U.S. patent application number 10/272269 was filed with the patent office on 2003-07-10 for oligoamine compounds and derivatives thereof for cancer therapy.
This patent application is currently assigned to SLIL Biomedical Corporation. Invention is credited to Basu, Hirak S., Blokhin, Andrei V., Frydman, Benjamin, Marton, Laurence J., Valasinas, Aldonia L..
Application Number | 20030130356 10/272269 |
Document ID | / |
Family ID | 23287844 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030130356 |
Kind Code |
A1 |
Frydman, Benjamin ; et
al. |
July 10, 2003 |
Oligoamine compounds and derivatives thereof for cancer therapy
Abstract
Oligoamine compounds with anti-cancer and anti-proliferative
activity are provided, as well as methods for making and using the
compounds. The compounds are shown to be active against prostate
cancer cell lines and against prostate cancer tumors in mice. The
compounds are also useful in treatment of breast cancer and other
cancers.
Inventors: |
Frydman, Benjamin; (Madison,
WI) ; Valasinas, Aldonia L.; (Madison, WI) ;
Blokhin, Andrei V.; (Fitchburg, WI) ; Basu, Hirak
S.; (Madison, WI) ; Marton, Laurence J.; (Palo
Alto, CA) |
Correspondence
Address: |
Robert K. Cerpa
Morrison & Foerster LLP
35th Floor
555 W. 5th Street
Los Angeles
CA
90013
US
|
Assignee: |
SLIL Biomedical Corporation
Madison
WI
|
Family ID: |
23287844 |
Appl. No.: |
10/272269 |
Filed: |
October 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60329982 |
Oct 16, 2001 |
|
|
|
Current U.S.
Class: |
514/667 ;
514/674; 564/503; 564/512 |
Current CPC
Class: |
C07C 211/14 20130101;
C07C 215/18 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/667 ;
514/674; 564/503; 564/512 |
International
Class: |
A61K 031/13; C07C
215/08 |
Claims
What is claimed is:
1. A compound of the formula 29where R.sub.10, R.sub.20, R.sub.60,
and R.sub.70 are independently selected from H, methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl;
each R.sub.80 and R.sub.90 are independently selected from H,
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,
and t-butyl; R.sub.30, each R.sub.40, and R.sub.50 are
independently selected from:
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2----CHOHCH.sub.2CH.sub.2CH.sub.2----CH.-
sub.2CHOHCH.sub.2CH.sub.2----CH.sub.2CH.sub.2CHOHCH.sub.2----CH.sub.2CH.su-
b.2CH.sub.2CHOH----CH.sub.2CH.sub.2CH.sub.2----CHOHCH.sub.2CH.sub.2----CH.-
sub.2CHOHCH.sub.2-- and --CH.sub.2CH.sub.2CHOH--; and where y is an
integer selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and all
salts thereof.
2. A compound according to claim 1, where each R.sub.40 is
independently selected from the group consisting of
--CH.sub.2CH.sub.2CH.sub.2-- and
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--.
3. A compound according to claim 2, where each R.sub.40 is
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--.
4. A compound according to claim 3, where R.sub.30 and R.sub.50 are
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--.
5. A compound according to claim 4, where R.sub.90 and each
R.sub.80 are H.
6. A compound according to claim 5, where R.sub.10 is H, R.sub.20
is ethyl, R60 is H, and R.sub.70 is ethyl.
7. A compound according to claim 6, where y is an integer selected
from 5, 7, 9, 11, and 13.
8. A compound according to claim 7, where y is an integer selected
from 5, 9, and 11.
9. A compound according to claim 1 of the formula 30and all salts
thereof.
10. A compound according to claim 1 of the formula 31and all salts
thereof.
11. A compound according to claim 1 of the formula 32and all salts
thereof.
12. A compound according to claim 1 of the formula 33and all salts
thereof.
13. A compound according to claim 1, where R.sub.90 and each
R.sub.80 are independently selected from the group consisting of H,
methyl, and ethyl.
14. A compound according to claim 1, where R.sub.90 and each
R.sub.80 are H.
15. A compound according to claim 1, where R.sub.30, each R.sub.40,
and R.sub.50 are independently selected from:
--CH.sub.2CH.sub.2CH.sub.2CH.su-
b.2----CHOHCH.sub.2CH.sub.2CH.sub.2----CH.sub.2CHOHCH.sub.2CH.sub.2----CH.-
sub.2CH.sub.2CHOHCH.sub.2--, and
--CH.sub.2CH.sub.2CH.sub.2CHOH--.
16. A compound according to claim 11, where each R.sub.40 is
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.30 and R.sub.50 are
independently selected from:
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2----CHOHCH-
.sub.2CH.sub.2CH.sub.2----CH.sub.2CHOHCH.sub.2CH.sub.2----CH.sub.2CH.sub.2-
CHOHCH.sub.2--, and --CH.sub.2CH.sub.2CH.sub.2CHOH--.
17. A compound according to claim 16, where R.sub.30 and R.sub.50
are independently selected from:
--CHOHCH.sub.2CH.sub.2CH.sub.2----CH.sub.2CH-
OHCH.sub.2CH.sub.2----CH.sub.2CH.sub.2CHOHCH.sub.2--, and
--CH.sub.2CH.sub.2CH.sub.2CHOH--.
18. A compound according to claim 1, where R.sub.10, R.sub.20,
R.sub.60, and R.sub.70 are independently selected from H, methyl,
or ethyl.
19. A compound according to claim 18, where R.sub.10 is H and
R.sub.60 is H.
20. A compound according to claim 19, where R.sub.20 and R.sub.70
are ethyl.
21. A compound according to claim 1, with the proviso that at least
one R.sub.30, R.sub.40, or R.sub.50 is independently selected from:
--CHOHCH.sub.2CH.sub.2CH.sub.2----CH.sub.2CHOHCH.sub.2CH.sub.2----CH.sub.-
2CH.sub.2CHOHCH.sub.2----CH.sub.2CH.sub.2CH.sub.2CHOH----CHOHCH.sub.2CH.su-
b.2----CH.sub.2CHOHCH.sub.2-- and --CH.sub.2CH.sub.2CHOH--.
22. A compound according to claim 21, with the proviso that at
least one R.sub.30, R.sub.40, or R.sub.50 is independently selected
from:
--CHOHCH.sub.2CH.sub.2CH.sub.2----CH.sub.2CHOHCH.sub.2CH.sub.2----CH.sub.-
2CH.sub.2CHOHCH.sub.2--; and --CH.sub.2CH.sub.2CH.sub.2CHOH--.
23. A compound according to claim 22, with the proviso that at
least one of R.sub.30 and R.sub.50 is independently selected from:
--CHOHCH.sub.2CH.sub.2CH.sub.2----CH.sub.2CHOHCH.sub.2CH.sub.2----CH.sub.-
2CH.sub.2CHOHCH.sub.2--; and --CH.sub.2CH.sub.2CH.sub.2CHOH--.
24. A method of treating cancer in an individual, comprising the
step of administering a therapeutic amount of a compound according
to claim 1.
25. A method of treating cancer in an individual, comprising the
step of administering a therapeutic amount of a compound according
to claim 7.
26. A method of making a compound according to claim 1, comprising
the steps of: a) providing a first compound of the form
H--N(R.sub.90)--R.sub.51--CON(R.sub.60)(R.sub.70) where R.sub.90 is
independently selected from H, methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, and t-butyl; R.sub.60 and R.sub.70
are independently selected from H, methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl; R.sub.51 is
selected from the group consisting of
--CH.sub.2CH.sub.2CH.sub.2----CHO(PG.sub.Hy)CH.sub.2C-
H.sub.2----CH.sub.2CHO(PG.sub.Hy)CH.sub.2----CH.sub.2CH.sub.2CHO(PG.sub.Hy-
)----CH.sub.2CH.sub.2----CHO(PG.sub.Hy)CH.sub.2-- and --C
H.sub.2CHO(PG.sub.Hy)--where PG.sub.Hy is a hydroxy protecting
group; b) providing a second compound of the form
BG.sub.NN(R.sub.80)--R.sub.41--CO- OH where blocking group BG.sub.N
is selected from the group consisting of an amino protecting group
and methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
sec-butyl, and t-butyl; R.sub.4 is selected from the group
consisting of
--CH.sub.2CH.sub.2CH.sub.2----CHO(PG.sub.Hy)CH.sub.2C-
H.sub.2----CH.sub.2CHO(PG.sub.Hy)CH.sub.2----CH.sub.2CH.sub.2CHO(PG.sub.Hy-
)----CH.sub.2CH.sub.2----CHO(PG.sub.Hy)CH.sub.2-- and
--CH.sub.2CHO(PG.sub.Hy)--; c) activating the carboxyl group of the
second compound; d) coupling the second compound to the first
compound to form a compound of the formula
BG.sub.N[N(R.sub.80)--R.sub.41--CO].sub.g--
-N(R.sub.90)--R.sub.51--CON(R.sub.60)(R.sub.70) where g is 1; e)
repeating step c) and repeating the coupling step of step d) for
(g-1) additional cycles to form a compound of the formula
BG.sub.N[N(R.sub.80)--R.sub.41---
CO].sub.g--N(R.sub.90)--R.sub.51--CON(R.sub.60)(R.sub.70) where g
is an integer from 7 to 15; f) reducing the amide groups to amine
groups; and g) removing any protecting groups BG.sub.N and
PG.sub.Hy that may be present in the compound.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit of U.S. provisional
patent application No. 60/329,982, filed Oct. 16, 2001. The content
of that application is hereby incorporated herein by reference in
its entirety.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Not applicable.
TECHNICAL FIELD
[0003] This invention is directed to compounds and methods useful
for treating cancer and other diseases caused by uncontrolled cell
proliferation, and for treating microsporidiosis and other
infectious diseases. More specifically, this invention is directed
to oligoamine compounds which display anti-tumor activity in vitro
and in vivo, and which display anti-microspora activity as well as
methods of making and using those compounds.
BACKGROUND ART
[0004] Cancer is one of the leading causes of death in the world.
According to the World Health Organization, cancer is the third
most common cause of death in the world, after heart disease and
infectious disease. Cancer is the second most common cause of death
(after heart disease) in the developed world. Accordingly,
discovery of new and effective treatments for cancer is a high
priority for health care researchers.
[0005] Cancer is often treated by using chemotherapy to selectively
kill or hinder the growth of cancer cells, while having a less
deleterious effect on normal cells. Chemotherapeutic agents often
kill rapidly dividing cells, such as cancer cells; cells which are
dividing less rapidly are affected to a lesser degree. Other
agents, such as antibodies attached to toxic agents, have been
evaluated for use against cancers. These agents target the cancer
cells by making use of a characteristic specific to the cancer, for
example, higher-than-normal rates of cell division, or unique
antigens expressed on the cancer cell surface.
[0006] Various naturally-occurring and synthetic amine-containing
compounds have been evaluated for anti-cancer and antiproliferative
activity. The following patents and patent applications, all of
which are hereby incorporated by reference in their entirety,
discuss certain of these compounds: U.S. Pat. Nos. 5,541,230,
5,880,161, and 5,889,061; and International Patent Cooperation
Treaty Applications WO 00/66587, WO 98/17624, and WO 95/18091.
Other publications which discuss various amine-containing
compounds, for a wide variety of applications, include U.S. Pat.
No. 3,956,502, to polyamine alcohols as microbicides; U.S. Pat. No.
4,013,507 and U.S. Pat. No. 5,866,016, which relate to the
compounds commonly called ionenes; CA 2,231,200, which discusses
polyalkylimines as gene transport carriers; EP 889 112, directed to
a lubricating oil composition for automatic transmissions; U.S.
Pat. No. 4,971,598, directed to reaction products of alkenyl
succinimides with ethylenediamine carboxy acids as fuel detergents;
U.S. Pat. No. 5,091,576, which discusses anti-neoplastic,
anti-viral, or anti-retroviral spermine derivatives, and U.S. Pat.
No. 5,393,757, which discusses polyamines and anti-diarrheal and
gastrointestinal anti-spasmodic pharmaceutical compositions and
methods of treatment; WO 93/04036 and U.S. Pat. No. 5,185,369,
directed to synthetic aryl and heteroaryl polyamines as excitatory
amino acid neurotransmitter antagonists; U.S. Pat. No. 5,530,092
and U.S. Pat. No. 5,698,662, directed to dendritic macromolecules
and the preparation thereof; U.S. Pat. No. 5,750,788, which
discusses preparation of amines from compounds having at least
three cyano groups; U.S. Pat. No. 5,847,190, to dendritic
nitrogen-containing organic compounds; U.S. Pat. No. 6,046,282, to
reactive diluents for polyamidoamine epoxy curatives; WO 95/20580,
to macrocyclic octaaza compounds; WO 96/38528, directed to betaine
esters for the delivery of alcohols; WO 97/07674, WO 98/51660, and
WO 99/17802, to ethyleneimine oligomers for selective modification
of nucleic acids; WO 00/09634, to diesel fuels comprising
hydrocarbyl amines; WO 01/64779, to polyamine polyoxides used as
asphalt emulsifiers; WO 01/79329, to oligopolysuccinimides; Bruice
T C et al., "A microgonotropen branched decaaza decabutylamine and
its DNA and DNA/transcription factor interactions," Bioorg Med
Chem. 5(4):685-92 (1997); Satz A L and Bruice T C, "Recognition in
the minor groove of double-stranded DNA by microgonotropens,: Acc.
Chem. Res. 35(2):86-95 (2002); Kroger N et al., "Species-specific
polyamines from diatoms control silica morphology," Proc. Natl.
Acad. Sci. USA 97(26):14133-8 (2000); Bacchi C J et al., "Novel
synthetic polyamines are effective in the treatment of experimental
microsporidiosis, an opportunistic AIDS-associated infection,"
Antimicrob. Agents Chemother. 46(1):55-61 (2002); and Bacchi C J et
al., "SL-11158, a synthetic oligoamine, inhibits polyamine
metabolism of Encephalitozoon cuniculi," J. Eukaryot. Microbiol.
Suppl:92S-94S (2001).
[0007] Despite intensive research aimed at finding effective
treatments for cancer, is well-known that, while some cancers can
be treated with relative success, no effective treatments exist for
other cancers. Thus, there is a need for additional pharmaceutical
agents to complement the medicinal remedies currently available for
treatment of cancer and diseases characterized by uncontrolled cell
proliferation.
[0008] In addition to treatment of cancer, the oligoamine compounds
of the present invention are also useful for treatment of diseases
caused by microorganisms such as bacteria, viruses, and parasites.
See Bacchi C J et al., "Novel synthetic polyamines are effective in
the treatment of experimental microsporidiosis, an opportunistic
AIDS-associated infection," Antimicrob. Agents Chemother.
46(1):55-61 (2002); and Bacchi C J et al., "SL-11158, a synthetic
oligoamine, inhibits polyamine metabolism of Encephalitozoon
cuniculi," J. Eukaryot. Microbiol. Suppl:92S-94S (2001).
Microsporidiosis refers to infections caused by any of the
parasitic protists of the phylum Microspora; over 140 genera and
1200 species of microsporidia are known, and at least 14 of these
species can cause pathology in humans: Enterocytozoon bieneusi,
Encephalitozoon intestinalis (previously known as Septata
intestinalis), Encephalitozoon hellem, Encephalitozoon cuniculi,
Pleistophora sp., Trachipleistophora hominis, T. anthropophthera,
Nosema ocularum, N. algerae, Vittaforma corneae, Microsporidium
ceylonensis, M. africanum, Brachiola vesicularum, and B. connori..
(See Centers for Disease Control information at World Wide Web URL
www.dpd.cdc.gov/dpdx/HTML/Microsporidiosis.htm). Microsporidiosis
is most prevalent in immunocompromised hosts, such as patients with
AIDS and HIV-related diseases, or transplant recipients on
immunosuppressive therapy. Healthy hosts appear to harbor
asymptomatic or self-limiting microsporidiosis. Symptoms of
microsporidiosis include diarrhea and other gastrointestinal
complications, muscle infections, genitourinary infections,
respiratory infections, and eye infections.
[0009] While pharmaceutical treatments currently exist for
microsporidiosis, such as albendazole and metronidazole (Flagyl),
not all treatments are effective against every pathogen, and
undesirable side effects to specific medicines can occur in certain
individuals. Thus, additional medicinal agents are needed to
complement the treatments currently available for microsporidiosis
and other diseases caused by microbes.
DISCLOSURE OF THE INVENTION
[0010] The invention is directed to oligoamine compounds and
derivatives thereof, methods of making them, and methods of using
them.
[0011] In particular, the invention embraces compounds of the
formula: 1
[0012] where
[0013] R.sub.1 is independently selected from the group consisting
of H and C.sub.1-C.sub.4 linear alkyl; R.sub.2 is independently
selected from the group consisting of H and C.sub.1-C.sub.4 linear
alkyl; each R.sub.3 is independently selected from the group
consisting of C.sub.1-C.sub.8 linear alkyl; each R.sub.4is
independently selected from the group consisting of H and
C.sub.1-C.sub.4 linear alkyl; R.sub.5 is independently selected
from the group consisting of H and C.sub.1-C.sub.4 linear alkyl; m
is an integer between 7 and 15, inclusive; and all salts
thereof.
[0014] In one embodiment, R.sub.1 is H. In another embodiment,
R.sub.2 is H. In another embodiment, both R.sub.1 and R.sub.2 are
H.
[0015] In another embodiment, at least one of R.sub.5 and the
R.sub.4 moiety bonded to the nitrogen to which R.sub.5 is also
bonded is H. In another embodiment, both R.sub.5 and the R.sub.4
moiety bonded to the nitrogen to which R.sub.5 is also bonded are
H. In another embodiment, each R.sub.4 is H. In another embodiment,
R.sub.1 is ethyl.
[0016] In another embodiment, R.sub.1 is ethyl and R.sub.2 is H. In
another embodiment, R.sub.5 is ethyl. In another embodiment,
R.sub.5 is ethyl and the R.sub.4 moiety bonded to the nitrogen to
which R.sub.5 is also bonded is H.
[0017] In another embodiment, R.sub.1 is ethyl, R.sub.2 is H,
R.sub.5 is ethyl and the R.sub.4 moiety bonded to the nitrogen to
which R.sub.5 is also bonded is H.
[0018] In yet another embodiment, each R.sub.3 is independently
selected from the group consisting of C.sub.3-C.sub.4 linear alkyl.
In yet another embodiment, each R.sub.3 is C.sub.3 linear alkyl. In
yet another embodiment, each R.sub.3 is C.sub.4 linear alkyl.
[0019] In other embodiments, m is 7. In other embodiments, m is 9.
In other embodiments, m is 11. In other embodiments, m is 13. In
other embodiments, m is 15.
[0020] The invention also embraces compounds of the formula: 2
[0021] where
[0022] R.sub.1 is independently selected from the group consisting
of H and C.sub.1-C.sub.4 linear alkyl; R.sub.2 is independently
selected from the group consisting of H and C.sub.1-C.sub.4 linear
alkyl; each R.sub.3 is independently selected from the group
consisting of C.sub.1-C.sub.8 linear hydroxyalkyl and
C.sub.1-C.sub.8 linear alkyl, with the proviso that at least one
R.sub.3 is C.sub.1-C.sub.8 linear hydroxyalkyl; each R.sub.4 is
independently selected from the group consisting of H and
C.sub.1-C.sub.4 linear alkyl; R.sub.5 is independently selected
from the group consisting of H and C.sub.1-C.sub.4 linear alkyl; m
is an integer between 7 and 15, inclusive; and all salts
thereof.
[0023] In one embodiment, at least one of R.sub.1 and R.sub.2 is H.
In another embodiment, both R.sub.1 and R.sub.2 are H. In another
embodiment, at least one of R.sub.5 and the R.sub.4 moiety bonded
to the nitrogen to which R.sub.5 is also bonded is H. In another
embodiment, each R.sub.4is H.
[0024] In another embodiment, R.sub.1 is ethyl. In another
embodiment, R.sub.1 is ethyl and R.sub.2 is H. In another
embodiment, R.sub.5 is ethyl. In another embodiment, R.sub.5 is
ethyl and the R.sub.4 moiety bonded to the nitrogen to which
R.sub.5 is also bonded is H.
[0025] In another embodiment, R.sub.1 is ethyl, R.sub.2 is H,
R.sub.5 is ethyl and the R.sub.4 moiety bonded to the nitrogen to
which R.sub.5 is also bonded is H.
[0026] In yet another embodiment, each R.sub.3 is independently
selected from the group consisting of C.sub.3-C.sub.4 linear
hydroxyalkyl and C.sub.3-C.sub.4 linear alkyl, with the proviso
that at least one R.sub.3 is C.sub.3-C.sub.4 linear hydroxyalkyl.
In yet another embodiment, each R.sub.3 is independently selected
from C.sub.3 linear hydroxyalkyl and C.sub.3 linear alkyl, with the
proviso that at least one R.sub.3 is C.sub.3 linear hydroxyalkyl.
In yet another embodiment, each R.sub.3 is independently selected
from C.sub.4 linear hydroxyalkyl and C.sub.4 linear alkyl, with the
proviso that at least one R.sub.3 is C.sub.4 linear
hydroxyalkyl.
[0027] In other embodiments, m is 7. In other embodiments, m is 9.
In other embodiments, m is 11. In other embodiments, m is 13. In
other embodiments, m is 15.
[0028] In additional embodiments, the alkyl segment flanked by the
two leftmost nitrogens of the compound contains a hydroxyalkyl
group. In additional embodiments, the alkyl segment flanked by the
two leftmost nitrogens of the compound contains the only
hydroxyalkyl group in the molecule. That is, when the structure is
drawn out in its entirety, the first R.sub.3 group encountered when
reading left to right is a hydroxyalkyl group, or the only
hydroxyalkyl group. In additional embodiments, the alkyl segment
flanked by the two rightmost nitrogens of the compound contains a
hydroxyalkyl group. In additional embodiments, the alkyl segment
flanked by the two rightmost nitrogens of the compound contains the
only hydroxyalkyl group in the molecule. That is, when the
structure is drawn out in its entirety, the last R.sub.3 group
encountered when reading left to right is a hydroxyalkyl group, or
the only hydroxyalkyl group.
[0029] In another embodiment, the invention embraces compounds of
the formula: 3
[0030] where
[0031] R.sub.10, R.sub.20, R.sub.69, and R.sub.70 are independently
selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl, and t-butyl;
[0032] where each R.sub.80 and R.sub.90 are independently selected
from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
sec.-butyl, and t-butyl;
[0033] where R.sub.30, each R.sub.40, and R.sub.50 are
independently selected from:
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--
--CHOHCH.sub.2CH.sub.2CH.sub.2--
--CH.sub.2CHOHCH.sub.2CH.sub.2--
--CH.sub.2CH.sub.2CHOHCH.sub.2--
--CH.sub.2CH.sub.2CH.sub.2CHOH--
--CH.sub.2CH.sub.2CH.sub.2--
--CHOHCH.sub.2CH.sub.2--
--CH.sub.2CHOHCH.sub.2--
--CH.sub.2CH.sub.2CHOH;--
[0034] and where y is an integer selected from 5, 6, 7, 8, 9, 10,
11, 12, and 13; and all salts thereof.
[0035] In one embodiment, at least one R.sub.30, R.sub.40, or
R.sub.50 is independently selected from:
--CHOHCH.sub.2CH.sub.2CH.sub.2--
--CH.sub.2CHOHCH.sub.2CH.sub.2--
--CH.sub.2CH.sub.2CHOHCH.sub.2--
--CH.sub.2CH.sub.2CH.sub.2CHOH--
--CHOHCH.sub.2CH.sub.2--
--CH.sub.2CHOHCH.sub.2-- and
--CH.sub.2CH.sub.2CHOH--.
[0036] In another embodiment, at least one R.sub.30, R.sub.40, or
R.sub.50 is independently selected from:
--CHOHCH.sub.2CH.sub.2CH.sub.2--
--CH.sub.2CHOHCH.sub.2CH.sub.2--
--CH.sub.2CH.sub.2CHOHCH.sub.2--; and
--CH.sub.2CH.sub.2CH.sub.2CHOH--.
[0037] In another embodiment, at least one of R.sub.30 and R.sub.50
is independently selected from:
--CHOHCH.sub.2CH.sub.2CH.sub.2--
--CH.sub.2CHOHCH.sub.2CH.sub.2--
--CH.sub.2CH.sub.2CHOHCH.sub.2--; and
--CH.sub.2CH.sub.2CH.sub.2CHOH--.
[0038] In another embodiment, each R.sub.40 is independently
selected from the group consisting of --CH.sub.2CH.sub.2CH.sub.2--
and --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. In another embodiment,
R.sub.30 is independently selected from the group consisting of
--CH.sub.2CH.sub.2CH.sub.2-- and
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. In another embodiment,
R.sub.50 is independently selected from the group consisting of
--CH.sub.2CH.sub.2CH.sub.2-- and --CH.sub.2CH.sub.2CH.sub.2-
CH.sub.2--. In another embodiment, R.sub.30 and R.sub.50 are
independently selected from the group consisting of
--CH.sub.2CH.sub.2CH.sub.2-- and
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. In another embodiment,
R.sub.40 and R.sub.50 are independently selected from the group
consisting of --CH.sub.2CH.sub.2CH.sub.2-- and
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. In another embodiment,
R.sub.30 and R.sub.40 are independently selected from the group
consisting of --CH.sub.2CH.sub.2CH.sub.2-- and
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. In another embodiment,
R.sub.30, each R.sub.40, and R.sub.50 are independently selected
from the group consisting of --CH.sub.2CH.sub.2CH.sub.2-- and
--CH.sub.2CH.sub.2CH.sub.2- CH.sub.2--. In one embodiment of the
foregoing embodiments, y=5, 7, 9, 11, or 13. In yet another
embodiment, y=6, 8, 10, or 12. In yet another embodiment, y=5, 7,
9, or 11. In yet another embodiment, y=5. In yet another
embodiment, y=7. In yet another embodiment, y=9. In yet another
embodiment, y=11.
[0039] In another embodiment, each R.sub.40 is
--CH.sub.2CH.sub.2CH.sub.2C- H.sub.2--. In another embodiment,
R.sub.30 is --CH.sub.2CH.sub.2CH.sub.2CH- .sub.2--. In another
embodiment, R.sub.50 is --CH.sub.2CH.sub.2CH.sub.2CH.- sub.2--. In
another embodiment, R.sub.30 and R.sub.50 are
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. In another embodiment, each
R.sub.40 and R.sub.50 are --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. In
another embodiment, R.sub.30 and each R.sub.40 are
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. In another embodiment,
R.sub.30, each R.sub.40, and R.sub.50 are
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. In one embodiment of the
foregoing embodiments, y=5, 7, 9, 11, or 13. In yet another
embodiment, y=6, 8, 10, or 12. In yet another embodiment, y=5, 7,
9, or 11. In yet another embodiment, y=5. In yet another
embodiment, y=7. In yet another embodiment, y=9. In yet another
embodiment, y=11.
[0040] In another embodiment, each R.sub.40 is
--CH.sub.2CH.sub.2CH.sub.2-- -. In another embodiment, R.sub.30 is
--CH.sub.2CH.sub.2CH.sub.2--. In another embodiment, R.sub.50 is
--CH.sub.2CH.sub.2CH.sub.2--. In another embodiment, R.sub.30 and
R.sub.50 are --CH.sub.2CH.sub.2CH.sub.2--. In another embodiment,
each R.sub.40 and R.sub.50 are --CH.sub.2CH.sub.2CH.sub.2--. In
another embodiment, R.sub.30 and each R.sub.40 are
--CH.sub.2CH.sub.2CH.sub.2--. In another embodiment, R.sub.30, each
R.sub.40, and R.sub.50 are --CH.sub.2CH.sub.2CH.sub.2--. In one
embodiment of the foregoing embodiments, y=5, 7, 9, 11, or 13. In
yet another embodiment, y=6, 8, 10, or 12. In yet another
embodiment, y=5, 7, 9, or 11. In yet another embodiment, y=5. In
yet another embodiment, y=7. In yet another embodiment, y=9. In yet
another embodiment, y=11.
[0041] In one embodiment, R.sub.10, R.sub.20, R.sub.60, and
R.sub.70 are independently selected from H, methyl, ethyl,
n-propyl, and n-butyl. In another embodiment, each R.sub.80 and
R.sub.90 are independently selected from H, methyl, ethyl,
n-propyl, and n-butyl. In another embodiment, R.sub.10, R.sub.20,
R60, and R.sub.70 are independently selected from H, methyl, ethyl,
n-propyl, and n-butyl, and each R.sub.80 and R.sub.90 are
independently selected from H, methyl, ethyl, n-propyl, and
n-butyl.
[0042] In another embodiment, R.sub.90 and each R.sub.80 are H. In
another embodiment, R.sub.10 is H, R.sub.20 is ethyl, R.sub.60 is
H, and R.sub.70 is ethyl. In one embodiment of the foregoing
embodiments, y=5, 7, 9, 11, or 13. In yet another embodiment, y=6,
8, 1 0, or 12. In yet another embodiment, y=5, 7, 9, or 11. In yet
another embodiment, y=5. In yet another embodiment, y=7. In yet
another embodiment, y=9. In yet another embodiment, y=11.
[0043] In another embodiment, R.sub.30, each R.sub.40, and R.sub.50
are --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and R.sub.90 and each
R.sub.80 are H. In another embodiment, R.sub.30, each R.sub.40, and
R.sub.50 are --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and R.sub.10 is
H, R.sub.20 is ethyl, R.sub.60 is H, and R.sub.70 is ethyl. In
another embodiment, R.sub.30, each R.sub.40, and R.sub.50 are
--CH.sub.2CH.sub.2CH.sub.2CH.su- b.2--, R.sub.90 and each R.sub.80
are H, and R.sub.10 is H, R.sub.20 is ethyl, R.sub.60 is H, and
R.sub.70 is ethyl. In one embodiment of the foregoing embodiments,
y=5, 7, 9, 11, or 13. In yet another embodiment, y=6, 8, 10, or 12.
In yet another embodiment, y=5, 7, 9, or 11. In yet another
embodiment, y=5. In yet another embodiment, y=7. In yet another
embodiment, y=9. In yet another embodiment, y=11.
[0044] In another embodiment, the invention embraces compounds of
the formula:
CH.sub.3CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2(NHCH.sub.2CH.-
sub.2CH.sub.2CH.sub.2).sub.yNHCH.sub.2CH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH-
.sub.3 where y=5, 6, 7, 8, 9, 10, 11, 12, or 13. In yet another
embodiment, y=5, 7, 9, 11, or 13. In yet another embodiment, y=6,
8, 10, or 12. In yet another embodiment, y=5, 7, 9, or 11. In yet
another embodiment, y=5. In yet another embodiment, y=7. In yet
another embodiment, y=9. In yet another embodiment, y=11.
[0045] In another embodiment, the invention embraces compounds of
the formula:
CH.sub.3CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2(NHCH.sub.2CH.sub.2CH.-
sub.2).sub.yNHCH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.3 where y=5,
6, 7, 8, 9, 10, 11, 12, or 13. In yet another embodiment, y=5, 7,
9, 11, or 13. In yet another embodiment, y=6, 8, 10, or 12. In yet
another embodiment, y=5, 7, 9, or 11. In yet another embodiment,
y=5. In yet another embodiment, y=7. In yet another embodiment,
y=9. In yet another embodiment, y=11.
[0046] In another embodiment, the invention embraces the
compounds
CH.sub.3CH.sub.2--NH--(CH.sub.2CH.sub.2CH.sub.2CH.sub.2--NH--).sub.9--CH.s-
ub.2CH.sub.3,
CH.sub.3CH.sub.2--NH--(CH.sub.2CH.sub.2CH.sub.2CH.sub.2--NH--).sub.7--CH.s-
ub.2CH.sub.3,
CH.sub.3CH.sub.2--NH--(CH.sub.2CH.sub.2CH.sub.2CH.sub.2--NH--).sub.13--CH.-
sub.2CH.sub.3, and
CH.sub.3CH.sub.2--NH--(CH.sub.2CH.sub.2CH.sub.2CH.sub.2--NH--).sub.11--CH.-
sub.2CH.sub.3, and all salts thereof.
[0047] The invention also embraces methods of making the compounds
described above, by protecting the amino group of an alkylamine in
such a manner that it can undergo one, and only one, alkylation
reaction at the amino group (such as protection with
mesitylenesulfonyl chloride), reacting the protected alkylamine
with a haloalkylnitrile compound, reducing the nitrile group to an
amino group, repeating the protection, reaction, and reduction
steps as desired until the desired chain length is generated, and
optionally reacting the last nitrogen to be added with an alkyl
halide to terminate the synthesis.
[0048] The invention also embraces methods of making the compounds
by preparing a linear amide chain, where the linear amide chain is
formed by protecting the nitrogen of an .omega.-amino acid,
reacting the carboxyl group of the protected co-amino acid with an
alkylamine to form an amino-protected co-amino amide; deprotecting
the amino group of the co-amino amide; and reacting the amino group
of the c-amino amide with the carboxyl group of a second co-amino
acid to form a peptide bond. This latter reaction can be repeated
as desired until the desired chain length is reached. The final
nitrogen added to the polyamide compound can optionally be reacted
with an alkanoic acid. Finally, the polyamide compound can be
reduced to a polyamine compound.
[0049] In another embodiment, the invention also embraces a method
of making a compound 4
[0050] where
[0051] R.sub.10, R.sub.20, R.sub.30, R.sub.40, R.sub.50, R.sub.60,
R.sub.70, R.sub.80, and R.sub.90 are as defined in the various
embodiments above, comprising the steps of:
[0052] a) providing a first compound of the form
H--N(R.sub.90)--R.sub.51--C(.dbd.O)N(R.sub.60)(R.sub.70)
[0053] where R.sub.51 is selected from the group consisting of
--CH.sub.2CH.sub.2CH.sub.2--
--CHO(PG.sub.Hy)CH.sub.2CH.sub.2--
--CH.sub.2CHO(PG.sub.Hy)CH.sub.2--
--CH.sub.2CH.sub.2CHO(PG.sub.Hy)--
--CH.sub.2CH.sub.2--
--CHO(PG.sub.Hy)CH.sub.2--and
--CH.sub.2CHO(PG.sub.Hy)--
[0054] where PG.sub.Hy is a hydroxy protecting group;
[0055] b) providing a second compound of the form
BG.sub.NN(R.sub.80)--R.sub.41--COOH
[0056] where blocking group BG.sub.N is selected from the group
consisting of an amino protecting group and methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl;
[0057] R.sub.80 is as defined in the various embodiments above;
[0058] R.sub.41 is selected from the group consisting of
--CH.sub.2CH.sub.2CH.sub.2--
--CHO(PG.sub.Hy)CH.sub.2CH.sub.2--
--CH.sub.2CHO(PG.sub.Hy)CH.sub.2--
--CH.sub.2CH.sub.2CHO(PG.sub.Hy)--
--CH.sub.2CH.sub.2--
--CHO(PG.sub.Hy)CH.sub.2-- and
--CH.sub.2CHO(PG.sub.Hy)--;
[0059] c) activating the carboxyl group of the second compound;
[0060] d) coupling the second compound to the first compound to
form a compound of the formula
BG.sub.N[N(R.sub.80)--R.sub.41--CO].sub.g--N(R.sub.90)--R.sub.51--CON(R.su-
b.60)(R.sub.70)
[0061] where g is 1;
[0062] e) repeating step c) and repeating the coupling step of step
d) for (g-1) additional cycles to form a compound of the
formula
BG.sub.N[N(R.sub.80)--R.sub.41--CO].sub.g--N(R.sub.90)--R.sub.51--CON(R.su-
b.60)(R.sub.70)
[0063] where g is an integer from 7 to 15;
[0064] f) reducing the amide groups to amine groups; and
[0065] g) removing any protecting groups BG.sub.N and PG.sub.Hy
that may be present in the compound. The resulting compound may
optionally be purified by any method known in the art, such as
column chromatography, ion-exchange chromatography, HPLC, or
thin-layer chromatography.
[0066] The invention also provides methods of treating diseases
characterized by uncontrolled cell proliferation, such as cancer,
including, but not limited to, prostate cancer and breast cancer,
by administration of one or more of the compounds described above.
The invention also includes compositions of one or more of the
compounds described above in combination with a
pharmaceutically-acceptable carrier, and/or with another
therapeutic agent. Examples of compounds of the invention which can
be used for the treatment of diseases characterized by uncontrolled
cell proliferation, e.g. cancer, such as prostate cancer and breast
cancer,
[0067] are
CH.sub.3CH.sub.2--NH--(CH.sub.2CH.sub.2CH.sub.2CH.sub.2--NH--).sub.9--CH.s-
ub.2CH.sub.3,
CH.sub.3CH.sub.2--NH--(CH.sub.2CH.sub.2CH.sub.2CH.sub.2--NH--).sub.7--CH.s-
ub.2CH.sub.3,
CH.sub.3CH.sub.2--NH--(CH.sub.2CH.sub.2CH.sub.2CH.sub.2--NH--).sub.13--CH.-
sub.2CH.sub.3, or
CH.sub.3CH.sub.2--NH--(CH.sub.2CH.sub.2CH.sub.2CH.sub.2--NH--).sub.11--CH.-
sub.2CH.sub.3,
[0068] or any salt thereof
[0069] The invention also embraces a method of treating
microsporidiosis and AIDS-associated infections, by administration
of one or more of the compounds described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] FIG. 1 depicts the effects of SL-11159, SL-11160, SL-11175,
and SL-11226 on survival of DuPro cancer cells.
[0071] FIG. 2 depicts the effect of SL-11159 cytotoxicity on PC-3
cancer cells after 5 days incubation.
BEST MODE FOR CARRYING OUT THE INVENTION
[0072] The invention is directed to various novel oligoamine
compounds and derivatives thereof as described herein. The
invention includes all salts of the compounds described herein.
Particularly preferred are pharmaceutically acceptable salts.
Pharmaceutically acceptable salts are those salts which retain the
biological activity of the free bases and which are not
biologically or otherwise undesirable. The desired salt may be
prepared by methods known to those of skill in the art by treating
the polyamine with an acid. Examples of inorganic acids include,
but are not limited to, hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, and phosphoric acid. Examples of
organic acids include, but are not limited to, formic acid, acetic
acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
sulfonic acids, and salicylic acid. Salts of the polyamines with
amino acids, such as aspartate salts and glutamate salts, can also
be prepared.
[0073] The invention also includes all stereoisomers of the
compounds, including diastereomers and enantiomers, as well as
mixtures of stereoisomers, including, but not limited to, racemic
mixtures. Unless stereochemistry is explicitly indicated in a
structure, the structure is intended to embrace all possible
stereoisomers of the compound depicted.
[0074] The term "alkyl" refers to saturated aliphatic groups
including straight-chain, branched-chain, cyclic groups, and
combinations thereof, having the number of carbon atoms specified,
or if no number is specified, having up to 12 carbon atoms.
"Straight-chain alkyl" or "linear alkyl" groups refers to alkyl
groups that are neither cyclic nor branched, commonly designated as
"n-alkyl" groups. Examples of alkyl groups include, but are not
limited to, groups such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, neopentyl,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl.
Cyclic groups can consist of one ring, including, but not limited
to, groups such as cycloheptyl, or multiple fused rings, including,
but not limited to, groups such as adamantyl or norbornyl.
[0075] "Substituted alkyl" refers to alkyl groups substituted with
one or more substituents including, but not limited to, groups such
as halogen (fluoro, chloro, bromo, and iodo), alkoxy, acyloxy,
amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl,
cyano, nitro, thioalkoxy, carboxaldehyde, carboalkoxy and
carboxamide, or a functionality that can be suitably blocked, if
necessary for purposes of the invention, with a protecting group.
Examples of substituted alkyl groups include, but are not limited
to, --CF.sub.3, --CF.sub.2--CF.sub.3, and other perfluoro and
perhalo groups.
[0076] "Hydroxyalkyl" specifically refers to alkyl groups having
the number of carbon atoms specified substituted with one --OH
group. Thus, "C.sub.3 linear hydroxyalkyl" refers to
--CH.sub.2CH.sub.2CHOH--, --CH.sub.2CHOHCH.sub.2--, and
--CHOHCH.sub.2CH.sub.2--. "C.sub.4 linear hydroxyalkyl" refers to
--CH.sub.2CH.sub.2CH.sub.2CHOH--, --CH.sub.2CH.sub.2CHOHCH.sub.2--,
--CH.sub.2CHOHCH.sub.2CH.sub.2--, and
--CHOHCH.sub.2CH.sub.2CH.sub.2--. Note that, for example,
--CH.sub.2CHOHCH.sub.2CH.sub.2-- is understood to include both the
fragment: 5
[0077] and the fragment: 6
[0078] where the wavy lines indicate the attachment of the fragment
to the remainder of the molecule; and similarly for the other
hydroxyalkyl diradicals.
[0079] The term "alkenyl" refers to unsaturated aliphatic groups
including straight-chain (linear), branched-chain, cyclic groups,
and combinations thereof, having the number of carbon atoms
specified, or if no number is specified, having up to 12 carbon
atoms, which contain at least one double bond (--C.dbd.C--).
Examples of alkenyl groups include, but are not limited to,
--CH.sub.2--CH.dbd.CH--CH.sub.3; and
--CH.sub.2--CH.sub.2-cyclohexenyl, where the ethyl group can be
attached to the cyclohexenyl moiety at any available carbon
valence. The term "alkynyl" refers to unsaturated aliphatic groups
including straight-chain (linear), branched-chain, cyclic groups,
and combinations thereof, having the number of carbon atoms
specified, or if no number is specified, having up to 12 carbon
atoms, which contain at least one triple bond (--C.ident.C--).
"Hydrocarbon chain" or "hydrocarbyl" refers to any combination of
straight-chain, branched-chain, or cyclic alkyl, alkenyl, or
alkynyl groups, and any combination thereof. "Substituted alkenyl,"
"substituted alkynyl," and "substituted hydrocarbon chain" or
"substituted hydrocarbyl" refer to the respective group substituted
with one or more substituents, including, but not limited to,
groups such as halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto,
carboxy, benzyloxy, phenyl, benzyl, cyano, nitro, thioalkoxy,
carboxaldehyde, carboalkoxy and carboxamide, or a functionality
that can be suitably blocked, if necessary for purposes of the
invention, with a protecting group.
[0080] "Aryl" or "Ar" refers to an aromatic carbocyclic group
having a single ring (including, but not limited to, groups such as
phenyl) or multiple condensed rings (including, but not limited to,
groups such as naphthyl or anthryl), and includes both
unsubstituted and substituted aryl groups. "Substituted aryls"
refers to aryls substituted with one or more substituents,
including, but not limited to, groups such as alkyl, alkenyl,
alkynyl, hydrocarbon chains, halogen, alkoxy, acyloxy, amino,
hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano,
nitro, thioalkoxy, carboxaldehyde, carboalkoxy and carboxamide, or
a functionality that can be suitably blocked, if necessary for
purposes of the invention, with a protecting group.
[0081] "Heteroalkyl," "heteroalkenyl," and "heteroalkynyl" refer to
alkyl, alkenyl, and alkynyl groups, respectively, that contain the
number of carbon atoms specified (or if no number is specified,
having up to 12 carbon atoms) which contain one or more heteroatoms
as part of the main, branched, or cyclic chains in the group.
Heteroatoms include, but are not limited to, N, S, O, and P; N and
O are preferred. Heteroalkyl, heteroalkenyl, and heteroalkynyl
groups may be attached to the remainder of the molecule either at a
heteroatom (if a valence is available) or at a carbon atom.
Examples of heteroalkyl groups include, but are not limited to,
groups such as --O--CH.sub.3, --CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--O--CH.sub.3,
--S--CH.sub.2--CH.sub.2--CH.sub.3,
--CH.sub.2--CH(CH.sub.3)--S--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.2- --CH.sub.2--,
1-ethyl-6-propylpiperidino, 2-ethylthiophenyl, and morpholino.
Examples of heteroalkenyl groups include, but are not limited to,
groups such as --CH.dbd.CH--NH--CH(CH.sub.3)--CH.sub.2--.
"Heteroaryl" or "HetAr" refers to an aromatic carbocyclic group
having a single ring (including, but not limited to, examples such
as pyridyl, thiophene, or furyl) or multiple condensed rings
(including, but not limited to, examples such as imidazolyl,
indolizinyl or benzothienyl) and having at least one hetero atom,
including, but not limited to, heteroatoms such as N, O, P, or S,
within the ring. Unless otherwise specified, heteroalkyl,
heteroalkenyl, heteroalkynyl, and heteroaryl groups have between
one and five heteroatoms and between one and twenty carbon atoms.
"Substituted heteroalkyl," "substituted heteroalkenyl,"
"substituted heteroalkynyl," and "substituted heteroaryl" groups
refer to heteroalkyl, heteroalkenyl, heteroalkynyl, and heteroaryl
groups substituted with one or more substituents, including, but
not limited to, groups such as alkyl, alkenyl, alkynyl, benzyl,
hydrocarbon chains, halogen, alkoxy, acyloxy, amino, hydroxyl,
mercapto, carboxy, benzyloxy, phenyl, benzyl, cyano, nitro,
thioalkoxy, carboxaldehyde, carboalkoxy and carboxamide, or a
functionality that can be suitably blocked, if necessary for
purposes of the invention, with a protecting group. Examples of
such substituted heteroalkyl groups include, but are not limited
to, piperazine, substituted at a nitrogen or carbon by a phenyl or
benzyl group, and attached to the remainder of the molecule by any
available valence on a carbon or nitrogen, --NH--SO.sub.2-phenyl,
--NH--(C.dbd.O)O-alkyl, --NH--(C.dbd.O)O-alkyl-aryl, and
--NH--(C.dbd.O)-alkyl. If chemically possible, the heteroatom(s) as
well as the carbon atoms of the group can be substituted. The
heteroatom(s) can also be in oxidized form, if chemically
possible.
[0082] The term "alkylaryl" refers to an alkyl group having the
number of carbon atoms designated, appended to one, two, or three
aryl groups.
[0083] The term "alkoxy" as used herein refers to an alkyl,
alkenyl, alkynyl, or hydrocarbon chain linked to an oxygen atom and
having the number of carbon atoms specified, or if no number is
specified, having up to 12 carbon atoms. Examples of alkoxy groups
include, but are not limited to, groups such as methoxy, ethoxy,
and t-butoxy.
[0084] The term "alkanoate" as used herein refers to an ionized
carboxylic acid group, such as acetate
(CH.sub.3C(.dbd.O)--O.sup.(-1)), propionate
(CH.sub.3CH.sub.2C(.dbd.O)--O.sup.(-1)), and the like. "Alkyl
alkanoate" refers to a carboxylic acid esterified with an alkoxy
group, such as ethyl acetate
(CH.sub.3C(.dbd.O)--O--CH.sub.2CH.sub.3). "co-haloalkyl alkanoate"
refers to an alkyl alkanoate bearing a halogen atom on the
alkanoate carbon atom furthest from the carboxyl group; thus, ethyl
.omega.-bromo propionate refers to ethyl 3-bromopropionate, methyl
.omega.-chloro n-butanoate refers to methyl 4-chloro n-butanoate,
etc.
[0085] The terms "halo" and "halogen" as used herein refer to Cl,
Br, F or I substituents.
[0086] "Protecting group" refers to a chemical group that exhibits
the following characteristics: 1) reacts selectively with the
desired functionality in good yield to give a protected substrate
that is stable to the projected reactions for which protection is
desired; 2) is selectively removable from the protected substrate
to yield the desired functionality; and 3) is removable in good
yield by reagents compatible with the other functional group(s)
present or generated in such projected reactions. Examples of
suitable protecting groups can be found in Greene et al. (1991)
Protective Groups in Organic Synthesis, 2nd Ed. (John Wiley &
Sons, Inc., New York). Amino protecting groups include, but are not
limited to, mesitylenesulfonyl (Mes), benzyloxycarbonyl (CBz or Z),
t-butyloxycarbonyl (Boc), t-butyldimethylsilyl (TBDIMS or TBDMS),
9-fluorenylmethyloxycarbonyl (Fmoc), tosyl, benzenesulfonyl,
2-pyridyl sulfonyl, or suitable photolabile protecting groups such
as 6-nitroveratryloxy carbonyl (Nvoc), nitropiperonyl,
pyrenylmethoxycarbonyl, nitrobenzyl, dimethyl dimethoxybenzil,
5-bromo-7-nitroindolinyl, and the like. Hydroxyl protecting groups
include, but are not limited to, t-butyl, benzyl, trityl, Fmoc,
TBDIMS, photolabile protecting groups (such as nitroveratryl
oxymethyl ether (Nvom)), Mom (methoxy methyl ether), and Mem
(methoxy ethoxy methyl ether), NPEOC (4-nitrophenethyloxycarbonyl)
and NPEOM (4-nitrophenethyloxymethyloxycarbonyl).
Synthetic Methods for Production of Oligoamine Compounds
[0087] Three general methods are presented herein for synthesizing
the oligoamine compounds of the invention.
[0088] 1. The first method involves repetitive addition of
bromocyanoalkyl compounds to mesitylsulfonylated alkylamines as
follows: the amino group of an alkylamine compound IA (such as
methylamine, ethylamine, n-propylamine, n-butylamine,
sec-butylamine, t-butylamine, and other alkylamine compounds, which
are commercially available from Aldrich Chemical Company,
Milwaukee, Wis., and other suppliers) is protected, e.g. by
reaction with mesitylenesulfonyl chloride (available from Aldrich
Chemical Company and other suppliers) to form a protected
alkylamine IIA. The protected alkylamine IIA is treated with base,
e.g.. sodium hydride in anhydrous dimethylformamide, and then
reacted with a haloalkylnitrile compound, such as
4-bromobutyronitrile or 3-bromopropionitrile (Aldrich). The nitrile
moiety of the resulting product IIIA is reduced by various methods
known in the art, such as treatment with hydrogen gas and palladium
or platinum metal, to yield the free amine IVA. The forgoing
steps--protection of the --NH.sub.2 group (such as with the
mesitylenesulfonyl group), and reaction of the --NH(Mes) moiety
with a haloalkylnitrile followed by reduction with H.sub.2 or other
methods known in the art, is repeated until the desired number of
nitrogen groups has been added. If a terminal alkyl group is
desired, e.g. an ethyl group, it is added to the last nitrogen by
simply reacting the last --NH(Mes) moiety with an alkyl halide
(e.g., bromoethane, n-butyl chloride) instead of a
haloalkylnitrile. 7
[0089] 2. The second general method of preparing the saturated
oligoamine compounds of the invention involves using chemistry
analogous to that used for peptide synthesis to generate a
polyamide chain, followed by reduction of the amide linkages to
amines. A non-naturally occurring amino acid, such as
H.sub.2NCH.sub.2CH.sub.2CH.sub.2COOH or another compound where an
amino group and carboxyl group are separated by a linear alkyl
chain of one to twelve, one to eleven, one to eight, or one to
seven CH.sub.2 groups, can be converted into its N-protected
derivative by using Boc, Fmoc, Cbz, or other amino protecting
groups well-known in the art, as illustrated below by conversion of
compound IB into compound IIB (PG indicates a protecting
group).
[0090] In certain methods for synthesizing the compounds of the
invention, the carboxyl group of the N-protected amino acid can
then be converted into an amide group, which functions to prevent
undesired reactions at the carboxyl group, and which will
ultimately be converted into an amine group bearing the outermost
alkyl groups of the final compound. That is, in certain methods for
synthesizing the compounds of the invention, such as
CH.sub.3CH.sub.2(NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2).sub.9NHCH.sub.2CH-
.sub.3, converting the carboxyl group of
Boc-NHCH.sub.2CH.sub.2CH.sub.2COO- H into
Boc-NHCH.sub.2CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.3 ultimately
provides the ethyl groups flanking the repeating core of the
molecule. This is shown for the general case for compound IIIB
below. It should be noted that the designation "alkyl" in the
scheme below can refer to groups which are identical or different,
that is, alkyl can be used to designate both the
--CH.sub.2CH.sub.2CH.sub.2-- group and a --CH.sub.2CH.sub.3 group
as in Boc-NHCH.sub.2CH.sub.2CH.sub.2CONHCH.sub.2- CH.sub.3.
[0091] Once the carboxyl group of the N-protected amino acid is
converted into an amide group, the N-terminus can be deprotected
(compound IVB below). Another N-protected amino acid (which can be,
but is not necessarily, identical to the amino acid used in the
previous step), can then be added. The step is repeated until a
desired length is reached, as in compound VB below. 8
[0092] Once compound VB has been generated, it can be used in
various further methods to synthesize the compounds of the
invention. One such method is to reduce the amide groups of
compound VB by various methods known in the art, using reagents
such as borane (e.g., borane-tetrahydrofuran complex) or lithium
aluminum hydride, to the compound VIB1 as follows. 9
[0093] Two units of the reduced, appropriately protected compound
VIIB1 (note that the protecting group designated "PG" can be the
same or different from the protecting groups used in previous
syntheses) are then condensed with a central alkyl group VIIIB1
(where "LG" designates a leaving group, which is displaced by the
secondary nitrogen of VIIV1). 10
[0094] VIIIB1.
LG-(alkyl)-LG
[0095] While "alkyl" is used to designate the groups in the
intermediates of the synthesis, unsaturated segments (alkenyl,
alkynyl) groups can be used as well, provided that they are reduced
at the end of the synthesis to alkyl groups.
[0096] Alternatively, if the desired number of alkyl links and
nitrogens are present in intermediate VB, the compound can be
acylated as depicted in VIB2: 11
[0097] followed by reduction of the amides to amino groups. Amides
can be reduced to amino groups using various methods known in the
art, using reagents such as borane (e.g., borane-tetrahydrofuran
complex) or lithium aluminum hydride. (Amine compound VIB1 can also
be acylated and reduced to form the final compound, with
appropriate protecting groups to prevent acylation of the secondary
amines.)
[0098] While the synthetic scheme described above is performed in
solution phase, solid-phase organic synthesis can also be employed.
Since many of the reactions above are similar to reactions
performed during solid-phase peptide synthesis, the reaction scheme
above is readily adapted to solid-phase synthesis using techniques
for peptide synthesis well-known in the art, as described in, for
example, Atherton and Sheppard, Solid Phase Peptide Synthesis: A
Practical Approach, New York: IRL Press, 1989; Stewart and Young:
Solid-Phase Peptide Synthesis 2nd Ed., Rockford, Ill.: Pierce
Chemical Co., 1984; and Jones, The Chemical Synthesis of Peptides,
Oxford: Clarendon Press, 1994. Automated synthesis of the
polyamides can be performed using an automated polypeptide
synthesizer employing the solid phase method, such as those sold by
Perkin Elmer-Applied Biosystems, Foster City, Calif. Following
completion of the entire polyamide precursor, the polyamide is
cleaved from the solid support and the amide groups are reduced to
amine groups as described above.
[0099] 3. The third general method of preparing the saturated
oligoamine compounds of the invention comprises reduction of
unsaturated polyamine compounds. Polyamine compounds which are
conformationally restricted due to the presence of one or more
double or triple bonds, such as those described in U.S. Pat. No.
5,889,061, WO 00/66587, and WO,98/17624, are reduced under a
hydrogen atmosphere in the presence of finely divided platinum,
palladium, nickel, or other hydrogenation catalysts well-known in
the art (e.g., platinum dioxide or "Adam's catalyst;" platinum
black; palladium on charcoal, Pd/C; Raney nickel).
Synthetic Methods for Production of Hydroxyoligoamine Compounds
[0100] Hydroxy groups can be readily incorporated into one or more
locations of the oligoamine compounds. Example 11 below indicates a
synthetic scheme for preparing an oligoamine with hydroxyl groups
in two alkyl segments of the oligoamine. The method can be readily
adapted to prepare oligoamines with hydroxy groups at any location
in any segment of the molecule, simply by using the appropriate
protected hydroxy amino acid. For example,
ZHN(CH.sub.2)(CHOR)(CH.sub.2)COOH can be used in place of
ZHN(CH.sub.2)(CH.sub.2)(CH.sub.2)COOH for one of the segments of
the oligoamine in any of the preceding general schemes, or in the
schemes in the examples. This compound is readily synthesized from
commercially available 4-amino-3-hydroxybutyric acid, by protecting
the amino group with the Z group and then protecting the hydroxy
group. The protecting group R on the hydroxy group can be TBDMS or
any other hydroxy protecting group stable to the subsequent
reaction conditions. As an additional example,
ZHN(CH.sub.2)(CH.sub.2)(CHOR)COOH (readily synthesized from
commercially available 4-amino-2-hydroxybutyric acid) can be used
to place the hydroxy group in an alternate location. The
hydroxy-containing aminobutyric acids are available in
enantiomerically pure R and S forms should stereospecific synthesis
be desired.
Therapeutic Use of Oligoamine Compounds
[0101] Oligoamine compounds of the present invention are useful for
treatment of a variety of diseases caused by uncontrolled
proliferation of cells, including cancer, particularly prostate
cancer, breast cancer, and other cancers. The oligoamine compounds
of the present invention are also useful for treatment of
infectious and microbial diseases, which can be caused by
microorganisms, including, but not limited to, microorganisms such
as bacteria, viruses, and parasites. In particular, the oligoamine
compounds of the present invention are useful in treating diseases
in immunocompromised patients. The oligoamine compounds of the
present invention are particularly useful for treating
microsporidiosis. (See Bacchi C J et al., "Novel synthetic
polyamines are effective in the treatment of experimental
microsporidiosis, an opportunistic AIDS-associated infection,"
Antimicrob. Agents Chemother. 46(1):55-61 (2002); and Bacchi C J et
al., "SL-l 1158, a synthetic oligoamine, inhibits polyamine
metabolism of Encephalitozoon cuniculi," J. Eukaryot. Microbiol.
Suppl:92S-94S (2001)). The compounds are used to treat mammals,
preferably humans. "Treating" a disease using an oligoamine
compound of the invention is defined as administering one or more
oligoamine compounds of the invention, with or without additional
therapeutic agents, in order to prevent, reduce, or eliminate
either the disease or the symptoms of the disease, or to retard the
progression of the disease or of symptoms of the disease.
"Therapeutic use" of the oligoamine compounds of the invention is
defined as using one or more oligoamine compounds of the invention
to treat a disease, as defined above. A "therapeutic amount" of the
oligoamine compounds of the invention is an amount sufficient to
treat a disease, as defined above.
[0102] In order to evaluate the efficacy of a particular oligoamine
compound for a particular medicinal application, the compounds can
be first tested against appropriately chosen test cells in vitro.
In a non-limiting example, oligoamine compounds can be tested
against tumor cells, for example, prostate tumor cells. Exemplary
experiments can utilize cell lines capable,of growing in culture as
well as in vivo in athymic nude mice, such as LNCaP. Horoszewicz et
al. (1983) Cancer Res. 43:1809-1818. Culturing and treatment of
carcinoma cell lines, cell cycle and cell death determinations
based on flow cytometry; enzyme assays including ODC, SAMDC and
SSAT activities; and high pressure liquid chromatography detection
and quantitation of natural polyamines and polyamine analogs are
described in the art, for example, Mi et al. (1998) Prostate
34:51-60; Kramer et al. (1997) Cancer Res. 57:5521-27; and Kramer
et al. (1995) J. Biol. Chem. 270:2124-2132. Evaluations can also be
made of the effects of the oligoamine compound on cell growth and
metabolism.
[0103] Analysis begins with IC.sub.50 determinations based on
dose-response curves ranging from 0.1 to 1000 .mu.M performed at 72
hr. From these studies, conditions can be defined which produce
about 50% growth inhibition and used to: (a) follow time-dependence
of growth inhibition for up to 6 days, with particular attention to
decreases in cell number, which may indicate drug-induced cell
death; (b) characterize oligoamine compound effects on cell cycle
progression and cell death using flow cytometry (analysis to be
performed on attached and detached cells); (c) examine oligoamine
compound effects on cellular metabolic parameters. Oligoamine
compound effects can be normalized to intracellular concentrations
(by HPLC analysis), which also provide an indication of their
relative ability to penetrate cells. Marked differences in
oligoamine compound uptake can be further characterized by studying
the compound's ability to utilize and regulate the polyamine
transporter, as assessed by competition studies using radiolabeled
spermidine, as previously described in Mi et al. (1998). Oligoamine
compounds could also enter the cells by a diffusion mechanism.
In vivo Testing of Oligoamine Compounds
[0104] Oligoamine compounds found to have potent anti-proliferative
activity in vitro towards cultured carcinoma cells can be evaluated
in in vivo model systems. In a non-limiting protocol, the first
goal is to determine the relative toxicity of the compounds in
non-tumor-bearing animals, such as DBA/2 mice. Groups of three
animals each can be injected intraperitoneally with increasing
concentrations of an oligoamine compound, beginning at, for
example, 10 mg/kg. Toxicity as indicated by morbidity is closely
monitored over the first 24 hr. A well-characterized polyamine
analog compound, such as BE-333, can be used as an internal
standard in these studies, since a data base has already been
established regarding acute toxicity via a single dose treatment
relative to chronic toxicity via a daily.times.5 d schedule. Thus,
in the case of oligoamine compounds, single dose toxicity relative
to BE-333 is used to project the range of doses to be used on a
daily.times.5 d schedule.
[0105] After the highest tolerated dosage on a daily.times.5 d
schedule is deduced, antitumor activity is determined. Typically,
tumors can be subcutaneously implanted into nude athymic mice by
trocar and allowed to reach 100-200 mm.sup.3 before initiating
treatment by intraperitoneal injection daily.times.5 d. Most
oligoamine compounds can be given in a range between 10 and 200
mg/kg. Oligoamine compounds can be evaluated at three treatment
dosages with 10-15 animals per group (a minimum of three from each
can be used for pharmacodynamic studies, described below). Mice can
be monitored and weighed twice weekly to determine tumor size and
toxicity. Tumor size is determined by multi-directional measurement
from which volume in mm.sup.3 is calculated. Tumors can be followed
until median tumor volume of each group reaches 1500 mm.sup.3
(i.e., 20% of body weight), at which time the animals can be
sacrificed. Although the initial anti-tumor studies focuses on a
daily.times.5 d schedule, constant infusion can be performed via
Alzet pump delivery for 5 days since this schedule dramatically
improves the anti-tumor activity of BE-333 against A549 human large
cell hung carcinoma. Sharma et al. (1997) Clin. Cancer Res.
3:1239-1244. In addition to assessing anti-tumor activity, free
oligoamine compound levels in tumor and normal tissues can be
determined in test animals.
Methods of Administration of Oligoamine Compounds
[0106] The oligoamine compounds of the present invention can be
administered to a mammalian, preferably human, subject via any
route known in the art, including, but not limited to, those
disclosed herein. Methods of administration include but are not
limited to, intravenous, oral, intraarterial, intratumoral,
intramuscular, topical, inhalation, subcutaneous, intraperitoneal,
gastrointestinal, and directly to a specific or affected organ. The
oligoamine compounds described herein are administratable in the
form of tablets, pills, powder mixtures, capsules, granules,
injectables, creams, solutions, suppositories, emulsions,
dispersions, food premixes, and in other suitable forms. The
compounds can also be administered in liposome formulations. The
compounds can also be administered as prodrugs, where the prodrug
undergoes transformation in the treated subject to a form which is
therapeutically effective. Additional methods of administration are
known in the art.
[0107] The pharmaceutical dosage form which contains the compounds
described herein is conveniently admixed with a non-toxic
pharmaceutical organic carrier or a non-toxic pharmaceutical
inorganic carrier. Typical pharmaceutically-acceptable carriers
include, for example, mannitol, urea, dextrans, lactose, potato and
maize starches, magnesium stearate, talc, vegetable oils,
polyalkylene glycols, ethyl cellulose, poly(vinylpyrrolidone),
calcium carbonate, ethyl oleate, isopropyl myristate, benzyl
benzoate, sodium carbonate, gelatin, potassium carbonate, silicic
acid, and other conventionally employed acceptable carriers. The
pharmaceutical dosage form can also contain non-toxic auxiliary
substances such as emulsifying, preserving, or wetting agents, and
the like. A suitable carrier is one which does not cause an
intolerable side effect, but which allows the novel oligoamine
compound(s) to retain its pharmacological activity in the body.
Formulations for parenteral and nonparenteral drug delivery are
known in the art and are set forth in Remington 's Pharmaceutical
Sciences, 18th Edition, Mack Publishing (1990) and Remington, The
Science and Practice of Pharmacy, Lippincott Williams & Wilkins
(2000). Solid forms, such as tablets, capsules and powders, can be
fabricated using conventional tableting and capsule-filling
machinery, which is well known in the art. Solid dosage forms,
including tablets and capsules for oral administration in unit dose
presentation form, can contain any number of additional non-active
ingredients known to the art, including such conventional additives
as excipients; desiccants; colorants; binding agents, for example
syrup, acacia, gelatin, sorbitol, tragacanth, or
polyvinylpyrollidone; fillers, for example lactose, sugar,
maize-starch, calcium phosphate, sorbitol or glycine; tableting
lubricants, for example magnesium stearate, talc, polyethylene
glycol or silica; disintegrants, for example potato starch; or
acceptable wetting agents such as sodium lauryl sulfate. The
tablets can be coated according to methods well known in standard
pharmaceutical practice. Liquid forms for ingestion can be
formulated using known liquid carriers, including aqueous and
non-aqueous carriers, suspensions, oil-in-water and/or water-in-oil
emulsions, and the like. Liquid formulations can also contain any
number of additional non-active ingredients, including colorants,
fragrance, flavorings, viscosity modifiers, preservatives,
stabilizers, and the like. For parenteral administration,
oligoamine compounds can be administered as injectable dosages of a
solution or suspension of the compound in a physiologically
acceptable diluent or sterile liquid carrier such as water or oil,
with or without additional surfactants or adjuvants. An
illustrative list of carrier oils would include animal and
vegetable oils (e.g., peanut oil, soy bean oil), petroleum-derived
oils (e.g., mineral oil), and synthetic oils. In general, for
injectable unit doses, water, saline, aqueous dextrose and related
sugar solutions, and ethanol and glycol solutions such as propylene
glycol or polyethylene glycol are preferred liquid carriers. The
pharmaceutical unit dosage chosen is preferably fabricated and
administered to provide a final concentration of drug at the point
of contact with the cancer cell of from 1 .mu.M to 10 mM. More
preferred is a concentration of from 1 to 100 .mu.M. The optimal
effective concentration of oligoamine compounds can be determined
empirically and will depend on the type and severity of the
disease, route of administration, disease progression and health
and mass or body area of the patient. Such determinations are
within the skill of one in the art. Oligoamine compounds can be
administered as the sole active ingredient, or can be administered
in combination with another active ingredient, including, but not
limited to, cytotoxic agents, antibiotics, antimetabolites,
nitrosourea, vinca alkaloids, polypeptides, antibodies, cytokines,
etc.
EXAMPLES
Chemical Synthesis Examples
[0108] The following examples are illustrative of the manufacture
of several compounds according to the present invention, and are
not intended to limit the invention disclosed and claimed herein in
any fashion. The Examples are included herein solely to aid in a
more complete understanding of the present invention.
[0109] All commercially available reagents were used without
further purification. All reactions were followed by TLC (silica
gel F.sub.264 precoated, Merck); column chromatography was carried
out with silica gel (Merck 60, 0.040-0.063 mesh). The detection was
performed either with UV light or the following reagents:
KMnO.sub.4 soln. (1:1 mixture of 1% aq. KMnO.sub.4 soln. and 5% aq.
Na.sub.2CO.sub.3 soln.); Schlittler reagent (iodine platinate) (1 g
H.sub.2PtCl.sub.6 in 6 ml H.sub.2O, 20 ml IN HCl and 25.5 g KI in
225 ml H.sub.2O diluted to 1 L) for amides and amines. IR
measurements are presented in units of [cm.sup.-1] and were
recorded on a Perkin-Elmer 781 instrument. NMR spectra were
recorded on Bruker-300 or Bruker AMX-600 instruments with .delta.
in ppm and using the appropriate solvent as internal standard. MS
spectra were generated on Finnigan MAT SSO 700 or Finnigan MAT 90
instruments using chemical ionization (CI) with NH.sub.3 and
electron impact (El; 70 eV), and on a Finnigan TSQ 700 instrument
using electrospray ionization (ESI).
Example 1
[0110] 12
[0111] N-Boc-.gamma.-Aminobutyric acid (1): (This compound can also
be purchased commercially from Sigma-Aldrich Chemical Company,
Saint Louis, Mo., USA, product B 1892). A solution of Boc.sub.2O
(95 g, 435 mmol) in 600 ml of dioxane was added at 0.degree. C. to
a stirred mixture of NaHCO.sub.3 (73 g, 870 mmol) in H.sub.2O (500
ml) and .gamma.-aminobutyric acid (30 g, 291 mmol), stirred for 1 h
at 0.degree. C. and for 10 h at 20.degree. C. The reaction mixture
was diluted with H.sub.2O (500 ml), extracted 3 times with
CHCl.sub.3, the aqueous layer was acidified with 3% HCl to pH 7 and
then with KHSO.sub.4 (20% aq. solution) to pH 2. The product was
extracted 5 times with CHCl.sub.3, dried (Na.sub.2SO.sub.4)
concentrated in vacuo, and crystallized from Et.sub.2O-petr. ether.
Yield 54.05 g (97%). mp: 58-59.degree. C. NMR (CDCl.sub.3): 1.44
(s, 9H), 1.83 (m, 2H), 2.40 (t, J=7.15, 2H), 3.10-3.30 (m, 2H), 4.7
(bs, 1H).
Example 2
[0112] 13
[0113] 1-Hydroxybenzotriazole derivative of
N-Boc-.gamma.-Aminobutyric acid (2): 1-Hydroxybenzotriazole (70.3
g, 520 mmol; abbreviated as "HOBt" as individual reagent and as
"Bt" indicating ester) and dicyclohexylcarbodiimide (DCC, 107.41 g.
520 mmol) were added into an ice cold solution of the acid 1 (105.5
g, 519 mmol) in DMF (700 ml), the cooling bath was removed and the
reaction mixture was stirred overnight at 20.degree. C. DMF was
evaporated in vacuo at 40.degree. C., the residue was suspended in
CH.sub.2Cl.sub.2/H.sub.2O (2:1) mixture (1.5 liter), filtered, and
the precipitate was washed with CH.sub.2Cl.sub.2 The washings and
filtrate were combined, washed 4 times with H.sub.2O, washed with
brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The
product was re-precipitated from hot CH.sub.2Cl.sub.2 with
Et.sub.2O. The mother liquor was concentrated and the residue was
re-precipitated again from hot CH.sub.2Cl.sub.2 with Et.sub.2O.
Both crops were combined, dried in vacuo to obtain 151 g (87% of 2
as a mixture of N-and O-isomers, which was used in the following
steps without further purification. mp: 52-55.degree. C. .sup.1H
NMR (CDCl.sub.3): 1.41 (s) 1.47 (s), 1.98-2.10 (m), 2.87 (t,
J=7.16), 3.18 (t, J=7.15), 3.27-3.36 (m), 4.71 (bs), 7.42-7.44 (m),
7.47-7.60 (m), 7.76-7.79 (m), 7.99-8.04 (m), 8.05-8.08 (m),
8.39-8.43 (m). ESI-MS: 663.2 (M.sub.2Na.sup.+), 641.4
(M.sub.2H.sup.+), 456.2, 342.0 (MNa.sup.+), 321.2 (MH.sup.+).
Example 3
[0114] 14
[0115] (3-Ethylcarbamoyl-propyl)-carbamic acid tert-butyl ester
(3): An aqueous solution of ethylamine (70%, 41 ml) was added into
an ice cold solution of benzotriazole derivative 2 (50 g, 156 mmol)
in CH.sub.2Cl.sub.2 (500 ml), stirred for 1 h at room temperature,
diluted 2 times with CH.sub.2Cl.sub.2, washed with H.sub.2O, then
brine, dried (Na.sub.2SO.sub.4), concentrated and dried in vacuo.
Yield 32.845 g (91%). mp: 82-83.degree. C. .sup.1H NMR
(CDCl.sub.3): 1.15 (t, J=7.27, 3H), 1.44 (s, 9H), 1.75-1.85 (m,
2H), 2.20 (t, J=7.10, 2H), 3.17 (q, J=6.49, 2H), 3.24-3.34 (m, 2H),
4.82 (s, 1H), 6.13 (bs, 1 H). .sup.13C NMR (CDCl.sub.3): 14.72,
24.91, 26.33, 28.35, 33.66, 33.92, 34.32, 39.78, 79.21, 156.44,
172.43. ESI-MS: 483.4 (M.sub.2Na.sup.+), 461.4 (M.sub.2H.sup.+),
321.2 (MH.sup.+).
Example 4
[0116] 15
[0117] [3-(3-Ethylcarbamoyl-propylcarbamoyl)-propyl]-carbamic acid
tert-butyl ester 4. Hydrochloric acid (12%, 64 ml) was added to a
cold solution of Boc-derivative 3 (21.1 g, 91.7 mmol) in MeOH (190
ml) and stirred overnight at room temperature. The next day
additional acid (30 ml) was added and stirring was continued for
another 10 h. The reaction mixture was filtered, washed with
CHCl.sub.3, concentrated and dried in vacuo overnight. The product
was suspended in DMF (300 ml) with DIEA (42 ml), cooled to
0.degree. C. and a solution of Bt-derivative 2 (29 g, 90.5 mmol) in
DMF (100 ml) was added into the reaction mixture. The cooling bath
was removed and the stirring was continued overnight. The solvent
and DIEA were removed in vacuo at 45.degree. C., the residue was
suspended in CHCl.sub.3/H.sub.2O mixture, washed with H.sub.2O, aq.
KHSO.sub.4 (20%), H.sub.2O, aq. NaHCO.sub.3 (2 times with each),
dried (Na.sub.2SO.sub.4), concentrated in vacuo, and triturated
with petr. ether. Yield 22.32 g (78%). mp: 134-1350. H
(CDCl.sub.3): 1.15 (t, J=7.27, 3H), 1.44 (s, 9H), 1.65-1.95 (m,
4H), 2.20-2.26 (m, 4H), 3.19 (q, J=6.15, 2H), 3.23-3.35 (m, 4H),
4.82 (bs, 1H), 6.40 (bs, 1H), 6.68 (bs, 1H). .sup.13C (CDCl.sub.3):
14.77, 24.92, 25.66, 26.43, 28.38, 33.59, 33.84, 33.92, 34.35,
38.83, 39.66, 79.35, 156.56,172.65,173.12. ESI-MS: 338.2
(MNa.sup.+), 316.0 (MH.sup.+), 216.0 (MH.sup.+-Boc).
Example 5
[0118] 16
[0119]
{3-[3-(3-Ethylcarbamoyl-propylcarbamoyl)-propylcarbamoyl]-propyl}-c-
arbamic acid tert-butyl ester 5. Boc-Protected derivative 4 (22.3
-g, 70.7 mmol) was stirred in a mixture of MeOH (200 ml) and HCl
(12%, 81 ml) for 48 h, concentrated to dryness in vacuo, dissolved
in H.sub.2O, and washed 3 times with CHCl.sub.3. Water was removed
on a rotary evaporator at 45.degree. C. The resulting hydrochloride
was dissolved in a mixture of CH.sub.2Cl.sub.2 (600 ml) and aq.
Na.sub.2CO.sub.3 (20%, 100 ml), cooled on an ice bath, and
Bt-derivative 2 (22.47 g, 70.15 mmol) was added. The cooling bath
was removed and the mixture was stirred for 18 h with a mechanical
stirrer. The main part of the CH.sub.2Cl.sub.2 was removed on a
rotary evaporator, the reaction mixture was suspended in water and
filtered. The product was triturated in EtOAc, filtered, washed
with petr. ether and dried in vacuo. Yield 25.46 g (90%). mp:
170-171.degree. C. .sup.1H NMR (CDCl.sub.3/CD.sub.3OD): 1.14 (t,
J=7.30, 3H), 1.44 (s, 9H), 1.72-1.85 (m, 6H), 2.10-2.23 (m, 6H),
3.06-3.12 (m, 2H), 3.18-3.26 (m, 6H), 5.57 (bs, 1H), 7.32 (bs, 1H),
7.46 (bs, 1H), 7.60 (bs, 1H). .sup.13C NMR (CDCl.sub.3/CD.sub.3OD):
14.22, 24.73, 25.32, 25.39, 25.96, 28.15, 33.33, 33.59, 34.12,
34.25, 38.60, 39.64, 79.29, 156.68, 173.39, 173.88. ESI-MS: 423.4
(MNa.sup.+), 401.4 (MH.sup.+).
Example 6
[0120] 17
[0121]
{3-{3-13-(3-Ethylcarbamoyl-propylcarbamoyl)-propylcarbamoyl]-propyl-
carbamoyl}-propyl}-carbamic acid tert-butyl ester 6 was prepared
from Boc derivative 5 (25.175 g) employing the same procedure as
for 5 with the yield 24.79 g, (81%). mp: 196-197.degree. C. .sup.1H
NMR (CDCl.sub.3/CD.sub.3OD): 1.14 (t, J=7.28, 3H), 1.44 (s, 9H),
1.74-1.85 (m, 8H), 2.15-2.24 (m, 8H), 3.10 (q, J=6.22, 2H),
3.18-3.26 (m, 8H), 5.55 (bs, 1H), 7.28 (bs, 1H), 7.43 (bs, 1H),
7.55 (bs, 1H), 7.62 (bs, 1H). .sup.13C NMR (CDCl.sub.3/CD.sub.3OD):
13.88, 24.56, 25.14, 25.23, 25.75, 27.90, 33.07, 33.37, 33.94,
38.34, 39.35, 79.04, 156.67, 173.37, 173.76, 173.86. ESI-MS: 508.4
(MNa.sup.+), 486.4 (MH.sup.+).
Example 7
[0122] 18
[0123] .sup.1N, .sup.6N, .sup.11N, .sup.16N,
.sup.21N-Pentakis(mesitylenes- ulfonyl)-1, 6, 11, 16,
21-pentazatricosane 7. Boc derivative 6 (24.80 g, 51.06 mmol) was
dissolved in a cold MeOH (370 ml)/aq. HCl (12%, 150 ml) solution,
stirred for 48 h at room temperature, concentrated on a rotary
evaporator at 45.degree. C., dissolved in a minimum of H.sub.2O,
washed 2 times with CHCl.sub.3, transferred into a 2 L round-bottom
flask, and dried in vacuo. The product was stirred with
BH.sub.3-THF (1 M, 1 L) at 65.degree. C. for 24 h, 200 ml more of
BH.sub.3-THF solution was added, and the reaction was continued for
another 72 h. The reaction mixture was cooled to 0.degree. C.,
quenched with HBr (30% in AcOH) until evolution of H.sub.2 stops
(approx. 300 ml), half of the solvent was removed on a rotary
evaporator (5 mm, 55.degree. C.), more HBr/AcOH (500 ml) was added,
and left overnight. The reaction mixture was concentrated again on
a rotary evaporator (5 mm, 55.degree. C.) until it became very
viscous, and triturated with a mixture HCl (6%, 1L) and CHCl.sub.3
(200 ml), and filtered. The CHCl.sub.3 layer was extracted 3 times
with H.sub.2O, the aqueous phases were combined, washed again with
CHCl.sub.3, and concentrated to dryness on a rotary evaporator at
55.degree. C.
[0124] The residue was suspended in a mixture of CH.sub.2Cl.sub.2
(400 ml) and NaOH (2N, 560 ml), cooled on an ice bath, and sulfonyl
chloride (61.2 g, 280 mmol) in CH.sub.2Cl.sub.2 (400 ml) was added
in a few portions into the stirred reaction mixture. The cooling
bath was removed and the stirring was continued for 10 h. The
reaction mixture was diluted twice with CHCl.sub.3, mixed with 200
ml of H.sub.2O, filtered, and the precipitate was washed with
CHCl.sub.3 and H.sub.2O. The filtrate and washings were combined,
washed 4 times with H.sub.2O, brine, dried (Na.sub.2SO.sub.4),
concentrated and purified on a column (SiO.sub.2, EtOAc:hexane=1:
1). Yield 36.8 g (77%). mp: 58-60.degree. C. .sup.1H NMR
(CDCl.sub.3): 0.96 (t, J=7.14, 3H), 1.23-1.40 (m, 16H), 2.29 (s,
15H), 2.54, 2.56, 2.58 (s, together 30H), 4.55 (t, J=6.30, 1H),
6.93 (s, 10H).
[0125] MALDI-MS: 1278.618 (MK.sup.+), 1262.600 (MNa.sup.+),
1240.712 (M.sup.+).
Example 8
[0126] 19
[0127] .sup.3N, .sup.8N, .sup.13N, .sup.18N .sup.23N, .sup.28N
.sup.33N, .sup.38N, .sup.43N,
.sup.48N-N-Decakis(mesitylenesulfonyl)-3, 8, 13, 18, 23, 28, 33,
38, 43, 48-decaazapentacontene-25 (8). Sodium hydride (60%
suspension in oil, 712 mg, 17.8 mmol) was added to an ice cold
stirred solution of the product 6 (18.4 g, 14.83 mmol) in DMF (185
ml), stirred for 10 min and 2-butene-1,4-diyl
bis[mesitylenesulfonate] (3.356 g, 7.42 mmol) was added into the
reaction mixture. The stirring was continued for 3 h at 0.degree.
C. and left overnight. After the mixture was cooled to 0.degree.
C., it was quenched with ice water, concentrated in vacuo at
50.degree. C., dissolved in CHCl.sub.3, washed 4 times with aq.
NH.sub.4Cl, dried (Na.sub.2SO.sub.4), filtered and concentrated on
a rotary evaporator. Yield: 16.9 g (90%). The product was utilized
in the following steps without further purification.
[0128] mp: 73-74.degree. C. .sup.1H NMR (CDCl.sub.3): 0.96 (t,
J=7.19, 6H), 7.15-7.40 (m, 32H), 2.29 (s, 30H), 2.52, 2.53, 2.54,
2.55 (s, 60H), 2.85-3.19 (m, 36H), 4.13 (d, J=3.93, 4H), 5.42 (bs,
2H), 6.92 (s, 20H). .sup.13C NMR (CDCl.sub.3): 12.70, 20.92, 22.76,
24.44, 24.61, 24.73, 40.02, 42.07, 44.53, 44.92, 45.59, 128.30,
131.95, 132.89, 133.35, 139.95, 140.09, 142.29, 142.59. 20
[0129] 3, 8, 13, 18, 23, 28, 33, 38, 43, 48-Decaazapentacontane
decahydrochloride 9. The product 8 (32.2 g 12.7 mmol) was dissolved
in 500 ml of CH.sub.2Cl.sub.2, cooled to 0.degree. C., and PhOH
(119.7 g, 1.27 mol) followed by 510 ml of HBr (30% in AcOH) were
added into the solution. The mixture was stirred for 15 h at
20.degree. C., quenched with 1000 ml of ice water, and the organic
layer was separated and extracted one time with 150 ml of H.sub.2O.
The aqueous phases were combined, washed with CH.sub.2Cl.sub.2
(8.times.150 ml), concentrated on a rotary evaporator at 50.degree.
C. to the volume of 600 ml, and stiffed overnight with 1 g of
activated carbon. Following filtration through a CELITE cake
(CELITE is a registered trademark for diatomaceous earth of the
Celite Corporation) and rinsing the cake with H.sub.2O, the
filtrate was transferred into a Parr apparatus and subjected
hydrogenation with 3 g of Pd on C (10%) for 48 h at 50 psi. The
catalyst was removed by filtration through a CELITE cake and rinsed
with H.sub.2O; the H.sub.2O was removed on a rotary evaporator at
50.degree. C. The residue was dissolved in EtOH, cooled to
0.degree. C. and product was precipitated with 35% HCl. Finally it
was filtered, triturated with EtOH and dried in vacuo. Yield 11 g
(80%). mp: above 210.degree. C. .sup.1H NMR (D.sub.2O): 1.27 (t,
J=7.37, 6H), 1.62-1.88 (m, 36 H), 2.95-3.25 (m, 40 H). .sup.13C NMR
(D.sub.2O): 13.31, 25.59, 45.68, 49.04, 49.66. MALDI-MS: 850.2
(MH.sup.+HCl), 714.0 (MH.sup.+), 515.2, 543.8, 357.8.
[0130] Example 10
[0131] 1,4-Bis(mesitylenesulfonyloxy)butane (11) 1,4-Butanediol
(4.5 g, 50 mmol) was dissolved in dioxane (30 ml), and a 50%
solution of KOH (45 ml) and benzyl triethylammonium bromide (675
mg, 2.5 mmol) were added. The mixture was stirred and cooled at
5.degree. C., and mesitylenesulfonyl chloride (26 g, 120 mmol) was
added in small portions. The mixture was kept for 5 hr at 5.degree.
C., excess water was added and the mixture was stirred for 18 h at
25.degree. C. The solid was filtered, dried and crystallized from
ethyl acetate/hexane; 14.6 g (64%) of 11 were obtained; mp
108.6.degree. C.; .sup.1H-NMR (CDCl.sub.3) .delta. 1.75 (t, 4H),
2.30 (s, 6H), 2.60 (s, 12H), 3.95 (t, 4H), 6.95 (s, 4H).
.sup.13CNMR(CDCl.sub.3) .delta. 20.94,22.48,25.15, 68.30, 131.70,
139.72, 143.29. MS-MALDI (m/z) 477.2 (M.sup.++Na), 493.1
(M.sup.++K). 21
[0132] Compound 16. Amide 16 was prepared starting with pentamide
12 (described in International Patent Application WO 00/66587)
following the sequence of reactions described in WO 00/66587,
namely, alkylation with 4-bromobutyronitrile, followed by reduction
of the nitrile 13 and protection with mesitylene chloride to give
14. Repeating the sequence of reactions gave compound 15, then
compound 16. 22
[0133] Compound 17. Amide 16 (350 mg, 0.2 mmol) was dissolved in
DMF (10 ml), the solution stirred at 5.degree. C. under N.sub.2,
and 60% NaH (10 mg) was added. Compound 11 was added, the mixture
was allowed to reach 25.degree. C. and was kept for 18 h. The
solvent was evaporated and the residue was extracted with
chloroform, the extracts were washed twice with saturated ammonium
chloride, dried (Na.sub.2SO.sub.4) and evaporated. The residue was
purified by filtration through a silica gel column using
hexane/ethyl acetate (6:5) as eluant; 140 mg of 17 (39%) were
obtained. .sup.1H NMR (CDCl.sub.3), .delta. 1.0 (t, 6H), 1.30 (m,
52 H), 2.30 (s, 42 H), 2.55 (s, 84H), 3.0 (m, 56H), 10 6.95 (s,
28H). .sup.13C NMR .delta. 12.68, 20.89, 22.67, 22.74, 24.38,
24.46, 24.74, 40.03, 44.55, 44.85, 44.96, 131.86, 133.37, 139.95,
142.28. 23
[0134] Compound 17 was deprotected to give tetradecamine 18 using
HBr/AcOH as 15 described in International Patent Application WO
00/66587 for compound 43 of that document, at pages 42-43.
Example 11
[0135] 24
[0136] Preparation of
5,46-Dihydroxy-3,8,13,18,23,28,33,38,43,48-decaazape- ntacontane
decahydrochloride 28. 2-Hydroxy-.gamma.-butyrolactone 20 was used
as a starting material. (This compound is commercially available as
both R and S isomers.) Following the protection of hydroxy group
with t-butyldimethylsilyl chloride (TBDMSCl), the resulting lactone
21 was treated with a THF solution of EtNH.sub.2. The resulting
hydroxy amide 22 was subjected to a Mitsunobu reaction with
phthalimide (PhTh), diethylazodicarboxylate (DEAD), and
triphenylphosphine, followed by deprotection with hydrazine to
obtain amine 23. Amine 23 was repeatedly acylated with
.gamma.-carbobenzyloxy-aminobutyric acid in presence of ethyl
chloroformate and the carbobenzyloxy (Z) group was removed by
hydrogenation over Pd/C catalyst to yield products 24-26. Reduction
of tetramide 26 with the borane-THF complex, followed by treatment
of the resulting tetramine with Boc-anhydride, gave 27. This
product was acylated with succinic acid after removal of the Z
group by hydrogenation, then Boc-deprotected with aqueous HCl and
the amide groups were again reduced using BH.sub.3-THF reagent to
yield desired product 28 as a decahydrochloride. The 5R, 46R and
5S, 46S analogues are prepared by using the R or S isomers of
2-hydroxy-.gamma.-butyrolactone, respectively, as the starting
material.
Example 12
[0137] Calf-thymus DNA aggregation by spermine and oligoamines. The
oligoamines of the invention are very efficient in producing DNA
aggregation. The concentrations required for polyamines at the
start of DNA aggregation along with the concentration required for
spermine to achieve the same can be seen in Table 1. The
oligoamines aggregated DNA 20 to 40 times more efficiently than
spermine under identical conditions.
[0138] Test for DNA Aggregation: DNA aggregation was studied using
a Perkin-Elmer Lambda 25 UV/visible spectrophotometer connected to
a PTP 6 heating unit using a previously published procedure (see
Basu, H S and Marton, L J, "The interaction of spermine and
pentamines with DNA," Biochemical Journal 244:243-246 (1987)).
Aggregation was determined in 50 mM NaCl, 1 mM Na-cacodylate pH 7.0
buffer by observing the increase in DNA absorbance (approximately
0.5 A.sub.260 units) at 320 nm.
1TABLE 1 DNA Aggregation Conc. (.mu.M) at the start of DNA
Polyamine Aggregation Spermine 88.5 SL-11159 2.5 SL-11160 4.5
SL-11175 2.0 SL-11226 2.2
Example 13
[0139] Effect of Saturated Oligoamines on Human Prostate Tumor Cell
Growth by the MTT assay. Saturated oligoamines inhibited prostate
cancer cell growth in vitro. DU-145 cells were most sensitive and
PC-3 cells were less sensitive to the 10 effects of oligoamines. In
general, ID.sub.50 values of less than 0.5 .mu.M were obtained
(Table 2). Even though the PC-3 cells were relatively more
resistant to the oligoamines, at a 5 .mu.M concentration the
oligoamines reduced the cell number to less than 1% of the control
on day 6 of incubation. Tissue cultures and the MTT assay were
performed as follows.
[0140] Tissue Culture. Cells were seeded into 75 cm.sup.2 culture
flasks with 15 ml of Eagle's minimal essential medium supplemented
with 10% fetal calf serum and nonessential amino acids. The flasks
were incubated in a humidified 95% air/5% CO.sub.2 atmosphere. The
cells were grown for at least 24 h to ensure that they are in the
log phase of growth and then they were treated with the
oligoamines. Cells were harvested by treatment for 5 min with STV
(saline A, 0.05% trypsin, 0.02% EDTA) at 37.degree. C. The flasks
were rapped on the lab bench, pipetted several times and aliquots
of cell suspension were withdrawn and counted using a Coulter
particle counter that has been standardized for counting each cell
line using a hemacytometer.
[0141] MTT Assay: Trypsinized cell suspensions were diluted to seed
80 .mu.l suspensions containing 500 cells in each well of a 96 well
Corning microtiter plate and incubated overnight at 37.degree. C.
in a humidified incubator in 5% CO.sub.2. 20 .mu.l of appropriately
diluted stock solution of each drug were added to the middle 8
columns of cell suspension in the microtiter plates. Each drug
concentration was run in quadruplicate. Outer columns of the plates
were used for buffer controls. Cells were incubated with the drug
for 6 days at 37.degree. C. in 5% CO.sub.2/H.sub.2O atmosphere. 25
.mu.l of 5 mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyl tetrazolium bromide (MTT) were added to each well and
incubated for 4 hours at 37.degree. C. in 5% CO.sub.2/H.sub.2O
incubator. Cells were lysed by incubating overnight with 100 .mu.l
lysis buffer (500 ml of the lysis buffer contains: 100 g lauryl
sulfate (SDS), 250 ml of N, N-dimethylformamide, and 2 ml of
glacial acetic acid, made up to volume with water; pH 4.8).
[0142] The color was monitored at room temperature at 570 nm in a
E-max Precision Microplate Reader (Molecular Devices Corporation,
Sunnyvale, Calif.) and data was analyzed using cell survival
software supplied by Molecular Devices Corporation.
2TABLE 2 ID.sub.50 (.mu.M) values for Human Oligo- Tumor Cell Lines
amines Structures of Oligoamines LnCap DU145 DuPro PC-3 SL-11159 25
0.25 0.15 0.30 0.60 SL-11160 26 0.15 0.13 0.55 0.40 SL-11175 27
0.32 0.24 0.35 0.5 SL-11226 28 0.15 0.08 0.11 0.14
Example 14
[0143] Cellular Uptake of Oligoamines. There was considerable
uptake of oligoamines by the cancer cell lines; see Table 3A (DuPro
cells) and Table 3B (PC-3 cells). In most cases, only a minor
decrease in intracellular polyamine levels were observed in both
cell lines even at conditions where the oligoamines exhibit
considerable growth inhibition and cytotoxicity. Therefore, these
data suggest that the mechanism of cytotoxicity of oligoamines did
not involve depletion of intracellular polyamine pools. While not
wishing to be limited by any particular theory of operation, the
cytotoxicity is likely related to their strong aggregation effect
on DNA.
[0144] Polyamine Analysis. An appropriate number of cells were
taken from harvested samples and centrifuged at 1000 rpm at
4.degree. C. for 5 min. The cells were washed twice with chilled
Dulbecco's isotonic phosphate buffer (pH 7.4) by centrifugation at
1000 rpm at 4.degree. C. and resuspended in the same buffer. After
the final centrifugation, the supernatant was decanted, and 250 ml
of 8% sulfosalycilic acid was added to the cell pellet. The cells
were sonicated, and the mixture was kept at 4.degree. C. for at
least 1 h. After centrifugation at 8000 g for 5 min, the
supernatant was removed for analysis. An appropriate volume (50-100
.mu.l) was fluorescence-labeled by derivatizing with dansyl
chloride. Labeled polyamines were loaded onto a C-18
high-performance liquid chromatography column and separated by
gradient elution with acetonitrile/water at 50.degree. C. Peaks
were detected and quantitated using Shimadzu HPLC fluorescence
monitor coupled with a Spectra-Physics peak integrator. Because
polyamine levels vary with environmental conditions, control
cultures were sampled for each experiment.
3TABLE 3A Cellular Polyamine and Oligoamine levels of DuPro Cells
treated with Oligoamines Analog Polyamine levels (nmoles/10.sup.6
Polyamine levels (nmoles/10.sup.6 Conc. cells) on Day 4 of
Treamtment cells) on Day 6 on Treatment Treatment (.mu.M) Put Spd
Spm Analog Put Spd Spm Analog Control -- 0.966 3.870 1.006 -- 0.567
2.930 0.680 -- SL- 0.8 0.560 ND ND 0.525 * * * * 11159 1.6 0.137 ND
ND 0.513 * * * * SL- 0.8 0.430 0.897 0.222 0.575 0.675 1.470 0.290
0.410 11160 1.6 0.412 ND 0.195 0.775 * * * * SL- 0.8 0.157 ND 0.050
0.145 0.155 ND ND 0.023 11175 1.6 0.410 ND ND 0.150 * * * * SL- 0.8
0.593 0.017 0.067 0.032 0.548 ND ND 0.022 11226 1.6 * * * * * * * *
ND = Not detected; * = Cell yield is too low for accurate
measurement
[0145]
4TABLE 3B Cellular Polyamine and Oligoamine levels of PC-3 Cells
treated with Oligoamines. Analog Polyamine levels (nmoles/10.sup.6
Polyamine levels (nmoles/10.sup.6 Conc. cells) on Day 4 of
Treatment cells) on Day 6 of Treatment Treatment (.mu.M) Put Spd
Spm Analog Put Spd Spm Analog Control -- 0.567 2.790 0.660 -- 0.300
0.565 0.234 -- SL- 1 0.525 0.531 0.300 0.513 0.640 ND ND 1.026
11159 5 0.670 ND 0.240 0.388 * * * * SL- 1 ND 0.097 0.502 0.005 ND
0.085 0.180 0.007 11160 5 ND 0.077 0.525 0.011 * * * * SL- 1 0.515
ND ND 0.055 0.76 ND ND 0.08 11175 5 0.519 ND ND 0.128 * * * * SL- 1
0.573 ND ND 0.021 * * * * 11226 5 0.985 0.071 ND 0.026 * * * * ND =
Not detected; * = Cell yield is too low for accurate
measurement
Example 15
[0146] Assessment of Oligoamine Cytotoxicity by Colony Forming
Assay. The cytotoxity of the oligoamines was further assayed by the
colony forming assay (CFE). Four saturated oligoamines were chosen
for their ability to kill DuPro cells after 5 days of incubation.
Oligoamines;SL-11159, SL-11175 and SL-11226 had over 4 logs of cell
kill on day 5 of treatment at 1-2 .mu.M concentrations; see FIG. 1.
Although the prostate tumor PC-3 line was somewhat less susceptible
to Oligoamine treatment (see Table 2 of ID.sub.50 values), a colony
survival assay showed that at very low concentrations (ca. 1.5-2
.mu.M) SL-11159 kills almost four logs of PC-3 cells; see FIG. 2.
The colony forming assay was performed as follows.
[0147] Colony Forming Efficiency Assay. All cell lines used in this
assay have already been optimized with respect to the number of
feeder cells and length of incubation time for observable colony
formation. Cells were washed, harvested, and replated in
quadruplicate at appropriate dilution into 60 mm plastic Petri
dishes. The Petri dishes were prepared not more than 24 hr in
advance with 4 ml of supplemented Eagle's minimum essential medium
containing 5-10% fetal bovine serum (standardized for each cell
line) for all cell lines. Cells were incubated for the previously
standardized number of days in a 95% air/5% CO.sub.2 atmosphere.
The plates were stained with 0.125% crystal violet in methanol and
counted. Results are expressed as the surviving fraction of an
appropriate control.
Example 16
[0148] Response of implanted sc DU-145 prostate tumors to treatment
with SL-11159 and SL-11226. This experiment evaluates the antitumor
efficacy of SLIL Biomedical Corporation compounds (SL-11159,
SL-11226) against subcutaneously-implanted DU-145 human prostate
tumor xenografts in male NCr-nu (line 3A10F17T1) mice. All
compounds were tested at a single dose level when administered
intravenously (iv), at 6.25 mg/kg. Both SL-11159 and SL-11226 were
prepared in water for injection (soluble); the injection volume was
0.1 mL/10 g body weight.
[0149] All compounds were administered for two rounds of five daily
treatments with a nine-day rest period between the rounds. The
control group was treated with vehicle (water for injection) for
two rounds on a Q1D.times.5 schedule. There were sixteen mice in
the control group and eight mice in each of the treatment groups.
All treatments were initiated on day 14 postimplant when all mice
had established tumors ranging in size from 75 to 198 mg. The
experiment was terminated on day 53 after tumor implantation. The
individual animal's time to reach the evaluation size (time to
reach four doublings) was used in the calculations of the median
tumor growth delay [(T-C)/C.times.100%] and as the endpoint in a
life tables analysis (stratified Kaplan-Meier estimation followed
by the Mantel-Haenszel log-rank test) in order to statistically
compare the growth data between groups (see Table 5; group 1 is the
control group, group 2 was treated with SL-l 1159, group 3 was
treated with SL-11226).
[0150] The time required for a tumor to double in mass is
calculated based on the initial tumor weight at the beginning of
the treatment period. When the initial tumor weight has been
selected, tumor weights are then examined, beginning with the last
recorded value, until a doubling is calculated. Examination from
the last recorded value is to ensure that the doubling time is
calculated during the final phase of tumor growth and not prior to
a tumor regression. Values between measurements are calculated by
exponential extrapolation, and a value may be estimated after the
final measured weight provided the extrapolated value occurs prior
to the animal's death.)
[0151] Control tumors grew well in fourteen out of sixteen mice.
There was one xenograft failure (no-take). One animal died during
the second round of the vehicle treatment, on day 29, with a tumor
weight of 770 mg. Tumors reached the evaluation size of four mass
doublings in 17.1 days, which covers the period of the first round
of treatment and three days of the second round of treatment. The
effect of each round of treatment was evaluated by the comparison
of the median tumor weight of the treated groups on days 21 or 35
(three days after the end of the first or the second round of
treatment, respectively) to the median tumor weight of the control
group on the same day (T/C.times.100%, see Table 4A).
[0152] The first round of SL-11159 treatment was well tolerated
without deaths and an average maximum body weight loss of 4% (1 g).
The second round of treatment resulted in the death of one animal
and a weight loss of 11% (3 g). The treatment resulted in a
statistically significant growth delay of >40%. The SL-11226
treatment was well tolerated without deaths and body weight losses
ranging from 0 to 10% (0-3 g). The treatment produced statistically
significant growth delays of>37%. Additional details are
provided in Table 4B.
[0153] In summary, the tested compounds, SL-11159 and SL-11226,
exhibited measurable antitumor activity at a dosage which was well
tolerated.
5TABLE 4A Treatment Treatment Results Non- Dose Specific Group
(mg/kg) % T/C % T/C Deaths/ No. Agent [route] Schedule Day 21 Day
35 Total 1 control -- Q 1D .times. 5 day -- -- 13 14, 28 2 SL- 6.25
Q 1D .times. 5 day 65 63 1/8 11159 [IV] 14, 28 3 SL- 6.25 Q 1D
.times. 5 day 46 61 0/8 11226 [IV] 14, 28
[0154]
6TABLE 4B Treatment Results, cont. Tumor Regression Duration Tumor
Free Days Number of Number of Med/Range Survival/ Days to 4 Delay
Partial Complete (Days) Total Doublings (T-C) -- -- -- 1/16* 17.1
-- 0 0 -- 0/8 >24.0 >6.9 0 0 -- 0/8 >23.5 >6.4 *one
tumor failure
[0155] Notes to Tables 4A and 4B:
[0156] Nonspecific deaths: a treated, tumored animal was presumed
to be a nonspecific death if its day of death was significantly
less (p<0.05) than the corresponding day of death in the treated
control group and its tumor was less than 400 mg, or if it died
with a tumor of 400 mg or less prior to 45 days after the last day
of treatment, or with a regressing tumor prior to 15 days after the
last day of treatment, or if the treated animal was uniquely
specified as a nonspecific death on data input.
[0157] Tumor regression was scored (excluding nonspecific deaths),
according to the smallest tumor size attained after the beginning
of treatment relative to the size at first treatment:
[0158] partial: <50 percent of its size at 1 st rx, but not
complete.
[0159] complete: tumor becomes unpalpable.
[0160] Duration of regression: the interval during which a tumor
classified as a partial or complete regressor was below 50 percent
of its size at first treatment.
[0161] Evaluation size: this value is the tumor mass selected at
four mass doublings beginning with the initial tumor size at the
start of treatment.
[0162] T-C (days): the difference in the median of times
postimplant for tumors of the treated groups to attain an
evaluation size compared to the median of the control group. The
T-C value is measured excluding nonspecific deaths and any other
animal that dies whose tumor failed to attain the evaluation
size.
7TABLE 5 Summary Of The Statistical Analysis GROUP PAIRS P VALUE* 1
vs. 2 0.0016 1 vs. 3 0.0082 *Mantel-Haenszel Log-Rank Test
[0163] All references, publications, patents and patent
applications mentioned herein are hereby incorporated by reference
herein in their entirety.
[0164] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding, it will be apparent to those skilled in the art
that certain changes and modifications may be practical. Therefore,
the description and examples should not be construed as limiting
the scope of the invention, which is delineated by the appended
claims.
* * * * *
References