Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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BIOCONJUGATES OF MANGANESE COMPLEXES AND THEIR
APPLICATION AS CATALYSTS
BACKGROUND OF THE INVENTION
This present invention relates to compounds
effective as catalysts for dismutating superoxide. This
invention relates to manganese(II) or manganese(III)
complexes of nitrogen-containing fifteen-membered
macrocyclic ligands which catalytically dismutate
superoxide. In another aspect, this invention relates
to manganese complexes of nitrogen-containing fifteen-
membered macrocyclic ligands which are conjugated to a
targeting biomolecule.
2. Related Art
The enzyme superoxide dismutase catalyzes the
conversion of superoxide into oxygen and hydrogen
peroxide according to equation (1) (hereinafter referred
to as dismutation). Reactive oxygen metabolites derived
from superoxide are postulated to contribute to the
tissue pathology in a number of
~2 ~ + ~2 ~ + 2H~ - Oz + HzOz (1)
inflammatory diseases and disorders, such as reperfusion
injury to the ischemic myocardium, inflammatory bowel
disease, rheumatoid arthritis, osteoarthritis,
atherosclerosis, hypertension, metastasis, psoriasis,
organ transplant rejections, radiation-induced injury,
asthma, influenza, stroke, burns and trauma. See, for
example, Bulkley, G.B., Reactive oxygen metabolites and
reperfusion injury: aberrant triggering of
reticuloendothelial function, The Lancet, Vol. 344, pp.
934-36, October 1, 1994; Grisham, M.B., Oxidants and
free radicals in inflammatory bowel disease, The Lancet,
Vol. 344, pp. 859-861, September 24, 1994; Cross, C.E.
et al., Reactive oxygen species and the lung, The
,
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Lancet, Vol. 344, pp. 930-33, October 1, 1994; Jenner,
P., Oxidative damage in neurodegenerative disease, T~e
Lancet, Vol. 344, pp. 796-798, September 17, 1994;
Cerutti, P.A., Oxy-radicals and cancer, The Lancet , Vol.
344, pp. 862-863, Sep~h~r 24, 1994 Simic, M. G., et
al, Oxygen Radicals in Biology and Medicine, Basic Life
Sciences, Vol. 49, Plenum Press, New York and London,
1988; Weiss J. Cell. Biochem., 1991 Suppl. 15C, 216
Abstract C110 (l991); Petkau, A., Cancer Treat. Rev. 13,
17 (1986); McCord, J. Free Radicals Biol. Med., 2, 307
(1986); and Bannister, J.V. et al, Crit. Rev. Biochem.,
22, 111 (1987). The above-identified references from
The Lancet teach the nexus between free radicals
derived from superoxide and a variety of diseases. In
particular, the Bulkley and Grisham r-eferences
specifically teach that there is a nexus between the
dismutation of superoxide and the final disease
treatment.
It is also known that superoxide is involved in
the breakdown of endothelium-derived vascular relaxing
factor (EDRF), which has been identified as nitric oxide
(NO), and that EDRF is protected from breakdown by
superoxide dismutase. This suggests a central role for
activated oxygen species derived from superoxide in the
pathogenesis of vasospasm, thrombosis and
atherosclerosis. See, for example, Gryglewski, R.J. et
al., "Superoxide Anion is Involved in the Breakdown of
Endothelium-derived Vascular Relaxing Factor", Nature,
Vol. 320, pp. 454-56 (1986) and Palmer, R.M.J. et al.,
"Nitric Oxide Release Accounts for the Biological
Activity of Endothelium Derived Relaxing Factor",
Nature, Vol. 327, pp. 523-26 (1987).
Clinical trials and animal studies with natural,
recombinant and modified superoxide dismutase enzymes
have been completed or are ongoing to demonstrate the
therapeutic efficacy of reducing superoxide levels in
-
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the disease states noted above. However, numerous
problems have arisen with the use of the enzymes as
potential therapeutic agents, including lack of oral
activity, short half-live5 in vivo, immunogenicity with
nonhuman derived enzymes, and poor tissue distribution.
The manganese complexes of nitrogen-containing
fifteen-membered macrocyclic ligands that are low
molecular weight mimics of superoxide ~ ase (SOD)
are useful as therapeutic agents and avoid many of the
problems associated with SOD enzymes. However, it would
be desirable to be able to direct the SOD mimics to a
desired target in the body where the compound can be
concentrated for optimal effect. Without some way to
render the compounds "targeting", increased dosages are
sometimes necessary in order to obta n an efficacious
concentration at the site of interest. Such increased
dosages can sometimes result in undesirable side effects
in the patient.
It has now been found that the macrocycles or
manganese complexes of the present invention can be
attached, i.e. conjugated, to one or more targeting
biomolecule(s) via a linker group to form a targeting
biomolecule-macrocycle or targeting biomolecule-
manganese complex conjugate.
SUMMA~Y OF THE INVENTION
It is an object of the invention to provide
bioconjugates of manganese (II) or manganese (III)
complexes of nitrogen-containing fifteen-membered
macrocyclic ligands that are low molecular weight mimics
of superoxide dismutase (SOD) which are useful as
therapeutic agents for inflammatory disease states or
disorders which are mediated, at least in part, by
superoxide. It is a further object of the invention to
provide bioconjugates of manganese (II) complexes of
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nitrogen-containing fifteen-membered macrocyclic ligands
which are useful as magnetic resonance imaging (MRI)
contrast agents having improved kinetic stability,
improved oxidative stability and improved hydrogen
bonding. It is yet a further object of the invention to
provide bioconjugates of manganese complexes of
nitrogen-containing fifteen-membered macrocyclic ligands
that can be targeted to a specific site in the body.
According to the invention, bioconjugates of
manganese (II) or manganese (III) complexes of nitrogen-
containing fifteen-membered macrocyclic ligands are
provided wherein (1) one to five of the "R" groups are
attached to biomolecules via a linker group, (2) one of
X, Y and Z is attached to a biomolecule via a linker
group, or (3) one to five of the "R" ~roups and one of
X, Y and Z are attached to biomolecules via a linker
group; and biomolecules are independently selected from
the group consisting of steroids, carbohydrates, fatty
acids, amino acids, peptides, proteins, antibodies,
vitamins, lipids, phospholipids, phosphates,
phosphonates, nucleic acids, enzyme substrates, enzyme
inhibitors and enzyme receptor substrates and the linker
group is derived from a substituent attached to the "R"
group or X, Y and Z which is reactive with the
biomolecule and is selected from the group consisting of
-NH2, -NHRIo, -SH, -OH, -COOH, -COORlo, -CONH2, -NCO,
-NCS, -cooX~, alkenyl, alkynyl, halide, tosylate,
mesylate, tresylate, triflate and phenol, wherein Rlo is
alkyl, aryl, or alkylaryl and Xn is a halide.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to
bioconjugates of manganese(II) or manganese(III)
complexes of nitrogen-containing fifteen-membered
macrocyclic ligands which catalyze the conversion of
-
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superoxide into oxygen and hydrogen peroxide. These
complexes can be represented by the formula:
2 '~n
~ R3
wherein R, R', Rl, Rl', R2, R2', R3, R3', R4, R,', R5, R5',
R6, R6', R7, R7', R8, R8', R9 and R9' independently
represents hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkylalkyl,
cycloalkylcycloalkyl, cycloalkenylalkyl,
alkylcycloalkyl, alkenylcycloalkyl, alkylcycloalkenyl,
alkenylcycloalkenyl, heterocyclic, aryl and aralkyl
radicals and radicals attached to the
~-carbon of c~-amino acids; or Rl or R'l and R2 or R'2, R3
or R' 3 and R4 or R' 4, R5 or R'5 and R6 or R' 6 ~ R7 or R'7 and
R8 or R' 8 ~ and Rg or R'9 and R or R' together with the
carbon atoms to which they are attached independently
form a saturated, partially saturated or unsaturated
cyclic having 3 to 20 carbon atoms; or R or R' and Rl or
R ~I~ R2 or R' 2 and R3 or R'3, R4 or R'4 and R5 or R' 5, R6 or
R' 6 and R7 or R'7, and R8 or R'8 and R9 or R'9 together
with the carbon atoms to which they are attached
independently form a nitrogen containing heterocycle
having 2 to 20 carbon atoms provided that when the
nitrogen containing heterocycle is an aromatic
heterocycle which does not contain a hydrogen attached
to the nitrogen, the hydrogen attached to the nitrogen
in said formula, which nitrogen is also in the
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macrocycle and the R groups attached to the same carbon
atoms of the macrocycle are absent; and combinations
thereof; and wherein (1) one to five of the "R" groups
are attached to biomolecules via a linker group, (2) one
of X, Y and Z is attached to a biomolecule via a linker
group, or (3) one to five of the "R" groups and one of
X, Y and Z are attached to biomolecules via a linker
group; and biomolecules are independently selected from
the group consisting of steroids, carbohydrates, fatty
acids, amino acids, peptides, proteins, antibodies,
VitA~i nC, lipids, phospholipids, phosphates,
phosphonates, nucleic acids, enzyme substrates, enzyme
inhibitors and enzyme receptor substrates and the linker
group is derived from a substituent attached to the "R"
group or X, Y and Z which is reactive-with the
biomolecule and is selected from the group consisting of
-NH2, -NHRlo, -SH, -OH, -COOH, -COORIo, -CONH2, -NCO,
-NCS, -COOXn, alkenyl, alkynyl, halide, tosylate,
mesylate, tresylate, triflate and phenol, wherein Rlo is
alkyl, aryl, or alkylaryl and X~ is a halide.
X, Y and Z represent suitable ligands or charge-
neutralizing anions which are derived from any
monodentate or polydentate coordinating ligand or ligand
system or the corresponding anion thereof (for example
benzoic acid or benzoate anion, phenol or phenoxide
anion, alcohol or alkoxide anion). X, Y and Z are
independently selected from the group consisting of
halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen,
peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia,
alkylamino, arylamino, heterocycloalkyl amino,
heterocycloaryl amino, amine oxides, hydrazine, alkyl
hydrazine, aryl hydrazine, nitric oxide, cyanide,
cyanate, thiocyanate, isocyanate, isothiocyanate, alkyl
nitrile, aryl nitrile, alkyl isonitrile, aryl
isonitrile, nitrate, nitrite, azido, alkyl sulfonic
acid, aryl sulfonic acid, alkyl sulfoxide, aryl
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sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid,
aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic
acid, alkyl thiol carboxylic acid, aryl thiol carboxylic
acid, alkyl thiol thiocarboxylic acid, aryl thiol
thiocarboxylic acid, alkyl carboxylic acid (such as
acetic acid, trifluoroacetic acid, oxalic acid), aryl
carboxylic acid (such as benzoic acid, phthalic acid),
urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea,
alkyl thiourea, aryl thiourea,alkyl aryl thiourea,
sulfate, sulfite, bisulfate, bisulfite, thiosulfate,
thiosulfite, hydrosulfite, alkyl phosphine, aryl
phosphine, alkyl phosphine oxide, aryl phosphine oxide,
alkyl aryl phosphine oxide, alkyl phosphine sulfide,
aryl phosphine sulfide, alkyl aryl phosphine sulfide,
alkyl phosphonic acid, aryl phosphon~c acid, alkyl
phosphinic acid, aryl phosphinic acid, alkyl phosphinous
acid, aryl phosphinous acid, phosphate, thiophosphate,
phosphite, pyrophosphite, triphosphate, hydrogen
phosphate, dihydrogen phosphate, alkyl guanidino, aryl
guanidino, alkyl aryl guanidino, alkyl carbamate, aryl
carbamate, alkyl aryl carbamate, alkyl thiocarbamate
aryl thiocarbamate, alkyl aryl thiocarbamate, alkyl
dithiocarbamate, aryl dithiocarbamate, alkyl aryl
dithiocarbamate, bicarbonate, carbonate, perchlorate,
chlorate, chlorite, hypochlorite, perbromate, bromate,
bromite, hypobromite, tetrahalomanganate,
tetrafluoroborate, hexafluorophosphate,
hexafluoroantimonate, hypophosphite, iodate, periodate,
metaborate, tetraaryl borate, tetra alkyl borate,
tartrate, salicylate, succinate, citrate, ascorbate,
saccharinate, amino acid, hydroxamic acid,
thiotosylate, and anions of ion exchange resins, or
systems where one or more of X,Y and Z are independently
attached to one or more of the "R" groups, wherein n is
0 or l. The preferred ligands from which X, Y and Z are
selected include halide, organic acid, nitrate and
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bicarbonate anions.
The linker groups, also termed herein "linker",
are derived from the specified functional groups
attached to the "R" groups or X, Y and Z, and function
to link the biomolecule to the "R" groups or X, Y and Z.
The functional groups are selected from the group
consisting of -NH2, -NHRlo, -SH, -OH, -COOH, -COORIo~
-CONH2, -NCO, -NCS, -COOX~, alkenyl, alkynyl, halide,
tosylate, mesylate, tresylate, triflate and phenol
wherein Rlo is alkyl, aryl, or alkaryl and X~ is a
halide. Currently, the preferred alkenyl group is
ethenyl and the preferred alkynyl group is ethynyl. The
functional groups on the "R" groups or X, Y and Z are
reactive with the biomolecule, i.e. reactive with a
functional group on the steroids, carbohydrates, fatty
acids, amino acids, peptides, proteins, antibodies,
Vit;~ ;n~:, lipids, phospholipids, phosphates,
phosphonates, nucleic acids, enzyme substrates, enzyme
inhibitors, enzyme receptor substrates and other
targeting biomolecules of interest. When the functional
group attached to the "R" groups or X, Y and Z reacts
with the biomolecule, the functional group is modified
and it is this derived functional group which is the
linker. For example, when an -NH2 functional group
attached to an "R" group is reacted with a steroid such
as in Example 1, the linker is -NH-. The exact
structure of specific linker groups will be readily
apparent to those of ordinary skill in the art and will
depend on the specific functional group and biomolecule
selected. The specific reaction conditions for reacting
a functional group attached to "R" groups or X, Y and Z
with a biomolecule will be readily apparent to those of
ordinary skill in the art.
The functional group useful to form the linker,
defined herein as a "linker precursor", may be present
on the "R" groups at the time the macrocycle is prepared
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W O 97/06824
or it may be added or modified after preparation of the
macrocycle or manganese complex thereof. Similarly, the
linker precursor can be present on an axial ligand, i.e.
X, Y or Z, when the manganese complex is prepared or an
exchange reaction of the axial ligands is conducted to
exchange the axial ligands present in the manganese
complex.
The macrocycle of the present invention can be
complexed with manganese either before or after
conjugation with the targeting biomolecule depending on
the specific biomolecule utilized. The conjugate of the
macrocyclic complex and the targeting biomolecule is
defined herein as a "bioconjugate".
Targeting of drugs is well known to those of
ordinary skill in the art. See, for-example, J. A.
Katzenellenbogen et al, ~ournal of Nuclear Medicine,
Vol. 33, No. 4, 1992, 558, and J.A. Katzenellenbogen et
al, Bioconjugate Chemistry, 1991, 2, 353.
Targeting agents are typically biomolecules. The
biomolecules of the invention are biologically active
molecules that are site specific, i.e. known to
concentrate in the particular organ or tissue of
interest. The biomolecules are selected to direct the
tissue distribution of the bioconjugate via receptor
binding, membrane association, membrane solubility, and
the like. These biomolecules include, for example,
steroids, carbohydrates (including monosaccharides,
disaccharides and polysaccharides), fatty acids, amino
acids, peptides, proteins, antibodies (including
polyclonal and monoclonal and fragments thereof),
vitamins, lipids, phospholipids, phosphates,
phosphonates, nucleic acids, enzyme substrates, enzyme
inhibitors and enzyme receptor substrates. The
biomolecules also include those biomolecules which are
combinations of the above biomolecules, such as a
combination of a steroid with a carbohydrate, e.g.
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--10--
digitonin.
The particular biomolecules which can be utilized
to target a desired organ or tissue are known in the art
or it will be readily apparent to those of ordinary
skill in the art. The biomolecules of the invention are
commercially available or can readily be prepared by one
of ordinary skill in the art using conventional methods.
It is currently preferred that a ~-Y; ~11~ of one
"R" group attached to the carbon atoms located between
nitrogen atoms in the macrocycle has a biomolecule
attached via a linker. In addition, the preferred
compounds are those which have one to five, most
preferably one to two, of the "R" groups attached to
biomolecules and none of X, Y and Z attached to a
biomolecule, or those which have one ~f X, Y and Z
attached to a biomolecule and none of the "R" groups
attached to a biomolecule.
Currently, the preferred compounds are those
wherein at least one, more preferably at least two, of
the "R" groups, in addition to the "R" groups which are
attached to a biomolecule, represent alkyl, cycloalkyl
alkyl and aralkyl radicals and the remaining "R" groups
not attached to a biomolecule represent hydrogen, a
saturated, partially saturated or unsaturated cyclic or
a nitrogen containing heterocycle. Other preferred
groups of compounds are those wherein at least one,
preferably two, of R~ or R'l and R2 or R' 2~ R3 or R'3 and
R4 or R' 4, R5 or R'5 and R6 or R' 6~ R7 or R' 7 and R~ or R'",
and R9 or R'9 and R or R' together with the carbon atoms
to which they are attached represent a saturated,
partially saturated or unsaturated cyclic having 3 to 20
carbon atoms and the remaining "R" groups in addition to
the "R" groups which are attached to a biomolecule via a
linker are hydrogen, nitrogen containing heterocycles or
alkyl groups, and those wherein at least one, preferably
two, of R or R' and R, or R~" R2 or R'2 and R3 or R'3, R4
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--11--
or R' 4 and R5 or R' 5, R6 or R' 6 ~ and R~ or R'~, and R8 or
R' 8 and R9 or R'9 together with the carbon atoms to which
they are attached are bound to form a nitrogen
containing heterocycle having 2 to 20 carbon atoms and
the remaining "R" groups in addition to the "R" groups
which are attached to a biomolecule via a linker are
independently selected from hydrogen, saturated,
partially saturated or unsaturated cyclics or alkyl
groups.
As used herein, "R" groups means all of the R
groups attached to the carbon atoms of the macrocycle,
R R' R R' R R' R R' R R' R R'
l-e~ 2/ 2~ 3~ 3~ 4~ 4~ 5~ 5~ R6~
R'6, R~, R'~, R8, R'8, R9 and R'g.
Another embodiment of the invention is a
pharmaceutical composition in unit d~sage form useful
for dismutating superoxide comprising (a) a
therapeutically or prophylactically effective amount of
a complex as described above and (b~ a nontoxic,
pharmaceutically acceptable carrier, adjuvant or
vehicle.
The commonly accepted ~ch~nism of action of the
manganese-based SOD enzymes involves the'cycling of the
manganese center between the two oxidation states
(II,III). See J. V. Bannister, W. H. Bannister, and G.
Rotilio, Crit. Rev. Biochem., 22, 111-180 (1987).
1) Mn(II) + HO2 ----> Mn(III) + HO2
2) Mn(III) + ~2 - - - - - > Mn(II) + ~2
The formal redox potentials for the ~2/~2 - and HO2/H2O2
couples at pH = 7 are -0.33 v and 0.87 v, respectively.
See A. E. G. Cass, in Metalloproteins: Part 1, Metal
Proteins with Redox Roles, ed. P. Harrison, P. 121.
Verlag Chemie (Weinheim, GDR) (1985). For the above
disclosed mechanism, these potentials require that a
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putative SOD catalyst be able to rapidly undergo
oxidation state changes in the range of -0.33 v to 0.87
v.
The complexes derived from Mn(II) and the general
class of C-substituted [15]aneN5 ligands described herein
have all been characterized using cyclic voltammetry to
measure their redox potential. The C-substituted
complexes described herein have reversible oxidations of
about +0.7 v (SHE). Coulometry shows that this
oxidation is a one-electron process; namely it is the
oxidation of the Mn(II) complex to the Mn(III) complex.
Thus, for these complexes to function as SOD catalysts,
the Mn(III) oxidation state is involved in the catalytic
cycle. This means that the Mn(III) complexes of all
these ligands are e~ually competent ~s SOD catalysts,
since it does not matter which form (Mn(II) or Mn(III))
is present when superoxide is present because superoxide
will simply reduce Mn(III) to Mn(II) liberating oxygen.
As utilized herein, the term "alkyl", alone or in
combination, means a straight-chain or branched-chain
alkyl radical containing from 1 to about 22 carbon
atoms, preferably from about 1 to about 18 carbon atoms,
and most preferably from about 1 to about 12 carbon
atoms which optionally carries one or more substituents
selected from (1) -NR30R3l wherein R30 and R3l are
independently selected from hydrogen, alkyl, aryl or
aralkyl ; or R30is hydrogen, alkyl, aryl or aralkyl and
R3l is selected from the group consisting of -NR32R33, -OH,
-OR34,
- C-Z, ~ bs. -g-~6, --S-~7-~d-~-t~~8)(~~s)i
wherein R32 and R33 are independently hydrogen, alkyl,
aryl or acyl, R34 is alkyl, aryl or alkaryl, Z is
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--13--
hydrogen, alkyl, aryl, alkaryl, -OR34, -SR34 or -NR40R
wherein R40 and R4l are independently selected from
hydrogen, alkyl, aryl or alkaryl, Z is alkyl, aryl,
alkaryl, -OR34, -SR34 or -NR40R4~, R35 is alkyl, aryl, -OR34,
or -NR40R4" R36 is alkyl, aryl or -NR40R4l R3, is alkyl,
aryl or alkaryl, X is oxygen or sulfur, and R38 and R39
are independently selected from hydrogen, alkyl or aryl;
(2) -SR42 wherein R42is hydrogen, alkyl, aryl, alkaryl,
--SR34, --NR32R33,
X' o o
- C -Z~ P~3, ~ -P~
wherein R43 is -OH, -OR34 or -NR32R33, and A and B are
independently --OR34, -SR34 or -NR32R33
--S~~k
15 (3~
wherein x is 1 or 2, and R44 is halide, alkyl, aryl,
alkaryl, --OH, --OR34, --SR34 or --NR32R33;
(4 ) -OR45 wherein R45 is hydrogen, alkyl, aryl, alkaryl,
--NR32R33 ~
X' R41 0 0
--11 _Z~,--S ~~k,--P ~)~), C~r --1l ~R34)~0R~4);
wherein D and E are independently --OR34 or --NR32R33;
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C7-21(1~573)A
-14-
X'
(5)
wherein R,6 is halide, -OH, --SH, -OR3", --SR3~ or -NR3~33;
or (6) amine oxides o~ the ~ormula
I R ~ 3:
o
provided R30 and R3~ are not hydroqen; or
t7)
wherein F and G are independently --OH,--sX,--OR34, --SR34
or -NR32R3~; or
(8) -O-(-(CH2)~-O)b-R~o wherein Rlo is hydrogen or alkyl,
and a and b an integers independently selected ~rom
1 + 6; or
(g) halo~en, cyano, nitro, or azido. Alkyl, aryl and
alkaryl groups on the substituents of the above-defined
alkyl groups may contain one additional substituent but
are pre~erably unsubstituted. Examples o~ such radicals
include methyl, ethyl,
n-propyl, isopropyl, n--butyl, isobutyl, sec-butyl, tert-
butyl, pentyl, isoamyl, hexyl, octyl, nonyl, decyl,
dodecyl, tetradecyl, hexadecyl, octadecyl and eicosyl.
The term "alkenyl", alone or in combination, means an
alkyl radical having one or more double bonds. Examples
o~ such alkenyl radicals include
ethenyl, propenyl, 1-butenyl, cis-2-butenyl, trans-
2-butenyl, iso-butylenyl, cis-2-pentenyl, trans-2-
pentenyl, 3-methyl-1-butenyl, 2,3-dimethyl-2-butenyl,
AM5~N~E0 SHEET
I P~/FP
CA 02229799 l998-02-l7
~7-21 12523)A
l-pentenyl, 1-hexenyl, 1-octenyl, decenyl, dodecenyl,
tetradecenyl, hexadecenyl, cis- and trans-
9-octadecenyl, 1,3-pentadienyl, 2,4-pentadienyl,
2,3-pentadienyl, 1~3-hexadienyl~ 2,4-hexadienyl,
5,8,11,14-eicosatetraenyl, and 9,12,15-octadecatrienyl.
The term "alkynyl", alone or in combination, means an
alkyl radical having one or more triple bonds. Examples
of such alkynyl groups include
ethynyl, propynyl (propar~yl), 1-butynyl, l-octynyl,
9-octadecynyl, 1,3-pentadiynyl, 2,4-pentadiynyl, 1,3-
hexadiynyl, and 2,4-hexadiynyl. The term "cycloalkyl",
alone or in combination means a cycloalkyl radical
containing from 3 to about 10, preferably from 3 to
about 8, and most preferably from 3 to about 6, carbon
atoms. Examples of such cycloalkyl ~adicals include
cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and
perhydronaphthyl. The term "cycloalkylalkyl" means an
alkyl radical as defined above which is substituted by a
cycloalkyl radical as defined above. Examples of
cycloalkylalkyl radicals include
cyclohexylmethyl, cyclopentylmethyl,
(4-isopropylcyclohexyl)methyl,
(4-t-butyl-cyclohexyl)methyl,
3-cyclohexylpropyl, 2-cyclo-hexylmethylpentyl,
3-cyclopentylmethylhexyl,
1-(4-neopentylcyclohexyl)methylhexyl, and
1-(4-isopropylcyclohexyl)methylheptyl. The term
"cycloalkylcycloalkyl" means a cycloalkyl radical as
defined above which is substituted by another cycloalkyl
radical as de~ined above. Examples o~
cycloalkylcycloalkyl radicals include
cyclohexylcyclopentyl and
cyclohexylcyclohexyl. The term "cycloalkenyl", alone or
in combination, means a cycloalkyl radical having one or
more double bonds. Examples of cycloalkenyl radicals
~P'--.A'-~
CA 02229799 l998-02-l7
~7-21tl2523)A
-16-
include cyclopentenyl,
cyclohexenyl, cyclooctenyl, cyclopentadienyl,
cyclohexadienyl and cyclooctadienyl. The term
"cycloalkenylalkyl" means an alkyl radical as defined
above which is subs.ituted by a cycloalkenyl radical as
defined above. Examples of cycloalkenylalkyl radicals
include
2-cyclohexen-1-ylmethyl, l-cyclopenten-}-ylmethyl,
2-(1-cyclohexen-1-yl)ethyl,
3-(1-cyclopenten-i-yl)propyl, 1-(1-cyclohexen-1-
ylmethyl)pentyl, 1-(l-cyclopenten-l-yl)hexyl~
6-(1-cyclohexen-1-yl)hexyl, 1-(1-cyclopenten-1-yl?nonyl
and l-(1-cyclohexen-1-yl)nonyl. The terms
"alkylcycloalkyl" and "alkenylcycloalkyl" mean a
cycloalkyl radical as defined above which is substituted
by an alkyl or alkenyl radical as defined above.
Examples of alkylcycloalkyl and alkenylcycloalkyl
radicals include
2-ethylcyclobutyl, l-~ethylcyclopentyl,
1-hexylcyclopentyl, 1-methylcyclohexyl,
1-(9-octadecenyl)cyclopentyl and
1-(9-octadecenyl)cyclohexyl. The terms
"alkylcycioalkenyl" and "alkenylcycloalkenyl" means a
cycloalkenyl radical as defined above which is
substituted by an alkyl or alkenyl radical as defined
above. Examples of alkylcycloalkenyl and
alkenylcycloalkenyl radicals include
1-methyl-2-cyclopentenyl,
1-hexyl-2-cyclopentenyl, 1-ethyl-2-cyclohexenyl,
1-butyl-2-cyclohexenyl, l-(9-octadecenyl)-2-cyclohexenyl
and l-(2-pentenyl)-2-cyclohexenyl. The term "aryl",
alone or in combina~ion, means a phenyl or naphthyl
radical which optionally carries one or more
substituents selected ~rom alkyl, cycloalkyl,
cycloalkenyl, aryl, heterocycle, alkoxyaryl, alkaryl,
alkoxy, haloqen, hydroxy, amine, cyano, nitro,
~,,, , . , . -- ; ~ . . . ;.
. .~. _
CA 02229799 l998-02-l7
~7-21~12523)A
-
-17-
alkylthio, phenoxy, ether, tri~luoromethyl and the like,
such-as phenyl, p-tolyl, 4-methoxyphenyl,
4-(tert-butoxy)phenyl, 4-fluorophenyl, 4-chlorophenyl,
4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, and the like.
S The term "aralkyl", alone or in combination, means an
al~yl or cycloalkyl radical as defined above in which
one hydrogen atom is replaced by an aryl radical as
defined above, such as benzyl, 2-phenylethyl, and the
like. The term "heterocyclic" means ring structures
containing at least one other kind of atom, in addition
; to carbon, in the ring. The most common of the other
~inds o~ atoms include nitroqen, oxygen and sulCur.
Examples o~ heterocyclics include
pyrrolidinyl, piperidyl, imidazolidinyl,
tetrahydrofuryl, tetrahydrothienyl, furyl, thienyl,
pyridyl, ~uinolyl, isoquinolyl, pyridazinyl, pyrazinyl,
indolyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl,
pyridinyl, benzoxadiazolyl, benzothiadiazolyl, triazolyl
and tetrazolyl groups. The term "saturated, partially
saturated or unsaturated cyclic" means fused ring
structures in which 2 carbons of the ring are also part
of the fifteen-membered macrocyclic ligand. The ring
structure can contain 3 to 20 carbon atoms, pre~erably
5 to lO carbon atoms, and can also contain one or more
other kinds o~ atoms in addition to carbon. ~he most
common of the other kinds o~ atoms include ni~roqen,
oxyqen and sulfur. The ring structure can also contain
more than one ring. The term "saturated, partially
saturated or unsaturated ring structure" means a ring
structure in which one carbon of the ring is also part
of the fifteen-membered macrocyclic ligand. The rinq
structure can contain 3 to 20, pre~erably S to 10,
carbon atoms and can also contain nitrogen, oxygen
and/or sul~ur atoms. The term "nitrogen containing
heterocycle" means ring structureS in which 2 carbons
and a nitrogen o~ the ring are also part of the ~i~teen-
A~ SHEET
IP~A/EP
CA 02229799 1998-02-17
WO 97/06824 PCT~US96/12767
-18-
membered macrocyclic ligand. The ring structure can
contain 2 to 20, preferably 4 to 10, carbon atoms, can
be partially or fully unsaturated or saturated and can
also contain nitrogen, oxygen and/or sulfur atoms in the
portion of the ring which is not also part of the
fifteen-membered macrocyclic ligand. The term "organic
acid anion" refers to carboxylic acid anions having from
about 1 to about 18 carbon atoms. The term "halide"
means chloride or bromide.
The macrocyclic ligands useful in the complexes
of the present invention can be prepared according to
the general procedure shown in Scheme A set forth below.
Thus, an amino acid amide, which is the corresponding
amide derivative of a naturally or non-naturally
occurring ~-amino acid, is reduced t~ form the
corresponding substituted ethylenediamine. Such amino
acid amide can be the amide derivative of any one of
many well known amino acids. Preferred amino acid
amides are those represented by the formula:
E~ ~ ~nH2
wherein R is derived from the D or L forms of the amino
acids Alanine, Aspartic acid, Arginine, Asparagine,
Cysteine, Glycine, Glutamic acid, Glutamine, Histidine,
Isoleucine, Leucine, Lysine, Methionine, Proline,
Phenylalanine, Serine, Tryptophan, Threonine, Tyrosine,
Valine and /or the R groups of unnatural ~-amino acids
such as alkyl, ethyl, butyl, tert-butyl, cycloalkyl,
phenyl, alkenyl, allyl, alkynyl, aryl, heteroaryl,
polycycloalkyl, polycycloaryl, polycycloheteroaryl,
imines, aminoalkyl, hydroxyalkyl, hydroxyl, phenol,
amine oxides, thioalkyl, carboalkoxyalkyl, carboxylic
CA 02229799 1998-02-17
WO 97/06824 PCT~US96/12767
--19--
acids and their derivatives, keto, ether, aldehyde,
amine, nitrile, halo, thiol, sulfoxide, sulfone,
sulfonic acid, sulfide, disulfide, phosphonic acid,
phosphinic acid, phosphine oxides, sulfonamides, amides,
amino acids, peptides, proteins, carbohydrates, nucleic
acids, fatty acids, lipids, nitro, hydroxylamines,
hydroxamic acids, thiocarbonyls, borates, boranes,
boraza, silyl, siloxy, silaza, and combinations thereof.
Most preferred are those wherein R represents hydrogen,
alkyl, cycloalkylalkyl, and aralkyl radicals. The
diamine is then tosylated to produce the di-N-tosyl
derivative which is reacted with a di-O-tosylated
tris-N-tosylated triazaalkane diol to produce the
corresponding substituted
N-pentatosylpentaazacycloalkane. Th~ tosyl groups are
then removed and the resulting compound is reacted with
a manganese(II) compound under essentially anhydrous and
anaerobic conditions to form the corresponding
substituted manganese(II) pentaazacycloalkane complex.
When the ligands or charge-neutralizing anions, i.e. X,
Y and Z, are anions or ligands that cannot be introduced
directly from the manganese compound, the complex with
those anions or ligands can be formed by conducting an
exchange reaction with a complex that has been prepared
by reacting the macrocycle with a manganese compound.
The complexes of the present invention, wherein
R9, and R2 are alkyl, and R3, R~3, R4, R'4, R5, R'S, R6,
R'6, R7, R'~, RB and R'8 can be alkyl, arylalkyl or
cycloalkylalkyl and R or R~ and Rl or R~ I together with
the carbon atoms they are attached to are bound to form
a nitrogen containing heterocycle, can also be prepared
according to the general procedure shown in Scheme B set
forth below utilizing methods known in the art for
preparing the manganese(II)
pentaazabicyclo[12.3.1]octadecapentaene complex
precursor. See, for example, Alexander et al., Inorg.
CA 02229799 1998-02-17
W O 97/06824 PCTAUS96/12767
-20-
Nucl. Chem. Lett., 6, 445 (1970). Thus a
2,6-diketopyridine is condensed with triethylene
tetraamine in the presence of a manganese(II) compound
to produce the manganese(II)
pentaazabicyclo[12.3.1]octadecapentaene complex. The
manganese(II) pentaazabicyclotl2~3~l]octadecapentaene
complex is hydrogenated with platinum oxide at a
pressure of 10-1000 psi to give the corresponding
manganese(II) pentaazabicyclotl2~3~l]octadecatriene
complex.
The macrocyclic ligands useful in the complexes
of the present invention can also be prepared by the
diacid dichloride route shown in Scheme C set forth
below. Thus, a triazaalkane is tosylated in a suitable
solvent system to produce the corres~onding tris
(N-tosyl) derivative. Such a derivative is treated with
a suitable base to produce the corresponding
disulfonamide anion. The disulfonamide anion is
dialkylated with a suitable electrophile to produce a
derivative of a dicarboxylic acid. This derivative of a
dicarboxylic acid is treated to produce the dicarboxylic
acid, which is then treated with a suitable reagent to
form the diacid dichloride. The desired vicinal diamine
is obtained in any of several ways. One way which is
useful is the preparation from an aldehyde by reaction
with cyanide in the presence of ammonium chloride
followed by treatment with acid to produce the alpha
ammonium nitrile. The latter compound is reduced in the
presence of acid and then treated with a suitable base
to produce the vicinal diamine. Condensation of the
diacid dichloride with the vicinal diamine in the
presence of a suitable base forms the tris(tosyl)diamide
macrocycle. The tosyl groups are removed and the amides
are reduced and the resulting compound is reacted with a
manganese (II) compound under essentially anhydrous and
anaerobic conditions to form the corresponding
-
CA 02229799 1998-02-17
W O 97/06824 PCTAJS96/12767
substituted pentaazacycloalkane manganese (II) complex.
The vicinal diamines have been prepared by the
route shown (known as the Strecker synthesis) and
vicinal diamines were purchased when commercially
available. Any method of vicinal diamine preparation
could be used.
The macrocyclic ligands useful in the complexes
of the present invention can also be prepared by the
pyridine diamide route shown in Scheme D as set forth
below. Thus, a polyamine, such as a tetraaza compound,
containing two primary amines is condensed with dimethyl
2,6-pyridine dicarboxylate by heating in an appropriate
solvent, e.g., methanol, to produce a macrocycle
incorporating the pyridine ring as the
2,6-dicarboxamide. The pyridine rin~ in the macrocycle
is reduced to the corresponding piperidine ring in the
macrocycle, and then the diamides are reduced and the
resulting compound is reacted with a manganese (II)
compound under essentially anhydrous and anaerobic
conditions to form the corresponding substituted
pentaazacycloalkane manganese (II) complex.
The macrocyclic ligands useful in the complexes
of the present invention can also be prepared by the
bis(haloacetamide) route shown in Scheme E set ~orth
below. Thus a triazaalkane is tosylated in a suitable
solvent system to produce the corresponding tris
(N-tosyl) derivative. Such a derivative is treated with
a suitable base to produce the corresponding
disulfonamide anion. A bis(haloacetamide), e.g., a
bis(chloroacetamide), of a vicinal diamine is prepared
by reaction of the diamine with an excess of haloacetyl
halide, e.g., chloroacetyl chloride, in the presence of
a base. The disulfonamide anion of the tris(N-tosyl)
- triazaalkane is then reacted with the
bis(chloroacetamide) of the diamine to produce the
substituted tris(N-tosyl)diamide macrocycle. The tosyl
CA 02229799 1998-02-17
WO 97/06824 PCT~US96/12767
-22-
groups are removed and the amides are reduced and the
resulting compound is reacted with a manganese (II)
compound under essentially anhydrous and anaerobic
conditions to form the corresponding substituted
pentaazacycloalkane manganese (II) complex.
The macrocyclic ligands useful in the complexes
of the present invention, wherein R~, R l~ R2, R 2 are
derived from a ~i~;no starting material and R5, R s, R"
R , and R9, R 9 can be H or any functionality previously
described, can be prepared according to the pseudo-
peptide method shown in Scheme F set forth below. A
substituted 1,2-diaminoethane represented by the formula
gl R2
'2
H2~ ~ 2
, wherein R" R 1, R2 and R 2 are the substituents on
adjacent carbon atoms in the product macrocyclic ligand
as set forth above, can be used in this method in
combination with any amino acids. The diamine can ~e
produced by any conventional method known to those
skilled in the art. The R groups in the macrocycle
derived from substituents on the ~-carbon of ~-amino
acids, i.e. R5, R s~ R" R 7, R9 and R 9, could be derived
from the D or L forms of the amino acids Alanine,
Aspartic acid, Arginine, Asparagine, Cysteine, Glycine,
Glutamic acid, Glutamine, Histidine, Isoleucine,
Leucine, Lysine, Methionine, Proline, Phenylalanine,
Serine, Tryptophan, Threonine, Tyrosine, Valine and/or
the R groups of unnatural ~-amino acids such as alkyl,
ethyl, butyl, tert-butyl, cycloalkyl, phenyl, alkenyl,
allyl, alkynyl, aryl, heteroaryl, polycycloalkyl,
CA 02229799 1998-02-17
W O 97/06824 PCT~US96/12767
-23-
polycycloaryl, polycycloheteroaryl, imines, aminoalkyl,
hydroxyalkyl, hydroxyl, phenol, amine oxides, thioalkyl,
carboalkoxyalkyl, carboxylic acids and their
derivatives, keto, ether, aldehyde, amine, nitrile,
halo, thiol, sulfoxide, sulfone, sulfonic acid, sulfide,
disulfide, phosphonic acid, phosphinic acid, phosphine
oxides, sulfonamides, amides, amino acids, peptides,
proteins, carbohydrates, nucleic acids, fatty acids,
lipids, nitro, hydroxylamines, hydroxamic acids,
thiocarbonyls, borates, boranes, boraza, silyl, siloxy,
silaza, and combinations thereof. As an example
1,8-dihydroxy, 4,5-diaminooctane is monotosylated and
reacted with Boc anhydride to afford the differentiated
N-Boc, N-tosyl derivative. The sulfonamide was
alkylated with methyl bromoacetate using sodium hydride
as the base and saponified to the free acid. The
diamine containing N-tosylglycine serves as a dipeptide
surrogate in standard solution-phase peptide synthesis.
Thus, coupling with a functionalized amino acid ester
affords the corresponding pseudo-tripeptide. Two
sequential TFA cleavage-couplings affords the pseudo-
pentapeptide which can be N- and C-terminus deprotected
in one step using HCl/AcOH. DPPA mediated cyclization
followed by LiAlH4or Borane reduction affords the
corresponding macrocylic ligand. This ligand system is
reacted with a manganese (II) compound, such as
manganese (II) chloride under essentially anaerobic
conditions to form the corresponding functionalized
manganese (II) pentaazacycloalkane complex. When the
ligands or charge-neutralizing anions, i.e. X, Y and Z,
are anions or ligands that cannot be introduced directly
from the manganese compound, the complex with those
anions or ligands can be formed by conducting an
exchange reaction with a complex that has been prepared
by reacting the macrocycle with a manganese compound.
The macrocyclic ligands useful in the complexes
CA 02229799 1998-02-17
WO 97/06824 PCTAUS96/12767
of the present invention, wherein Rl, R'l, R3, R'3, R5,
R'5, R7, R'~, Rg and R'g can be H or any functionality as
previously described, can be prepared according to the
general peptide method shown in Scheme G set forth
below. The R groups in the macrocycle derived from
substitutents on the ~-carbon of ~-amino acids, i.e. Rl,
R'l, R3, R'3, Rs~ R'5, R" R'" Rg and R'9, are defined
above in Scheme F. The procedure for preparing the
cyclic peptide precursors from the corresponding linear
peptides are the same or significant modifications of
methods known in the art. See, for example, Veber, D.F.
et al., J. Org. Chem., 44, 3101 (1979). The general
method outlined in Scheme G below is an example
utilizing the sequential solution-phase preparation of
the functionalized linear pentapepti~e from N-terminus
to C-terminus. Alternatively, the reaction sequence to
prepare the linear pentapeptide can be carried out by
solid-phase preparation utilizing methods known in the
art. The reaction sequence could be conducted from
C-terminus to N-terminus and by convergent approaches
such as the coupling of di- and tri-peptides as needed.
Thus a Boc-protected amino acid is coupléd with an amino
acid ester using standard peptide coupling reagents.
The new Boc-dipeptide ester is then saponified to the
free acid which is coupled again to another amino acid
ester. The resulting Boc-tri-peptide ester is again
saponified and this method is continued until the Boc-
protected pentapeptide free acid has been prepared. The
Boc protecting group is removed under standard
conditions and the resulting pentapeptide or salt
thereof is converted to the cyclic pentapeptide. The
cyclic pentapeptide is then reduced to the
pentaazacyclopentadecane with lithium aluminum hydride
or borane. The final ligand is then reacted with a
manganese (II) compound under essentially anaerobic
conditions to form the corresponding manganese (II)
CA 02229799 1998-02-17
Wo97/06824 PCT~S96/12767
pentaazacyclopentadecane complex. When the ligands or
charge-neutralizing anions, e.g. X,Y and Z, are anions
or ligands that cannot be introduced directly from the
manganese compound, the complex with those anions or
S ligands can be formed by conducting an exchange reaction
with a complex that has been prepared by reacting the
macrocycle with a manganese compound.
CA 02229799 1998-02-17
PCT~US96/12767
W O 97/06824
-26-
O_ < R8CXE~E A ~ ~ NTs
H2~ NH2N I Ts Na~
~~ .D MF
R~ R ~ R
~ 3R3 R~R'R~ 6
H2~ N~2 R ~ ~ ~ R7
TSa ~ OH Ho~R.
Tsa / Et3N
R CP2CI2
T~IIN NSIT~ ~6
OTs T
N~H /D~fF,100qC
R~
R5 R'5 Ts ~ R',
/1. }3Br or H2SO~ or Na~CloHt]
/ 2. NaOH R
~N~
s R3
CA 02229799 1998-02-17
W O 97/06824
PCT~US96/12767
-27-
B
~R~N~
MnCI2
MeOH
F~O2
H2, 100 psi
~" MeOH, 100~C
Rg /~\
CA 02229799 1998-02-17
WO 97/06824 PCT~US96/12767
-28-
8C~EME C
F~R~ pTyS~ln Na N--Ts
Rg--cHo d 43~cNaoc2Hs ~s ~
" 2 HCI4 BrCR2COOCH3
R 9 0~_OCH3 CH30_ ?
H N) R9 ~ H R7~ N_, T~; 2
H2. PtO2, HCI R.~S~R,R'
1. NaOH
~ ~ R ~ 2. HCI
Rg- ) ~ RR, R ~ H O H HO_
Cl HJN + +NH3 Cl ~ R7 ~~ N~,2
base ~.>~j~R,
R ~ 9 ~ ~' ~ Cl Cl ~
bas~ TSR~N ~RR,2
o~o s R.~
RTS~ ,~RR2~ R9~R
~s~ R~ RR2
--R~R. ~
CA 02229799 1998-02-17
WO 97/06824 PCT/US96/12767
CH~0CH 3
CH 30H
renLK
h,~= \~ R~
H2, ~t~2
CH ~OH Ha
R2>~ N' ~R~
~ ~ R,
\ LIAIH "
\~thf
~n N RR ~,~N ' R'7
A~N~
CA 02229799 1998-02-17
W O 97/06824 . PCTAUS96/12767
-30-
Scheme E
R,
R~~~~f~2
LW~
\ dme or thf
MnC~ ~ R,
CA O 2 2 2 9 7 9 9 19 9 8 0 PC~lus96ll2767
WO 97/068~4
_3 1--
SC~ F s ~ ~OCE~
5 ~5
}~L~_ RL TS O R~
~L ~ R~ N ~N~ a
KL;~ cP,CO~
Rg X9
~2 ~ ~ ~0 ~ TPJ~
~>~
1~ o R5 X5 OC~3X~ ' ~I~OC~3
R3~ ~-- ~ ~C3t~O~c~ NN~--r~ ~7
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WO 97/06824 PCTAUS96112767
-32-
8~M~ F (cont.)
E~A
or
BE~
MC
CA 02229799 1998-02-17
WO 97/06824 PCT/US96/12767
--33--
8C~EME G
R9 R, O EDC-HCI, HOBT, Rg R H O
>~ OH H2N Jl~ DMF, TEA, RT
BocNH ~ R >~ OEt a J~, ~ '~OEI
DMF,TEA, O ~C O R, Rl
Rg R', IH o EDC-HCI, HOBT,
CH30H >~N>~I~OH + H2N>:J~OEt DMF, TEA, RT
R' 'R ~' Ethyl cl '~ '~ ,.. t~"
~ ,~lEt NaOH, H20
Rs R, IH ~ R3 ~R, H O EDC-HCI, HOBT, o
~ ~ DMF, TEA, RT
eacNH ~N~ l ~N~ N~ ~OEt ~ H2N~.~ l ~
¦¦ R~ Ethyl r'~ Illdlr, /~ OEt
O ~ R1 H o R S R5 DMF,TEA,0~CR 5 R5
NaOH, H20
CH30H
y
Rg R ~ H o R H O EDC-HCI, HOBT,
>~ >~ ~ ~OH + ~OEt
" Ethyl cl ,l~
O R I R1 H o R 5 R5 R, R7 DMF, TEA,0~C
CA 02229799 1998-02-17
W O 97/06824 PCT~US96/12767
F!M~;! G ~ cont. )
R9~?~1
NaOH. H20
CH ,OH
BocNA~ H O
~ CH2CI2 or HCI, ethet
H2N>~ ~N>~ ?~N~ R j~ R~ R,
o R, R1 H o R~ H O DPPA DMF TE~, R7 NH ~
o~ ~R3
LiAlH,
THF. .
Ot
BH3, THF
F. ~R, MDCIZ MeOH ~ f
CA 02229799 1998-02-17
W O 97/06824 PCT~US96/12767
The pentaazamacrocycles of the present invention
can possess one or more asymmetric carbon atoms and are
thus capable of existing in the form of optical isomers
as well as in the form of racemic or nonracemic mixtures
thereof. The optical isomers can be obtained by
resolution of the racemic mixtures according to
conventional processes, for example by formation of
diastereoisomeric salts by treatment with an optically
active acid. Examples of appropriate acids are
tartaric, diacetyltartaric, dibenzoyltartaric,
ditoluoyltartaric and camphorsulfonic acid and then
separation of the mixture of diastereoisomers by
crystallization followed by liberation of the optically
active bases from these salts. A different process for
separation of optical isomers involves the use of a
chiral chromatography column optimally chosen to
~ ; ;ze the separation of the enantiomers. Still
another available method involves synthesis of covalent
diastereoisomeric molecules by reacting one or more
secondary amine group(s) of the compounds of the
invention with an optically pure acid in an activated
form or an optically pure isocyanate. The synthesized
diastereoisomers can be separated by conventional means
such as chromatography, distillation, crystallization or
sublimation, and then hydrolyzed to deliver the
enantiomerically pure ligand. The optically active
compounds of the invention can likewise be obtained by
utilizing optically active starting materials, such as
natural amino acids.
The compounds or complexes of the present
invention are novel and can be utilized to treat
numerous inflammatory disease states and disorders. For
example, reperfusion injury to an ischemic organ, e.g.,
reperfusion injury to the ischemic myocardium,
surgically-induced ischemia, inflammatory bowel disease,
rheumatoid arthritis, osteoarthritis, psoriasis, organ
CA 02229799 l998-02-l7
~-21(-2523)A
-36-
transplant rejections, radiation-induced injury,
oxidant-induced tissue in~uries and damage,
atherosclerosis, thrombosis, platelet aggregation,
stroke, acute pancreatitis, insulin-dependent diabetes
mellitus, disseminated intravascular coagulation, .atty
embolism, adult and infantile respiratory distress,
metastasis and carcinogenesis.
Activity of the compounds or complexes of the
present invention for catalyzing the dismutation of
superoxide can be demonstrated using the stopped-flow
kinetic analysis technique as descri~ed in ~iley, D.P.,
Rivers, W.J. and Weiss, R.H., "Stopped-Flow Kinetic
Analysis for Monitoring Superoxide Decay in Aqueous
Systems," ~nal. 3iochem., 196, 344-349 (1991)~
St-opped-~low kinetic
analysis is an accurate and direct method for
~uantitatively monitoring the decay rates of superoxide
in water. The stopped-flow kinetic analysis is suitable
~or screening compounds ~or SOD activity and catalytic
activity of the compounds or complexes o~ the present
invention for dismutating superoxide, as shown by
stopped-flow analysis, correlate to treating the above
disease states and disorders.
Total daily dose administered to a host in single
or divided doses may ~e in amounts, for example, from
about l to about 100 mg/kg body weight daily and ~ore
usually about 3 to 30 mg/kg. Unit dosage compositions
may contain such amounts of submultiples thereof to make
up the daily dose.
The amount of active ingredient that may be
combined with the carrier materials to produce a single
dosage form will vary depending upon the host treated
and .he particular mode of administration.
The dosage regimen ~or treating a disease
condition with the compounds and/or compositions of this
invention is selected in accordance with a variety of
CA 02229799 1998-02-17
Wo97/06824 PCT~S96/12767
-37-
factors, including the type, age, weight, sex, diet and
medical condition of the patient, the severity of the
disease, the route of administration, pharmacological
considerations such as the activity, efficacy,
pharmacokinetic and toxicology profiles of the
particular compound employed, whether a drug delivery
system is utilized and whether the compound is
a~m;ni~tered as part of a drug combination. Thus, the
dosage regimen actually employed may vary widely and
therefore may deviate from the preferred dosage regimen
set forth above.
The compounds of the present invention may be
a~ini~tered orally, parenterally, by inhAl~tion spray,
rectally, or topically in dosage unit formulations
containing conventional nontoxic pha~maceutically
acceptable carriers, adjuvants, and vehicles as desired.
Topical administration may also involve the use of
transdermal a~in;~tration such as transdermal patches
or iontophoresis devices. The term parenteral as used
herein includes subcutaneous injections, intravenous,
intramuscular, intrasternal injection, or infusion
techniques.
Injectable preparations, for example, sterile
injectable aqueous or oleaginous suspensions may be
formulated according to the known art using suitable
dispersing or wetting agents and suspending agents. The
sterile injectable preparation may also be a sterile
injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent, for example,
as a solution in l,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water,
Ringer's solution, and isotonic sodium chloride
solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending
medium. For this purpose any bland fixed oil may be
employed including synthetic mono- or diglycerides. In
CA 02229799 l998-02-l7
~-21(-252~)A
-38-
addition, ~atty acids such as oleic acid find use in the
preparation of injectables.
Suppositories ~or rectal administration of the
drug can be prepared by mixing the drug with a suitable
nonirritating excipient such as cocoa butter and
polyethylene glycols which are solid at ordinary
temperatures but liquid at the rectal temperature and
will therefore melt in the rectum and release the drug.
Solid dosage ~orms for oral administration may
include capsules, tablets, pills, powders, granules and
gels. In such solid dosage ~orms, the active compound
may be admixed with at least one inert diluent such as
sucrose lactose or starch. Such dosage forms may also
comprise, as in normal practice, additional substances
other than inert diluents, e.g., lubr-icating agents such
as magnesium stearate. In the case of capsules,
tablets, and pills, the dosage forms may also comprise
buffering agents. Tablets and pills can additionally be
pre?ared with enteric coatings.
Liquid dosage forms for oral administration may
include pharmaceutically acceptable emulsions,
solutions, suspensions, syrups, and elixirs containing
inert diluents commonly used in the art, such as water.
Such compositions may also comprise adjuvants, such as
wetting agents, emulsifying and suspending agents, and
sweetening, flavoring, and perfuming agents.
While the compounds of the invention can be
administered as the sole active pharmaceutical agent,
they can also be used in combination with one or more
compounds which are known to be ef~ective against the
specific disease state that one is targeting for
treatment.
The compounds or complexes o~ the invention can
also be utilized as MRI contrast agents. A discussion
of the use of contrast agents in MRI can be ~ound in
patent application Serial No. 08/397,469~
A~/IENO~D S~EET
IPEA/EP
CA 02229799 l998-02-l7
07-21~12523)~
.
-39-
Contemplated equivalents of the general formulas
set forth above for the compounds and derivatives as
well as the intermediates are compounds otherwise
corresponding thereto and having the same general
properties such as tautomers of the compounds and such
as wherein one or more of the various R groups are
simple variations of the substituents as defined
therein, e.g., wherein R is a higher alkyl group than
that indicated, or where the tosyl groups are other
nitrogen or oxygen protectinq groups or wherein the
O-tosyl is a halide. Anions having a char~e other than
1, e.g., carbonate, phosphate, and hydrogen phos~hate,
can be used instead of anions having a charge of 1, so
long as they do not adversely affect-the overall
activity of the complex. However, using anions having a
charge other than 1 will result in a slight modification
of the general ~ormula for the complex set forth above.
In addition, where a substituent is designated as, or
ZO can be, a hydrogen, the exact chemical nature of a
substituent which is other than hydrogen at that
position, e.g., a hydrocarbyl radical or a haloqen,
hydroxy, amino and the like functional group, is not
critical so long as it does not adversely a~fect r he
overall activity and/or synthesis procedure. Further,
it is contemplated that manganese(III) complexes will be
equivalent to the subject manganese(II) complexes.
The chemical reactions described above are
generally disclosed in terms of their broadest
application to the preparation of the compounds of this
invention. Occasionally, the reactions may not be
applicable as described to each compound included within
the disclosed scope. The compounds ~or which this
occurs will be readily recognized by those skilled in
3~ the art. In all such cases, either the reactions can be
success~ully performed by conventional modifications
~MÇ~N~ D ~ ~ET
-
CA 02229799 1998-02-17
~7-2~(125Z3)~
-40-
known to those skilled in the art, e.g., by appropriate
protection o~ inter~ering groups, ~y changing to
alternative conventional reagents, by routine
modi~ication o~ reaction conditions, and the like, or
other reactions disclosed herein or otherwise
conventional, will be applicable to the preparation o~
the corresponding compounds o~ this invention. In all
preparative methods, all starting materials are known or
readily preparable ~rom known starting materials.
EXAMPLES
All reagents were used as received without
purification unless otherwise indicated. All NMR
spectra were obtained on a Varian VXR-300 or VXR-400
nuclear magnetic resonance spectrometer. Qualitative
and quantitative mass spectroscopy was run on a Finigan
MATso, a Finigan 4500 and a VG40-250T using
m-nitrobenzyl alcohol (NBA), m-nitrobenzyl alcohol/LiCl
(NBA - Li). Melting points (mp) are uncorrected.
The ~ollowing abbreviations relatinq to amino
acids and their protective groups are in accordance with
the recommendation by IUPAC-IUB Commission on
Biochemical Nomenclature (Biochemistry 1972, 11, 1726)
and common usage.
T C
. _ , .. .
CA 02229799 1998-02-17
W 097/06824 PCTrUS96/12767
Ala L-Alanine
DAla D-Alanine
Gly Glycine
Ser L-Serine
5 DSer D-Serine
Bzl Benzyl
Boc tert-Butoxycarbonyl
Et Ethyl
TFA Trifluoroacetic acid
10 DMF Dimethylformamide
H0BT-H20 l-Hydroxy- (lH) -benzotriazole
monohydrate
EDC-HCl 1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride
15 TEA Triethylamine -
DMS0 Dimethylsulfoxide
THF Tetrahydrofuran
DPPA Diphenylphosphoryl azide
*The abbreviation Cyc represents 1,2-cyclohexanediamine
(stereochemistry, i.e. R,R or S,S, is indicated as
such). This allows three letter code peptide
nomenclature to be used in pseudopeptides containing the
1,2-cyclohexane diamine "residue".
ExamPle 1
A. Svnthesis of N-(P-toluenesulfonvl)-fR.R)-l 2-
diaminocYclohexane
To a stirred solution of (R,R)-1,2-
diaminocyclohexane (300 g, 2.63 mole) in CH2Cl2 (5.00 1)
at -10~ C was added a solution of
p-toluenesulfonylchloride (209 g, 1.10 mole) in CH2Cl2
(5.00 1) dropwise over a 7 h period, maintaining the
temp at -5 to -10~ C. The mixture was allowed to warm
to room temp while stirring overnight. The mixture was
concentrated in vacuo to a volume of 3 1 and the white
_
CA 02229799 1998-02-17
PCT~US96/12767
W O 97/06824
solid was removed by filtration. The solution was then
washed with H20 (10 x 1 1) and was dried over MgSO~.
Removal of the solvent in vacuo gave 286 g (97.5 ~
yield) of the product as a yellow crystalline solid: 'H
NMR (CDCl3) ~ 0.98 - 1.27 (m, 4 H), 1.54 - 1.66 (m, 2
H), 1.81 - 1.93 (m, 2 H), 2.34 (dt, J - 4.0, 10.7 Hz, 1
H), 2.42 (s, 3 H), 2.62 (dt, J = 4.2, 9.9 Hz, 1 H), 7.29
(d, J = 8.1 Hz, 2 H), 7.77 (d, J = 8.3 Hz, 2 H); MS
(LRFAB - DTT - DTE) m/z 269 [M + H]+.
B. SYnthesis of N-(~-toluenesulfonyl)-N -(Boc)-(R R)-
1.2-diaminocvclohexane
To a stirred solution of N-(p-toluenesulfonyl)-
(R,R)-1,2-~; A~; nocyclohexane prepared as in Example lA
(256 g, 0.955 mole) in THF (1.15 1) was added a 1 N
solution of aqueous NaOH (1.15 1, 1.15 mole). Di-t-
butyldicarbonate (229 g, 1.05 mole) was then added and
the resulting mixture was stirred overnight. The layers
were separated and the a~ueous layer was adjusted to pH
2 with 1 N HCl and saturated with NaCl. The aqueous
solution was then extracted with CH2Cl2 (2 x 500 mL) and
the extracts and THF layer were combined and dried over
MgSO4. The solvent was removed in vacuo to give a yellow
solid. The crude product was purified by
crystallization from a THF-ether-heXanes mixture to give
310 g (88.1% yield) of the product as a white
crystalline solid: mp: 137 - 139~ C; lH NMR (CDCl3) ~
1.04 - 1.28 (m, 4 H), 1.44 (s, 9 H), 1.61 - 1.69 (m, 2
H), 1.94 - 2.01 (m, 2 H), 2.43 (s, 3 H), 2.86 (brs, 1
H), 3.30 (br d, J = 9.6 Hz, 1 H), 4.37 (br d, J = 6.7
Hz, 1 H), 5.48 (br d, J = 4.6 Hz, 1 H), 7.27 (d, J = 9.7
Hz, 2 H), 7.73 (d, J = 8.1 Hz, 2 H); MS (LRFAB, NBA -
Li) m/z 37S [M + Li]t.
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-43-
C. Svnthesis of Boc-(R R)-Cvc(Ts)-qlv-OMe
To a stirred solution of N-(p-toluenesulfonyl)-
N -(Boc)-(R,R)-1,2-~ nocyclohexane prepared as in
Example lB (310 g, 0.841 mole) in anhydrous DMF (3.11 1)
at 0~ C was added NaH (37.4 g - 60 % in oil, 0.934 mole)
in portions and the resulting mixture was stirred for 30
min. Methyl bromoacetate (142 g, 0.925 mole) was then
added dropwise over 45 min and the mixture was allowed
to warm to room temp while stirring overnight. After
10 stirring for 17 h, the solvent was removed in vacuo and
the residue was dissolved in ethyl acetate(3 1) and H2O
(1 1). The ethyl acetate solution was washed with
saturated NaHCO3 (1 1), saturated NaCl (500 mL) and was
dried over MgSO4. The solvent was removed in vacuo and
15 the resulting oil was dissolved in ether.
Crystallization by the addition of hexanes gave 364 g
(98 % yield) of the product (TLC (98:2 CHCl3--MeOH/silica
gel/W detn) showed that the product contained about 5%
starting material) as colorless needles: mp of pure
20 sample lS1 - 2~ C; ~H NMR (CDCl3) ~ 1.11 - 1.22 (m, 4
H), 1.45 (s, 9 H), 1.64 -- 1.70 (m, 3 H), 2.16 -- 2.19 (m,
1 H), 2.43 (s, 3 H), 3.34 - 3.40 (m, 2 H), 3.68 (s, 3
H), 4.06 (ABq, J = 18.5 Hz, ~ = 155 Hz, 2H), 4.77 (br
s 1 H), 7.30 (d, J = 8.3 Hz, 2 H), 7.82 (d, J = 8.3 Hz,
25 2 H); MS (LRFAB, DTT - DTE) m/z 441 [M + H]t.
D. Svnthesis of Boc-(R,R)--Cvc(Ts)--Glv--OH
To a stirred solution of impure Boc--(R,R)--
Cyc(Ts)--Gly-OMe prepared as in Example lC (217 g, 0.492
30 mole) in MeOH (1.05 1) was slowly added a 2.5N solution
of aqueous NaOH (295 mL, 0.737 mole) and the resulting
solution was stirred for 2 h. The solvent was removed
in vacuo and the residue was dissolved in H2O (1.5 1).
The solution was filtered to remove a small amount of
35 solid and was washed with ether (7 x 1 1) to remove the
-
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W O 97/06824 PCT~US96/12767
impurity (compound lB) which upon drying of the combined
washes over MgSO4 and removal of the solvent in vacuo
resulted in recovery of 8.37 g. The pH of the aqueous
solution was then adjusted to 2 with 1 N HCl and the
s product was extracted with ethyl acetate (3 x 1 l).
The extracts were combined, washed with saturated NaCl
(500 mL) and dried over MgSO~. The solvent was removed
in vacuo and the residual ethyl acetate removed by
coevaporation with ether (500 mL) and then CH2Cl2 (500
mL) to give 205 g (97.6 ~ yield) of the product as a
white foam: lH NMR (CDCl3) ~ 1.15 - 1.22 (m, 4 H), 1.48
(s, 9 H), 1.55 - 1.68 (m, 3 H), 2.12 - 2.15 (m, 1 H),
2.43 (s, 3 H), 3.41 - 3.49 (m, 2 H), 3.97 (ABq, J = 17.9
Hz, A u = 69.6 Hz, 2 H), 4.79 (br s, 1 H), 7.31 (d, J =
8.1 Hz, 2 H), 7.77 (d, J = 8.3 Hz, 2-H), 8.81 (br s, 1
H); MS (LRFAB, NBA - Li) m/z 433 [M + Li]+.
. Svnthesis of Boc-(R.R)-Cyc(Ts) -G1Y - G1Y - OEt
To Boc-(R,R)-Cyc(Ts)-Gly-OH (18.1 g, 43.1 mmol)
in DMF (480 mL) was added HOBt-H2O (7.92 g, 51.7 mmol)
and EDC-HCl (9.91 g, 51.7 mmol) and the resulting
mixture was allowed to stir for 20 min at RT. To this
solution was added GlyOEt-HCl (6.0 g, 43.1 mmol) and TEA
(7.2 mL, 51.7 mmol) and the resulting mixture was
allowed to stir for 16 h thereafter. The DMF was
evaporated and the residue was partitioned between water
(250 mL) and EtOAc (400 mL). The EtOAc layer was
separated and washed with lN KHSo4 (250 mL), water (250
mL), sat. NaHCO3 (250 mL) and brine (250 mL) and dried~
(Na2SO4). Filtration and concentration afforded 21.9 g
(99 % yield) of pure product as a white foam: lH NMR
(DMSO-d6) ~ 1.00 - 1.10 (m, 1 H), 1.19 (t, J = 7.6 Hz, 3
H), 1.38 (s, 9 H), 1.50 - 1.56 (m, 3 H), 1.75 - 1.84 (m,
1 H), 2.38 (s, 3 H), 3.30 -3.40 (bs, 2 H), 3.75 -4.01
(complex m, 4H), 4.08 (q, J = 7.6 Hz, 2 H), 6.05 (bs, 1
_
CA 02229799 1998-02-17
W O 97/06824 PCTAJS96/12767
H), 7.32 (d, J = 8.0 Hz, 2 H), 7.77 (d, J = 8.0 Hz, 2
H), 8.32 (t , J = 7.2 Hz, 1 H); MS(HRFAB) m/z 518.2551
(M + Li) t; 518.2512 calculated for C24H3~N3O~SLi.
F. Svnthesis of CYc(Ts)-GlY-GlY-OEt TFA salt
To a solution of Boc-Cyc(Ts)-Gly-Gly-OEt (21.2 g,
41.4 mmol) in CH2Cl2 (180 mL) was added TFA (44 mL) and
the resulting mixture was stirred at RT for 30 min. The
solution was concentrated and the residue was dissolved
in ether (50 mL) and precipitated with hexanes (500 mL).
The solvents were decanted and the residue was washed
with 10:1 hexanes/ether (500 mL). The final residue was
dried thoroughly at high vacuum to afford 20.7 g (95%
yield) of the product as a tan foam: IH NMR (DMSO-d6) ~
0.85 - 0.96 (m, 1 H), 1.03 - 1.31 (complex m, 7 H), 1.09
(t, J = 7.6 Hz, 3 H), 2.00 (m, 1 H), 2.39 (s, 3 H), 3.02
(bs, 1 H), 3.62 (m, 1 H), 3.82 - 4.05 (m, 4 H), 4.10 (q,
J = 7.6, 2 H), 7.41 (d, J = 8.0 Hz, 2 H), 7.67 (d, J =
8.0 Hz, 2 H), 8.25 (bs, 3 H), 9.09 (t, J = 5.63 Hz, 1
H). MS(HRFAB) m/z 418.1990 (M -TFA + Li)+; 418.1988
calculated for Cl9H29N3O5S.
G. SYnthesis of Boc-Orn(Z)-Cvc(Ts)-Glv-Glv-OEt
To Boc-Orn(Z)-OH (8.37 g, 22.8 mmol) in DMF (200
mL) was added HOBt-H2O (4.29 g, 27.4 mmol) and EDC-HCl
(5.25 g, 27.4 mmol) and the resulting solution was
stirred for 20 min at RT. To this solution was added
Cyc(Ts)-Gly-Gly-OEt TFA salt (12.0 g, 22.8 mmol) and TEA
(3.82 mL, 27.4 mmol) and stirring was maintained for 16
h thereafter. The DMF was evaporated and the residue
was partitioned between water (200 mL) and EtOAc (250
mL). The ETOAc layer was separated and washed with lN
KHSO4 (150 mL), water (150 mL), sat. NaHCO3 (150 mL) and
brine (150 mL) and dried (MgSO4). Filtration and
concentration afforded 15.1 g (87 % yield) of the
. .
CA 02229799 l998-02-l7
W 097106824 PCT~US96/12767
product as a white foam: ~H NMR (DMSO-d6) ~ 1.00 - 1.94
(complex m, 12 H), 1.15 (t, J = 7.4 Hz, 3 H), 2.38 (s, 3
H), 2.98 (bs, 2 H), 3.30 - 3.46 (m, 2 H), 3.70 - 3.82
(m, 4 H), 3.90 ~.02 (m, 1 H), 4.05 (t, J = 7.4 Hz, 2 H),
5.00 (s, 2 H), 6.43 (m, 1 H), 7.17 (m, 1 H), 7.20 - 7.37
(m, 8 H), 7.78 (m, 2 H), 8.30 (bs, 1 H); MS(LRFAB, NBA +
HCl) m/z 760 (M + H) t-
H. Svnthesis of Orn(Z)-CYc(Ts)-Glv-GlY-OEt TFA salt
To a solution of Boc-Orn(Z)-Cyc(Ts)-Gly-Gly-oEt
(14.5 g, 19.1 mmol) in CH2Cl2 (120 mL) was added TFA (30
mL) and the resulting solution was stirred at RT for 30
min. The solution was evaporated and the residue was
triturated with ether (100 mL). The ether was decanted
and the residue was dried thoroughly-at high vacuum to
a~ford 15.5 g (>100 % yield, contains TFA) of the
product as an orange foam: IH NMR (DMSO-d6) ~ 0.97 -
1.93 (comples m, 12 H), 1.16 (t, J = 7.4 Hz, 3 H), 2.38
(s, 3 H), 2.98 (bs, 2 H), 3.31 - 3.50 (m, 2 H), 3.71 -
3.91 (m, 4 H), 3.97 - 4.04 (m, 1 H), 4.08 (q, J 5 7.4
Hz, 2 H), 5.00 (s, 2H), 7.23 - 7.39 (m, 8 H), 7.77 -
7.81 (m, 2H), 8.18 (bs, 3 H), 8.41 (bs, 1 H); MS(LRFAB,
NBA ~ HCl) m/z 660 (M - TFA)~.
I. Svnthesis of Boc-Glv-Orn(Z)-Cvc(Ts)-Glv-Gly-OEt
To a solution of Boc-Gly-OH (3.36 g, 19.2 mmol)
in DMF (220 mL) was added HOBt-H2O (3.52 g, 23.0 mmol)
and EDC-HCl (4.41 g, 23.0 mmol) and the resulting
solution was stirred for 20 min at RT. To this solution
was added Orn(Z)-Cyc(Ts)-Gly-Gly-OEt TFA salt (14.8 g,
19.2 mmol) and TEA (3.20 mL, 23.0 mmol) and stirring was
maintained for 12 h thereafter. The DMF was evaporated
and the residue was partitioned between water (200 mL)
and EtOAc (350 mL). The layers were separated and the
EtOAc layer was washed with lN KHS04 (150 mL), water (150
CA 02229799 l998-02-l7
Wo97/06824 PCT~S96/12767
-~7-
mL), sat. NaHCO3 (150 mL) and brine (150 mL) and dried
(MgSO4). Filtration and concentration afforded 13.7 g
(87% yield) of the product as a white foam: lH NMR (DMSO-
d6) ~ 0.96 - 1.10 (m, 2 H), 1.17 (t, J = 7.4 Hz , 3 H),
5 1.38 (s, 9H), 1.35 - 2.00 (complex m, lO H), 2.97 (m, 2
H), 3.60 (bs, 2 H), 3.67 - 3.84 (m, 4 H), 3.93 - 4.03
(m, 3 H), 4.06 (q, J = 7.4 Hz, 2 H), 6.92 (bs, lH), 7.19
(m, 1 H), 7.24 - 7.37 (m, 7 H), 7.60 (d, J = 8.3 Hz, 1
H), 7.76 (m, 2 H), 7.38 (bs, 1 H). MS(LRFAB, NBA + Li)+
m/z 823 (M+Li)'.
J. SYnthesis of Boc-GlY-OrnfZ)-CYc(Ts)-Glv-GlY-OH
To a solution of Boc-Gly-Orn(Z)-Cyc(Ts)-Gly-Gly-
OEt (13.3 g, 16.3 mmol) in methanol (lOO mL) was added 1
15 N NaOH (25 mL). The resulting mixtufe was stirred at RT
and monitored by TLC. After 2 h the reaction was
complete. The methanol was evaporated and water (50 mL)
was added to the residue. This aqueous phase was washed
with EtOAc ( 2 x 100 mL) and the EtOAc layers were
20 discarded. The pH was lowered to 3.5 with lN KHSO4and
the aqueous phase was extracted with EtOAc ( 3 x 100 mL).
The combined EtOAc layers were dried (MgSO~), filtered
and concentrated to afforded 11. 7 g (91 ~ yield) of the
product as a white foam: IH NMR (CDCl3) ~ O. 98 - 1.25
25 (m, 2 H), 1.38 (s, 9 H), 1.40 - 1.92 (m, lO H), 2.38 (s,
3 H), 2.97 (m, 2 H), 3.62 (bs, 2 H), 3.75 - 3.85 (m, 3
H), 3.95 - 4.05 (m, 2 H), 5.01 (s, 2 H), 6.96 (bs, 1 H),
7.28 (m, 1 H), 7.25 - 7.38 (m, 7 H), 7.61 (d, J = 8.4
Hz, 1 H), 7.78 (m, 2 H), 8.25 (bs, 1 H).
K. Svnthesis of GlY-Orn(Z)-Cvc(Ts)-Glv-GlY-OH TFA salt
~o a solution of Boc-Gly-Orn(Z)-Cyc(Ts)-Gly-Gly-
OH (11.2 g, 14.3 mmol) in CH2Cl2 (lOO mL) was added TFA
(24 mL) and the resulting solution was stirred for 30
35 min at RT. The solution was concentrated and triturated
CA 02229799 1998-02-17
W O 97/06824 PCT~US96/12767
-48-
with ethyl ether (500 mL). Filtration of afforded 11.3
g (99 % yield) of the product as a white powder: lH NMR .
(DMSO-d6) ~ 0.95 - 1.98 (complex m, 12 H), 2.39 (s, 3 H),
3.01 (m, 2 H), 3.38 (m, 1 H), 3.65 - 4.10 (complex m, 7
H), 4.18 (q, J = 7.4 Hz, 1 H), 5.02 (s, 2 H), 7.24 -
7.40 (m, 9 H), 7.77 - 7.85 (m, 2 H), 8.13 (bs, 3 H),8.31
(bs, 1 H), 8.42 (d, J = 8.3 Hz, 1 H); MS(HRFAB) 689.2953
(M-TFA)~; 689.2969 calculated for C32H45N6O9S.
L. Svnthesis of cyclo-(Glv-Orn(Z)-Cvc(Ts)-Glv-Glv-)
A solution of Gly-Orn(Z)-Cvc(Ts)-Glv-GlY-OH TFA
salt (5.0 g, 6.23 mmol) in dry degassed DMF (1520 mL)
was treated with TEA (1.74 mL, 12.5 mmol) and cooled to
-40 ~C. DPPA (1.64 mL, 7.60 mmol was added dropwise
over 10 min and the reaction was stirred at -40 ~C for 3
hr therea~ter. A~ter this time the reaction was place
in a -2 ~C bath and allowed to stand at this temperature
for 16 h thereafter. Water (1520 mL) was added and the
resulting solution was stirred with mixed bed ion-
exchange resin (750 g ) for 6 h at RT. The resin wasfiltered and the solution was concentrated to a volume
of -100 mL (DMF). The addition of ethyl ether (500 mL)
produced a solid residue which was redissolved in
methanol (100 mL) and again precipitated by the addition
of ethyl ether (500 mL). Filtration afforded 3.26 g (78
% yield) of product as a white powder: IH NMR (CDCl3)
0.96 - 2. 10 (complex m, 14 H), 2.37 (bs, 3 H), 2.68 -
3.05 (m, 3 H), 3.42 - 3.90 (complex m, 8 H), 4.14 (m, 1
H), 4.20 (m, 1 H), 4.97 - 5.08 (m, 3 H), 6.42 (d, J =
8.4 Hz, 1 H), 7.19 (d, J = 8.0 Hz, 1 H), 7.20 - 7.39 (m,
7 H), 7.65 - 7.78 (m, 2 H), 9.15 (bs, 1 H), 9.22 (bs, 1
H); MS(HRFAB) m/z 671.2842 (M + H)'; 671.2863 calculated
for C32H43N6O8S-
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M. Svnthesis of cYclo-(GlY-Orn-CYc(Ts)-Glv-GlY-t
To a solution of cyclo-(Gly-Orn(Z)-Cyc(Ts)-Gly-
Gly-) (3.94 g, 5.90 mmol) in methanol (40 mL) was added
Pd (black) (1.0 g) and ammonium formate (2.0 g). The
reaction was refluxed for 2 h and allowed to cool. The
mixture was filtered under Argon through a pad of celite
and the filtrate was concentrated to afford 2.86 g (89 %
yield) of product as a white foam: lH NMR (DMSO-d6)
0.94 - 2.22 (complex m, 12 H), 2.39 (s, 3 H), 2.55 -
2.95 (m, 7 H), 3.42 - 3.89 (complex m, 9 H), 4.11 (m, 1
H), 4.39 (m, 1 H), 6,43 (d, J = 8.4 Hz, 1 H), 7.27 (d, J
- 9.3 Hz, 1 H), 7.25 - 7.45 (m, 2 H), 7.64 - 7.80 (m, 2
H), 9.12 - 9.29 (m, 2 H); MS (HRFAB) m/z 537.2511 (M +
H)'; 537.2495 calculated for C24H36N6SO6.
N. SYnthesis of cYclo-(Glv-Orn(Lithocholvl)-Cyc(Ts)-
ÇlY-Gly-)
To a solution of cyclo-(Gly-Orn-Cyc(Ts)-Gly-Gly-)
(1.0 g, 1.9 mmol) in CHCl3 (25 mL) was added lithocholic
acid NHS active ester (881 mg, 1,9 mmol) and the
resulting mixture was stirred for 16 h thereafter.
Addition of ethyl ether (50 mL) produced a solid.
Filtration afforded 946 mg (56 % yield) of the product
as a tan powder: 'H NMR (CD30D) ~ 0.66 (m, 3 H), 0.93
(bs, 6 H), 0.94 - 2.37 (complex m, 48 H), 2.43 (s, 3H),
2.80 - 4.60 (bm, 14 H), 7.39 (bs, 2 H), 7.80 (bs, 2 H);
MS (HRFAB) m/z 895.5432 (M + H)+; 895.5367 calculated
for C~H~5N6O~S.
O. Svnthesis of 2.3-(R.R)-Cvclohexano-6-(5)-~3-
(lithocholylamino)Propyl~-l 4 7 10 13-Penta-
azaccloDentadecane
To a suspension of cyclo-(Gly-Orn(Lithocholyl)-
Cyc(Ts)-Gly-Gly-) (2.70 g, 3.00 mmol) in THF (50 mL) was
added lithium aluminum hydride (51.0 mL of a 1.0 M
solution). The resulting mixture was refluxed for 16 h
CA 02229799 1998-02-17
WO 97/06824 PCT/US96/12767
--50--
thereafter. The reaction mixture was cooled to --20 ~C
and ~uenched (cautiously) with 5 % Na2S04 (30 mL)
followed by methanol (30 mL). This solution was stirred
at RT for 1 h and concentrated to a dry powder. The
powder was triturated with ethyl ether (3 x 200 mL) and
filtered. The ether was concentrated and the oil was
recrystallized from acetonitrile to afford 800 mg (40 %
yield) of product as a colorless oil: IH NMR (C6D6) ~
0.64 (s, 3 H), 0.67 (s, 3 H), 0.88 (d, J = 3.0 Hz, 3 H),
0.84 - 2.61 (complex m, 52 H), 2.38 - 2.95 (complex m,
14 H), 3.49 (m, 3 H); 13C NMR (CDCl3) ~ 71.4, 63.1, 62.6,
61.8, 58.2, 56.5, 56.1, 51.5, 50.4, 50.1, 48.3, 47.9,
46.1, 45.7, 42.6, 42.1, 40.4, 40.1, 36.4, 35.8, 35.7,
35.6, 35.4, 34.5, 31.9, 31.7, 31.6, 30.8, 30.5,29.4,
28.3, 27.2, 26.4, 26.2, 24.9, 24.2, ~3.4, 20.8, 18.6,
12.0; MS(LRFAB, NBA + Li~ m/z 677 (M+Li)+.
P. Synthesis of rManqanese (II) dichloro 2 3-(R.R)-
CYC1 ohexano-6-(S)-~3-(lithocho 1Y1 amino)-~ro~vl~-
1,4 7,10.13-~enta-azaccloDentadecane]
2,3-(R,R)-Cyclohexano-6-(S)-{3-
(lithocholylamino)propyl}-1,4,7,10,13-penta-
azacclopentadecane prepared as in example 10 (547 mg,
0.817 mmol) was added to a hot anhydrous methanol
solution (50 mL) containing manganese (II) chloride
(103 mg, 0.818 mmol) under a dry nitrogen atmosphere.
After refluxing for 2 h the solution was reduced to
dryness and the residue was dissolved in a solvent
mixture of THF (35 mL) and ethyl ether (5 m~) and
filtered through a pad of celite. Concentration and
trituration with ethyl ether afforded after filtration
512 mg (79 % yield) of the complex as a white solid: FAB
mass spectrum (NBA) m/z 760 tM-Cl]+; Anal. Calculated. !'
for C41H78N60MnC12: C, 61.79; H, 9.87; N, 10.55; Cl,
8.90. Found: C, 62.67; H, 9.84; N, 8.04; Cl, 8.29.
CA 02229799 1998-02-17
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Ex~mPle 2
StoPPed-Flow Kinetic AnalYsis
Stopped-flow kinetic analysis has been utilized
to determine whether a compound can catalyze the
dismutation of superoxide (Riley, D.P., Rivers, W.J. and
Weiss, R.H., "Stopped-Flow Kinetic Analysis for
Monitoring Superoxide Decay in Aqueous Systems," Anal.
Biochem, 196, 344-349 [1991]). For the attainment of
consistent and accurate measurements all reagents were
biologically clean and metal-free. To achieve this, all
buffers (Calbiochem) were biological grade, metal-free
buffers and were handled with utensils which had been
washed first with 0.1 N HCl, followed by purified water,
followed by a rinse in a 104 M EDTA bath at pH 8,
followed by a rinse with purified water and dried at
65~C for several hours. Dry DMS0 solutions of potassium
superoxide (Aldrich) were prepared under a dry, inert
atmosphere of argon in a Vacuum Atmospheres dry glovebox
using dried glassware. The DMS0 solutions were prepared
immediately before every stopped-flow experiment. A
mortar and pestle were used to grind the yellow solid
potassium superoxide (-100 mg). The powder was then
ground with a few drops of DMS0 and the slurry
transferred to a flask containing an additional 25 ml of
DMS0. The resultant slurry was stirred for 1/2 h and
then filtered. This procedure gave reproducibly -2 mM
concentrations of superoxide in DMS0. These solutions
were transferred to a glovebag under nitrogen in sealed
vials prior to loading the syringe under nitrogen. It
should be noted that the DMS0/superoxide solutions are
extremely sensitive to water, heat, air, and extraneous
metals. A fresh, pure solution has a very slight
yellowish tint.
Water for buffer solutions was delivered from an
in-house deionized water system to a Barnstead Nanopure
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Ultrapure Series 550 water system and then double
distilled, first from alkaline potassium permanganate
and then from a dilute EDTA solution. For example,
a solution containing 1.0 g of potassium permanganate,
2 liters of water and additional sodium hydroxide
n~C~ccAry to bring the pH to 9.0 were added to a 2-liter
flask fitted with a solvent distillation head. This
distillation will oxidize any trace of organic compounds
in the water. The final distillation was carried out
under nitrogen in a 2.5-liter flask cont~;n;ng 1500 ml
of water from the first still and 1.0 x 106M EDTA. This
step will remove remaining trace metals from the
ultrapure water. To prevent EDTA mist from volatilizing
over the reflux arm to the still head, the 40-cm
vertical arm was packed with glass beads and wrapped
with insulation. This system produces deoxygenated
water that can be measured to have a conductivity of
less than 2.0 nanomhos/cm2.
The stopped-flow spectrometer system was designed
and manufactured by Kinetic Instruments Inc. (Ann Arbor,
MI) and was interfaced to a MAC IICX personal computer.
The software for the stopped-flow analysis was provided
by Kinetics Instrument Inc. and was written in
QuickBasic with MacAdios drivers. Typical injector
volumes (0.10 ml of buffer and 0.006 ml of DMS0) were
calibrated so that a large excess of water over the DMS0
solution were mixed together. The actual ratio was
approximately 19/1 so that the initial concentration of
superoxide in the aqueous solution was in the range 60-
120 ~M. Since the published extinction coefficient ofsuperoxide in H20 at 245 nm is -2250 Ml cm~l (1~, an
initial absorbance value of approximately 0.3-0.5 would
be expected for a 2-cm path length cell, and this was
observed experimentally. Aqueous solutions to be mixed
with the DMS0 solution of superoxide were prepared using
80 mM concentrations of the Hepes buffer, pH 8.1 (free
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acid + Na form). One of the reservoir syringes was
filled with 5 ml of the DMSO solution while the other
was filled with 5 ml of the aqueous buffer solution.
The entire injection block, mixer, and spectrometer cell
were immersed in a thermostatted circulating water bath
with a temperature of 21.0 + 0.5~C.
Prior to initiating data collection for a
superoxide decay, a baseline average was obtained by
injecting several shots of the buffer and DMSO solutions
into the mixing chamber. These shots were averaged and
stored as the baseline. The first shots to be collected
during a series of runs were with aqueous solutions that
did not contain catalyst. This assures that each series
of trials were free of contamination capable of
generating first-order superoxide deeay profiles. If
the decays observed for several shots of the buffer
solution were second-order, solutions of manganese(II)
complexes could be utilized. In general, the potential
SOD catalyst was screened over a wide range of
concentrations. Since the initial concentration of
superoxide upon mixing the DMSO with the aqueous buffer
was -1.2 x 1o-4 M, we wanted to use a manganese (II)
complex concentration that was at least 20 times less
than the substrate superoxide. Consequently, we
generally screened compounds for SOD activity using
concentrations ranging from 5 x 10 7 to 8 x 10-6 M. Data
acquired from the experiment was imported into a
suitable math program (e.g., Cricket Graph) so that
standard kinetic data analyses could be performed.
The catalytic rate constant for dismutation of
superoxide by the manganese(II) complex of Example 1 was
determined from the linear plot of observed rate
constants (kob~) versus the concentration of the
manganese(II) complexes. kob, values were obtained from
the liner plots of ln absorbance at 245 nm versus time
for the dismutation of superoxide by the manganese(II)
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-54-
complex. The kc~, (M~~secl) of the manganese (II) complex
of Example 1 at pH c 8.1 and 21~C was determined to be
0.77 x 10'7 M-lsec-l.
The manganese(II) complex of the nitrogen-
con~i n; ~g macrocyclic ligand in Example 1 is an
effective catalyst for the dismutation of superoxide, as
can be seen from the kc,, above.
