U.S. patent application number 13/897801 was filed with the patent office on 2013-12-19 for mitochondria targeted cationic anti-oxidant compounds for prevention, therapy or treatment of hyper-proliferative disease, neoplasias and cancers.
The applicant listed for this patent is COLBY PHARMACEUTICAL COMPANY, MEDICAL COLLEGE OF WISCONSIN, INC.. Invention is credited to Hirak S. BASU, Joy JOSEPH, Balaraman KALYANARAMAN, David A. ZARLING.
Application Number | 20130338110 13/897801 |
Document ID | / |
Family ID | 43648225 |
Filed Date | 2013-12-19 |
United States Patent
Application |
20130338110 |
Kind Code |
A1 |
ZARLING; David A. ; et
al. |
December 19, 2013 |
MITOCHONDRIA TARGETED CATIONIC ANTI-OXIDANT COMPOUNDS FOR
PREVENTION, THERAPY OR TREATMENT OF HYPER-PROLIFERATIVE DISEASE,
NEOPLASIAS AND CANCERS
Abstract
The inventions disclosed include methods of treating cancers and
related neoplasias, especially prostate cancer, with
pharmaceutically acceptable salts comprising lipophilic cation
moieties linked to nitroxide or linked to hydroxylamine
anti-oxidant groups.
Inventors: |
ZARLING; David A.; (Menlo
Park, CA) ; BASU; Hirak S.; (Madison, WI) ;
KALYANARAMAN; Balaraman; (Wauwatosa, WI) ; JOSEPH;
Joy; (New Berlin, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEDICAL COLLEGE OF WISCONSIN, INC.
COLBY PHARMACEUTICAL COMPANY |
Milwaukee
Menlo Park |
WI
CA |
US
US |
|
|
Family ID: |
43648225 |
Appl. No.: |
13/897801 |
Filed: |
May 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12554476 |
Sep 4, 2009 |
8466130 |
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13897801 |
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PCT/US2008/056035 |
Mar 6, 2008 |
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12554476 |
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60905237 |
Mar 6, 2007 |
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Current U.S.
Class: |
514/89 ; 514/315;
514/91; 546/22; 546/24; 546/242; 548/413 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 31/675 20130101; C07D 211/94 20130101; A61K 31/445 20130101;
C07D 207/46 20130101; A61P 35/00 20180101; C07F 9/59 20130101; C07F
9/572 20130101 |
Class at
Publication: |
514/89 ; 546/24;
546/242; 546/22; 548/413; 514/91; 514/315 |
International
Class: |
C07F 9/59 20060101
C07F009/59; C07F 9/572 20060101 C07F009/572; C07D 211/94 20060101
C07D211/94 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
No. 63119 awarded by the National Institutes of Health
(NIH)/National Heart, Lung, and Blood Institute (NHLBI). The
government has certain rights in the invention.
Claims
1. A salt comprising a cation having the formula: ##STR00053##
wherein A is an antioxidant moiety selected from ##STR00054## E is
a nitrogen or phosphorus atom; R.sub.1', R.sub.1'', and R.sub.1'''
are each independently organic moieties comprising between 1 and 12
carbon atoms, and E, R.sub.1', R.sub.1'', and R.sub.1''' together
form a quaternary ammonium or phosphonium cation; each R.sup.2 is
independently C.sub.1-C.sub.4 alkyl; Lg is --CH.sub.2--; L
comprises a polyalkylene chain having 1 to 21 carbon chain atoms,
wherein any one or more of the hydrogens bonded to said carbon
chain atoms can be optionally substituted with one or two
independently selected hydroxyl, halogen, amino, methylamino,
dimethylamino, or C.sub.1-C.sub.4 organic moieties selected from
alkyl, hydroxyalkyl, alkoxy, alkoxylalkyl, carboxy, or carboxyalkyl
moieties; and wherein the salt further comprises one or more anions
X.sup.m- wherein m is an integer from 1 to 4, to form the salt.
2. The salt of claim 1, wherein X.sup.m- is a halide, sulfate,
hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen
phosphate, carbonate, hydrogen carbonate, fumarate, maleate,
maltolate, succinate, acetate, benzoate, oxalate, citrate, or
tartrate anion.
3. The salt of claim 1, wherein X.sup.m- is a bromide anion.
4. The salt of claim 1, wherein all R.sup.2 are methyl.
5. The salt of claim 1, wherein L is --(CH.sub.2)n-, wherein n is
from 5 to 20.
6. The salt of claim 5, wherein n is 10.
7. The salt of claim 1, wherein L further comprises, in the
polyalkylene, one to 10 groups independently selected from --O--,
--S--, --S(O)--, --S(O).sub.2--, --NH--, --NCH.sub.3--, --C(O)--,
or --CO.sub.2--.
8. The salt of claim 7, wherein L is
--(CH.sub.2CH.sub.2O)m-CH.sub.2CH.sub.2O--, wherein m is an integer
from 0 to 3.
9. The salt of claim 1, wherein R.sub.1', R.sub.1'', and R.sub.1'''
are independently C.sub.4-C.sub.10 alkyl or phenyl moieties, each
of which is optionally substituted with one or two independently
selected hydroxyl, halogen, amino, cyano, methylamino,
dimethylamino, alkyl, hydroxyalkyl, alkoxy, alkoxylalkyl, carboxy,
or carboxyalkyl moieties.
10. The salt of claim 9, wherein R.sub.1', R.sub.1'', and
R.sub.1''' are each independently selected from the group
consisting of n-C.sub.4H.sub.9 moieties, phenyl moieties, and
benzyl moieties.
11. The salt of claim 10, wherein R.sub.1', R.sub.1'', and
R.sub.1''' are all phenyl.
12. The salt of claim 1, wherein E is a phosphorus atom.
13. The salt of claim 12, wherein E is a phosphorus atom and
R.sub.1', R.sub.1'', and R.sub.1''' are all phenyl or benzyl.
14. The salt of claim 1, wherein A together with L.sub.g is
##STR00055##
15. The salt of claim 1, wherein A together with L.sub.g is
##STR00056## L is --(CH.sub.2)n-, wherein n is from 5 to 24; and E
is a phosphorus atom; and R.sub.1', R.sub.1'', and R.sub.1''' are
all phenyl.
16. A pharmaceutical composition comprising one or more
pharmaceutically acceptable salts of claim 1 and a pharmaceutically
acceptable carrier.
17. A method for treating or inhibiting the occurrence, recurrence,
progression or metastasis, of a cancer or a neoplasia precursor
thereof, consisting of administering to a mammal diagnosed as
having a cancer or precursor neoplasia thereof, in an amount
effective to treat the cancer or neoplasia precursor thereof or
inhibit the occurrence, recurrence, progression, or metastasis of
the cancer or precursor neoplasia thereof, a salt of claim 1.
18. The method of claim 17, wherein the cancer is prostate
cancer.
19. A method for treating or inhibiting the occurrence, recurrence,
or progression of a non-cancerous inflammation or hyperplasia,
comprising administering to a mammal diagnosed as having a
non-cancerous inflammation or hyperplasia, in an amount effective
to treat the non-cancerous inflammation or hyperplasia or inhibit
the occurrence, recurrence, or progression of the non-cancerous
inflammation or hyperplasia, a salt of claim 1.
20. The method of claim 19, wherein the non-cancerous hyperplasia
is prostate hyperplasia.
21. A salt comprising a cation having the formula: ##STR00057##
wherein A is an antioxidant moiety selected from ##STR00058## E is
a nitrogen or phosphorus atom; R.sub.1', R.sub.1'', and R.sub.1'''
are each independently organic moieties comprising between 1 and 12
carbon atoms, and E, R.sub.1', R.sub.1'', and R.sub.1''' together
form a quaternary ammonium or phosphonium cation; L is an organic
moiety comprising from 1 to 8 carbon atoms; and wherein the salt
further comprises one or more anions X.sup.m- wherein m is an
integer from 1 to 4, to form the salt.
22. The salt of claim 21 wherein E is a phosphorus atom.
23. The salt of claim 21, wherein R.sub.1', R.sub.1'', and
R.sub.1''' are independently C.sub.4-C.sub.10 alkyl or phenyl
moieties, each of which is optionally substituted with one or two
independently selected hydroxyl, halogen, amino, cyano,
methylamino, dimethylamino, alkyl, hydroxyalkyl, alkoxy,
alkoxylalkyl, carboxy, or carboxyalkyl moieties.
24. The slat of claim 23, wherein R.sub.1', R.sub.1'', and
R.sub.1''' are each independently selected from the group
consisting of n-C.sub.4H.sub.9 moieties, phenyl moieties, and
benzyl moieties.
25. The salt of claim 24, wherein R.sub.1', R.sub.1'', and
R.sub.1''' are all phenyl
26. The salt of claim 21, wherein L is --(CH.sub.2)n-, wherein n is
3, 4, 5, 6, 7, 8, or 9.
27. The salt of claim 21, wherein L is
--(CH.sub.2CH.sub.2O)m-CH.sub.2CH.sub.2O--, wherein m is an integer
from 0 to 3.
28. The salt of claim 21, wherein A is ##STR00059##
29. The salt of claim 21, wherein the cation is of formula:
##STR00060## wherein n is 3, 4, 5, 6, 7, 8, or 9;
30. The salt of claim 21, wherein the salt is: ##STR00061##
31. A pharmaceutical composition comprising one or more
pharmaceutically acceptable salts of claim 21 and a
pharmaceutically acceptable carrier.
32. A method for treating or inhibiting the occurrence, recurrence,
progression or metastasis, of a cancer or a neoplasia precursor
thereof, consisting of administering to a mammal diagnosed as
having a cancer or precursor neoplasia thereof, in an amount
effective to treat the cancer or neoplasia precursor thereof or
inhibit the occurrence, recurrence, progression, or metastasis of
the cancer or precursor neoplasia thereof, a salt of claim 1.
33. The method of claim 32, wherein the cancer is prostate
cancer.
34. A method for treating or inhibiting the occurrence, recurrence,
or progression of a non-cancerous inflammation or hyperplasia,
comprising administering to a mammal diagnosed as having a
non-cancerous inflammation or hyperplasia, in an amount effective
to treat the non-cancerous inflammation or hyperplasia or inhibit
the occurrence, recurrence, or progression of the non-cancerous
inflammation or hyperplasia, a salt of claim 1.
35. The method of claim 34, wherein the non-cancerous hyperplasia
is prostate hyperplasia.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/554,476, filed Sep. 4, 2009, which claims
benefit under 35 U.S.C. .sctn.120 and is a Continuation of
International PCT Application No. PCT/US2008/056035, filed Mar. 6,
2008, which claims benefit under 35 U.S.C. .sctn.119(e) of U.S.
Provisional Application No. 60/905,237, filed Mar. 6, 2007. Content
of all of which is incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The inventions described herein relate to the treatment of
various forms of cancer, or related neoplasias, with compounds that
comprise one or more quaternary cationic moieties chemically linked
to one or more anti-oxidant moieties comprising nitroxide or amine
oxide moieties.
BACKGROUND
[0004] Oxidative stress has been known to contribute to a variety
of human degenerative diseases associated with aging, such as
Parkinson's disease and Alzheimer's disease, as well as to
Huntington's Chorea, diabetes and Friedreich's Ataxia (Allen, S.,
et al., J. Biol. Chem. 278:6371-6383, 2003; Hirai, K., et al., J.
Neurosci. 21:3017-3023, 2001) and to non-specific cellular damage
that accumulates with aging.
[0005] Mitochondria are the intracellular organelles primarily
responsible for energy metabolism, and are also the major source of
the free radicals and reactive oxygen species ("ROS", such as
hydrogen peroxide and the superoxide radical anion
(O.sub.2.sup.-.)) that cause oxidative stress and/or damage inside
most cells (Murphy M P, Smith R A., Ann. Rev Pharmacol Toxicol.
2006 Oct. 2; Epub ahead of print). Mitochondria are equipped to
detoxify hydrogen peroxide due to the presence of antioxidant
enzymes (peroxiredoxins, thioredoxins, and GSH-dependent
peroxidases (Chang, T. S., J. Biol. Chem. 279, 41975-41984, 2004).
Typically, mitochondrial superoxide (, the radical anion produced
by one electron reduction of O.sub.2) is dismutated according to
the stoichiometry shown below, by manganese superoxide dismutase
(MnSOD) that is localized within the mitochondrial matrix.
2+2H.sup.+.fwdarw.O.sub.2+H.sub.2O.sub.2
[0006] However, when cellular ROS production exceeds the cell's
detoxification capacity, oxidative damage can occur. This damage
disrupts mitochondrial function and oxidative phosphorylation and
leads to significant cellular damage to mitochondrial, cytoplasmic
or nuclear cellular proteins, DNA, RNA and phospholipids and thus
induces cell damage, death and/or disease. Superoxide can also
react with nitric oxide at a diffusion-controlled reaction rate,
forming a highly potent oxidant and peroxynitrite that can modify
proteins and DNA through oxidation and nitration reactions
(Beckman, J. S., et al., Nature 364, 584, 1993). In addition to
these damaging and pathological roles, ROS also act as a redox
signaling molecule(s) and promotes cell proliferation, DNA damage
repair errors and mutation leading to inflammatory
hyper-proliferation, neoplasia and malignancy (see Michikawa et.
al., "Aging-dependent large accumulation of point mutations in the
human mtDNA control region for replication." Science. October 22;
286(5440):774-9).
[0007] Naturally occurring exogenous and endogenous tissue reactive
oxygen species (ROS) are known to play a major role in prostate,
colorectal, lymphoma and pancreatic carcinogenesis (De Marzo et
al., J Cell Biochem. 2004 Feb. 15; 91(3):459-77; Reliene R,
Schiestl R H. Antioxidants suppress lymphoma and increase longevity
in ATM-deficient mice. J Nutr. 2007 January; 137(1):229S-32S). ROS
alters the activity of thiol-dependent enzymes, changes the
cellular redox balance and covalently modifies proteins and
modifies and mutagenizes DNA (Sikka S C., Curr Med Chem. 2001 June;
8(7):851-62. Kamat A M, Lamm D L. W V Med J. 2000; 96: 449-54.). It
has also been shown that increased lipid peroxidation and
production of high levels of unregulated ROS in men with high fat
diets is one of the major reasons for the higher incidence of
prostate cancer in industrialized nations, as compared to that in
developing countries (Dargel, R., Exp Toxic Pathol 1992,
44:169-181). In recent years, direct experimental evidence has
linked increased ROS levels with the corresponding increase in
mutations and tumor development in various tissues, including in
the pancreas and the prostate organs (Oberley et. al., The
Prostate, 2000:44:144-155). For example, Oberley and his colleagues
monitored oxidative stress induced enzymes and oxidative damage to
DNA bases of malignant and normal human prostate tissues. Malignant
prostate tumor tissues showed significantly higher oxidative stress
and ROS-induced DNA modifications as compared to normal prostate
tissues. Ho and her coworkers (Tam et al., Prostate. 2006 Jan. 1;
66(1):57-69) demonstrated the presence of high oxidative stress
induced DNA modifications in the pre-neoplastic lesions occurring
in the well studied TRAMP (Transgenic Adenocarcinoma of Mouse
Prostate) and Noble rat (Tam et al., Sex Hormones induce direct
epithelial and inflammation-mediated oxidative/nitrosative stress
that favors prostatic carcinogenesis in the Noble rat. Am. J.
Pathol. 2007 October 117(4); 1334-41, Epub. 2007 Aug. 23.) prostate
cancer mouse model of human prostate cancer.
[0008] Hence, unregulated mitochondrial ROS production, the
resulting oxidative cellular damage-induced-carcinogenesis
represent unsolved problems in the art, and present a compelling
target for pharmacological drug interventions with pharmaceutical
anti-superoxide small molecule drug formulations.
[0009] To prevent the cellular damage caused by oxidative stress a
number of prior art anti-oxidant therapies have been developed for
treating various diseases resulting from oxidative stress. However,
most of those inventions are not targeted to other organelles
within cells or to the mitochondria and are therefore less than
optimally effective.
[0010] In recent years, there has been interest in
mitochondria-targeting technologies (see Murphy M P. "Selective
targeting of bioactive compounds to mitochondria." Trends
Biotechnol. 1997 August; 15(8):326-30). In these approaches,
"warhead" groups are covalently coupled via linker groups to a
bulky and/or lipophilic cationic moiety such as a quaternary
ammonium or phosphonium cationic moiety. These compounds are
initially absorbed and accumulate in the cytoplasmic region of
cells in response to the negative plasma membrane potential of
30-60 mV (3). Additionally, within a few minutes after drug
treatment, the lipophilic cations with a positive 30-60 mV
potential permeate through the mitochondrion's lipid layers and
selectively accumulate within mitochondria due to the larger
mitochondrial membrane potential of 150-170 mV; (negative
inside).
[0011] Mitochondria-targeted compounds in this class of agents are
shown below and include a mitochondria-directed ubiquinone (MitoQ)
reported by Murphy and coworkers (U.S. Pat. Nos. 6,331,532 and
7,232,809, and EP Patent 1 047 701 B1, all of which are herein
incorporated by reference in their entirety).
##STR00001##
[0012] MitoQ has shown promise in the treatment of only some, but
not all, oxidative stress induced diseases. MitoQ is currently
undergoing Phase II clinical trials for the treatment of
Parkinson's disease, but it has relatively minor activity against
other oxidative stress-induced neurodegenerative diseases such as
Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease). This
class of compounds is also disclosed in U.S. Published Application
No. U.S. 2008/0032940, herein incorporated by reference in its
entirety, in the context of methods for treating cancer.
[0013] Other classes of mitochondria-targeted compounds include
mitochondria-targeted nitroxides, which have been used in method
for treating neurodegenerative disorders (see U.S. Published
Application No. 2007/0066572, herein incorporated by reference in
its entirety) and mitochondria-targeted antioxidants, which have
been used in methods for treating cancer (see U.S. application Ser.
No. 11/834,799, entitled "Methods for Reducing
Anthracycline-Induced Toxicity," filed Aug. 7, 2007, herein
incorporated by reference in its entirety).
[0014] Accordingly, there remains a need for mitochondrially
targeted anti-inflammatory, anti-proliferative anti-cancer agents
with improved properties and/or toxicity profiles and it is towards
the provision of such anti-oxidants, which may be targeted to
mitochondria that the various inventions disclosed and described
below are directed.
SUMMARY
[0015] One aspect of the disclosure relates to methods for treating
or inhibiting the occurrence, recurrence, progression or
metastasis, of a cancer or a neoplastic or hyper-proliferative
precursor thereof, consisting of administering to a mammal
diagnosed as having a cancer or precursor neoplasia or
hyper-proliferative disorder thereof, in an amount effective to
treat the cancer or inhibit the occurrence recurrence, progression,
or metastasis of the cancer or precursor hyperplasia or neoplasia
thereof, a pharmaceutically acceptable salt comprising one or more
cations having the formula:
##STR00002##
[0016] wherein [0017] a) A is an antioxidant moiety comprising one
or more nitroxide or hydroxylamine moieties, or a pro-drug thereof,
having from three to 16 carbon atoms, [0018] b) L is an organic
linking moiety comprising 4 to 30 carbon atoms, [0019] c) E is a
nitrogen or phosphorus atom, [0020] d) R.sub.1', R.sub.1'', and
R.sub.1''' are each independently selected organic moieties
comprising between 1 and 12 carbon atoms, [0021] wherein E,
R.sub.1', R.sub.1'', and R.sub.1''' together form a quaternary
ammonium or phosphonium cation; [0022] and wherein the salt further
comprises one or more X.sup.m- pharmaceutically acceptable anions,
where m is an integer from 1 to 4, in sufficient quantities to form
the pharmaceutically acceptable salt.
[0023] A related aspect of the disclosure relates to a method for
treating, regulating or inhibiting the prostate's inflammation,
hyperplasia or enlargement and the occurrence, recurrence,
progression or metastasis of prostate cancer, consisting of
administering to a mammal diagnosed as having prostate disease or
cancer or precursor neoplasia thereof, in an amount effective to
treat the cancer or modulate the inflammation, hyperplasia, or
enlargement of the prostate or the occurrence, recurrence,
progression, or metastasis of prostate cancer or precursor
neoplasia thereof, with one or more pharmaceutically acceptable
salts having one or more cations having the formula
##STR00003##
[0024] wherein [0025] a) E is a nitrogen or phosphorus atom, [0026]
b) R.sub.1', R.sub.1'', and R.sub.1''' are each independently
selected organic moieties comprising between 1 and 12 carbon atoms,
[0027] c) n is an integer between 8 and 12, and [0028] d) R.sub.1',
R.sub.1'', and R.sub.1''' are each independently selected organic
moieties comprising between 1 and 12 carbon atoms, [0029] wherein
E, R.sub.1', R.sub.1'', and R.sub.1''' together form a quaternary
ammonium or phosphonium cation; [0030] wherein the salt further
comprises one or more pharmaceutically acceptable anions X.sup.m-,
wherein m is an integer from 1 to 4, in sufficient amount to form
the pharmaceutically acceptable salt.
[0031] In other related aspects, the inventions described and
disclosed herein relate to salts comprising one or more cations
having the formula:
##STR00004##
[0032] wherein [0033] a) E is a nitrogen or phosphorus atom, [0034]
b) R.sub.1', R.sub.1'', and R.sub.1''' are each independently
selected organic moieties comprising between 1 and 12 carbon atoms,
[0035] c) n is an integer between 5 and 12, and [0036] wherein E,
R.sub.1', R.sub.1'', and R.sub.1''' together form a quaternary
ammonium or phosphonium cation; and [0037] wherein the salt further
comprises is one or more pharmaceutically acceptable anions
X.sup.m, wherein m is an integer from 1 to 4, in sufficient amount
to form the pharmaceutically acceptable salt.
[0038] Another aspect of the disclosure relates to salts comprising
one or more cations having the formula:
##STR00005##
[0039] wherein [0040] a) E is a nitrogen or phosphorus atom, [0041]
b) R.sub.1', R.sub.1'', and R.sub.1''' are each independently
selected organic moieties comprising between 1 and 12 carbon atoms,
[0042] c) n is an integer between 5 and 12, and [0043] wherein E,
R.sub.1', R.sub.1'', and R.sub.1''' together form a quaternary
ammonium or phosphonium cation; and [0044] wherein the salt further
comprises one or more anions X.sup.m- in a sufficient amount to
form the pharmaceutically acceptable salt.
[0045] It is understood that the examples and embodiments described
above are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended
claims.
[0046] Additional aspects of the inventions and advantages thereof
will be set forth in part in the description which follows and in
part will be obvious from the description, or may be learned by
practice of the aspects described below. The advantages described
below will be realized and attained by means of the elements and
combinations particularly pointed out in the appended claims. It is
to be understood that both the foregoing general description and
the following detailed description are exemplary and explanatory
only and are not restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several aspects
described below. Like numbers represent the same elements
throughout the figures.
[0048] FIG. 1 shows the inhibitory effect of varying concentrations
of MitoT on the proliferation and growth of human prostate tumor
LNCaP and PC-3 cells, as determined by Hoechst dye-DNA fluorescence
assays.
[0049] FIG. 2 shows a microscopic picture of DCF fluorescence
(green) and Hoechst dye-DNA fluorescence (blue) in human prostate
tumor LNCaP cells, which have been placed under high oxidative
stress due to treatment of the cells with a 1 nM solution of the
synthetic androgen analog, R1881.
[0050] FIG. 3 shows the inhibitory effect of treatment with varying
concentrations of MitoT on the oxidative stress levels in LNCaP
human prostate tumor cells, as determined by the ratio of DCF
fluorescence/Hoechst dye-DNA fluorescence.
[0051] FIG. 4 shows that MitoT treatment at sufficient
concentrations reduces the ROS level and causes inhibition of cell
growth and proliferation in LNCaP human prostate cancer cells (A)
Plot of % control of MTT absorbance at increasing MitoT
concentration shows MitoT absorbs mitochondrial electrons. (B) Plot
of the ratio of % of control cell MTT absorbance/DNA fluorescence
shows that the decrease in MTT oxidation is related to cell growth
arrest.
[0052] FIG. 5 shows that synthetic androgen (R1881)
treatment-induced oxidative stress in LNCaP human prostate cancer
cells, as determined by the ratio of DCF fluorescence/DNA
fluorescence, is strongly inhibited by pre-treatment of the cells
with 1 .mu.M MitoT.
[0053] FIG. 6 shows the pharamacokinetics of MitoT absorption in
serum and in kidney tissues of white mice given one intraperitoneal
(i.p.) injection of MitoT (5 mg/kg) showing serum clearance and
tissue accumulation of the drug with the passage of time.
[0054] FIG. 7 shows pictorial microscopic evidence of Hydroethidine
dye fluorescence due to ROS oxidation in kidney tissue of (A)
untreated mice and (B) mice treated with 5 mg/kg MitoT for 3
hours.
[0055] FIG. 8 shows the inhibitory effect of varying concentrations
of MitoT-10 on the growth and proliferation of LNCaP and PC-3 human
prostate cancer cells, as determined by Hoechst dye-DNA
fluorescence assays
DETAILED DESCRIPTION
[0056] Before the disclosure is described in detail, it is
understood that scope of this disclosure is not limited to the
particular methodology, protocols, cell lines, and reagents
described, as these may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only and is not intended to limit the scope of the
disclosure, which will be limited only by the appended claims.
[0057] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and equivalents thereof known to those skilled in the art, and so
forth. As well, the terms "a" (or "an"), "one or more" and "at
least one" can be used interchangeably herein. It is also to be
noted that the terms "comprising", "including", and "having" can be
used interchangeably.
[0058] Often, ranges are expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint.
[0059] "Optional" or "optionally" means that the subsequently
described event or circumstance can or cannot occur, and that the
description includes instances where the event or circumstance
occurs and instances where it does not. For example, the phrase
"optionally substituted lower alkyl" means that the lower alkyl
group can or can not be substituted and that the description
includes both unsubstituted lower alkyl and lower alkyl where there
is substitution.
[0060] A cell can be in vitro. Alternatively, a cell can be in vivo
and can be found in a subject. A "cell" can be a cell from any
organism including, but not limited to, a bacterium or a mammalian
cell or a tumor cell.
[0061] As used throughout, by a "subject" is meant an individual.
Thus, the "subject" can include domesticated animals, such as cats,
dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats,
rabbits, etc.), laboratory animals (e.g., mouse, rabbit, rat,
guinea pig, ferret, mink, etc.) and birds. In one aspect, the
subject is a higher mammal such as a primate or a human.
[0062] In one aspect, the compounds described herein can be
administered to a subject comprising a human or an animal
including, but not limited to, a murine, canine, feline, equine,
bovine, porcine, caprine or ovine species and the like, that is in
need of alleviation or amelioration from a recognized medical
condition.
[0063] References in the specification and concluding claims to
parts by weight, of a particular element or component in a
composition or article, denotes the weight relationship between the
element or component and any other elements or components in the
composition or article for which a part by weight is expressed.
Thus, in a compound containing 2 parts by weight of component X and
5 parts by weight component Y, X and Y are present at a weight
ratio of 2:5, and are present in such ratio regardless of whether
additional components are contained in the compound.
[0064] A weight percent of a component, unless specifically stated
to the contrary, is based on the total weight of the formulation or
composition in which the component is included.
[0065] The term "moiety" defines a carbon containing residue, i.e.
a moiety comprising at least one carbon atom, and includes but is
not limited to the carbon-containing groups defined hereinabove.
Organic moieties can contain various heteroatoms, or be bonded to
another molecule through a heteroatom, including oxygen, nitrogen,
sulfur, phosphorus, or the like. Examples of organic moieties
include but are not limited alkyl or substituted alkyls, alkoxy or
substituted alkoxy, mono or di-substituted amino, amide groups,
etc. Organic moieties can preferably comprise 1 to 21 carbon atoms,
1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms,
1 to 8 carbon atoms, or 1 to 4 carbon atoms.
[0066] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing the
chemicals, cell lines, vectors, animals, instruments, statistical
analysis and methodologies which are reported in the publications
which might be used in connection with the invention. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0067] The term "alkyl" denotes a moiety containing a saturated,
straight or branched hydrocarbon residue having from 1 to 18
carbons, or preferably 4 to 14 carbons, 5 to 13 carbons, or 6 to 10
carbons. An alkyl is structurally similar to a non-cyclic alkane
compound modified by the removal of one hydrogen from the
non-cyclic alkane and the substitution, therefore, with a
non-hydrogen group or moiety. Alkyl moieties can be branched or
unbranched. Lower alkyl moieties have 1 to 4 carbon atoms. Examples
of alkyl moieties include methyl, ethyl, n-propyl, iso-propyl,
n-butyl, sec-butyl, t-butyl, amyl, t-amyl, n-pentyl and the
like.
[0068] The term "substituted alkyl" denotes an alkyl moiety
analogous to the above definition that is substituted with one or
more organic or inorganic substituent moieties. In some
embodiments, 1 or 2 organic or inorganic substituent moieties are
employed. In some embodiments, each organic substituent moiety
comprises between 1 and 4, or between 5 and 8 carbon atoms.
Suitable organic and inorganic substituent moieties include, but
are not limited to, hydroxyl, halogens, cycloalkyl, amino,
mono-substituted amino, di-substituted amino, acyloxy, nitro,
cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted
alkylcarboxamide, dialkylcarboxamide, substituted
dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl,
thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy,
heteroaryl, substituted heteroaryl, aryl or substituted aryl. When
more than one substituent group is present then they can be the
same or different.
Abbreviations Used Herein Include:
[0069] The term "alkoxy" as used herein denotes a moiety alkyl,
defined above, attached directly to a oxygen to form an ether
residue. Examples include methoxy, ethoxy, n-propoxy, iso-propoxy,
n-butoxy, t-butoxy, iso-butoxy and the like.
[0070] The term "substituted alkoxy" denotes a alkoxy moiety of the
above definition that is substituted with one or more groups, but
preferably one or two substituent groups including hydroxyl,
cycloalkyl, amino, mono-substituted amino, di-substituted amino,
acyloxy, nitro, cyano, carboxy, carboalkoxy, alkylcarboxamide,
substituted alkylcarboxamide, dialkylcarboxamide, substituted
dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl,
thiohaloalkyl, alkoxy, substituted alkoxy or haloalkoxy. When more
than one group is present then they can be the same or
different.
[0071] The term "mono-substituted amino" denotes an amino
(--NH.sub.2) group substituted with one group selected from alkyl,
substituted alkyl or arylalkyl wherein the terms have the same
definitions found throughout.
[0072] The term "di-substituted amino" denotes an amino substituted
with two moieties that can be same or different selected from aryl,
substituted aryl, alkyl, substituted alkyl or arylalkyl wherein the
terms have the same definitions found throughout. Some examples
include dimethylamino, methylethylamino, diethylamino and the
like.
[0073] The term "haloalkyl" denotes a alkyl moiety, defined above,
substituted with one or more halogens, preferably fluorine, such as
a trifluoromethyl, pentafluoroethyl and the like.
[0074] The term "haloalkoxy" denotes a haloalkyl, as defined above,
that is directly attached to an oxygen to form a halogenated ether
residue, including trifluoromethoxy, pentafluoroethoxy and the
like.
[0075] The term "acyl" denotes a moiety of the formula --C(O)--R
that comprises a carbonyl (C.dbd.O) group, wherein the R moiety is
an organic moiety having a carbon atom bonded to the carbonyl
group. Acyl moieties contain 1 to 8 or 1 to 4 carbon atoms.
Examples of acyl moieties include but are not limited to formyl,
acetyl, propionyl, butanoyl, iso-butanoyl, pentanoyl, hexanoyl,
heptanoyl, benzoyl and like moieties.
[0076] The term "acyloxy" denotes a moiety containing 1 to 8
carbons of an acyl group defined above directly attached to an
oxygen such as acetyloxy, propionyloxy, butanoyloxy,
iso-butanoyloxy, benzoyloxy and the like.
[0077] The term "aryl" denotes an unsaturated and conjugated
aromatic ring moiety containing 6 to 18 ring carbons, or preferably
6 to 12 ring carbons. Many aryl moieties have at least one
six-membered aromatic "benzene" moiety therein. Examples of such
aryl moieties include phenyl and naphthyl.
[0078] The term "substituted aryl" denotes an aryl ring moiety as
defined above that is substituted with or fused to one or more
organic or inorganic substituent moieties, which include but are
not limited to a halogen, alkyl, substituted alkyl, haloalky,
hydroxyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, amino, mono-substituted amino,
di-substituted amino, acyloxy, nitro, cyano, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,
substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl,
thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy or haloalkoxy,
aryl, substituted aryl, heteroaryl, heterocyclic ring, substituted
heterocyclic ring moiety, wherein the terms are defined herein.
Substituted aryl moieties can have one, two, three, four, five, or
more substituent moieties. The substituent moieties can be not be
of unlimited size or molecular weight, and each organic moiety can
comprise 15 or fewer, 10 or fewer, or 4 or fewer carbon atoms
unless otherwise expressly contemplated by the claims
[0079] The term "heteroaryl" denotes an aryl ring moiety as defined
above, wherein at least one of the carbons of the aromatic ring has
been replaced with a heteroatom, which include but are not limited
to nitrogen, oxygen, and sulfur atoms. Heteroaryl moieties include
6 membered aromatic ring moieties, and can also comprise 5 or 7
membered aromatic rings, or bicyclic or polycyclic heteroaromatic
rings as well. Examples of heteroaryl moieties include pyridyl,
bipyridyl, furanyl, and thiofuranyl residues. It is to be
understood that the heteroaryl moieties can optionally be
substituted with one or more organic or inorganic substituent
moieties bound to the carbon atoms of the heteroaromatic rings, as
described hereinabove for substituted aryl moieties. Substituted
heteroaryl moieties can have one, two, three, four, five, or more
substituent organic or inorganic moieties, in a manner analogous to
the substituted aryl moieties defined herein. The substituent
moieties cannot be of unlimited size or molecular weight, and each
organic substituent moiety can comprise 15 or fewer, 10 or fewer,
or four or fewer carbon atoms unless otherwise expressly
contemplated by the claims.
[0080] The term "halo," "halide," or "halogen" refers to a fluoro,
chloro, bromo or iodo atom or ion.
[0081] The term "heterocycle" or "heterocyclic", as used in the
specification and concluding claims, refers to a moiety having a
closed ring structure comprising 3 to 10 ring atoms, in which at
least one of the atoms in the ring is an element other than carbon,
such as, for example, nitrogen, sulfur, oxygen, silicon,
phosphorus, or the like. Heterocyclic compounds having rings with
5, 6, or 7 members are common, and the ring can be saturated, or
partially or completely unsaturated. The heterocyclic compound can
be monocyclic, bicyclic, or polycyclic. Examples of heterocyclic
compounds include but are not limited to pyridine, piperidine,
thiophene, furan, tetrahydrofuran, and the like. The term
"substituted heterocyclic" refers to a heterocyclic moiety as
defined above having one or more organic or inorganic substituent
moieties bonded to one of the ring atoms.
[0082] The term "carboxy", as used in the specification and
concluding claims, refers to the --C(O)OH moiety that is
characteristic of carboxylic acids. The hydrogen of the carboxy
moieties is often acidic and (depending on the pH) often partially
or completely dissociates, to form an acid H+ ion and a carboxylate
anion (--CO.sub.2.sup.-), wherein the carboxylate anion is also
sometimes referred to as a "carboxy" moiety.
[0083] It is understood that when a chiral atom is present in a
compound disclosed herein, both separated enantiomers, racemic
mixtures and mixtures of enantiomeric excess are within the scope
of the invention. Separation of such mixtures may be done using
techniques known in the art. As defined herein, a racemic mixture
is an equal ratio of each of the enantiomers, whereas an
enantiomeric excess is when the percent of one enantiomer is
greater than the other enantiomer, all percentages are within the
scope of the invention. Furthermore, when more than one chiral atom
is present in a compound, then the enantiomers, racemic mixtures,
mixtures of enantiomeric excess and diastereomic mixtures are
within the scope of the invention.
II. The Inventions
Compounds Employed in the Inventions
[0084] The compounds described below are salts, and can be used for
the treatment of various diseases as disclosed elsewhere herein. As
will be appreciated by those of ordinary skill in the art, the
salts comprise a mixture of cations and anions whose total number
of positive and negative charges are electrically balanced. More
particularly, however, the salts disclosed herein have one or more
cations having the Formula (I) illustrated below
##STR00006##
[0085] wherein [0086] a) A is an antioxidant moiety comprising one
or more nitroxide or hydroxylamine moieties, or a prodrug thereof,
[0087] b) L is an organic linking moiety, [0088] c) E is a nitrogen
or phosphorus atom, [0089] d) R.sub.1', R.sub.1'', and R.sub.1'''
are each independently selected from organic moieties comprising
between 1 and 12 carbon atoms, [0090] wherein E, R.sub.1',
R.sub.1'', and R.sub.1''' together form a quaternary ammonium or
phosphonium cation; and [0091] wherein the salt further comprises
is one or more pharmaceutically acceptable anions X.sup.m-, wherein
m is an integer from 1 to 4, in sufficient amount to form the
pharmaceutically acceptable salt. The various genera, subgenera,
and species of the compounds of Formula (I) share at least the
features disclosed above, and have related functions and utilities,
but can differ in specific structural features, as described
below.
The Antioxidant "A" Moieties
[0092] The compounds of the invention all comprise at least one
antioxidant moiety "A" which comprises at least one or more
nitroxide radial or hydroxylamine moieties bonded therein or
thereto.
[0093] Nitroxides and relevant hydroxylamines have the chemical
structures shown below:
##STR00007##
[0094] It is well known, as explained above, that superoxide anion
is formed in relatively large concentrations in the mitochondria of
cells, and that excess concentrations of superoxide are deleterious
to the mitochondria and other parts of the cell. It is well known
in the art that nitroxides and/or hydroxylamines can function to
catalyze the dismutation of superoxide anion in the fashion
illustrated below.
##STR00008##
[0095] Accordingly, the "A" moieties of the salts described herein,
which comprise one or more nitroxide or hydroxylamine moieties, can
catalyze the dismutation of superoxide radical anions in the cell,
to form hydrogen peroxide which can be dealt with by enzymes in the
cell, and therefore serve to function as "anti-oxidants." The
nitroxide or hydroxylamine moieties are part of a larger A moiety,
which in many embodiments can comprise between 4 and 30 carbon
atoms, or, 6 to 24 carbon atoms, or 7 to 18 carbon atoms, or from 8
to 12 carbon atoms.
[0096] In some embodiments, the A moieties have 5 or 6-member rings
containing a nitroxide free radical therein having the
formulas:
##STR00009##
5 and 6-member rings containing a hydroxylamine moiety having the
formula:
##STR00010##
wherein the R.sup.2 moieties are organic substitutent moieties. In
some embodiments, the R.sup.2 moieties can be C.sub.1-C.sub.4
alkyls, aryls, alkoxides, and the like. In some embodiments, the
R.sup.2 moieties can be C.sub.1-C.sub.4 linear or branched alkyl,
for example, methyl (C.sub.1), ethyl (C.sub.2), n-propyl (C.sub.3),
iso-propyl (C.sub.3), n-butyl (C.sub.4), sec-butyl (C.sub.4),
iso-butyl (C.sub.4), and tert-butyl (C.sub.4).
[0097] In many embodiments, the R.sup.2 moieties are all methyl
groups as shown below, which are
[2,2,5,5-tetramethylpyrrolidinyloxy] free radical having the
formula:
##STR00011##
[0098] Similar A moieties having corresponding hydroxylamine
moieties are 2,2,5,5-tetramethylpyrrolidin-1-ols having the
formula:
##STR00012##
[0099] Such compounds comprising the five membered nitroxide or
hydroxylamine moieties incorporated into such five-membered
heterocycles are often referred to in the art as "Proxyl"
derivatives.
[0100] In other embodiments, the "A" moieties are
[2,2,6,6-tetramethylpiperidinyloxy] free radical moieties the
formula:
##STR00013##
or 2,2,6,6-tetramethylpiperidin-1-ol moieties having the
formula:
##STR00014##
[0101] Such compounds comprising a nitroxide or hydroxylamine
moieties incorporated into such six-membered heterocycles are often
referred to in the art as "Tempol" derivatives.
[0102] In the Proxyl or Tempol analogs shown above, L.sub.g can be
any of a variety of moieties that form a bond to the "L" linking
group. In many embodiments, L.sub.g can be one of the following
moieties: --O, --CH.sub.2--; --CH.sub.2--O--, --CH.sub.2--NH--,
--NR.sup.3--, --NR.sup.3C(O)--; --C(O)NR.sup.3--, --C(O)O--,
--C(O)--, --OC(O)--, --NHC(O)NH--, or --NH(.dbd.NH)NH--, wherein
R.sup.3 is hydrogen or a linear or branched C.sub.1-C.sub.4 alkyl,
for example, methyl (C.sub.1), ethyl (C.sub.2), n-propyl (C.sub.3),
iso-propyl (C.sub.3), n-butyl (C.sub.4), sec-butyl (C.sub.4),
iso-butyl (C.sub.4), and tert-butyl (C.sub.4).
[0103] In some embodiments, the L.sub.g moieties taken together
with nitroxide free radical moieties can provide "Tempol" analog
moieties having the formula:
##STR00015##
or any of the similar hydroxylamine moieties having the
formulas;
##STR00016##
[0104] In some embodiments, the L.sub.g moieties taken together
with nitroxide free radical moieties can provide "Proxyl" analog
moieties having the formula:
##STR00017##
or any of the similar hydroxylamine moieties having the
formulas:
##STR00018##
[0105] L moieties serve to connect the quaternary ammonium and/or
phosphonium salt moieties with the A moieties. L moieties can
comprise a polyalkylene chain having 5 to 30 carbon chain atoms,
wherein any one or more of the hydrogens bonded to said carbon
chain atoms can be optionally substituted with one or two
independently selected hydroxyl, halogen, amino, methylamino,
dimethylamino, or C.sub.1-C.sub.4 organic moieties selected from
alkyl, hydroxyalkyl, alkoxy, alkoxylalkyl, carboxy, or carboxyalkyl
moieties.
The Ammonium or Phosphonium Cationic Moieties
[0106] The compounds useful for the methods of the disclosure
comprise one or more cationic moieties. The cationic moieties carry
a positive charge, which, while not being bound by theory, is
believed to cause the desirable selective accumulation of the
resultant compounds in the mitochondria, because of the large
mitochondrial membrane potential of 150-170 mV, and the resulting
electrostatic attractions. Again, while not being bound by theory,
it has been found that the selective accumulation of the cationic
salts disclosed herein is also improved if the cationic moieties
comprise relatively large and/or lipophilic organic substitutent
moieties, so that the resulting cationic group is relatively
lipophilic when considered as a whole. One of ordinary skill in the
art will recognize that many relatively lipophilic cationic groups
can be synthesized, especially from compounds comprising nitrogen
or phosphorus atoms, and it is evident that many such cationic
moieties could be linked in various ways to the anti-oxidant A
moieties, and provide a cation that might be useful in the practice
of the methods described herein. More particularly however, in many
embodiments of the salts and/or cationic compounds of Formula (I)
have quaternary ammonium or phosphonium moieties, having the
formula:
##STR00019##
wherein: E is a nitrogen or phosphorus atom; and R.sub.1',
R.sub.1'', and R.sub.1''' are each independently organic moieties
comprising from 1 to 12 carbon atoms.
[0107] In many embodiments, the compounds of Formula (I) can have
R.sup.1', R.sup.1'', and R.sup.1''' are each independently selected
from alkyl, aryl, heteroaryl, or aralkyl moieties, which may be
unsubstituted, or optionally substituted with one or two
independently selected substituent moieties, such as those
described above, which include but are not limited to hydroxyl,
halogen, amino, amino, dimethylamino, alkyl, hydroxyalkyl, alkoxy,
alkoxylalkyl, carboxy, or carboxyalkyl moieties. Non-limiting
examples of the optional substituents for R.sup.1', R.sup.1'', and
R.sup.1''' include: [0108] i) C.sub.1-C.sub.4 linear branched
alkyl; for example, methyl (C.sub.1), ethyl (C.sub.2), n-propyl
(C.sub.3), iso-propyl (C.sub.3), n-butyl (C.sub.4), sec-butyl
(C.sub.4), iso-butyl (C.sub.4), and tert-butyl (C.sub.4); [0109]
ii) C.sub.1-C.sub.4 linear or branched alkoxy; for example, methoxy
(C.sub.1), ethoxy (C.sub.2), n-propoxy (C.sub.3), iso-propoxy
(C.sub.3), n-butoxy (C.sub.4), sec-butoxy (C.sub.4), iso-butoxy
(C.sub.4), and tert-butoxy (C.sub.4); [0110] iii) halogen; for
example, --F, --Cl, --Br, --I, and mixtures thereof; [0111] iv)
amino and substituted amino; for example, --NH.sub.2, --NH.sub.2,
--NHCH.sub.3, --NHCH.sub.3, and --N(CH.sub.3).sub.2; [0112] v)
hydroxyl; --OH; [0113] vi) C.sub.1-C.sub.4 linear or branched
hydroxyalkyl; for example, --CH.sub.2OH, --CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2OH, and --CH.sub.2CHOHCH.sub.3; [0114]
vii) C.sub.1-C.sub.4 linear or branched alkoxyalkyl; for example,
--CH.sub.2OCH.sub.3, --CH.sub.2CH.sub.2OCH.sub.3,
--CH.sub.2CH.sub.2CH.sub.2OCH.sub.3, and
--CH.sub.2CH(OCH.sub.3)CH.sub.3; [0115] viii) carboxy or
carboxylate, for example, --CO.sub.2H or the anionic equivalent
carboxylate moieties --CO.sub.2.sup.-; and [0116] xi) carboxyalkyl,
for example, --CH.sub.2CO.sub.2H, --CH.sub.2CH.sub.2CO.sub.2H,
--CH.sub.2CO.sub.2CH.sub.3, --CH.sub.2CH.sub.2CO.sub.2CH.sub.3, and
--CH.sub.2CH.sub.2CH.sub.2CO.sub.2CH.sub.3.
[0117] In related embodiments, R.sub.1', R.sub.1'', and R.sub.1'''
can be each independently selected from alkyl, aryl, or benzyl
moieties optionally substituted with one or two independently
selected hydroxyl, halogen, amino, diamino, dimethylamino, alkyl,
hydroxyalkyl, alkoxy, alkoxylalkyl, carboxy, or carboxyalkyl
moieties.
[0118] In other related embodiments, R.sup.1', R.sup.1'', and
R.sup.1''' can be independently selected from C.sub.4-C.sub.10
alkyl or phenyl moieties, which can optionally be substituted with
one or two independently selected substituent moieties, which can
include but are not limited to hydroxyl, halogen, amino, diamino,
dimethylamino, alkyl, hydroxyalkyl, alkoxy, alkoxylalkyl, cyano,
carboxy, or carboxyalkyl moieties. In additional embodiments,
R.sup.1', R.sup.1'', and R.sup.1''' can be independently selected
from C.sub.4-C.sub.10 alkyl or phenyl moieties. In some additional
embodiments R.sup.1', R.sup.1'', and R.sup.1''' are independently
selected from C.sub.4-C.sub.10 alkyl. In yet other related
embodiments R.sup.1', R.sup.1'', and R.sup.1''' are each
n-C.sub.4H.sub.9 moieties.
[0119] In some embodiments of the compounds of Formula (I) having
phosphonium cations, R.sup.1', R.sup.1'', and R.sup.1''' are each
phenyl moieties, to produce triphenyl phosphonium cations having
the formula:
##STR00020##
[0120] In alternative but related embodiments, R.sup.1', R.sup.1'',
and R.sup.1''' are each benzyl moieties, to produce tribenzyl
phosphonium cations having the formula:
##STR00021##
[0121] Other embodiments of the cations of Formula (I) relates to
quaternary ammonium cations, i.e. wherein E is a nitrogen atom. In
some such embodiments, R.sup.1', R.sup.1'', and R.sup.1''' are each
independently selected from alkyl, aryl, heteroaryl, or aralkyl
moieties, which can be optionally substituted with one or two
independently selected substituent moieties, which include but are
not limited to hydroxyl, halogen, amino, dimethylamino, alkyl,
hydroxyalkyl, alkoxy, alkoxylalkyl, cyano, carboxy, or carboxyalkyl
moieties. Non-limiting examples of the R.sup.1', R.sup.1'', and
R.sup.1''' substituents include: [0122] i) C.sub.1-C.sub.4 linear
branched alkyl; for example, methyl (C.sub.1, ethyl (C.sub.2),
n-propyl (C.sub.3), iso-propyl (C.sub.3), n-butyl (C.sub.4),
sec-butyl (C.sub.4), iso-butyl (C.sub.4), and tert-butyl (C.sub.4);
[0123] ii) C.sub.1-C.sub.4 linear or branched alkoxy; for example,
methoxy (C.sub.1), ethoxy (C.sub.2), n-propoxy (C.sub.3),
iso-propoxy (C.sub.3), n-butoxy (C.sub.4), sec-butoxy (C.sub.4),
iso-butoxy (C.sub.4), and tert-butoxy (C.sub.4); [0124] iii)
halogen; for example, --F, --Cl, --Br, --I, and mixtures thereof;
[0125] iv) amino and substituted amino; for example, --NH.sub.2,
--NH.sub.2, --NHCH.sub.3, --NHCH.sub.3, and --N(CH.sub.3).sub.2;
[0126] v) hydroxyl; --OH; [0127] vi) C.sub.1-C.sub.4 linear or
branched hydroxyalkyl; for example, --CH.sub.2OH,
--CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2OH, and
--CH.sub.2CHOHCH.sub.3; [0128] vii) C.sub.1-C.sub.4 linear or
branched alkoxyalkyl; for example, --CH.sub.2OCH.sub.3,
--CH.sub.2CH.sub.2OCH.sub.3, --CH.sub.2CH.sub.2CH.sub.2OCH.sub.3,
and --CH.sub.2CH(OCH.sub.3)CH.sub.3; [0129] viii) carboxy; or
carboxylate, for example, --CO.sub.2H or the anionic equivalent
carboxylate moieties --CO.sub.2.sup.-; and [0130] xi) carboxyalkyl,
for example, --CH.sub.2CO.sub.2H, --CH.sub.2CH.sub.2CO.sub.2H,
--CH.sub.2CO.sub.2CH.sub.3, --CH.sub.2CH.sub.2CO.sub.2CH.sub.3, and
--CH.sub.2CH.sub.2CH.sub.2CO.sub.2CH.sub.3.
[0131] In additional embodiments of the cations of Formula (I),
wherein E is nitrogen, R.sub.1', R.sub.1'', and R.sub.1''' are each
independently selected from alkyl aryl, or benzyl moieties, which
can be optionally substituted with one or two independently chosen
substitutent moieties, such as those described above, which include
but are not limited hydroxyl, halogen, amino, dimethylamino, alkyl,
hydroxyalkyl, alkoxy, alkoxylalkyl, carboxy, or carboxyalkyl
moieties.
[0132] In another embodiment R.sup.1', R.sup.1'', and R.sup.1'''
are independently selected from C.sub.4-C.sub.10 alkyl or phenyl
moieties optionally substituted with one or two independently
selected hydroxyl, halogen, amino, dimethylamino, alkyl,
hydroxyalkyl, alkoxy, alkoxylalkyl, carboxy, or carboxyalkyl
moieties. In one further aspect of this embodiment R.sup.1',
R.sup.1'', and R.sup.1''' are independently selected from
C.sub.4-C.sub.10 alkyl or phenyl moieties; and in one further
embodiment R.sup.1', R.sup.1'', and R.sup.1''' are independently
selected from C.sub.4-C.sub.10 alkyl.
[0133] In yet another embodiment of cations wherein E is nitrogen,
R.sup.1', R.sup.1'', and R.sup.1''' are each n-C.sub.4H.sub.9
moieties.
The "L" Linker Moiety
[0134] The cations of Formula (I) comprise a linker moiety "L",
which connects the "A" moiety and the cationic moiety. The exact
structure and size of the L moieties can vary considerably, and
many variations of the L moieties are within the scope of the
inventions disclosed herein. In some the L moieties are often
organic moieties, and can comprise a wide variety of structures. In
many embodiments it is desirable that the L moiety be of sufficient
size and character that it provides some space and/or flexibility
in the connection between the A and cation groups, but does not
become of such high molecular weight so as to impair the water
solubility or trans-membrane absorbability of the resulting
cations.
[0135] Accordingly, in some embodiments, the L moiety, when
considered as a whole, comprises from about 4 and 30 carbon atoms,
or from about 4 and 20 carbon atoms. In some embodiments, the L
moiety comprises from 6 to 18 carbon atoms, or from 8 to 12 carbon
atoms.
[0136] In some embodiments, the L moieties can comprise only
methylene or polymethylene moieties, i.e. --(CH.sub.2).sub.n--
moieties. Some embodiments provide L having from 5 to 24 carbon
chain atoms, for example, --(CH.sub.2).sub.n--, wherein the index n
is from 5 to 24. Another embodiment relates to L having from 5 to
20 carbon chain atoms, for example, --(CH.sub.2).sub.n--, wherein
the index n is from 5 to 20. A further embodiment relates to L
having from 6 to 16 carbon chain atoms, for example,
--(CH.sub.2).sub.n--, wherein the index n is from 6 to 16. A yet
further embodiment relates to L having from 7 to 16 carbon chain
atoms, for example, --(CH.sub.2).sub.n--, wherein the index n is
from 7 to 16. A still yet further embodiment relates to L having
from 8 to 12 carbon chain atoms, for example, --(CH.sub.2).sub.n--,
wherein the index n is from 8 to 12. One particular example is an L
unit comprising 10 methylene moieties having the formula:
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.-
2CH.sub.2--, wherein n=10.
[0137] Nevertheless, the L moieties can further comprise in the
carbon chain from 1 to 10 additional atoms or groups independently
selected from --O--, --S--, --S(O)--, --S(O).sub.2--, --NH--,
--NCH.sub.3--, --C(O)--, or --CO.sub.2-- moieties. For example, in
some embodiments, L can be a polyalkylene glycol moiety, or a
polyethylene glycol moiety, having the structure
##STR00022##
wherein n is an integer from 0 to 3.
[0138] It is to be understood that in some embodiments, the L
moiety can comprise therein or thereon an additional ionic
substituent moiety, so the electrical charge of the cation of
Formula (I) could be increased or decreased. In particular, the L
moiety can comprise an additional one or two quaternary ammonium or
phosphonium moiety therein, so that the overall electrical charge
of the cation of Formula I could be 2+ or 3+, so as to be a
dication, or trication. Such dicationic or tricationic compounds
can provide even higher water solubility and/or selectivity of
absorption into mitochondria than compounds of Formula (I) that are
only monocationic.
The X.sup.m- Anions
[0139] The salt compounds comprising the cations of Formula (I)
also comprise an anion X.sup.m, wherein m is an integer from 1 to
4, corresponding to monoanions, di-anions, tri-anions, and
tetra-anions. The first iteration of X.sup.- relates to inorganic
anion moieties. Mono-anionic inorganic anions include any halide
anion, such as fluoride, chloride, bromide, or iodide; nitrate,
hydrogen sulfate; dihydrogen phosphate, and the like. Dianionic
inorganic cations can include carbonate, sulfate or
hydrogenphosphate, and tri-anionic inorganic anions include
phosphates.
[0140] In other embodiments of the X.sup.m- anions, the anions are
organic anions. Non-limiting examples of organic anion moieties
that can be employed to form the salts from the cations of Formula
(I) include organosulphates such as methylsulphonate (mesylate),
trifluoromethylsulfonate (triflate), benzenesulphonate,
toluenesulphonate (tosylate), or purely organic anions, often
formed by the neutralization of organic acids, such as fumarate,
maleate, maltolate, succinate, acetate, benzoate, oxalate, citrate,
or tartrate anions.
[0141] Those of ordinary skill in the art will recognize that both
the cations of Formula (I) and the corresponding X.sup.m- anions
must be combined in appropriate ratios so as to produce isolated
and electrically neutral salt compounds that can be isolated and
used in the methods and compositions disclosed herein. Accordingly,
one way of expressing the condition of electrical neutrality when
applied to the salt compounds as a whole is to recognize that such
salt compounds can have the formula:
N[cation].sup.p+M[anion].sup.m-
wherein the indices M, N, m and p are each independently from 1 to
4, provided that the product (M.times.m)=(p.times.N) thereby
forming a neutral salt.
Making the Compounds of the Inventions
[0142] Various synthetic methods and/or strategies can be employed
in the synthesis or production of salts having cations of Formula
(I) and X.sup.m- anions, as described above and in the examples
below. Several such synthetic methods and/or strategies will be
disclosed herein below.
Compounds wherein L.sub.E is Oxygen
[0143] The
[4-(tri-organophosphonium)acyloxy-2,2,6,6-tetraalkylpiperidinyl-
oxy, free radical] salts of the present invention have the
formula:
##STR00023##
wherein R.sub.1', R.sub.1'', R.sub.1''', R.sup.2, L, and X are
defined elsewhere. These compounds can be synthesized by the
following generic procedure outlined in Scheme I.
##STR00024##
[0144] The 4-substituted-2,2,6,6-tetraalkylpiperidinyloxy moieties
used as starting materials for step (a) of Scheme I are well known
in the art as "Tempol" derivatives and many such compounds are
known in the art and/or readily commercially available, especially
compounds wherein all R.sup.2 groups are methyls.
[0145] Step (a) encompasses removal of an H.sup.+ cation from the
starting material with a strong base, with generation of the
corresponding 4-alkoxy anion. The proton can be removed by any
strong organic or inorganic base which is convenient and which does
not affect the R.sup.2 substituents. Non-limiting examples of
organic bases include alkyl or aryl lithium reagents, such as
phenyl lithium, methyl lithium, n-butyl lithium, tert-butyl
lithium, sodium amide, lithium amide, lithium diisopropylamide,
lithium dimethylamide, lithium diethylamide, and the like. The
formation of the 4-alkoxy anion can be conducted in the cold, i.e.
at room temperature or from 0.degree. C. to -78.degree. C.
[0146] Non-limiting examples of inorganic bases include NaOH, KOH,
LiOH, Ca(OH).sub.2, Na.sub.2CO.sub.3, K.sub.2CO.sub.3, and the
like. One embodiment which is described herein below utilizes NaH
as the base. Other hydride bases, inter alia, KH can be utilized
especially when low temperature conditions and solvent
compatibility cause more reactive bases to be used. In addition,
strong inorganic bases can be used to form organic bases from polar
aprotic solvents, inter alia, dimethyl sulfoxide,
N,N-dimethylacetamide, and hexamethylphosphoric triamide, for
example, a dimsyl anion from dimethyl sulfoxide.
[0147] Any non-reactive or aprotic solvent or mixtures of solvents
can be used for Step (a). Non-limiting examples of solvents include
alkane solvents, inter alia, pentane, iso-pentane, hexane, heptane,
octane, isooctane, and the like; aromatic hydrocarbon solvents,
inter alia, benzene, toluene, and xylene (all isomers); ethers,
inter alia, diethyl ether, tetrahydro-furan, and dioxane; and
various fluorinated hydrocarbons. In addition, polar aprotic
solvents can be used, inter alia, dimethyl sulfoxide,
dimethylformamide, N,N-dimethylacetamide, and hexamethylphosphoric
triamide.
[0148] In addition, combinations of solvents can be used. For
example, typically once the anion is generated, the formulator can
proceed to Step (b) without necessarily having to isolate the
alkoxide, therefore, a second co-solvent may be used which enhances
the solubility of the reagents used in Step (b) without affecting
the formation of the anion.
[0149] Step (b) encompasses attaching a linker group L to the
alkoxide generated in Step (a). The 4-alkoxide is reacted with a
linker molecule L having two reactive leaving groups; Z and X. The
leaving groups may be the same or different.
[0150] L is a linking group comprising from 4 to 30 carbon atoms as
described herein. Z and X can be any suitable leaving group which
is sufficiently reactive to allow attachment of the tether without
loss of the free radical moiety. Non-limiting examples of suitable
leaving groups include halogens: iodine, bromine, and chlorine;
sulphonyl leaving groups, inter alia, methylsulphonyl (mesyl),
ethylsulphonyl, benzenesulphonyl, toluenesulphonyl (tosyl), and the
like.
[0151] Non-limiting examples of compounds having two leaving groups
that can be used to as reagents in forming the linking group
between the free radical portion of the molecule and the quaternary
ammonium or phosphonium moiety include: [0152] i)
.alpha.,.omega.-di-bromoalkanes, inter alia, 1,4-dibromobutane,
1,5-dibromopentane, 1,6-dibromohexane, 1,7-dibromoheptane,
1,8-dibromooctane, 1,9-di-bromononane, and 1,10-dibromodecane;
[0153] ii) .alpha.,.omega.-di-chloroalkanes, inter alia,
1,4-dichlorobutane, 1,5-dichloropentane, 1,6-dichlorohexane,
1,7-dichloroheptane, 1,8-dichlorooctane, 1,9-di-chlorononane, and
1,10-dichlorodecane; [0154] iii) .alpha.,.omega.-di-iodoalkanes,
inter alia, 1,4-diiodobutane, 1,5-diiodopentane, 1,6-diiodohexane,
1,7-diiodoheptane, 1,8-diiodooctane, 1,9-di-iodononane, and
1,10-diiododecane; [0155] iv) .alpha.,.omega.-mixed leaving group
alkanes, inter alia, 1-bromo-4-chlorobutane,
1-bromo-5-chloropentane, 1-bromo-6-chlorohexane,
1-bromo-10-chloro-decane, 1-iodo-4-chlorobutane,
1-iodo-5-chloropentane, 1-iodo-6-chloro-hexane,
1-iodo-10-chlorodecane, 1-iodo-4-bromobutane,
1-iodo-5-bromo-pentane, 1-iodo-6-bromohexane,
1-iodo-10-bromodecane, 1-toluene-sulphonyl-4-chlorobutane,
1-toluenesulphonyl-5-chloropentane,
1-toluene-sulphonyl-6-chlorohexane,
1-toluenesulphonyl-10-chlorodecane,
1-bromo-4-toluenesulphonylbutane,
1-bromo-5-toluenesulphonylpentane,
1-bromo-6-toluenesulphonylhexane,
1-bromo-10-toluenesulphonyldecane, 1-azido-4-chlorobutane,
1-azido-5-chloropentane, 1-azido-6-chlorohexane, and
1-azido-10-chlorodecane.
[0156] The formulator can take advantage of the differential
reactivity of leaving groups when using .alpha.,.omega.-mixed
leaving group alkanes for the formation of the tether. However, the
formulator may wish to insert an optional Step (b)(ii) into the
process for preparing the compounds of the present invention. For
example, the formulator can, once intermediate B is formed,
increase the reactivity of leaving group X in order to adjust for
any lesser reactivity that the reagent which introduces the
quaternary ammonium or phosphonium salt has. For example, in the
following step 1-iodo-4-chlorobutane is used to attach the first
end of the tether to the free radical containing unit taking
advantage of the increased reactivity of iodine as a preferred
leaving group over chlorine.
##STR00025##
wherein chlorine is replaced by iodine.
[0157] Non-limiting examples of solvents which can be used for Step
(b), as well as any optional steps, inter alia, Step (b)(i),
include alkane solvents, inter alia, pentane, iso-pentane, hexane,
heptane, octane, isooctane, and the like; aromatic hydrocarbon
solvents, inter alia, benzene, toluene, and xylene (all isomers);
ethers, inter alia, diethyl ether, tetrahydro-furan, and dioxane;
and various fluorinated hydrocarbons. In addition, polar aprotic
solvents can be used, inter alia, dimethyl sulfoxide,
dimethylformamide, N,N-dimethylacetamide, and hexamethylphosphoric
triamide.
[0158] Step (c) encompasses forming a quaternary ammonium or
phosphonium salt at the end of the linking unit opposite the end
which contains the nitroxide or amine oxide moiety. For example,
formation of a phosphonium salt:
##STR00026##
or formation of an quaternary ammonium salt:
##STR00027##
However, in the case of quaternary ammonium salt containing
products, Step (c) may optionally be divided into two separate
steps, Step (c)(i) and Step (c)(ii), which can be conducted either
without isolation of the intermediate C or which can be conducted
as two discrete steps, wherein intermediate C is isolated prior to
quaternization, for example:
[0159] Step (c)(i):
[0160] Formation of a Tertiary Amine by Displacement of the Leaving
Group X to Form Intermediate C:
##STR00028##
which can be isolated if desired, followed by quaternization of the
amine to form the final quaternary ammonium salt:
[0161] Step (c)(ii):
##STR00029##
The formulator can chose the leaving group X which comprises the
alkylating agent in Step (c)(ii) to be the same as the leaving
group that is displaced in Step (c)(i) or to take the opportunity
to provide a different leaving group which results in the potential
source of a different counter ion for the quaternary ammonium ion.
However, the formulator can alternatively choose to provide the
final anion by exchanging the anion present as a result of the
reaction which occurs in Step (c) with another anion, and this can
be done by exposing the final product to a counter ion exchange
system, for example, an ion exchange resin.
[0162] In the case of forming a phosphonium salt, a tri-substituted
phosphine can be directly reacted with compounds such as
intermediate B to form final compounds. Examples of this reaction
are found in Examples 1 and 2 herein below.
[0163] Depending upon the reactivity of the leaving group X and how
nucleophilic the phosphine adduct is toward leaving group X, the
reaction can be conducted over a wide range of temperatures. A
first embodiment of this phosphonium forming step encompasses the
reaction taking place in a refluxing solvent. Because a salt if
formed, it is advantageous for the formulator to select a solvent
wherein the starting materials have good solubility, but the
product which forms is either insoluble or has a reduced
solubility. In this way when a stoichiometric excess of one
starting reagent is used, the excess will potentially remain in
solution while the desired product precipitates as an insoluble
material. In addition, a co-solvent in which the product is known
to be insoluble but which affords solubility to any unreacted
starting material may be added to cause precipitation of the
product. These same principles apply to the formation and isolation
of quaternary ammonium salts formed whether in Step (c), or by way
of alternative Step (c)(i) and Step (c)(ii).
[0164] Step (c) can be conducted in any non-nucleophilic solvent
which allows for the adequate formation of the desired product.
[0165] Suitable solvents or mixtures of solvents can be used for
Step (c). Non-limiting examples of solvents include alkane
solvents, inter alia, pentane, iso-pentane, hexane, heptane,
octane, isooctane, and the like; aromatic hydrocarbon solvents,
inter alia, benzene, toluene, and xylene (all isomers); ethers,
inter alia, diethyl ether, tetrahydro-furan, and dioxane; ketones,
inter alia, acetone, methyl ethyl ketone, 3-pentanone; halogenated
solvents, inter alia, dichloromethane, chloroform, carbon
tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane,
1,1,1-trichloroethane, and various fluorinated hydrocarbons. In
addition, polar aprotic solvents can be used, inter alia, dimethyl
sulfoxide, dimethylformamide, N,N-dimethylacetamide, and
hexamethylphosphoric triamide.
[0166] In some instances reactivity may be increased by the
addition of a protic solvent, inter alia, water, methanol, ethanol,
and the like, which can aid in solvation and separation of ion
pairs, however, these adjustments to Step (c) are within the scope
of the artisan and can be made without undue experimentation.
[0167] Scheme II outlines the preparation of a compound disclosed
herein which is further described in detail in Example 1.
##STR00030##
Reagents and conditions: (a) NaH, benzene; reflux, 24 hr.
##STR00031##
Reagents and conditions: (b) 1,10-dibromodecane, benzene; reflux,
48-72 hr.
##STR00032##
Reagents and conditions: (c) P(C.sub.6H.sub.5).sub.3, n-propanol;
reflux, 72 hr.
Example 1
[4-(10-triphenylphosphonium)decyloxy-2,2,6,6-tetramethylpiperidinyloxy,
free radical] bromide (2) (also known as MitoT-10, or CPC-410)
[0168] Tempol (0.01 mol) was added to a three-neck flask containing
100 ml dry benzene that is maintained at nitrogen atmosphere. To
the flask, sodium hydride (0.015 mol) was added and kept refluxed
for 24 hrs. The flask was cooled in ice bath and added
1,10-dibromodecane (0.02 mol) in one portion. The refluxing was
then resumed for another 72 hrs. The contents of the flask was
cooled in ice bath and added 25 ml water and transferred to a
separatory funnel. The red upper benzene layer was separated, dried
over anhydrous magnesium sulfate and solvent removed by rotory
evaporation to get a red oil. The oil was purified by column
chromatography on silica gel 60. The material was added to the
column and eluted first with about 150 ml hexane that removed the
excess of dibromodecane. The desired bromodecanoyl ether of Tempol
was eluted with a mixture of hexane and ether (90:10). The red
eluate was collected and was found to be pure on thin layer
chromatography plates developed using the same solvent mixture. The
yield was 0.008 mol (80%).
[0169] The bromoether of Tempol 0.008 mol and triphenyl phosphine
(0.01 mol) were taken in a flask and added 20 ml of n-propanol. The
contents of the flask was kept refluxed under nitrogen for 72 hrs.
The flask was cooled and the solvent was removed by rotory
evaporation. The residue was dissolved in 10 ml dichloromethane and
added to 100 ml ether with stirring. The precipitated product was
collected by decantation of the solvent. The residual semisolid was
then purified on silica gel 60 column eluting first with
dichloromethane and the desired product was eluted with a mixture
of dichloromethane and methanol (90:10). Homogeneous fractions
combined and solvent removed to get a red-brown semisolid with a
yield of 65%. Purity was ascertained by LC-MS (mass=573.4).
[0170] Scheme III outlines the preparation of a compound disclosed
herein which is further described in detail in Example 2.
##STR00033##
Reagents and conditions: (a) 1,4-dibromobutane, benzene; reflux,
48-72 hr.
##STR00034##
Reagents and conditions: (b) P(C.sub.6H.sub.5).sub.3, dioxane;
reflux, 72 hr.
Example 2
[4-(4-triphenylphosphonium)butoxy-2,2,6,6-tetramethylpiperidinyloxy,
free radical] bromide (4)
[0171] Preparation of
4-(4-bromobutoxy)-2,2,6,6-tetramethylpiperidinyloxy, free radical
(3): To a 250-mL 3-neck flask equipped with a reflux condenser and
nitrogen inlet line was charged
4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical [tempol]
(3.44 g, 20 mmol) and dry benzene (100 mL). Sodium hydride (0.72 g,
30 mmol) was slowly added. Once the addition was complete, the
reaction was brought to reflux for 24 hours. The resulting
suspension was cooled in an ice bath and 1,4-dibromobutane (8.6 g,
40 mmol) was added and the solution was again brought to reflux for
24 hours. After the reaction was complete the reaction solution was
cooled and water (50 mL) was added. The contents of the reaction
vessel were transferred to a separatory funnel and extracted with
diethyl ether (100 mL). The organic layer was decanted and dried
over MgSO.sub.4 then concentrated under reduced pressure to afford
the crude product containing unreacted di-bromide. The product was
purified over silica [gradient eluetion: 100% hexane to 1:1
hexane/diethyl ether]. The solvent was removed in vacuo to afford
5.0 g (82% yield) of the desired product as a red oil. TLC
(hexane/diethyl ether 1:1) one spot at R.sub.f 0.75).
[0172] Preparation of
[4-(4-triphenylphosphonium)dibutoxy-2,2,6,6-tetramethyl-piperidinyloxy,
free radical] bromide (4): To a 3-neck flask equipped with a reflux
condenser and nitrogen inlet tube was charged
4-(10-bromobutoxy)-2,2,6,6-tetramethylpiperidinyloxy, free radical,
3, (5 g, 16.4 mmol) and dioxane (50 mL). The solution was stirred
and triphenylphosphine (8 g, 30.5 mmol) was added. The mixture was
then brought to reflux for 24 hours. The reaction solution was
cooled and the solvent removed under reduced pressure. The residue
was treated with diethyl ether (200 mL), the organic layer was
decanted and the residue dissolved in CH.sub.2Cl.sub.2 (10 mL) then
added to a flask containing diethyl ether (100 mL). The precipitate
was collected, dissolved in CH.sub.2Cl.sub.2 and re-precipitated
with diethyl ether. This procedure was repeated 3 additional times
to afford 6.5 g (60% yield) of the desired product as a fluffy
brown solid that is hydroscopic. The presence of the free radical
was confirmed by EPR spectroscopy. LCMS MH.sup.+ 489.5.
[0173] Scheme IV outlines an example of the preparation of
compounds disclosed herein wherein the linking unit is tethered to
the free radical portion of the final compound by way of an ester
linkage.
##STR00035##
Reagents and conditions: (a) 4-bromobutyryl chloride, benzene;
reflux.
##STR00036##
Reagents and conditions: (b) P(C.sub.6H.sub.5).sub.3, dioxane;
reflux.
Example 3
4-[4-(Triphenylphosphonium)butyryloxy-2,2,6,6-tetramethylpiperidinyloxy,
free radical] bromide (6)
[0174] Preparation of
4-(4-bromobutyryloxy)-2,2,6,6-tetramethylpiperidinyloxy, free
radical (5): To a 250 mL 3-neck flask equipped with a reflux
condenser and nitrogen inlet line was charged
4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical [tempol]
(3.44 g, 20 mmol), triethylamine (2.22 g, 22 mmol) and dry benzene
(100 mL). The solution was cooled in an ice bath to 0.degree. C.
4-Bromobutyryl chloride (3.9 g, 21 mmol) was added slowly. After
the addition is complete, the solution was allowed to warm to room
temperature and stir over night. The contents of the reaction
vessel were transferred to a separatory funnel and extracted with
water (50 mL), brine (50 mL), then dried over Na.sub.2SO.sub.4. The
solvent was removed in vacuo to afford the desired product which
can be used for the next step without further purification.
[0175] Preparation of
4-[4-(triphenylphosphonium)butyryloxy-2,2,6,6-tetramethylpiperidinyloxy,
free radical] bromide (6): To a 3-neck flask equipped with a reflux
condenser and nitrogen inlet tube was charged
4-(4-bromobutyryloxy)-2,2,6,6-tetramethylpiperidinyloxy, free
radical, 5, (6.42 g, 20 mmol) and dioxane (50 mL). The solution was
stirred and triphenylphosphine (10.5 g, 40 mmol) was added. The
mixture was then brought to reflux for 24 hours. The reaction
solution was cooled and the solvent removed under reduced pressure.
The residue was treated with diethyl ether (200 mL), the organic
layer was decanted and the residue dissolved in CH.sub.2Cl.sub.2
(10 mL) then added to a flask containing diethyl ether (100 mL).
The precipitate was collected, dissolved in CH.sub.2Cl.sub.2 and
re-precipitated with diethyl ether. This procedure was repeated 3
additional times to afford the desired product.
[0176] Scheme V outlines an example of the preparation of exemplary
compounds disclosed herein which is further described in detail in
Example 4.
##STR00037##
Reagents and conditions: (a) NaI, acetone; reflux, 24 hr.
##STR00038##
Reagents and conditions: (b) triethylamine, dioxane; reflux.
Example 4
{4-[4-(N,N,N-Triethylammonium)butoxy]-2,2,6,6-tetramethylpiperidinyloxy,
free radical} iodide
[0177] Preparation of
4-(4-iodobutoxy)-2,2,6,6-tetramethylpiperidinyloxy, free radical
(7): To a 250 mL 3-neck flask equipped with a reflux condenser and
a nitrogen inlet line is charged
4-(4-bromobutoxy)-2,2,6,6-tetramethylpiperidinyloxy, free radical,
3, (6.12 g, 20 mmol) and anhydrous acetone (50 mL). Sodium iodide
(15 g, 100 mmol) that has been dried in an oven and stored in a
desiccator is added in one portion. The reaction is brought to
reflux for 1 hour then allowed to cool and stir overnight. Water (5
mL) is added and the volume of solvent is reduced in vacuo to
approximately 20 mL. The resulting solution is extracted with
CH.sub.2Cl.sub.2 (3.times.25 mL), the organic layers combined,
dried over MgSO.sub.4 and concentrated in vacuo to afford the
desired product which is used without further purification.
[0178] Preparation of
{4-[4-(N,N,N-triethylammonium)butoxy]-2,2,6,6-tetramethylpiperidinyloxy,
free radical} iodide (8): To a 250 mL 3-neck flask equipped with a
reflux condenser is charged
4-(4-iodobutoxy)-2,2,6,6-tetramethyl-piperidinyloxy, free radical,
7, (7.08 g, 20 mmol), triethylamine (20.2 g, 27.8 mL, 200 mmol),
and benzene (100 mL). The reaction is warmed to 60.degree. C. and
allowed to stir overnight. The reaction is cooled to room
temperature after which the reaction is poured into diethyl ether
(200 mL). The resulting solution is stirred and the organic layer
decanted. The solid that remains is triturated with diethyl ether,
the solid collected and dried to afford the desired compound.
Compounds Wherein L.sub.g is --NH--
[0179] The
[4-(triphenylphosphonium)acylamino-2,2,6,6-tetraalkylpiperidiny-
loxy, free radical] salts of the present invention have the
formula:
##STR00039##
wherein R.sub.1', R.sub.1'', R.sub.1''', R.sup.2, L, and X are
further defined herein above. These compounds can be made by the
following generic procedure outlined in Scheme VI.
##STR00040##
[0180] Step (d) encompasses attaching a tether to the free radical
containing portion of the molecule. The
4-amino-2,2,6,6-tetraalkylpiperidinyloxy free radical is reacted
with a molecule having two reactive leaving groups; Z and X. The
leaving groups may be the same or different, however, Z is the
group which reacts first to form the
4-acyloxy-2,2,6,6-tetraalkylpiperidinyloxy free radical
intermediate, D, as depicted in Step (d) of Scheme VI.
[0181] Because in Step (e) described herein, a quaternary ammonium
salt or phosphonium salt is formed, it is convenient for the
formulator to utilize as a leaving group X, a unit that can form a
stable anion and therefore act as the counter ion for the
quaternary ammonium or phosphonium salt that is formed.
[0182] L is a linking group comprising from 4 to 30 carbon atoms as
described elsewhere herein
[0183] Reaction procedures, conditions, solvents, reaction times,
etc are similar to those employed with similar compounds wherein Lg
is oxygen, and adjusting such parameters to produce useable yields
of the desired products is within the level of skill of those
skilled in the art of organic synthesis.
[0184] Scheme VII outlines an example of the preparation of amide
compounds disclosed herein wherein the linking unit is tethered to
the free radical portion of the final compound by way of an amide
linkage. These compounds can be prepared by the following procedure
outlined herein below.
##STR00041##
##STR00042##
Reagents and conditions: (a) 4-bromobutyryl chloride,
CH.sub.2Cl.sub.2; rt, 48-72 hr.
##STR00043##
Reagents and conditions: (b) P(C.sub.6H.sub.5).sub.3, dioxane;
reflux, 72 hr.
Example 5
{4-[4-(Triphenylphosphonium)butyrylamide-2,2,6,6-tetramethylpiperidinyloxy-
] free radical}bromide
[0185] Preparation of
4-(4-bromobutyrylamide)-2,2,6,6-tetramethylpiperidinyloxy, free
radical (9): To a 250 mL 3-neck flask equipped with a reflux
condenser and nitrogen inlet line is charge
4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, free radical
[4-amino-tempo] (3.44 g, 20 mmol), triethylamine (2.22 g, 22 mmol)
and dry benzene (100 mL). The solution is cooled in an ice bath to
0.degree. C. 4-Bromobutyryl chloride (3.9 g, 21 mmol) is added
slowly. After the addition is complete, the solution is allowed to
warm to room temperature and stir over night. The contents of the
reaction vessel is transferred to a separatory funnel and extracted
with water (50 mL), brine (50 mL), then dried over
Na.sub.2SO.sub.4. The solvent is removed in vacuo to afford the
desired product which can be used for the next step without further
purification.
[0186] Preparation of
{4-[4-(triphenylphosphonium)butyrylamide-2,2,6,6-tetramethylpiperidinylox-
y] free radical}bromide (10): To a 3-neck flask equipped with a
reflux condenser and nitrogen inlet tube is charged
4-(4-bromobutyrylamide)-2,2,6,6-tetramethylpiperidinyloxy, free
radical, 9, (6.4 g, 20 mmol) and dioxane (50 mL). The solution is
stirred and triphenylphosphine (10.5 g, 40 mmol) is added. The
mixture is then brought to reflux for 24 hours. The reaction
solution is cooled and the solvent removed under reduced pressure.
The residue is treated with diethyl ether (200 mL), the organic
layer is decanted and the residue dissolved in CH.sub.2Cl.sub.2 (10
mL) then added to a flask containing diethyl ether (100 mL). The
precipitate is collect, dissolved in CH.sub.2Cl.sub.2 and
re-precipitated with diethyl ether. This procedure is repeated 3
additional times to afford the desired product.
[0187] The examples herein above encompass free radical moieties
derived from 6-member ring starting materials. The disclosed
compounds also encompass free radical moieties derived from
5-member ring starting materials, for example:
##STR00044##
wherein R.sub.1', R.sub.1'', R.sub.1''', R.sup.2, L, L.sub.g, E,
and X are further defined herein above. Such compounds, wherein are
well known in the art as "Proxyl" compounds and many suitable
precursors of the Proxyl "A" moiety are readily commercially
available from standard suppliers of fine chemicals, such as
Aldrich-Sigma of Milwaukee Wis. For example, the following
precursors of a Proxyl moiety are commercially available:
##STR00045##
[0188] The preparation of compounds starting from such "Proxyl
starting materials can be carried out in analogy to the procedures
outlined in Schemes I and II above, and other similar procedures as
would be well within the capabilities of one of ordinary skill in
the art of synthetic organic chemistry.
[0189] Scheme VIII outlines an example of the preparation of
compounds disclosed herein wherein the free radical moiety is
derived from a 5-member ring starting material.
##STR00046##
Reagents and conditions: (a) 4-bromobutyryl chloride,
CH.sub.2Cl.sub.2; rt, 48-72 hr.
##STR00047##
Reagents and conditions: (b) P(C.sub.6H.sub.5).sub.3, dioxane;
reflux, 72 hr.
Example 6
({3-[4-(Triphenylphosphonium)butyrylamide]methyl-2,2,5,5-tetramethyl-pyrro-
lidinyloxy} free radical) bromide
[0190] Preparation of
3-(4-bromobutyrylamide)methyl-2,2,5,5-tetramethylpyrrolidin-yloxy,
free radical (11): To a 250 mL 3-neck flask equipped with a reflux
condenser and nitrogen inlet line is charge
3-aminomethyl-2,2,6,6-tetramethylpiperidine-1-oxyl, free radical
[3-aminomethyl-proxyl] (3.42 g, 20 mmol), triethylamine (2.22 g, 22
mmol) and dry benzene (100 mL). The solution is cooled in an ice
bath to 0.degree. C. 4-Bromobutyryl chloride (3.9 g, 21 mmol) is
added slowly. After the addition is complete, the solution is
allowed to warm to room temperature and stir over night. The
contents of the reaction vessel is transferred to a separatory
funnel and extracted with water (50 mL), brine (50 mL), then dried
over Na.sub.2SO.sub.4. The solvent is removed in vacuo to afford
the desired product which can be used for the next step without
further purification.
[0191] Preparation of
({3-[4-(triphenylphosphonium)butyrylamide]methyl-2,2,5,5-tetramethyl-pyrr-
olidinyloxy} free radical) bromide (12): To a 3-neck flask equipped
with a reflux condenser and nitrogen inlet tube is charged
3-(4-bromobutyrylamide)methyl-2,2,5,5-tetramethylpyrrolidin-yloxy,
free radical, 11, (6.68 g, 20 mmol) and dioxane (50 mL). The
solution is stirred and triphenylphosphine (10.5 g, 40 mmol) is
added. The mixture is then brought to reflux for 24 hours. The
reaction solution is cooled and the solvent removed under reduced
pressure. The residue is treated with diethyl ether (200 mL), the
organic layer is decanted and the residue dissolved in
CH.sub.2Cl.sub.2 (10 mL) then added to a flask containing diethyl
ether (100 mL). The precipitate is collect, dissolved in
CH.sub.2Cl.sub.2 and re-precipitated with diethyl ether. This
procedure is repeated 3 additional times to afford the desired
product.
[0192] Another example of a related synthesis of compounds wherein
Proxyl groups can be linked to phosphonium cations by ether groups
derived from glycols, to form compounds within the scope of the
invention is shown below in Scheme IX:
##STR00048##
[0193] Another example of a related synthesis of compounds wherein
Proxyl groups can be linked to phosphonium cations by moieties
derived from glycols, to form compounds within the scope of the
invention is shown below in Scheme X:
##STR00049##
[0194] Those of ordinary skill in the art of synthetic chemistry
can readily devise other related methods for synthesizing compound
comprising Proxyl or Tempol antioxidant groups linked by various
moieties to quaternary phosphonium or ammonium cations.
Biological Activity of the Compounds
[0195] The salts described above have been found to be potent
compounds in a number of in vitro biological assays that correlate
to or are representative of human diseases, especially diseases of
uncontrolled inflammation and/or cellular proliferation, including
various cancers.
[0196] The biological activity of the compounds described herein
can be measured, screened, and/or optimized by testing the salts
for their relative ability to kill or inhibit the growth of various
human tumor cell lines and primary tumor cell cultures.
[0197] Tumor cell lines that can be employed for such tests
include, but are not limited to, known cell lines that model
cancers and/or diseases of uncontrolled cellular proliferation,
such as:
[0198] For Leukemia: CCRF-CEM, HL-60 (TB), K-562, MOLT-4,
RPMI-8226, and SR. Lung Cancer: A549/ATCC, EKVX, HOP-62, HOP-92,
NCI-H226, NCI-H23, NCI-H322M, NCI-H460, and NCI-H522.
[0199] Colon Cancer: COLO 205, HCC-2998, HCT-116, HCT-15, HT-29,
KM-12, and SW-620.
[0200] CNS Cancer: SF-268, SF-295, SF-539, SNB-19, SNB-75, U-231,
U-235 and U-251.
[0201] Melanoma: LOX-IMVI, MALME-3M, M-14, SK-MEL-2, SK-MEL-28,
SK-MEL-5, UACC-257, and UACC-62.
[0202] Ovarian Cancer: IGR-OVI, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8,
and SK-OV-3.
[0203] Renal Cancer: 786-0, A-498, ACHN, CAKI-1, RXF-393, RXF-631,
SN12C, TK-10, and U0-31.
[0204] Prostate Cancer: DU-145, PC-3 and LNCaP.
[0205] Breast Cancer: MDA-MB-468, MCF 7, MCF7/ADR-RES,
MDA-MB-231/ATCC, HS578T, MDA-MB-435, MDA-N, BT-549, and T-47D.
[0206] Pancreatic Cancer: PANC-1, Bx-PC3 AsPC-1.
[0207] After the compounds to be screened have been applied to one
or more of the above cancer cell lines, the anti-cancer
effectiveness can be gauged using a variety of assay procedures
known to those of ordinary skill in the art for measuring the
number of live cells in the cultures as a function of time.
[0208] One well known procedure employs
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
("MTT") to differentiate live cells from dead cells. The MTT assay
is based on the production of a dark blue formazan product by
active dehydrogenase in the mitochondria of live tumor cells (see
M. C. Alley, D. A. Scudiero, A. Monks, M. L. Hursey, M. J.
Czerwinski, D. L. Fine, B. J. Abbout, J. G. Mayo, R. H. Shoemaker
and M. R. Boyd, Cancer Res., 48, 589, 1988). After exposure of
cancer cells to the compounds to be screened for a fixed number of
days, only living cells contain active dehydrogenases, and produce
dark blue formazan from MTT and are stained. The numbers of live
cells can be measured by absorbance of visible light by the
formazan at 595 nm. Anti-cancer activity can be reported as percent
of the tumor cell growth in a culture treated with a placebo. These
MTT assay procedures have an advantage over an in vivo assay with
common laboratory animals such as mice, in that results are
obtained within a week as opposed to requiring several weeks or
months.
[0209] These MTT anti-cancer activity screening assay provides data
regarding the general cytotoxicity of an individual compound. In
particular, as described in the examples herein, active anticancer
compounds can be identified by applying the compounds at a
concentration of about 10 .mu.M to one or more cultured human tumor
cell lines, such as for example leukemia, lung cancer, colon
cancer, CNS cancer, melanoma, ovarian cancer, renal cancer,
prostate cancer, breast cancer, or pancreatic cancer, so as to kill
or inhibit cell growth of the tumor cells.
[0210] In some embodiments of the invention, the compounds of the
invention are considered to be biologically active for the
treatment of a particular cancer if, when they are applied to a
culture of one of the above cancer cell lines at a concentration of
about 10 .mu.M or less, for a period of at least about 5 days, the
growth of the cancer cells is inhibited, or the cancer cells killed
to the extent of about 50% or more, as compared to a control not
comprising the compound of the invention.
[0211] For DNA assay, each culture plate was thawed and
equilibrated to room temperature under protection from light.
Hoechst dye was then added to each well in 200 .mu.L of high salt
THE buffer (10 mM Tris, 1 mM EDTA, 2 M NaCl [pH 7.4]) at a final
concentration of 6.7 .mu.g/mL. After further incubation at room
temperature for 2 hours under protection from light, culture plates
were scanned on the CytoFluor 2350.TM. scanner using the 360/460 nm
filter excitation and emission set. The DNA fluorescence intensity
was used as a measure of cell growth.
[0212] In particular, the biological activity of two particular
salts whose structures are shown below were assayed for their
relevance to the treatment or inhibition of the growth of prostate
cancers.
##STR00050##
[0213] The effects of varying concentrations of Mito-T drug on the
growth of LNCaP and PC-3 cells over a period of 4 days was assayed
using the Hoechst dye-DNA fluorescence assay described above. In
these and all subsequent cell culture studies described below, each
data point and its associated error bar are respectively, an
average value and the standard deviation of data obtained from six
wells of a 96-well plate run in duplicate in three separate sets of
experiments.
[0214] The results are shown in FIG. 1. Mito-T treatment inhibits
the growth of both LNCaP and PC-3 cells, with estimated IC.sub.50
values of approximately 4 .mu.M and 60 .mu.M, respectively.
[0215] The inhibitory effect of MitoT on the oxidative stress level
in LNCaP prostate tumor cells can also be determined by the ratio
of DCF fluorescence/Hoechst dye-DNA fluorescence (Ripple M O, Henry
W F, Rago R P, Wilding G. Prooxidant-antioxidant shift induced by
androgen treatment of human prostate carcinoma cells. J Natl Cancer
Inst. 1997 Jan. 1; 89(1):40-8). DCFH is oxidized to DCF by ROS to
yield easily quantifiable ROS levels monitored by the green
fluorescence of the DCF (6-carboxy-2',7'-dichlorofluorescin
diacetate) dye, as can be seen in FIG. 2. The DCF fluorescence in
LNCaP cells treated with 1 nM of the androgen analog R1881 and
normalized with the blue fluorescence of the Hoechst dye-DNA
complex in the same cells at varying concentrations of MitoT in
order to evaluate the level the oxidative stress per individual
cell.
[0216] The inhibitory effect of MitoT on the oxidative stress level
in LNCaP prostate tumor cells can be determined by the ratio of DCF
fluorescence/Hoechst dye-DNA fluorescence. MitoT treatment markedly
reduced the oxidative stress in LNCaP cells as determined by DCF
fluorescence/DNA fluorescence assay shown in FIG. 3. MitoT
treatment effectively and reproducibly reduced the ROS levels in
LNCaP cells at concentrations at or above about 1-10 .mu.M. It
should be noted that MitoT treatment induced a reduction of
oxidative stress determined by DCF assay and mitochondrial function
determined by MTT assay, and is parallel to MitoT's effect in the
inhibition of prostate tumor cell growth as determined by DNA
assay, as shown in FIG. 4, This oxidative stress is probably due to
increased lipid peroxidation during apoptotic and/or necrotic cell
death (reviewed in 1,2).
[0217] Results shown in FIG. 5 clearly demonstrates that MitoT
pretreatment at a sublethal dose (1 .mu.M) can also completely
block the oxidative stress induced by androgen (R1881) treatment in
LNCaP cells. As most published literature demonstrates that
androgen is the leading cause of oxidative stress generation and
changes (Reviewed by Attia S and Wilding G, Novel Antioxidant
Technology for Prostate Cancer Chemoprevention and Treatment.
Expert opinion on therapeutic patents, 16(9) 1255-67), which is a
primary causative agent of prostate cancer and other prostatic
diseases, including, but not limited, to benign prostatic
hyperplasia. Thus, the anti-oxidant spin trapping effect of MitoT
treatment is capable of removing one of the most important
metabolic products that causes cancer in general and prostate
cancer for specific.
[0218] FIG. 6 shows that when animals are treated with MitoT, the
serum level of MitoT within the first hour of treatment is between
10-20 .mu.g/ml, which is 10-20 times above the level of MitoT
required to block androgen induced oxidative stress in LNCaP cells
(FIG. 5) and in the concentration range of completely blocking all
oxidative stress in the cancer cells. Also, under these treatment
conditions, MitoT markedly reduced the oxidative stress in the
prostatic lumen of live animals, as observed by Hydroethidine dye
oxidation assay in the animal's prostate. Hydroethidine dye was
injected into the tail vein of male mice one hour before sacrifice.
Hydroethidine shows a typical red fluorescence upon oxidation by
ROS. The resected prostate glands of the sacrificed animals were
processed and observed under fluorescence microscopy. The data are
shown in FIG. 7. As in the case of cell culture studies, the MitoT
treated animals did not show any evidence of oxidative stress in
the prostate gland
Using the Compositions
[0219] In view of their ability to inhibit the growth of, at least
some cancer cell lines in vitro, the compounds described herein can
be used to prevent, alleviate or otherwise treat diseases of
uncontrolled proliferation in mammals, including humans, such as
cancer or precancerous diseases. The compounds described herein can
be used for the preparation of medicaments for treating diseases of
uncontrolled inflammation, proliferation, cancers, and prostate
cancer.
[0220] Therefore, in some embodiments, the invention relates to
methods of treatment for a disease of uncontrolled cellular
inflammation, proliferation, wherein the method comprises
administering to a mammal diagnosed as having a disease of
uncontrolled cellular inflammation and/or proliferation a compound
of the invention or a pharmaceutical composition thereof comprising
one or more of the compounds of the invention, in an amount that is
effective to treat the disease of uncontrolled cellular
inflammation and/or proliferation.
[0221] The disease of uncontrolled cellular inflammation and/or
proliferation treated can be a hyperplasia or a carcinoma,
lymphoma, leukemia, or sarcoma. The types of diseases treated by
methods of the invention include but are not limited to Hodgkin's
Disease, myeloid leukemia, polycystic kidney disease, bladder
cancer, brain cancer, head and neck cancer, kidney cancer, lung
cancer, myeloma, neuroblastoma, glioblastoma, ovarian cancer,
pancreatic cancer, prostate cancer, skin cancer, liver cancer,
melanoma, colon cancer, cervical carcinoma, breast cancer,
metastases to the brain or bone, epithelial cancer, and leukemia.
The compositions can also be used as regulators in diseases of
uncontrolled inflammation and/or proliferation and/or precancerous
conditions such as cervical and anal dysplasias, other dysplasias,
severe dysplasias, hyperplasias, atypical hyperplasias, and
neoplasias.
[0222] The compounds of the invention have been found to
particularly effective for the treatment of prostate cancers and
related neoplasias, including pancreas adenocarcinomas or prostate
adenocarcinomas, and/or inhibiting the growth of prostate
hyperplasias (non-cancerous) and/or cancers and related
neoplasias.
Methods of Treating Diseases with the Salts and/or Compositions
[0223] In some embodiments, the inventions described herein relate
to methods for treating or inhibiting the recurrence, progression
or metastasis, of a cancer or a neoplasia precursor thereof,
consisting of administering to a mammal diagnosed as having a
cancer or precursor neoplasia thereof, in an amount effective to
treat the cancer or inhibit the recurrence, progression, or
metastasis of the cancer or precursor neoplasia thereof, one or
more pharmaceutically acceptable salts having a cation having the
formula
##STR00051##
[0224] wherein [0225] a) A is an antioxidant moiety comprising one
or more nitroxide or hydroxylamine moieties, or a prodrug thereof,
having from three to 16 carbon atoms, [0226] b) L is an organic
linking moiety comprising 4 to 30 carbon atoms, [0227] c) E is a
nitrogen or phosphorus atom, [0228] d) R.sub.1', R.sub.1'', and
R.sub.1''' are each independently selected organic moieties
comprising between 1 and 12 carbon atoms, [0229] wherein E,
R.sub.1', R.sub.1'', and R.sub.1''' together form a quaternary
ammonium or phosphonium cation; [0230] and wherein the salt further
comprises is one or more pharmaceutically acceptable anions
X.sup.m-, wherein m is an integer from 1 to 4, in sufficient amount
to form the pharmaceutically acceptable salt.
[0231] The pharmaceutically acceptable salts of the invention have
been found to be particularly effective in treating certain forms
inflammation, organ enlargement or cancer, including, but not
limited to prostate hyperplasia and/or cancer, colorectal cancer,
gastric cancer, renal cancer, skin cancer, head and neck cancer,
brain cancer, pancreatic cancer, lung cancer, ovarian cancer,
uterine cancer, liver cancer, and breast cancer.
[0232] In some embodiments, the invention relates to method for
treating, or inhibiting inflammation and benign prostatic
hyperplasia or the occurrence, recurrence, progression or
metastasis of prostate cancer, consisting of administering to a
mammal diagnosed as having prostate disease or cancer or
hyperplasia or precursor neoplasia thereof, in an amount effective
to treat the disease or cancer or inhibit the occurrence,
recurrence, progression, or metastasis of the prostate disease or
cancer or precursor neoplasia thereof, one or more pharmaceutically
acceptable salts of the invention comprising a cation of Formula
(I). In some favored embodiments of the invention, the
pharmaceutically acceptable salts have a cation having the
formula:
##STR00052##
[0233] wherein [0234] e) E is a nitrogen or phosphorus atom, [0235]
f) R.sub.1', R.sub.1'', and R.sub.1''' are each independently
selected organic moieties comprising between 1 and 12 carbon atoms,
[0236] g) n is an integer between 8 and 12, and [0237] wherein E,
R.sub.1', R.sub.1'', and R.sub.1''' together form a quaternary
ammonium or phosphonium cation; and [0238] the salt also comprises
one or more pharmaceutically acceptable anions X.sup.m- wherein m
is an integer from 1 to 4, sufficient to form the pharmaceutically
acceptable salt.
Preparing and Administering the Compounds and Compositions as
Pharmaceutical Compositions
[0239] Although the compounds described herein can be administered
as pure chemicals either singularly or plurally, it is preferable
to present the active ingredient as a pharmaceutical composition.
Thus, another embodiment of the invention is the use of a
pharmaceutical composition comprising one or more compounds and/or
a pharmaceutically acceptable salt thereof, together with one or
more pharmaceutically acceptable carriers thereof and, optionally,
other therapeutic and/or prophylactic ingredients. The carrier(s)
should be "acceptable" in the sense of being compatible with the
other ingredients of the composition and not overly deleterious to
the recipient thereof. The pharmaceutical composition, is
administered to an animal diagnosed as in need of treatment for a
disease of uncontrolled cellular inflammation and/or proliferation,
in an amount effective to treat the disease of uncontrolled
cellular inflammation and/or proliferation, such as the various
cancers and precancerous conditions described herein.
[0240] As used herein, "pharmaceutical composition" means
therapeutically effective amounts of a pharmaceutically effective
compound together with suitable combination of one or more
pharmaceutically-acceptable carriers, many of which are known in
the art, including diluents, preservatives, solubilizers,
emulsifiers, and adjuvants, collectively".
[0241] As used herein, the terms "effective amount" and
"therapeutically effective amount" refer to the quantity of active
therapeutic agent sufficient to yield a desired therapeutic
response, without undue adverse side effects, such as toxicity,
irritation, or allergic response. The specific "effective amount"
will, obviously, vary with such factors as the particular condition
being treated, the physical condition of the patient, the type of
animal being treated, the duration of the treatment, the nature of
concurrent therapy (if any), and the specific formulations employed
and the structure of the compounds or its derivatives. In this
case, an amount would be deemed therapeutically effective if it
resulted in one or more of the following: (a) the prevention of an
androgen-mediated or androgen-independent disorder (e.g., prostate
cancer); and (b) the reversal or stabilization of an
androgen-mediated or androgen-independent disorder (e.g., prostate
disease and cancer). The optimum effective amounts can be readily
determined by one of ordinary skill in the art using routine
experimentation.
[0242] Pharmaceutical compositions can be liquids or lyophilized or
otherwise dried formulations and include diluents of various buffer
content (e.g., Tris-HCl, acetate, phosphate), pH and ionic
strength, additives such as albumin or gelatin to prevent
absorption to surfaces, detergents (e.g., Tween 20, Tween 80,
Pluronic F68, bile acid salts), solubilizing agents (e.g.,
glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic
acid, sodium metabisulfite), preservatives (e.g., Thiomersal,
benzyl alcohol, parabens), bulking substances or tonicity modifiers
(e.g., lactose, mannitol), covalent attachment of polymers such as
polyethylene glycol to the protein, complexation with metal ions,
or incorporation of the material into or onto particulate
preparations of polymeric compounds such as polylactic acid,
polyglycolic acid, gels, hydrogels, etc, or onto liposomes,
microemulsions, micelles, etc.
[0243] Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's and fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers such as those based on Ringer's dextrose,
and the like. Preservatives and other additives may also be
present, such as, for example, antimicrobials, antioxidants,
collating agents, inert gases and the like.
[0244] Controlled or sustained release compositions administrable
according to the invention include formulation in lipophilic depots
(e.g. fatty acids, waxes, oils). Also comprehended by the invention
are particulate compositions coated with polymers (e.g. poloxamers
or poloxamines) and the compound coupled to antibodies directed
against tissue-specific receptors, ligands or antigens or coupled
to ligands of tissue-specific receptors.
[0245] Other embodiments of the compositions administered according
to the invention incorporate particulate forms, protective
coatings, protease inhibitors or permeation enhancers for various
routes of administration, including parenteral, pulmonary, nasal
and oral.
[0246] Compounds modified by the covalent attachment of
water-soluble polymers such as polyethylene glycol, copolymers of
polyethylene glycol and polypropylene glycol, carboxymethyl
cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or
polyproline are known to exhibit substantially longer half-lives in
blood following intravenous injection than do the corresponding
modified compounds (Abuchowski et al., 1981; Newmark et al., 1982;
and Katre et al., 1987). Such modifications may also increase the
compound's solubility in aqueous solution, eliminate aggregation,
enhance the physical and chemical stability of the compound, and
greatly reduce the immunogenicity and reactivity of the compound.
As a result, the desired in vivo biological activity may be
achieved by the administration of such polymer-compound abducts
less frequently or in lower doses than with the unmodified
compound.
[0247] In yet another method according to the invention, a
pharmaceutical composition can be delivered in a controlled release
system. For example, the agent may be administered using
intravenous infusion, an implantable osmotic pump, a transdermal
patch, liposomes, or other modes of administration. In one
embodiment, a pump may be used (see Langer, supra; Sefton, CRC
Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery
88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989). In
another embodiment, polymeric materials can be used. In yet another
embodiment, a controlled release system can be placed in proximity
to the therapeutic target, i.e., the prostate, thus requiring only
a fraction of the systemic dose (see, e.g., Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-138
(1984). Other controlled release systems are discussed in the
review by Langer (Science 249: 1527-1533 (1990).
[0248] The pharmaceutical preparation can comprise the
anti-androgen compound alone, or can further include a
pharmaceutically acceptable carrier, and can be in solid or liquid
form such as tablets, powders, capsules, pellets, solutions,
suspensions, elixirs, emulsions, gels, creams, or suppositories,
including rectal and urethral suppositories.
[0249] Pharmaceutically acceptable carriers include gums, starches,
sugars, cellulosic materials, and mixtures thereof. The
pharmaceutical preparation containing the anti-androgen compound
can be administered to a patient by, for example, subcutaneous
implantation of a pellet. In a further embodiment, a pellet
provides for controlled release of anti-androgen compound over a
period of time. The preparation can also be administered by
intravenous, intra-arterial, or intramuscular injection of a liquid
preparation oral administration of a liquid or solid preparation,
or by topical application. Administration can also be accomplished
by use of a rectal suppository or a urethral suppository.
[0250] Though it is not possible to specify a single predetermined
pharmaceutically effective amount of the compounds of the
invention, and/or their pharmaceutical compositions, for each and
every disease condition to be treated, determining such
pharmaceutically effective amounts are within the skill of, and
ultimately at the discretion of an attendant physician or clinician
of ordinary skill. In some embodiments, the active compounds of the
invention are administered to achieve peak plasma concentrations of
the active compound of from typically about 0.1 to about 100 .mu.M,
about 1 to 50 .mu.M, or about 2 to about 30 .mu.M. This can be
achieved, for example, by the intravenous injection of a 0.05% to
5% solution of the active ingredient, optionally in saline, or
orally administered as a bolus containing about 0.5-500 mg of the
active ingredient. Desirable blood levels can be maintained by
continuous infusion to provide about 0.01-5.0 mg/kg/hr or by
intermittent infusions containing about 0.4-15 mg/kg of the active
compounds of the invention.
[0251] Pharmaceutical compositions include those suitable for oral,
enteral, parental (including intramuscular, subcutaneous and
intravenous), topical, nasal, vaginal, ophthalinical, sublingual,
nasal or by inhalation administration. The compositions can, where
appropriate, be conveniently presented in discrete unit dosage
forms and can be prepared by any of the methods well known in the
art of pharmacy. Such methods include the step of bringing into
association the active compound with liquid carriers, solid
matrices, semi-solid carriers, finely divided solid carriers or
combination thereof, and then, if necessary, shaping the product
into the desired delivery system.
[0252] The compounds of the invention can have oral bioavailability
as exhibited by blood levels after oral dosing, either alone or in
the presence of an excipient. Oral bioavailability allows oral
dosing for use in chronic diseases, with the advantage of
self-administration and decreased cost over other means of
administration. Pharmaceutical compositions suitable for oral
administration can be presented as discrete unit dosage forms such
as hard or soft gelatin capsules, cachets or tablets each
containing a predetermined amount of the active ingredient; as a
powder or as granules; as a solution, a suspension or as an
emulsion. The active ingredient can also be presented as a bolus,
electuary or paste. Tablets and capsules for oral administration
can contain conventional excipients such as binding agents,
fillers, lubricants, disintegrants, or wetting agents. The tablets
can be coated according to methods well known in the art, e.g.,
with enteric coatings.
[0253] Oral liquid preparations can be in the form of, for example,
aqueous or oily suspensions, solutions, emulsions, syrups or
elixirs, or can be presented as a dry product for constitution with
water or other suitable vehicle before use. Such liquid
preparations can contain conventional additives such as suspending
agents, emulsifying agents, non-aqueous vehicles (which can include
edible oils), or one or more preservative.
[0254] The pharmaceutical preparations administrable by the
invention can be prepared by known dissolving, mixing, granulating,
or tablet-forming processes. For oral administration, the compounds
or their physiologically tolerated derivatives such as salts,
esters, N-oxides, and the like are mixed with additives customary
for this purpose, such as vehicles, stabilizers, or inert diluents,
and converted by customary methods into suitable forms for
administration, such as tablets, coated tablets, hard or soft
gelatin capsules, aqueous, alcoholic or oily solutions. Examples of
suitable inert vehicles are conventional tablet bases such as
lactose, sucrose, or cornstarch in combination with binders such as
acacia, cornstarch, gelatin, with disintegrating agents such as
cornstarch, potato starch, alginic acid, or with a lubricant such
as stearic acid or magnesium stearate.
[0255] Examples of suitable oily vehicles or solvents are vegetable
or animal oils such as sunflower oil or fish-liver oil.
Preparations can be effected both as dry and as wet granules or
supercritically formulated nanoparticles.
[0256] The compounds can also be formulated for parenteral
administration (e.g., by injection, for example, bolus injection or
continuous infusion) and can be presented in unit dose form in
ampules, pre-filled syringes, small bolus infusion containers or in
multi-does containers with an added preservative. The compositions
can take such forms as suspensions, solutions, or emulsions in oily
or aqueous vehicles, and can contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient can be in powder form, obtained by aseptic
isolation of sterile solid or by lyophilization from solution, for
constitution with a suitable vehicle, e.g., sterile, pyrogen-free
water, before use.
[0257] For parenteral administration (subcutaneous, intravenous,
intraarterial, or intramuscular injection), the compounds or their
physiologically tolerated derivatives such as salts, esters,
N-oxides, and the like are converted into a solution, suspension,
or expulsion, if desired with the substances customary and suitable
for this purpose, for example, solubilizers or other auxiliaries.
Examples are sterile liquids such as water and oils, with or
without the addition of a surfactant and other pharmaceutically
acceptable adjuvants. Illustrative oils are those of petroleum,
animal, vegetable, or synthetic origin, for example, peanut oil,
soybean oil, or mineral oil. In general, water, saline, aqueous
dextrose and related sugar solutions, and glycols such as propylene
glycols or polyethylene glycol are preferred liquid carriers,
particularly for injectable solutions.
[0258] The preparation of pharmaceutical compositions which contain
an active component is well understood in the art. Such
compositions may be prepared as aerosols delivered to the
nasopharynx or as injectables, either as liquid solutions or
suspensions; however, solid forms suitable for solution in, or
suspension in, liquid prior to injection can also be prepared. The
preparation can also be emulsified. The active therapeutic
ingredient is often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like or any combination
thereof.
[0259] In addition, the composition can contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents which enhance the effectiveness of the active
ingredient.
[0260] The compounds of the invention comprise cationic
anti-oxidants in the form of a pharmaceutically acceptable salt
with pharmaceutically acceptable anions. Pharmaceutically
acceptable salts include pharmaceutically acceptable halides such
as fluoride, chloride, bromide, or iodide, tribasic phosphate,
dibasic hydrogen phosphate, monobasic dihydrogen phosphate, or the
anionic forms of pharmaceutically acceptable organic carboxylic
acids as acetates, oxalates, tartrates, mandelates, succinates,
citrates, and the like. Such pharmaceutically acceptable salts can
be readily synthesizes from other salts used for the initial
synthesis of the compounds by ion exchange reactions and
technologies well known to those of ordinary skill in the art.
[0261] Salts formed from any free carboxyl groups on the cationic
antioxidant moieties can also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases asisopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the
like.
[0262] For use in medicine, the salts of the anti-androgen compound
may be pharmaceutically acceptable salts. Other salts may, however,
be useful in the commercial or laboratory preparation of the
compounds according to the invention or of their pharmaceutically
acceptable salts. Suitable pharmaceutically acceptable salts of the
compounds include acid addition salts which may, for example, be
formed by mixing a solution of the compound according to the
invention with a solution of a pharmaceutically acceptable acid
such as hydrochloric acid, sulphuric acid, methanesulphonic acid,
fumaric acid, maleic acid, succinic acid, acetic acid, benzoic
acid, oxalic acid, citric acid, tartaric acid, carbonic acid or
phosphoric acid.
[0263] In addition, the salts described herein may be provided in
the form of nutraceutical compositions where the anti-oxidant
properties of the salts prevents the onset of or reduces or
stabilizes various disorders, e.g., including inhibiting the
occurrence of various forms of cancer, including prostate cancer.
The term "nutraceutical," or "nutraceutical composition," for the
purposes of this specification, refers to a food item, or a part of
a food item, that offers medical health benefits, including
prevention and/or treatment of disease. A nutraceutical composition
according to the present invention may contain only a cationic
antioxidant compound according to the present invention as an
active ingredient or, alternatively, may further comprise, in
admixture with the aforesaid cationic antioxidant compound, dietary
supplements including vitamins, co-enzymes, minerals. herbs, amino
acids and the like which supplement the diet by increasing the
total intake of that substance.
[0264] Therefore, the present invention provides methods of
providing nutraceutical benefits to a patient, comprising the step
of administering to the patient a nutraceutical composition
containing a compound having Formula I or a pharmaceutically
acceptable salt thereof. Such compositions generally include a
"nutraceutically-acceptable carrier" which, as referred to herein,
is any carrier suitable for oral delivery including, but not
limited to, the aforementioned pharmaceutically-acceptable
carriers. In certain embodiments, nutraceutical compositions
according to the invention comprise dietary supplements which,
defined on a functional basis, include immune boosting agents,
anti-inflammatory agents, anti-oxidant agents, or mixtures
thereof.
[0265] Although some of the supplements listed above have been
described as to their pharmacological effects, other supplements
may also be utilized in the present invention and their effects are
well documented in the scientific literature.
[0266] In general, one of skill in the art understands how to
extrapolate in vivo data obtained in a model organism, such as
athymic nude mice inoculated with human tumor cell lines, to
another mammal, such as a human. These extrapolations are not
simply based on the weights of the two organisms, but rather
incorporate differences in rates of metabolism, differences in
pharmacological delivery, and administrative routes. Based on these
types of considerations, a suitable dose will, in alternative
embodiments, typically be in the range of from about 0.5 to about
10 mg/kg/day, or from about 1 to about 20 mg/kg of body weight per
day, or from about 5 to about 50 mg/kg/day.
[0267] The desired dose can conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose, as necessary by one skilled in the art, can itself be
further divided, e.g., into a number of discrete loosely spaced
administrations.
[0268] One skilled in the art will recognize that dosage and dosage
forms outside these typical ranges can be tested and, where
appropriate, be used in the methods of this invention.
Combinations with Other Active Agents
[0269] According to another aspect of the invention, pharmaceutical
compositions of matter useful for the treatment of cancer and/or
benign prostatic hyperplasia are provided that contain, in addition
to the aforementioned compounds, an additional therapeutic agent.
Such agents can be chemotherapeutic agents, ablation or other
therapeutic hormones, antineoplastic agents, monoclonal antibodies
useful against cancers and angiogenesis inhibitors. The following
discussion highlights some agents in this respect, which are
illustrative, not limitative. A wide variety of other effective
agents also can be used.
[0270] Among hormones which can be used in combination with the
present inventive compounds, diethylstilbestrol (DES), leuprolide,
flutamide, hydroxyflutamide, bicalutamide, cyproterone acetate,
ketoconazole, abiraterone acetate and amino glutethimide.
[0271] Among antineoplastic and anticancer agents that can be used
in combination with the inventive compounds, 5-fluorouracil,
vinblastine sulfate, estramustine phosphate, suramin and
strontium-89. Other chemotherapeutics useful in combination and
within the scope of the present invention are buserelin,
chlorotranisene, chromic phosphate, cisplatin, cyclophosphamide,
dexamethasone, doxorubicin, estradiol, estradiol valerate,
estrogens conjugated and esterified, estrone, ethinyl estradiol,
floxuridine, goserelin, hydroxyurea, melphalan, methotrexate,
mitomycin and prednisone.
[0272] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
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