U.S. patent application number 16/689930 was filed with the patent office on 2020-10-08 for targeted nitroxide agents.
This patent application is currently assigned to University of Pittsburgh - Of the Commonwealth System of Higher Education. The applicant listed for this patent is University of Pittsburgh - Of the Commonwealth System of Higher Education. Invention is credited to Marie-Celine Frantz, Peter Wipf.
Application Number | 20200317730 16/689930 |
Document ID | / |
Family ID | 1000004901430 |
Filed Date | 2020-10-08 |
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United States Patent
Application |
20200317730 |
Kind Code |
A1 |
Wipf; Peter ; et
al. |
October 8, 2020 |
TARGETED NITROXIDE AGENTS
Abstract
Provided herein are compositions and related methods useful for
free radical scavenging, with particular selectivity for
mitochondria. The compounds comprise a nitroxide-containing group
attached to a mitochondria-targeting group. The compounds can be
cross-linked into dimers without loss of activity. Also provided
herein are methods, for preventing, mitigating and treating damage
caused by radiation. The method comprises delivering a compound, as
described herein, to a patient in an amount and dosage regimen
effective to prevent, mitigate or treat damage caused by
radiation.
Inventors: |
Wipf; Peter; (Pittsburgh,
PA) ; Frantz; Marie-Celine; (Seine Saint Denis,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Pittsburgh - Of the Commonwealth System of Higher
Education |
Pittsburgh |
PA |
US |
|
|
Assignee: |
University of Pittsburgh - Of the
Commonwealth System of Higher Education
Pittsburgh
PA
|
Family ID: |
1000004901430 |
Appl. No.: |
16/689930 |
Filed: |
November 20, 2019 |
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10526368 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/64 20170801;
C07D 209/46 20130101; C07D 471/08 20130101; A61K 31/454 20130101;
C07D 401/12 20130101; C07C 237/16 20130101; C07K 5/08 20130101;
C07D 211/58 20130101; C07D 279/06 20130101; C07D 417/12 20130101;
C07D 209/44 20130101; C07D 221/22 20130101; C07D 211/94
20130101 |
International
Class: |
C07K 5/08 20060101
C07K005/08; A61K 31/454 20060101 A61K031/454; C07D 211/58 20060101
C07D211/58; C07D 401/12 20060101 C07D401/12; A61K 47/64 20060101
A61K047/64; C07D 417/12 20060101 C07D417/12; C07C 237/16 20060101
C07C237/16; C07D 209/46 20060101 C07D209/46; C07D 211/94 20060101
C07D211/94; C07D 221/22 20060101 C07D221/22; C07D 209/44 20060101
C07D209/44; C07D 279/06 20060101 C07D279/06; C07D 471/08 20060101
C07D471/08 |
Goverment Interests
STATEMENT REGARDING FEDERAL FUNDING
[0002] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of Grant No. NIAID U19 AI068021, awarded by the National Institutes
of Health.
Claims
1-23. (canceled)
24. A compound having the structure ##STR00119## wherein X is one
of ##STR00120## and R.sub.1 is hydrogen, C.sub.1-C.sub.6 straight
or branched-chain alkyl, or a C.sub.1-C.sub.6 straight or
branched-chain alkyl further comprising a phenyl (C.sub.6H.sub.5)
group, that is unsubstituted or is methyl-, hydroxyl- or
fluoro-substituted; R.sub.4 is hydrogen, C.sub.1-C.sub.6 straight
or branched-chain alkyl, or a C.sub.1-C.sub.6 straight or
branched-chain alkyl further comprising a phenyl (C.sub.6H.sub.5)
group, that is unsubstituted or is methyl-, hydroxyl- or
fluoro-substituted; R.sub.3 is --NH--R.sub.5, --O--R.sub.5 or
--CH.sub.2--R.sub.5, and R.sub.5 is an --N--O, --N--OH or N.dbd.O
containing group; and R is --C(O)--R.sub.6, --C(O)O--R.sub.6, or
--P(O)--(R.sub.6).sub.2, wherein R.sub.6 is C.sub.1-C.sub.6
straight or branched-chain alkyl or C.sub.1-C.sub.6 straight or
branched-chain alkyl further comprising one or more phenyl
(--C.sub.6H.sub.5) groups that are independently unsubstituted, or
methyl-, ethyl-, hydroxyl- or fluoro-substituted.
25. The compound of claim 24 having the structure ##STR00121##
26. The compound of claim 24 having the structure ##STR00122##
wherein R.sub.4 is hydrogen or methyl.
27-39. (canceled)
40. The compound of claim 24, wherein R.sub.5 has the structure
##STR00123##
41. The compound of claim 24, wherein R is Ac, Hoc, Cbz, or
--P(O)-Ph.sub.2.
42. The compound of claim 24, in which R.sub.1, R.sub.4 and R.sub.6
are independently chosen from hydrogen, methyl, ethyl, propyl,
2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl, and
hydroxybenzyl.
43. The compound of claim 24 wherein X is ##STR00124##
44. The compound of claim 24, wherein X is ##STR00125##
45. The compound of claim 24, wherein R.sub.5 has the structure:
##STR00126##
46. The compound of claim 25, wherein R.sub.5 has the structure:
##STR00127##
47. The compound of claim 26, wherein R.sub.5 has the structure:
##STR00128##
48. The compound of claim 42, wherein R.sub.5 has the structure:
##STR00129##
49. A compound of structure: ##STR00130##
50. A compound of structure: ##STR00131##
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) to United States Provisional Patent Application Nos.
61/081,585, filed on Jul. 17, 2008; 61/158,569, filed on Mar. 9,
2009; and 61/178,570, filed on May 15, 2009, each of which is
incorporated herein by reference in its entirety.
[0003] Provided herein are novel compounds and compositions of
matter comprising a nitroxide group-containing cargo (or "nitroxide
containing group") and a mitochondria-targeting group (or
"targeting group"). The targeting group is believed, without any
intent to be bound, to have the ability to selectively deliver the
composition to mitochondrial, delivering the antioxidant and
free-radical scavenging activity of the nitroxide group to cells,
including but not limited to an enrichment in mitochondria. These
compounds are useful, generally, for their anti-oxidant and
free-radical scavenging capacity, and more specifically, for
example and without limitation, for their radioprotective abilities
and prevention as well as mitigation of degenerative diseases.
[0004] Oxidation stress in cells typically manifests itself by way
of generating reactive oxygen species ("ROS") and reactive nitrogen
species ("RNS"). Specifically, the cellular respiration pathway
generates ROS and RNS within the mitochondrial membrane of the
cell, see Kelso et al., Selective Targeting of a Redox-active
Ubiquinone to Mitochondria within Cells: Antioxidant and
Antiapoptotic Properties, J Biol Chem. 276:4588 (2001). Reactive
oxygen species include free radicals, reactive anions containing
oxygen atoms, and molecules containing oxygen atoms that can either
produce free radicals or are chemically activated by them. Specific
examples include superoxide anion, hydroxyl radical, and
hydroperoxides. In many disease states, the normal response to ROS
and RNS generation is impaired.
[0005] Naturally occurring enzymes, such as superoxide dismutase
("SOD") and catalase salvage ROS and RNS radicals to allow normal
metabolic activity to occur. Significant deviations from cell
homeostasis, such as hemorrhagic shock, lead to an oxidative stress
state, thereby causing "electron leakage" from the mitochondrial
membrane, this "electron leakage" produces an excess amount of ROS
for which the cell's natural antioxidants cannot compensate.
Specifically, SOD cannot accommodate the excess production of ROS
associated with hemorrhagic shock which ultimately leads to
premature mitochondria dysfunction and cell death via apoptosis,
see Kentner et al., Early Antioxidant Therapy with TEMPOL during
Hemorrhagic Shock Increases Survival in Rats, J Trauma Inj Infect
Crit Care., 968 (2002).
[0006] Cardiolipin ("CL") is an anionic phospholipid exclusively
found in the inner mitochondrial membrane of eukaryotic cells, sec
Iverson, S. L. and S. Orrenius, The cardiolipincytochrome c
interaction and the mitochondria) regulation of apoptosis, Arch
Biochem. 423:37-46 (2003). Under normal conditions, the
pro-apoptotic protein cytochrome c is anchored to the mitochondrial
inner membrane by binding with CL, see Tuominen, E. K. J., et al.
Phospholipid cytochrome c interaction: evidence for the extended
lipid anchorage, J Biol Chem., 277:8822-8826 (2002). The acyl
moieties of CL are susceptible to peroxidation by reactive oxygen
species. When ROS are generated within mitochondria in excess
quantities, cytochrome C bound to CL can function as an oxidase and
induces extensive peroxidation of CL in the mitochondrial membrane,
see Kagan, V. E. et al., Cytochrome c acts as a cardiolipin
oxygenase required, for release of proapoptotic, factors, Nat Chem
Biol. 1:223-232 (2005); also Kagan, V. E. et al., Oxidative
lipidomics of apoptosis: redox catalytic interactions of cytochrome
c with cardiolipin and phosphatidylserine, Free Rad Biol Med.
37:1963-1985 (2005).
[0007] The peroxidation of the CL weakens the binding between the
CL and cytochrome C, see Shidoji, Y. et al., Loss of molecular
interaction between cytochrome C and cardiolipin due to lipid
peroxidation. Biochem Biophys Res Comm. 264:343-347 (1999). This
leads to the release of the cytochrome C into the mitochondrial
intermembrane space, inducing apoptotic cell death. Further, the
peroxidation of CL has the effect of opening the mitochondrial
permeability transition pore ("MPTP"), see Dolder, M. et al.,
Mitochondria creatine kinase in contact sites: Interaction with
porin and adenine nucleotide translocase, role in permeability
transition and sensitivity to oxidative damage, Biol Sign Recept,
10:93-111 (2001); also Imai, H. et al., Protection from
inactivation of the adenine nucleotide translocator during
hypoglycaemia-induced apoptosis by mitochondria/phospholipid
hydroperoxide glutathione peroxidase, Biochem J., 371:799-809
(2003). Accordingly, the mitochondrial membrane swells and releases
the cytochrome C into the cytosol. Excess cytochrome C in the
cytosol leads to cellular apoptosis, see Iverson, S. L. et al. The
cardiolipin-cytochrome c interaction and the mitochondria
regulation of apoptosis, Arch Biochem Biophys. 423:37-46
(2003).
[0008] Moreover, mitochondrial dysfunction and cell death may
ultimately lead to multiple organ failure despite resuscitative
efforts or supplemental oxygen supply, see Cairns, C., Rude
Unhinging of the Machinery of Life: Metabolic approaches to
hemorrhagic Shock, Curr Crit Care., 7:437 (2001). Accordingly,
there is a need in the art for an antioxidant which scavenges the
ROS, thereby reducing oxidative stress. Reduction of oxidative
stress delays, even inhibits, physiological conditions that
otherwise might occur, such as hypoxia.
[0009] Also, there is also a need to improve the permeability of
antioxidants' penetration of the cellular membrane. One of the
limitations of SOD is that it cannot easily penetrate the cell
membrane. However, nitroxide radicals, such as TEMPO
(2,2,6,6-tetramethylpiperidine-N-oxyl) and its derivatives, have
been shown to penetrate the cell membrane better than SOD. Further,
nitroxide radicals like, for example and without limitation, TEMPO
prevent the formation of ROS, particularly superoxide, due to their
reduction by the mitochondrial electron transport chain to hydroxyl
amine radical scavengers, see Wipf. P. et al., Mitochondria
targeting of selective electron scavengers: synthesis and
biological analysis of hemigramicidin-TEMPO conjugates, J Am Chem
Soc. 127:12460-12461. Accordingly, selective delivery of TEMPO
derivatives may lead to a therapeutically beneficial reduction of
ROS and may delay or inhibit cell death due to the reduction of
oxidative stress on the cell.
[0010] Selective delivery may be accomplished by way of a number of
different pathways--e.g., by a biological or chemical moiety having
a specific targeting sequence for penetration of the cell membrane,
ultimately being taken up by the mitochondrial membrane. Selective
delivery of a nitroxide SOD mimic into the mitochondrial membrane
has proven difficult. Accordingly, there is a need in the art for
effective and selective delivery of antioxidants that specifically
target the mitochondria and its membranes as well as inter-membrane
space to help reduce the ROS and RNS species. The antioxidants also
help prevent cellular and mitochondria apoptotic activity which
often results due to increased ROS species, see Kelso et al.,
Selective Targeting of a Redox-active Ubiquinone to Mitochondria
within Cells: Antioxidant and Antiapoptotic Properties, J Biol
Chem., 276: 4588 (2001). Examples of mitochondria-targeting
antioxidants are described in United States Patent Publication Nos.
20070161573 and 20070161544.
[0011] There remains a very real need for a composition and
associated methods for delivering cargo of various types to
mitochondria. In one embodiment, a composition comprising membrane
active peptidyl fragments having a high affinity with the
mitochondria linked to cargo is provided. The cargo may be selected
from a large group of candidates. There is a need for compositions
and methods for effectively treating a condition that is caused by
excessive mitochondria production of ROS and RNS in the
mitochondrial membrane. There also is a need for compounds that can
protect cells and tissues of animals against radiation damage.
Radiation Exposure
[0012] The biologic consequences of exposure to ionizing radiation
(IR) include genomic instability and cell death (Little J B,
Nagasawa H, Pfenning T, et al. Radiation-induced genomic
instability: Delayed mutagenic and cytogenetic effects of X rays
and alpha particles. Radial Res 1997; 148:299-307). It is assumed
that radiolytically generated radicals are the primary cause of
damage from 1R. Direct radiolysis of water and the secondary
reactive intermediates with a short lifetime (10.sup.-10-10.sup.6
seconds) mediate the chemical reactions that trigger the damage of
cellular macromolecules, including DNA and proteins, as well as
phospholipids in membranes (Mitchell J B, Russo A, Kuppusamy P, et
al. Radiation, radicals, and images. Ann N Y Acad Sci 2000;
899:28-43). The DNA is believed to be the primary target for the
radical attack, resulting in single and double DNA strand breaks
(Bryant P E. Enzymatic restriction of mammalian cell DNA: Evidence
for double-strand breaks as potentially lethal lesions. Int J
Radial Biol 1985; 48:55-60). To maintain the genomic integrity,
multiple pathways of DNA repair and cell-cycle checkpoint control
are activated in response to irradiation-induced DNA damage
(Elledge S J. Cell cycle checkpoints: Preventing an identity
crisis. Science 1996; 274:1664-1672). Failure of these repair and
regulatory systems leads to genotoxicity, malignant transformation,
and cell death (Sachs R K, Chen A M, Brenner D J. Proximity effects
in the production of chromosome aberrations by ionizing radiation.
Int J Radiat Biol 1997; 71:1-19).
[0013] One of the major mechanisms of IR-induced cell death is
apoptosis, most commonly realized through a mitochondria-dependent
intrinsic pathway (Newton K, Strasser A. Ionizing radiation and
chemotherapeutic drugs induce apoptosis in lymphocytes in the
absence of Fas or FADD/MORT1 signaling. Implications for cancer
therapy. J Exp Med 2000; 191:195-200). The latter includes
permeabilization of mitochondria followed by the release of
cytochrome (cyt) c and other proapoptotic factors (Smac/Diablo
[second mitochondrial-derived activator of caspase/direct inhibitor
of apoptosis-binding protein with low pI], EndoG [endonuclease G],
Omi/HtrA2, and AIF [apoplosisinducing factor]) into the cytosol as
the key events in the execution of the death program. The released
cyt c facilitates the formation of apoptosomes by interacting with
apoptotic protease activating factor 1 (Apaf-1) and then recruits
and activates procaspase-9 and triggers the proteolytic cascade
that ultimately leads to cell disintegration. Release of
proapoptotic factors and caspase activation designate the
commencement of irreversible stages of apoptosis. Therefore,
significant drug discovery efforts were directed toward the
prevention of these events, particularly of the mitochondrial
injury representing an important point of no return (Szewczyk A,
Wojtczak L. Mitochondria as a pharmacological target. Pharmacol Rev
2002; 54:101-127). However, the exact mechanisms of cyt c release
from mitochondria are still poorly understood. It was postulated
that generation of reactive oxygen species (ROS), likely by means
of disrupted electron transport, has a crucial role in promoting
cyt c release from mitochondria (Kowaltowski A J, Castilho R F,
Vercesi A E. Opening of the mitochondrial permeability transition
pore by uncoupling or inorganic phosphate in the presence of Ca2+
is dependent on mitochondrial-generated reactive oxygen species.
FEBS Lett 1996; 378:150-152). Notably, ROS can induce mitochondria
membrane permeabilization both in vitro and in vivo, and the
mitochondrial membrane transition pore was shown to be redox
sensitive (Kroemer G, Reed J C. Mitochondrial control of cell
death. Nat Med 2000; 6:513-519).
[0014] Conversely, antioxidants and reductants, overexpression of
manganese superoxide dismutase (MnSOD) (Wong G H, Elwell J H,
Oberley L W, et al. Manganous superoxide dismutase is essential for
cellular resistance to cytotoxicity of tumor necrosis factor. Cell
1989; 58:923-931), and thioredoxin (Iwata S, Hori T, Sato N, et al.
Adult T cell leukemia (ATL)-derived factor/human thioredoxin
prevents apoptosis of lymphoid cells induced by L-cystine and
glutathione depletion: Possible involvement of thiol-mediated redox
regulation in apoptosis caused by pro-oxidant stale. J Immunol
1997; 158:3108-3117) can delay or inhibit apoptosis. Previous
studies showed that early in apoptosis, a mitochondria-specific
phospholipid-cardiolipin (CL) translocated from the inner to the
outer mitochondrial membrane and activated cyt c into a CL-specific
peroxidase (Fernandez M G, Troiano L, Moretti L, et al. Early
changes in intramitochondrial cardiolipin distribution during
apoptosis. Cell Growth Differ 2002; 13:449-455 and Kagan V E,
Tyurin V A, Jiang J, et al. Cytochrome c acts as a cardiolipin
oxygenase required for release of proapoptotic factors. Nat Chem
Biol 2005; 1:223-232). The activated cyt c further catalyzed the
oxidation of CL by using mitochondrially generated ROS (Kagan V E,
Tyurin V A, Jiang J, et al. Cytochrome c acts as a cardiolipin
oxygenase required for release of proapoptotic factors. Nat Chem
Biol 2005; 1:223-232). Most importantly, oxidized CL is an
important contributor to the release of cyt c from mitochondria
(Kagan V E, Tyurin V A, Jiang J, et al. Cytochrome c acts as a
cardiolipin oxygenase required for release of proapoptotic factors.
Nat Chem Biol 2005; 1:223-232 and Petrosillo G, Casanova G, Matera
M, et al. Interaction of peroxidized cardiolipin with rat-heart
mitochondrial membranes: Induction of permeability transition and
cytochrome c release. FEBS Lett 2006; 580:6311-6316), which might
be attributed to changes in microenvironment for the interaction
between this phospholipid and cyt c (Ott M, Robertson J D, Gogvadze
V, et al. Cytochrome c release from mitochondria proceeds by a
two-step process. Proc Nad Acad Sci USA 2002; 99:1259-1263 and
Garrido C, Galluzzi L, Brunet M. et al. Mechanisms of cytochrome c
release from mitochondria. Cell Death Differ 2006; 13:1423-1433)
and/or participation of oxidized CL in the formation of
mitochondrial permeability transition pones (MTP) in coordination
with Bcl-2 family proteins (Bid, Bax/Bak), as well as adenine
nucleotide translocator (ANT) and voltage-dependent anion channel
(VDAC) (Petrosillo G, Casanova G, Matera M. et al. Interaction of
peroxidized cardiolipin with rat-heart mitochondrial membranes:
Induction of permeability transition and cytochrome c release. FEBS
Lett 2006; 580:6311-6316 and Gonzalvez F, Gottlieb E. Cardiolipin:
Setting the beat of apoptosis. Apoptosis 2007; 12:877-885). In
addition to their essential role in the apoptotic signaling
pathway, ROS were also implicated in perpetuation of the bystander
effect (Narayanan P K, Goodwin E H, Lehnert B E. Alpha particles
initiate biological production of superoxide anions and hydrogen
peroxide in human cells. Cancer Res 1997; 57:3963-3971 and Iyer R,
Lehnert B E. Factors underlying the cell growth-related bystander
responses to alpha particles. Cancer Res 2000; 60:1290-1298) and
genomic instability after irradiation exposure (Spitz D R, Azzam E
I, Li J J, et al. Metabolic oxidation/reduction reactions and
cellular responses to ionizing radiation: A unifying concept in
stress response biology. Cancer Metastasis Rev 2004; 23:311-322;
Limoli C L, Giedzinski E, Morgan W F, et al. Persistent oxidative
stress in chromosomally unstable cells. Cancer Res 2003;
63:3107-3111; and Kim G J, Chandrasekaran K, Morgan W F.
Mitochondrial dysfunction, persistently elevated levels of reactive
oxygen species and radiation-induced genomic instability: A review.
Mutagenesis 2006; 21:361-367). Hence, elimination of intracellular
ROS, particularly its major source, mitochondrial ROS, by
antioxidants may be an important opportunity for developing
radioprotectors and radiomitigators. Protection by antioxidants
against IR has been studied for more than 50 years (Weiss J F,
Landauer M R. Radioprotection by antioxidants. Ann N Y Acad Sci
2000; 899:44-60).
[0015] One of the major mechanisms of ionizing irradiation induced
cell death is apoptosis, most commonly realized through a
mitochondria dependent intrinsic pathway. Oxidation of cardiolipin
catalyzed by cytochrome c ("cyt c"), release of cytochrome c and
other pro-apoptotic factors into the cytosol and subsequent caspase
activation are the key events in the execution of the death program
designating the commencement of irreversible stages of
apoptosis.
[0016] In Belikova, N A, et al., (Cardiolipin-Specific Peroxidase
Reactions of Cytochrome C in Mitochondria During
Irradiation-Induced Apoptosis, Int. J. Radiation Oncology Biol.
Phys., Vol. 69, No. 1, pp. 176-186, 2007), a small interfering RNA
(siRNA) approach was used to engineer HeLa cells with lowered
contents of cyt c (14%, HeLa 1.2 cells). Cells were treated by
.gamma.-irradiation (in doses of 5-40 Gy). Lipid oxidation was
characterized by electrospray ionization mass spectrometry analysis
and fluorescence highperformance liquid chromatography-based Amplex
Red assay. Release of a proapoptotic factor (cyt c, Smac/DIABLO)
was detected by Western blotting. Apoptosis was revealed by
caspase-3/7 activation and phosphatidylserine externalization. They
showed that irradiation caused selective accumulation of
hydroperoxides in cardiolipin (CL) but not in other phospholipids.
HeLa 1.2 cells responded by a lower irradiation-induced
accumulation of CL oxidation products than parental HeLa cells.
Proportionally decreased release of a proapoptotic factor,
Smac/DIABLO, was detected in cyt c-deficient cells after
irradiation. Caspase-3/7 activation and phosphatidylserine
externalization were proportional to the cyt c content in cells.
They concluded that cytochrome c is an important catalyst of CL
peroxidation, critical to the execution of the apoptotic program.
This new role of cyt c in irradiation-induced apoptosis is
essential for the development of new radioprotectors and
radiosensitizers.
[0017] Significant drug discovery efforts have been directed
towards prevention of these events, particularly of the
mitochondrial injury that represents an important point of no
return. Although the exact mechanisms are still not well
understood, generation of reactive oxygen species (ROS) and
oxidation of cardiolipin by the peroxidase function of cytochrome
c/cardiolipin complexes are believed to play a critical role in
promoting cytochrome c release from mitochondria. ROS--superoxide
radicals dismutating to H.sub.2O.sub.2--feed the peroxidase cycle
and facilitate accumulation of oxidized cardiolipin. Hence,
elimination of intracellular ROS, particularly its major source,
mitochondrial ROS, by electron and radical scavengers is a
promising opportunity for developing radioprotectors and
radiomitigators. Significant research has been conducted in the
field of radiation protection to use antioxidants against ionizing
irradiation (Weiss et al. Radioprotection by Antioxidants. Ann N Y
Acad Sci 2000; 899:44-60).
[0018] A new class of antioxidants, stable nitroxide radicals, has
been suggested as potent radioprotectors due to multiplicity of
their direct radical scavenging properties as well as catalytic
enzyme-like mechanisms (Saito et al. Two reaction sites of a spin
label, TEMPOL with hydroxyl radical. J Pharm Sci 2003; 92:275-280;
Mitchell et al. Biologically active metal-independent superoxide
dismutase mimics. Biochemistry 1990; 29:2802-2807). TEMPOL
(4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) is a nitroxide
whose properties as a radioprotector in vitro and in vivo have been
extensively studied (Mitchell et al. Nitroxides as radiation
protectors. Mil Med 2002; 167:49-50; Hahn et al. In vivo
radioprotection and effects on blood pressure of the stable free
radical nitroxides. Int J Radiat Oncol Biol Phys 1998; 42:839-842.
Mitchell et al. Inhibition of oxygen-dependent radiation-induced
damage by the nitroxide superoxide dismutase mimic, tempol. Arch
Biochem Biophys 1991; 289:62-70; Hahn et al. Tempol, a stable free
radical, is a novel murine radiation protector. Cancer Res 1992;
52:1750-1753). Currently, TEMPOL is in clinical trials as a topical
radiation protector to prevent hair loss during cancer
radiotherapy. While found promising and relatively effective, the
required high millimolar concentrations of TEMPOL, mainly due to
its poor partitioning into cells and mitochondria, set a limit for
its broader applications (Gariboldi et al. Study of in vitro and in
vivo effects of the piperidine nitroxide Tempol--a potential new
therapeutic agent for gliomas. Eur J Cancer 2003; 39:829-837). In
addition, it has been demonstrated that TEMPOL must be present
during irradiation to exert its radioprotective effect (Mitchell et
al. Radiation, radicals, and images. Ann N Y Acad Sci 2000;
899:28-43; Mitchell et al. Inhibition of oxygen-dependent
radiation-induced damage by the nitroxide superoxide dismutase
mimic, tempol. Arch Biochem Biophys 1991; 289:62-70), This suggests
that the protective mechanisms of TEMPOL are limited to its
interactions with short-lived radiolytic intermediates produced by
irradiation.
[0019] Sufficient concentrations of antioxidants at the sites of
free radical generation are critical to optimized protection
strategies. A great deal of research has indicated that
mitochondria are both the primary source and major target of ROS
(Reviewed in Orrenius S. Reactive oxygen species in
mitochondria-mediated cell death. Drug Metab Rev 2007; 39:443-455).
In fact, mitochondria have been long considered as an important
target for drug discovery (Szewczyk et al., Mitochondria as a
pharmacological target. 221 Pharmacol. Rev. 54:101-127; 2002;
Garber K. Targeting mitochondria emerges as therapeutic strategy.
J. Natl. Cancer Inst. 97:1800-1801; 2005).
[0020] Chemistry-based approaches to targeting of compounds to
mitochondria include the use of proteins and peptides, as well as
the attachment of payloads to lipophilic cationic compounds,
triphenyl phosphonium phosphate, sulfonylureas, anthracyclines, and
other agents with proven or hypothetical affinities for
mitochondria (Murphy M P. Targeting bioactive compounds to
mitochondria. Trends Biotechnol, 15:326-330; 1997; Dhanasekaran et
al., Mitochondria superoxide dismutase mimetic inhibits
peroxideinduced oxidative damage and apoptosis: role of
mitochondrial superoxide. Free Radic. Biol. Med. 157 39:567-583;
2005; Hoye et al., Targeting Mitochondria. Ace. Chem. Res. 41:
87-97, 2008). However, at the time of this writing, no evidence has
been presented that GS-nitroxides
SUMMARY
[0021] There remains a very real need for a composition and
associated methods for delivering cargo of various types to
mitochondria, specifically antioxidants. Provided herein are
compounds comprising a targeting group and a cargo that is a
nitroxide-containing group and compositions comprising the
compounds. As illustrated in the Examples, below, compounds and
compositions described herein have use in the prophylaxis and
treatment of exposure to ionizing radiation, in anti-ageing
therapies and, generally, in treating conditions that benefit from
antioxidant treatment. Examples of these compounds are provided
below and in the claims.
[0022] For example, the effective mitochondrial concentration of
mitochondria targeted conjugated nitroxides (--N-0., --N--OH or
N.dbd.O containing compounds and groups) against
.gamma.-irradiation could be increased up to 1,000 times (and their
required tissues concentrations can be reduced 1,000 times from 10
mM to 10 .mu.M) compared with parent non-conjugated nitroxides.
Enrichment in mitochondria of mitochondria targeted nitroxides has
been demonstrated by EPR spectroscopy as well as by MS analysis of
their content in mitochondria obtained from cells incubated with
mitochondria targeted nitroxides. Delivery of mitochondria
targeted-nitroxides into mitochondria does not depend on the
mitochondrial membrane potential. Therefore, mitochondria targeted
nitroxides can accumulate not only in intact but also in
de-energized or damaged mitochondria with low membrane potential.
Moreover, mitochondria targeted nitroxide conjugates are delivered
into mitochondria without affecting the mitochondrial membrane
potential. Hence, they do not impair the major mitochondrial
function, the energy production, in cells. In addition, the
conjugated nitroxides provide a new important feature, post
irradiation protection.
[0023] Like other nitroxides, conjugated mitochondria targeted
nitroxides might potentially lower blood pressure and sympathetic
nerve activity. However, the dramatically reduced dose of
mitochondria targeted nitroxides (about 1,000-fold), compared to
non-conjugated parental nitroxides, may be significantly below of
those inducing side effects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIGS. 1-1A to 1-2G provide non-limiting examples of certain
nitroxides. The log P values were estimated using the online
calculator of molecular properties and drug likeness on the
Molinspirations Web site
(www.molinspiration.com/cgi-bin/properties). TIPNO=tert-butyl
isopropyl phenyl nitroxide.
[0025] FIG. 2-1A to 2-3F provide examples of structures of certain
mitochondria-targeting antioxidant compounds referenced herein, and
the structure of TEMPOL.
[0026] FIG. 3 depicts an example of a synthetic pathway for the
TEMPO-hemigramicidin conjugates.
[0027] FIGS. 4A-4B show an EPR-based analysis of integration and
reduction of nitroxide Gramicidin S peptidyl-TEMPO conjugates in
MECs.
[0028] FIGS. 5A-5H show a fluorescein isothiocyanate-dextran (FD4)
read-out which reflects the effect of Gramicidin-S TEMPO conjugates
on rat ileal mucosal permeability following profound hemorrhagic
shock. Data are expressed as a percentage of the change
permeability relative to that observed in simultaneously assayed
control segments loaded during shock with normal saline solution.
FIG. 5A shows an FD4 read-out of TEMPOL which is used as a
"positive control" for the gut mucosal protection assay. FIG. 5B
shows an FD4 read-out of TEMPO conjugate XJB-5-208 reflecting gut
mucosal protection. FIG. 5C shows an FD4 read-out of XJB-5-125
which has the TEMPO payload, but fails to provide protection
against gut barrier dysfunction induced by hemorrhage. FIG. 5D
shows an FD4 read-out of XJB-5-127 which lacks the TEMPO payload
and fails to provide protection against gut barrier dysfunction
induced by hemorrhage. FIG. 5E shows an FD4 read-out of TEMPO
conjugate XJB-5-131 reflecting gut mucosal protection. FIG. 5F
shows an FD4 read-out of XJB-5-133 which lacks the TEMPO payload
even though it possesses the same hemigramicidin mitochondria
targeting moiety as the most active compound, XJB-5-131. FIG. 5G
shows an FD4 read-out of XJB-5-197 which has the TEMPO payload, but
fails to provide protection against gut barrier dysfunction induced
by hemorrhage. FIG. 5H shows an FD4 read-out of XJB-5-194 which
lacks the TEMPO payload and fails to provide protection against gut
barrier dysfunction induced by hemorrhage.
[0029] FIGS. 6A-6F show graphical representations of the effect of
nitroxide conjugates on ActD-induced apoptosis. FIG. 6A is a
graphical representation of superoxide production based upon mean
fluorescence intensity from 10,000 ileal cells. FIG. 6B is a
graphical representation of phosphatidylserine (PS) externalization
as indicated by the percentage of annexin V-positive cells. FIG. 6C
is a graphical representation of caspase-3 activity as indicated by
amount of its specific substrate present, Z-DVED-AMC, in nmol/mg
protein. FIG. 6D is a graphical representation of DNA fragmentation
as indicated by propidium iodide fluorescence. FIG. 6E is a
graphical representation of PS externalization at different
concentrations of the compound 5a (as shown in FIG. 3). FIG. 6F is
a graphical representation of adenosine triphosphate (ATP) levels
in mitochondria in the presence or absence of 5a or
2-deoxyglucose.
[0030] FIG. 7 illustrates the effects of intraluminal XJB-5-131 on
hemorrhage-induced peroxidation of phospholipids in intestinal
mucosa. FIG. 7A is a graphical representation of the peroxidation
of phosphatidylcholine ("PC"). FIG. 7B is a graphical
representation of peroxidation activity with respect to
phosphatidylethanolamine ("PE"). FIG. 7C is a graphical
representation of peroxidation activity with respect to
phosphatidylserine ("PS"). FIG. 7D is a graphical representation of
peroxidation activity with respect to cardiolipin ("CL").
[0031] FIG. 8 is a graphical representation of caspase 3 and 7
activity that illustrates the effects of intraluminal
XJB-5-131.
[0032] FIG. 9 is a graphical representation of permeability of
XJB-5-131 with respect to Caco-2BBe human enterocyte-like
monolayers subjected to oxidative stress. The permeability of the
monolayers is expressed as a clearance (pLh-1cm2).
[0033] FIG. 10A is a graphical representation of the effects of
intravenous treatment with XJB-5-131 on MAP (mean arterial
pressure, mm Hg) of rates subjected to volume controlled
hemorrhagic shock. FIG. 10B is a graphical representation of the
effects of intravenous treatment with XJB-5-131 on survival
probability of rates subjected to volume controlled hemorrhagic
shock.
[0034] FIG. 11A is a schematic of a synthesis protocol for JP4-039.
FIG. 11B provides a synthesis route for a compound of Formula 4,
below.
[0035] FIGS. 12A-12B show that nitroxide conjugate XJB-5-125
integrates into cells and mitochondria much more efficiently than
their parent non-conjugated 4-amino-TEMPO in mouse embryonic cells.
FIG. 12A shows their cellular and mitochondrial integration
efficiencies in mouse embryonic cells, and FIG. 12B shows
representative EPR spectrum of nitroxides recovered from
mitochondria.
[0036] FIGS. 13A-13B reveal that nitroxide conjugate XJB-5-125
protects mouse embryonic cells against gamma irradiation induced
superoxide generation and cardiolipin peroxidation. FIG. 13A
superoxide generation. Cells were exposed to 10 Gy of
.gamma.-irradiation. XJB-5-125 (20 .mu.M) was added to cells either
10-min before or 1-h after irradiation and removed after 5-h
incubation. Cells were incubated with 5 .mu.M DHE for 30 min at the
indicated time points. Ethidium fluorescence was analyzed using a
FACScan flow cytometer supplied with CellQuest software. Mean
fluorescence intensity from 10,000 cells was acquired using a
585-nm bandpass filter. FIG. 13B Cardiolipin oxidation. Cardiolipin
hydroperoxides were determined using a fluorescent HPLC-based
Amplex Red assay. Data presented are means.+-.S.E. (n=3).
*p<0.01 vs non-irradiated cells; *p<0.01(0.05) vs irradiated
cells without XJB-5-125 treatment under the same condition. Insert
is a typical 2D-HPTLC profile of phospholipids from cells.
[0037] FIGS. 14A-14E reveal that nitroxide conjugate XJB-5-125
protects cells against gamma irradiation induced apoptosis. FIG.
14A XJB-5-125 blocks 7-irradiation induced accumulation of
cytochrome c in the cytosol of mouse embryonic cells. FIG. 14B
Densitometry ratio of cytochrome c/actin. Semi-quantitation of the
bands was carried out by densitometry using Labworks Image
Acquisition and Analysis Software (UVP, Upland, Calif.). The level
of cytochrome c release was expressed as the mean densitometry
ratio of cytochrome c over actin. FIG. 14C Dose (5, 10 and 20
.mu.M) dependent radioprotective effect of XJB-5-125
(pre-treatment) on .gamma.-irradiation (10 Gy) induced
phosphatidylserine (PS) externalization. After 48 h
post-irradiation incubation, cells were harvested and stained with
annexin-V-FITC and propodium iodide (PI) prior to flow cytometry
analysis. FIG. 14D Time (2, 3, 4, 5, and 6 h) dependent
radioprotective effect of XJB-5-125 (201.04) on .gamma.-irradiation
(10 Gy) induced PS externalization (48 h post irradiation) in mouse
embryonic cells. FIG. 14E Effect of XJB-5-125 on
.gamma.-irradiation (10 Gy) induced PS externalization in human
bronchial epithelial cell line BEAS-2B cells. Cells were treated
with 5-125 (5 or 10 .mu.M) before (10-min) or after (1-h)
irradiation. Externalization of PS was analyzed 72 h
post-irradiation exposure. Data shown are means.+-.S.E. (n=3).
*(&)p<0.01(0.05) vs irradiated cells without 5-125
treatment, # p<0.05 vs cells pre-treated with 5-125.
[0038] FIG. 15 shows the effect of nitroxide conjugate XJB-5-125 on
gamma-irradiation dose survival curves of mouse embryonic cells.
Cells were pre- (10-min) or post-treated (1-h) with XJB-5-125 (20
.mu.M), which was removed after 4-h incubation period. The
surviving fraction was calculated as the plating efficiency of the
samples relative to that of the control. The data was fitted to a
single-hit multitarget model using SigmaPlot 9.0 (Systat Software).
Data presented are the mean.+-.S.E. (n=3).
[0039] FIG. 16 illustrates the effect of GS conjugated nitroxide,
XJB-5-125, on gamma-irradiation dose survival curves of 32D cl 3
murine hematopoietic cells. The cells incubated in XJB-5-125 or
Tempol had an increased Do (1.138 or 1.209 Gy, respectively)
compared to the 32D cl 3 cells (0.797 Gy). The cells incubated in
XJB-5-125 had an increased shoulder on the survival curve with an n
of 18.24 compared to 5.82 for the cells incubated in tempol.
[0040] FIGS. 17A and 17B are graphs showing GS-nitroxide compound
JP4-039 increases survival of mice exposed to 9.75 Gy total body
irradiation.
[0041] FIG. 18 is a graph showing that GS-nitroxide compound
JP4-039 increases survival of mice exposed to 9.5 Gy total body
irradiation.
[0042] FIG. 19 is a graph showing that GS-nitroxide JP4-039 is an
effective hematopoietic cell radiation mitigator when delivered 24
hr after irradiation.
[0043] FIG. 20 is a graph showing that JP4-039 is an effective
mitigator of irradiation damage to KM101 human marrow stromal
cells.
[0044] FIGS. 21A-21B show results with detection of human cells in
NOD/SCID mouse marrow harvested 27 days after cord blood
transplanted I.V, showing flow cytometric analysis and
identification of human CD45+(light gray) hematopoietic cells in
NOD/SCED mouse BM following irradiation, proximal tibia bone
drilling (see below), and human cord blood injection.
[0045] FIG. 21C is a photomicrograph of cross-section through a
tibial wound 7-days after surgical construction with a drill bit of
a unicortical 2-mm diameter wound in the lateral aspect of the
tibia 2-mm below the proximal epiphyseal plate.
[0046] FIG. 22 is a schematic diagram of a Bronaugh diffusion
system for studying in vitro transdermal flux.
[0047] FIG. 23 is a graph showing delivery of XJB-5-125 into mouse
skin after 24 hours.
[0048] FIGS. 24A-24D show typical EPR spectra of GS-nitroxides
recorded from different fractions obtained after the filtration
through the mouse skin. 1--donor fluid, 2--receiver fluid after 6 h
of solution A filtration, 3--receiver fluid after 6 h of solution B
filtration, 4--skin after 24 h exposure to solution A. The EPR
spectra of GS-nitroxide radicals in medium, or skin homogenates
were recorded in 28.5% of acetonitrile with addition of 2 mM
K.sub.3Fe(CN).sub.6
[0049] FIG. 25 is a graph showing cumulative transdermal absorption
of XJB-5-125 through mouse skin over 24 hours
[0050] FIGS. 26A and 26B provide structures for compounds
JED-E71-37 and JED-E71-58, respectively.
[0051] FIG. 27 shows a treatment paradigm for study to determine
the impact of XJB-5-131 on the age at onset of signs of aging in
progeroid Ercc1.sup.-/.DELTA. mice. XJB-5-131 was 2 mg/kg prepared
from a 10 .mu.g/.mu.L stock in DMSO mixed with 50 .mu.L, of
sunflower seed oil and injected intraperitoneally. As control
littermate Ercc1.sup.-/.DELTA. mice were treated with an equal
volume of sunflower seed oil only, in double-blind twin study.
[0052] FIG. 28 is a summary table showing effects of treatment with
XJB-5-131 ("XJB" in this figure), relative to control (sunflower
seed oil) on the age at onset (in weeks) of various indicia of
aging in Ercc1.sup.-/.DELTA. mice, using the protocol of FIG. 27.
The duration of treatment of mice in this figure was three times
per week, beginning at 5 wks of age and continuing throughout their
lifespan. Cells highlighted in the XJB column indicate a
significant delay in onset of the age-related degenerative change
in mice treated with XJB relative to isogenic controls treated with
vehicle only.
[0053] FIG. 29 is a bar graph showing glycosaminoglycan (an
extracellular matrix protein that is essential for disc maintenance
and flexibility) content of intervertebral discs of
Ercc1.sup.-/.DELTA. mice either treated with XJB-5-131 ("XJB" in
this figure) or vehicle (sunflower seed oil) according to the
protocol shown in FIG. 27. The duration of treatment of mice in
this figure was three times per week, beginning at 5 wks of age and
continuing throughout their lifespan.
[0054] FIG. 30 provides photographs showing the effects of
(photo)aging in Ercc1.sup.-/.DELTA. mice either treated with
XJB-5-131 (80 .mu.g emulsified in a topical cream) or cream only,
according to the protocol shown in FIG. 27. The duration of
treatment of mice in this figure was daily for five days post-UV
irradiation.
[0055] FIGS. 31A-B are graphs showing weights as a function of age
of FIG. 31A male and FIG. 31B female Ercc1.sup.-/.DELTA. mice
either treated with XJB-5-131 or vehicle (sunflower seed oil)
according to the protocol shown in FIG. 27. XJB-5-131 does not
cause weight loss as does the parental compound TEMPO.
[0056] FIG. 32 provides photomicrographs of SA-.beta. galactosidase
(a marker of cellular senescence) staining in mouse embryonic
fibroblast ("MEF") cells prepared from Ercc1.sup.-/- mice, where
the MEF cells were either treated with XJB-5-131 ("XJB" in this
figure; 500 nM dissolved in media) or media alone continuously for
48 hr prior to fixing and staining the cells.
[0057] FIG. 33 provides photomicrographs of .gamma.H2.LAMBDA.X
immunostaining (a marker of DNA double strand breaks and cellular
senescence) of mouse embryonic fibroblast ("MEF") cells prepared
from Ercc1.sup.-/- mice, where the MEF cells were either treated
with XJB-5-131 ("XJB" in this figure; 500 nM dissolved in media) or
media alone continuously for 48 hr prior to fixing and staining the
cells.
[0058] FIG. 34 is a graph showing apoptosis in mouse embryonic
fibroblast ("MEF") cells prepared from Ercc1.sup.-/- mice, where
the MEF cells were either treated with XJB-5-131 ("XJB" in this
figure; 500 nM dissolved in media) or media alone continuously for
48 hr prior to fixing and staining the cells.
[0059] FIG. 35 provides photomicrographs showing the effects of
varying doses of JP4-039 on proliferation and growth of mouse
embryonic fibroblast ("MEP") cells prepared from Ercc1.sup.-/-
mice. JP4-039 is not toxic to cells at concentrations as high as 10
.mu.M.
[0060] FIG. 36 provides photomicrographs showing the effects of
varying doses of JP4-039 on proliferation and growth of mouse
embryonic fibroblast ("MEF") cells prepared from wild-type mice.
JP4-039 is not toxic to cells at concentrations as high as 10
.mu.M.
[0061] FIG. 37 provides photomicrographs showing levels of p16, a
marker of irreversible cellular senescence, in mouse embryonic
fibroblast ("MEF") cells prepared from Ercc1.sup.-/.DELTA. mice,
where the MEF cells were either treated with either JP4-039 ("0-39"
in this figure; 10 .mu.M dissolved in media) or media alone for 48
hrs prior to fixing and immunostaining the cells.
[0062] FIG. 38 provides photomicrographs showing cell proliferation
of primary mouse embryonic fibroblast ("MEF") cells prepared from
Ercc1.sup.-/- mice and grown in conditions of oxidative stress (20%
oxygen), where the MEF cells were either treated with either
JED-E71-37, JED-E71-58 91 uM dissolved in media), or media alone
for a duration of 48 hrs prior to fixing and staining the
cells.
[0063] FIG. 39 provides photomicrographs showing .gamma.H2AX
immunostaining (a marker of DNA double strand breaks and cellular
senescence) of mouse embryonic fibroblast ("MEF") cells prepared
from Ercc1.sup.--.DELTA. mice and grown in conditions of oxidative
stress (20% oxygen), where the MEF cells were either treated with
either JED-E71-58 (1 .mu.M dissolved in media, or media alone for a
duration of 48 hrs prior to fixing and staining the cells.
[0064] FIG. 40 is a schematic showing alternative designs of
nitroxide analogues.
[0065] FIG. 41 is a schematic of a synthesis protocol for various
alternative designs of nitroxide analogues.
[0066] FIG. 42 is a schematic of a synthesis protocol for an
alternative nitroxide moiety of
1,133-tetramethylisoindolin-2-yloxyl (TMIO).
[0067] FIG. 43 is a schematic of a synthesis protocol for an
alternative nitroxide moiety of 1-methyl 2-azaadamantane N-oxyl
(1-Me-AZADO).
DETAILED DESCRIPTION
[0068] As used herein, the term "subject" refers to members of the
animal kingdom including but not limited to human beings. The term
"reactive oxygen species" ("ROS") includes, but is not limited to,
superoxide anion, hydroxyl, and hydroperoxide radicals.
[0069] An antioxidant compound is defined herein as a compound that
decreases the rate of oxidation of other compounds or prevents a
substance from reacting with oxygen or oxygen containing compounds.
A compound may be determined to be an antioxidant compound by
assessing its ability to decrease molecular oxidation and/or
cellular sequellae of oxidative stress, for example, and without
limitation, the ability to decrease lipid peroxidation and/or
decrease oxidative damage to protein or nucleic acid. In one
embodiment, an antioxidant has a level of antioxidant activity
between 0.01 and 1000 times the antioxidant activity of ascorbic
acid in at least one assay that measures antioxidant activity.
[0070] Provided herein are compounds and compositions comprising a
targeting group and a cargo, such as a nitroxide-containing group.
The cargo may be any useful compound, such as an antioxidant, as
are well known in the medical and chemical arts. The cargo may
comprise a factor having anti-microbial activity. For example, the
targeting groups may be cross-linked to antibacterial enzymes, such
as lysozyme, or antibiotics, such as penicillin. Other methods for
attaching the targeting groups to a cargo are well known in the
art. In one embodiment, the cargo is an antioxidant, such as a
nitroxide-containing group. In another embodiment, the cargo
transported by mitochondria-selective targeting agents may include
an inhibitor of NOS activity. The cargo may have a property
selected from the group consisting of antioxidant, radioprotective,
protective, anti-apoptotic, therapeutic, ameliorative, NOS
antagonist and combinations thereof. In another embodiment, the
cargo may have the ability to inhibit nitric oxide synthase enzyme
activity. It will be appreciated that a wide variety of cargos may
be employed in the composition described herein. Non-limiting
examples of cargos include: a 2-amino-6-methyl-thiazine, a
ubiquinone analog, a ubiquinone analog fragment moiety, a
ubiquinone analog fragment moiety lacking a hydrophilic tail, a
superoxide dismutase mimetic, a superoxide dismutase biomimetic and
a salen-manganese compound.
[0071] While the generation of ROS in small amounts is a typical
byproduct of the cellular respiration pathway, certain conditions,
including a disease or other medical condition, may occur in the
patient when the amount of ROS is excessive to the point where
natural enzyme mechanisms cannot scavenge the amount of ROS being
produced. Therefore, compounds, compositions and methods that
scavenge reactive oxygen species that are present within the
mitochondrial membrane of the cell are useful and are provided
herein. These compounds, compositions and methods have the utility
of being able to scavenge an excess amount of ROS being produced
that naturally occurring enzymes SOD and catalase, among others,
cannot cope with.
[0072] In one non-limiting embodiment, the compound has the
structure:
##STR00001##
wherein X is one of
##STR00002##
and R.sub.1, R.sub.2 and R.sub.4 are, independently, hydrogen,
C.sub.1-C.sub.6 straight or branched-chain alkyl, optionally
including a phenyl (C.sub.6H.sub.5) group, that optionally is
methyl-, hydroxyl- or fluoro-substituted, including: methyl, ethyl,
propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl,
hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl and hydroxyphenyl.
R.sub.3 is --NH--R.sub.5, --O--R.sub.5 or --CH.sub.2--R.sub.5,
where R.sub.5 is an --N--O., --N--OH or N.dbd.O containing group.
In one embodiment, R.sub.3 is
##STR00003##
(I-Me-AZADO or 1-methyl 2-azaadamantane N-oxyl). In another
embodiment R.sub.3 is
##STR00004##
(TMIO; 1,1,3,3-tetramethylisoindolin-2-yloxyl). R is
--C(O)--R.sub.6, --C(O)O--R.sub.6, or --P(O)--(R.sub.6).sub.2,
wherein R.sub.6 is C.sub.1-C.sub.6 straight or branched-chain alkyl
optionally comprising one or more phenyl (--C.sub.6H.sub.5) groups,
and that optionally are methyl-, ethyl-, hydroxyl- or
fluoro-substituted, including Ac (Acetyl, R.dbd.--C(O)--CH.sub.3),
Boc (R.dbd.--C(O)O-tert-butyl), Cbz (R.dbd.--C(O)O-benzyl (Bn))
groups. R also may be a diphenylphosphate group, that is,
##STR00005##
Excluded from this is the enantiomer XJB-5-208. In certain
embodiments, R.sub.1 is t-butyl and R.sub.2 and R.sub.4 are H; for
instance:
##STR00006##
[0073] As used herein, unless indicated otherwise, for instance in
a structure, all compounds and/or structures described herein
comprise all possible stereoisomers, individually or mixtures
thereof.
[0074] As indicated above, R.sub.5 is an --N--O., --N--OH or
--N.dbd.O containing group (not --N--O., --N--OH or --N.dbd.O
alone, but groups containing those moieties, such as TEMPO, etc, as
described herein). As is known to one ordinarily skilled in the
art, nitroxide and nitroxide derivatives, including TEMPOL and
associated TEMPO derivatives are stable radicals that can withstand
biological environments. Therefore, the presence of the
4-amino-TEMPO, TEMPOL or another nitroxide "payload" within the
mitochondria membrane can serve as an effective and efficient
electron scavenger of the ROS being produced within the membrane.
Non-limiting examples of this include TEMPO
(2,2,6,6-Tetramethyl-4-piperidine 1-oxyl) and TEMPOL
(4-Hydroxy-TEMPO), in which, when incorporated into the compound
described herein, for example, when R.sub.3 is --NH--R.sub.5,
--O--R.sub.5:
##STR00007##
[0075] Additional non-limiting examples of --N--O.RTM., --N--OH or
N.dbd.O containing group are provided in Table 1 and in FIG. 1
(from Jiang, J., et al. "Structural Requirements for Optimized
Delivery, Inhibition of Oxidative Stress, and Antiapoptotic
Activity of Targeted Nitroxides", J Pharmacol Exp Therap. 2007,
320(3):1050-60). A person of ordinary skill in the art would be
able to conjugate (covalently attach) any of these compounds to the
rest of the compound using common linkers and/or conjugation
chemistries, such as the chemistries described herein. Table 1
provides a non-limiting excerpt from a list of over 300 identified
commercially-available --N--O., --N--OH or N.dbd.O containing
compounds that may be useful in preparation of the compounds or
compositions described herein.
TABLE-US-00001 TABLE 1 Commercially-available --N--O.cndot.,
--N--OH or N.dbd.O containing groups Structure Name CAS No.
##STR00008## Trimethylamine N-Oxide 1184-78-7 ##STR00009##
N,N-Dimethyldodecylamine N-Oxide 1643-20- 570592-80-2 ##STR00010##
N-Benzoyl-N- Phenylhydroxylamine 304-88-1 ##STR00011##
N,N-Diethylhydroxylamine 3710-84-7 ##STR00012## N,N-
Dibenylhydroxylamine 14165-27- 6621-07-8 ##STR00013## Di-Tert-Butyl
Nitroxide 2406-25-9 ##STR00014## N,N- Dimethylhydroxylamine
Hydrochloride 16645-06-0 ##STR00015## Metobromuron 3060-89-7
##STR00016## Benzyl-Di-Beta-Hydroxy Ethylamine-N-Oxide ##STR00017##
Bis(Trifluoromethyl)Nitroxide 2154-71-4 ##STR00018## Triethylamine
N-Oxide 2687-45-8 ##STR00019## ##STR00020## N-Methoxy-N-
Methylcarbamate 6919-62-6 ##STR00021## N,N-Bis(2-Chloro-6-
Fluorobenzyl)-N-[(([2,2- Dichloro-1-(1,4-Thiazinan-
4-yl+)ethylidene] amino)carbonyl)oxy]amine ##STR00022##
Tri-N-Octylamine N-Oxide 13103-04-3 ##STR00023## Diethyl
(N-Methoxy-N- Methylcarbamoylmethyl)Phos- phonate 124931-12- 0
##STR00024## N-Methoxy-N-Methyl-2- (Triphenylphosphoranylidene)
Acetamide 129986-67- 0 ##STR00025## N-Methoxy-N-Methyl-N'-
[5-Oxo-2- (Trifluoromethyl)-5h- Chromeno[2,3-B]Pyridi+ N-3-yl]Urea
##STR00026## N-[(4-Chlorobenzyl)Oxy]- N-([5-Oxo-2-Phenyl-1,3-
Oxazol-4(5h)- Yliden]Methyl+N Acetamide ##STR00027##
N-Methylfurohydroxamic Acid 109531-96- 6 ##STR00028##
N,N-Dimethylnonylamine N-Oxide 2536-13-2 ##STR00029##
N-(Tert-Butoxycarbonyl)-L- Alanine N'-Methoxy-N'- Methylamide
87694-49-3 ##STR00030## 1-(4-Bromophenyl)-3- (Methyl([3-
(Trifluoromethyl)Benzoyl] Oxy)Amino)-2-Prop+ En-1- One ##STR00031##
2- ([[(Anilinocarbonyl)Oxy](Meth- yl)Amino]Methylene)-
5-(4-Chlorophenyl)-1,3+- Cyclohexanedione ##STR00032##
N-Methoxy-N-Methyl-2- (Trifluoromethyl)-1,8- Naphthyridine-3-
Carboxamide ##STR00033## N-Methoxy-N-Methyl- Indole-6-Carboxamide
##STR00034## ##STR00035## AKOS 91254 127408-31- 5 ##STR00036##
N-[(3s,4r)-6-Cyano-3,4- Dihydro-3-Hydroxy-2,2-
Dimethyl-2h-1-Benzopyran- 4-Y+ L]-N-Hydroxyacetamide 127408-31- 5
##STR00037## N-Methoxy-N-Methyl-1,2- Dihydro-4-Oxo-
Pyrrolo[3,2,1-1j]Quinoline- 5-Carboxa+ Mide ##STR00038## Fr-900098
##STR00039## 2,2'-(Hydroxyimino)Bis- Ethanesulfonic Acid Disodium
Salt 133986-51- 3 ##STR00040## Fmoc-N-Ethyl- Hydroxylamine
##STR00041## Bis(N,N- Dimethylhydroxamido)Hydrox- ooxovanadate
##STR00042## Pyraclostrobin 175013-18- 0 ##STR00043##
1-Boc-5-Chloro-3- (Methoxy-Methyl- Carbamoyl)Indazole ##STR00044##
N-Methoxy-N-Methyl- Thiazole-2-Carboxamide ##STR00045##
4,4-Difluoro-N-Methyl-N- Methoxy-L-Prolinamide Hcl ##STR00046##
3-Fluoro-4- (Methoxy(Methyl)Carbamoyl) Phenylboronic Acid
913835-59- 3 ##STR00047## 1-Isopropyl-N-Methoxy-N- Methyl-1h-
Benzo[D][1,2,3]Triazole-6- Carboxamide 467235-06- 9 ##STR00048##
(Trans)-2-(4-Chlorophenyl)- N-Methoxy-N- Methylcyclopropanecarboxa-
mide ##STR00049## Bicyclo[2.2.1]Heptane-2- Carboxylic Acid Methoxy-
Methyl-Amide ##STR00050## Akos Bc-0582 ##STR00051## 3-(N,O-
Dimethylhydroxylaminocarbo- nyl)Phenylboronic Acid, Pinacol Ester
##STR00052## 1-Triisopropylsilanyl-1h- Pyrrolo[2,3-B]Pyridine-5-
Carboxylic Acid Methoxy+- Methyl-Amide
[0076] According to one embodiment, the compound has the
structure
##STR00053##
or the structure
##STR00054##
wherein R is --NH--R.sub.1, --O--R.sub.1 or --CH.sub.2--R.sub.1,
and R.sub.1 is an --N--O., --N--OH or N.dbd.O) containing group. In
one embodiment, R is --NH--R.sub.1, and in another R is
--NH-TEMPO.
[0077] According to another embodiment, the compound has the
structure:
##STR00055##
in which R1, R2 and R3 are, independently, hydrogen,
C.sub.1-C.sub.6 straight or branched-chain alkyl, optionally
including a phenyl (C.sub.6H.sub.5) group, that optionally is
methyl-, hydroxyl- or fluoro-substituted, including 2-methyl
propyl, benzyl, methyl-, hydroxyl- or fluoro-substituted benzyl,
such as 4-hydroxybenzyl. R4 is an --N--O., --N--OH or N.dbd.O
containing group. In one embodiment, R4 is
##STR00056##
(1-Me-AZADO or 1-methyl 2-azaadamantane N-oxyl). In another
embodiment R4 is
##STR00057##
(TMIO; 1,1,3,3-tetramethylisoindolin-2-yloxyl). R is --C(O)--R5,
--C(O)O--R5, or --P(O)--(R5).sub.2, wherein R5 is C.sub.1-C.sub.6
straight or branched-chain alkyl, optionally comprising one or more
phenyl (--C.sub.6H.sub.5) groups, and that optionally are methyl-,
ethyl-, hydroxyl- or fluoro-substituted, including Ac, Boc, and Cbz
groups. R also may be a diphenylphosphate group, that is
##STR00058##
[0078] In certain specific embodiments, in which R4 is TEMPO, the
compound has one of the structures A, A1, A2, or A3
(Ac=Acetyl=CH.sub.3C(O)--):
##STR00059## [0079] According to another embodiment, the compound
has the structure
##STR00060##
[0079] In which R1, R2 and R3 are, independently, hydrogen,
C.sub.1-C.sub.6 straight or branched-chain alkyl, optionally
including a phenyl (C.sub.6H.sub.5) group, that optionally is
methyl-, hydroxyl- or fluoro-substituted, including 2-methyl
propyl, benzyl, methyl-, hydroxyl- or fluoro-substituted benzyl,
such as 4-hydroxybenzyl. R4 is an --N--O., --N--OH or N.dbd.O
containing group. In one embodiment, R4 is
##STR00061##
(1-Me-AZADO or 1-methyl 2-azaadamantane N-oxyl). In another
embodiment R4 is
##STR00062##
(TMIO; 1,1,3,3-tetramethylisoindolin-2-yloxyl). R is --C(O)--R5,
--C(O)O--R5, or --P(O)--(R5).sub.2, wherein R5 is C.sub.1-C.sub.6
straight or branched-chain alkyl, optionally comprising one or more
phenyl (--C.sub.6H.sub.5) groups, and that optionally are methyl-,
ethyl-, hydroxyl- or fluoro-substituted, including Ac, Boc, and Cbz
groups. R also may be a diphenylphosphate group, that is, R=
##STR00063##
In certain specific embodiments, in which R4 is TEMPO, the compound
has one of the structures D, D1, D2, or D3
(Ac=Acetyl=CH.sub.3C(O)--):
##STR00064##
[0080] In another non-limiting embodiment, the compound has the
structure:
##STR00065##
wherein X is one of
##STR00066##
L and
[0081] R.sub.1 and R.sub.4 are, independently, hydrogen,
C.sub.1-C.sub.6 straight or branched-chain alkyl, optionally
including a phenyl (C.sub.6H.sub.5) group, that optionally is
methyl-, hydroxyl- or fluoro-substituted, including: methyl, ethyl,
propyl, 2-propyl, butyl, l-butyl, pentyl, hexyl, benzyl,
hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl and hydroxyphenyl.
R.sub.3 is --NH--R.sub.5, --O--R.sub.5 or --CH.sub.2--R.sub.5,
where R.sub.5 is an --N--O., --N--OH or N.dbd.O containing group.
In one embodiment, R.sub.3 is
##STR00067##
(1-Me-AZADO or 1-methyl azaadamantane N-oxyl). In another
embodiment R.sub.3 is
##STR00068##
(TMIO; 1,1,3,3-tetramethylisoindolin-2-yloxyl). R is
--C(O)--R.sub.6, --C(O)O--R.sub.6, or --P(O)--(R.sub.6).sub.2,
wherein R.sub.6 is C.sub.1-C.sub.6 straight or branched-chain alkyl
optionally comprising one or more phenyl (--C.sub.6H.sub.5) groups,
and that optionally are methyl-, ethyl-, hydroxyl- or
fluoro-substituted, including Ac (Acetyl, R.dbd.--C(O)--CH.sub.3),
Boc (R.dbd.--C(O)O-tert-butyl), Cbz (R.dbd.--C(O)O-benzyl (Bn))
groups. R also may be a diphenylphosphate group, that is.
##STR00069##
[0082] In one non-limiting embodiment, the compound has one of the
structures
##STR00070##
[0083] In yet another non-limiting embodiment, the compound has the
structure
##STR00071##
in which R.sub.4 is hydrogen or methyl.
[0084] The compounds described above, such as the compound of
Formula 1, can be synthesized by any useful method. The compound
JP4-039 was synthesized by the method of Example 8. In one
embodiment, a method of making a compound of Formula 1 is provided.
The compounds are synthesized by the following steps:
reacting an aldehyde of structure R.sub.1--C(O)--, wherein, for
example and without limitation, R.sub.1 is C.sub.1-C.sub.6 straight
or branched-chain alkyl, optionally including a phenyl
(C.sub.6H.sub.5) group, that optionally is methyl-, hydroxyl- or
fluoro-substituted, including: methyl, ethyl, propyl, 2-propyl,
butyl, t-butyl, pentyl, hexyl, benzyl, hydroxybenzyl (e.g.,
4-hydroxybenzyl), phenyl and hydroxyphenyl, with
(R)-2-methylpropane-2-sulfinamide to form an inline, for
example
##STR00072##
reacting a terminal alkyne-1-ol (HCC--R.sub.2--CH.sub.2--OH),
wherein, for example and without limitation, R.sub.2 is not present
or is branched or straight-chained alkylene, including methyl,
ethyl, propyl, etc., with a tert-butyl diphenylsilane salt to
produce an alkyne, for example
##STR00073##
reacting (by hydrozirconation) the alkyne with the imine in the
presence of an organozirconium catalyst to produce an alkene, for
example
##STR00074##
[0085] D1. acylating the alkene to produce a carbamate, for
example
##STR00075##
wherein, for example and without limitation, R.sub.1 is
C.sub.1-C.sub.6 straight or branched-chain alkyl, optionally
including a phenyl (C.sub.6H.sub.5) group, that optionally is
methyl-, hydroxyl- or fluoro-substituted, including including:
methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl,
benzyl, hydroxybenzyl (e.g., 4-hydroxy benzyl), phenyl and
hydroxyphenyl;
[0086] D2. optionally, cyclopropanating the alkene and then
acylating the alkene to produce a carbamate, for example
##STR00076##
wherein, for example and without limitation, R.sub.3 is
C.sub.1-C.sub.6 straight or branched-chain alkyl, optionally
including a phenyl (C.sub.6H.sub.5) group, that optionally is
methyl-, hydroxyl- or fluoro-substituted, including including:
methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl,
benzyl, hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl and
hydroxyphenyl; removing the t-butyldiphenylsilyl group from the
carbamate to produce an alcohol, for example
##STR00077##
oxidizing the alcohol to produce a carboxylic acid, for example
##STR00078##
and reacting the carboxylic acid with a nitroxide-containing
compound comprising one of a hydroxyl or amine in a condensation
reaction to produce the antioxidant compound, for example
##STR00079##
wherein R.sub.4 is --NH--R.sub.4 or --O--R.sub.4, and R, is an
--N--O., --N--OH or N.dbd.O containing group, such as described
above.
[0087] In another non-limiting embodiment, a compound is provided
having the structure R1-R2-R3 in which R1 and R3 are a group having
the structure --R4-R5, in which R4 is a mitochondria targeting
group and R5 is --NH--R6, --O--R6 or --CH.sub.2--R6, wherein R6 is
an --N--O., --N--OH or N.dbd.O containing group, such as TEMPO. R1
and R2 may be the same or different. Likewise, R4 and R5 for each
of R1 and R3 may be the same or different. R2 is a linker that, in
one non-limiting embodiment, is symmetrical. FIGS. 26A and 26B
depicts two examples of such compounds. In one embodiment, R1 and
R2 have the structure shown in formulas 1, 2, or 3, above, with all
groups as defined above, including structures A, A1, A2, A3, D, D1,
D2 and D3, above, an example of which is compound JED-E71-58, shown
in FIG. 26B. In another embodiment, R1 and R2 are, independently, a
gramicidin derivative, for example, as in the compound JED-E71-37,
shown in FIG. 26A. Examples of gramicidin derivatives are provided
herein, such as XJB-5-131 and XJB-5-125 (see. FIG. 2), and are
further described both structurally and functionally in United
States Patent Publication Nos. 20070161573 and 20070161544 as well
as in Jiang, J, et al. (Structural Requirements for Optimized
Delivery, Inhibition of Oxidative Stress, and Antiapoptotic
Activity of Targeted Nitroxides, J Pharmacol Exp Therap. 2007,
320(3):1050-60, see also, Hoye, A T et al., Targeting Mitochondria,
Acc Chem Res. 2008, 41(1):87-97, see also, Wipf, P, et al.,
Mitochondrial Targeting of Selective Electron Scavengers: Synthesis
and Biological Analysis of Hemigramicidin-TEMPO Conjugates, (2005)
J Am Chem Soc. 2005, 127:12460-12461). The XJB compounds can be
linked into a dimer, for example and without limitation, by
reaction with the nitrogen of the BocHN groups (e.g., as in
XJB-5-131), or with an amine, if present, for instance, if one or
more amine groups of the compound is not acylated to form an amide
(such as NHBoc or NHCbx).
[0088] In Jiang, J, et al. (J Pharmacol Exp Therap. 2007,
320(3):1050-60), using a model of ActD-induced apoptosis in mouse
embryonic cells, the authors screened a library of nitroxides to
explore structure-activity relationships between their
antioxidant/antiapoptotic properties and chemical composition and
three-dimensional (3D) structure. High hydrophobicity and effective
mitochondrial integration were deemed necessary but not sufficient
for high antiapoptotic/antioxidant activity of a nitroxide
conjugate. By designing conformationally preorganized peptidyl
nitroxide conjugates and characterizing their 3D structure
experimentally (circular dichroism and NMR) and theoretically
(molecular dynamics), they established that the presence of the
.beta.-turn/.beta.-sheet secondary structure is essential for the
desired activity. Monte Carlo simulations in model lipid membranes
confirmed that the conservation of the D-Phe-Pto reverse turn in
hemi-GS analogs ensures the specific positioning of the nitroxide
moiety at the mitochondrial membrane interface and maximizes their
protective effects. These insights into the structure-activity
relationships of nitroxide-peptide and -peptide isostere conjugates
are helpful in the development of new mechanism-based
therapeutically effective agents, such as those described
herein.
[0089] Targeting group R4 may be a membrane active peptide fragment
derived from an antibiotic molecule that acts by targeting the
bacterial cell wall. Examples of such antibiotics include:
bacitracins, gramicidins, valinomycins, enniatins, alamethicins,
beauvericin, serratomolide, sporidesmolide, tyrocidins, polymyxins,
monamycins, and lissoclinum peptides. The membrane-active peptide
fragment derived from an antibiotic may include the complete
antibiotic polypeptide, or portions thereof having membrane, and
preferably mitochondria-targeting abilities, which is readily
determined, for example, by cellular partitioning experiments using
radiolabeled peptides Examples of useful gramicidin-derived
membrane active peptide fragments are the Leu-D-Phe-Pro-Val-Orn and
D-Phe-Pro-Val-Orn-Leu hemigramicidin fragments. As gramicidin is
cyclic, any hemigramicidin 5-mer is expected to be useful as a
membrane active peptide fragment, including Leu-D-Phe-Pro-Val-Orn,
D-Phe-Pro-Val-Orn-Leu, Pro-Val-Orn-Leu-D-Phe, Val-Orn-Leu-D-Phe-Pro
and Orn-Leu-D-Phe-Pro-Val (from Gramicidin S). Any larger or
smaller fragment of gramicidin, or even larger fragments containing
repeated gramicidin sequences (e.g.,
Leu-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro) are
expected to be useful for membrane targeting, and can readily
tested for such activity, in one embodiment, the Gramicidin
S-derived peptide comprises a .beta.-turn, which appears to confer
to the peptide a high affinity for mitochondria. Derivatives of
Gramicidin, or other antibiotic fragments, include isosteres
(molecules or ions with the same number of atoms and the same
number of valence electrons--as a result, they can exhibit similar
pharmacokinetic and pharmacodynamic properties), such as (E)-alkene
isosteres (see. United Stales Patent Publication Nos. 20070161573
and 20070161544 for exemplary synthesis methods). As with
Gramicidin, the structure (amino acid sequence) of bacitracins,
oilier gramicidins, valinomycins, enniatins, alamethicins,
beauvericin, serratomolide, sporidesmolide, tyrocidins, polymyxins,
monamycins, and lissoclinum peptides are all known, and fragments
of these can be readily prepared and their membrane-targeting
abilities can easily be confirmed by a person of ordinary skill in
the art.
[0090] Alkene isosteres such as (E)-alkene isosteres of Gramicidin
S (i.e., hemigramicidin) were used as part of the targeting
sequence. See FIG. 3 for a synthetic pathway for (E)-alkene
isosteres and reference number 2 for the corresponding chemical
structure. First, hydrozirconation of alkyne (FIG. 3, compound 1)
with Cp.sub.2ZrHCl is followed by transmetalation to Me.sub.2Zn and
the addition of N-Boc-isovaleraldimine. The resulting compound (not
shown) was then worked up using a solution of tetrabutylammonium
fluoride ("TBAF") and diethyl ether with a 74% yield. The resulting
compound was then treated with acetic anhydride, triethylamine
(TEA), and 4-N,N1-(dimethylamino) pyridine ("DMAP") to provide a
mixture of diastereomeric allylic amides with a 94% yield which was
separated by chromatography. Finally, the product was worked up
with K.sub.2CO.sub.3 in methanol to yield the (E)-alkene, depicted
as compound 2. The (E)-alkene, depicted as compound 2 of FIG. 3,
was then oxidized in a multi-step process to yield the compound 3
(FIG. 3)--an example of the (E)-alkene isostere.
[0091] The compound 3 of FIG. 3 was then conjugated with the
peptide H-Pro-Val-Orn (Cbz)-OMe using
1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride) (EDC)
as a coupling agent. The peptide is an example of a suitable
targeting sequence having affinity for the mitochondria of a cell.
The resulting product is shown as compound 4a in FIG. 3.
Saponification of compound 4a followed by coupling with
4-amino-TEMPO (4-AT) afforded the resulting conjugate shown as
compound 5a in FIG. 3, in which the Leu-DPhe peptide bond has been
replaced with an (E)-alkene.
[0092] In an alternate embodiment, conjugates 5 b in FIG. 3 was
prepared by saponification and coupling of the peptide 4b
(Boc-Leu-DPhe-Pro-Val-Orn(Cbz)-OMe) with 4-AT. Similarly, conjugate
5c in FIG. 3 was prepared by coupling the (E)-alkene isostere as
indicated as compound 3 in FIG. 3 with 4-AT. These peptide and
peptide analogs are additional examples of suitable targeting
sequences having an affinity to the mitochondria of a cell.
[0093] In another embodiment, peptide isosteres may be employed as
the conjugate. Among the suitable peptide isosteres are
trisubstituted (E)-alkene peptide isosteres and cyclopropane
peptide isosteres, as well as all imine addition products of hydro-
or carbometalated internal and terminal alkynes for the synthesis
of d-i and trisubstituted (E)-alkene and cyclopropane peptide
isosteres. See Wipf et al. Imine additions of internal alkynes for
the synthesis of trisubstituted (E)-alkene and cyclopropane
isosteres. Adv Synth Catal. 2005, 347:1605-1613. These peptide
mimetics have been found to act as .beta.-turn promoters. See Wipf
et al. Convergent Approach to (E)-Alkene and Cyclopropane Peptide
Isosteres, Org Lett. 2005, 7(1):103-106.
[0094] The linker, R2, may be any useful linker, chosen for its
active groups, e.g., carboxyl, alkoxyl, amino, sulfhydryl, amide,
etc. Typically, aside from the active groups, the remainder is
non-reactive (such as saturated alkyl or phenyl), and does not
interfere, sterically or by any other physical or chemical
attribute, such as polarity or hydrophobicity/hydrophilicity, in a
negative (loss of function) capacity with the activity of the
overall compound. In one embodiment, aside from the active groups,
the linker comprises a linear or branched saturated
C.sub.4-C.sub.20 alkyl. In one embodiment, the linker. R2 has the
structure
##STR00080##
[0095] In which n is 4-18, including all integers therebetween, in
one embodiment, 8-12, and in another embodiment, 10.
[0096] A person skilled in the organic synthesis arts can
synthesize these compounds by crosslinking groups R1 and R3 by any
of the many chemistries available. In one embodiment, R1 and R3 are
to R2 by an amide linkage (peptide bond) formed by dehydration
synthesis (condensation) of terminal carboxyl groups on the linker
and an amine on R1 and R3 (or vice versa). In one embodiment, R1
and R3 are identical or different and are selected from the group
consisting of: XJB-5-131, XJB-5-125, XJB-7-75, XJB-2-70, XJB-2-300,
XJB-5-208, XJB-5-197, XJB-5-194, JP4-039 and JP4-049, attached in
the manner shown in FIGS. 26A and 26B.
[0097] In a therapeutic embodiment, a method of scavenging
free-radicals in a subject (e.g., a patient in need of treatment
with a free-radical scavenger) is provided, comprising
administering to the subject an amount of one or more compound
described herein and having a free-radical scavenging group, such
as a nitroxide-containing group effective to scavenge free
radicals. As described above, a number of diseases, conditions or
in juries can be ameliorated or otherwise treated or prevented by
administration of free radical scavenging compounds, such as those
described herein.
[0098] In any case, as used herein, any agent or agents used for
prevention, mitigation or treatment in a subject of injury caused
by radiation exposure is administered in an amount effective to
prevent, mitigate of treat such injury, namely in an amount and in
a dosage regimen effective to prevent injury or to reduce the
duration and/or severity of the injury resulting from radiation
exposure. According to one non-limiting embodiment, an effective
dose ranges from 0.1 or 1 mg/kg to 100 mg/kg, including any
increment or range therebetween, including 1 mg/kg, 5 mg/kg, 10
mg/kg, 20 mg/kg, 25 mg/kg, 50 mg/kg, and 75 mg/kg. However, for
each compound described herein, an effective dose or dose range is
expected to vary from that of other compounds described herein for
any number of reasons, including the molecular weight of the
compound, bioavailability, specific activity, etc. For example and
without limitation, where XJB-5-131 is the antioxidant, the dose
may be between about 0.1 and 20 mg/kg, or between about 0.3 and 10
mg/kg, or between about 2 and 8 mg/kg, or about 2 mg/kg and where
either JP4-039, JED-E71-37 or JED-E71-58 is the antioxidant, the
dose may be between about 0.01 and 50 mg/kg, or between about 0.1
and 20 mg/kg, or between about 0.3 and 10 mg/kg, or between about 2
and 8 mg/kg, or about 2 mg/kg. The therapeutic window between the
minimally-effective dose, and maximum tolerable dose in a subject
can be determined empirically by a person of skill in the art, with
end points being determinable by in vitro and in vivo assays, such
as those described herein and/or are acceptable in the
pharmaceutical and medical arts for obtaining such information
regarding radioprotective agents. Different concentrations of the
agents described herein are expected to achieve similar results,
with the drug product administered, for example and without
limitation, once prior to an expected radiation dose, such as prior
to radiation therapy or diagnostic exposure to ionizing radiation,
during exposure to radiation, or after exposure in any effective
dosage regimen. The compounds can be administered continuously,
such as intravenously, one or more times daily, once every two,
three, four, five or more days, weekly, monthly, etc., including
increments therebetween. A person of ordinary skill in the
pharmaceutical and medical arts will appreciate that it will be a
matter of simple design choice and optimization to identify a
suitable dosage regimen for prevention, mitigation or treatment of
injury due to exposure to radiation.
[0099] The compounds described herein also are useful in
preventing, mitigating (to make less severe) and/or treating injury
caused by radiation exposure. By radiation, in the context of this
disclosure, it is meant types of radiation that result in the
generation of free radicals, e.g., reactive oxygen species (ROS),
as described herein. The free radicals are produced, for example
and without limitation, by direct action of the radiation, as a
physiological response to the radiation and/or as a consequence of
damage/injury caused by the radiation. In one embodiment the
radiation is ionizing radiation. Ionizing radiation consists of
highly-energetic particles or waves that can detach (ionize) at
least one electron from an atom or molecule. Examples of ionizing
radiation are energetic beta particles, neutrons, and alpha
particles. The ability of light waves (photons) to ionize an atom
or molecule varies across the electromagnetic spectrum. X-rays and
gamma rays can ionize almost any molecule or atom; far ultraviolet
light can ionize many atoms and molecules; near ultraviolet and
visible light are ionizing to very few molecules. Microwaves and
radio waves typically are considered to be non-ionizing radiation,
though damage caused by, e.g., microwaves, may result in the
production of free-radicals as part of the injury and/or
physiological response to the injury.
[0100] The compounds typically are administered in an amount and
dosage regimen to prevent, mitigate or treat the effects of
exposure of a subject to radiation. The compounds may be
administered in any manner that is effective to treat, mitigate or
prevent damage caused by the radiation. Examples of delivery routes
include, without limitation: topical, for example, epicutaneous,
inhalational, enema, ocular, otic and intranasal delivery; enteral,
for example, orally, by gastric feeding tube and rectally; and
parenteral, such as, intravenous, intraarterial, intramuscular,
intracardiac, subcutaneous, intraosseous, intradermal, intrathecal,
intraperitoneal, transdermal, iontophoretic, transmucosal, epidural
and intravitreal, with oral, intravenous, intramuscular and
transdermal approaches being preferred in many instances.
[0101] The compounds may be compounded or otherwise manufactured
into a suitable composition for use, such as a pharmaceutical
dosage form or drug product in which the compound is an active
ingredient. Compositions may comprise a pharmaceutically acceptable
carrier, or excipient. An excipient is an inactive substance used
as a carrier for the active ingredients of a medication. Although
"inactive," excipients may facilitate and aid in increasing the
delivery or bioavailability of an active ingredient in a drug
product. Non-limiting examples of useful excipients include:
antiadherents, binders, rheology modifiers, coatings,
disintegrants, emulsifiers, oils, buffers, salts, acids, bases,
fillers, diluents, solvents, flavors, colorants, glidants,
lubricants, preservatives, antioxidants, sorbents, vitamins,
sweeteners, etc., as are available in the
pharmaceutical/compounding arts.
[0102] Useful dosage forms include: intravenous, intramuscular, or
intraperitoneal solutions, oral tablets or liquids, topical
ointments or creams and transdermal devices (e.g., patches). In one
embodiment, the compound is a sterile solution comprising the
active ingredient (drug, or compound), and a solvent, such as
water, saline, lactated Ringer's solution, or phosphate-buffered
saline (PBS). Additional excipients, such as polyethylene glycol,
emulsifiers, salts and buffers may be included in the solution.
[0103] In one embodiment, the dosage form is a transdermal device,
or "patch" The general structure of a transdermal patch is broadly
known in the pharmaceutical arts. A typical patch includes, without
limitation: a delivery reservoir for containing and delivering a
drug product to a subject, an occlusive backing to which the
reservoir as attached on a proximal side (toward the intended
subject's skin) of the backing and extending beyond, typically
completely surrounding the reservoir, and an adhesive on the
proximal side of the backing, surrounding the reservoir, typically
completely, for adhering the patch to the skin of a patient. The
reservoir typically comprises a matrix formed from a non-woven
(e.g., a gauze) or a hydrogel, such as a polyvinylpyrrolidone (PVP)
or polyvinyl acetate (PVA), as are broadly known. The reservoir
typically comprises the active ingredient absorbed into or adsorbed
onto the reservoir matrix, and skin permeation enhancers. The
choice of permeation enhancers typically depends on empirical
studies. As is shown in Example 12, below, certain formulations
that may be useful as permeation enhancers include, without
limitation: DMSO; 95% Propylene Glycol+5% Linoleic Acid; and 50%
EtOH+40% HSO+5% Propylene Glycol+5% Brij30.
[0104] Examples 1-7 are excerpts from United Stated patent
application Ser. No. 11/565,779, and are recited herein to provide
non-limiting illustrations of useful synthetic methods and
efficacies of certain mitochondria-targeting free-radical
scavenging compounds utilizing Gramicidin S-derived
mitochondria-targeting groups.
Example 1
[0105] Materials.
[0106] All chemicals were from Sigma-Aldrich (St Louis, Mo.) unless
otherwise noted. Heparin, ketamine HCl and sodium pentobarbital
were from Abbott Laboratories (North Chicago, Ill.). Dulbecco's
modified Eagle medium ("DMEM") was from BioWhittaker (Walkersville,
Md.). Fetal bovine serum (FBS; <0.05 endotoxin units/ml) was
from Hyclone (Logan, Utah). Pyrogen-free sterile normal saline
solution was from Baxter (Deerfield, Ill.).
[0107] General.
[0108] All moisture-sensitive reactions were performed using
syringe-septum cap techniques under an N2 atmosphere and all
glassware was dried in an oven at 150.degree. C. for 2 h prior to
use. Reactions carried out at -78.degree. C. employed a
CO.sub.2-acetone bath. Tetrahydrofuran (THF) was distilled over
sodium/benzophenone ketyl; CH.sub.2Cl.sub.2 (DCM), toluene and
Et.sub.3N were distilled from CaH.sub.2. Me.sub.2Zn was purchased
from Aldrich Company.
[0109] Reactions were monitored by thin layer chromatography
("TLC") analysis (EM Science pre-coated silica gel 60 F254 plates,
250 .mu.m layer thickness) and visualization was accomplished with
a 254 nm UV light and by staining with a Vaughn's reagent (4.8 g
(NH.sub.4).sub.6Mo7O.sub.24 4H.sub.2O, 0.2 g
Ce(SO.sub.4).sub.2.4H.sub.2O in 10 mL cone. H.sub.2SO.sub.4 and 90
mL H.sub.2O). Flash chromatography on SiO.sub.2 was used to purify
the crude reaction mixtures.
[0110] Melting points were determined using a Laboratory Devices
MeI-Temp II. Infrared spectra were determined on a Nicolet Avatar
360 FT-IR spectrometer. Mass spectra were obtained on a Waters
Autospec double focusing mass spectrometer ("EI") or a Waters Q-Tof
mass spectrometer ("ESI"). LC-MS data were obtained on an Agilent
1100 instrument, using a Waters Xterra MS CH 3.5 .mu.m RP column
(4.6.times.100 mm).
Synthesis Example 1
[0111] Prepared as a colorless oil (FIG. 3, compound 1) according
to the literature procedure, see Edmonds, M. K. et al. Design and
Synthesis of a Conformationally Restricted Trans Peptide Isostere
Based on the Bioactive Conformations of Saquinavir and Nelfinavir,
J Org Chem. 66:3747 (2001); see also Wipf P. et al., Org Lett.
7:103 (2005); see also Xiao, J. et al., Electrostatic versus Steric
Effects in Peptidomimicry: Synthesis and Secondary Structure
Analysis of Gramicidin S Analogues with (E)-Alkene Peptide
Isosteres, J Am Chem Soc. 127:5742 (2005).
[0112] A solution of 2.20 g (5.52 mmol) of compound 1 (FIG. 3) in
20.0 mL of dry CH.sub.2Cl.sub.2 was treated at room temperature
with 1.85 g (7.17 mmol) of Cp.sub.2ZrHCl. The reaction mixture was
stirred at room temperature for 5 min, CH.sub.2Cl.sub.2 was removed
in vacuo and 20.0 mL of toluene was added. The resulting yellow
solution was cooled to -78.degree. C. and treated over a period of
30 min with 2.76 mL (5.52 mmol) of Me-Zn (2.0 M solution in
toluene). The solution was stirred at -78.degree. C. for 30 min,
warmed to 0.degree. C. over a period of 5 min and treated in one
portion with 2.05 g (11.1 mmol) of N-Boc-isovaleraldimine, see
Edmonds, M. K. et al. J Org Chem. 66:3747 (2001); see also Wipf, P.
et al., J Org Lett. 7:103 (2005); see also Xiao et al., J Am Chem
Soc. 127:5742 (2005).
[0113] The resulting mixture was stirred at 0.degree. C. for 2 h,
quenched with saturated NH.sub.4Cl, diluted with EtOAc, filtered
through a thin pad of Celite, and extracted with EtOAc. The organic
layer was dried (MgSO.sub.4), concentrated in vacuo, and purified
by chromatography on SiO.sub.2 (20:1, hexane/EtOAc) to yield 3.13 g
(97%) as a colorless, oily 1:1 mixture of diastereomers.
[0114] A solution of 4.19 g (7.15 mmol) of product in 100 mL of dry
tetrahydrofuran (`THF`) was treated at 0.degree. C. with 9.30 ml.
(9.30 mmol) of tetrabutylammoniumfluoride (TBAF, 1.0 M solution in
THF). The reaction mixture was stirred at room temperature for 20
h. diluted with EtOAc, and washed with brine. The organic layer was
dried (MgSO.sub.4), concentrated in vacuo, and purified by
chromatography on SiO.sub.2 (4:1, hexane/EtOAc) to yield 1.89 g
(76%) as a light yellowish, foamy 1:1 mixture of diastereomers.
[0115] A solution of 1.86 g (5.23 mmol) of product in 40.0 mL of
dry CH.sub.2Cl.sub.2 was treated at 0.degree. C. with 1.46 mL (10.5
mmol) of triethylamine ("TEA"), 2.02 mL (21.4 mmol) of Ac.sub.2O,
and 63.9 mg (0.523 mmol) of 4-N,N.sup.1-(dimethylamino) pyridine
("DMAP"). The reaction mixture was stirred at 0.degree. C. for 15
min and at room temperature for 3 h, diluted with EtOAc, and washed
with brine. The organic layer was dried (MgSO.sub.4), concentrated
in vacuo, and purified by chromatography on SiO.sub.2 (20:1,
hexane/Et.sub.2O) to yield 1.97 g (94%) of acetic acid
(2S)-benzyl-(5R)-tert-butoxycarbonylamino-7-methyloct-(3E)-enyl
ester (807 mg, 38.7%), acetic acid
(2S)-benzyl-(5S)-tert-butoxycarbonylamino-7-methyloct-(3E)-enyl
ester (826 mg, 39.6%), and a mixture of the aforementioned species
(337 mg, 16.2%).
[0116] A solution of 350 mg (0.899 mmol) of acetic acid
(2S)-benzyl-(5S)-tert-butoxycarbonylamino-7-methyloct-(3E)-enyl
ester in 8.00 mL of MeOH was treated at 0.degree. C. with 62.0 mg
(0.449 mmol) of K.sub.2CO.sub.3. The reaction mixture was stirred
at 0.degree. C. for 1 h and at room temperature for an additional 4
h, diluted with EtOAc, and ashed with H.sub.2O. The organic layer
was dried (MgSO.sub.4), concentrated in vacuo, and purified by
chromatography on SiO.sub.2 (4:1, hexane/EtOAc) to yield 312 mg
(quant.) of compound 2 (FIG. 3) as a colorless oil.
[0117] A solution of 23.0 mg (66.2 .mu.mol) of compound 2 (FIG. 3)
in 2.00 mL of dry CH.sub.2Cl.sub.2 was treated at 0.degree. C. with
42.1 mg (99.3 .mu.mol) of Dess-Martin Periodinane. The reaction
mixture was stirred at 0.degree. C. for 1 h and at room temperature
for an additional 4 h, quenched with saturated
Na.sub.2S.sub.2O.sub.3 in a saturated NaHCO.sub.3 solution, stirred
for 30 min at room temperature, and extracted with
CH.sub.2Cl.sub.2. The organic layer was dried (Na.sub.2SO.sub.4),
concentrated in vacuo to give a colorless foam and subsequently
dissolved in 3.00 mL of THE, and treated at 0.degree. C. with 300
.mu.L (600 .mu.mol) of 2-methyl-2-butene (2.0 M solution in THF)
followed by another solution of 18.0 mg (199 .mu.mol) of
NaClO.sub.2 and 18.2 mg (132 .mu.mol) of NaH.sub.2PO.sub.4.H.sub.2O
in 3.00 mL of H.sub.2O. The reaction mixture was stirred at
0.degree. C. for 1 h and at room temperature for an additional 3 h,
extracted with EtOAc, and washed with H.sub.2O. The organic layer
was dried (Na.sub.2SO.sub.4) and concentrated in vacuo to yield
compound 3 (FIG. 3) as a crude colorless foam that was used for the
next step without purification.
[0118] A solution of crude compound 3 (FIG. 3) (66.2 .mu.mol) in
3.00 mL of CHCl.sub.3 was treated at 0.degree. C. with 10.7 mg
(79.2 .mu.mol) of 1-hydroxybenzotrizole ("HOBt") and 14.0 mg (73.0
.mu.mol) of 1-ethyl-3-(3-dimethylaminopropyl) carbodimide
hydrochloride ("EDC"), followed by a solution of 62.9 mg (132
.mu.mol) of H-Pro-Val-Orn(Cbz)-OMc, see Edmonds, M. K. et al. J Org
Chem. 66:3747 (2001); see also Wipf P. et al., J Org Lett. 7:103
(2005), see also Xiao, J. et al., J Am Chem Soc. 127:5742 (2005),
in 1.00 mL of CHCl.sub.3 and 0.8 mg (6.6 .mu.mol) of DMAP. The
reaction mixture was stirred at room temperature for 2 d, diluted
with CHCl.sub.3, and washed with H.sub.2O. The organic layer was
dried (Na.sub.2SO.sub.4), concentrated in vacuo, and purified by
chromatography on SiO.sub.2 (from 2:1, hexanes/EtOAc to 20:1,
CHCl.sub.3/MeOH) to yield 51.3 mg (94%) of compound 4a (FIG. 3) as
a colorless foam.
[0119] A solution of 53.7 mg (65.5 .mu.mol) of compound 4a (FIG. 3)
in 2.00 mL of MeOH was treated at 0.degree. C. with 655 .mu.L (655
.mu.mol) of 1 N NaOH. The reaction mixture was stirred at room
temperature for 6 h, and treated with 655 .mu.L (655 .mu.mol) of 1
N HCl. The solution was extracted with CHCl.sub.3 and the organic
layer was dried (Na.sub.2SO.sub.4) and concentrated in vacuo to
give the crude acid as a colorless form. This acid was dissolved in
5.00 mL of CHCl.sub.3 and treated at room temperature with 10.6 mg
(78.4 .mu.mol) of HOBt, 15.1 mg (78.8 nmol) of EDC, 20.2 mg (118
.mu.mol) of 4-amino-TEMPO and 8.0 mg (65.5 .mu.mol) of DMAP. The
reaction mixture was stirred at room temperature for 36 h, diluted
with CHCl.sub.3, and washed with H.sub.2O. The organic layer was
dried (Na.sub.2SO.sub.4), concentrated in vacuo, and purified by
chromatography on SiO.sub.2 (from 1:1, hexane/EtOAc to 20:1,
CHCl.sub.3/MeOH) to yield 62.0 mg (99%) of compound 5a (FIG. 3) as
a colorless solid. The following characterization data were
obtained: LC-MS (Rt 8.81 min, linear gradient 70% to 95% CH.sub.3CN
(H.sub.2O) in 10 min, 0.4 mL/min; m/z=959.5 [M+H].sup.+, 981.5
[M+Na].sup.+) and HRMS (ESI) m/z calculated for
C.sub.53H.sub.80N.sub.7O.sub.9Na (M+Na) 981.5915, found
981.5956.
[0120] A solution of 60.0 mg (71.7 .mu.mol) of compound 4b (FIG.
3), see Tamaki, M. et al. I. Bull Chem Soc Jpn., 66:3113 (1993), in
2.15 mL of MeOH was treated at room temperature with 717 tL (717
.mu.mol) of 1 N NaOH. The reaction mixture was stirred at room
temperature for 5 h, and treated at 0.degree. C. with 717 .mu.L
(717 .mu.mol) of 1 N HCl. The solution was extracted with
CHCl.sub.3 and the organic layer was dried (Na.sub.2SO.sub.4) and
concentrated in vacuo to give the crude acid as colorless foam. The
acid was dissolved in 6.04 mL of CHCl.sub.3 and treated at room
temperature with 11.6 mg (85.8 .mu.mol) of HOBt, 16.5 mg (85.1
.mu.mol) of EDC, 18.5 mg (108 .mu.mol) of 4-amino-TEMPO and 8.8 mg
(72.0 .mu.mol) of DMAP. The reaction mixture was stirred at room
temperature for 20 h, diluted with CHCl.sub.3, and washed with
H.sub.2O. The organic layer was dried (Na.sub.2SO.sub.4),
concentrated in vacuo, and purified by chromatography on SiO.sub.2
(from 2:1, hexane/EtOAc; to 20:1, CHCl.sub.3/MeOH) to yield 69.6 mg
(99%) of compound 5b (FIG. 3) as a yellowish solid. The following
characterization data were obtained: LC-MS (Rt 7.02 min, linear
gradient 70% to 95% CH.sub.3CN (H.sub.2O) in 10 min, 0.4 mL/min;
m/z=976.5 [M+H], 998.4 [M+Na].sup.+) and HRMS (ESI) m/z calculated
for C.sub.52H.sub.79N.sub.8O.sub.10Na (M+Na) 998.5817, found
998.5774.
[0121] A solution of crude compound 3 (FIG. 3) (40.3 .mu.mol) in
3.00 mL of CH.sub.2Cl.sub.2 was treated at 0.degree. C. with 10.4
mg (60.7 .mu.mol) of 4-amino-TEMPO, 7.7 mg (40.2 .mu.mol) of EDC,
and 5.4 mg (44.2 .mu.mol) of DMAP. The reaction mixture was stirred
at room temperature for 20 h, diluted with CHCl.sub.3, and washed
with H.sub.2O. The organic layer was dried (Na.sub.2SO.sub.4),
concentrated in vacuo, and purified by chromatography on SiO.sub.2
(from 4:1 to 1:1, hexane/EtOAc) to yield 18.8 mg (91%) of compound
5c (FIG. 3) as a yellowish solid. The following characterization
data were obtained: LC-MS (Rt 7.01 min, linear gradient 70% to 95%
CH.sub.3CN (H.sub.2O) in 10 min, 0.4 mL/min; m/z=537.3
[M+Na].sup.+) and HRMS (ESI) in/z calculated for C30H48N3O4Na
(M+Na) 537.3543, found 537.3509.
[0122] Determination of Intracellular Superoxide Radicals.
[0123] Oxidation-dependent fluorogenic dye, dihydroethidium ("DHE",
Molecular Probes) was used to evaluate intracellular production of
superoxide radicals. DHE is cell permeable and, in the presence of
superoxide, is oxidized to fluorescent ethidium which intercalates
into DNA. The fluorescence of ethidium was measured using a FACscan
(Becton-Dickinson, Rutherford, N.J.) flow cytometer, equipped with
a 488-nm argon ion laser and supplied with the Cell Quest software.
Mean fluorescence intensity from 10,000 cells were acquired using a
585-nm bandpass filter (FL-2 channel).
[0124] Determination of Intracellular ATP Levels.
[0125] Cells were incubated with 10 .mu.m of compound 5a (FIG. 3)
for indicated periods of time (2, 4, 6, 12, and 14 h). At the end
of incubation, cells were collected and the content of
intracellular ATP was determined using a bioluminescent somatic
cell assay kit (Sigma, St. Louis, Mass.). As a positive control,
cells were incubated with 2 mM of 2-dexy-glucose, a glucose
analogue which competitively inhibits cellular uptake and
utilization of glucose, for 12 and 14 h.
[0126] Cells.
[0127] Caco-2BBe human enterocyte-like epithelial cells were
obtained from the American Type Culture Collection (Manassas, Va.).
Cells were routinely maintained at 37.degree. C. in under a
humidified atmosphere containing 8% CO2 in air. The culture medium
was DMEM supplemented with 10% PBS, non-essential amino acids
supplement (Sigma-Aldrich catalogue # M7145), sodium pyruvate (2
mM), streptomycin (0.1 mg/ml), penicillin G (100 U/ml) and human
transferrin (0.01 mg/ml). The culture medium was changed 3 times
per week.
[0128] Surgical Procedures to Obtain Vascular Access.
[0129] All study protocols using rats followed the guidelines for
the use of experimental animals of the US National Institutes of
Health and were approved by the Institutional Animal Care and Use
Committee at the University of Pittsburgh.
[0130] Male specific pathogen-free Sprague Dawley rats (Charles
River Laboratories, Wilmington, Mass.), weighing 150-250 g, were
housed in a temperature-controlled environment with a 12-h
light/dark cycle. The rats had free access to food and water. For
experiments, rats were anesthetized with intramuscular ketamine HCl
(30 mg/kg) and intraperitoneal sodium pentobarbital (35 mg/kg).
Animals were kept in a supine position during the experiments.
Lidocaine (0.5 ml of a 0.5% solution) was injected subcutaneously
to provide local anesthesia at surgical cut-down sites. In order to
secure the airway, a tracheotomy was performed and polyethylene
tubing (PE 240; Becton Dickinson, Sparks, Md.) was introduced into
the trachea. Animals were allowed to breathe spontaneously.
[0131] The right femoral artery was cannulated with polyethylene
tubing (PE 10). This catheter was attached to a pressure transducer
that allowed instantaneous measurement of mean arterial pressure
(MAP) during the experiment. For experiments using the
pressure-controlled hemorrhagic shock (HS) model, the right jugular
vein was exposed, ligated distally, and cannulated with
polyethylene tubing (PE 10) in order to withdraw blood. For
experiments using the volume-controlled hemorrhagic shock (HS)
model, the jugular catheter was used to infuse the resuscitation
solution and the right femoral vein, which was cannulated with a
silicon catheter (Chronic-Cath, Norfolk Medical, Skokie, Ill.), was
used to withdraw blood.
[0132] All animals were instrumented within 30 min. Heparin (500
U/kg) was administered immediately after instrumentation through
the femoral vein. Animals were placed in a thermal blanket to
maintain their body temperature at 37.degree. C. The positioning of
the different devices aforementioned was checked postmortem.
[0133] Intestinal Mucosal Permeability Assay.
[0134] Animals were allowed access to water but not food for 24 h
prior to the experiment in order to decrease the volume of
intestinal contents. The rats were instrumented as described above.
A midline laparotomy was performed and the small intestine was
exteriorized from the duodenojejunal junction to the ileocecal
valve. A small incision was made on the antimesenteric aspect of
the proximal small intestine and saline solution (1.5 ml) was
injected. The bowel was ligated proximally and distally to the
incision with 4-0 silk (Look, Reading, Pa.).
[0135] The small intestine was compressed gently in aboral
direction along its length to displace intestinal contents into the
colon. Starting 5 cm from the ileocecal valve, the ileum was
partitioned into six contiguous water-tight segments. Each segment
was 3 cm long and was bounded proximally and distally by
constricting circumferential 4-0 silk sutures. Care was taken to
ensure that the vascular supply to intestine was not compromised,
and each segment was well-perfused.
[0136] Two randomly selected segments in each rat were injected
with 0.3 ml of vehicle and served as "no treatment" controls. In
order to fill the segments, a small incision was made and the
solution was injected using a Teflon catheter (Abbocath 16Ga, Abbot
Laboratories).
[0137] The remaining four other segments were injected with
solutions containing either
4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) or one of
the Gramicidin S-based compounds. Four different final
concentrations of TEMPOL in normal saline were evaluated: 0.1, 1, 5
and 20 mM. The hemigramicidin-based compounds were dissolved in a
mixture of dimethylsulfoxide (DMSO) and normal saline (1:99 v/v)
and injected at final concentrations of 0.1, 1, 10 or 100
.mu.M.
[0138] After the segments were loaded with saline or the test
compounds, the bowel was replaced inside the peritoneal cavity and
the abdominal incision was temporarily closed using Backhaus
forceps.
[0139] After a 5 min stabilization period, hemorrhagic shock was
induced by withdrawing blood via the jugular catheter. MAP was
maintained at 30.+-.3 mm Hg for 2 hours. The shed blood was
re-infused as needed to maintain MAP within the desired range.
[0140] After 2 h of shock, the animals were euthanized with an
intracardiac KCl bolus injection. The ileum was rapidly excised
from the ileocecal valve to the most proximal gut segment. The tips
of each segment were discarded. In order to assay caspases 3 and 7
activity and phospholipids peroxidation, mucosa samples were
collected from gut segments immediately after hemorrhage and stored
at -80.degree. C. For permeability measurements, each segment was
convened into an everted gut sac, as previously described by
Wattanasirichaigoon et al., see Wattanasirichaigoon, S. et al.
Effect of mesenteric ischemia and reperfusion or hemorrhagic shock
on intestinal mucosal permeability and ATP content in rats, Shock.
12:127-133 (1999).
[0141] Briefly, as per the Wattanasirichaigoon protocol referenced
above, the sacs were prepared in ice-cold modified Krebs-Henseleit
bicarbonate buffer ("KHBB"), pH 7.4. One end of the gut segment was
ligated with a 4-0 silk suture; the segment was then everted onto a
thin plastic rod. The resulting gut sac was mounted on a Teflon
catheter (Abbocath 16GA, Abbot Laboratories) connected to a 3 ml
plastic syringe containing 1.5 ml of KHBB. The sac was suspended in
a beaker containing KHBB plus fluorescein-isothiocyanate labeled
dextran (average molecular mass 4 kDa, FD4; 0.1 mg/ml). This
solution was maintained at 37.degree. C., and oxygenated by
bubbling with a gas mixture (O.sub.2 95%/CO.sub.2 5%). After 30
min, the fluid within the gut sac was collected. The samples were
cleared by centrifugation at 2000 g for 5 min.
[0142] Fluorescence of FD4 in the solution inside the beaker and
within each gut sac was measured using a fluorescence
spectrophotometer (LS-50, Perkin-Elmer, Palo Alto, Calif.) at an
excitation wavelength of 492 nm and an emission wavelength of 515
nm. Mucosal permeability was expressed as a clearance normalized by
the length of the gut sac with units of nLmin.sup.-1cm.sup.2, as
previously described, see Yang, R. et al., Ethyl pyruvate modulates
inflammatory gene expression in mice subjected to hemorrhagic
shock, Am J Physiol Gastrointest Liver Physiol 283:G212-G22
(2002).
[0143] Results for a specific experimental condition (i.e.,
specific test compound at a single concentration) were expressed as
relative change in permeability calculated according to this
equation: Relative change in permeability (%)=(C.sub.Hs
exp-C.sub.normal)/C.sub.Hs cont-C.sub.normal).times.100, where
C.sub.HS exp is the clearance of FD4 measured for a gut segment
loaded with the experimental compound, C.sub.normal is the
clearance of FD4 measured in 6 gut segments from 3 normal animals
not subjected to hemorrhagic shock, and C.sub.HS cont is the mean
clearance of FD4 measured in 2 gut segments filled with vehicle
from the same animal used to measure C.sub.HS exp.
[0144] Measurement of Permeability of Caco-2 Monolayers.
[0145] Caco-2.sub.BBe cells were plated at a density of
5.times.10.sup.4 cells/well on permeable filters (0.4 .mu.m pore
size) in 12-well bicameral chambers (Transwell, Costar, Corning,
N.Y.). After 21 to 24 days, paracellular permeability was
determined by measuring the apical-to-basolateral clearance of
FD4.
[0146] Briefly, the medium on the basolateral side was replaced
with control medium or medium containing menadione (50 .mu.M
final). Medium containing FIX (25 mg/ml) was applied to the apical
chamber. In some cases, one of the gramicidin S-based compounds,
XJB-5-131, also was added to the apical side at final
concentrations of 0.1, 1, 10 or 100 .mu.M. After 6 hours of
incubation, the medium was aspirated from both compartments.
Permeability of the monolayers was expressed as a clearance
(pLh.sup.-1cm.sup.-2), see Han, X. et al., Proinflammatory
cytokines cause NO dependent and independent changes in expression
and localization of tight junction proteins in intestinal
epithelial cells, Shock. 19:229-237 (2003).
[0147] Caspases 3 and 7 Activity Assay.
[0148] Caspases 3 and 7 activity was measured using a commercially
available assay kit, Caspase GIo.TM. 3/7 assay kit (Promega,
Madison, Wis.). Briefly, 50 .mu.l of rat gut mucosa homogenate (20
jug protein) was mixed with 50 .mu.l of Caspase-GIo.TM. reagent and
incubated at room temperature for 1 hour. At the end of incubation
period, the luminescence of each sample was measured using a plate
reading chemiluminometer (ML1000, Dynatech Laboratories, Horsham,
Pa.). Activity of caspases 3 and 7 was expressed as luminescence
intensity (arbitrary units per mg protein). Protein concentrations
were determined using the BioRad assay (Bio-Rad Laboratories, Inc.,
Hercules, Calif.).
[0149] Assay for Peroxidation of Phospholipids.
[0150] Gut mucosal samples were homogenized. Lipids were extracted
from homogenates using the Folch procedure, see M. Lees and G. H.
Sloan-Stanley, A simple method for isolation and purification of
total lipids from animal tissue. J. BIOL. CHEM. 226:497-509 (1957),
and resolved by 2D HPTLC (High Performance Thin Layer
Chromatography) as previously described, see Kagan, V. E. et al., A
role for oxidative stress in apoptosis: Oxidation and
externalization of phosphatidylserine is required for macrophage
clearance of cell undergoing Fas-mediated apoptosis, J Immunol.
169:487-489 (2002). Spots of phospholipids were scraped from HPTLC
plates and phospholipids were extracted from silica. Lipid
phosphorus was determined by a micro-method, see Bottcher, C. J. F.
et al., A rapid and sensitive sub-micro phosphorus determination.
Anal Chim Acta. 24: 203-204 (1961).
[0151] Oxidized phospholipids were hydrolyzed by pancreatic
phospholipase A2 (2 U/.mu.l) in 25 mM phosphate buffer containing 1
mM CaCl.sub.3, 0.5 mM EDTA and 0.5 mM sodium dodecyl sulfate (SDS)
(pH 8.0, at room temperature for 30 min). Fatty acid hydroperoxides
formed were determined by fluorescence HPLC of resorufin
stoichiometrically formed during their microperoxidase 11-catalized
reduction in presence of Amplex Red (for 40 min at 4.degree. C.)
(8). Fluorescence HPLC (Eclipse XDB-C18 column, 5 pin,
150.times.4.6 mm, mobile phase was composed of 25 mM disodium
phosphate buffer (pH 7.0)/methanol (60:40 v/v); excitation
wavelength 560 nm, emission wavelength 590 nm) was performed on a
Shimadzu LC-100AT HPLC system equipped with fluorescence detector
(RF-10Axl) and autosampler (SIL-10AD).
[0152] Survival of Rats Subjected to Volume-Controlled Hemorrhagic
Shock.
[0153] Following surgical preparation and a 5-min stabilization
period to obtain baseline readings, rats were subjected to
hemorrhagic shock. Bleeding was carried out in 2 phases.
[0154] Initially, 21 ml/kg of blood was withdrawn over 20 min.
Immediately thereafter, an additional 12.5 ml/kg of blood was
withdrawn over 40 min. Thus, hemorrhage occurred over a total
period of 60 min and the total blood loss was 33.5 ml/kg or
approximately 55% of the total blood volume. Rats were randomly
assigned to receive XJB-5-131 (2 .mu.mol/kg) or its vehicle, a
33:67 (v/v) mixture of DMSO and normal saline. XJB-5-131 solution
or vehicle alone was administered as a continuous infusion during
the last 20 min of the hemorrhage period. The total volume of fluid
infused was 2.8 ml/kg and it was administered intravenously using a
syringe pump (KD100, KD Scientific, New Hope, Pa.). Rats were
observed for 6 hours or until expiration (defined by apnea for
>1 min). At the end of the 6 hour observation period, animals
that were still alive were euthanized with an overdose of KCl.
[0155] Blood pressure was recorded continuously using a commercial
strain-gauge transducer, amplifier, and monitor (S90603a,
SpaceLabs, Redmond, Wash.). Blood samples (0.5 ml) were collected
from the jugular vein at the beginning of hemorrhage (baseline), at
the end of hemorrhage (shock) and at the end of resuscitation
(resuscitation). Hemoglobin concentration [Hb], lactate and glucose
concentration were determined using an auto-analyzer (Model ABL
725, Radiometer Copenhagen, Westlake, Ohio).
[0156] Data Presentation and Statistics.
[0157] All variables are presented as means+Standard Error Mean
(SEM). Statistical significance of differences among groups was
determined using ANOVA (analysis of variance) and LSD (Least
Significant Difference) tests, or Kruskal-Wallis and Mann-Whitney
tests as appropriate. Survival data were analyzed using the
log-rank test Significance was declared for p values less than
0.05.
Example 2
[0158] Selective delivery of TEMPO to mitochondria could lead to
therapeutically beneficial reduction of ROS; therefore,
investigation of the use of conjugates of 4-amino-TEMPO ("4-AT")
was explored. In order to selective target the mitochondria, a
targeting sequence using the membrane active antibiotic Gramicidin
S ("GS") as well as corresponding alkene isosteres, shown in FIGS.
2 and 3. Accordingly, using the Gramicidin S peptidyl fragments and
alkene isosteres as "anchors," the TEMPO "payload" could be guided
into the mitochondria.
[0159] The Leu-.sup.DPhe-Pro-Val-Orn fragment of hemigramicidin was
used as a targeting sequence. Alkene Isosteres such as (E)-alkene
isosteres of Gramicidin S (i.e., hemigramicidin) were used as part
of the targeting sequence. See FIG. 3 for the synthetic pathway for
(E)-alkene isosteres and compound 3 for the corresponding chemical
structure. The (E)-alkene as depicted in compound 2 of FIG. 3 was
then oxidized in a multi-step process to yield the compound as
depicted in compound 3 an example of the (E)-alkene isostere.
[0160] Then, the compound depicted as compound 3 of FIG. 3 was
conjugated with the tripeptide H-Pro-Val-Orn(Cbz)-OMe using
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
("EDC") as a coupling agent. The tripeptide is an example of a
suitable targeting sequence having affinity for the mitochondria of
a cell. The resulting product is shown as compound 4a in FIG. 3.
Saponification of compound 4a followed by coupling with
4-amino-TEMPO ("4-AT") afforded the resulting conjugates shown as
compound 5a in FIG. 3, in which the Leu-.sup.DPhe peptide bond has
been replaced with an (E)-alkene.
[0161] In an alternate embodiment, conjugates 5b and 5c in FIG. 3
by coupling the peptide 4b (Boc-Leu-.sup.DPhe-Pro-Val-Orn(Cbz)-OMe)
and the (E)-alkene isostere as indicated as compound 3 in FIG. 3 to
4-AT. The peptide is another example of a suitable targeting
sequence having an affinity with the mitochondria of a cell.
[0162] Electron paramagnetic resonance ("EPR") spectroscopy was
used to monitor the cellular delivery of compounds 5a and 5b shown
in FIG. 3 in mouse embryonic cells ("MEC").
[0163] The following conditions were used during the EPR-based
analysis of the integration and reduction of nitroxide Gramicidin
S-peptidyl conjugates in MECs. The MECs at a concentration of 10
million MECs per ml, were incubated with 10 .mu.M of 4-AT and
compound 5a, respectively. Recovered nitroxide radicals in whole
cells, mitochondria, and cytosol fractions were resuspended in
phosphate buffer saline ("PBS") in the presence and absence,
respectively, of 2 .mu.M K.sub.3Fe(CN).sub.6. In brief. FIG. 4A
shows a representative EPR spectra of compound 5a in different
fractions of MECs in the presence of K.sub.3Fe(CN).sub.6. Further,
FIG. 4B shows an assessment of integrated nitroxides.
[0164] Distinctive characteristic triplet signals of nitroxide
radicals were detected in MECs incubated with 10 .mu.M of compound
5a (FIG. 3) as well as in mitochondria isolated from these cells.
The cytosolic function did not elicit EPR signals of nitroxide
radicals; similar results were observed with conjugate 5b (FIG. 3)
(data not shown).
[0165] Incubation of MECs with compound 5a (FIG. 3) resulted in
integration and one-electron reduction of compound 5a, as evidenced
by a significant increase in magnitude of the EPR signal intensity
upon addition of a one-electron oxidant, ferricyanide (FIG. 4B)
(Note: EPR results for incubation of MECs with 5b are not shown in
FIG. 4; however, EPR results for 5b were similar when compared to
5a). In contrast to 5a and 5b, however, 4-amino-TEMPO (4-AT) did
not effectively permeate cells or the mitochondria, as shown by the
absence of significant amplitude change in the EPR results for
4-AT.
[0166] The ability of 5a, 5b (FIG. 3), and 4-AT to prevent
intracellular superoxide generation by flow cytometric monitoring
of oxidation of dihydroethidium ("DUE") to a fluorescent ethidium
was tested. The ability of 5a, 5b, and 4-AT to protect cells
against apoptosis triggered by actinomycin D ("ActD") was also
tested. MECs were pretreated with 10 .mu.M 4-AT, 5a, or 5b then
incubated with ActD at a concentration of 100 ng/mL. It was found
that 5a and 5b completely inhibited nearly two-fold intracellular
superoxide generation in MECs (sec FIG. 6A). 4-AT had no effect on
the superoxide production in MECs.
[0167] Apoptotic cell responses were documented using three
biomarkers: (1) externalization of phosphatidylserine ("PS") on the
cell surface (by flow cytometry using an FITC-labeled PS-binding
protein, annex in V, see FIGS. 6B and 6E); (2) activation of
caspase-3 by cleavage of the 7-DEVD-AMC substrate (sec FIG. 6C),
and, (3) DNA fragmentation by flow cytometry of propidiium iodide
stained DNA (see FIG. 6D).
[0168] Phosphatidylserine ("PS") is an acidic phospholipid located
exclusively on the inner leaflet of the plasma membrane; exposure
of PS on the cell surface is characteristic of cell apoptosis.
Externalization of PS was analyzed by flow cytometry using an
annexin V kit. Cells were harvested by trypsinization at the end of
incubation and then stained with annexin V-FITC and propidium
iodide ("PS"). Ten thousand cell events were collected on a FACScan
flow cytometer. Annexin V-positive and PI-negative cells were
considered apoptotic.
[0169] Activation of capase-3, a cysteine protease only activated
in the execution phase of apoptosis, was determined using an
EnzChek capsase-3 assay kit.
[0170] Further, calcium and magnesium dependent nucleases are
activated that degrade DNA during apoptosis. These DNA fragments
are eluted, stained with propidium iodide and analyzed using flow
cytometry. A cell population with decreased DNA content was
considered a fraction of apoptotic cells.
[0171] Anti-apoptotic effects of compounds 5a and 5b were observed
at relatively low concentrations of 10 .mu.M. Compounds 5a and 5b
(FIG. 3) reduced the number of annexin V-positive cells as shown in
FIG. 6B, prevented caspase-3 activation as shown in FIG. 6C, and
prevented DNA fragmentation as shown in FIG. 6D. At concentrations
in excess of 10 .mu.M, both 5a and 5b were either less protective
or exhibited cytotoxicity (FIG. 6E). In contrast, 4-AT afforded no
protection.
[0172] In contrast, compound 5c, which docs not have a complete
targeting moiety, was ineffective in protecting MECs against
ActD-induced apoptosis (FIGS. 6B and 6C) at low concentrations.
Accordingly, the hemigramicidin peptidyl targeting sequence is
essential for anti-apoptotic activity of nitroxide conjugates such
as those containing TEMPO.
[0173] Finally, the reduction of compounds 5a and 5b could also
cause inhibition of mitochondrial oxidative phosphorylation, so the
ATP levels of MECs treated with these compounds were tested. As is
known to one ordinarily skilled in the art, ATP serves as the
primary energy source in biological organisms; reduction of ATP
levels would greatly impair normal cell function. ATP levels in
MECs in the presence or absence of 5a or 2-deoxyglucose ("2-DG")
were used as a positive control (see FIG. 6F). At concentrations at
which anti-apoptotic effects were maximal (.about.10 .mu.M, FIG.
6E), nitroxide conjugates did not cause significant changes in the
cellular ATP level Therefore, synthetic GS-peptidyl conjugates
migrate into cells and mitochondria where they are reduced without
affecting the ability of the mitochondria to produce ATP.
Example 3
[0174] In an in vivo assay, the ileum of rats was divided into a
series of well-vascularized components in a manner akin to links of
sausage. The lumen of each ileal compartment was filled with a 3
.mu.L aliquot of test solution. Two of the ileal compartments were
filled with vehicle alone (i.e., a solution containing at least in
part the TEMPO derivative). These two components served as internal
controls to account for individualistic variations in the severity
of shock or the response of the mucosa to the shock.
[0175] Using this assay system, eight compounds were evaluated as
shown in FIG. 5: TEMPOL (FIG. 5A), one dipeptidic TEMPO analog
(FIG. 5B--XJB-5-208), 3 hemigramicidin-TEMPO conjugates (FIGS. 5C
XJB-5-125, 5E XJB-5-131, and 5G XJB-5-197), and 3 hemigramicidin
compounds that do not have the TEMPO moiety (FIGS. 5D--XJB-5-127,
5F--XJB-5-133, and 5H--XJB-5-194).
[0176] Hemorrhagic shock in rats leads to marked derangements in
intestinal mucosal barrier function--in other words, the mucosal
permeability of shocked intestinal segments was significantly
greater than the permeability of segments from normal rats
(52.3+0.5 versus 6.9+0.1 nLmin.sup.-1cm.sup.-2, respectively;
p<0.01), see Tuomincn, E. K. J., Phospholipid cytochrome c
interaction: evidence for the extended lipid anchorage, J. BIOL.
CHEM., 277:8822-8826 (2002); also Wipf P. et al., Mitochondria
targeting of selective electron scavengers: synthesis and
biological analysis of hemigramicidin-TEMPO conjugates, J. AM.
CHEM. SOC. 127:12460-12461. Accordingly, mice were subjected to 2
hours of shock (Mean Arterial Pressure ("MAP")=30 f 3 mm Hg), the
gut segments were harvested and mucosal permeability to flourescein
isothiocyanate-dextran ("FD4") measured ex vivo. Data in FIG. 5 are
expressed as a percentage of the change permeability relative to
that observed in simultaneously assayed control segments loaded
during shock with normal saline solution.
[0177] Accordingly, intraluminal TEMPOL was used as a "positive
control" for gut mucosal protection assay. TEMPOL concentrations
>1 mM in the gut lumen ameliorated hemorrhagic shock-induced
ileal mucosal hyperpermeability (FIG. 5A). Two of the TEMPO
conjugates, namely XJB-5-208 (FIG. 5B) and XJB-5-131 (FIG. 5C),
also significantly ameliorated hemorrhagic shock-induced ileal
mucosal hyperpermeability. The lowest effective concentration for
XJB-5-208 (FIG. 5B) and XJB-5-131 (FIG. 5E) was 1 .mu.M; i.e., both
of these compounds were .about.1000-fold more potent than TEMPOL.
Two other compounds carrying the TEMPO payload, XJB-5-125 (FIG. 5C)
and XJB-5-197 (FIG. 5G) failed to provide protection against gut
barrier dysfunction induced by hemorrhage. XJB-5-133 (FIG. 5F) has
the same (hemigramicidin-based) mitochondrial targeting moiety as
XJB-5-131 (FIG. 5E) but lacks the TEMPO payload. It is noteworthy,
therefore, that XJB-5-133 (FIG. 5F) did not afford protection from
the development of ileal mucosal hyperpermeability.
[0178] Ineffective as well were the two other hemigramicidin-based
compounds that also lacked the TEMPO payload, XJB-5-127 (FIG. 5D)
and XJB-5-194 (FIG. 5H). Of the compounds screened, XJB-5-131 (FIG.
5E) appeared to be the most effective, reducing hemorrhagic
shock-induced mucosal hyperpermeability to approximately 60% of the
control value.
[0179] Based upon the results as reflected in FIGS. 5A-51I, both
the TEMPO payload and the "anchoring" hemigramicidin fragment are
requisite moieties that should be present in order for effective
electron scavenging activity by the XJB-5-131 compound.
Accordingly, it was found that XJB-5-131 ameliorates peroxidation
of mitochondrial phospholipids (i.e., ROS activity) in gut mucosa
from rats subject to hemorrhagic shock.
[0180] In the subsequent series of in vivo studies, the affect of
intraluminal XJB-5-131 on hemorrhage-induced peroxidation of
phospholipids in intestinal mucosa was examined. Isolated segments
of the ileum of rats were divided into a series of
well-vascularized components in a manner akin to sausage and the
lumen of each ileal compartment was filled with the same volume of
test solution containing either vehicle or a 10 .mu.M solution of
XJB-5-131, which was previously indicated to be the most active of
the hemigramicidin-TEMPO conjugates. In a preferred embodiment, 0.3
mL of test solution filled the lumen of each ileal compartment.
[0181] After two hours of HS, samples of ileal mucosa from the gut
sacs filled with the vehicle and XJB-5-131 were obtained and
compared with ileal mucosa of normal MECs. All samples were assayed
with caspase 3 or caspase 7 activity as well as the peroxidation of
phosphatidylcholine ("PC"), phosphatidylethanolamine ("PE"),
phosphatidylserine ("PS"), and cardiolipin ("CL"), summarized in
FIG. 7.
[0182] As can be seen in FIGS. 7A-7D, treatment with XJB-5-131
significantly ameliorated hemorrhage-induced peroxidation of CL,
the only phospholipid tested found in mitochondria. However,
treatment with XJB-5-131 only had a small effect on PE peroxidation
and no effect on peroxidation of PC and PS. Based upon these
trends, hemorrhagic shock is associated with substantial oxidative
stress even in the absence of resuscitation. Further, this data
also establishes that XJB-5-131 is an effective ROS scavenger as it
localizes predominantly in mitochondria and protects CL from
peroxidation.
[0183] Relative to the activity measured in samples from normal
animals, the activity of caspases 3 and 7 was markedly increased in
vehicle-treated mucosal samples from hemorrhaged rats (FIG. 8).
However, when the ileal segments were filled with XJB-5-131
solution instead of its vehicle, the level of caspase 3 and 7
activity after hemorrhagic shock was significantly decreased.
Accordingly, hemorrhagic shock is associated with activation of
pro-apoptotic pathways in gut mucosal cells. Moreover, the data
support the view that this process is significantly ameliorated
following mitochondrial treatment with XJB-5-131.
Example 4
[0184] In another series of experiments, monolayers of
enterocyte-like cells, Caco-2.sub.BBe, were studied for
physiological and pathophysiological purposes for determining
intestinal barrier function. Just as with the prior Examples with
respect to ROS exposure, the permeability of Caco-2.sub.BBe
monolayers increases when the cells are incubated with the ROS,
hydrogen peroxide, or menadione (a redox-cycling quinine that
promotes the formation of superoxide anion radicals), see Baker, R.
D. et al., Polarized Caco-2 cells. Effect of reactive oxygen
metabolites on enterocyte barrier function, DIGESTIVE DIS. SCI.
40:510-518 (1995); also Banan, A. et al., Activation of
delta-isoform of protein kinase C is required for oxidant-induced
disruption of both the microtubule cytoskeleton and permeability
barrier of intestinal epithelia, J. PHARMACOL. EXP. THER. 303:17-28
(2002).
[0185] Due to the results with respect to XJB-5-131 and its
amelioration of hemorrhage-induced CL peroxidation in mucosal cells
in vivo (see aforementioned Examples 1 and 2), a possible treatment
using XJB-5-131 was investigated to determine if menadione-induced
epithelial hyperpermeability could be ameliorated in vitro.
Consistent with the prior in vivo observations. Caco-2.sub.BBe
monolayers were incubated in the absence and in the presence of
menadione, respectively. After 6 hours, incubation of
Caco-2.sub.BDc monolayers with menadione caused a marked increase
in the apical-basolateral clearance of FD4 (FIG. 9). Treatment with
10 .mu.M XJB-5-131 provided significant protection against
menadione-induced hyperpermeability.
Example 5
[0186] As reflected by the above in vivo and in vitro studies,
XJB-5-131 had significantly beneficial effects on several
biochemical and physiological read-outs. Accordingly, systemic
administration of XJB-5-131 was investigated with respect to
whether it would prolong survival of patients subjected to profound
periods of hemorrhagic shock with massive blood loss in the absence
of standard resuscitation with blood and crystalloid solution. As
in the above studies, rats were utilized as test patients.
[0187] A total of sixteen rats were tested in this study. Rats were
treated with 2.8 ml/kg of vehicle or the same volume of XJB-5-131
solution during the final 20 min of the bleeding protocol. The
total dose of XJB-5-131 infused was 2 .mu.mol/kg. Following
profound hemorrhagic shock consistent with the protocol described
above for the prior studies, thirteen survived for at least 60 min
and received the full dose of either XJB-5-131 solution or the
vehicle, a 33:67 (v/v) mixture of DMSO and normal saline. As shown
in Table 2, blood glucose, lactate and hemoglobin concentrations
were similar in both groups at baseline and before and immediately
after treatment. None of the between-group differences were
statistically significant.
TABLE-US-00002 TABLE 2 End of first End of second phase of phase of
Parameter Compound Baseline hemorrhage hemorrhage Blood glucose
Vehicle 143 .+-. 5 255 .+-. 30 219 .+-. 26 concentration XJB-5-131
134 .+-. 4 228 .+-. 24 201 .+-. 38 (mg/dL) Blood lactate Vehicle
1.8 .+-. 0.4 606 .+-. 0.8 5.9 .+-. 3 concentration XJB-5-131 1.8
.+-. 0.2 5.7 .+-. 0.8 5.6 .+-. 1.2 (mEq/L) Blood Hb Vehicle 12.7
.+-. 0.5 11.1 .+-. 0.3 9.4 .+-. 0.2 concentration XJB-5-131 12.7
.+-. 0.3 10.7 .+-. 0.3 9.4 .+-. 0.3 (g/dL)
[0188] Shortly after treatment was started, mean arterial pressure
("MAP") increased slightly in both groups (see FIG. 10A). In both
groups, mean arterial pressure ("MAP") decreased precipitously
during the first phase of the hemorrhage protocol and remained
nearly constant at 40 mm Hg during the beginning of the second
phase. Six of the seven animals in the vehicle-treated (control)
group died within one hour of the end of the bleeding protocol and
all were dead within 125 minutes (FIG. 10B). Rats treated with
intravenous XJB-5-131 survived significantly longer than those
treated with the vehicle. Three of the six rats survived longer
than 3 hours after completion of the hemorrhage protocol; one rat
survived the whole 6 hour post-bleeding observation period (FIG.
10B).
[0189] Accordingly, analysis of the XJB-5-131 studies indicate that
exposure of the patient to the compound prolongs the period of time
that patients can survive after losing large quantities of blood
due to traumatic injuries or other catastrophes (e.g., rupture of
an abdominal aortic aneurysm).
[0190] By extending the treatment window before irreversible shock
develops, treatment in the field with XJB-5-131 might "buy" enough
time to allow transport of more badly injured patients to locations
where definitive care, including control of bleeding and
resuscitation with blood products and non-sanguineous fluids, can
be provided. The results using a rodent model of hemorrhagic shock
also open up the possibility that drugs like XJB-5-131 might be
beneficial in other conditions associated with marked tissue
hypoperfusion, such as stroke and myocardial infarction.
[0191] The results presented here also support the general concept
that mitochondrial targeting of ROS scavengers is a reasonable
therapeutic strategy. Although previous studies have shown that
treatment with TEMPOL is beneficial in rodent HS situations, a
relatively large dose of the compound was required (30 mg/kg
bolus+30 mg/kg per h). In contrast, treatment with a dose of
XJB-5-131 that was about 300 fold smaller (.about.0.1 mg/kg) was
clearly beneficial. The greater potency of XJB-5-131 as compared to
TEMPOL presumably reflects the tendency of XJB-5-131 to localize in
mitochondrial membranes, a key embodiment of the invention. As
indicated above, two hemigramicidin-4-amino-TEMPO conjugates
(namely XJB-5-208 and XJB-5-131, see FIG. 2) are concentrated in
the mitochondria of cultures mouse embryonic cells following
incubation with solutions of the compounds.
[0192] Further, the use of XJB-5-131 significantly prolonged the
survival of the rats subjected to massive blood loss, even though
the animals were not resuscitated with either blood or other
non-sanguineous fluids and they remained profoundly
hypotensive.
[0193] In light of the above, synthetic hemigramicidin
peptidyl-TEMPO conjugates permeate through the cell membrane and
also the mitochondrial membrane where they act as free radical
scavengers for ROS such as, but not limited to, superoxide anion
radicals. The conjugates are then reduced within the mitochondria
by electron-transport proteins which are involved with the cellular
respiration pathway, thereby coupling the decoupled ROS species.
These conjugates also have the advantage, as discussed above, of
being anti-apoptotic, especially in the case of compounds such as
5a and 5b.
[0194] By effectively reducing the amount of ROS species, a
patient's condition, including an illness or other medical
condition, may be ameliorated and, in some cases, survival may be
prolonged as described in the Example IV study. Examples of such
conditions, including diseases and other medical conditions,
include (but are not limited to) the following medical conditions
which include diseases and conditions: myocardial ischemia and
reperfusion (e.g., after angioplasty and stenting for management of
unstable angina or myocardial infarction), solid organ (lung liver,
kidney, pancreas, intestine, heart) transplantation, hemorrhagic
shock, septic shock, stroke, tissue damage due to ionizing
radiation, lung injury, acute respiratory distress syndrome (ARDS),
necrotizing pancreatitis, and necrotizing enterocolitis.
Example 6
[0195] In a further embodiment, in support of the
inter-changeability of cargoes of the mitochondria-targeting
groups, a composition for scavenging radicals in a mitochondrial
membrane comprises a radical scavenging agent or an NOS inhibitor
and a membrane active peptidyl fragment having a high affinity with
the mitochondrial membrane. The membrane active peptidyl fragment
preferably has a property selected from the group consisting of
antioxidant, radioprotective, protective, anti-apoptotic,
therapeutic, ameliorative, NOS antagonist and combinations thereof.
In a related embodiment, with respect to compounds with antibiotic
properties, it is generally preferable to employ compounds whose
mode of action includes bacterial wall targets.
[0196] In another embodiment, the membrane active compound is
preferably selected from the group consisting of bacitracins,
gramicidins, valinomycins, enniatins, alamethicins, beauvericin,
serratomolide, sporidesmolide, tyrocidins, polymyxins, monamycins,
and lissoclinum peptides.
[0197] In a related embodiment, the NOS antagonist is selected from
the group consisting of XJB-5-234 (a), XJB-5-133 (b), XJB-5-241
(c), and XJB-5-127 (d), comprising AMT NOS antagonist cargos:
##STR00081##
Example 7
[0198] The following examples provide protocols for additional
cargo usable in compounds described herein which serve as NOS
antagonists.
##STR00082##
[0199] Compound (1) is
Boc-Leu-.psi.[(E)-C(CH.sub.3).dbd.CH]-.sup.DPhe-Pro-Val-Orn(Cbz)-AMT
(XJB-5-241) and was prepared according to the following protocol. A
solution of 11.0 mg (13.2 .mu.mol) of
Boc-leu-.psi.[(E)-C(CH.sub.3).dbd.CH]-.sup.DPhe-Pro-Val-Orn(Cbz)-OMc
(248) in 400 .mu.L of MeOH was treated at 0.degree. C. with 132
.mu.L (132 nmol) of 1 N NaOH. The reaction mixture was stirred at
room temperature for 8 h. and treated with 132 .mu.L (132 nmol) of
1 N HCl. The solution was extracted with CHCl.sub.3 and the organic
layer was dried (Na.sub.2SO.sub.4) and concentrated in vacuo to
give the crude acid as a colorless form. This acid was dissolved in
2.00 mL of CHCl.sub.3 and treated at room temperature with 2.1 mg
(16 nmol) of HOBt, 3.0 mg (16 nmol) of EDC, 3.3 mg (20 nmol) of
2-amino-5,6-dihydro-6-methy 1-4H-1,3-thiazine.HCl and 3.5 mg (27
nmol) of DMAP. The reaction mixture was stirred at room temperature
for 48 h, diluted with CHCl.sub.3, and washed with H.sub.2O. The
organic layer was dried (Na.sub.2SO.sub.4), concentrated in vacuo,
and purified by chromatography on SiO.sub.2 (1:1, hexanes/EtOAc
followed by 20:1, CHCl.sub.3/MeOH) to yield 11 mg (89%) of
XJB-5-241 as a colorless powder. The following characterization
data were obtained: LC-MS(R.sub.t 8.37 min, linear gradient 70% to
95% CH.sub.3CN (H.sub.2O) in 10 min. 0.4 mL/min; m/z=932.4
[M+H].sup.+) 954.3 [M+Na].sup.+) and HRMS (ESI) m/z calculated for
C.sub.50H.sub.74N.sub.7O.sub.8S (M+H) 932.5320, found 932.5318.
##STR00083##
[0200] Compound (2) is
Boc-leu-.psi.[(E)-CH.dbd.CH]-.sup.DPhe-Pro-Val-Orn(Cbz)-AMT
(XJB-5-133) and was prepared according to the following protocol. A
solution of 20.0 mg (24.3 nmol) of 2-85 (XJB-5-194) in 800 nL of
MeOH was treated at 0.degree. C. with 243 .mu.L (243 nmol) of 1 N
NaOH. The reaction mixture was stirred at room temperature for 6 h,
and treated with 243 .mu.L (243 .mu.mol) of 1 N HCl. The solution
was extracted with CHCl.sub.3 and the organic layer was dried
(Na.sub.2SO.sub.4) and concentrated in vacuo to give the crude acid
as a colorless form. This acid was dissolved in 1.00 mL of
CHCl.sub.3 and treated at room temperature with 3.9 mg (29 nmol) of
HOBt, 5.6 mg (29 .mu.mol) of EDC, 6.1 mg (37 nmol) of
2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 7.4 mg (61
.mu.mol) of DMAP. The reaction mixture was stirred at room
temperature for 20 h, diluted with CHCl.sub.3, and washed with
H.sub.2O. The organic layer was dried (Na.sub.2SO.sub.4),
concentrated in vacuo, and purified by chromatography on SiO.sub.2
(1:1, hexanes/EtOAc followed by 20:1, CHCl.sub.3/MeOH) and an
additional preparative Cm reverse phase HPLC purification was
performed: 80% to 100% CH.sub.3CN (H.sub.2O) in 20 min, 5.0 mL/mm)
to afford 12.9 mg (58%) of XJB-5-133 as a colorless powder. The
following characterization data were obtained: LC-MS (R.sub.t 7.89
min, linear gradient 70% to 95% CH.sub.3CN (H.sub.2O) in 10 min,
0.4 mL/min; m/z=918.3 [M+H].sup.+, 940.3 [M+Na].sup.+) and HRMS
(ESI) m/z calculated for C.sub.49H.sub.72N.sub.7O.sub.8S (M+H)
918.5163, found 918.5185.
##STR00084##
[0201] Compound (3) is Boc-Leu-.sup.DPhe-Pro-Val-Orn(Cbz)-AMT
(XJB-5-127) and was prepared according to the following protocol. A
solution of 24.0 mg (28.7 .mu.mol) of
Boc-Leu-.sup.DPhe-Pro-Val-Orn(Cbz)-OMe in 800 .mu.L of MeOH was
treated at room temperature with 287 .mu.L (287 .mu.mol) of 1 N
NaOH. The reaction mixture was stirred at room temperature for 5 h.
and treated at 0.degree. C. with 287 .mu.L (287 .mu.mol) of 1 N
HCl. The solution was extracted with CHCl.sub.3 and the organic
layer was dried (Na.sub.2SO.sub.4) and concentrated in vacuo to
give the crude acid as a colorless foam. The crude acid was
dissolved in 2.00 mL of CHCl.sub.3 and treated at room temperature
with 4.6 mg (34 .mu.mol) of HOBt, 6.6 mg (34 .mu.mol) of EDC, 5.7
mg (34 .mu.mol) of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl
and 8.8 mg (72.0 .mu.mol) of DMAP. The reaction mixture was stirred
at room temperature for 24 h, diluted with CHCl.sub.3, and washed
with H.sub.2O. The organic layer was dried (Na.sub.2SO.sub.4),
concentrated in vacuo, and purified by chromatography on SiO.sub.2
(2:1, hexanes/EtOAc followed by 20:1, CHCl.sub.3/MeOH) to yield
17.0 mg (63%) of XJB-5-127 as a colorless powder. The following
characterization data were obtained: LC-MS (R.sub.t 6.32 min,
linear gradient 70% to 95% CH.sub.3CN (H.sub.2O) in 10 min, 0.4
mL/min; m/z=935.3 [M+H].sup.+, 957.3 [M+Na].sup.+) and HRMS (ESI)
m/z calculated for C.sub.48H.sub.71N.sub.8O.sub.9S (M+H) 935.5065,
found 935.5044.
##STR00085##
[0202] Compound (4) is Boc-Leu-.omega.[(E)-CH.dbd.CH].sup.DPhe-AMT
(XJB-5-234) A solution of erode
Boc-Leu-.psi.[(E)-CH.dbd.CH]-.sup.DPhe-OH (2-84) (30.5 .mu.mol) in
2.00 mL of CH.sub.2Cl.sub.2 was treated at 0.degree. C. with 6.1 mg
(37 .mu.mol) of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl,
7.0 mg (37 .mu.mol) of EDC, 4.9 mg (37 .mu.mol) of HOBt, and 9.3 mg
(76 .mu.mol) of DMAP. The reaction mixture was stirred at room
temperature overnight, concentrated in vacuo, and purified by
chromatography on SiO.sub.2 (2:1, CH.sub.2Cl.sub.2/EtOAc) to yield
9.1 mg (63%) of XJB-5-234 as a colorless foam. The following
characterization data were obtained: LC-MS (R.sub.t 8.42 min,
linear gradient 70% to 95% CH.sub.3CN (H.sub.2O) in 10 min, 0.4
mL/min; m/z=474.5 [M+H].sup.+) and HRMS (ESI) m/z calculated for
C.sub.26H.sub.40N.sub.3O.sub.3S (M+H) 474.2790, found 474.2781.
##STR00086##
[0203] Compound (5) is
Boc-Leu-.psi.[(Z)--(CF.dbd.CH]-.sup.DPhe-Pro-Val-Orn(Cbz)-TEMPO
(XJB-7-53). A solution of 3.4 mg (4.1 .mu.mol) of
Boc-Leu-.psi.[(Z)--CF.dbd.CH]-.sup.DPhe-Pro-Val-Orn(Cbz)-OMe
XJB-5-66) in 400 .mu.L of MeOH was treated at 0.degree. C. with 41
.mu.L (41 .mu.mol) of 1 N NaOH. The reaction mixture was stirred at
room temperature for 12 h, and treated with 41 .mu.L (41 .mu.mol)
of 1 N HCl. The solution was extracted with CHCl.sub.3 and the
organic layer was dried (Na.sub.2SO.sub.4) and concentrated in
vacuo to give the crude acid as a colorless form. This acid was
dissolved in 400 .mu.L of CHCl.sub.3 and treated at room
temperature will) 0.7 mg (5 .mu.mol) of HOBt, 0.9 mg (5 .mu.mol) of
EDC, 0.5 mg (4 .mu.mol) of 4-amino-TEMPO and 1.1 mg (6 .mu.mol) of
DMAP. The reaction mixture was stirred at room temperature for 12
h, diluted with CHCl.sub.3, and washed with H.sub.2O. The organic
layer was dried (Na.sub.2SO.sub.4), concentrated in vacuo, and
purified by chromatography on SiO.sub.2 (1:1, hexanes/EtOAc
followed by 20:1, CHCl.sub.3/MeOH) to yield 3.6 mg (91%) of
XJB-7-53 as a colorless powder. The following characterization data
were obtained: LC-MS (R.sub.t 8.45 min, linear gradient 70% to 95%
CH.sub.3CN (H.sub.2O) in 10 min, 0.4 mL/min; m/z=977.5 [M+H].sup.+,
999.5 [M+Na].sup.+) and HRMS
[0204] (ESI) m/z calculated for C.sub.53H.sub.79FN.sub.7O.sub.9Na
(M+Na) 999.5821.
##STR00087##
[0205] Compound (6) is
Boc-.sup.DPhe-.psi.[(E)-C(CH.sub.3).dbd.CH]-Ala-Val-Orn(Cbz)-Leu-AMT
(XJB-7-42) and was prepared according to the following protocol. A
solution of 4.5 mg (5.6 .mu.mol) of
Boc-.sup.DPhe-.psi.[(E)-C(CH.sub.3).dbd.CH]-Ala-Val-Orn(Cbz)-Leu-OMe
(2-119) in 0.35 mL of MeOH was treated at 0.degree. C. with 56
.mu.L (56 .mu.mol) of 1 N NaOH. The reaction mixture was stirred at
room temperature for 12 h, and treated with 56 .mu.L (56 .mu.mol)
of 1 N HCl. The solution was extracted with CHCl.sub.3 and the
organic layer was dried (Na.sub.2SO.sub.4) and concentrated in
vacuo to give the crude acid as a colorless form. This acid was
dissolved in 0.80 mL of CHCl.sub.3 and treated at room temperature
with 0.9 mg (6.7 .mu.mol) of HOBt, 1.3 mg (6.7 .mu.mol) of EDC, 1.4
mg (8.4 .mu.mol) of
2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 1.7 mg (14
.mu.mol) of DMAP. The reaction mixture was stirred at room
temperature for 36 h, concentrated in vacuo, and purified by
chromatography on SiO.sub.2 (20:1, CHCl.sub.3/MeOH) to yield 5.0 mg
(99%) of XJB-7-42 as a colorless foam. The following
characterization data were obtained: LC-MS (R.sub.t 6.61 min,
linear gradient 70% to 95% CH.sub.3CN (H.sub.2O) in 10 min, 0.4
mL/min; m/z=907.3 [M+H].sup.+, 929.4 [M+Na].sup.+) and HRMS (ESI)
m/z calculated for C.sub.48H.sub.72N.sub.7O.sub.8S (M+H) 906.5163,
found 906.5190.
##STR00088##
[0206] Compound (7) is
Boc-.sup.DPhe-.psi.[(E)-C(CH.sub.3).dbd.CH]-Ala-Val-AMT (XJB-7-43).
A solution of 14.3 .mu.mol of crude
Boc-.sup.DPhe-v[(E)-C(CH.sub.3).dbd.CH]-Ala-Val-OMe (2-111) in 1.00
mL of CHCl.sub.3 was treated at room temperature with 2.3 mg (17
.mu.mol) of HOBt, 3.3 mg (17 .mu.mol) of EDC, 3.6 mg (22 .mu.mol)
of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 4.4 mg (36
.mu.mol) of DMAP. The reaction mixture was stirred at room
temperature for 36 h, concentrated in vacuo, and purified by
chromatography on SiO.sub.2 (20:1, CHCl.sub.3/MeOH) to yield 7.5 mg
(96%) of XJB-7-43 as a colorless foam. The following
characterization data were obtained: LC-MS (R.sub.t 5.41 min,
linear gradient 70% to 95% CH.sub.3CN (H.sub.2O) in 10 min, 0.4
mL/min; m/z=545.3 [M+H].sup.+, 567.3 [M+Na].sup.+) and HRMS (ESI)
m/z calculated for C.sub.29H.sub.44N.sub.4O.sub.4S (M+Na) 567.2981,
found 567.2971.
[0207] Among the preferred radical scavenging agents are a material
selected from the group consisting of a ubiquinone analog, a
ubiquinone analog fragment moiety, a ubiquinone analog fragment
moiety lacking a hydrophilic tail, a superoxide dismutase mimetic,
a superoxide dismutase biomimetic or a salen-manganese
compound.
[0208] As is known to one ordinarily skilled in the art, ionizing
radiation activates a mitochondrial nitric oxide synthase
("mtNOS"), leading to inhibition of the respiratory chain,
generation of excess superoxide radicals, peroxynitrite production
and nitrosativc damage. The damage done by ionizing radiation is
believed to be alleviated [See Kanai, A. J. et al., Am J Physiol.
383: F1304-F1312 (2002); and Kanai, A. J. et al. Am J Physiol. 286:
H13-H21 (2004)]. The composition of this embodiment is
characterized by the property of inhibiting mtNOS, thereby
resisting generation of excess superoxide radicals, peroxynitrite
and nitrosative damage.
[0209] Protection again irradiation damage using systemic drug
delivery can result in unwanted side effects. One approach to limit
or prevent these adverse side effects is to target drug delivery to
the mitochondria using a peptide carrier strategy.
[0210] In one embodiment, a potent NOS inhibitor, the non-arginine
analog of 2-amino-6-methyl-thiazine ("AMT"), was selected as a
cargo. Irradiation of the ureopithelium results in increased
production of superoxide and nitric oxide ("NO"), mouse bladders
were instilled with AMT or 4-amino-TEMPO to determine if inhibition
of N O or scavenging free radicals is more radioprotective.
[0211] An unconjugated and conjugated NOS antagonist, (AMT, 100
.mu.M) and an unconjugated and conjugated nitroxide derivative
(4-amino-TEMPO, 100 .mu.M) were incubated for two hours at
37.degree. C. with 32D c13 hemopoietic cells.
##STR00089##
[0212] Following incubation, the cells were lysed and the
mitochondria isolated for a mass spectrometry analysis where
compounds isolated from mitochondria were identified as Na+
adducts. The resulting spectra (not shown) demonstrate that
4-amino-TEMPO only permeate the mitochondrial membrane with the
assistance of the attached GS-derived targeting sequence. Further
spectra (not shown) indicate that unconjugated AMT do not enter the
mitochondria membrane in substantial quantities. Thus, the
targeting peptides successfully direct a NOS antagonist and a
nitroxide to the mitochondria.
[0213] Further physiological studies were conducted to determine
the effects of peptide-targeted AMT and 4-amino-TEMPO on NO and
peroxynitrite production in irradiated uroepithelial cells. The
cells were cultured in an 8-well slide chamber for 3 days and then
microsensor measurements were taken 24 hours after irradiation.
[0214] In untreated irradiated cells and cells treated with
unconjugated 4-amino-TEMPO (100 .mu.M) or unconjugated AMT (10
.mu.M), capsaicin evoked NO production and resulted in the
formation of comparable amount of peroxynitrite. In cells treated
with high-dose conjugated 4-amin-TEMPO (100 .mu.M), peroxynitrite
production was decreased by approximately 4-fold. In non-radiated
cells or cells treated with conjugated AMT (10 .mu.M), NO induced
peroxynitrite formation was nearly completely inhibited. This
suggested that peptides conjugates couple or covalently link with
membrane impermeant 4-amino-TEMPO or AMT and facilitate the
transport of 4-amin-TEMPO across the mitochondrial membrane.
Furthermore, this data suggests that the peptide conjugates do not
interfere with the NOS inhibitory activity of AMT or the free
radical scavenging activity of 4-amino-TEMPO and that AMT is a more
effective radioprotectant [Kanai, A. J. et al., Mitochondrial
Targeting of Radioprotectants Using Peptidyl Conjugates, ORGANIC
AND BIOMOLECULAR CHEMISTRY (in press)].
[0215] Quantitative mass spectrometry studies were used to compare
the effectiveness of several AMT peptide conjugates in permeating
the mitochondrial membrane, specifically XJB-5-234, XJB-5-133,
XJB-5-241, and XJB-5-127. The fmole/10 .mu.M mitochondrial protein
ratio provides a relative quantification of conjugate concentration
at the target site. Table 3 indicates that the most efficacious
conjugate was compound XJB-5-241.
TABLE-US-00003 TABLE 3 Compound fmole/10 .mu.M mitochondrial
protein XJB-5-234 1.45 XJB-5-133 89.8 XJB-5-241 103.3 XJB-5-127
50.8
[0216] The trisubstituted (E)-alkene moiety embedded in XJB-5-241
has a stronger conformational effect that the less biologically
active disubstituted (b)-alkene XJB-5-133 or the GS peptidyl
fragment XJB-5-127, see Wipf P. et al., Methyl- and
(Trifuoromethyl)alkene Peptide Isosteres: Synthesis and Evaluation
of Their Potential as .beta.-Turn Promoters and Peptide Mimetics J
ORG. CHEM. 63:6088-6089 (1998); also Wipf, P. et al., Imine
Additions of Internal Alkynes for the Synthesis of Trisubstituted
(E)-Alkene and Cyclopropane Peptide Isosteres ADV. SYNTH. CAT.
347:1605-1613 (2005). The data indicates that a defined secondary
structure and an appropriate conformational preorganization is
important in accomplishing mitochondrial permeation of compounds
that reduce nitrosative and oxidative effects.
[0217] The presence of a non-hydrolyzable alkene isostere functions
in place of labile peptide bonds and is significant for a prolonged
mechanism of action. The relatively rigid (E)-alkenes
(.psi.[(E)-C(R).dbd.CH]) represent useful, conformationally
preorganized structural mimetics and have been used as surrogates
of hydrolytically labile amide bonds in a number of enzyme
inhibitors. The primary objective of this strategy is the accurate
mimicry of the geometry of the peptide bond; however, (E)-alkenes
also modulate the physicochemical properties, solubility, and
lipophilicity, number of hydrogen donors and acceptors, etc, of the
parent structures, and therefore generally have a different
metabolic fate than simple peptides.
[0218] A targeted delivery strategy employed in this invention is
advantageous since some neuronal NOS (nNOS) antagonists and most
antioxidants, including nitroxide derivatives, are poorly
cell-permeable and require therapeutically effective concentrations
greater than 100 .mu.M if used without a conjugate.
[0219] The method related to this embodiment of the invention
delivers a composition to mitochondria comprising transporting to
said mitochondria a desired cargo which may, for example, be (a) a
radical scavenging agent by use of a membrane active peptidyl
fragment preferably having has a .beta.-turn motif having a high
affinity for the mitochondrial membrane or (b) a nitric oxide
synthase antagonist bonded to the membrane active peptidyl
fragment.
Example 8--Synthesis of JP4-039 (See FIG. 11)
[0220] Synthesis of JP4-039 was accomplished according to the
following.
##STR00090##
(R,E)-2-Methyl-N-(3-methylbutylidene)propane-2-sulfinamide (1)
[0221] (Staas, D. D.; Savage, K. L; Homnick, C. F.; Tsou, N.; Ball,
R. G. J. Org. Cham., 2002, 67, 8276)--To a solution of
isovaleraldehyde (3-Methylbutyraldehyde, 5.41 mL, 48.5 mmol) in
CH.sub.2Cl.sub.2 (250 mL) was added
(R)-2-methylpropane-2-sulfinamide (5.00 g, 40.4 mmol), MgSO.sub.4
(5.0 eq, 24.3 g, 202 mmol) and PPTS (10 mol %, 1.05 g, 4.04 mmol)
and the resulting suspension was stirred at RT (room temperature,
approximately 25.degree. C.) for 24 h. The reaction was filtered
through a pad of Celite.RTM. and the crude residue was purified by
chromatography on SiO.sub.2 (3:7, EtOAc:hexanes) to yield 6.75 g
(88%) as a colorless oil. .sup.1H NMR .delta. 8.07 (t, 1H, J=5.2
Hz), 2.47-2.38 (m, 2H), 2.18-1.90 (m, 1H), 1.21 (s, 9H), 1.00 (d,
6H, J=6.7 Hz). As an alternative, filtration through a pad of
SiO.sub.2 provides erode imine that functions equally well in
subsequent reactions.
##STR00091##
(But-3-ynyloxy)(tert-butyl)diphenylsilane (2)
[0222] (Nicolaou, K. C. et al., J. Am. Chem. Soc. 2006, 128,
4460)--To a solution of 3-butyn-1-ol (5.00 g, 71.3 mmol) in
CH.sub.2Cl.sub.2 (400 mL) was added imidazole (5.40 g, 78.5 mmol)
and TBDPSCl ((tert-butyl)diphenylsilane chloride) (22.0 g, 78.5
mmol) and the reaction was stirred at RT for 22 h. The reaction was
filtered through a pad a SiO.sub.2, the SiO.sub.2 washed with
CH.sub.2Cl.sub.2 and the colorless solution concentrated to yield
21.4 g (97%) of crude alkyne that was carried on without further
purification.
##STR00092##
(S,E)-8-(tert-Butyldiphenylsilyloxy)-2-methyloct-5-en-4-amine
hydrochloride (3)
[0223] To a solution of (2) (15.9 g, 51.5 mmol) in CH.sub.2Cl.sub.2
(300 mL) was added zirconocene hydrochloride (15.1 g. 58.4 mmol) in
3 portions and the resulting suspension was stirred at RT for 10
min. The resulting yellow solution was cooled to 0.degree. C. and
Me.sub.3Al (2.0 M in hexanes, 27.5 mL, 54.9 mmol) was added and
stirred for 5 minutes followed by addition of a solution of imine
(1) (6.50 g, 34.3 mmol) in CH.sub.2Cl.sub.2 (50 mL) and the orange
solution was stirred for an additional 4 h while allowed to warm to
rt. The reaction was quenched with MeOH, diluted with H.sub.2O and
CH.sub.2Cl.sub.2 and HCl (1 M) was added to break up the emulsion
(prolonged stirring with Rochelle's salt can also be utilized). The
organic layer was separated and the aqueous layer was washed with
CH.sub.2Cl.sub.2 (2.times.). The organic layers were combined,
washed with brine, dried (MgSO.sub.4), filtered though a pad of
Celite.RTM. and concentrated. Since the crude oil was contaminated
with metal salts, the oil was dissolved in Et2O (diethyl ether,
Et=ethyl), allowed to sit for 2 h, and then filtered though a pad
of Celite.RTM. and concentrated. Analysis of the crude residue by
1H NMR showed only 1 diastereomer (>95:5 dr).
[0224] To the crude residue in Et.sub.2O (800 mL) was added HCl
(4.0 M in dioxane, 17.2 mL, 68.7 mmol) and the reaction was stirred
for 30 minutes, during which time a white precipitate formed. The
precipitate was filtered, washed with dry Et.sub.2O, and dried to
afford 11.0 g (74% over 2 steps) of (3) as a colorless solid, mp
151-154.degree. C.; [.alpha.].sub.D-2.9 (c 1.0, CH.sub.2Cl.sub.2);
.sup.1H NMR .delta. 8.42 (bs, 3H), 7.70-7.55 (m, 4H), 7.48-7.30 (m,
6H), 5.90 (dt, 1H, J=14.9, 7.5 Hz), 5.52 (dd, 1H, J=15.4, 8.4 Hz),
3.69 (appt, 3H, J=6.5 Hz), 2.45-2.20 (m, 2H), 1.80-1.50 (m, 3H),
1.03 (s, 9H), 0.95-0.84 (m. 6H); .sup.13C NMR .delta. 135.5, 134.5,
133.7, 129.5, 127.6, 127.3, 63.0, 52.9, 42.1, 35.6, 26.7, 24.4,
22.9, 21.5, 19.1; EIMS m/z 395 ([M-HCl].sup.+, 40), 338 (86), 198
(100); HRMS (EI) m/z calcd for C.sub.25H.sub.37NOSi (M-HCl)
395.2644, found 395.2640.
##STR00093##
(S,E)-tert-Butyl
8-(tert-butyldiphenylsilyloxy)-2-methyloct-5-en-4-ylcarbamate
(4)
[0225] To a solution of (3) (10.5 g, 24.3 mmol) in CH.sub.2Cl.sub.2
(400 mL) was added Et.sub.3N (triethylamine) (3.0 eq, 10.3 mL, 72.9
mmol) and Boc.sub.2O (1.05 eq, 5.74 g, 25.5 mmol) and the resulting
suspension was stirred at RT for 14 h. The reaction was quenched
with sat. aq. NH.sub.4Cl, the organic layers separated, dried
(MgSO.sub.4), filtered and concentrated. The crude residue was
carried onto the next step without further purification.
##STR00094##
(S,E)-tert-Butyl 8-hydroxy-2-methyloct-5-en-4-ylcarbamate (5)
[0226] To a solution of crude (4) (12.0 g, 24.3 mmol) in THF (200
mL) at 0.degree. C. was added TBAF (1.0 M in THF, 1.25 eq. 30.4 mL,
30.4 mmol) and the reaction was warmed to RT and stirred for 2 h.
The reaction was quenched with sat. aq. NH.sub.4Cl, organic layer
washed with brine, dried (MgSO.sub.4), filtered and concentrated.
The crude residue was purified by chromatography on SiO.sub.2 (3:7,
EtOAc:hexanes) to yield 5.51 g (88%, 2 steps) as a colorless oil.
[.alpha.].sub.D-12.7 (c 1.0, CH.sub.2Cl.sub.3); .sup.1H NMR .delta.
5.56 (dt, 1H, J=15.3, 6.9 Hz). 5.41 (dd, 1H, J=15.4, 6.4 Hz), 4.41
(bs. 1H), 4.06 (bm, 1H), 3.65 (appbq, 2H, J=5.7 Hz), 2.29 (q, 2H,
J=6.3 Hz), 1.76 (bs, 1H), 1.68 (m, 1H), 1.44 (s, 9H), 1.33 (m, 2H),
0.92 (m, 6H); .sup.13C NMR .delta. 155.4, 134.3, 126.9, 79.2, 61.5,
50.9, 44.5, 35.6, 28.3, 24.6, 22.5, EIMS m/z 257 ([M].sup.+, 10),
227 (55), 171 (65); HRMS (EI) m/z calcd for
C.sub.14H.sub.27NO.sub.3 257.1991, found 257.1994.
##STR00095##
(S,E)-5-(tert-Butoxycarbonylamino)-7-methyloct-3-enoic acid (6)
[0227] To a solution of (5) (1.00 g, 3.89 mmol) in acetone (40 mL)
at 0.degree. C. was added a freshly prepared solution of Jones
Reagent (2.5 M, 3.89 mL, 9.71 mmol) and the reaction was stirred at
0.degree. C. for 1 h. The dark solution was extracted with
Et.sub.2O (3.times.50 mL), the organic layers washed with water
(2.times.75 mL), brine (1.times.50 mL), dried (Na.sub.2SO.sub.4),
filtered and concentrated to yield 990 mg (94% crude) of acid (6)
as a yellow oil that was used without further purification.
##STR00096##
TEMPO-4-yl-(S,E)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide
(7)
[0228] To a solution of (6) (678 mg, 2.50 mmol, crude) in
CH.sub.2Cl.sub.2 (35 mL) at 0.degree. C. was added 4-amino tempo
(1.5 eq, 662 mg, 3.75 mmol), EDCI (1.2 eq, 575 mg, 3.00 mmol), DMAP
(1.1 eq, 339 mg, 2.75 mmol) and HOBt-hydrate (1.1 eq, 377 mg, 2.75
mmol) and the resulting orange solution was stirred at RT for 14 h.
The reaction was diluted with CH.sub.2Cl.sub.2, washed with sat.
aq. NH.sub.4Cl and the organic layer dried (Na.sub.2SO.sub.4),
filtered and concentrated. The crude residue was purified by
chromatography on SiO.sub.2 (1:1 to 2:1, EtOAc/hexanes) to yield
857 mg (76%, 2 steps) as a peach colored solid, mp 61.degree. C.
(softening point: 51.degree. C.); [.alpha.].sub.D.sup.23+35.6 (c
0.5. DCM); ESIMS m/z 365 (40), 391 (50), 447 ([M+Na].sup.+, 100),
257 (20); HRMS (ESI) m/z calcd for C.sub.23H.sub.42N.sub.3O.sub.4Na
447.3073, found 447.3109.
[0229] The compounds shown as formula 4, above can be synthesized
as shown in FIG. 11B. Briefly, synthesis was accomplished as
follows: To a solution of compound (1) in CH.sub.2Cl.sub.2 was
added zirconocene hydrochloride, followed by addition of
Me.sub.2Zn, then a solution of
N-diphenylphosphoryl-1-phenylmethanimine (Imine). The reaction
mixture was refluxed, filtered, washed, and dried to afford (2).
Cleavage of the TBDPS protecting group was achieved by treating (2)
with TBAF, which resulted in the formation of (3). The terminal
alcohol (3) was dehydrated to alkene (4), which was further treated
by ozonolysis to afford ester (5). Protocols similar to that given
for the synthesis of JP4-039, above, were used to acylate the amino
group with the Boc protecting group and to react the terminal
carboxylic acid with 4-amino-TEMPO to afford (6).
Example 9--a Mitochondria-Targeted Nitroxide/Hemigramicidin S
Conjugate Protects Mouse Embryonic Cells Against Gamma
Irradiation
[0230] (see, Jiang, J, et al. Int. J. Radiation Oncology Biol.
Phys., Vol. 70, No. 3, pp. 816-825, 2008)
[0231] EPR-Based Analysis of Integration and Distribution of
Nitroxides.
[0232] To compare the integration efficiency, mouse embryonic cells
(1.times.10.sup.7/mL) were incubated with 10 .mu.M nitroxides for
10 min. ESR spectra of nitroxide radicals in the incubation medium,
cell suspension or mitochondrial suspension were recorded after
mixing with acetonitrile (1:1 v/v) after 5-min incubation with 2 mM
K.sub.3Fe(CN).sub.6 using JEOL-RE1X EPR spectrometer under the
following conditions: 3350 G center field; 25 G scan range; 0.79 G
field modulation, 20 mW microwave power; 0.1 s time constant; 4 min
scan time. Integration efficiency was calculated as
(E.sub.initial-E.sub.medium)/E.sub.initial.times.100%. Mitochondria
were isolated using a mitochondria isolation kit (Pierce, Rockford,
Ill.) according to the manufacturer's instruction. Amounts of
nitroxide radicals integrated into mitochondria were normalized to
the content of cytochrome c oxidase subunit IV.
[0233] Superoxide Generation.
[0234] Oxidation-dependent fluorogenic dye, DHE was used to
evaluate intracellular production of superoxide radicals. DHE is
cell permeable and, in the presence of superoxide, is oxidized to
fluorescent ethidium which intercalates into DNA. Briefly, cells
were treated with 5 .mu.M DHE for 30 min at the end of incubation.
Cells were then collected by trypsinization and resuspended in PBS.
The fluorescence of ethidium was measured using a FACScan flow
cytometer (Becton-Dickinson, Rutherford, N.J.) supplied with the
CellQuest software. Mean fluorescence intensity from 10,000 cells
was acquired using a 585/42 nm bandpass filter.
[0235] CL Oxidation.
[0236] CL hydroperoxides were determined by fluorescence HPLC of
products formed in MP-11-catalyzed reaction with a fluorogenic
substrate, Amplex Red. Oxidized phospholipids were hydrolyzed by
pancreatic phospholipase A.sub.2 (2 U/ml) in 25 mM phosphate buffer
containing 1 mM CaCl.sub.2, 0.5 mM EDTA and 0.5 mM SDS (pH 8.0 at
RT for 30 min). After that Amplex Red and MP-11 were added and
samples were incubated for 40 min at 4.degree. C. Shimadzu LC-100AT
vp HPLC system equipped with fluorescence detector (RF-10Axl,
Ex/Em=560/590 nm) and autosampler (SIL-10AD vp) were used for the
analysis of products separated by HPLC (Eclipse XDB-C18 column, 5
.mu.m, 150.times.4.6 mm). Mobile phase was composed of
NaH.sub.2PO.sub.4 (25 mM, pH 7.0)/methanol (60:40 v/v).
[0237] Phosphatidylserine (PS) Externalization.
[0238] Externalization of PS was analyzed by flow cytometry using
annexin-V kit. Briefly, harvested cells were stained with
annexin-V-FITC and PI for 5 min in dark prior to flow cytometry
analysis. Ten thousand events were collected on a FACScan flow
cytometer (Becton-Dickinson) supplied with CellQuest software.
[0239] Gamma-Irradiation Dose Survival Curves of Mouse Embryonic
Cells.
[0240] Cells were plated in 35-mm Petri dishes with 2 ml culture
medium at a density between 100 and 1000 cells per dish. Cells were
treated with GS-nitroxide (XJB-5-125) either before (10-min) or
after (1-h) .gamma.-irradiation. XJB-5-125 was removed from the
medium 4-h post-irradiation. Colonies were fixed and stained with
0.25% crystal violet and 10% formalin (35% v/v) in 80% methanol for
30 min after a 9-day incubation period, and those of .gtoreq.50
cells were counted as survivors. The surviving fraction was
calculated as the plating efficiency of the samples relative to
that of the control.
[0241] FIG. 12 shows that nitroxide conjugate XJB-5-125 integrates
into cells and mitochondria much more efficiently than their parent
non-conjugated 4-amino-TEMPO in mouse embryonic cells. (A) shows
their cellular and mitochondrial integration efficiencies in mouse
embryonic cells, and (B) shows representative EPR spectrum of
nitroxides recovered from mitochondria.
[0242] FIG. 13 reveals that nitroxide conjugate XJB-5-125 protects
mouse embryonic cells against gamma irradiation induced superoxide
generation and cardiolipin peroxidation. (A) superoxide generation.
Cells were exposed to 10 Gy of .gamma.-irradiation. XJB-5-125 (20
.mu.M) was added to cells either 10-min before or 1-h after
irradiation and removed after 5-h incubation. Cells were incubated
with 5 .mu.M DHE for 30 min at the indicated time points. Ethidium
fluorescence was analyzed using a FACScan flow cytometer supplied
with CellQuest software. Mean fluorescence intensity from 10,000
cells was acquired using a 585-nm bandpass filter. (B) Cardiolipin
oxidation. Cardiolipin hydroperoxides were determined using a
fluorescent HPLC-based Amplex Red assay. Data presented are
means.+-.S.E. (n=3). *p<0.01 vs non-irradiated cells; *p<0.01
(0.05) vs irradiated cells without XJB-5-125 treatment under the
same condition. Insert is a typical 2D-HPTLC profile of
phospholipids from cells.
[0243] FIG. 14 reveals that nitroxide conjugate XJB-5-125 protects
cells against gamma irradiation induced apoptosis. (A) XJB-5-125
blocks .gamma.-irradiation induced accumulation of cytochrome c in
the cytosol of mouse embryonic cells. (B) Densitometry ratio of
cytochrome c/actin. Semi-quantitation of the bands was carried out
by densitometry using Labworks Image Acquisition and Analysis
Software (UVP, Upland, Calif.) The level of cytochrome c release
was expressed as the mean densitometry ratio of cytochrome c over
actin. (C) Dose (5, 10 and 20 .mu.M) dependent radioprotective
effect of XJB-5-125 (pre-treatment) on .gamma.-irradiation (10 Gy)
induced phosphatidylserine (PS) externalization. After 48 h
post-irradiation incubation, cells were harvested and stained with
annexin-V-FITC and propodium iodide (PI) prior to flow cytometry
analysis. (D) Time (2, 3, 4, 5, and 6 h) dependent radioprotective
effect of XJB-5-125 (20 .mu.M) on .gamma.-irradiation (10 Gy)
induced PS externalization (48 h post irradiation) in mouse
embryonic cells, (E) Effect of XJB-5-125 on .gamma.-irradiation (10
Gy) induced PS externalization in human bronchial epithelial cell
line BEAS-2B cells. Cells were treated with 5-125 (5 or 10 .mu.M)
before (10-min) or after (1-h) irradiation. Externalization of PS
was analyzed 72 h post-irradiation exposure. Data shown are
means.+-.S.E. (n=3). *(&)p<0.01(0.05) vs irradiated cells
without 5-125 treatment, # p<0.05 vs cells pre-treated with
5-125.
[0244] FIG. 15 shows the effect of nitroxide conjugate XJB-5-125 on
gamma-irradiation dose survival curves of mouse embryonic cells.
Cells were pre- (10-min) or post-treated (1-h) with XJB-5-125 (20
.mu.M), which was removed after 4-h incubation period. The
surviving fraction was calculated as the plating efficiency of the
samples relative to that of the control. The data was fitted to a
single-hit multitarget model using SigmaPlot 9.0 (Systat Software).
Data presented are the mean.+-.S.E. (n=3).
[0245] FIG. 16 illustrates the effect of GS conjugated nitroxide,
XJB-5-125, on gamma-irradiation dose survival curves of 32D cl 3
murine hematopoietic cells. The cells incubated in XJB-5-125 or
Tempol had an increased Do (1.138 or 1.209 Gy, respectively)
compared to the 32D cl 3 cells (0.797 Gy). The cells incubated in
XJB-5-125 had an increased shoulder on the survival curve with an n
of 18.24 compared to 5.82 for the cells incubated in tempol.
Example 10--Testing of the Radioprotective Abilities of JP4-039
[0246] FIGS. 17A and 17B are graphs showing GS-nitroxide compound
JP4-039 increases survival of mice exposed to 9.75 Gy total body
irradiation. In FIG. 17A, mice received intraperitoneal injection
of 10 mg per kilogram of each of the chemicals indicated in FIG. 5,
then 24 hours later received 9.75 Gy total body irradiation
according to published methods. Mice were followed for survival
according to IACUC regulations. There was a significant increase in
survival of mice receiving JP4-039 compared to irradiated control
mice. (P=0.0008). In FIG. 17B, mice received intraperitoneal
injection of JP4-039 either 10 minutes before (square symbols) or 4
hours after (triangle symbols) irradiation with 9.75 Gy.
[0247] FIG. 18 is a graph showing that GS-nitroxide compound
JP4-039 increases survival of mice exposed to 9.5 Gy total body
irradiation. Groups of 15 mice received intraperitoneal injection
of 10 mg. per kilogram of each indicated GS-nitroxide compound or
carrier (Cremphora plus alcohol at 1 to 1 ratio, then diluted 1 to
10 in distilled water). Mice received 10 mg per kilogram
intra-peritoneal injection 24 hours prior to total body
irradiation. Control mice received radiation alone. There was a
statistically significant increase in survival in mice receiving
GS-nitroxide compounds. (P=0.0005)
[0248] FIG. 19 is a graph showing that GS-nitroxide JP4-039 is an
effective hematopoietic cell radiation mitigator when delivered 24
hr after irradiation. Irradiation survival curves were performed on
cells from the 32D cl 3 mouse hematopoietic progenitor cell line,
incubated in 10 .mu.M JP4-039 for 1 hour before irradiation, or
plated in methylcellulose containing 10 .mu.M JP4-030 after
irradiation. Cells were irradiated from 0 to 8 Gy, plated in 0.8%
methylcellulose containing media, and incubated for 7 days at
37.degree. C. Colonies of greater than 50 cells were counted and
data analyzed by linear quadratic and single-hit, multi-target
models. Cells incubated in JP4-039 were more resistant as
demonstrated by an increased shoulder on the survival curve with an
ft of 5.25.+-.0.84 if drug was added before irradiation or
4.55.+-.0.47 if drug was added after irradiation compared to
1.29+0.13 for 32D cl 3 cells alone (p=0.0109 or 0.0022,
respectively).
[0249] FIG. 20 is a graph showing that JP4-039 is an effective
mitigator of irradiation damage to KM101 human marrow stromal
cells. KM101 cells were incubated in media alone or in JP4-039 (10
.mu.M) for one hour before irradiation or 24 hours after
irradiation. The cells were irradiated to doses ranging from 0 to 6
Gy and plated in 4 well plates. Seven days later the cells were
stained with crystal violet and colonies of greater than 50 cells
counted. Cells incubated in JP4-039 either before or after
irradiation were more radioresistant as shown by an increased
shoulder of n=2.3.+-.0.2 or 2.2.+-.0.2, respectively compared to n
of 1.1.+-.0.1 for the KM101 cells (p=0.0309 or 0.0386,
respectively). There was no significant change in the Do for the
different conditions.
Example 11--NOD/SCID Mouse Model to Optimize JP4-039 for a Clinical
Trial
[0250] We have significant preliminary data on use of NOD/SCID mice
to test the effects of JP4-039 on human marrow stromal cell and
hematopoietic stem cell recovery from total body irradiation to
doses that cause the hematopoietic syndrome. FIG. 21A shows results
with detection of human cells in NOD/SCID mouse marrow harvested 27
days after cord blood transplanted I.V, showing flow cytometric
analysis and identification of human CD45+(light gray)
hematopoietic cells in NOD/SCID mouse BM following irradiation,
proximal tibia bone drilling (see below), and human cord blood
injection. [0251] Six NOD/SCID mice were irradiated to 350cGy and
injected with 1.times.10.sup.7 human cord blood (CB) mononuclear
cells (MNC). Five months after the CB MNC cells were initially
injected, the right leg of 6 mice was irradiated to 10 Gy. 24 hours
post-irradiation holes were drilled in the tibiae. (See FIG. 21B)
Drill bit size 1 mm. diameter (Dremel Corp). 24 hours post-bone
drilling 1.times.10.sup.7 CB MNC was injected into 3 of the 6 mice.
Control mice (3) received no CB. 27 days after the CB was injected,
the bones were harvested for histochemical analysis and flow
cytometric analysis for human CD45+ cells (light grey) in the BM
using a PE-conjugated anti-CD45 antibody (BD Biosciences). Analysis
was performed on a BD LSR II flow cytometer (BD Biosciences). Human
CD45+ cells were delectable in all of the mice (numbers 1-3) that
received human CB MNC when compared to control mice (mouse 4). The
percent of CD45+ cells ranged from 0.045-3.288 percent in the non
boosted leg and from 0.028-0.892 percent in the high dose
irradiated leg. There was no difference between the
boost-irradiated and non boosted leg in these mice. Although the
data suggest that there is a trend (the percent of human CD45+
cells was lower in the high dose irradiated leg), there was no
statistically significant difference the total body irradiated non
boosted compared to 1000 cGy boosted leg (p=0.25). Day 7 bone photo
shown in FIG. 21B.
[0252] FIG. 21B is a photomicrograph of cross-section through a
tibial wound 7-days after surgical construction with a drill bit of
a unicortical 2-mm diameter wound in the lateral aspect of the
tibia 2-mm below the proximal epiphyseal plate. Robust trabecular
bone fills the intramedullary canal as well as die cortical window
in this intermediate phase of spontaneous wound repair. This time
point is optimal for assessing inhibition of marrow stromal cell
mediated osteogenesis by irradiation and restoration by JP4-039, as
proposed in this application. Arrows indicate margins of the wound.
(Toluidine blue stain, .times.35)(58)
Example 12--Topical and Transdermal Absorption of GS-Nitroxide
[0253] A practical skin patch is planned for delivery of JP4-039 or
other compounds delivered herein. The patch can be administered to
a subject before, during or after exposure to radiation, including
24 hr or later after irradiation exposure of the subject. In
preliminary studies, we sought to characterize the
absorption/penetration of a topically applied representative
GS-Nitroxide XJB-5-125 in mouse skin. XJB-5-125 was selected as a
potential topical agent based on its ability to inhibit ROS
generation, inhibit apoptosis and suppress oxidative damage to
mitochondrial lipids. XJB-5-125 comprises the
(Leu-D-Phe-Pro-Val-Orn) segment of XJB-5-125 and has been shown to
attenuate ActD-induced PS externalization in a dose-dependent
manner of 2.5-20 .mu.M. It can also inhibit the release of
cytochrome c from mitochondria and suppress CL peroxidation. The
physical properties of a chemical are critical to its ability to
penetrate into and through the skin. Two important factors are the
log octanal/water (Ko/w) partition coefficient and the molecular
weight. For XJB-5-125, the log Ko/w=4.5 and molecular weight is
956. The lipophilicity "rule" is based on the need for a compound
to partition out of the lipophilic stratum corneum and into the
more hydrophilic epidermis and dermis. The log Ko/w and MW of
XJB-5-125 are similar to ketaconazole (log Ko/w=4.34, MW=532),
clotrimazole (log k/ow=6.27, MW=902), and Indomethecin (log
Ko/w=4.23, MW=358) suggesting feasibility of delivery using
formulations similar to those used to effectively deliver these
agents. Like JP4-039, XJB-5-125 is a radiation mitigator as well as
a protector (see FIG. 15).
[0254] A small piece of skin (2 cm.sup.2) was placed in a Bronaugh
style flow-through diffusion cell system (PermeGear, Riegelsville,
Pa.) (FIG. 22). It was then sandwiched between two pieces of the
inert polymer Kel-F and clamped shut to prevent leakage. The
epidermal side faces upward and is exposed to the donor solution
(test solution), and the dermal side is in contact with the
receptor fluid. The exposed surface area is 0.79 cm.sup.2 (circular
chamber with 1 cm diameter). The skin forms a water-tight seal in
the flow through chamber so the receiving fluid (PBS+25% ethanol)
on the dermal side will contain the XJB-5-125 only if it has
penetrated through the skin. The receiver chamber was perfused with
this buffer that then passes to a fraction collector via Teflon
tubing. The PBS+25% ethanol was used because it is an effective
sink for hydrophobic compounds and produces better in vitrolin vivo
correlations than other receiver solutions. The skin was maintained
at 32.degree. C. by placing the chamber in a metal block heated via
a recirculating water bath. The skin was equilibrated for 60
minutes prior to introduction of the test compound. Seventy five
.mu.L of XJB-5-125 was placed on the skin and was allowed to remain
for the course of the experiment. The efflux was collected for 24
hours. (FIGS. 23-25).
[0255] To evaluate XJB-5-125 penetration in mouse skin, C57/BL6
mice were shaved using animal clippers (#40 blade), followed by a
brief treatment with Nair (depilatory) to remove remaining hair.
The skin was washed immediately after hair removal to prevent
further irritation. The skin was allowed to recover for 24 hours
prior to study. This reduces interference by hair and allows time
for small abrasions to heal prior to dermal penetration
studies.
[0256] Upon completion of the study, the skin was removed from the
diffusion chamber. The stratum corneum, which will contain the
majority of the topically applied compound, but is not relevant
from a therapeutic standpoint, was removed by sequential
tape-stripping (15 times) using Brookman Tape (3M, Minneapolis,
Minn.). The remaining skin (viable epidermis and dermis) and
transdermal effluent were assayed for XJB-5-125 via ESR.
[0257] Mouse skin was homogenized in 400 .mu.L 50 mM PBS pH 7.4.
EPR measurements were performed m gas-permeable Teflon tubing (0.8
mm internal diameter, 0.013 mm thickness) obtained from Alpha Wire
Corp. (Elizabeth, N.J., USA) on a JEOL JES-REIX spectrometer at
25.degree. C. The Teflon tube (approximately 8 cm in length) was
filled with 70 .mu.L of sample containing 28.5% of acetonitrile and
2 mM K.sub.3Fe(CN).sub.6, folded in half, and placed into an open
EPR quartz tube (inner diameter of 3.0 mm). (FIG. 24)
[0258] EPR spectra were recorded at 334.7 mT, center field; 20 mW,
power; 0.079 mT, field modulation; 5 mT, sweep width; 400 and 4000,
receiver gain; 0.1 s, time constraint. Spectra were collected using
EPRware software (Scientific Software Services, Bloomington, Ill.,
USA).
[0259] These preliminary experiments demonstrate that XJB-5-125 can
sufficiently penetrate intact skin. Farther, the total transdermal
absorption after 24 hours and the level of XJB-5-125 present in the
viable skin can be successfully measured using the techniques
described herein. The effect of formulation on topical delivery was
examined by using three different donor solutions (FIG. 25). Donor
A=1 mM XJB-5-125 in DMSO, donor B=1 mM XJB-5-125 in 95% Propylene
Glycol+5% Linoleic Acid, and Donor C=1 mM XJB-5-125 in 50% EtOH+40%
HSO+5% Propylene Glycol+5% Brij30. A total of 75 nmole was placed
on top of each piece of skin to begin these experiments (75 ul of
100 mM). The delivery of XJB-5-125 into the skin resulted in
between 0.07% and 0.46% remaining within the skin after 24 hours.
The higher delivery rate is in the range of other topical
products.
[0260] Given the observation that XJB-5-125 is active in cells in
the concentration range from 2.5-20 .mu.M and assuming a tissue
density of 1 g/cm.sup.3, an order of magnitude analysis based on
these data indicates that the topical delivery of XJB-5-125 method
to enhance systemic blood levels to protect bone marrow is
feasible.
[0261] Additionally, the fact that the total skin absorption is
generally regarded as linearly related to the donor concentration
implies that topical delivery will be greatly enhanced by
increasing the donor concentration. These preliminary studies
demonstrate feasibility of XJB-5-125 delivery to therapeutic levels
and indicate that the smaller JP4-039 molecule, as well as other
compounds described herein, may be useful as a skin
patch-deliverable radiation mitigator of the hematopoietic
syndrome.
Example 13 (Proposed)
[0262] The following can be used to select and optimize the best
GS-nitroxide JP4-039 (radiation damage mitigator drug) that can
enhance human bone marrow stromal cell and fresh human stromal cell
line seeding efficiency into irradiated limbs of NOD/SCID mice.
MnSOD-overexpressing cells are a positive control.
[0263] Experiments with KM101-MnSOD/ds-red (control KM101-ds-red)
clonal cell lines. Groups of 12 NOD/SCID mice receive 300 cGy total
body irradiation (low dose leg) and a 1000 cGy boost to the left
hind leg (high dose leg), then 24 hours later intravenous injection
of 1.times.10.sup.5 or 1.times.10.sup.6 cells of each cell line
(groups 1 and 2). Group 3 is mice that receive MnSOD-PL
intravenously 24 hours prior to irradiation and then injection of
KM101-MnSOD/ds-red. Group 4 is MnSOD-PL intravenously 24 hours
prior to irradiation, then control KM101/ds-red cells. This
experiment may be repeated twice. Mice will have bone marrow
flushed from the hind limbs at days 1, 3, 7, 14 after cell
transplantation, and scoring of the percent of total cells and
number of colony forming cells recoverable which are ds-red
positive thus of human origin. The scoring may be by ds-red
positivity, and then by colony formation in vitro by stromal cells.
We may score the total, then the percent of stromal cells of human
origin.
[0264] Experiments demonstrating improvement in human bone marrow
stromal cell line KM101 seeding by mitochondrial targeted radiation
protection/mitigation JP4-039 (GS-nitroxide) administration. This
experiment may be conducted essentially as described above (A),
with all groups, but with a sub-group receiving JP4-039 (24 hours)
after radiation (same day as cell lines are injected, or a
sub-group receiving intraperitoneal JP4-039 (daily or weekly after
cell line transplantation). Cells may be explanted from the high
dose and low dose irradiated femurs at days 7, 14, 21, and cultured
in vitro for human stromal colony forming progenitor cells (CFU-F).
The percent and total number of human cells entering the high dose
and low dose irradiated limbs can be quantitated by cell sorting
for ds-red. Each experiment can be completed twice.
[0265] Experiments as in (A) above, but substituting fresh human
marrow Stro1+ stromal cells from a 45 y.o. donor.
[0266] Experiments as in (B) above substituting Stro1+ human marrow
stromal cells.
[0267] Statistical Considerations
[0268] In (A), we propose comparing at 4 different time points
between 4 groups where either MnSOD or no MnSOD, and either
10.sup.5 or 10.sup.6 KM101 cells are injected, in terms of the
number of DsRed-KM101 cells. In (B), we propose comparing at 3
different time points between 10 groups where different doses and
schedules of the experimental compound will be used, in terms of
the same endpoint as in (A). (C) and (D) are the same as (A) and
(B) respectively, except that human stromal cells are used in place
of KM101 cells. All the comparisons in this task are performed
separately for high and low dose radiated legs. ANOVA followed by
Tukey's test can be used for these analyses. Sample size can be
estimated by the two sample t-test for pairwise comparisons. Due to
the lack of preliminary data, sample size estimation is based on
the expected difference to detect between groups in terms of the
common standard deviation .sigma.. Six mice per group can be
sacrificed per time point. With this sample size, there will be 82%
power to detect a difference of 1.8.sigma. between groups using the
two sided two sample t test with significance level 0.05.
[0269] As the secondary endpoint, the number of colony forming unit
fibroblast (human) CFU-F can also be compared between groups with
the same method as the primary endpoint.
[0270] It is expected that MnSOD overexpression in
KM101-MnSOD/ds-red cells will lead to a higher seeding efficiency
into both the high and low dose irradiated limbs of NOD/SCID mice.
We expect that MnSOD-PL treatment of the hematopoietic
microenvironment prior to KM101 clonal line cell line infusion will
further enhance engraftment of both KM101-MnSOD/ds-red and
KM101-ds-red cell lines. We expect the highest percent of seeding
efficiency will be detected in the mice receiving MnSOD-PL prior to
irradiation and injection of KM101-MnSOD/ds-red cells.
[0271] We expect that JP4-039 administration daily after cell
transplantation will facilitate improved stability of engraftment
of all stromal cell lines by decreasing free radical production by
the irradiated marrow microenvironment.
[0272] An inactive control compound for JP4-039 may be used,
(JP4-039 absent the nitroxide active moiety). Based upon the
results of these experiments, the optimal condition for bone marrow
stromal cell seeding can be derived, and these conditions may be
used in experiments described below.
Example 14 (Proposed)
[0273] Selection and optimization of a GS-nitroxide JP4-039 therapy
to enhance human CD34+ cord blood multilineage hematopoietic stem
cell progenitor cell seeding into irradiated limbs of NOD/SCID mice
that have been prepared by engraftment of human marrow stromal
cells. [0274] i. Experiments with TBI treated C57BL/6J mice and
mouse marrow screening, (preliminary system test) [0275] ii.
Experiments using the optimal seeding protocol for human KM101
cells into irradiated NOD/SCID mice (anticipated to be those mice
receiving MnSOD-PL prior to irradiation, and then injection with
KM101-MnSOD/ds-red, supplemented with JP4-039 daily). Mice can then
receive intravenous injection of 1.times.10.sup.5 or
1.times.10.sup.5 CD34+ LIN- cells from human umbilical cord blood
origin. Control cells may be CD34+ LIN+(differentiated progenitor)
cells 10.sup.5 or 10.sup.6 per injection. Groups of 12 mice.
[0276] These experiments may be carried out in two schedules.
[0277] Injection of cord blood cells at the same time as
KM101-MnSOD/ds-red cells. [0278] Injection of cord blood cells at
time of optimal recovery of KM101-MnSOD/ds-red cells from the
explant experiments of Example 13. This should be at day 7 or day
14 after stromal cell injection.
[0279] In these experiments, mice can be followed and tested at
serial time points out to two months after cord blood stem cell
transplantation. The percent of human peripheral blood
hematopoietic cells can be scored in weekly peripheral blood
samples and number of cells forming CFU-GEMM colonies can be tested
in explanted bones from sacrificed mice.
[0280] At days 7, 14, 21, 28, or 60 after cord blood
transplantation, mice in sub-groups may be sacrificed, and all
cells flushed from the high dose and low dose irradiated femurs,
and assays carried out for human multilineage hematopoietic
progenitors-CFU-GEMM. Assays may be carried out by two methods:
[0281] i. Sorting human CD34+ cells with monoclonal antibodies
specific for human. [0282] ii. Colony formation in human CFU-GEMM
culture medium and then secondary scoring of human colonies as the
subset of total mouse and human colony forming cells detected at
days 7 and days 14 in vitro.
[0283] In vitro experiments may be carried out in parallel as
follows:
[0284] KM101-MnSOD-PL plateau phase stromal cells may be irradiated
in vitro to 100, 200, 500, 1000 cGy, and then CD34+ LIN- human cord
blood cells co-cultivated with the stromal cells in vitro. Controls
can include unirradiated KM101-MnSOD/ds-red, irradiated
KM101-ds-red cells, unirradiated KM101-ds-red.
[0285] We can score human cobblestone islands (stem cell colonies)
on these cultures weekly, plot cumulative cobblestone island
formation, cumulative non-adherent cell production with weekly cell
harvest, and assay of weekly cell harvest for CFU-GEMM formation.
These studies may be carried out over two-three weeks. In vitro
co-cultivation studies can only partially duplicate the in vivo
hematopoietic microenvironment, and thus two weeks should be the
maximum efficient time for detection of whether MnSOD-PL expression
in the adherent KM101 layer will increase engraftment of cord blood
stem cells.
[0286] 3. Experiments with JP4-039 supplementation of the cord
blood transplantation program as in (1) above to increase homing,
stable quiescence, and repopulation capacity of human cord blood
stem cells by removing ROS production in the irradiated marrow
stromal cell environment.
[0287] Experiments in vitro supplementing in co-cultivation culture
media the drug JP4-039 daily. The experiments with irradiated KM101
subclonal lines, co-cultivated with cord blood stem cells may be
carried out with the addition of JP4-039, or an active analog
JP4-039 daily. Control experiments can include addition of CD34+
LIN+ differentiated cord blood cells that are expected to produce
fewer CFU-GEMM over time. Stromal cell cultures may be irradiated,
cord blood cells added, and cultures scored as above.
[0288] Groups of 12 mice can receive the optimal protocol for human
CFU-GEMM cell engraftment from the experiment above, and then
sub-groups can be treated as follows: [0289] i. JP4-039 twice
weekly. [0290] ii. JP4-039 daily. [0291] iii. Inactive JP4-039
analog daily.
[0292] 4. Experiments as in (1) above substituting fresh human
Stro1+ marrow cells for KM101 subclonal lines.
[0293] 5. Experiments as in (2) above substituting human Stro1+
marrow cells for KM101 subclonal lines.
[0294] Statistical Considerations
[0295] In (1), we can compare at 5 different time points between 7
groups where we use MnSOD-KM101 and/or 10.sup.5/10.sup.6 CD34+
cells, in terms of the number of CD45+ cells. In (2), we can
compare at 5 different lime points between 7 groups that use KM101,
CD34+ cells, KM101 plus CD34+ cells, the experimental compound
single or double administrations, or inactive analog of the
experimental compound single or double administrations, in terms of
the same endpoint as in (1). Tasks (3) and (4) are the same as (A)
and (B) of Example 13, respectively, except that we can use human
Stro1+ marrow cells in place of KM101 cells. All the comparisons in
this task can be performed separately for high and low dose
radiated legs. ANOVA followed by Tukey's test can be used for these
analyses. Similar to the sample size considerations in Example 13,
we will use 6 mice per group at each time point. As the secondary
endpoint, the number of CFU-GEMM can also be compared between
groups with the same method as the primary endpoint.
[0296] Likely Outcomes
[0297] Based on the results of Example 13, we expect that cord
blood stem cell and human bone marrow stromal cell homing in vitro
will be optimized by MnSOD-PL treatment of the mouse
microenvironment prior to stromal cell transplantation, and that
MnSOD-PL overexpressing KM101 cells will show further stability in
the irradiated microenvironment. We expect that JP4-039 treatment
will further enhance hematopoietic cell survival and increase
CFU-GEMM in numbers.
Example 15 (Proposed)
[0298] These experiments utilize osteogenesis by human stromal
cells as a measure of effective mitigation of marrow injury by
JP4-039. JP4-039 can be tested for repair of artificial fracture of
the proximal tibiae in NOD/SCID mice by human stromal cell derived
osteoblasts producing human collagen and can show enhanced fracture
healing by antioxidant JP4-039 treatment.
[0299] Experiments with mice engrafted with KM101-MnSOD/ds-red
compared to KM101 cells. Mice may have holes drilled in both
proximal tibias as described above, then irradiation 300 cGy total
body dose, 1000 cGy to one hind limb, and then 24 hours later
injection of 1.times.10.sup.5 bone marrow stromal cells of each
line. Mice can be followed for 21 days and at serial seven days
time points tibias explanted and assayed for relative content of
human collagen in the healed bones.
[0300] JP4-039 weekly or daily supplemented injections in a repeat
experiment of experiment described in (A) (12 mice per group).
[0301] Mice receiving MnSOD-PL intravenously 24 hours prior to
irradiation (on the day of bone drilling), and then injection of
either KM101-MnSOD/ds-red or KM101-ds-red.
[0302] Mice receiving scrambled sequence MnSOD-PL injection prior
to cell line injection as described in (C) above.
[0303] ) Experiments as in (A-D) substituting fresh Stro1+ stromal
cells for KM101 subclones.
[0304] Statistical Considerations
[0305] In (A), we compare at 3 different time points 17 groups that
use KM101 cells, MnSOD, the experimental compound single or double
administrations, scrambled MnSOD, or a combination of some of
these, in terms of the percent of human collagen. (B) is the same
as (A) except that human Stro1+ marrow cells are used in place of
KM101 cells. All the comparisons in this task can be performed
separately for high and low dose radiated legs. ANOVA followed by
Tukey's test can be used for these analyses. Similar to the sample
size considerations in Example 13, we can use 6 mice per group at
each time point.
[0306] Likely Outcomes
[0307] We expect that KM101-MnSOD/ds-red will demonstrate improved
osteogenic capacity in vivo. We anticipate that MnSOD-PL
administration to mice 24 hours prior to irradiation will further
enhance homing and osteoblast differentiation of
KM101-MnSOD/ds-red.
[0308] Preliminary data show radiation survival curves of bone
marrow stromal cell lines and enhancement of survival by MnSOD
overexpression. Other preliminary data are expected to show that
each Stro1+ cell transfected with MnSOD-PL and KM101-MnSOD/ds-red
as well as KM101-ds-red are capable to differentiation to
osteoblasts in vitro (osteogenic media experiments in progress) and
in vivo in hole drilled NOD/SCID mice. Radiation survival curves of
KM101-MnSOD/ds-red and KM101-ds-red treated with JP4-039, but not
the inactive analog of JP4-039 are shown above. We anticipate that
three conditions: 1) MnSOD-PL administration to the
microenvironment, 2) overexpression of MnSOD in bone marrow stromal
cell lines of human origin, and 3) supplementation of JP4-039
antioxidant therapy will lead to maximum osteogenic differentiation
by human origin collagen producing cells. As further controls for
the experiments, we can determine whether hematopoietic cells of
human origin are required for optimal functioning of bone marrow
stromal cells. KM101-MnSOD/ds-red stromal cell seeded NOD/SCID mice
can be supplemented with injection of human cord blood CD34+ LIN-,
or CD34+ LIN+ cells administered either with the stromal cells, or
24 hours later, to see if these cells produce optimal colony
formation. Other controls can be CD34+ LIN-, CD34+ LIN+
hematopoietic cells only. Other controls may include STRO1+ stromal
cells progenitors from cord blood alone or whole cord blood
controls.
Example 16
[0309] To assess the effectiveness of XJB-5-131 in inhibiting
degeneration and/or signs of aging, the compound was administered,
over an 18-21 week period, progeroid Ercc1.sup.-/.DELTA. mice, at a
dose of 2 mg/kg in sunflower oil carrier (to promote solubility)
administered intraperitoneally three times per week (FIG. 32).
Sunflower seed oil was administered to twin Ercc1.sup.-/.DELTA.
mice according to the same schedule as a control. The treated and
control mice were monitored twice a week for weight and
symptom/sign development.
[0310] FIG. 33 presents a summary table showing the results of the
treatment with XJB-5-131 ("XJB" in this figure), relative to
control (sunflower seed oil) after treatment from 5 wks of life
until death. The numbers indicate the average age at onset of each
age-related symptom for mice treated with XJB-5-131 or vehicle only
(oil) (n=5 mice per group). Cells highlighted in yellow indicate
symptoms that were significantly delayed by XJB-5-131. In addition
to improvement in most signs measured, the overall aging score was
significantly improved in the XJB-5-131-treated mice. Of note, all
of the signs of neurodegeneration, including dystonia, trembling,
ataxia, wasting and urinary incontinence were delayed in the
treated animals, providing strong evidence that XJB-5-131 protects
neurons against degenerative changes caused by oxidative
stress.
[0311] To assess the ability of XJB-5-131 to inhibit deterioration
of intervertebral discs (an index of degenerative disease of the
vertebra), the level of glycosaminoglycan in the discs in treated
and control mice were measured, and the results are shown in FIG.
34. The intervertebral discs of treated mice contained
approximately 30 percent more glycosaminoglycan relative to control
mice, indicating inhibition of disc degeneration.
[0312] As a measure of the effect of XJB-5-131 on photoaging,
Ercc1.sup.-/cond; K14-Cre mice, which are missing ERCC1 only in the
skin, were shaved, treated with a depilatory then irradiated with
UV-B light to induce a sunburn (500 J/m.sup.2, the median erythemal
dose). Subsequently, the mice were treated with XJB-5-131 (80
.mu.g) emulsified in a cream daily for five days. The results,
shown in FIG. 35, indicate that the skin of treated mice appeared
much more smooth and healthy relative to control (mice treated with
cream only).
[0313] At a macroscopic level, administration of XJB-5-131 appears
to have been well-tolerated by the animals, as indicated by the
fact that they did not lose weight as a result of treatment. Graphs
showing weight over time of treated, untreated and control animals
are shown in FIG. 36A-B. To assess the impact of XJB-5-131 at a
cellular level a number of experiments were carried out using mouse
embryonic fibroblasts ("MEF") cells harvested from Ercc1.sup.-/-
mouse embryos. As shown in FIG. 37, such MEF cultures were prepared
and grown under ambient oxygen (oxidative stress) conditions, and
then either untreated (media only) or treated with a concentration
of 500 nM (in media) XJB-5-131, and then tested for SA-.beta.
galactosidase staining (a marker of cellular senescence). The
amount of staining was notably less in the treated cells. In
addition, XJB-5-131 treatment was found to reduce the number of
.gamma.H2AX foci in DNA (a second marker of cellular senescence as
well as DNA double-strand breaks) (FIG. 38), although it did not
reduce the amount of apoptosis (FIG. 39).
Example 17--Protective Effects of JP4-039
[0314] To assess the therapeutic potential of JP4-039, tests for
safety and protective activity were performed. FIGS. 35 and 36 show
the results of tests to evaluate whether varying concentrations of
JP4-039 produce toxic effects after 48 hours in cultures of MEF
cells prepared from Ercc1.sup.-/- or wild-type mouse embryos,
respectively. Even under the highest concentrations tested (10
.mu.M), no signs of toxicity were observed in either culture system
and cellular proliferation is enhanced relative to untreated
control cells (media only).
[0315] To test the protective activity of JP4-039, cultures of
primary MEFs were prepared from Ercc1.sup.-/- mouse embryos and
grown under 20% oxygen (ambient air), which creates oxidative
stress in these cells that are hypersensitive to reactive oxygen
species. The cells were then either treated with a concentration of
1 uM XJB-5-131, JP4-039, JED-E71-37 or JED-E71-58, or left
untreated (media), and then after 48 hrs the level of p16, a marker
of irreversible cellular senescence, was measured by
immunofluorescence staining. As seen in FIG. 37, the level of p16
was much lower in MEF cells treated with JP4-039 relative to its
level in untreated cells, whereas XJB-5-131, JED-E71-37 and
JED-E71-58 were observed to be less effective at this
concentration.
Example 18--Protective Effects of Antioxidants in Cell Culture
[0316] To evaluate the protective activities of JED-E71-37 and
JED-E71-58, primary MEF cells were prepared from Ercc1.sup.-/- mice
and grown under conditions of oxidative stress (ambient air, 20%
oxygen). The cells were then either untreated or treated with 1
.mu.M JED-E71-37 or JED-E71-58 for a period of 48 hours. As can be
seen in FIG. 38, both compounds improved cell proliferation despite
the oxidative stress.
[0317] Next, the abilities of these two agents, as well as
XJB-5-131 and JP4-039, each at a concentration of 1 .mu.M, were
tested for their ability to prevent oxidation-induced DNA
double-strand breaks in cell cultures prepared, and oxidatively
stressed, as in the preceding paragraph. Treated as well as
untreated cells were, after 48 hours of treatment, immunostained
for .gamma.-H2AX, a marker of DNA double-strand breaks as well as
cellular senescence. Results for JED-E71-58 are shown in FIG. 39,
showing a distinct decrease in .gamma.-H2AX. JP4-039 was also
effective, but XJB-5-131 and JED-E71-37 were observed to be less
effective at this concentration.
Example 19--Alternative Designs of Nitroxide Analogues
[0318] To further investigate the structural requirements for high
activity of GS-nitroxide compound JP4-039, we have designed several
nitroxide analogues. FIG. 40 shows a schematic of alternative
designs of nitroxide analogues. The design can encompass one or
both of: modification of the targeting group to optimize the
drug-like properties and/or investigation of alternative nitroxide
containing groups to improve their oxidant efficiency (for example
and without limitation, see Reid, D. A. et al. The synthesis of
water soluble isoindoline nitroxides and a pronitroxide
hydroxylamine hydrochloride UV-VIS probe for free radicals. Chem
Comm. 1998, 17:1907-8; Iwabuchi, Y. J., Exploration and
Exploitation of Synthetic Use of Oxoammonium Ions in Alcohol
Oxidation. J. Synth. Org. Chem. Jpn. 2008, 66(11):1076-84).
Modification of the targeting group can include replacement of Boc
for alternative protecting groups, such as Ac (--C(O)CH.sub.3), Cbz
(--C(O)O-Bn, where Bn is a benzyl group) or dialkylphosphates.
Dialkylphosphates include --P(O)-Ph.sub.2, where Ph is a phenyl
group. Other modifications also include isosteric replacement of
the alkene group within the targeting group, such as with a
cyclopropane group. The nitroxide containing group includes TEMPO
and TEMPOL, as well as alternative nitroxide moieties, such as TMIO
(1,1,3,3-tetramethylisoindolin-2-yloxyl) or 1-Me-AZADO (1-methyl
2-azaadamantane N-oxyl). Synthesis protocols of these alternative
nitroxide moieties are provided below.
[0319] FIG. 41 shows a synthetic protocol that can be used to
produce various alternative designs of nitroxide analogues,
including JP4-039, compounds according to Formula 2, compounds
according to Formula 3, and other analogues. The specific synthesis
of JP4-039 has been described above in Example 8. JP4-039 and its
analogues were prepared via an efficient method for the asymmetric
synthesis of allylic amines, previously developed in our laboratory
(Wipf P. & Pierce J. G. Expedient Synthesis of the
.alpha.-C-Glycoside Analogue of the Imnmnostimulant
Galactosylceramide (KRN7000), Org. Lett. 2006, 8(15):3375-8). One
key step in FIG. 41 includes use of the zirconium methodology to
produce a diastereomeric allylic amine (7). This methodology
includes hydrozirconation of alkyne (5) with Cp.sub.2ZrHCl,
transmetalation to Me.sub.3Al, and addition to AZ-tBu-sulfinyl
amine (3). The Smith cyclopropanation of the alkene (8b) with
Zn(CH.sub.2I).sub.2 is another key step in FIG. 41. In tins latter
step, the stereochemistry around the cyclopropane ring is to be
determined after the reaction.
[0320] Synthesis of compounds (10a, JP4-039), (10b), (10c), (14a),
and (14b) (shown in FIG. 41) was accomplished according to the
following.
(R,E)-2-Methyl-N-(3-methylbutylidene)propane-2-sulfinamide (3)
[0321] The synthesis of the title compound has already been
described in Example 8 (compound 1).
(But-3-ynyloxy)(tert-butyl)diphenylsilane (5)
[0322] The synthesis of the title compound has already been
described in Example 8 (compound 2).
(S,E)-8-(tert-Butyldiphenylsilyloxy)-2-methyloct-5-en-4-amine
hydrochloride (7)
[0323] The synthesis of the title compound has already been
described in Example 8 (compound 3).
##STR00097##
(S,E)-tert-Butyl
8-(tert-butyldiphenylsilyloxy)-2-methyloct-5-en-4-ylcarbamate
(8a)
[0324] The synthesis of the title compound has already been
described in Example 8 (compound 4).
(S,E)-Benzyl
8-(tert-butyldiphenylsilyloxy)-2-methyloct-5-en-4-ylcarbamate
(8b)
[0325] To a mixture of the amine 7 (1.50 g, 3.79 mmol) in dry THF
(15 mL) were added Et.sub.3N (1.65 mL, 11.75 mmol), and then a
solution of benzyl chloroformate (CbzCl, 0.59 mL. 4.17 mmol) in dry
THF (4 mL) at 0.degree. C. The resulting white suspension was
allowed to warm to rt and stirred for 5 h, then diluted with DCM
and water. The aqueous phase was extracted with DCM (2.times.), and
the combined organic layers were washed with 10% HCl and sat.
NaHCO.sub.3, dried (MgSO.sub.4), filtered and concentrated in
vacuo. Flash chromatography (SiO.sub.2, 8:2, hexanes/EtOAc)
afforded 1.45 g (72%) of the title compound as a yellow oil.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.75-7.65 (m, 4H),
7.50-7.28 (m, 11H), 5.70-5.55 (m, 1H), 5.40 (dd, 1H, J=15.4, 6.2
Hz), 5.11 (s, 2H), 4.58 (m, 1H), 4.21 (m, 1H), 3.71 (t, 2H, J=6.6
Hz), 2.30 (q, 2H, J=6.6 Hz), 1.67 (m, 1H), 1.40-1.22 (m, 2H), 1.07
(s, 9H), 0.92 (m, 6H); HRMS (ESI) m/z calcd for
C.sub.33H.sub.43NO.sub.3SiNa 552.2910, found 552.2930.
(S,E)-N-(8-(tert-Butyldiphenylsilyloxy)-2-methyloct-5-en-4-yl)-P,P-dipheny-
lphosphinic amide (8c)
[0326] To a solution of the amine 7 (400 mg, 1.01 mmol) in dry DCM
(7 mL) were added Et.sub.3N (0.44 mL, 3.13 mmol), and then a
solution of diphenylphosphinic chloride (Ph.sub.2POCl, 0.22 mL,
1.11 mmol) in dry DCM (3 mL) at 0.degree. C. After being stirred at
0.degree. C. for 15 min, the reaction mixture was allowed to warm
to rt and stirred for 4 h, then diluted with DCM and 10% HCl. The
aqueous phase was extracted with DCM and the combined organic
layers were washed with sat. NaHCO.sub.3, dried (MgSO.sub.4),
filtered and concentrated in vacuo to afford 720 mg of the crude
title compound as a pale yellow solidified oil, which was used for
the next step without further purification.
##STR00098##
(S,E)-tert-Butyl 8-hydroxy-2-methyloct-5-en-4-ylcarbamate (9a)
[0327] The synthesis of the title compound has already been
described in Example 8 (compound 5).
(S,E)-Benzyl 8-hydroxy-2-methyloct-5-en-4-ylcarbamate (9b)
[0328] To a solution of the TBDPS-protected alcohol 8b (584 mg,
1.10 mmol, crude) in dry THF (9 mL) at 0.degree. C. was added TBAF
(1.0M/THF, 1.38 mL, 1.38 mmol), and the reaction mixture was
allowed to warm to rt while stirring under argon for 3.5 h, then
quenched with sat. aq. NH.sub.4Cl and diluted with EtOAc. The
aqueous phase was separated and extracted with EtOAc. The combined
organic layers were washed with brine, dried (Na.sub.2SO.sub.4),
filtered and concentrated in vacuo. Flash chromatography
(SiO.sub.2, 5:5, hexanes/EtOAc) afforded 194 mg (60%, 2 steps) of
the title compound as a colorless oil. [.alpha.].sub.D.sup.23 -6.4
(c 1.0, DCM); .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.20-7.40
(m, 5H), 5.65-5.49 (m, 1H), 5.44 (dd, 1H, 7=15.3, 6.6 Hz), 5.09 (s,
2H), 4.67 (bs, 1H), 4.16 (m, 1H), 3.63 (bs, 2H), 2.28 (q, 2H, J=6.0
Hz), 1.82 (bs, 1H), 1.65 (m, 1H), 1.40-1.25 (m, 2H), 0.80-1.00 (m,
6H); HRMS (ESI) m/z calcd for C.sub.17H.sub.25NO.sub.3Na 314.1732,
found 314.1739.
(S,E)-N-(8-Hydroxy-2-methyloct-5-en-4-yl)-P,P-diphenylphosphinic
amide (9c)
[0329] To a solution of the TBDPS-protected alcohol 8c (700 mg,
0.983 mmol, crude) in dry THF (8 mL) at 0.degree. C. was added TBAF
(1.0M/THF, 1.23 mL, 1.23 mmol), and the reaction mixture was
allowed to warm to rt while stirring under argon. As completion was
not reached after 4 h, 0.75 eq of TBAF (0.75 mL) was added at
0.degree. C. The reaction mixture was stirred further at rt for 3
h, then quenched with sat. aq. NH.sub.4Cl and diluted with EtOAc.
The aqueous phase was separated and extracted with EtOAc. The
combined organic layers were washed with brine, dried
(Na.sub.2SO.sub.4), filtered and concentrated in vacuo. Hash
chromatography (SiO.sub.2, 95:5, EtOAc/MeOH) afforded 272 mg (77%,
2 steps) of the title compound as a white solid, mp
124.0-124.2.degree. C.; [.alpha.].sub.D.sup.23 -12.1 (c 1.0, DCM);
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.00-7.83 (m, 4H),
7.58-7.35 (m, 6H), 5.52 (dd, 1H, J=15.3, 9.0 Hz), 5.24 (m, 1H),
4.58 (bs, 1H), 3.78-3.47 (m, 3H), 2.80 (appdd, 1H, J=9.2, 3.8 Hz),
2.16 (m, 2H), 1.68 (bs, 1H), 1.55-1.43 (m, 1H), 1.43-1.31 (m, 1H),
0.87 (dd, 6H, J=8.6, 6.4 Hz); HRMS (ESI) m/z calcd for
C.sub.21H.sub.28NO.sub.2PNa 380.1755, found 380.1725.
##STR00099##
TEMPO-4-yl-(S,E)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide
(10a, JP4-039)
[0330] The synthesis of the title compound has already been
described in Example 8 (compound 7).
TEMPO-4-yl-(S,E)-5-(benzyloxycarbonylamino)-7-methyloct-3-enamide
(10b)
[0331] To a solution of the alcohol 9b (158 mg, 0.543 mmol) in
acetone (5 mL) at 0.degree. C. was added slowly a freshly prepared
solution of Jones reagent (2.5M, 0.54 mL, 1.358 mmol). The
resulting dark suspension was stirred at 0.degree. C. for 1 h, then
diluted with Et.sub.2O and water. The aqueous phase was separated
and extracted with Et.sub.2O (2.times.). The combined organic
layers were washed with water (2.times.) and brine (1.times.),
dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo to
yield 166 mg (quant.) of the crude acid as a slightly yellow oil,
that was used for the next step without further purification.
[0332] To a solution of this acid (160 mg, 0.524 mmol, crude) in
dry DCM (7 mL) at 0.degree. C. were added successively a solution
of 4-amino-TEMPO (139 mg, 0.786 mmol) in dry DCM (0.5 mL), DMAP (71
mg, 0.576 mmol), HOBt H.sub.2O (78 mg, 0.576 mmol) and EDCI (123
mg, 0.629 mmol). The resulting orange solution was stirred at rt
under argon for 15 h, and then washed with sat. NH.sub.4Cl. The
aqueous phase was separated and extracted once with DCM, and the
combined organic layers were dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo. Hash chromatography (SiO.sub.2, 5:5 to 3:7,
hexanes/EtOAc) afforded 171 mg (71%) of the title compound as a
peach colored foam, mp 60.5.degree. C. (softening point: 44.degree.
C.); [.alpha.].sub.D.sup.23+26.5 (c 0.5, DCM); EIMS m/z 458
([M].sup.+, 37), 281 (19), 154 (28), 124 (47), 91 (100), 84 (41);
HRMS (EI) m/z calcd for C.sub.26H.sub.40N.sub.3O.sub.4 458.3019,
found 458.3035.
TEMPO-4-yl-(S,E)-5-(diphenylphosphorylamino)-7-methyloct-3-enamide
(10c)
[0333] To a solution of the alcohol 9c (166.5 mg, 0.466 mmol) in
acetone (5 mL) at 0.degree. C. was slowly added a freshly prepared
solution of Jones reagent (2.5M, 0.47 mL, 1.165 mmol). The
resulting dark suspension was stirred at 0.degree. C. for 2 h, then
diluted with Et.sub.2O and water. The aqueous phase was separated
and extracted with Et.sub.2O (2.times.). The combined organic
layers were washed with water (2.times.) and brine (1.times.),
dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo to
yield 114 mg (66%) of the crude acid as a white foam, that was used
for the next step without further purification.
[0334] To a solution of this acid (110 mg, 0.296 mmol, crude) in
dry DCM (3.5 mL) at 0.degree. C. were added successively a solution
of 4-amino-TEMPO (78.4 mg, 0.444 mmol) in dry DCM (0.5 mL), DMAP
(40.2 mg, 0.326 mmol), HOBt.H.sub.2O (44.0 mg, 0.326 mmol) and EDCI
(69.5 mg, 0.355 mmol). The resulting orange solution was stirred at
rt under argon for 13 h, and then washed with sat. NH.sub.4Cl. The
aqueous phase was separated and extracted once with DCM, and the
combined organic layers were dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo. Flash chromatography (SiO.sub.2, EtOAc to
97:3, EtOAc/MeOH) afforded 91.2 mg (59%) of the title compound as
an orange oil which solidified very slowly upon high vacuum, mp
168.0-168.8.degree. C. (softening point: .about.75.degree. C.);
[.alpha.].sub.D.sup.23 -14.1 (c 0.5, DCM); EIMS m/z 525
([M+H].sup.+, 10), 371 (27), 218 (28), 201 (74), 124 (100), 91
(35), 84 (26); HRMS (EI) m/z calcd for
C.sub.30H.sub.43N.sub.3O.sub.3P 524.3042, found 524.3040.
##STR00100##
Benzyl
(1S)-1-(2-(2-(tert-butyldiphenylsilyloxy)ethyl)cyclopropyl)-3-meth-
ylbutylcarbamate (11b)
[0335] To a solution of ZnEt.sub.2 (110 mg, 0.844 mmol) in dry DCM
(2 mL) was added DME (distilled, 0.088 mL, 844 mmol). The reaction
mixture was stirred at rt for 10 min under N.sub.2, then cooled to
-20.degree. C. and CH.sub.2I.sub.2 (0.137 mL, 1.687 mmol) was added
dropwise over 4 min. After stirring for 10 min, a solution of the
alkene 8b (149 mg, 0.281 mmol) in dry DCM (1 mL) was added dropwise
over 5 min. The reaction mixture was allowed to warm to rt while
stirring. After 10 h, the reaction mixture was quenched with sat.
aq. NH.sub.4Cl and diluted with DCM and water, the aqueous phase
was separated and extracted with EtOAc. The combined organic layers
were dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo.
Rash chromatography (SiO.sub.3, 9:1, hexanes/Et.sub.2O) afforded
785 mg (68%) of the title compound as a colorless oil. .sup.1H NMR
analysis showed only 1 diastereomer (>95:5 dr),
[.alpha.].sub.D.sup.23 -26.8 (r 1.0, DCM); .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 7.73-7.66 (m, 4H), 7.48-7.28 (m. 11H),
5.13-4.96 (m, 2H), 4.62 (appbd, 1H, J=8.4 Hz), 3.72 (appbt, 2H,
J=6.4 Hz), 3.21 (m, 1H), 1.80-1.63 (m, 1H). 1.60-1.25 (m, 4H), 1.08
(s, 9H), 0.92 (appd, 6H, J=6.3 Hz), 0.79 (m, 1H), 0.51 (m, 1H),
0.40 (m, 1H), 0.30 (m, 1H); HRMS (ESI) m/z calcd for
C.sub.34H.sub.45NO.sub.3SiNa 566.3066, found 566.3103.
(1S)-1-(2-(2-(Tert-butyldiphenylsilyloxy)ethyl)cyclopropyl)-3-methylbutan--
1-amine (12)
[0336] A flask containing a solution of the Cbz-protected amine 11b
(460 mg, 0.846 mmol) in a 5:1 MeOH/EtOAc mixture (12 mL) was purged
and filled 3 limes with argon, then 10% Pd/C (50 mg) was added. The
flask was purged and filled 3 times with H.sub.2, and the resulting
black suspension was stirred at rt under H.sub.2 (1 atm). Since the
reaction did not reach completion after 3 h, an additional amount
of 10% Pd/C (30 mg) was added and stirring under H.sub.2 was
continued for 5 h. The reaction mixture was then filtered through a
pad of Celite, the Celite washed with MeOH and AcOEt, and the
solution concentrated in vacuo to yield 317 mg (92%) of the crude
title compound as a pale yellow oil, that was used for the next
step without further purification.
Tert-butyl
(1S)-1-(2-(2-(tert-butyldiphenylsilyloxy)ethyl)cyclopropyl)-3-m-
ethylbutylcarbamate (11a)
[0337] To a solution of the amine 12 (309 mg, 0.755 mmol) in dry
DCM (12 mL) was added Et.sub.3N (0.21 mL, 0153 mmol) and then
Boc.sub.2O (183 mg, 0.830 mmol) at 0.degree. C. The reaction
mixture was stirred at rt under N.sub.2 for 28 h. The reaction was
quenched with sat. aq. NH.sub.4Cl and the aqueous phase extracted
with DCM The combined organic layers were dried (Na.sub.2SO.sub.4),
filtered and concentrated in vacuo to yield 471 mg of the crude
title compound as a colorless oil, that was used for the next step
without further purification.
##STR00101##
Tert-butyl
(1S)-1-(2-(2-hydroxyethyl)cyclopropyl)-3-methylbutylcarbamate
(13a)
[0338] To a solution of the crude TBDPS-protected alcohol 11a (464
mg, 0.742 mmol) in dry THE (6 mL) at 0.degree. C. was added TBAF
(1.0M/THE, 0.93 mL, 0.927 mmol), and the reaction mixture was
allowed to warm to rt while stirring under N.sub.2. Since TLC
showed uncomplete reaction after 5 h. 0.75 eq. TBAF (0.56 mL) was
added. After 9 h, the reaction mixture was quenched with sat. aq.
NH.sub.4Cl and diluted with EtOAc. The aqueous phase was separated
and extracted with EtOAc. The combined organic layers were washed
with brine, dried (Na.sub.2SO.sub.4), filtered and concentrated in
vacuo. Flash chromatography (SiO.sub.1, 5:5, hexanes/EtOAc)
afforded 177 mg (88%) of the title compound as a colorless oil
which solidified upon high vacuum to give a white powder, mp
49.8-50.2.degree. C.; [.alpha.].sub.D.sup.23 -30.8 (c 1.0, DCM),
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 4.50 (appbd, 1H, J=4.5
Hz), 3.66 (bs, 2H), 2.94 (m, 1H), 2.36 (bs, 1H), 1.82 (bs, 1H),
1.71 (m, 1H), 1.45 (s, 9H), 1.39 (t, 2H, J=7.2 Hz), 1.01 (bs, 2H),
0.90 (dd, 6H, J=10.2, 6.6 Hz), 0.50 (m, 1H), 0.43-0.27 (m, 2H);
HRMS (ESI) m/z calcd for C.sub.15H.sub.29NO.sub.3Na 294.2045, found
294.2064.
Benzyl
(1S)-1-(2-(2-hydroxyethyl)cyclopropyl)-3-methylbutylcarbamate
(13b)
[0339] To a solution of the TBDPS-protected alcohol 11b (320 mg,
0.588 mmol) in dry THE (5 mL) at 0.degree. C. was added TBAF
(1.0M/THF, 0.74 mL, 0.735 mmol), and the reaction mixture was
allowed to warm to rt while stirring under argon for 7 h, then
quenched with sat. aq. NH.sub.4Cl and diluted with EtOAc. The
aqueous phase was separated and extracted with EtOAc. The combined
organic layers were washed with brine, dried (Na.sub.2SO.sub.4),
filtered and concentrated in vacuo. Hash chromatography (SiO.sub.2,
5:5, hexanes/EtOAc) afforded 166 mg (92%) of the title compound as
a colorless oil. [.alpha.].sub.D.sup.23 -21.6 (c 1.0, DCM); .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 7.42-7.28 (m, 5H), 5.10 (m, 2H),
4.76 (appbd, 1H, J=5.7 Hz), 3.63 (bs, 2H), 3.04 (m, 1H), 2.12-1.98
(bs, 1H), 1.83-1.62 (m, 2H), 1.42 (t, 2H, J=7.0 Hz), 1.16-0.95 (m,
2H), 0.90 (appt, 6H, J=7.0 Hz), 0.53 (sept, 1H, J=4.3 Hz), 0.42
(dt, 1H, J=8.4, 4.5 Hz), 0.34 (dt, 1H, J=8.4, 5.0 Hz); HRMS (ESI)
m/z calcd for C.sub.18H.sub.27NO.sub.3Na 328.1889, found
328.1860.
##STR00102##
TEMPO-4-yl-2-(2-((S)-1-(tert-butoxycarbonylamino)-3-methylbutyl)cycloprop-
yl)acetamide (14a)
[0340] To a solution of the alcohol 13a (130 mg, 0.477 mmol) in
acetone (5 mL) at 0.degree. C. was slowly added a solution of Jones
reagent (2.5M, 0.48 mL, 1.194 mmol). The resulting dark suspension
was stirred at 0.degree. C. for 1 h, then diluted with Et.sub.2O
and water. The aqueous phase was separated and extracted with
Et.sub.2O (2.times.). The combined organic layers were washed with
water (2.times.) and brine (1.times.), dried (Na.sub.2SO.sub.4),
filtered and concentrated in vacuo to yield 133 mg (97%) of the
crude title compound as a colorless oil, that was used for the next
step without further purification.
[0341] To a solution of this acid (127.6 mg, 0.447 mmol, crude) in
dry DCM (5.5 mL) at 0.degree. C. were added successively a solution
of 4-amino-TEMPO (118.4 mg, 0.671 mmol) in dry DCM (0.5 mL), DMAP
(60.7 mg, 0.492 mmol), HOBt.H.sub.2O (66.4 mg, 0.492 mmol) and EDCI
(105.0 mg, 0.536 mmol). The resulting orange solution was stirred
at rt under argon for 15 h, and then washed with sat. NH.sub.4Cl.
The aqueous phase was separated and extracted once with DCM, and
the combined organic layers were dried (Na.sub.2SO.sub.4), filtered
and concentrated in vacuo. Flash chromatography (SiO.sub.2, 5:5 to
3:7, hexanes/EtOAc) afforded 150.0 mg (76%) of the title compound
as a peach colored foam, mp 139.5.degree. C.;
[.alpha.].sub.D.sup.23 -15.7 (c 0.5, DCM); EIMS m/z 438 ([M].sup.+,
6), 252 (57), 140 (67), 124 (80), 91 (48), 84 (59), 57 (100); HRMS
(ED m/z calcd for C.sub.24H.sub.44N.sub.3O.sub.4 438.3332, found
438.3352.
TEMPO-4-yl-2-(2-((S)-1-(benzyoxycarbonylamino)-3-methylbutyl)cyclopropyl)a-
cetamide (14b)
[0342] To a solution of the alcohol 13b (110.5 mg, 0.362 mmol) in
acetone (5 mL) at 0.degree. C. was slowly added a solution of Jones
reagent (2.5M, 0.36 mL, 0.904 mmol). The resulting dark suspension
was stirred at 0.degree. C. for 1 h, then diluted with Et.sub.2O
and water. The aqueous phase was separated and extracted with
Et.sub.2O (2.times.). The combined organic layers were washed with
water (2.times.) and brine (1.times.), dried (Na.sub.2SO.sub.4),
filtered and concentrated in vacuo to yield 113.5 mg (98%) of the
erode title compound as a colorless oil, that was used for the next
step without further purification.
[0343] To a solution of this acid (110 mg, 0.344 mmol, crude) in
dry DCM (4.5 mL) at 0.degree. C. were added successively a solution
of 4-amino-TEMPO (91.2 mg, 0.517 mmol) in dry DCM (0.5 mL), DMAP
(46.7 mg, 0.379 mmol), HOBt.H.sub.2O (51.2 mg, 0.379 mmol) and EDCI
(80.8 mg, 0.413 mmol). The resulting orange solution was stirred at
rt under argon for 18 b, and then washed with sat. NH.sub.4Cl. The
aqueous phase was separated and extracted once with DCM, and the
combined organic layers were dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo. Flash chromatography (SiO.sub.2, 4:6,
hexanes/EtOAc) afforded 123 mg (75%) of the title compound as a
peach colored foam, mp 51.8.degree. C. (softening point: 44.degree.
C.); [.alpha.].sub.D.sup.23 -15.3 (c 0.5, DCM); EIMS m/z 472
([M].sup.+, 42), 415 (58), 322 (43), 168 (47), 140(46), 124 (75),
91 (100), 84(53); HRMS (EI) m/z calcd for
C.sub.27H.sub.42N.sub.3O.sub.4 472.3175, found 472.3165.
Example 20--Synthesis of Alternative Nitroxide Moieties
[0344] Schematics are shown for alternative nitroxide moieties,
where FIG. 42 shows a synthesis protocol for
5-amino-1,1,3,3-tetramethylisoindolin-2-yloxyl (5-amino-TMIO) and
FIG. 43 shows a synthesis protocol for 6-amino-1-methyl
2-azaadamantane N-oxyl (6-amino-1-Me-AZADO).
[0345] Compounds 5-amino-1,1,3,3-tetramethylisoindolin-2-yloxyl
(5-amino-TMIO) and (20) are shown m FIG. 42 and were prepared
according to the following.
[0346] Synthesis of 5-amino-TMIO was previously described by Reid,
D. A. et al (The synthesis of water soluble isoindoline nitroxides
and a pronitroxide hydroxylamine hydrochloride UV-VIS probe for
free radicals. Chem Comm. 1998, 77, 1907-8) and references cited
therein.
##STR00103##
2-Benzyl-1,1,3,3-tetramethylisoindoline (16)
[0347] (First step: Org. Synth. 1998, 9, 649; second step:
Griffiths, P. G et al. Synthesis of the radical scavenger
1,1,3,3-tetramethylisoindolin-2-yloxyl. Aust J. Chem. 1983, 36,
397-401). An oven-dried 250 mL, three-necked, round-bottom flask
was flushed with nitrogen, and magnesium turnings (3.84 g, 156.5
mmol) were introduced, that were covered with dry Et.sub.2O (9 mL).
A solution of MeI (9.45 mL, 150.2 mmol) in dry Et.sub.2O (80 mL)
was then added dropwise via a dropping funnel while stirring over a
period of 50 min. The resulting reaction mixture was then stirred
for an additional 30 min, and then concentrated by slow
distillation of solvent until the internal temperature reached
80.degree. C. The residue was allowed to cool to 60.degree. C., and
a solution of N-benzylphthalimide (6.00 g, 25.04 mmol) in dry
toluene (76 mL) was added dropwise via a dropping funnel with
stirring at a sufficient rate to maintain this temperature. When
the addition was complete, solvent was distilled slowly from the
mixture until the temperature reached 108-110.degree. C. The
reaction mixture was refluxed at 110.degree. C. for 4 h, then
concentrated again by further solvent distillation. It was then
cooled and diluted with hexanes (turned purple). The resulting
slurry was filtered through Celite and washed with hexanes. The
combined yellow filtrate turned dark red-purple after standing in
air overnight. It was then concentrated in vacuo. The resulting
purple residue was passed through a short column of basic alumina
(grade I, 70-230 mesh), eluting with hexanes (.about.1 L), to
afford 2.585 g (39%) of the title compound as a colorless oil which
solidified to give a white solid, mp 61.0-61.4.degree. C. .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 7.48 (appd, 2H, J=7.2 Hz),
7.34-7.19 (m, 5H), 7.18-7.11 (m, 2H), 4.00 (s, 2H), 1.31 (s, 12H);
HRMS (EI) m/z calcd for C.sub.19H.sub.23N 265.1830, found
265.1824.
##STR00104##
1,1,3,3-Tetramethylisoindoline (17)
[0348] (Griffiths, P. G. et al. Synthesis of the radical scavenger
1,1,3,3-tetramethylisoindolin-2-yloxyl. Aust. J. Chem. 1983, 36,
397-401; Chan, K. S. et al. Reactions of nitroxides with
metalloporphyrin alkyls bearing beta hydrogens: aliphatic
carbon-carbon bond activation by metal centered radicals. J.
Organomet. Chem. 2008, 693, 399407). The protected benzyl-amine 16
(1.864 g, 7.02 mmol) was dissolved in AcOH (34 mL) in a Parr flask,
and 10% Pd/C (169.5 mg) was added. (The reaction was splited in 3
batches.) The flask was placed in a high pressure reactor. The
reactor was charged with H.sub.2 and purged for 5 cycles and was
finally pressurized with H.sub.2 at 4 bars (60 psi). After stirring
at rt for 3 h. the reaction mixture was filtered through Celite,
and the solvent removed in vacuo. The resulting residue was
dissolved in water (5 mL) and the solution neutralized with 2.5N
NaOH (pH 11.5), and extracted with Et.sub.2O (3.times.50 mL). The
combined organic layers were dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo to yield 1.165 g (95%) of the crude title
compound as slightly yellow crystals, mp 36.0-36.5.degree. C.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.30-7.23 (m, 2H),
7.18-7.11 (m, 2H), 1.86 (bs, 1H), 1.48 (s, 12H).
##STR00105##
1,1,3,3-Tetramethylisoindolin-2-yloxyl (18)
[0349] (Griffiths, P. G. et al. Synthesis of the radical scavenger
1,1,3,3-tetramethylisoindolin-2-yloxyl. Aust. J. Chem. 1983, 36,
397-401; Chan, K. S. et al. Reactions of nitroxides with
metalloporphyrin alkyls bearing beta hydrogens: aliphatic
carbon-carbon bond activation by metal centered radicals. J.
Organomet Chem. 2008, 693, 399-407). To a solution of the amine 17
(1.46 g, 8.33 mmol) in a 14:1 mixture of MeOH/MeCN (16.6 mL) were
added successively NaHCO.sub.3 (560 mg, 6.67 mmol),
Na.sub.2WO.sub.4.2H.sub.2O (83.3 mg, 0.25 mmol) and 30% aq. (3.12
mL, 27.50 mmol). The resulting suspension was stirred at rt. After
18 h, a bright yellow suspension formed and 30% aq. H.sub.2O.sub.2
(3.00 mL, 26.44 mmol) was added. The reaction mixture was stirred
for 2 days, then diluted with water and extracted with hexanes
(2.times.). The combined organic layers were washed with 1M
H.sub.2SO.sub.4 and brine, dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo to yield 1.55 g (98% crude) of the title
compound as a yellow crystalline powder, that was used for the next
step without further purification, mp 122-125.degree. C. (softening
point: 108.degree. C.); HRMS (EI) m/z calcd for C.sub.12H.sub.17NO
191.1310, found 191.1306.
##STR00106##
5-Nitro-1,1,3,3-tetramethylisoindolin-2-yloxyl (19)
[0350] (Bolton, R. et al. An EPR and NMR study of some
tetramethylisoindolin-2-yloxyl free radicals. J. Chem. Sac. Perkin
Hans. 2, 1993, 2049-52). Conc. H.sub.2SO.sub.4 (13.5 mL) was added
dropwise to 18 (1.345 g, 7.07 mmol) cooled in an ice-water bath,
forming a dark-red solution which was then warmed to 60.degree. C.
for 15 min and then cooled to 0.degree. C. Conc. HNO.sub.3 (0.90
mL, 19.09 mmol) was added dropwise. When the reaction appeared
complete, the yellow-orange solution was heated at 100.degree. C.
for 10 min, the color turning to red-orange. After cooling to rt,
the reaction mixture was neutralized by careful addition to
ice-cooled 2.5N NaOH (30 mL). This aqueous phase was extracted with
Et.sub.2O until it became colorless and the combined organic layers
were dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo
to yield 1.64 g (98%) of the crude title compound as a
yellow-orange powder, that was used for the next step without
further purification.
##STR00107##
5-amino-1,1,3,3-tetramethylisoindolin-2-yloxyl (5-amino-TMIO)
[0351] (First step: Reid, D. A. et al. The synthesis of water
soluble isoindoline nitroxides and a pronitroxide hydroxylamine
hydrochloride UV-VIS probe for free radicals. Chem Comm. 1998, 77,
1907-8; Giroud, A. M. and Rassat, A. Nitroxydes LXXX: syntheses de
mono et biradicaux nilroxydes derives de l'isoindoline. Bull. Soc.
Chim. Fr. 1979, 11, 48-55; second step: Keana, J. F. W. and Lee, T.
D. Versatile synthesis of doxyl spin labels bypassing the usual
ketone precursors. J. Am. Chem. Soc. 1975, 97, 1273-4). A flask
containing a solution of 19 (1.50 g, 6.38 mmol, crude) in MeOH (75
mL) was purged and filled with argon, then 10% Pd/C (150 mg) was
added. The flask was purged and filled 3 times with H.sub.2, and
the resulting black suspension was stirred at rt under H.sub.2 (1
atm) for 4 h. the reaction mixture was then filtered through
Celite, the Celite washed with MeOH, and the solution concentrated
in vacuo to yield 1.38 g of the crude title compound as a yellow
solid, that was used for the next step without further
purification. .sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 6.89 (d,
1H, 7=8.1 Hz), 6.25 (dd, 1H, J=8.1, 2.1 Hz), 6.54 (d, 1H, J=2.1
Hz), 3.35 (s, 2H), 1.34 (appd, 12H, J=5.7 Hz).
[0352] To a solution of the crude hydroxylamine (1.38 g, 6.38 mmol)
in MeOH (75 mL) was added Cu(OAc).sub.2.H.sub.2O (26 mg, 0.128
mmol). The reaction mixture was stirred at rt under air for 1.5 h,
the color, turning to dark brown. The solvent was then removed in
vacuo, the residue taken up in CHCl.sub.3 and a small amount of
MeOH to dissolve the insoluble material, and washed with water. The
aqueous phase was extracted twice with CHCl.sub.3, and the combined
organic layers were washed with brine, dried (Na.sub.2SO.sub.4),
filtered and concentrated in vacuo. Flash chromatography
(SiO.sub.2, 6:4 to 5:5, hexanes/EtOAc) afforded 1.126 g (86%) of
the title compound as a yellow powder, mp 192-194.degree. C.
(softening point: 189.degree. C.; HRMS (EI) m/z calcd for
C.sub.12H.sub.17N.sub.2O 205.1341, found 205.1336.
##STR00108##
TMIO-5-yl-(S,E)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide
(20)
[0353] To a solution of the alcohol 9a (187 mg, 0.728 mmol,
prepared according to previous examples) in acetone (7 mL) at
0.degree. C. was slowly added a solution of Jones reagent (2.5M,
0.73 mL, 1.821 mmol). The resulting dark suspension was stirred at
0.degree. C. for 1 h, then diluted with Et.sub.2O and water. The
aqueous phase was separated and extracted with Et.sub.2O
(2.times.). The combined organic layers were washed with water
(2.times.) and brine (1.times.), dried (Na.sub.2SO.sub.4), filtered
and concentrated in vacuo to yield 190 mg (96%) of the crude title
compound as a slightly yellow oil, that was used for the next step
without further purification.
[0354] To a solution of this acid (187.4 mg, 0.691 mmol, crude) in
dry DCM (8 mL) at 0.degree. C. were added successively 5-amino-TMIO
(212.6 mg, 1.036 mmol), DMAP (93.7 mg, 0.760 mmol), HOBt.H.sub.2O
(102.6 mg, 0.760 mmol) and EDCI (162.1 mg, 0.829 mmol). The
resulting yellowish solution was stirred at rt under argon for 16
h. and then washed with sat. NH.sub.4Cl. The aqueous phase was
separated and extracted once with DCM, and the combined organic
layers were washed twice with 1 N HCl and once with sat.
NaHCO.sub.3, dried (Na.sub.2SO.sub.4), filtered and concentrated in
vacuo. Flash chromatography (SiO.sub.2, 6:4, hexanes/EtOAc)
afforded 221.0 mg (70%) of the title compound as a pale orange
foam, mp 78-79.degree. C. (softening point: 70.degree. C.);
[.alpha.].sub.D.sup.22 +72.2 (c 0.5, DCM); ESIMS m/z 481
[M+Na].sup.+, 50), 939 ([2M+Na].sup.+, 100).
[0355] Compound 6-amino-1-methyl 2-azaadamantane N-oxyl
(6-amino-1-Me-AZADO) and (30) are shown in FIG. 43 and were
prepared according to the following.
##STR00109##
2-Adamantanecarbonitrile
(tricyclo[3.3.1.13,7]decane-2-carbonitrile, 22)
[0356] (Oldenziel, O. H. et al. 2-Adamantanecarbonitrile. Org.
Synth. 1977, 57, 8; Rohde. J. J. et al. Discovery and Metabolic
Stabilization of Potent and Selective 2-Amino-N-(adamant-2-yl)
Acetamide 11.beta.-Hydroxysteroid Dehydrogenase Type 1 inhibitors.
J. Med. Chem. 2007, 50, 149-64). A 3-5.degree. C. solution of
2-adamantanone (tricyclo[3.3.1.13,7]decan-2-one, 21) (21.0 g, 137
mmol), p-tolylsulfanylmethyl isocyanide (TosMIC, 35.5 g, 178 mmol)
and EtOH (14 mL, 233 mmol) in 1,2-dimethoxyethane (DME, 470 mL) was
treated with portionwise addition of solid t-BuOK (39.2 g, 342
mmol), maintaining the internal temperature below 10.degree. C.
After the addition, the resulting slurry reaction mixture was
stirred at rt for 30 min and then al 35-40.degree. C. for 30 min.
The heterogeneous reaction mixture was filtered and the solid
washed with DME. The filtrate was concentrated in vacuo, loaded to
a short Al.sub.2O.sub.3 column (activated, neutral, Brockmann I,
150 mesh, 7 cm thick.times.15 cm height), and washed off with a 5:1
mixture of hexanes/DCM (.about.1.5 L). The solution was
concentrated in vacuo to afford 19.0 g (86%) of the title compound
as a white powder, .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 2.91
(s, 1H), 2.23-2.08 (m, 4H), 2.00-1.80 (m, 4H), 1.80-1.66 (m,
6H).
##STR00110##
2-Adamantane carboxylic Acid (23)
[0357] (Rohde, J. J. et al. Discovery and Metabolic Stabilization
of Potent and Selective 2-Amino-N-(adamant-2-yl) Acetamide
11.beta.-Hydroxysteroid Dehydrogenase Type 1 Inhibitors. J. Med.
Chem. 2007.50, 149-64). A mixture of the nitrile 22 (18.9 g, 117
mmol) in AcOH (56 mL) and 48% HBr (224 mL) was stirred at
120.degree. C. overnight. The reaction mixture was cooled at
4.degree. C., standing for 4 h, then filtered. The solid was washed
with water and dried in vacuum over silica gel overnight, to yield
20.6 g (98%) of the title compound as off-white crystals, .sup.1H
NMR (300 MHz, DMSO-4) .delta. 12.09 (s, 1H), 2.55-2.47 (m, 1H),
2.20 (bs, 2H), 1.87-1.64 (m, 10H), 1.60-1.50 (m, 2H).
##STR00111##
5,7-Dibromo-2-adamantane carboxylic acid (24)
[0358] (Adapted from Rohde, J. J. et al. Discovery and Metabolic
Stabilization of Potent and Selective 2-Amino-N-(adamant-2-yl)
Acetamide 11.beta.-Hydroxysteroid Dehydrogenase Type 1 Inhibitors.
J. Med Chan. 2007, 50, 149-64). A vigorously stirred 0.degree. C.
solution of AlBr.sub.3 (18.9 g, 69.6 mmol), BBr.sub.3 (2.40 g, 9.49
mmol) and Br.sub.2 (40 mL) was treated portionwise with the acid 23
(5.70 g, 31.6 mmol). Upon completion of the addition, the reaction
mixture was stirred at 70.degree. C. for 48 h, then cooled in an
ice bath, and quenched carefully with sat. sodium bisulfite.
Stirring was continued at rt overnight. The resultant pale brown
suspension was filtered, the solid washed with water and dried
overnight under vacuum at 60.degree. C. to yield 10.95 g (quant.)
of the crude title compound as a beige powder. .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta. 12.56 (bs, 0.3H), 2.85 (appd, 211,
7=12.9 Hz), 2.75-2.55 (m, 2H), 2.50-2.35 (m, 2H), 2.35-2.10 (m,
7H).
##STR00112##
(5,7-Dibromo-adamantan-2-yl)-carbamic acid tert-butyl ester
(25)
[0359] A suspension of the acid 24 (2.00 g, 5.92 mmol) in dry
toluene (30 mL) was treated successively with Et.sub.3N (1.0 mL,
7.10 mmol) and diphenylphosphoryl azide (DPPA, 1.6 mL, 7.10 mmol).
The resulting mixture was stirred at 85.degree. C. for 15 h. To a
separated flask containing a solution of t-BuOK (1.35 g, 11.8 mmol)
in dry THF (80 mL) al 0.degree. C. was added the isocyanate
solution dropwise via a dropping funnel. The resulting reaction
mixture was allowed to warm to rt over 30 min, and then it was
quenched with water. The THF was removed in vacuo, and the
resulting material was diluted with EtOAc. The organic layer was
washed with 1 N HCl, sat. NaHCO.sub.3 and brine, dried
(Na.sub.2SO.sub.4), filtered and concentrated in vacuo. Flash
chromatography (SiO.sub.2, 95:5 to 8:2, hexanes/EtOAc) afforded
1.20 g (50%, 2 steps) of the title compound as a white powder.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 4.68 (bs, 1H), 3.76 (bs,
1H), 2.87 (s, 2H), 2.47-2.13 (m, 10H), 1.46 (s, 9H).
##STR00113##
(7-Methylene-bicyclo[3.3.1]nonan-3-one-9-yl)-carbamic acid
tert-butyl ester (26)
[0360] (First step: Rohde, J. J. et al. Discovery and Metabolic
Stabilization of Potent and Selective 2-Amino-N-(adamant-2-yl)
Acetamide 11.beta.-Hydroxysteroid Dehydrogenase Type 1 Inhibitors.
J. Med. Chem. 2007, 50, 149-64). A solution of 25 (125 mg, 0.305
mmol) in dioxane (0.80 mL) was treated with 2N NaOH (0.70 mL, 1.37
mmol) and irradiated under microwaves (.mu.w, Biotage) for 15 min
at 180.degree. C. The dioxane was removed in vacuo. The residue was
dissolved in DCM, washed with water, dried (Na.sub.2SO.sub.4),
filtered and concentrated in vacuo to afford 82.5 mg of crude
9-amino-7-methylene-bicyclo[3.3.1]nonan-3-one as a yellow oil, that
was used for the next step without further purification.
[0361] To a solution of this crude amine in dry DCM (5 mL) was
added Et.sub.3N (0.13 mL, 0.913 mmol) and then Boc.sub.2O (73.8 mg,
0.335 mmol) at 0.degree. C. The reaction mixture was stirred at rt
under N.sub.2 for 14 h. The reaction was quenched with sat. aq.
NH.sub.4Cl and the aqueous phase extracted twice with DCM. The
combined organic layers were dried (Na.sub.2SO.sub.4), filtered and
concentrated in vacuo. Flash chromatography (SiO.sub.2, 7:3,
hexanes/EtOAc) afforded 48.0 mg (59%, 2 steps) of the title
compound as a white powder. .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 4.93 (bs, 0.25H), 4.84 (s, 2H), 4.81 (bs, 0.75H), 4.12 (bs,
0.25H), 3.91 (appbd, 0.75H, J=3.6 Hz), 2.64-2.37 (m, 6H), 2.37-2.23
(m, 3.25H), 2.17 (appbd, 0.75H, J=13.8 Hz), 1-48 and 1.46 (2 s,
9H).
##STR00114##
(7-Methylene-bicyclo[33.1]nonan-3-one oxime-9-yl)-carbamic acid
tert-butyl ester (27)
[0362] To a solution of ketone 26 (137 mg, 0.515 mmol) in dry
pyridine (1 mL) was added NH.sub.2OH.HCl (109 mg, 1.54 mmol). The
reaction mixture was stirred at rt under argon for 23 h. The
solvent was then removed in vacuo, and the residue was diluted with
EtOAc and diet) water was added. The layers were separated and the
aqueous phase extracted with EtOAc. The combined organic layers
were washed with 5% aq. CuSO.sub.4 (3.times.), brine (1.times.),
dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. Flash
chromatography (SiO.sub.2, 4:6, hexanes/EtOAc) afforded 133 mg
(92%) of the title compound as a colorless gum. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 7.02 (bs, 0.6H), 4.90 (bs, 0.25H), 4.80
(d, 1H, 7=2.1 Hz), 4.76 (bs, 0.75H), 4.69 (d, 1H, J=2.1 Hz), 3.87
(bs, 1H), 3.26 (d, 0.25H, J=16.8 Hz), 3.11 (d, 0.75H, J=16.8 Hz),
2.55-2.48 (m, 4H), 2.48-2.20 (m, 4H). 2.16 (appd, 0.25H, J=17.1
Hz), 2.04 (dd, 0.75H, J=17.1, 5.4 Hz), 1.47 (s, 9H).
##STR00115##
(1-Iodomethyl-2-azaadamantan-6-yl)-carbamic acid tert-butyl ester
(28)
[0363] To a mixture of oxime 27 (130 mg, 0.464 mmol) and MoO.sub.3
(94 mg, 0.649 mmol) in dry MeOH (4.6 mL) at 0.degree. C. under
argon was added NaBH.sub.4 (179 mg, 4.64 mmol) portion wise. The
reaction mixture was stirred at 0.degree. C., and 2 additional
amounts of NaBH.sub.4 (179 mg, 4.64 mmol) were added portionwise
after 2.5 h and after 5.5 h. After 7 h, the dark brown reaction
mixture was quenched with acetone and then filtered through Celite,
and the Celite rinsed with acetone. The filtrate was concentrated
in vacuo. The resulting residue was diluted with water and
extracted twice with EtOAc. The combined organic layers were washed
with brine, dried (K.sub.2CO.sub.3), filtered and concentrated in
vacuo to afford 136 mg of the crude amine as a yellow oil, that was
used for the next step without further purification.
[0364] To a suspension of this crude amine in dry acetonitrile
(MeCN, 2.3 mL) at 0.degree. C. under argon was added I.sub.2 (117
mg, 0.462 mmol). The reaction mixture was allowed to stir at rt for
4 h and then quenched with sat. NaHCO.sub.3 and sat.
Na.sub.2S.sub.2O.sub.3. The resulting mixture was extracted twice
with DCM/CHCl.sub.3, and the organic layer was dried
(K.sub.2CO.sub.3), filtered and concentrated in vacuo. Mash
chromatography (SiO.sub.2, 95:5 to 9:1, DCM/MeOH) afforded 76.5 mg
(42%) of the title compound as a brown oil. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 4.83 (bs, 1H), 3.77 (bs, 1H), 3.30 (bs. 1H),
3.24 (apps, 2H), 2.14 (appbs, 2H), 1.94 (appbd, 2H, J=13.5 Hz),
1.75 (m, 6H), 1.46 (s, 9H).
##STR00116##
(1-Methyl-2-azaadamantane-N-oxyl-6-yl)-carbamic acid tert-butyl
ester (29)
[0365] Deiodination of the amine 28 can be achieved by treating 28
with a reducing agent, such as LiAlH.sub.4 or NaBH.sub.4, possibly
in the presence of a catalyst, such as InCl.sub.3, and in a polar
aprotic solvent such as THF or MeCN.
[0366] Oxidation of the resulting amine to afford the corresponding
nitroxide 29 can be achieved by treating the said amine with
H.sub.2O.sub.2 in the presence of a catalytic amount of
Na.sub.2WO.sub.4.2H.sub.2O, in a solvent mixture of MeOH and
H.sub.2O.
##STR00117##
6-Amino-1-methyl-2-azaadamantane-N-oxyl (6-amino-1-Me-AZADO)
[0367] Cleavage of the Boc-protecting group can be achieved by
treating the protected amine 29 with trifluoroacetic acid (TFA) in
DCM, to afford the free amine 6-amino-1-Me-AZADO.
##STR00118##
(1-Me-AZADO-6-yl)-(S,E)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamid-
e (30)
[0368] Jones oxidation of (S,E)-tert-butyl
8-hydroxy-2-methyloct-5-en-4-ylcarbamate (9a) affords the
corresponding acid as described above. Compound (9a) is prepared
according to previous examples.
[0369] Amide coupling of the said acid with 6-amino-1-Me-AZADO is
achieved following the conditions described above, using the
coupling agents EDCI, DMAP, and HOBt-hydrate in CH.sub.2Cl.sub.2
(DCM), to yield compound (30).
[0370] Whereas particular embodiments of this invention have been
described above for purposes of illustration, it will be evident to
those skilled in the art that numerous variations of the details of
the present invention may be made without departing from the
invention as defined in the appended claims.
[0371] The use of numerical values in the various ranges specified
in this application, unless expressly indicated otherwise, are
stated as approximations as though the minimum and maximum values
within the slated ranges are both preceded by the word "about". In
this manner, slight variations above and below the stated ranges
can be used to achieve substantially the same results as values
within the ranges. Also, unless indicated otherwise, the disclosure
of these ranges is intended as a continuous range including every
value between the minimum and maximum values.
* * * * *