, NHC.sub.3-6cycloalkyl, N(C.sub.3-6
cycloalkyl).sub.2, NHC(O)C.sub.1-6 alkyl,
NHC(O)C.sub.3-6cycloalkyl, NHC(O)NHC.sub.1-6 alkyl,
NHC(O)NHC.sub.3-6 cycloalkyl, SO.sub.2NH.sub.2, SO.sub.2NHC.sub.1-6
alkyl, SO.sub.2NHC.sub.3-6 cycloalkyl, SO.sub.2N(C.sub.1-6
alkyl).sub.2, SO.sub.2N(C.sub.3-6 cycloalkyl).sub.2,
NHSO.sub.2C.sub.1-6 alkyl, NHSO.sub.2C.sub.3-6 cycloalkyl,
CO.sub.2C.sub.1-6 alkyl, CO.sub.2C.sub.3-6 cycloalkyl,
CONHC.sub.1-6 alkyl, CONHC.sub.3-6 cycloalkyl, CON(C.sub.1-6
alkyl).sub.2, CON(C.sub.3-6 cycloalkyl).sub.2OH, OC.sub.1-3 alkyl,
C.sub.1-3 fluorinated-alkyl, OC.sub.3-6 cycloalkyl, OC.sub.3-6
cycloalkyl-C.sub.1-3 alkyl, SH, SO.sub.xC.sub.1-3 alkyl, C.sub.3-6
cycloalkyl, or SO.sub.xC.sub.3-6cycloalkyl-C.sub.1-3 alkyl, where x
is 0, 1, or 2.
[0110] "Heteroaryl" refers to mono and bicyclic aromatic groups of
4 to 10 atoms containing at least one heteroatom. Heteroatom as
used in the term heteroaryl refers to oxygen, sulfur and nitrogen.
Examples of monocyclic heteroaryls include, but are not limited to,
oxazinyl, thiazinyl, triazinyl, tetrazinyl, imidazolyl, tetrazolyl,
isoxazolyl, furanyl, furazanyl, oxazolyl, thiazolyl, thiophenyl,
pyrazolyl, triazolyl, and pyrimidinyl. Examples of bicyclic
heteroaryls include but are not limited to, benzimidazolyl,
indolyl, isoquinolinyl, indazolyl, quinolinyl, quinazolinyl,
purinyl, benzisoxazoryl, benzoxazolyl, benzthiazolyl,
benzodiazolyl, benzotriazolyl, isoindolyl and indazolyl. A
heteroaryl group can be unsubstituted or substituted with one or
more of the following groups: H, halogen, CN, OH, aryl, arylalkyl,
heteroaryl, heteroarylalkyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.1-3 fluorinated-alkyl, C.sub.3-6
cycloalkyl, C.sub.3-6cycloalkyl-C.sub.1-3alkyl, NO.sub.2, NH.sub.2,
NHC.sub.1-6 alkyl, N(C.sub.1-6 alkyl).sub.2, NHC.sub.3-6
cycloalkyl, N(C.sub.3-6 cycloalkyl).sub.2, NHC(O)C.sub.1-6 alkyl,
NHC(O)C.sub.3-6 cycloalkyl, NHC(O)NHC.sub.1-6 alkyl,
NHC(O)NHC.sub.3-6 cycloalkyl, SO.sub.2NH.sub.2, SO.sub.2NHC.sub.1-6
alkyl, SO.sub.2NHC.sub.3-6 cycloalkyl,
SO.sub.2N(C.sub.1-6alkyl).sub.2, SO.sub.2N(C.sub.3-6
cycloalkyl).sub.2, NHSO.sub.2C.sub.1-6 alkyl, NHSO.sub.2C.sub.3-6
cycloalkyl, CO.sub.2C.sub.1-6 alkyl, CO.sub.2C.sub.3-6 cycloalkyl,
CONHC.sub.1-6 alkyl, CONHC.sub.3-6 cycloalkyl, CON(C.sub.1-6
alkyl).sub.2, CON(C.sub.3-6 cycloalkyl).sub.2OH, OC.sub.1-3 alkyl,
C.sub.1-3 fluorinated-alkyl, OC.sub.3-6 cycloalkyl, OC.sub.3-6
cycloalkyl-C.sub.1-3 alkyl, SH, SO.sub.xC.sub.1-3 alkyl, C.sub.3-6
cycloalkyl, or SO.sub.xC.sub.3-6 cycloalkyl-C.sub.1-3 alkyl, where
x is 0, 1, or 2.
[0111] "Arylalkyl" refers to an aryl group with at least one alkyl
substitution. Examples of arylalkyl include, but are not limited
to, toluenyl, phenylethyl, xylenyl, phenylbutyl, phenylpentyl, and
ethylnaphthyl. An arylalkyl group can be unsubstituted or
substituted with one or more of the following groups: H, halogen,
CN, OH, aryl, arylalkyl, heteroaryl, heteroarylalkyl, C.sub.1-6
alkyl, alkenyl, C.sub.2-6 alkynyl, C.sub.1-3 fluorinated-alkyl,
C.sub.3-6 cycloalkyl, C.sub.3-6cycloalkyl-C.sub.1-3alkyl, NO.sub.2,
NH.sub.2, NHC.sub.1-6 alkyl, N(C.sub.1-6 alkyl).sub.2, NHC.sub.3-6
cycloalkyl, N(C.sub.3-6 cycloalkyl).sub.2, NHC(O)C.sub.1-6 alkyl,
NHC(O)C.sub.3-6 cycloalkyl, NHC(O)NHC.sub.1-6 alkyl,
NHC(O)NHC.sub.3-6 cycloalkyl, SO.sub.2NH.sub.2, SO.sub.2NHC.sub.1-6
alkyl, SO.sub.2NHC.sub.3-6 cycloalkyl, SO.sub.2N(C.sub.1-6
alkyl).sub.2, SO.sub.2N(C.sub.3-6 cycloalkyl).sub.2,
NHSO.sub.2C.sub.1-6 alkyl, NHSO.sub.2C.sub.3-6 CO.sub.2C.sub.1-6
alkyl, CO.sub.2C.sub.3-6 cycloalkyl, CONHC.sub.1-6 alkyl,
CONHC.sub.3-6 cycloalkyl, CON(C.sub.1-6 CON(C.sub.3-6
cycloalkyl).sub.2OH, OC.sub.1-3 alkyl, C.sub.1-3 fluorinated-alkyl,
OC.sub.3-6 cycloalkyl, OC.sub.3-6 cycloalkyl-C.sub.1-3 alkyl, SH,
SO.sub.xC.sub.1-3 alkyl, C.sub.3-6 cycloalkyl, or SO.sub.xC.sub.3-6
cycloalkyl-C.sub.1-3 alkyl, where x is 0, 1, or 2.
[0112] "Heteroarylalkyl" refers to a heteroaryl group with at least
one alkyl substitution. A heteroarylalkyl group can be
unsubstituted or substituted with one or more of the following: H,
halogen, CN, OH, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-3
fluorinated-alkyl, C.sub.3-6 cycloalkyl,
C.sub.3-6cycloalkyl-C.sub.1-3alkyl, NO.sub.2, NH.sub.2, NHC.sub.1-6
alkyl, N(C.sub.1-6 alkyl).sub.2, NHC.sub.3-6cycloalkyl, N(C.sub.3-6
cycloalkyl).sub.2, NHC(O)C.sub.1-6 alkyl, NHC(O)C.sub.3-6
cycloalkyl, NHC(O)NHC.sub.1-6 alkyl, NHC(O)NHC.sub.3-6 cycloalkyl,
SO.sub.2NH.sub.2, SO.sub.2NHC.sub.1-6 alkyl, SO.sub.2NHC.sub.3-6
cycloalkyl, SO.sub.2N(C.sub.1-6 alkyl).sub.2, SO.sub.2N(C.sub.3-6
cycloalkyl).sub.2, NHSO.sub.2C.sub.1-6 alkyl, NHSO.sub.2C.sub.3-6
cycloalkyl, CO.sub.2C.sub.1-6 alkyl, CO.sub.2C.sub.3-6 cycloalkyl,
CONHC.sub.1-6 alkyl, CONHC.sub.3-6 cycloalkyl, CON(C.sub.1-6
alkyl).sub.2, CON(C.sub.3-6 cycloalkyl).sub.2OH, OC.sub.1-3 alkyl,
C.sub.1-3 fluorinated-alkyl, OC.sub.3-6 cycloalkyl, OC.sub.3-6
cycloalkyl-C.sub.1-3 alkyl, SH, SO.sub.xC.sub.1-3 alkyl, C.sub.3-6
cycloalkyl, or SO.sub.xC.sub.3-6 cycloalkyl-C.sub.1-3 alkyl, where
x is 0, 1, or 2.
[0113] "C.sub.1-C.sub.6 alkyl" refers to a straight or branched
chain saturated hydrocarbon containing 1-6 carbon atoms. Examples
of a C.sub.1-C.sub.6 alkyl group include, but are not limited to,
methyl, ethyl, propyl, isopropyl, n-pentyl, isopentyl, neopentyl,
and hexyl.
[0114] "C.sub.2-C.sub.6 alkenyl" refers to a straight or branched
chain unsaturated hydrocarbon containing 2-6 carbon atoms and at
least one double bond. Examples of a C.sub.2---C.sub.6 alkenyl
group include, but are not limited to, ethylene, propylene,
1-butylene, 2-butylene, isobutylene, sec-butylene, 1-pentene,
2-pentene, isopentene, 1-hexene, 2-hexene, 3-hexene, and
isohexene.
[0115] "C.sub.3-C.sub.6 alkenyl" refers to a straight or branched
chain unsaturated hydrocarbon containing 3-6 carbon atoms and at
least one double bond. Examples of a C.sub.3-C.sub.6 alkenyl group
include, but are not limited to, propylene, 1-butylene, 2-butylene,
isobutylene, sec-butylene, 1-pentene, 2-pentene, isopentene,
1-hexene, 2-hexene, 3-hexene, and isohexene.
[0116] "C.sub.1-C.sub.6 alkoxy" refers to a straight or branched
chain saturated or unsaturated hydrocarbon containing 1-6 carbon
atoms and at least one oxygen atom. Examples of a
C.sub.1-C.sub.6-alkoxy include, but are not limited to, methoxy,
ethoxy, isopropoxy, butoxy, n-pentoxy, isopentoxy, neopentoxy, and
hexoxy.
[0117] "Cycloalkyl" refers to a cyclic saturated hydrocarbon.
Examples of a cycloalkyl group include, but are not limited to,
cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane,
and cyclooctane.
[0118] "C.sub.1-C.sub.3 fluorinated alkyl" refers to a saturated
straight or branched chain hydrocarbon containing 1-3 carbon atoms
that can be further substituted with other functional groups.
Examples of a C.sub.1-C.sub.3 fluorinated alkyl are
trifluoromethyl, 2,2,2-trifluoroethyl, and trifluoroacetyl.
[0119] "Halogen" refers to an atom of fluorine, chlorine, bromine,
or iodine.
[0120] "Bicycloalkyl" refers to a saturated hydrocarbon two ring
system sharing a pair of bridgehead carbons that can be further
substituted with other functional groups. Examples of a
bicycloalkyl include, but are not limited to, bicyclo[2.2.1]heptane
(norbornane), bicyclo[4.3.2]undecane, bicyclo[4.1.0]heptane,
bicyclo[4.4.0]decane (decalin), and bicyclo[2.2.2]octane.
[0121] "Heterobicycloalkyl" refers to a saturated hydrocarbon two
ring system sharing two bridgehead atoms with at least one
heteroatom. Heteroatom as used in the term heteroaryl refers to
oxygen, sulfur, and nitrogen. Examples of heterobicycloalkyl
include, but are not limited to, 2,5-diazabicyclo[2.2.1]heptanyl,
2-azabicyclo[2.2.1]heptanyl, 3-aza-bicyclo[4.1.0]heptanyl, and
1,4-dioxaspiro[4.4]nonyl.
[0122] "Cyclized imide" and "cyclic-imide" refer to either
saturated or unsaturated cyclic or heterocyclic compounds that
contain the imide functional group which consists of two carbonyl
groups bound to a nitrogen atom. Cyclic-imides can be further
substituted with other functional groups. Examples of a
cyclic-imide include, but are not limited to, piperidyl-2,6-dione,
morpholyl-3,5-dione, and pyrrolidyl-2,5-dione.
[0123] The term "optionally substituted NH" or "optionally
substituted NH.sub.2" when used herein means that one or more of
the hydrogen atoms can be substituted for one of the following
groups: C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.3 fluorinated alkyl,
SO.sub.2NH.sub.2, SO.sub.2NHC.sub.1-C.sub.6 alkyl,
SO.sub.2N(C.sub.1-C.sub.6 alkyl).sub.2, C(O)OC.sub.1-C.sub.6 alkyl,
CONHC.sub.1-C.sub.6 alkyl, CON(C.sub.1-C.sub.6 alkyl).sub.2,
--C(O)--CH(R'')NH.sub.2, wherein R'' in this context is the side
chain of any naturally occurring amino acid.
[0124] The phrase "one or more labile bonds capable of enzymatic or
chemical hydrolysis" as used herein means a bond that is chemically
reactive in the biological environment of the cell or blood or a
bond known to be hydrolyzed by enzymes. Non-Limiting examples of
labile bonds capable of chemical or enzymatic hydrolysis are
esters, amides, imides, thioethers, anhydrides, azides, carbamates,
and carbonates.
[0125] The term "optionally substituted CH.sub.2" when used herein
means that one or both hydrogen atoms may be substituted with one
or more of the following groups: OH, halogen, CN, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl,
C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.3 fluorinated alkyl,
NO.sub.2, NH.sub.2, NHC.sub.1-6 alkyl, N(C.sub.1-C.sub.6
alkyl).sub.2, NHC(O)C.sub.1-C.sub.6 alkyl, NHC(O)NHC.sub.1-C.sub.6
alkyl, SO.sub.2NH.sub.2, SO.sub.2NHC.sub.1-C.sub.6 alkyl,
SO.sub.2N(C.sub.1-C.sub.6 alkyl).sub.2, NHSO.sub.2C.sub.1-C.sub.6
alkyl, C(O)OC.sub.1-C.sub.6 alkyl, CONHC.sub.1-C.sub.6 alkyl,
CON(C.sub.1-C.sub.6 alkyl).sub.2, C.sub.1-C.sub.6 alkyl, or both
hydrogen atoms may be substituted and the substituted groups when
taken together with the carbon to which they are attached, form a
cycloalkyl or heterocycloalkyl, each optionally substituted with
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.1-C.sub.6 alkoxy, CO.sub.2C.sub.1-C.sub.6 alkyl, CN,
OH, cycloalkyl, CONH.sub.2, aryl, heteroaryl, COaryl, or
trifluoroacetyl;
[0126] A "subject" is a mammal. e.g., a human, mouse, rat, guinea
pig, dog, cat, horse, cow, pig, or non-human primate, such as a
monkey, chimpanzee, baboon, or rhesus.
[0127] The term "pharmaceutically acceptable salts" include, e.g.,
water-soluble and water-insoluble salts, such as the acetate,
amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate,
benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide,
butyrate, calcium edetate, camsylate, carbonate, chloride, citrate,
clavulariate, dihydrochloride, edetate, edisylate, estolate,
esylate, fumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexafluorophosphate, hexylresorcinate,
hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate,
isothionate, lactate, lactobionate, laurate, malate, maleate,
mandelate, mesylate, methylbromide, methylnitrate, methylsulfate,
mucate, napsylate, nitrate, N-methylglucamine ammonium salt,
3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate
(1,1-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate,
phosphate/diphosphate, picrate, polygalacturonate, propionate,
p-toluenesulfonate, salicylate, stearate, subacetate, succinate,
sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate,
tosylate, triethiodide, and valerate salts.
[0128] An "effective amount" when used in connection with a
fumagillin analog conjugate or a fumagillin analog is an amount
effective for treating or preventing an angiogenic disease.
[0129] An "aminopeptidase" includes, but is not limited to, protein
methionine aminopeptidase type 2.
[0130] The term "fumagillin core structure which demonstrates
MetAP-2 inhibition" means any fumagalin core structure that
inhibits the ability of MetAP-2 to remove NH.sub.2-terminal
methionines from proteins as described in Rodeschini et al., J.
Org. Client., 69, 357-373, 2004 and Liu, et al., Science 282,
1324-1327, 1998. Nonlimiting examples of "fumagillin core
structures" are disclosed in J. Org. Chem., 69, 357, 2004; J. Org.
Chem., 70, 6870, 2005; European Patent Application 0 354 787; J.
Med. Chem., 49, 5645, 2006; Bioorg. Med. Chem., 11, 5051, 2003;
Bioorg. Med. Chem., 14, 91, 2004; Tet. Lett. 40, 4797, 1999;
WO99/61432; U.S. Pat. No. 6,603,812; U.S. Pat. No. 5,789,405; U.S.
Pat. No. 5,767,293; U.S. Pat. No. 6,566,541; and U.S. Pat. No.
6,207,704.
[0131] The phrase "reduced toxicity" as used herein has its
ordinary meaning as understood by persons of skill in the art.
Merely by way of example, and by no means as a limitation on the
meaning of the term, the administration of the fumagillin analog
conjugate causes less side effects in open field tests with mice,
as compared to the fumagillin analog alone.
[0132] The phrase "improved water solubility" has its ordinary
meaning as understood by persons of skill in the art, Merely by way
of example, and by no means as a limitation on the meaning of the
term, the following description of the term is informative: an
increased amount of a fumagillin analog will dissolve in water as a
result of its covalent incorporation into a conjugate as compared
to the amount of the unconjugated fumagillin analog that will
dissolve in water alone.
[0133] The phrase "longer half-life" has its ordinary meaning as
understood by persons of skill in the art. Merely by way of
example, and by no means as a limitation on the meaning of the
term, the following description of the term is informative: any
appreciable increase in the length of time required to deactivate
fumagillin conjugate either in vivo or in vitro as compared to the
half-life of the fumagillin analog alone either in vivo or in
vitro.
[0134] The term "polyal" means a polymer having at least one acetal
or ketal oxygen atom in each monomer unit positioned within the
main chain. Examples of polyals can be found in U.S. Pat. No.
5,811,510, U.S. Pat. No. 5,863,990, U.S. Pat. No. 5,958,398 and
international application PCT/US2004/029130 which are incorporated
herein by reference in their entirety. In certain embodiments,
biodegradable biocompatible polymer carriers, useful for
preparation of polymer conjugates described herein, are naturally
occurring polysaccharides, glycopolysaccharides, and synthetic
polymers of polyglycoside, polyacetal, polyamide, polyether, and
polyester origin and products of their oxidation,
functionalization, modification, cross-linking, and
conjugation.
[0135] The term "fumagillin analog" as used herein includes a
fumagillin core structure which demonstrates MetAP-2 inhibition (as
defined above) along with a chemical spacer moiety that allows the
fumagillin core structure to be covalently attached through the C-6
of the fumagillin core structure to an OH of the polyal. The
chemical spacer moiety has a molecular weight range of between
about 100 and about 1000.
[0136] As used herein, the term "angiogenic disease" includes a
disease, disorder, or condition characterized or caused by aberrant
or unwanted, e.g., stimulated or suppressed, formation of blood
vessels (angiogenesis). Aberrant or unwanted angiogenesis may
either cause a particular disease directly or exacerbate an
existing pathological condition. Examples of angiogenic diseases
include cancer, e.g., carcinomas and sarcomas, where progressive
growth is dependent upon the continuous induction of angiogenesis
by these tumor cells; pediatric disorders, e.g., angiofibroma, and
hemophiliac joints; blood vessel diseases such as hemangiomas, and
capillary proliferation within atherosclerotic plaques; disorders
associated with surgery, e.g., hypertrophic scars, wound
granulation and vascular adhesions; autoimmune diseases such as
rheumatoid, immune and degenerative arthritis, where new vessels in
the joint may destroy articular cartilage; and sclerodermaocular
disorders and ocular disorders, e.g. diabetic retinopathy,
retinopathy of prematurity, corneal graft rejection, retrolental
fibroplasia, neovascular glaucoma, rubeosis, retinal
neovascularization due to macular degeneration, hypoxia,
angiogenesis in the eye associated with infection or surgical
intervention, ocular tumors and trachoma, and other abnormal
neovascularization conditions of the eye, where neovascularization
may lead to blindness; and disorders affecting the skin, e.g.,
psoriasis and pyogenic granuloma, obesity, where adipogenesis is
associated with neovascularization, and activated adipocytes
produce multiple pro-angiogenic factors which can stimulate
neovascularization during fat mass expansion; and endometriosis,
where the endometriotic lesion is supported by the growth of new
blood vessels, and the endometrium of patients with endometriosis
shows enhanced endothelial cell proliferation.
[0137] The term angiogenic disease also includes diseases
characterized by excessive or abnormal stimulation of endothelial
cells, including but not limited to intestinal adhesions. Crohn's
disease, atherosclerosis, scleroderma, and hypertrophic scars,
i.e., keloids; diseases that have angiogenesis as a pathologic
consequence such as cat scratch disease (Rochele ninalia quiritosa)
and ulcers (Helicobacter pylori). In addition, the angiogenesis
inhibitor compounds of the present invention are useful as birth
control agents (by virtue of their ability to inhibit the
angiogenesis dependent ovulation and establishment of the placenta)
and may also be used to reduce bleeding by administration to a
subject prior to surgery.
[0138] The following abbreviations are used herein and have the
indicated definitions: EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), HOBt
(N-hydroxybenzotriazole hydrate), CH.sub.2Cl.sub.2
(dichloromethane), MeCN (acetonitrile), MeOH (methanol), THF
(tetrahydrofuran), DMAP (dimethylamino pyridine), EtOAc (ethyl
acetate), PHE-CA (poly(1-hydroxymethylethylene
hydroxymethyl-formal) conjugated to glutaric acid), DMF (dimethyl
formamide), DME (dimethyl ether), LCMS (liquid chromatography-mass
spectrometry).
[0139] The Fumagillin Analog Conjugates of Formulas I, II, and IV
and the Fumagillin Analogs of Formula V.
[0140] In one aspect, biocompatible biodegradable conjugates are
provided which comprise at least one fumagillin analog conjugated
to a water soluble polyal, the conjugate having the structure of
Formula I:
[fumagillin analog].sub.m-polyal (I)
[0141] wherein fumagillin analog is any fumagillin core structure
which demonstrates MetAP-2 inhibition;
[0142] m is an integer from 1 to 40; and
[0143] polyal is any polymer having at least one acetal oxygen atom
or ketal oxygen atom in each monomer unit positioned within the
main chain of the polymer.
[0144] Non-limiting examples of compounds capable of inhibiting
MetAP-2 in removing NH.sub.2-terminal methionines from proteins are
represented by Formula IIA:
##STR00024##
[0145] wherein
[0146] X is O, S(.dbd.O).sub.q, optionally substituted CH.sub.2, or
optionally substituted NH;
[0147] R is (2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane,
2-methylhepta-2,5-diene, (2R,3S)-2-isopentyl-3-methyloxirane,
6-methylhept-2-ene, (Z)-acetaldehyde O-benzyl oxime,
C.sub.2-heterocyclic-C.sub.1-C.sub.6 alkyl,
C.sub.2-heterocyclic-C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-heterocyclic-C.sub.1-C.sub.6-alkenyl-COO--C.sub.1-C.sub.6
alkyl, C.sub.2-heterocyclic-C.sub.1-C.sub.6
alkyl-COO--C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6
alkyl=N--O--C.sub.1-C.sub.6 alkyl-aryl, C(O)C.sub.1-C.sub.6 alkyl,
CN, or halogen;
[0148] M is O, or
##STR00025##
[0149] q is 0, 1, or 2; and
[0150] the fumagillin core structure can be optionally substituted
at C-7 and C-8, independently, with C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, OH, ketone, or
alkoxy.
[0151] In some embodiments, the fumagillin analog of the conjugate
has the core structure
##STR00026##
[0152] wherein C-7 and C-8 are optionally substituted;
[0153] X is O, S(.dbd.O).sub.q, optionally substituted NH, or
optionally substituted CH.sub.2;
[0154] M is O, or
##STR00027##
[0155] q is 0, 1, or 2; and
[0156] the fumagillin analog is directly or indirectly attached to
the polyal through X.
[0157] In other embodiments, the fumagillin analog of the conjugate
has the core structure B:
##STR00028##
[0158] wherein C-7 and C-8 are optionally substituted;
[0159] X is O, S(.dbd.O).sub.q, optionally substituted NH, or
optionally substituted CH.sub.2;
[0160] M is O, or
##STR00029##
[0161] q is 0, 1, or 2; and
[0162] the fumagillin analog is directly or indirectly attached to
the polyal through X.
[0163] In still other embodiments, the fumagillin analog of the
conjugate has the core structure C:
##STR00030##
[0164] wherein C-7 and C-8 are optionally substituted;
[0165] X is O, S(.dbd.O).sub.q, optionally substituted NH, or
optionally substituted CH.sub.2;
[0166] M is O, or
##STR00031##
[0167] q is 0, 1, or 2; and
[0168] the fumagillin analog is directly or indirectly attached to
the polyal through X.
[0169] In some embodiments, the polyal has the structure
##STR00032## [0170] wherein for each occurrence of the n bracketed
structure, either
[0171] one of R.sub.1 and R.sub.2 is hydrogen, and the other is a
biocompatible group and includes a carbon atom covalently attached
to C.sub.1 or
[0172] each occurrence of R.sub.1 and R.sub.2 is a biocompatible
group and includes a carbon atom covalently attached to
C.sub.1;
[0173] R.sup.x includes a carbon atom covalently attached to
C.sub.2;
[0174] n is an integer;
[0175] each occurrence of R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is
a biocompatible group and is independently hydrogen or an organic
moiety; and
[0176] for each occurrence of the bracketed structure n, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6
comprises a functional group suitable for coupling directly or
indirectly with the fumagillin analog.
[0177] In another aspect, biocompatible biodegradable conjugates
are provided which comprise at least one fumagillin analog of the
formula II conjugated to a water soluble polyal
##STR00033##
[0178] wherein
[0179] X is O, S(O).sub.q, optionally substituted CH.sub.2, or
optionally substituted NH;
[0180] R is (2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane,
2-methylhepta-2,5-diene, (2R,3S)-2-isopentyl-3-methyloxirane,
6-methylhept-2-ene, (Z)-acetaldehyde O-benzyl oxime,
C.sub.2-heterocyclic-C.sub.1-C.sub.6 alkyl,
C.sub.2-heterocyclic-C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-heterocyclic-C.sub.1-C.sub.6-alkenyl-COO--C.sub.1-C.sub.6
alkyl, C.sub.2-heterocyclic-C.sub.1-C.sub.6
alkyl-COO--C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6
alkylN-O--C.sub.1-C.sub.6 alkyl-aryl, C(O)C.sub.1-C.sub.6 alkyl,
CN, or halogen;
[0181] M is O, or
##STR00034##
[0182] q is 0, 1, or 2;
[0183] wherein the fumagillin core structure can be optionally
substituted at C-7 and C-8, independently, with C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, OH,
ketone, or alkoxy;
[0184] wherein the fumagillin analog is conjugated by covalent
attachment of X to a free hydroxyl of the polyal through a spacer
moiety of the Formula III
##STR00035##
[0185] wherein Tether is an organic moiety with a molecular weight
between about null and about 1000 covalently attached to both the X
of Formula II and to Linker;
[0186] wherein Linker is an organic moiety covalently attached to
both Tether and to a free hydroxyl of the polyal, having a
molecular weight between about null and about 1000; and
[0187] wherein the spacer moiety of Formula III comprises one or
more labile bonds capable of enzymatic or chemical cleavage, so as
to provide a fumagillin analog conjugate having either a higher
water solubility than, a longer biological half-life than, or less
neurotoxicity than, the unconjugated fumagillin analog.
[0188] Without being limited by any theory, the chemical
composition of Linker can allow the fumagillin analog to be
released from the polyal either through chemical or enzymatic
cleavage.
[0189] In some embodiments, R is
(2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane.
[0190] In some embodiments, R is 2-methylhepta-2,5-diene.
[0191] In other embodiments, R is
(2R,3S)-2-isopentyl-3-methyloxirane.
[0192] In still other embodiments, R is (Z)-acetaldehyde O-benzyl
oxime.
[0193] In some embodiments, Linker is an oxalic, malonic, succinic,
glutaric, adipic, pimelic, suberic, azelaic, sebacic, phthalic,
isophthalic, terephthalic, diglycolic acid, tartaric, glutamic,
fumaric, or aspartic moiety, including amide, imide, or
cyclic-imide derivatives of each thereof, and each optionally
substituted.
[0194] In some embodiments, Linker is
##STR00036##
[0195] wherein
[0196] L is a bond, optionally substituted --C.sub.2--, --CH(OH)--,
optionally substituted --NH--, --O--, --S--, --SO--, --SO.sub.2--,
--C(CH.sub.3).sub.2, --CHO--, or --COCH.sub.2--;
[0197] the dashed line between the carbon atoms at positions a and
h represents a carbon-carbon single bond or a carbon-carbon double
bond; and
[0198] the methylene units adjacent to L can be optionally
substituted.
[0199] In some embodiments, L is --CH.sub.2--.
[0200] In other embodiments, L is --O--.
[0201] In other embodiments, the polyal has the structure:
##STR00037## [0202] wherein for each occurrence of the n bracketed
structure, either
[0203] one of R.sub.1 and R.sub.2 is hydrogen, and the other is a
biocompatible group and includes a carbon atom covalently attached
to C.sub.1 or
[0204] each occurrence of R.sub.1 and R.sub.2 is a biocompatible
group and includes a carbon atom covalently attached to
C.sub.1;
[0205] R.sub.x includes a carbon atom covalently attached to
C.sub.2;
[0206] n is an integer;
[0207] each occurrence of R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is
a biocompatible group and is independently hydrogen or an organic
moiety; and
[0208] for each occurrence of the bracketed structure n, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6
comprises a functional group suitable for coupling with Linker.
[0209] In another embodiment, the polyal is
poly(1-hydroxymethylethylene hydroxymethyl-formal) (hereinafter
abbreviated "PHF").
[0210] In another embodiment, the PHF has a molecular weight from
about 40 kDa to about 100 kDa.
[0211] In another embodiment, PHF has a molecular weight from about
45 kDa to about 95 kDa.
[0212] In another embodiment, PHF has a molecular weight from about
50 kDa to about 90 kDa,
[0213] In another embodiment, PHF has a molecular weight from about
55 kDa to about 85 kDa.
[0214] In another embodiment, PHF has a molecular weight from about
60 kDa to about 80 kDa.
[0215] In another embodiment, PHF has a molecular weight from about
65 kDa to about 75 kDa.
[0216] In another embodiment, PHF has a molecular weight from about
68 kDa to about 72 kDa.
[0217] In another embodiment, PHF has a molecular weight of about
70 kDa.
[0218] In another aspect, biocompatible, biodegradable conjugates
of the Formula IV are provided:
##STR00038##
[0219] wherein R, X, W, Q, and Y are as defined above;
[0220] Z is a polyal with the structure:
##STR00039##
[0221] wherein for each occurrence of the n bracketed structure,
one of R.sub.1 and R.sub.2 is hydrogen, and the other is a
biocompatible group and includes a carbon atom covalently attached
to C.sub.1;
[0222] R.sub.x includes a carbon atom covalently attached to
C.sub.2;
[0223] n is an integer from 1 to 3000; each occurrence of R.sub.3,
R.sub.4, R.sub.5, and R.sub.6 is a biocompatible group and is
independently hydrogen or a fumagillin analog; and
[0224] for each occurrence of the bracketed structure n, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.5, and R.sub.6 comprises a
functional group suitable for coupling with W, Y, or Q.
[0225] In one embodiment, Z is PHF.
[0226] In one embodiment, the PHF has a molecular weight of about
70 kDa.
[0227] In one embodiment, X is O.
[0228] In one embodiment, X is NH.
[0229] In one embodiment, Y is C(O)CH.sub.2CH.sub.2(O)C--.
[0230] In some embodiments, Y is
C(O)CH.sub.2CH.sub.2OCH.sub.2CH.sub.2(O)C--.
[0231] In some embodiments, Y is
C(O)CH.sub.2CH.sub.2CH.sub.2(O)C--.
[0232] In some embodiments, R is
(2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane.
[0233] In some embodiments, R is 2-methylhepta-2,5-diene.
[0234] In other embodiments, R is
(2R,3S)-2-isopentyl-3-methyloxirane.
[0235] In still other embodiments, R is (Z)-acetaldehyde O-benzyl
oxime.
[0236] In some embodiments, Q is --NH--.
[0237] In some embodiments, Q is amino acid.
[0238] In some embodiments, Q is NH-amino acid.
[0239] In some embodiments, W is --C(O)NH--C.sub.1-C.sub.6
alkyl-aryl-.
[0240] In some embodiments, W is --C(O)--NH--C(O)--C.sub.0-C.sub.6
alkyl-5-aryl-.
[0241] In some embodiments, W is
C(O)-heterocycloalkyl-C.sub.1-C.sub.6 alkyl-O--.
[0242] In another embodiment,
##STR00040##
is selected from the group consisting of
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047## ##STR00048## ##STR00049##
##STR00050##
[0243] Illustrative Conjugates of Formula IV are listed below:
TABLE-US-00001 Conjugate No. Z ##STR00051## 1 PHF ##STR00052## 2
PHF ##STR00053## 3 PHF ##STR00054## 4 PHF ##STR00055## 5 PHF
##STR00056## 6 PHF ##STR00057## 7 PHF ##STR00058## 8 PHF
##STR00059## 9 PHF ##STR00060## 10 PHF ##STR00061## 11 PHF
##STR00062## 12 PHF ##STR00063## 13 PHF ##STR00064## 14 PHF
##STR00065## 15 PHF ##STR00066## 16 PHF ##STR00067## 17 PHF
##STR00068## 18 PHF ##STR00069## 19 PHF ##STR00070## 20 PHF
##STR00071## 21 PHF ##STR00072## 22 PHF ##STR00073## 23 PHF
##STR00074## 24 PHF ##STR00075## 25 PHF ##STR00076## 26 PHF
##STR00077## 27 PHF ##STR00078## 28 PHF ##STR00079## 29 PHF
##STR00080## 30 PHF ##STR00081## 31 PHF ##STR00082## 32 PHF
##STR00083## 33 PHF ##STR00084## 34 PHF ##STR00085## 35 PHF
##STR00086## 36 PHF ##STR00087## 37 PHF ##STR00088## 38 PHF
##STR00089## 39 PHF ##STR00090## 40 PHF ##STR00091## 41 PHF
##STR00092## 42 PHF ##STR00093## 43 PHF ##STR00094## 44 PHF
##STR00095##
[0244] In another aspect, compounds of the Formula V are
described:
##STR00096##
[0245] wherein,
[0246] X is O, S(.dbd.O).sub.q, optionally substituted CH.sub.2, or
optionally substituted NH;
[0247] q is 0, 1, or 2;
[0248] R is (2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane,
2-methylhepta-2,5-diene, (2R,3S)-2-isopentyl-3-methyloxirane,
6-methylhept-2-ene, (Z)-acetaldehyde O-benzyl oxime,
C.sub.2-heterocyclic-C.sub.1-C.sub.6 alkyl,
C.sub.2-heterocyclic-C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-heterocyclic-C.sub.1-C.sub.6-alkenyl-COO--C.sub.1-C.sub.6
alkyl, C.sub.2-heterocyclic-C.sub.1-C.sub.6
alkyl-COO--C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6
alkyl=N--O--C.sub.1-C.sub.6 alkyl-aryl, C(O)C.sub.1-C.sub.6 alkyl,
CN, or halogen;
[0249] R.sub.8 is selected from the group consisting of VI, VII,
VIII, IX, X, XI, XII, and MIA whose formulas are represented
below:
##STR00097##
[0250] R' is --CO.sub.2H, optionally substituted --NH.sub.2, or --N
cyclic imide, NHC(O)(C.sub.1-C.sub.6 alkyl)-C(O)R'', R' is meta or
para in relation to the --S-- atom; and
[0251] R'' is --OH, --O--C.sub.1-C.sub.6 alkyl, or --NH.sub.2
optionally acylated through the carboxyl group of an amino
acid;
##STR00098##
[0252] wherein R.sub.9 is H or C(O)R.sub.11;
[0253] R.sub.10 is --NH.sub.2, --NHCH(C.sub.1-C.sub.6alkyl)-,
--NHC(O)(C.sub.1-C.sub.6 alkyl), N-cyclized imide; --NH acylated
through the carboxyl group of an amino acid, wherein the nitrogen
of the amino group of the amino acid is optionally protected,
and
[0254] R.sub.11 is OH, OC.sub.1-C.sub.6 alkyl, or optionally
substituted --NH.sub.2;
##STR00099##
[0255] wherein R.sub.12 is H, C.sub.1-C.sub.6 alkyl,
--(C.sub.1-C.sub.6)--COOH,
--(C.sub.1-C.sub.6)--C(O)O--(C.sub.1-C.sub.6),
--CH.sub.2CH.sub.2O--R.sub.13, --C(O)(C.sub.1-C.sub.6-alkyl), or an
amino acid attached through the carboxyl group of the amino
acid;
[0256] R.sub.13 is --H or an amino acid attached through the
carboxyl group of the amino acid, wherein the nitrogen of the amino
acid is optionally protected, or C(O)(C.sub.1-C.sub.6
alkyl)-COR'';
[0257] R'' is --OH, --OC.sub.1-C.sub.6 alkyl, or --NH.sub.2
optionally acylated through the carboxyl group of an amino
acid;
[0258] [-------] represents an optional methylene bridge
(--CH.sub.2--) between carbons 2 and 5 of the piperazine moiety;
and
[0259] Z' is a bond, --C.sub.1-C.sub.6 alkyl, --NHC(O)--, or
--NHSO.sub.2--;
##STR00100##
[0260] wherein R.sub.14 is --H, --CO.sub.2H,
--CO.sub.2(C.sub.1-C.sub.6 alkyl), --C(O)NH--C.sub.1-C.sub.6
alkyl-OH, wherein the O of --C(O)NH--C.sub.1-C.sub.6 alkyl-OH is
optionally acylated with the carboxyl group of an amino acid;
optionally substituted --NH.sub.2, C.sub.1-C.sub.6-alkyl-NH.sub.2,
wherein the NH.sub.2 is optionally substituted; and
[0261] [-------] represents an optional ethylene bridge
(--CH.sub.2CH.sub.2--) between carbons 1 and 4 of the cyclohexane
moiety;
##STR00101##
[0262] Z' is a bond, --CH.sub.2--, CH.sub.2--S--,
CH.sub.2CH.sub.2--, --C(H)(Me)-, NHCH.sub.2--, --NHCH(CH.sub.3)--,
--NHCH.sub.2CH.sub.2--; and
[0263] R.sub.15 is H, optionally substituted --NH.sub.2,
--NHC(O)(C.sub.1-C.sub.6-alkyl), --N cyclized imide optionally
containing a heteroatom within the cyclic structure,
--NHC(O)CH.sub.2OCH.sub.2C(O)OH, NHC(O)CH(C.sub.1-C.sub.6 alkyl)-N
cyclized imide, --NHC(O)CH(R'')NHC(O)--(C.sub.1-C.sub.6
alkyl)-C(O)OH, --NHC(O)--(C.sub.1-C.sub.6 alkyl)-C(O)OH,
--C(O)O(C.sub.1-C.sub.6 alkyl), --C(O)N(H)(C.sub.1-C.sub.6
alkyl)-OH, or NO.sub.2:
[0264] R'' is H, or --C.sub.1-C.sub.6 alkyl;
##STR00102##
[0265] wherein Y' is C.sub.1-C.sub.3 alkyl, or NH--C.sub.1-C.sub.3
alkyl and is attached to positions 1, 2, or 3 of the indole;
and
[0266] R.sub.16 is H, C.sub.1-C.sub.6 alkyl, --CH.sub.2COOH, or
--CH.sub.2CH.sub.2OH, wherein the 0 of --CH.sub.2CH.sub.2OH can be
optionally acylated with an amino acid;
##STR00103##
[0267] wherein R'' is --OH, --OC.sub.1-C.sub.6 alkyl, or --NH.sub.2
optionally acylated through the carboxyl group of an amino acid;
and
##STR00104##
[0268] wherein Y'' is C(O)N(CH.sub.3)(OCH.sub.3), C(O)OCH.sub.3,
CH.sub.2Cl, or NHC(O)CH.sub.2Cl.
[0269] In one embodiment, R in Formula V is
(2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane.
[0270] Illustrative examples of the Compounds of Formula V include
the following compounds selected from the group consisting of:
##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109##
##STR00110##
[0271] In another embodiment, illustrative compounds of Formula V
which are released by the fumagillin conjugates described herein
include the compounds selected from the group consisting of:
##STR00111## ##STR00112##
[0272] Methods for Using the Fumagillin Analog Conjugates
[0273] In another aspect, compositions comprising at least one
fumagillin analog conjugate or a pharmaceutically acceptable salt
of a fumagillin analog conjugate and a pharmaceutically acceptable
carrier are provided.
[0274] In another aspect, methods of treating an angiogenic
disease, comprising administering to a subject in need thereof a
fumagillin analog conjugate or a pharmaceutically acceptable salt
of a fumagillin analog conjugate in an amount effective to inhibit
angiogenesis is described.
[0275] In some embodiments, the angiogenic disease is selected from
the group consisting of cancer, retinal neovascularization due to
macular degeneration, psoriasis and pyogenic granuloma, rheumatoid,
immune, and degenerative arthritis.
[0276] In another aspect, methods of treating cancer, comprising
administering to a subject in need thereof a fumagillin analog
conjugate or a pharmaceutically acceptable salt of a fumagillin
analog conjugate in an amount effective to treat the cancer are
described.
[0277] In some embodiments, the cancer is selected from the group
consisting of anal, astrocytoma, leukemia, lymphoma, head and neck,
liver, testicular, cervical, sarcoma, hemangioma, esophageal, eye,
laryngeal, mouth, mesothelioma, skin, myeloma, oral, rectal,
throat, bladder, breast, uterus, ovary, prostate, lung, colon,
pancreas, renal, and gastric.
[0278] In another aspect, a method of treating cancer, comprising
co-administering to a subject in need thereof a fumagillin analog
conjugate or a pharmaceutically acceptable salt of a fumagillin
analog conjugate and a known anti-cancer agent, whereby the
conjugate and the agent act synergistically is described.
[0279] In another aspect, a method of treating cancer is described,
which comprises co-administering to a subject in need thereof a
fumagillin analog conjugate or a pharmaceutically acceptable salt
of a fumagillin analog conjugate and a known anti-cancer agent,
whereby the conjugate and the agent act additively is
described.
[0280] In some embodiments, the anti-cancer agent can be
administered first and the fumagillin analog conjugate second or
the fumagillin analog conjugate can be administered first and the
anti-cancer agent second.
[0281] In another aspect, a method of treating cancer, comprising
administering by metronomic dosing to a subject in need thereof a
fumagillin analog conjugate or a pharmaceutically acceptable salt
of a fumagillin analog conjugate is described. In other
embodiments, thalidomide, interferon-.alpha., interferon-0, or a
COX-2 inhibitor can also be administered.
[0282] In another aspect, a method of treating cancer, comprising
administering to a subject in need thereof a fumagillin analog
conjugate or a pharmaceutically acceptable salt of a fumagillin
analog conjugate, either of which deactivates an aminopeptidase is
described.
[0283] In one embodiment, the aminopeptidase is methionine
aminopeptidase type 2 (MetAP-2).
[0284] In another aspect, a method of reducing the central nervous
system effect or toxicity in vivo or in vitro of a fumagillin
analog is described which comprises conjugating said fumagillin
analog to a polyal, including, but not limited to, PHF prior to
administering the fumagillin analog to a subject.
[0285] In another aspect, a method of reducing the central nervous
system effect or toxicity in vivo or in vitro of a fumagillin
analog, as compared to TNP-470, comprising conjugating said
fumagillin analog to PHF prior to administering the fumagillin
analog to a subject is described.
[0286] Benefits of drug association with carrier macromolecules
relate, in part, to the following factors: (1) solubilization of
the drug substance; (2) restricted drug substance access to normal
interstitium due to the large hydrodynamic size of the conjugate,
(3) conjugate delivery to the tumor tissues via the Enhanced
Permeability and Retention (EPR) effect, and (4) maintenance of
sustained drug levels over periods exceeding cancer cell cycle.
[0287] Methods for Using the Fumagillin Analogs
[0288] In another aspect, compositions comprising at least one
fumagillin analog or a pharmaceutically acceptable salt thereof and
a pharmaceutically acceptable carrier are described.
[0289] In another aspect, methods of treating an angiogenic
disease, comprising administering to a subject in need thereof a
fumagillin analog or a pharmaceutically acceptable salt of a
fumagillin analog in an amount effective to inhibit angiogenesis is
described.
[0290] In some embodiments, the angiogenic disease is selected from
the group consisting of cancer, retinal neovascularization due to
macular degeneration, psoriasis and pyogenic granuloma, rheumatoid,
immune, and degenerative arthritis.
[0291] In another aspect, methods of treating cancer, comprising
administering to a subject in need thereof a fumagillin analog or a
pharmaceutically acceptable salt of a fumagillin analog in an
amount effective to treat the cancer are provided.
[0292] In some embodiments, the cancer is selected from the group
consisting of: anal, astrocytoma, leukemia, lymphoma, head and
neck, liver, testicular, cervical, sarcoma, hemangioma, esophageal,
eye, laryngeal, mouth, mesothelioma, skin, myeloma, oral, rectal,
throat, bladder, breast, uterus, ovary, prostate, lung, colon,
pancreas, renal, gastric, and brain.
[0293] In another aspect, a method of inhibiting angiogenesis,
comprising administering to a subject in need thereof a fumagillin
analog or a pharmaceutically acceptable salt of a fumagillin analog
in an amount effective to inhibit angiogenesis is described.
[0294] In another aspect, a method of treating cancer, comprising
co-administering to a subject in need thereof a fumagillin analog
or a pharmaceutically acceptable salt of a fumagillin analog and a
known anti-cancer agent, whereby the conjugate and the agent act
synergistically is described.
[0295] In another aspect, a method of treating cancer is described,
which comprises co-administering to a subject in need thereof a
fumagillin analog or a pharmaceutically acceptable salt of a
fumagillin analog and a known anti-cancer agent, whereby the
conjugate and the agent act additively is described.
[0296] In some embodiments, the anti-cancer agent can be
administered first and the fumagillin analog second or the
fumagillin analog can be administered first and the anti-cancer
agent second.
[0297] In another aspect, a method of treating cancer, comprising
administering by metronomic dosing to a subject in need thereof a
fumagillin analog or a pharmaceutically acceptable salt of a
fumagillin analog is described. In other embodiments, thalidomide,
interferon-.alpha., interferon-.beta., or a COX-2 inhibitor can
also be administered.
[0298] In another aspect, a method of treating cancer, comprising
administering to a subject in need thereof a fumagillin analog or a
pharmaceutically acceptable salt of a fumagillin analog, either of
which deactivates an aminopeptidase is described.
[0299] In one embodiment, the aminopeptidase is methionine
aminopeptidase type 2 (MetAP-2).
[0300] Therapeutic Administration of the Fumagillin Analog
Conjugates
[0301] When administered to a subject, the fumagillin analog
conjugates or pharmaceutically acceptable salts of the fumagillin
analog conjugates can be administered as a component of a
composition that comprises a physiologically acceptable carrier or
vehicle. The compositions described herein can be prepared using a
method comprising admixing the fumagillin analog conjugates or a
pharmaceutically acceptable salt of the fumagillin analog
conjugates and a physiologically acceptable carrier, excipient, or
diluent. Admixing can be accomplished using methods well known for
admixing a fumagillin analog conjugate or a pharmaceutically
acceptable salt of the fumagillin analog conjugate and a
physiologically acceptable carrier, excipients, or diluents.
[0302] The fumagillin analog conjugates or pharmaceutically
acceptable salts of fumagillin analog conjugates can be
administered by any convenient route, for example, by infusion or
bolus injection and can be administered together with another
therapeutic agent. Administration of the fumagillin analog
conjugate will result in release of a fumagillin analog into the
bloodstream.
[0303] In one embodiment, the fumagillin analog conjugate or a
pharmaceutically acceptable salt of the fumagillin analog conjugate
is administered intravenously.
[0304] The present compositions can optionally comprise a suitable
amount of physiologically acceptable excipients.
[0305] In variations of these embodiments, it may be desirable to
include other pharmaceutically active compounds, such as
anti-inflammatories or steroids which are used to reduce swelling,
antibiotics, antivirals, or antibodies. Other compounds which can
be included are preservatives, antioxidants, and fillers, coatings
or bulking agents which may also be utilized to alter polymer
matrix stability and/or drug release rates.
[0306] Buffers, acids and bases are used to adjust the pH of the
composition.
[0307] Fillers are water soluble or insoluble materials
incorporated into the formulation to add bulk. Types of fillers
include sugars, starches and celluloses. The amount of filler in
the formulation will typically be in the range of between about and
about 90% by weight.
[0308] Such physiologically acceptable excipients can be liquids,
such as water and oils, including those of petroleum, animal,
vegetable, or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. The physiologically
acceptable excipients can be saline, gum acacia, gelatin, starch
paste, talc, keratin, colloidal silica, urea and the like. In
addition, auxiliary, stabilizing, thickening, lubricating, and
coloring agents can be used. In one embodiment, the physiologically
acceptable excipients are sterile when administered to a subject.
The physiologically acceptable excipient should be stable under the
conditions of manufacture and storage and should be preserved
against the contaminating action of microorganisms. Water is a
particularly useful excipient when the fumagillin analog conjugate
or a pharmaceutically acceptable salt of the fumagillin analog
conjugate is administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid excipients, particularly for injectable solutions. Suitable
physiologically acceptable excipients also include bulking agents
like sorbitol and mannitol and surfactants like polysorbates,
hydroxypropyl-.beta.-cyclodextrin, and poloxamer. The present
compositions, if desired, can also contain minor amounts of pH
buffering agents.
[0309] Liquid carriers may be used in preparing solutions. The
fumagillin analog conjugate or pharmaceutically acceptable salt of
the fumagillin analog conjugate can be dissolved or suspended in a
pharmaceutically acceptable liquid carrier such as water, an
organic solvent, a mixture of both, or pharmaceutically acceptable
oils or fat. The liquid carrier can contain other suitable
pharmaceutical additives including solubilizers, emulsifiers,
buffers, preservatives, colors, viscosity regulators, stabilizers,
or osmo-regulators.
[0310] The present compositions can take the form of solutions for
injection, or any other form suitable for use. Other examples of
suitable physiologically acceptable excipients are described in
Remington's Pharmaceutical Sciences pp. 1447-1676 (Alfonso R.
Gennaro, Ed., 19th ed. 1995).
[0311] In another embodiment, the fumagillin analog conjugate or a
pharmaceutically acceptable salt of the fumagillin analog conjugate
can be formulated for intravenous administration. Typically,
compositions for intravenous administration comprise sterile
isotonic aqueous buffer. Where necessary, the compositions can also
include a solubilizing agent. Compositions for intravenous
administration can optionally include a local anesthetic such as
lignocaine to lessen pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or
water-free concentrate in a hermetically sealed container such as
an ampule or sachette indicating the quantity of active agent.
Where the fumagillin analog conjugate or a pharmaceutically
acceptable salt of the fumagillin analog conjugate is to be
administered by infusion, it can be dispensed, for example, with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the fumagillin analog conjugate or a pharmaceutically
acceptable salt of the fumagillin analog conjugate is administered
by injection, an ampule of sterile water for injection or saline
can be provided so that the ingredients can be mixed prior to
administration.
[0312] The amount of the fumagillin analog conjugate or a
pharmaceutically acceptable salt of the fumagillin analog conjugate
that is effective for treating or preventing cancer, or inhibiting
angiogenesis can be determined using standard clinical techniques.
In addition, in vitro or in vivo assays can optionally be employed
to help identify optimal dosage ranges. The precise dose to be
employed can also depend on the route of administration, the
condition, the seriousness of the condition being treated, as well
as various physical factors related to the individual being
treated, and can be decided according to the judgment of a
health-care practitioner. The typical dose will range from about
0.001 mg/kg to about 250 mg/kg of body weight per day, in one
embodiment, from about 1 mg/kg to about 250 mg/kg body weight per
day, in another embodiment, from about 1 mg/kg to about 50 mg/kg
body weight per day, and in another embodiment, from about 1 mg/kg
to about 20 mg/kg of body weight per day. Dosages are based on the
amount of the equivalents of fumagillin analog present on the
conjugate. Equivalent dosages may be administered over various time
periods including, but not limited to, about every 2 hours, about
every 6 hours, about every 8 hours, about every 12 hours, about
every 24 hours, about every 36 hours, about every 48 hours, about
every 72 hours, about every week, about every two weeks, about
every three weeks, about every month, and about every two months.
The number and frequency of dosages corresponding to a completed
course of therapy can be determined according to the judgment of a
health-care practitioner. The effective dosage amounts described
herein refer to total amounts administered; that is, if more than
one fumagillin analog conjugate or more than one pharmaceutically
acceptable salt of the fumagillin analog conjugate is administered,
the effective dosage amounts correspond to the total amount
administered.
[0313] Effective amounts of the other prophylactic or therapeutic
agents are well known to those skilled in the art. However, it is
well within the skilled artisan's purview to determine the other
prophylactic or therapeutic agent's optimal effective amount range.
In one embodiment, where another prophylactic or therapeutic agent
is administered to a subject, the effective amount of the
fumagillin conjugate is less than its effective amount would be if
the other prophylactic or therapeutic agent is not administered. In
this case, without being bound by theory, it is believed that
fumagillin analog conjugate and the other prophylactic or
therapeutic agent act in an additive or synergistic way to treat
cancer, inflammatory diseases, or inhibit angiogenesis.
[0314] The present methods for treating cancer, or inhibiting
angiogenesis, can further comprise administering another
therapeutic agent to the subject being administered the fumagillin
analog conjugate. In one embodiment, the other therapeutic agent is
administered in an effective amount.
[0315] Suitable other therapeutic agents useful in the methods and
compositions include, but are not limited to, anti-cancer agents,
anti-angiogenesis agents, anti-inflammatory agents, and irritable
bowel syndrome agents.
[0316] Suitable anti-cancer agents useful in the methods and
compositions include, but are not limited to, temozolomide, a
topoisomerase I inhibitor, procarbazine, dacarbazine, gemcitabine,
capecitabine, methotrexate, taxol, taxotere, mercaptopurine,
thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide,
nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine,
procarbizine, etoposide, teniposide, camptothecins, bleomycin,
doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin,
mitoxantrone, L-asparaginase, epirubicin, 5-fluorouracil, taxanes
such as docetaxel and paclitaxel, leucovorin, levamisole,
irinotecan, estramustine, etoposide, nitrogen mustards,
1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), nitrosoureas such as
carmustine and lomustine, vinca alkaloids such as vinblastine,
vincristine and vinorelbine, platinum complexes such as cisplatin,
carboplatin and oxaliplatin, imatinib mesylate, hexamethylmelamine,
topotecan, tyrosine kinase inhibitors, tyrphostins, herbimycin A,
genistein, erbstatin, and lavendustin A.
[0317] Other therapeutic agents useful in the methods and
compositions include, but are not limited to, hydroxyzine,
glatiramer acetate, interferon .beta.-1a, interferon .beta.-1b, and
natalizumab.
[0318] Anti-angiogenesis agents are also therapeutic agents useful
in the methods and compositions described herein. Non-limiting
examples of anti-angiogenesis agents include bevacizumab
(Avastin.RTM.), sunitinib (Sutent.RTM.), sorafenib (Nexavar.RTM.),
thalidomide (Thalomid.RTM.), lenalidomide (Revlimid.RTM.),
panitumumab (Vectibix.RTM.), cetuximab (Erbitux.RTM.), and
erlotinib (Tarceva.RTM.).
[0319] In one embodiment, the fumagillin analog conjugate or a
pharmaceutically acceptable salt of the fumagillin analog conjugate
is administered concurrently with another therapeutic agent.
[0320] In one embodiment, a composition comprising an effective
amount of the fumagillin analog conjugate or a pharmaceutically
acceptable salt of the fumagillin analog conjugate and an effective
amount of another therapeutic agent within the same composition can
be administered.
[0321] In another embodiment, a composition comprising art
effective amount of the fumagillin analog conjugate or a
pharmaceutically acceptable salt of the fumagillin analog conjugate
and a separate composition comprising an effective amount of
another therapeutic agent can be concurrently administered.
[0322] In another embodiment, an effective amount of the fumagillin
analog conjugate or a pharmaceutically acceptable salt of the
fumagillin analog conjugate is administered prior to or subsequent
to administration of an effective amount of another therapeutic
agent. In this embodiment, the fumagillin analog conjugate or a
pharmaceutically acceptable salt of the fumagillin analog conjugate
is administered while the other therapeutic agent exerts its
therapeutic effect, or the other therapeutic agent is administered
while the fumagillin analog conjugate or a pharmaceutically
acceptable salt of the fumagillin analog conjugate exerts its
preventative or therapeutic effect in treating cancer, or
inhibiting angiogenesis.
[0323] Therapeutic Administration of the Fumagillin Analogs
[0324] When administered to a subject, the fumagillin analog or
pharmaceutically acceptable salt of the fumagillin analog can be
administered as a component of a composition that comprises a
physiologically acceptable carrier or vehicle. A composition can be
prepared using a method comprising admixing the fumagillin analog
or a pharmaceutically acceptable salt of the fumagillin analog and
a physiologically acceptable carrier, excipient, or diluent.
[0325] Methods of administration of the analogs themselves include,
but are not limited to, intradermal, intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal, epidural,
oral, sublingual, intracerebral, intravaginal, transdermal, rectal,
by inhalation, or topical, particularly to the ears, nose, eyes, or
skin.
[0326] The fumagillin analog or pharmaceutically acceptable salt of
fumagillin analog can be administered by any other convenient
route, for example, by infusion or bolus injection and can be
administered together with another therapeutic agent,
Administration can be systemic or local. Various known delivery
systems, including encapsulation in Liposomes, microparticles,
microcapsules, and capsules, can be used.
[0327] In one embodiment, the fumagillin analog or a
pharmaceutically acceptable salt of the fumagillin analog is
administered orally.
[0328] In one embodiment, the fumagillin analog or a
pharmaceutically acceptable salt of the fumagillin analog is
administered intravenously.
[0329] In another embodiment, the fumagillin analog or a
pharmaceutically acceptable salt of the fumagillin analog can be
administered locally. This can be achieved, for example, by local
infusion during surgery, topical application, e.g., in conjunction
with a wound dressing after surgery, by injection, by means of a
catheter, by means of a suppository or enema, or by means of an
implant, said implant being of a porous, non-porous, or gelatinous
material, or fibers.
[0330] In yet another embodiment, the fumagillin analog or a
pharmaceutically acceptable salt of the fumagillin analog can be
delivered in a controlled-release system or sustained-release
system (see, e.g., Goodson, in Medical Applications of Controlled
Release, vol, 2, pp. 115-138 (1984)). Other controlled or
sustained-release systems discussed in the review by Langer, R.,
(1990) Science, 249:1527-1533 can be used. In one embodiment, a
pump can be used (Langer, R., (1990) Science, 249:1527-1533;
Sefton, M., (1987) CRC Grit. Ref Biomed & Eng., 14: 201;
Buchwald H., et al., (1980) Surgery, 88: 507; and Saudek C., et
al., (1989) N. Engl. J. Med., 321: 574). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, (Langer and Wise eds., 1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, (Smolen and
Ball eds., 1984); Ranger and Peppas, (1983) J. Macromol Sci. Rev.
Macromol. Chem., 2: 61; Levy et al., (1985) Science, 228: 190;
During et al., (1989) Ann. Neural., 25: 351; and Howard et al.,
(1989) J. Neurosurg., 71: 105).
[0331] The present compositions can optionally comprise a suitable
amount of physiologically acceptable excipients.
[0332] In variations of these embodiments, it may be desirable to
include other pharmaceutically active compounds, such as
antiinflammatories or steroids which are used to reduce swelling,
antibiotics, antivirals, or antibodies. Other compounds which can
be included are preservatives, antioxidants, and fillers, coatings
or bulking agents which may also be utilized to alter polymer
matrix stability and/or drug release rates.
[0333] Fillers are water soluble or insoluble materials
incorporated into the formulation to add bulk. Types of fillers
include sugars, starches and celluloses. The amount of filler in
the formulation will typically be in the range of between about 1
and about 90% by weight.
[0334] Such physiologically acceptable excipients can be liquids,
such as water and oils, including those of petroleum, animal,
vegetable, or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. The physiologically
acceptable excipients can be saline, gum acacia, gelatin, starch
paste, talc, keratin, colloidal silica, urea and the like. In
addition, auxiliary, stabilizing, thickening, lubricating, and
coloring agents can be used. In one embodiment, the physiologically
acceptable excipients are sterile when administered to a subject.
The physiologically acceptable excipient should be stable under the
conditions of manufacture and storage and should be preserved
against the contaminating action of microorganisms. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid excipients, particularly for injectable
solutions. Suitable physiologically acceptable excipients also
include bulking agents like sorbitol and mannitol and surfactants
like polysorbates, hydroxypropyl-.beta.-cyclodextrin, and
poloxamer. The present compositions, if desired, can also contain
minor amounts of pH buffering agents.
[0335] Liquid carriers may be used in preparing solutions,
suspensions, emulsions, syrups, and elixirs. The fumagillin analogs
or pharmaceutically acceptable salts of the fumagillin analogs can
be dissolved or suspended in a pharmaceutically acceptable liquid
carrier such as water, an organic solvent, a mixture of both, or
pharmaceutically acceptable oils or fat. The liquid carrier can
contain other suitable pharmaceutical additives including
solubilizers, emulsifiers, buffers, preservatives, sweeteners,
flavoring agents, suspending agents, thickening agents, colors,
viscosity regulators, stabilizers, or osmo-regulators. Suitable
examples of liquid carriers for oral and parenteral administration
include water (particular containing additives as above, e.g.,
cellulose derivatives, including sodium carboxymethyl cellulose
solution), alcohols (including monohydric alcohols and polyhydric
alcohols, e.g., glycols) and their derivatives, and oils (e.g.,
fractionated coconut oil and arachis oil). For parenteral
administration the carrier can also be an oily ester such as ethyl
oleate and isopropyl myristate. Sterile liquid carriers are used in
sterile liquid form compositions for parenteral administration. The
liquid carrier for pressurized compositions can be halogenated
hydrocarbon or other pharmaceutically acceptable propellant.
[0336] The present compositions can take the form of solutions for
injection, or any other form suitable for use. Other examples of
suitable physiologically acceptable excipients are described in
Remington's Pharmaceutical Sciences pp. 1447-1676 (Alfonso R.
Gennaro, ed., 19th ed. 1995).
[0337] Surface modifying agents include nonionic and anionic
surface modifying agents. Representative examples of surface
modifying agents include, but are not limited to, poloxamer 188,
benzalkonium chloride, calcium stearate, cetostearyl alcohol,
cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon
dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum
silicate, and triethanolamine.
[0338] In another embodiment, the fumagillin analog or a
pharmaceutically acceptable salt of the fumagillin analog can be
formulated for intravenous administration. Typically, compositions
for intravenous administration comprise sterile isotonic aqueous
buffer. Where necessary, the compositions can also include a
solubilizing agent. Compositions for intravenous administration can
optionally include a local anesthetic such as lignocaine to lessen
pain at the site of the injection. Generally, the ingredients are
supplied either separately or mixed together in unit dosage form,
for example, as a dry lyophilized powder or water-free concentrate
in a hermetically sealed container such as an ampule or sachette
indicating the quantity of active agent. Where the fumagillin
analog or a pharmaceutically acceptable salt of the fumagillin
analog is to be administered by infusion, it can be dispensed, for
example, with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the fumagillin analog or a
pharmaceutically acceptable salt of the fumagillin analog is
administered by injection, an ampule of sterile water for injection
or saline can be provided so that the ingredients can be mixed
prior to administration.
[0339] The amount of the fumagillin analog or a pharmaceutically
acceptable salt of the fumagillin analog that is effective for
treating or preventing treating cancer, or inhibiting angiogenesis
can be determined using standard clinical techniques. In addition,
in vitro or in vivo assays can optionally be employed to help
identify optimal dosage ranges. The precise dose to be employed can
also depend on the route of administration, the condition, the
seriousness of the condition being treated, as well as various
physical factors related to the individual being treated, and can
be decided according to the judgment of a health-care practitioner.
The typical dose will range from about 0.001 mg/kg to about 250
mg/kg of body weight per day, in one embodiment, from about 1 mg/kg
to about 250 mg/kg body weight per day, in another embodiment, from
about 1 mg/kg to about 50 mg/kg body weight per day, and in another
embodiment, from about 1 mg/kg to about 20 mg/kg of body weight per
day. Equivalent dosages may be administered over various time
periods including, but not limited to, about every 2 hours, about
every 6 hours, about every 8 hours, about every 12 hours, about
every 24 hours, about every 36 hours, about every 48 hours, about
every 72 hours, about every week, about every two weeks, about
every three weeks, about every month, and about every two months.
The number and frequency of dosages corresponding to a completed
course of therapy can be determined according to the judgment of a
health-care practitioner. The effective dosage amounts described
herein refer to total amounts administered; that is, if more than
one fumagillin analog or more than one pharmaceutically acceptable
salt of the fumagillin analog is administered, the effective dosage
amounts correspond to the total amount administered.
[0340] Effective amounts of the other prophylactic or therapeutic
agents are well known to those skilled in the art. However, it is
well within the skilled artisan's purview to determine the other
prophylactic or therapeutic agent's optimal effective amount range.
In one embodiment, when another prophylactic or therapeutic agent
is administered to a subject, the effective amount of the
fumagillin analog is less than its effective amount would be where
the other prophylactic or therapeutic agent is not administered. In
this case, without being bound by theory, it is believed that
fumagillin analog and the other prophylactic or therapeutic agent
act in an additive or synergistic way to treat cancer, inflammatory
diseases, or inhibit angiogenesis.
[0341] In one embodiment, the pharmaceutical composition comprising
a fumagillin analog is in unit dosage form, e.g., as a tablet,
capsule, powder, solution, suspension, emulsion, granule, or
suppository. In such form, the composition is sub-divided in unit
dose containing appropriate quantities of the active ingredient;
the unit dosage form can be packaged compositions, for example,
packeted powders, vials, ampoules, pre-filled syringes or sachets
containing liquids. The unit dosage form can be, for example, a
capsule or tablet itself, or it can be the appropriate number of
any such compositions in package form. Such unit dosage form may
contain from about 1 mg/kg to about 250 mg/kg of fumagillin analog,
and may be given in a single dose or in two or more divided
doses.
[0342] The fumagillin analog or a pharmaceutically acceptable salt
of the fumagillin analog can be assayed in vitro or in vivo for the
desired therapeutic or prophylactic activity prior to use in
humans. Animal model systems can be used to demonstrate safety and
biological activity.
[0343] The present methods for treating cancer, or inhibiting
angiogenesis, can further comprise administering another
therapeutic agent to the subject being administered the fumagillin
analog. In one embodiment, the other therapeutic agent is
administered in an effective amount.
[0344] Suitable other therapeutic agents useful in the methods and
compositions described herein include, but are not limited to,
anti-cancer agents, anti-angiogenesis agents, anti-inflammatory
agents, and irritable bowel syndrome agents.
[0345] Suitable anti-cancer agents useful in the methods and
compositions described herein include, but are not limited to,
temozolomide, a topoisomerase I inhibitor, procarbazine,
dacarbazine, gemcitabine, capecitabine, methotrexate, taxol,
taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,
mitomycin, dacarbazine, procarbizine, etoposide, teniposide,
camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin,
dactinomycin, plicamycin, mitoxantrone, L-asparaginase, epirubicin,
5-fluorouracil, taxanes such as docetaxel and paclitaxel,
leucovorin, levamisole, irinotecan, estramustine, etoposide,
nitrogen mustards, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU),
nitrosoureas such as carmustine and lomustine, vinca alkaloids such
as vinblastine, vincristine and vinorelbine, platinum complexes
such as cisplatin, carboplatin and oxaliplatin, imatinib mesylate,
hexamethylmelamine, topotecan, tyrosine kinase inhibitors,
tyrphostins, herbimycin A, genistein, erbstatin, and lavendustin
A.
[0346] Other therapeutic agents useful in the methods and
compositions include, but are not limited to, hydroxyzine,
glatiramer acetate, interferon beta-1a, interferon beta-1b, and
natalizumab.
[0347] Suitable anti-angiogenesis agents useful in the methods and
compositions include, bevacizumab (Avastin.RTM.), sunitinib
(Sutent.RTM.), sorafenib (Nexavar.RTM.), thalidomide
(Thalomid.RTM.), and lenalidomide (Revlimid.RTM.), panitumumab,
erbitux, and erlotinib (Tarceva.RTM.).
[0348] In one embodiment, the fumagillin analog or a
pharmaceutically acceptable salt of the fumagillin analog is
administered concurrently with another therapeutic agent.
[0349] In one embodiment, a composition comprising an effective
amount of the fumagillin analog or a pharmaceutically acceptable
salt of the fumagillin analog and an effective amount of another
therapeutic agent within the same composition can be
administered.
[0350] In another embodiment, a composition comprising an effective
amount of the fumagillin analog or a pharmaceutically acceptable
salt of the fumagillin analog and a separate composition comprising
an effective amount of another therapeutic agent can be
concurrently administered.
[0351] In another embodiment, an effective amount of the fumagillin
analog or a pharmaceutically acceptable salt of the fumagillin
analog is administered prior to or subsequent to administration of
an effective amount of another therapeutic agent. In this
embodiment, the fumagillin analog or a pharmaceutically acceptable
salt of the fumagillin analog is administered while the other
therapeutic agent exerts its therapeutic effect, or the other
therapeutic agent is administered while the fumagillin analog or a
pharmaceutically acceptable salt of the fumagillin analog exerts
its preventative or therapeutic effect in treating cancer, or
inhibiting angiogenesis.
[0352] The fumagillin analog conjugates and pharmaceutically
acceptable salts of fumagillin analog conjugates can be prepared
using a variety of methods starting from commercially available
compounds, known compounds, or compounds prepared by known methods.
The fumagillin analogs and pharmaceutically acceptable salts of
fumagillin analogs can be prepared using a variety of methods
starting from commercially available compounds, known compounds, or
compounds prepared by known methods. General synthetic routes to
many of the compounds described are included in the following
schemes. It is understood by those skilled in the art that
protection and deprotection steps not shown in the Schemes may be
required for these syntheses, and that the order of steps may be
changed to accommodate functionality in the target molecule.
[0353] Methods useful for making the fumagillin analog conjugates
and the fumagillin analogs are set forth in the Examples below and
generalized in the following schemes,
##STR00113##
[0354] As set forth in Scheme 1, fumagillol can be produced by
suspending fumagillin dicyclohexylammonium salt in Et.sub.2O and
stirring in 0.5 N aqueous solution of NaOH. After standard workup
and column purification fumagillol is isolated in about 70%
yield.
##STR00114##
[0355] wherein R is as defined above for Formula II.
[0356] As set forth in Scheme 2, a compound of formula XIII can be
reacted with p-nitrophenyl chloroformate in an organic solvent such
as methylene chloride with a base such as pyridine at about
0.degree. C. and allowed to stir. After quenching the reaction,
standard workup gives a compound of the formula XIV. Alternatively,
substituting p-nitrophenyl chloroformate with carbonyl diimidazole
gives a compound of the formula XV.
##STR00115##
[0357] wherein R is as defined above for Formula II.
[0358] As set forth in Scheme 3, a compound of the formula XIV can
be reacted with an amine such as ethyl 3-(piperazin-1-yl)
propanoate at room temperature for 12 hours. Standard extraction
and purification of the reaction mixture gives a compound of the
formula XVI.
##STR00116##
[0359] wherein R is as defined above for Formula
[0360] As set forth in Scheme 4, a compound of the formula XV can
be reacted with a primary amine such as 4-(aminomethyl)aniline at
65.degree. C. in an aprotic organic solvent such as acetonitrile to
give a compound of the formula XVII.
##STR00117##
[0361] wherein p=m, and m and R are as defined above for Formula I
and II, respectively.
[0362] As set forth in Scheme 5 conjugates can be synthesized by
dissolving PHF-GA (made according to U.S. 2007/0190018) with a
compound of the formula XVII in a solvent mixture of an organic
solvent such as DMF or acetonitrile and water. The pH is adjusted
to between 5.9 and 6.0 and the reaction mixture cooled to 0.degree.
C. Afterwards 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)
is added, and the mixture stirred for about 2.5 to 4 hours. The
reaction mixture is acidified to pH 5 with 1.0 N HCl and the
mixture filtered through a 0.2 n membrane and purified by size
exclusion chromatography to give, for example, Conjugate 12 when R
is (2S,3R)-2-methyl-3-(3-methylbut-2-enyl) oxirane.
##STR00118##
[0363] wherein R is as defined above for Formula II.
[0364] As set forth in Scheme 6, a compound of the formula XVII-A
can be coupled to an organic acid, such as an amino acid, by
dissolving it together with an organic acid, such as Fmoc protected
L-alanine, and EDC in an aprotic organic solvent such as methylene
chloride. Subsequently, the Fmoc protecting group can be removed by
reaction with piperidine in an aprotic solvent such as
acetonitrile. Extraction and chromatographic purification gives a
compound of the formula XVIII.
##STR00119##
[0365] wherein p=m, and m and R are as defined above for Formula I
and II, respectively.
[0366] As set forth in Scheme 7, an alternative method for coupling
a fumagillin analog to a polyal involves dissolving a compound of
the formula XVI-A in THF and cooling the reaction to 0.degree. C. A
base such as triethylamine and isobutyl chloroformate are added and
the mixture stirred. In a second vessel PHF is dissolved in
anhydrous pyridine. Afterwards, the contents of both vessels are
combined and allowed to stir and warm to room temperature over 12
hours. The mixture is filtered, as described above, and purified by
size exclusion, giving for example Conjugate 4 when R is
(2S,3R)-2-methyl-3-(3-methylbut-2-enyl)oxirane.
##STR00120##
[0367] wherein R is as defined above for Formula II.
[0368] As set forth in Scheme 8, fumagillone and its C-4 analogs
can be produced by reacting fumagillol and its C-4 analogs with an
oxidizing agent such as Chromium (VI) oxide in pyridine with
stirring overnight at room temperature. Standard workup by
extraction and chromatography on silica gel provides fumagillone
and its C-4 analogs.
##STR00121##
[0369] wherein R is as defined above for Formula II.
[0370] As set forth in Scheme 9, fumagillamine and its C-4 analogs
can be produced by reacting a fumagillone analog in anhydrous MeOH
with ammonium acetate and sodium cyanoborohydride.
##STR00122##
[0371] wherein R is as defined above for Formula II.
[0372] As set forth in Scheme 10, a compound of formula XIX can be
reacted with p-nitrophenyl chloroformate in an organic solvent such
as methylene chloride with a base such as triethylamine at
0.degree. C. and allowed to stir and warm to room temperature.
Afterwards a solution of 2-(4-aminophenyl)ethylamine is added and
after stirring for a further 2 hours the reaction is quenched which
after workup gives a compound of the formula XX.
##STR00123##
[0373] wherein R is as defined above for Formula II.
[0374] As set forth in Scheme 11, a compound of the formula XXI can
be reacted with an anhydride, such as succinic anhydride, in the
presence of DMAP in CH.sub.2Cl.sub.2 to give a compound of the
formula XXII.
##STR00124##
[0375] wherein R is as defined above for Formula II.
[0376] As set forth in Scheme 12, the synthesis of a C-6 carbo
derivative of fumagillin can be accomplished starting with
fumagillone where the spiro-epoxide is protected as a chlorohydrin
XXIII (Bioorg. Med. Chem. Lett 2003, 11, 5051-5058). Subsequently,
a Homer-Wittig reaction (Journal of Chem. Soc. 1988, 17, 1184-1186)
gives a compound such as XXIV, followed by hydrolysis of the
resulting methyl enol ether (Synth. Commun. 2001, 31, 939-946) and
lastly, reformation of the spiro-epoxide with potassium
tert-butoxide (Bioorg. Med. Chem. Lett. 2004, 14, 91-94) will
afford a fumagillin analogue with a C-6 methylene such as a
compound of formula XXV.
##STR00125##
[0377] wherein R is as defined above for Formula II.
[0378] As set forth in Scheme 13, the C-6 thio analog XXVI can be
synthesized by reacting the C-6 keto analog with Lawesson's reagent
in dimethoxyethane (DME) followed by reduction using sodium
borohydride. Standard workup gives the C-6 thio analog of
fumagillin.
EXAMPLES
General
LCMS Methods
[0379] LCMS data was collected on an Agilent 1200 Series LC/MSD-SL
system equipped with 1100/1200 diode array detector and C18 Luna
2.5 .mu.m 100.times.3.0 mm column. Mobile Phase A: 0.1% formic acid
in water; Mobile Phase B: 0.1% formic acid in 98% aqueous MeCN. A 1
ml/min linear gradient of 10-90% mobile phase B with a column
temperature of 45.degree. C. was used for separation. Detection was
performed at 254 nm and 215 nm. Method A: gradient time 10 minutes,
Method B: gradient time 12 minutes, Method C: gradient time 15
minutes.
Example 1
Procedure for the Preparation of Fumagillol
##STR00126##
[0381] Fumagillin dicyclohexylammonium salt (15.0 g, 23.4 mmol) was
suspended in Et.sub.2O (300 ml) and vigorously stirred. To the
vigorously stirring suspension was added a 0.5 N aqueous solution
of NaOH (300 ml) over 15 min. The resulting biphasic reaction
mixture was stirred vigorously at 23.degree. C. for 1 h after which
it was diluted with Et.sub.2O (500 ml) and water (500 ml) and the
organic layer separated. The aqueous layer was washed with
Et.sub.2O (2.times.100 ml), and the combined organic layers were
washed with an aqueous saturated solution of NaCl, dried with
MgSO.sub.4, and concentrated in vacuo to yield an orange oil.
Purification through silica gel chromatography (120 g:
0.fwdarw.100%, EtOAc in hexanes, 35 min.) delivered fumagillol
(4.68 g, 16.6 mmol, 71%) as an orange oil or solid. LC/MS method A:
m/z 283 [M+H].sup.+, R.sub.t 9.7 min.
Example 2
Synthesis of Compound BB
##STR00127##
[0383] Carbonyl diimidazole (45.7 g, 282 mmol) was suspended in
CH.sub.2Cl.sub.2 (469 ml) and cooled to 0.degree. C. with vigorous
stirring. Using a pressure equalizing addition funnel, a solution
of fumagillol (26.5 g, 94.0 mmol) in CH.sub.2Cl.sub.2 (100 ml) was
added over 30 min. while maintaining a reaction temperature of
0.degree. C. The mixture was allowed to stir at 0.degree. C. for an
additional 2 h after which it was quenched through slow addition of
water (100 ml, 1 h) to the cooled reaction mixture, Careful
attention was paid to maintain the crude mixture at 0.degree. C.
during the exothermic process of water addition. The organic layer
was washed with water (3.times.100 ml) and the combined aqueous
layers were washed with CH.sub.2Cl.sub.2. The combined organic
layers were dried with MgSO.sub.4 and concentrated in vacuo to
yield compound AA (38.5 g, 102 mmol, >98%) as viscous brown oil
which was used in subsequent coupling reactions without
purification, LC/MS method B: m/z 377 [M+H].sup.+, R.sub.t 5.9
min.
##STR00128##
[0384] To a stirring solution of Compound AA (10.2 g, 27.2 mmol) in
MeCN (175 ml) was added 4-aminobenzylamine (3.32 g, 27.2 mmol) and
the solution heated to 65.degree. C. for 2 h. The solution was then
allowed to cool to 23.degree. C. and concentrated in vacuo to yield
a viscous orange oil. Purification through silica gel
chromatography (80 g: 0.fwdarw.100% EtOAc in hexanes, 30 min)
delivered BB as a white solid (7.50 g, 17.4 mmol, 64%). LC/MS
method B: m/z 453 [M+Na].sup.+, R.sub.t 4.75 min. .sup.1H NMR (400
MHz): .delta. 7.07 (d, 2H); 6.63 (d, 2H); 5.51 (br s, 1H, NH); 5.19
(dd, 1H); 5.01-4.99 (m, 1H); 4.27 (dd, 1H), 4.17 (dd, 1H), 4.14
(dd, 1H); 3.68 (br s, 2H, NH.sub.2); 3.64 (dd, 1H); 3.45 (s, 3H);
2.96 (d, 1H); 2.54 (d, 1H); 2.53 (d, 1H); 2.38-2.31 (m, 1H);
2.19-2.12 (m, 1H); 2.08-1.99 (m, 2H); 1.91 (d, 1H); 1.85-1.75 (m,
1H); 1.73 (s, 3H); 1.64 (s, 3H); 1.20 (s, 3H);
Example 3
Synthesis of Compound CC
##STR00129##
[0386] Fmoc-L-Ala (15.4 g, 49.5 mmol), EDC (9.49 g, 49.5 mmol), and
HOBt (7.58 g, 49.5 mmol) were taken up in CH.sub.2Cl.sub.2 (225 ml)
and stirred at 23.degree. C. To the suspension was added a solution
of compound BB (20.0 g, 45.0 mmol) in CH.sub.2Cl.sub.2 (50 ml). The
reaction mixture was allowed to stir at 23.degree. C. for 3 h after
which it was quenched through addition of an aqueous 10% solution
of citric acid (100 ml). The organic layer was separated and the
aqueous layer washed with CH.sub.2Cl.sub.2 (3.times.100 ml). The
combined organic layers were washed sequentially with an aqueous
10% solution of citric acid, an aqueous saturated solution of
NaHCO.sub.3, and an aqueous saturated solution of NaCl. The organic
layers were dried with MgSO.sub.4 and concentrated in vacuo to
yield a white solid. Purification through silica gel chromatography
(330 g: 0.fwdarw.75% EtOAc in hexanes, 45 min) delivered the
Fmoc-protected derivative of compound CC as a white solid (8.64 g,
11.7 mmol, 42%) LC/MS method B: m/z 760 [M+Na].sup.+, R.sub.t 10.0
mM.
[0387] The Fmoc-protected derivative of compound CC (8.50 g, 11.5
mmol was dissolved in MeCN (115 ml) and the solution was cooled to
0.degree. C. To the solution was added piperidine (11.4 ml, 115
mmol) and the reaction was allowed to stir at 0.degree. C. for 1 h.
Concentration of the reaction mixture in vacuo yielded a white
solid. Purification through silica gel chromatography (330 g:
0.fwdarw.20% MeOH (0.5% Et.sub.3N) in CH.sub.2Cl.sub.2 (0.5%
Et.sub.3N), 45 mM) delivered compound CC as white solid (2.73 g,
5.30 mmol, 46%). .sup.1H NMR (400 MHz): .delta. 9.43 (br s, 1H,
NH), 7.53 (d, 2H), 7.22 (d, 2H), 5.47 (br s, 1H, NH), 5.20 (dd,
1H), 4.78 (dd, 1H), 3.64-3.58 (m, 2H), 3.44 (s, 3H), 3.44-3.38 (m,
2H), 2.96 (d, 1H), 2.77 (dd, 2H), 2.57-2.52 (m, 2H), 2.40 (ddd,
1H), 2.16 (ddd, 1H), 2.06-1.99 (m, 2H), 1.90 (d, 1H), 1.84-1.80 (m,
1H), 1.74 (s, 3H), 1.65 (s, 3H), 1.42 (d, 3H), 1.20 (s, 3H),
1.14-1.03 (m, 1H). LC/MS method B: m/z 516 [M+H].sup.+, R.sub.t 4.4
min.
Example 4
Synthesis of Compound DD
##STR00130##
[0389] Fumagillol (10.0 g, 35.4 mmol) was dissolved in
C.sub.1-12Cl.sub.2 (120 ml). Pyridine was added (8.59 ml, 106 mmol)
and the solution was cooled to 0.degree. C. with constant stirring.
To the stirring solution at 0.degree. C., p-nitrophenyl
chloroformate (9.77 g, 46.0 mmol) in CH.sub.2Cl.sub.2 (55 ml) was
added dropwise and in portions over 1.5 h after which the reaction
mixture was allowed to stir an additional 30 min. at 0.degree. C.
Thereafter, the suspension was diluted with EtOAc and washed
sequentially with an aqueous 10% solution of citric acid
(3.times.150 ml, 100 ml, 50 ml), water, and an aqueous saturated
solution of NaCl. The organic layers were dried with MgSO.sub.4 and
concentrated in vacuo to yield tan solid. Purification through
silica gel chromatography (330 g: 10.fwdarw.30% EtOAc in hexanes,
45 mM) delivered fumagillol p-nitrophenyl carbonate Compound DD
(12.3 mg, 27.6 mmol, 78%) as white solid. LC/MS method C: m/z 448
[M+H].sup.+, R.sub.t 18.4 mM.
Example 5
Synthesis of Compound EE
##STR00131##
[0391] To a stirring suspension of compound DD (100 mg, 0.223 mmol)
was added a solution of 3-(piperazin-1-yl)propionic acid ethyl
ester (41.6 mg, 0.223 mmol) in MeCN (1 ml), and the solution was
allowed to stir 12 h at 23.degree. C. The reaction mixture was
diluted with CH.sub.2Cl.sub.2 (50 ml), washed with a 0.5 M aqueous
solution of NaOH, dried with MgSO.sub.4, and concentrated in vacuo
to yield yellow oil. Purification through silica gel chromatography
(12 g: 0.fwdarw.20% MeOH in CH.sub.2Cl.sub.2, 20 min.) delivered
Compound EE (42.5 mg, 0.086 mmol, 38%) as a colorless oil. LC/MS
method C: m/z 495 [M+H].sup.+, R.sub.t 10.5 min.
Example 6
Procedure for the Preparation of Fumagillone
##STR00132##
[0393] Chromium (VI) oxide (6.37 g, 63.7 mmol) was added to a
stirred solution of pyridine (15 ml, 185 mmol) in CH.sub.2Cl.sub.2
(70 ml) in several portions causing an immediate color change to
yellow before turning a deep red color. The slurry was stirred for
10 min at 0.degree. C. then at 25.degree. C. for 20 mM, during
which time most of the solids had dissolved. Fumagillol (3.00 g,
10.6 mmol) in C.sub.2Cl.sub.2 (20.0 ml) was then added to the
mixture causing an immediate color change to dull brown with
formation of a ppt. After stirring overnight at 25.degree. C., the
solution was decanted into a beaker and the tarry residue rinsed
with diethyl ether (200 ml). The combined organics were washed with
1 N aq. NaOH (50 ml), 10% aqueous citric acid (2.times.50 ml) and
brine (50 ml). The organic extract was dried over MgSO.sub.4,
filtered and concentrated in vacuo to provide 3.8 g of the crude
product as a yellow oil. Purification by chromatography on silica
gel (40 g) eluting with an ethyl acetate/hexane gradient
(0-->30%, 5 mM; 30%, 5 min; 30-->40%, 5 min; 40%, 10 min;
40-50%, 3 mM) provided 2.297 g (77%) of the desired product as a
yellow oil. LC/MS Method B: m/z 281 [M+H]+, R.sub.t 8.40 min.
Example 7
Procedure for the Preparation of Fumagillamine
##STR00133##
[0395] To a solution of fumagillone (903 mg, 3.07 mmol) in
anhydrous MeOH (20.0 ml) cooled to 0.degree. C. was added ammonium
acetate (2.49 g, 31.3 mmol) and sodium cyanoborohydride (254 mg,
3.84 mmol). The resultant yellow solution was stirred at 0 After 3
h, the reaction was concentrated in vacuo to a light yellow liquid
which was diluted with EtOAc and washed with sat. aq. NaHCO.sub.3
(10 ml). The aq. layer was salted with solid NaCl and back
extracted with EtOAc (4.times.25 ml). The combined organic extracts
were dried (MgSO.sub.4), filtered and concentrated in vacuo to
provide 1.047 g of a light yellow powder that was used immediately
in the next step without purification.
Example 8
Synthesis of Compound FF
##STR00134##
[0397] To a stirring solution of fumagillamine (1.00 g, 3.55 mmol)
in CH.sub.2Cl.sub.2 (15 ml) at 0.degree. C. was added Et.sub.3N
(0.545 ml, 3.91 mmol) followed by a solution of p-nitrophenyl
chloroformate (0.788 g, 3.91 mmol) in CH.sub.2Cl.sub.2 (2 ml). The
reaction mixture was allowed to warm to 23.degree. C. over 1.5 h
after which a solution of 2-(4-aminophenyl)ethylamine (0.458 ml,
3.55 mmol) in CH.sub.2Cl.sub.2 (2 ml) was added and the mixture
allowed to stir at 23.degree. C. for an additional 2 h. The
reaction mixture was diluted with CH.sub.2Cl.sub.2, washed with an
aqueous saturated solution of NaHCO.sub.3, dried over MgSO.sub.4,
and concentrated in vacuo to yield yellow solid. Purification
through silica gel chromatography (12 g: 0.fwdarw.5% MeOH (0.05%
Et.sub.3N) in CH.sub.2Cl.sub.z (0.05% Et.sub.3N), 26 min) delivered
Compound FF (0.890 g, 2.00 mmol, 57%) as light yellow solid. LC/MS
method A: m/z 444 [M+H].sup.+, R.sub.t 4.1 min.
Example 9
Synthesis of Compound GG
##STR00135##
[0399] Compound FF (0.890 g, 2.01 mmol), Fmoc-L-Ala monohydrate
(0.687 g, 2.21 mmol), HOBt (0.338 g, 2.21), and EDC (0.423 g, 2.21
mmol) were dissolved in CH.sub.2Cl.sub.2 (5 ml) and allowed to stir
at 23.degree. C. for 4 h. The reaction mixture was quenched through
addition of an aqueous 10% solution of citric acid (5 ml) and the
aqueous layer was washed with CH.sub.2Cl.sub.2 (2.times.10 ml). The
combined organic layers were washed sequentially with an aqueous
10% solution of citric acid (20 ml), an aqueous saturated solution
NaHCO.sub.3, and an aqueous saturated solution of NaCl, after which
they were dried with MgSO.sub.4 and concentrated in vacuo to yield
yellow oil. Purification through silica gel chromatography (12 g:
0.fwdarw.5% MeOH in CH.sub.2Cl.sub.2, 26 min) delivered the
Fmoc-protected derivative of Compound GG (0.634 g, 0.860 mmol, 43%)
as white solid. LC/MS method A: m/z 737 [M+H].sup.+, R.sub.t 8.1
min.
[0400] The Fmoc-protected derivative of Compound GG (0.634 g, 0.860
mmol) was dissolved in DMF (5 ml) and the solution cooled to
0.degree. C. To the stirring solution was added piperidine (0.852
ml, 8.60 mmol) and the mixture was allowed to stir at 0.degree. C.
for 1 h after which it was concentrated in vacuo to yield white
solid. Purification through silica gel chromatography (4 g:
0.fwdarw.50% MeOH (0.5% Et.sub.3N) in CH.sub.2Cl.sub.2 (0.5%
Et.sub.3N), 30 mM) delivered Compound GG (0.336 g, 0.653 mmol, 76%)
as white solid. LC/MS method A: m/z 515 [M+H].sup.+, R.sub.t 3.9
min.
Example 10
Synthesis of Compound JJ
##STR00136##
[0402] Compound HH (0.459 g, 1.05 mmol), which was prepared in a
procedure analogous to that of compound FF, described above, was
dissolved in CH.sub.2Cl.sub.2 (10.5 ml) and succinic anhydride
(0.105 g, 1.05 mmol) was added followed by DMAP (0.013 g, 0.105
mmol). The mixture was allowed to stir at 23.degree. C. for 24 h
after which additional succinic anhydride was added (0.105 g, 1.05
mmol) and the mixture was allowed to stir an additional 1 h
23.degree. C. The reaction mixture was concentrated in vacuo to
yield Compound JJ (0.564 g, 1.05 mmol, >98%) as a pale yellow
oil which was used in subsequent coupling reactions without
purification. LC/MS method A: m/z 538 [M+H].sup.+, R.sub.r 3.7
mM.
Example 11
Representative Procedure
Preparation of Conjugate 12: (by Using the Appropriate Starting
Materials/Reactants, Conjugates 3, 6, 11, 15, 16, 24, 25, 26, 27,
28, 33, 37 and 43 can be Prepared in an Analogous Manner)
##STR00137##
[0403] p=m, and m is as defined above for Formula I.
[0404] PHF-GA (11.4 g, 78.0 mmol) was dissolved in a solution of
20% DMF in water (114 ml). To the stirring mixture at 23.degree. C.
was added a solution of Compound BB (1.02 g, 2.72 mmol) in DMF (20
ml) and the pH adjusted to 5.9-6.0. The reaction mixture was cooled
to 0.degree. C. with constant stirring after which EDC (1.04 g,
5.44 mmol) was added in three portions over 30 minute intervals.
Upon addition of the last portion of EDC, stirring was continued
for 2.5-4.0 h at 0.degree. C. The pH was adjusted to 4.5-5.0 using
1.0 N HCl and the mixture filtered through a 0.2.mu. membrane and
purified by size exclusion chromatography. Fractions that contained
desired product were collected and the resulting solution was
lyophilized to yield Conjugate 12 as white solid (12.6 g). NMR
indicated a loading of 8.0% (w/w) of BB.
Example 12
Representative Procedure
Preparation of Conjugate 4. (by Using the Appropriate Starting
Materials/Reactants, Conjugates 5, 10, 14 and 41 can be Prepared in
an Analogous Manner)
##STR00138##
[0405] wherein p=m as defined above for Formula I.
[0406] Compound KK (200 mg, 0.429 mmol) was dissolved in THF (15
ml) and cooled to 0.degree. C. Et.sub.3N (0.119 ml, 0.857 mmol) was
added followed by isobutyl chloroformate (0.051 ml, 0.386 mmol) and
the mixture was allowed to stir 2 h at 0.degree. C. In a separate
vessel, PHF (579 mg, 4.29 mmol) was dissolved in anhydrous
pyridine. The two solutions were combined and allowed to stir and
warm to 23.degree. C. over 12 h. The crude reaction mixture was
concentrated in vacuo and diluted with 100 ml water. The mixture
was filtered through a 0.2.mu. membrane and purified by size
exclusion. Fractions that contained desired product were collected
and the resulting solution was lyophilized to yield conjugate 4 as
white solid (550 mg). .sup.1H NMR indicated a loading of 5.0% (w/w)
of KR.
Example 13
Synthesis of Compound LL
##STR00139##
[0408] To a solution of fumagillone (6.0 g, 21.4 mmol) in anhydrous
MeOH (150 ml) cooled to 0.degree. C. was added ammonium acetate
(17.0 g, 214 mmol) and sodium cyanoborohydride (2831 mg, 42.8 mmol)
forming a light yellow solution that was stirred at 0.degree. C.
After 1 h 40 min, another portion of sodium cyanoborohydride (500
mg, 7.56 mmol) was added to the reaction. Stirring was continued
for a total of 2.5 h at 0.degree. C. followed by warming to
25.degree. C. over 15 min. This was followed by dilution with EtOAc
(100 ml), concentration in vacuo to remove MeOH, adding additional
EtOAc (100 ml) and washing with sat aq. NaHCO.sub.3 (50 ml). The
aqueous layer was salted with NaCl(s) then back extracted with
EtOAc (3.times.70 ml). The combined organic extracts were dried
(MgSO.sub.4), filtered and concentrated in vacuo to provide 6.153 g
of a light-yellow solid. The solid was taken up in CH.sub.2Cl.sub.2
(130 ml) and cooled to 0.degree. C. followed by addition of
chloroacetylisocyanate (3.60 ml, 42 mmol) forming a yellow solution
which was allowed to warm slowly to 25.degree. C. After overnight
stirring, the reaction was diluted with sat aq. NH.sub.4Cl (50 ml),
stirred vigorously for 10 min, then diluted with additional EtOAc
(100 ml) and concentrated in vacuo to remove CH.sub.2Cl.sub.2. The
layers were separated and the aqueous layer was salted with NaCl
(s) then back extracted with EtOAc (2.times.70 ml). The combined
organic extracts were washed with brine (30 ml), dried
(MgSO.sub.4), filtered and concentrated in vacuo to provide an
off-white solid. Purification by chromatography on silica gel (120
g) eluting with an EtOAc/hexane gradient [0-->30%, 7 min; 30%, 7
min; 30-->50%, 7 min; 50%, 10 min; 50-->100%, 5 min; 100%, 3
min] provided 4.17 g (49%) of the desired product as a white solid.
LC/MS Method B: m/z 282 [M-C.dbd.ONHC.dbd.OCH.sub.2Cl].sup.+,
R.sub.t 7.36 min.
Example 14
Synthesis of Compound MM
##STR00140##
[0410] To a solution of the alkyl chloride (3810 mg, 9.50 mmol) in
anhydrous DMF (70 ml) cooled to 0.degree. C. and under inert
atmosphere was added Hunig's base (2.00 ml, 11.40 mmol) followed by
4-aminothiophenol (1360 mg, 10.45 mmol), generating a clear, yellow
solution. Stirring was maintained at 0.degree. C. for 1.5 h, then
at 25.degree. C. for 15 min. The solution was then diluted with
EtOAc (100 ml) and H.sub.2O (70 ml), layers separated, and aqueous
layer extracted further with EtOAc (2.times.100 ml). The combined
organic extracts were washed with brine (25 ml), dried, filtered
and concentrate in vacuo to yield a thick, yellow oil. Purification
by chromatography on silica gel (120 g) eluting with an
EtOAc/hexane gradient [0-->40%, 5 min; 40%, 4 min; 40-->60%,
4 min; 60%, 10 min; 60-->80%, 3 min; 80%, 5 min; 80-100%, 3 min]
provided 2.05 g (44%) of MM. LC/MS Method B: m/z 490 [M+H].sup.+,
R.sub.t 7.134 min.
Example 15
Synthesis of Compound NN
##STR00141##
[0412] To a solution of the carboxylic acid (607 mg, 2.86 mmol) in
anhydrous DMF (13.0 ml), under inert atmosphere, was added
di-isopropylethylamine (DIPEA) (1.50 ml, 8.61 mmol) followed by
benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate (BOP) (1.44 g, 3.12 mmol). The resultant clear
solution was stirred at 25.degree. C. for 5 min, then cooled to
0.degree. C. followed by addition of fumagillamine (875 mg, 3.11
mmol, freshly prepared from fumagillone and used without further
purification) in DMF (3.0 ml), generating a yellow solution. The
solution was stirred at 0.degree. C. and allowed to warm slowly to
25.degree. C. overnight. After 14 h, the reaction was diluted with
EtOAc and water (25 ml each), layers separated and the aqueous
layer extracted with additional EtOAc (2.times.50 ml). The combined
organic extracts were washed with water (2.times.15 ml), sat aq.
NaHCO.sub.3 and brine (15 ml each), dried (MgSO4), filtered and
concentrated in vacuo to provide an orange-yellow oil. Purification
by chromatography on silica gel (40 g) eluting with an EtOAc/hexane
gradient [0-->30%, 5 min; 30%, 4 min; 30-->50%, 4 min; 50%, 5
min; 50-->80%, 3 min] provided 924 mg (69%) of NN as a light
yellow oil. LC/MS Method B: m/z 476 [M+H].sup.+, R.sub.t 9.85
min.
Example 16
Synthesis of Compound OO
##STR00142##
[0414] To a solution of the methyl ester (1.2 g, 2.52 mmol) in
tert-butanol (16.0 ml) and water (8.0 ml) cooled to 0.degree. C.
was added LiOH (215 mg, 5.12 mmol), in one portion, forming a
slurry. After 30 min, the reaction mixture had thickened and become
opaque. The reaction mixture was allowed to warm slowly to
25.degree. C. overnight. After 22 h, the reaction was quenched by
addition of 1.0 M NaHSO.sub.4 (7.0 ml, 7.0 mmol) to pH 4. The
volatiles were then removed in vacuo, leaving a few milliliters of
opaque liquid, which was diluted with water (25 ml) and EtOAc (50
ml) and layers separated. The aqueous layer was salted with NaCl
(s) and extracted further with EtOAc (2.times.50 ml). The combined
organic extracts were dried (MgSO.sub.4), filtered and concentrated
in vacuo to provide 1.38 g of 00 as yellow oil. Purification by
chromatography on silica gel (40 g) eluting with a
MeOH/CH.sub.2Cl.sub.2 gradient [2%, 5 min; 2-->10%, 10 min]
provided 943 mg (81%) of the OO as white solid. LC/MS Method B: m/z
462 [M+H].sup.+, R.sub.t 7.76 min.
Example 17
Synthesis of Compound PP
##STR00143##
[0416] To a solution of the carboxylic acid OO (440 mg, 0.953 mmol)
in anhydrous DMF (4.0 ml) was added diisopropylethyl amine (500
.mu.L, 2.86 mmol) and HATU (448 mg, 1.144 mmol) at 25.degree. C.
under inert atmosphere. The resultant yellow solution was stirred
at 25.degree. C. for 10 mM, then cooled to 0.degree. C. for
addition of 3-amino-1-propanol (80 .mu.L, 1.050 mmol), causing the
solution to become bright yellow. The solution was stirred at
0.degree. C. for 10 min then warmed to 25.degree. C. and stirring
continued. After 2 h, the reaction was diluted with EtOAc and
H.sub.2O (10 ml each), layers separated and aqueous layer extracted
with EtOAc (2.times.20 ml). The combined organic extracts were
dried (MgSO.sub.4), filtered and concentrated in vacuo to an orange
oil. Purification by chromatography on silica gel (12 g) eluting
with a MeOH/EtOAc gradient [0%, 1 min; 0-->5%, 1 min; 5%, 3 mM;
5-10%, 10 min] provided 411 mg (83%) of the desired product PP as
yellow oil. LC/MS Method B: m/z 519 [M+H].sup.+, R.sub.t 6.76
min.
Example 18
Synthesis of Compound QQ
##STR00144##
[0418] To a solution of the alcohol PP (404 mg, 0.779 mmol) in
CH.sub.2Cl.sub.2 (5.0 ml) was added the Fmoc-D-Proline amino acid
(312 mg, 0.925 mmol), in one portion, and DMAP (27 mg, 0.221 mmol),
forming a clear solution. To this was added
di-2-(pyridyl)thiocarbonate (DPTC) (366 mg, 1.542 mmol) and the
resultant orange solution was stirred at 25.degree. C. under inert
atmosphere. After 2 min, the solution became a deep orange-red
color. After 2.5 h, an additional portion of DPTC (50 mg, 0.211
mmol) was added to the reaction. After 5.5 h, the reaction was
diluted with EtOAc and H.sub.2O (10 ml each), layers separated, and
the aqueous layer extracted with EtOAc (2.times.20 ml). The
combined organic extracts were washed with sat aq. NaHCO.sub.3 and
brine (10 ml each), dried, filtered and concentrated in metro to
provide an orange-brown oil. Purification by chromatography on
silica gel (12 g) eluting with an EtOAc/hexane gradient
[0-->50%, 5 min; 50%, 5 min; 50-->100%, 5 min] provided 431
mg (67%) of the desired Fmoc-protected derivative of QQ as a dark
yellow oil. LC/MS Method B: m/z 838 [M+H].sup.+, R.sub.t 10.19
min.
[0419] Fmoc-protected QQ (277 mg, 0.331 mmol) was dissolved in DMF
(1.30 ml) and the solution cooled to 0.degree. C. To the stirring
solution was added piperidine (0.330 ml, 3.34 mmol) and the
solution stirred at 0.degree. C. A precipitate formed after 20 min.
After 4 h, the solids were filtered off on a frit, rinsed with
methanol and the filtrate concentrated in vacuo to provide a white
solid. Purification by chromatography on silica gel (4 g) eluting
with a gradient of MeOH/CH.sub.2Cl.sub.2+0.5% Et.sub.3N
[0-->50%, 30 min] provided 185 mg (91%) of Compound QQ as a
yellow oil. LC/MS Method B: m/z 616 [M+H].sup.+, R.sub.t 4.44
min.
Example 19
Representative Procedure
Preparation of Conjugate 19 (by Using the Appropriate Starting
Materials/Reactants, Conjugates 1, 2, 7, 8, 9, 13, 17, 18, 19, 20,
21, 22, 23, 30, 34, 35, 36, 39, 40, 42, And 44 can be Prepared in
an Analogous Manner)
##STR00145##
[0420] where p=m and is as defined above for Formula I.
[0421] Water (140 ml) was added to PHF-GA (7.30 g) and the
resulting mixture was stirred vigorously for 1 h to yield a yellow
solution. The fumagillin derivative MM (852 mg) in MeCN (8.3 ml)
was then added at 25.degree. C. Additional MeCN (33.7 ml) was added
to make 42 ml total. The solution was cooled to 0.degree. C.
followed by addition of EDC (475 mg) portionwise over 5 min after
which the pH of the reaction was adjusted to 6.0. After 30 min, the
solution was warmed to 25.degree. C. Additional portions of EDC
were added at 2 h (200 mg) and 3 h (100 mg). At 4.5 h, the reaction
was acidified with 1.0 N HCl to pH 5.0. The reaction was diluted to
.about.300 ml with water, filtered thru a 0.2.mu. membrane and
purified by size exclusion chromatography. Fractions that contained
the desired product cleanly were pooled and lyophilized to provide
6.6 g of conjugate 19 as white solid, UV analysis (254 nm)
indicated a 9.8% loading of MM.
Example 20
[0422] METAP-2 Inhibition by Fumagillin Core Structure
[0423] The test samples were evaluated for their ability to inhibit
human MetAP-2 enzyme. The compounds were added at varying
concentrations to human MetAP-2 enzyme (6 nM) in a buffered medium
containing 20 mM Hepes, pH 7.4, 100 mM KCl, 0.1 mM Co(II), 10%
glycerol, 5 mg/ml of L-Amino acid oxidase, 10 mg/ml of peroxidase,
10 mg/ml of o-dianisidine, and 2 mM of Met-Ser-Ala at 37.degree. C.
After 10 minutes incubation the reaction is started by adding the
substrate Met-Gly-Pro-AMC to 2 mM and the release of AMC is
measured with a Decan Plate reader (Excitation at 345 nm, emission
at 445 nm). The kinetic data is recorded at 30 sec intervals for 30
min. Table 1 lists some of the conjugates and compounds tested
along with their approximate IC.sub.50 values for inhibiting
Met-AP2.
TABLE-US-00002 TABLE 1 Compound or Conjugate IC.sub.50 Compound 8
<10 nM Conjugate 12 <1000 nM Compound 63 <10 nM Compound
39 <10 nM Conjugate 19 <50 nM Compound 65 <10 nM Compound
8 <10 nM Conjugate 15 <50 nM Compound 60 <10 nM Compound
61 <10 nM Compound 51 <10 nM Conjugate 24 <50 nM Compound
59 <10 nM Compound 58 <10 nM TNP-470* <10 nM PPI-2458**
<10 nM *The chemical structure of TNP-470: ##STR00146## **The
chemical structure of PPI-2458 is: ##STR00147##
Example 21
Inhibitory Effect of Fumagillin Conjugates and Analogs on Cell
Growth
[0424] Using human umbilical vein endothelial cells (HUVECs), the
effect of the fumagillin analog conjugates and the fumagillin
analogs on cell growth was evaluated.
[0425] The HUVECs were added to M199 medium supplemented with 20%
FBS, after which the cells were incubated at 37.degree. C. under 5%
CO.sub.2. 96-well plates were seeded at 5,000 cells/well in a total
volume of 100 .mu.L. The cells were left overnight to attach to the
plate. Cells were subcultured a maximum of 15 times before being
discarded.
[0426] To evaluate the inhibitory activity of the fumagillin
conjugate or the fumagillin analog alone on the growth of HUVEC
cells, the conjugate or fumagillin analog was dissolved in the
required amount of 0.9% saline, vortexed briefly to wet and
disperse the solids, followed by vortexing for a total of 30 min.
2.times. stock solutions for each conjugate and fumagillin analog
were made ranging from 12 .mu.M to 0.06 .mu.M. The cells were
treated with the conjugate solution or fumagillin analog solution
at various concentrations ranging from 30 nM to 0.01 nM, and
cultured for 5 days at 37.degree. C.
[0427] Colorimetry was performed to determine the extent of cell
growth by adding 20 .mu.L of MTS/PES reagent to each well,
incubation for 4 h at 37.degree. C. followed by measuring the
absorbance at 490 nm. Table 2 tabulates the results of the tests
performed on some of the conjugates and compounds of the invention
and other reference compounds.
TABLE-US-00003 TABLE 2 Inhibitory Effect of Fumagillin Conjugates
and Analogs on Cell Growth Agent IC.sub.50 (nM) Conjugate 1 nd
Conjugate 2 nd Conjugate 3 nd Conjugate 4 <25 Conjugate 5 nd
Conjugate 6 <25 Conjugate 7 <25 Conjugate 8 nd Conjugate 9 nd
Conjugate 10 nd Conjugate 11 <25 Conjugate 12 25-100 Conjugate
13 25-100 Conjugate 14 nd Conjugate 15 100-300 Conjugate 16 >300
Conjugate 17 nd Conjugate 18 <25 Conjugate 19 >300 Conjugate
20 100-300 Conjugate 21 nd Conjugate 22 >300 Conjugate 23
>300 Conjugate 24 100-300 Conjugate 25 25-100 Conjugate 26 nd
Conjugate 27 nd Conjugate 28 nd Conjugate 29 100-300 Conjugate 30
>300 Conjugate 31 <25 Conjugate 32 <25 Conjugate 33
>300 Conjugate 34 >300 Compound 1 <25 Compound 2 <25
Compound 3 <25 Compound 4 >30 Compound 5 <25 Compound 6
<25 Compound 7 <25 Compound 8 <25 Compound 9 <25
Compound 10 <25 Compound 11 <25 Compound 12 <25 Compound
13 >30 Compound 14 <25 Compound 15 >30 Compound 16 >30
Compound 17 <25 Compound 18 <25 Compound 19 <25 Compound
20 <25 Compound 21 <25 Compound 22 <25 Compound 23 <25
Compound 24 <25 Compound 25 >30 Compound 26 <25 Compound
27 <25 Compound 28 <25 Compound 29 <25 Compound 30 <25
Compound 31 <25 Compound 32 <25 Compound 33 >30 Compound
34 <25 Compound 35 <25 Compound 36 <25 Compound 37 25-100
Compound 38 25-100 Compound 39 <25 Compound 40 25-100 Compound
41 100-300 Compound 42 100-300 Compound 43 <25 Compound 44
<25 Compound 45 <25 Compound 46 <25 Compound 47 <25
Compound 48 <25 Compound 49 100-300 Compound 50 <25 Compound
51 <25 Compound 52 <25 Compound 53 25-100 Compound 54 <25
Compound 55 <25 Compound 56 <25 Compound 57 <25 nd = not
determined
[0428] The structures of the Conjugates tested can be found in the
listing of the illustrative conjugates of Formula IV. The
structures of the compounds tested in Table 2 are as set forth
below:
TABLE-US-00004 Compound No. Structure 1 ##STR00148## 2 ##STR00149##
3 ##STR00150## 4 ##STR00151## 5 ##STR00152## 6 ##STR00153## 7
##STR00154## 8 ##STR00155## 9 ##STR00156## 10 ##STR00157## 11
##STR00158## 12 ##STR00159## 13 ##STR00160## 14 ##STR00161## 15
##STR00162## 16 ##STR00163## 17 ##STR00164## 18 ##STR00165## 19
##STR00166## 20 ##STR00167## 21 ##STR00168## 22 ##STR00169## 23
##STR00170## 24 ##STR00171## 25 ##STR00172## 26 ##STR00173## 27
##STR00174## 28 ##STR00175## 29 ##STR00176## 30 ##STR00177## 31
##STR00178## 32 ##STR00179## 33 ##STR00180## 34 ##STR00181## 35
##STR00182## 36 ##STR00183## 37 ##STR00184## 38 ##STR00185## 39
##STR00186## 40 ##STR00187## 41 ##STR00188## 42 ##STR00189## 43
##STR00190## 44 ##STR00191## 45 ##STR00192## 46 ##STR00193## 47
##STR00194## 48 ##STR00195## 49 ##STR00196## 50 ##STR00197## 51
##STR00198## 52 ##STR00199## 53 ##STR00200## 54 ##STR00201## 55
##STR00202## 56 ##STR00203## 57 ##STR00204##
Example 22
Inhibitory Effects of Fumagillin Conjugates on Tumor Growth Rates
in B16F10 Mouse Melanoma
[0429] BDF1 female mice (N=6) with 1.times.10.sup.6B16F10 tumor
cells positioned subcutaneous in the flank were treated with
various fumagillin conjugates of the invention. Tumor growth was
monitored in parallel with positive and negative controls of
cyclophosphamide and saline respectively. Treatment began when
tumors reached an average size of 80-120 mg and tumor volumes were
measured two times per week until animals reached an endpoint tumor
volume of 2 grams or 45 days, whichever came first. Conjugates were
administered as solutions in saline intravenously at dose levels of
30-100 mg/kg (expressed as fumagillin analog equivalents) on a
schedule of q3dx4, Treatment outcomes were assessed in terms of
percent tumor growth delay (% TGD), (see FIG. 1) defined as the
percent increase in median time to endpoint for mice treated with
an agent compared to those treated with saline.
[0430] Conjugates represented in Table 3 and FIG. 1 exhibited tumor
growth delays in the ranges of 5-20, 20-50, and >50 percent. A
number of fumagillin conjugates notably delayed tumor growth in
mice bearing B16F10 tumors, thus illustrating the biological
activity of these agents against mouse melanoma.
TABLE-US-00005 TABLE 3 Effect of Fumagillin Analog Conjugates on
Tumor Growth Delay in a B16 Melanoma Cell Line Conjugate No.
mg/kg.sup.1 Route Schedule % TGD 6 80 IV q3d .times. 4 5-20 7 80 IV
q3d .times. 4 5-20 9 100 IV q3d .times. 4 20-50 10 60 IV q3d
.times. 4 20-50 11 65 IV q3d .times. 4 20-50 12 80 IV q3d .times. 4
5-20 13 80 IV q3d .times. 4 5-20 15 80 IV q3d .times. 4 >50 16
80 IV q3d .times. 4 20-50 18 80 IV q3d .times. 4 5-20 19 40 IV q3d
.times. 4 20-50 22 30 IV q3d .times. 4 5-20 23 80 IV q3d .times. 4
5-20 25 80 IV q3d .times. 4 20-50 26 60 IV q3d .times. 4 5-20 27 50
IV q3d .times. 4 5-20 31 80 IV q3d .times. 4 20-50 32 80 IV q3d
.times. 4 20-50 33 65 IV q3d .times. 4 5-20 35 80 IV q3d .times. 4
20-50 38 80 IV q3d .times. 4 5-20 40 80 IV qod .times. 4 20-50 44
80 IV q3d .times. 4 5-20 .sup.1The range of doses is 30-100 mpk
fumagillin analog-equiv. which corresponds to 300 mg-1.2 g
conjugate.
Example 23
Inhibitory Effects of Fumagillin Conjugates on Tumor Growth Rates
in A2058 Human Melanoma Xenograft Studies
[0431] HRLN female mice (N=8) with 1.times.10.sup.7 A2058 tumor
cells positioned subcutaneous in flank were treated with fumagillin
conjugates. Tumor growth was monitored in parallel with positive
and negative controls of dacarbazine, and saline respectively.
Treatment began when tumors reached an average size of 80-120 mg
and tumor volumes were measured twice per week until animals
reached an endpoint tumor size of 2 grams or 45 days, whichever
came first. Conjugates were administered as solutions in saline
intravenously at dose levels of 10-60 mg/kg (expressed in
fumagillin analog equivalents) on a schedule of q4dx5. Treatment
outcomes were assessed in terms of percent tumor growth delay (%
TGD), defined as the percent increase in median time to endpoint
for mice treated with an agent compared to those treated with
saline.
[0432] As shown in FIG. 2, conjugates exhibited tumor growth delays
in the range of 0-40 percent. A number of fumagillin conjugates
notably delayed tumor growth in mice bearing A2058 tumors, thus
illustrating the biological activity of these agents against human
A2058 melanoma in mice.
Example 24
Inhibitory Effects of Fumagillin Conjugates on Tumor Growth Rates
in PC3 Human Prostate Cancer Xenograft Studies
[0433] HRLN female mice (N=8) with 1 mm.sup.3 PC3 tumor fragments
positioned subcutaneous in flank were treated with fumagillin
conjugates. Tumor growth was monitored in parallel with positive
and negative controls of docetaxel and saline respectively.
Treatment began when tumors reached an average size of 80-120 mg
and tumor volumes were measured twice per week until animals
reached an endpoint tumor size of 2 grams or 45 days, whichever
came first. Conjugates were administered as solutions in saline
intravenously at dose levels of 20-60 mg/kg (expressed in
fumagillin analog equivalents) on a schedule of q4dx5. Treatment
outcomes were assessed in terms of percent tumor growth delay (%
TGD), defined as the percent increase in median time to endpoint
for mice treated with an agent compared to those treated with
saline.
[0434] Conjugates represented in FIG. 3 exhibited tumor growth
delays in the range of 20-50 percent. A number of fumagillin
conjugates notably delayed tumor growth in mice bearing human PC3
prostate cancer tumors, thus illustrating the biological activity
of these agents against human PC3 prostate cancer in mice.
Example 25
Effects of Fumagillin Conjugates on Activity Levels in Mice
Assessed through Open Field Studies
[0435] C57B1/6 female mice (N=8) were housed until they reached 11
weeks of age. Animals were then regularly administered with
fumagillin conjugates or TNP-470 and their activity levels
monitored in an open field arena. Activity levels were assessed
with saline as a control. Conjugates were administered as solutions
in saline intravenously at dose levels of 50-100 mg/kg (expressed
as fumagillin-analog equivalents) and TNP-470 was administered
subcutaneously at dose levels of 50-100 mg/kg. Dosing was performed
for four weeks on schedules ranging from q2d to once weekly, and
activity levels were observed four times throughout the study: (1)
prior to compound administration, (2) after one week of dosing, (3)
after two weeks of dosing, and (4) following the final week of
dosing. Animals were placed in the open field arena for 10 minutes
per trial and their activity monitored in terms of travel distance,
peripheral squares crossed, center squares crossed, and rearing
events.
[0436] As represented in FIGS. 4 and 5 animals that were treated
with fumagillin conjugates exhibited rearing-event activity levels
similar to those of saline treated animals. In contrast, increased
rearing-event activity levels were observed in mice that received
TNP-470.
[0437] In the open field neurobehavioral models, animals that were
treated with fumagillin conjugates performed in a manner similar to
those that were treated with saline. Changes in activity observed
with TNP-470 in the above described neurobehavioral models may be
consistent with clinically observed TNP-470 side effects. Because
increased rearing-event activity was not observed in fumagillin
conjugate treated animals, these findings are testament to the
potential increased neurological safety of fumagillin conjugates
compared to their small molecule competitors/predeccssors such as
TNP-470.
Example 26
Fumagillin Analog Conjugates
Plasma and Cerebrospinal Fluid Pharmacokinetics Parameters
[0438] Male Sprague Dawley rats (Hsd: Sprague Dawley SD) were
catheterized independently for blood and CSF collection and
administered with a fumagillin conjugate or TNP-470, with a
targeted active compound dose of 40 mg/kg (expressed in
fumagillin-analog equivalents) and a dose volume of 10 mL/kg. The
plasma samples were analyzed for conjugated and small molecule
active fumagillin compounds. For TNP-470, both the parent TNP-470,
and the known metabolite M-IV, were assessed. CSF samples were
analyzed for small molecule fumagillin analogs only, i.e. the
fumagillin-analog released from the corresponding conjugate.
Analyses for fumagillin conjugates were carried out after their
isolation from plasma by co-precipitation with plasma proteins and
subsequent conversion into low molecular weight fumagillin products
by basic hydrolysis. Liquid chromatography tandem mass-spectrometry
(LC/MS/MS) was used for fumagillin compound detection in biological
fluids.
[0439] Data for conjugates 12, 15, 19, and 24 and TNP-470 are
provided below. The protocol design is outlined in Table 4, and
sampling timepoints are tabulated in Table 5.
TABLE-US-00006 TABLE 4 Protocol Design Treatment Regimen Group n
Agent Catheter Type mg/kg Route Schedule 1 3 No treatment
Intra-cisternal -- IV qd .times. 1 2 3 Conjugate 12 Jugular 40 IV
qd .times. 1 3 4 Conjugate 12 Intra-cisternal 40* IV qd .times. 1 4
4 Conjugate 12 Intra-cisternal 40* IV qd .times. 1 2' 3 Conjugate
15 Jugular 40 IV qd .times. 1 3' 4 Conjugate 15 Intra-cisternal 40
IV qd .times. 1 4' 4 Conjugate 15 Intra-cisternal 40 IV qd .times.
1 2'' 3 Conjugate 19 Jugular 10 IV qd .times. 1 3'' 4 Conjugate 19
Intra-cisternal 10 IV qd .times. 1 4'' 4 Conjugate 19
Intra-cisternal 10 IV qd .times. 1 2''' 3 Conjugate 24 Jugular 40
IV qd .times. 1 3''' 4 Conjugate 24 Intra-cisternal 40 IV qd
.times. 1 4''' 4 Conjugate 24 Intra-cisternal 40 IV qd .times. 1 5
3 TNP-470 Jugular 40 IV qd .times. 1 6 4 TNP-470 Intra-cisternal 40
IV qd .times. 1 7 4 TNP-470 Intra-cisternal 40 IV qd .times. 1
Pharmacokinetic sampling was performed for each group as described
in Table 5. *In some cases the dose was adjusted to 10 mg/kg due to
viscosity and the volume was increased to 20 mL/kg
TABLE-US-00007 TABLE 5 Sampling Timepoints by Group and Tissue
Timepoint Plasma CSF Any time G1 G1 5 minutes G2, G2', G2'', G2'''
10 minutes G5 G3, G3', G3'', G3''', G6 1 hour G2, G2', G2'', G2''',
G5 2 hours G2, G2', G2'', G2''', G5 G4, G4', G4'', G4''', G7 4
hours G2, G2', G2'', G2''', G5 6 hours G2, G2', G2'', G2''' 8 hours
G2, G2', G2'', G2''', G5 G3, G3', G3'', G3''', G6 10 hours G2, G2',
G2'', G2''' 12 hours G2, G2', G2'', G2''' 24 hours G2, G2', G2'',
G2''' G4, G4', G4'', G4''', G7
[0440] The results of the tests described in Example 26 are shown
in Tables 6 and 7.
TABLE-US-00008 TABLE 6 PK Characteristics for Conjugated and
Conjugate Released Fumagillin Derivatives.sup.# Conjugated
Fumagillin Analog PK parameters Released/free Fumagillin analog
plasma PK parameters T.sub.max t1/2 T.sub.max C.sub.max t1/2 AUC
0-t AUC 0-24 AUC 0-inf Conjugate/Compound (h) (h) (h) (ng/ml) (h)
(.mu.g*h/ml) (.mu.g*h/ml) (.mu.g*h/ml) Conjugate 12 0.083 19.4
0.083 421 20.4 2.9 2.9 5.4 Conjugate 15 0.083 3.5 0.083 20203 3.7
40.5 40.5 41.6 Conjugate 19 0.083 12.2 0.083 1775 8.1 1.8 1.8 2.2
Conjugate 24 0.083 5.0 0.083 7067 5.3 17.4 17.4 19.1 TNP-470
metabolite M-IV n/a n/a 1 318 0.6 0.4 0.4 0.4 .sup.#(mean, N = 3).
Data expressed in fumagillin-analog equivalents and dose normalized
to 40 mg/kg fumagillin analog equivalents.
[0441] As represented by the data in Table 6, the fumagillin
conjugates have a significantly longer half-life in comparison to
TNP-470. As a result of conjugation, the fumagillin analog is
delivered in a sustained fashion in the plasma, resulting in
significantly increased exposure in plasma relative to TNP-470.
TABLE-US-00009 TABLE 7 Cerebral Spinal Fluid Pharmacokinetics
Parameters Fumagillin analog CSF accumulation Conjugate/ AUC AUC
AUC Compound T.sub.max C.sub.max 0-t 0-24 0-inf Dosed.sup.## (h)
(ng/ml) (.mu.g*h/ml) (.mu.g*h/ml) (.mu.g*h/ml) Conjugate 12 2.0 26
n/a* n/a* n/a* Conjugate 15 0.17 1084 n/a* n/a* n/a* Conjugate 19
0.17 n/a* n/a* n/a* n/a* Conjugate 24 0.17 128 1.10 1.10 1.29
TNP-470 0.17 1642 1.67 1.72 1.72 metabolite MIV *The lower limit of
quantitation (LLOQ) for fumagillin analogs was 5 ng/mL. In cases
where the concentrations at various timepoints were below the LLOQ
Cmax and/or AUC could not be calculated. .sup.##Data represent the
fumagillin-analog released from the corresponding conjugate; data
is normalized to an administered dose of 40 mg/kg, expressed in
fumagillin-analog equivalents
[0442] As represented by the data in Table 7, after dosing 40 mg/kg
in rats, the concentrations of fumagillin analogs detected in the
CSF are very low, with C.sub.max values below the level observed
for the TNP-470 metabolite MIV. A graphical representation of the
CSF levels observed is depicted in FIG. 6.
[0443] The very low amounts of fumagillin analog observed in the
CSF of rats after dosing of the corresponding conjugate is
consistent with the polymer conjugate not crossing the blood-brain
barrier, resulting in very low levels (in many instances below the
LLOQ) of the corresponding fumagillin analog detected in the CSF.
These data demonstrate that conjugation of a fumagillin analog can
result in very low levels of the fumagillin analog entering the CNS
compartment, and consequently result in decreased CNS toxicity in
comparison to the small-molecule fumagillin analog TNP-470.
[0444] While particular embodiments described herein have been
illustrated and described, it would be obvious to those skilled in
the art that various other changes and modifications can be made
without departing from the spirit and scope of the disclosure. It
is therefore intended to cover in the appended claims all such
changes and modifications that are within the scope of this
invention.
* * * * *