O2-SUBSTITUTED 1-[(2-CARBOXYLATO) PYRROLIDIN-1-YL] DIAZEN-1-IUM-1,2-DIOLATES

1-[(2-CARBOXYLATO) PYRROLIDIN-1-YL]DIAZEN-1-IUM-1,2-DIOLATES 02-SUBSITUES

English Abstract

Diazeniumdiolates, wherein the N1 position is substituted by an inorganic or organic moiety and the O2-oxygen is bound to a substituted or unsubstituted aromatic group, are provided. Also provided are O2-glycosylated 1-substituted diazen-1-ium-1,2-diolates (O2-glycosylated diazeniumdiolates) and O2-substituted 1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates (1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates). The O2-aryl diazeniumdiolates are stable with respect to the hydrolytic generation of nitric oxide in neutral to acidic solutions and generate nitric oxide in basic or nucleophilic environments or microenvironments. Also provided are compositions, including pharmaceutical compositions, comprising such compounds and methods of using such compounds.

French Abstract

Des diazéniumdiolates, dans lesquels la position N1 est substituée par une fraction inorganique ou organique et l'oxygène O2 est lié à un groupe aromatique substitué ou insubstitué. Sont également présentés des diazén-1-ium-1,2-diolates 1-substitués O2-glycosylés (diazéniumdiolates O2-glycosylés) et des 1-[(2-carboxylato)pyrrolidin-1-yl]diazén-1-ium-1,2-diolates O2-substitués (1-[(2-carboxylato)pyrrolidin-1-yl]diazéniumdiolates). Les diazéniumdiolates O2-aryle sont stables par rapport à la production hydrolytique d'oxyde nitrique dans des solutions neutres à acides et génèrent de l'oxyde nitrique dans des environnements ou des microenvironnements basiques ou nucléophiles. Des compositions sont également présentées, y compris des compositions pharmaceutiques, comprenant ces composés, ainsi que des méthodes d'utilisation de ces composés.

Claims

78
CLAIMS:
1. An O2-substituted 1-[(2-carboxylato)pyrrolidin-1-
yl]diazen-1-ium-1,2-diolate derivative of formula II:
Image
(II)
wherein
R is optionally substituted and is a C1-12 straight
chain alkyl, a C3-12 branched chain alkyl, a C2-12 straight
chain or a C3-12 branched chain olefinic, a C1-12 acyl,
sulfonyl, carboxamido, a glycosyl group, a C3-C30 aryl, or a
group of the formula -(CH2)n-ON=N(O)NR28R29, wherein n is an
integer of 2-8, and R28 and R29 are independently a C1-12
straight chain alkyl, a C3-12 branched chain alkyl, or a C2-12
straight chain or a C3-12 branched chain olefinic, or R28 and
R29, together with the nitrogen atom to which they are
bonded, form a heterocyclic group selected from the group
consisting of a pyrrolidino, a piperidino, a piperazino and
a morpholino group; and
R22 is hydrogen, hydroxyl, OM, wherein M is a cation, a
halo, X1R23R24, wherein X1 is O, N or S, and R23 and R24 are
independently a C1-24 alkyl, a C3-24 cycloalkyl, a C2-24
olefinic, a C3-C30 aryl, or a heterocyclic group, and, when
X1 is O or S. there is no R24.


79
2. The diazeniumdiolate of claim 1, wherein said moiety R
is substituted with moieties selected from the group
consisting of hydroxy, halo, acyloxy, alkoxy, acylthio and
benzyl.
3. The diazeniumdiolate of claim 1, wherein, when X1 is
nitrogen, R23 and R24, together with the nitrogen to which
they are bonded, form a heterocyclic ring selected from the
group consisting of:
Image
in which A is O, NH or S, w is 1-12, y is 1 or 2, z is
1-5, R8, R9, R25 and R26 are hydrogen, a C1-8 straight chain
alkyl, a C3-8 branched chain alkyl, a C3-8 cycloalkyl, or a
C3 - C30 aryl.


80
4. The diazeniumdiolate of any one of claims 1 to 3,
wherein said aryl is selected from the group consisting of
an acridine, an anthracene, a benzene, a benzofuran, a
benzothiophene, a benzoxazole, a benzopyrazole, a
benzothiazole, a carbazole, a chlorophyll, a cinnoline, a
furan, an imidazole, an indole, an isobenzofuran, an
isoindole, an isoxazole, an isothiazole, an isoquinoline, a
naphthalene, an oxazole, a phenanthrene, a phenanthridine,
a phenothiazine, a phenoxazine, a phthalimide, a
phthalazine, a phthalocyanine, a porphin, a pteridine, a
purine, a pyrazine, a pyrazole, a pyridazine, a pyridine, a
pyrimidine, a pyrrocoline, a pyrrole, a quinolizinium ion,
a quinoline, a quinoxaline, a quinazoline, a sydnone, a
tetrazole, a thiazole, a thiophene, a thyroxine, a
triazine, and a triazole.
5. The diazeniumdiolate of claim 3 or 4, wherein, when X1
is nitrogen and R23 and R24, together with the nitrogen to
which they are bonded, form the heterocyclic ring
Image
R25 is hydrogen, a C1-C8 straight chain alkyl, a C3-C8
branched chain alkyl, a C3-C8 cycloalkyl or a C3-C30 aryl, R26
is hydrogen, a C1-C8 alkyl, a C3-C30 aryl, or C(O) - YR27,
wherein Y is sulfur, oxygen or nitrogen and R27 is CH2OCH3,
vinyl, a C1-C8 straight chain alkyl, a C3-C8 branched chain
alkyl, a C3-C8 cycloalkyl, polyethylene glycol, a
polysaccharide, a peptide or a protein.

81
6. A compound having the formula:
Image
wherein R is a C1-12 straight chain alkyl, a C3-12
branched chain alkyl, a C2-12 straight chain or a C3-12
branched chain olefinic, a C1-12 acyl, sulfonyl, carboxamido,
a glycosyl group, a C1-C30 aryl group or a group of the
formula -(CH2)n-ON=N(O)NR28R29, wherein n is an integer of 2-
8, and R28 and R29 are independently a C1-12 straight chain
alkyl, a C3-12 branched chain alkyl, or a C2-12 straight chain
or a C3-12 branched chain olefinic, or R28 and R29, together
with the nitrogen atom to which they are bonded, form a
heterocyclic group selected from the group consisting of a
pyrrolidino, a piperidino, a piperazino and a morpholino
group; and R22 is hydrogen, hydroxyl, OM, wherein M is a
cation, a halo, X1R23R24, wherein X1 is O, N or S, and R23 and
R24 are independently a C1-24 alkyl, a C3-24 cycloalkyl, a C2-24
olefinic, a C3-C30 aryl, or a heterocyclic group, and, when
X1 is O or S, there is no R24.
7. The compound of claim 6, wherein R is substituted with
a hydroxy, halo, acyloxy, alkoxy, acylthio or benzyl.

82
8. The compound of claim 6, wherein, when X1 is nitrogen,
R23 and R24, together with the nitrogen to which they are
bonded, form a heterocyclic ring selected from the group
consisting of:
Image
wherein A is O, NH or S, w is 1-12, y is 1 or 2, z is
1-5, R8, R9, R25, and R26 are hydrogen, a C1-8 straight chain
alkyl, a C3-8 branched chain alkyl, a C3-8 cycloalkyl, or a
C3-C30 aryl.
9. The compound of claim 6, wherein the aryl is selected
from the group consisting of an acridine, an anthracene, a
benzene, a benzofuran, a benzothiophene, a benzoxazole, a
benzopyrazole, a benzothiazole, a carbazole, a chlorophyll,
a cinnoline, a furan, an imidazole, an indole, an
isobenzofuran, an isoindole, an isoxazole, an isothiazole,
an isoquinoline, a naphthalene, an oxazole, a phenanthrene,
a phenanthridine, a phenothiazine, a phenoxazine, a
phthalimide, a phthalazine, a phthalocyanine, a porphin, a
pteridine, a purine, a pyrazine, a pyrazole, a pyridazine,
a pyridine, a pyrimidine, a pyrrocoline, a pyrrole, a
quinolizinium ion, a quinoline, a quinoxaline, a


83
quinazoline, a sydnone, a tetrazole, a thiazole, a
thiophene, a thyroxine, a triazine, and a triazole.
10. The compound of claim 8, wherein, when X1 is nitrogen
and R23 and R24, together with the nitrogen to which they are
bonded, form the heterocyclic ring
Image
R25 is hydrogen, a C1-C8 straight chain alkyl, a C3-C8
branched chain alkyl, a C3-C8 cycloalkyl or a C3-C30 aryl, R26
is hydrogen, a C1-C8 alkyl, a C3-C30 aryl, or C(O) -YR27,
wherein Y is sulfur, oxygen or nitrogen and R27 is CH2OCH3,
vinyl, a C1-C8 straight chain alkyl, a C3-C6 branched chain
alkyl, a C3-C8 cycloalkyl, polyethylene glycol, a
polysaccharide, a peptide or a protein.
11. Use of an O2-substituted 1-[(2-carboxylato)pyrrolidin-
1-yl]diazen-1-ium-1,2-diolate derivative of formula II:
Image
(II)
wherein
R is a C1-12 straight chain alkyl, a C3-12 branched chain
alkyl, a C2-12 straight chain or a C3-12 branched chain
olefinic, a C1-12 acyl, sulfonyl, carboxamido, a glycosyl
group, a C3-C30 aryl, or a group of the formula -(CH2)n-


84
ON=N(O)NR28R29, wherein n is an integer of 2-8, and R28 and
R29 are independently a C1-12 straight chain alkyl, a C3-12
branched chain alkyl, or a C2-12 straight chain or a C3-12
branched chain olefinic, or R28 and R29, together with the
nitrogen atom to which they are bonded, form a heterocyclic
group selected from the group consisting of a pyrrolidino,
a piperidino, a piperazino and a morpholino group; and
R22 is hydrogen, hydroxyl, OM, wherein M is a cation, a
halo, X1R23R24, wherein X1 is O, N or S, and R23 and R24 are
independently a C1-24 alkyl, a C3-24 cycloalkyl, a C2-24
olefinic, a C3-C30 aryl, or a heterocyclic group, and, when
X1 is O or S, there is no R24, in the manufacture of a
medicament in the treatment or prevention of a biological
disorder treatable or preventable by nitric oxide.
12. The use of claim 11, wherein said moiety R is
substituted with moieties selected from the group
consisting of hydroxy, halo, acyloxy, alkoxy, acylthio and
benzyl.
13. The use of claim 11, wherein, when X1 is nitrogen, R23
and R24, together with the nitrogen to which they are
bonded, form a heterocyclic ring selected from the group
consisting of:
Image


85
Image and Image
in which A is O, NH or S, w is 1-12, y is 1 or 2, z is
1-5, R8, R9, R25, and R26 are hydrogen, a C1-8 straight chain
alkyl, a C3-8 branched chain alkyl, a C3-8 cycloalkyl, or a
C3-C30 aryl.
14. The use of any one of claims 11 to 13, wherein said
aryl is selected from the group consisting of an acridine,
an anthracene, a benzene, a benzofuran, a benzothiophene, a
benzoxazole, a benzopyrazole, a benzothiazole, a carbazole,
a chlorophyll, a cinnoline, a furan, an imidazole, an
indole, an isobenzofuran, an isoindole, an isoxazole, an
isothiazole, an isoquinoline, a naphthalene, an oxazole, a
phenanthrene, a phenanthridine, a phenothiazine, a
phenoxazine, a phthalimide, a phthalazine, a
phthalocyanine, a porphin, a pteridine, a purine, a
pyrazine, a pyrazole, a pyridazine, a pyridine, a
pyrimidine, a pyrrocoline, a pyrrole, a quinolizinium ion,
a quinoline, a quinoxaline, a quinazoline, a sydnone, a
tetrazole, a thiazole, a thiophene, a thyroxine, a
triazine, and a triazole.


86
15. The use of claim 13 or 14, wherein, when X1 is nitrogen
and R23 and R24, together with the nitrogen to which they are
bonded, form the heterocyclic ring
Image
R25 is hydrogen, a C1-C8 straight chain alkyl, a C3-C8
branched chain alkyl, a C3-C8 cycloalkyl or a C3-C30 aryl, R26
is hydrogen, a C1-C8 alkyl, a C3-C30 aryl, or C(O) - YR27,
wherein Y is sulfur, oxygen or nitrogen and R27 is CH2OCH3,
vinyl, a C1-C9 straight chain alkyl, a C3-C6 branched chain
alkyl, a C3-C8 cycloalkyl, polyethylene glycol, a
polysaccharide, a peptide or a protein.
16. The use of any one of claims 11 to 15, wherein said
biological disorder is selected from infection,
inflammation, metastasis, fulminant liver failure, malaria,
a respiratory disorder, impotence, a cardiovascular
disorder, and a hematologic disorder.
17. Use of a diazeniumdiolate of any one of claims 1 to
10, in the manufacture of a medicament for treating an
animal with an infectious agent comprising a zinc finger
protein that can be inactivated by a diazeniumdiolate.
18. The use of claim 17, wherein the zinc finger protein
comprises a C-X2-C-X4-H-X4-C domain.


87
19. Use of a diazeniumdiolate of any one of claims 1 to
10, in the manufacture of a medicament for treating an
animal for cancer, wherein said cancer involves a zinc
finger protein that can be inactivated by said
diazeniumdiolate.
20. A method of treating a plant, a plant cell or a tissue
culture thereof, which is infected with an infectious agent
comprising a zinc finger protein that can be inactivated by
a diazeniumdiolate of any of claims 1 to 10, which method
comprises contacting the plant, plant cell or tissue
culture thereof with an amount of the diazeniumdiolate
sufficient to inactivate the zinc finger protein in the
infectious agent so as to treat the infection in the plant,
plant cell or tissue culture thereof.
21. The method of claim 20, wherein the infectious agent
is a virus.
22. The method of claim 21, wherein the virus is selected
from tobacco streak virus and alfalfa mosaic virus.
23. Use of a diazeniumdiolate of any of claims 1 to 10 in
the manufacture of a medicament for modulating steroid
hormone activity in a mammal, which is in need of
modulation of steroid hormone activity and which comprises
a steroid hormone receptor protein that comprises a zinc
finger that can be inactivated by the diazeniumdiolate.


88
24. A method of reducing on an inanimate object the
presence of an infectious agent comprising a zinc finger
protein that can be inactivated by a diazeniumdiolate of
any of claims 1 to 10, which method comprises contacting
the inanimate object with an amount sufficient to
inactivate the zinc finger protein so as to reduce the
presence of the infectious agent on the inanimate object.
25. The method of claim 24, wherein the infectious agent
is selected from a virus, a bacterium, and a parasite.

Description

CA 02814748 2013-05-02
02-SUBSTITUTED 1-[(2-CARBOXYLATO)PYRROLIDIN-1-YL]
DIAZEN-1-IUM-1,2-DIOLATES
TECHNICAL FIELD OF THE INVENTION
The present invention relates to 02-aryl 1-
substituted diazen-l-ium-1,2-diolates (02-aryl
diazeniumdiolates) 02-glycosylated 1-substituted
diazeniumdiolates, and 02-substituted 1-((2-
carboxylato)pyrrolidin-l-ylldiazeniumdiolates,
compositions comprising such diazeniumdiolates, methods
of using such diazeniumdiolates, and methods of preparing
02-aryl diazeniumdiolates.
BACKGROUND OF THE INVENTION
Nitric oxide (NO) has been implicated in a wide
variety of bioregulatory processes, and compounds, which
contain nitric oxide or are capable of releasing nitric
oxide, have been identified as useful in regulating these
processes. Many classes of nitric oxide-containing
and/or -releasing adducts are known in the art, such as
glyceryl trinitrate and nitroprusside (reviewed in U.S.
Patent No. 5,405,919 (Keefer et al.), including
limitations of their use in biological applications).
The limited utility of such compounds has, in part, given
rise to the development of another class of nitric oxide-
generating compounds, diazeniumdiolates, which are
especially useful biologically.
Diazeniumdiolates include compounds containing an
N202 functional group and are structurally and V.
functionally distinct from nitrosamines (see, e.g.,
Reilly, U.S. Patent No. 3,153,094). The known
diazeniumdiolates are disclosed in recently issued
patents. U.S. Patent Nos.-5,039,705 (Keefer et al.) and
5,208,233 (Keefer et al.) disclose secondary amine-nitric
oxide adducts and salts thereof. U.S. Patent Nos.
5,155,137 (Keefer et al.) and 5,250,550 (Keefer et al.)
disclose complexes of nitric oxide and polyamines. U.S.

CA 02814748 2013-05-02
2
Patent No. 5,389,675 (Christodoulou et al.) discloses
mixed ligand metal complexes of nitric oxide-nucleophile
adducts and U.S. Patent Nos. 5,525,357 (Keefer et al.)
and 5,405,919 (Keefer et al.) disclose polymer-bound
nitric oxide/nucleophile adduct compositions. U.S.
Patent Nos. 4,954,526 (Keefer et al.; the '526 patent)
and 5,212,204 (Keefer et al.) disclose the use of ionic
diazeniumdiolates as cardiovascular agents. In addition,
the '526 patent discloses 02-substituted and metal-bound
diazeniumdiolates. Keefer et al., U.S. Patent No.
5,366,997 ('997), discloses diazeniumdiolates having the
formula:
R1R2N-N-->0
II
N-OR3
in which the 02-oxygen of the N202- group is bonded to the
functional group R3. When the R3 group is cleaved from
the 02-oxygen, NO can be released spontaneously.
Although Keefer et al. ('997) discloses that (i) RI
and R2, together with the nitrogen atom to which they are
bonded, can form a pyrrolidinyl, piperazino or other
heterocyclic group, (ii) R3 is a C1-12 straight-chain or
C3-17 branched-chain alkyl, optionally olefinic and/or
substituted with hydroxy, halo, acyloxy or alkoxy, a C1-12
unsubstituted/substituted acyl, sulfonyl, carboxamido,
sulfinyl, sulfenyl, a carbonate derivative or a carbamate
derivative, and (iii) the pyrrolidinyl group can have the
structure:
(CH) N---
\12C 4
wherein w=4, and R4=hydrogen, a C1.43 straight or branched
chain alkyl, a C3-8 cycloalkyl, or a substituted or an

ak 02814748 2013-05-02
3
unsubstituted aryl, Keefer et al. ('997) does not
disclose that R3 is an aryl or a substituted aryl or that
the pyrrolidino group can be substituted with a
substituted or unsubstituted carboxyl group (see, also,
Example 1 of U.S. Patent No. 5,632,981) at position 2.
Similarly, Keefer et al. ('997) does not disclose 02-
glycosylation of diazeniumdiolates.
Heretofore it was not known that 02-aryl
substitutions of the diazeniumdiolates was possible.
Further, chemical studies of previously disclosed
diazeniumdiolates led to the conclusion that they are
generally at least as stable at high pH as they are at
low pH, and that, unlike certain other classes of
"nitrovasodilator" drugs, their rates of NO release are
not affected by the presence of nucleophilic thiols.
Thus, there remains a need for such classes of
diazeniumdiolates, which offer advantages over other
currently available diazeniumdiolates. In this regard,
the 02-aryl substituted diazeniumdiolates are advantageous
in that they can release NO spontaneously under alkaline
conditions or after nucleophilic attack. 02-Aryl
substituted diazeniumdiolates also can release NO
spontaneously after a combination of oxidative or
electophilic activation and nucleophilic attack.
It is, therefore, a principal object of the present
invention to provide a nitric oxide/nucleophile adduct in
which the 02-oxygen of the N202- group is derivatized with
an aryl or substituted aryl group to protect the
diazeniumdiolate against the spontaneous release of NO.
It is another object of the invention to provide a novel
class of diazeniumdiolates, which resists releasing
nitric oxide in neutral or acidic solutions, but releases
NO on nucleophilic attack or on increasing the pH. It is
still another object of the present invention to provide
02-glycosylated 1-substituted diazen-1-ium-1,2-diolates
and 02-substituted 1-((2-carboxylato)pyrrolidin-1-
yl)diazen-1-ium-1,2-diolates. It is a further object of

ak 02814748 2013-05-02
4
the present invention to provide compo8itions comprising
such compounds, including compositions comprising a
nitric oxide/nucleophile adduct comprising a novel
targeting moiety. It is a related object to provide 02-
aryl substituted diazeniumdiolates, which are amenable to
biological tissue-targeting strategies, which offer
greater flexibility and specificity for targeting NO
release. It is a still further object of the present
invention to provide methods of using such compounds.
These and other objects of the present invention, as well
as additional inventive features, will be apparent from
the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides an 02-aryl substituted
diazeniumdiolate (i.e., 02-aryl diazeniumdiolate)
illustrated by the formula:
N-O-Q (I),
wherein X is an inorganic or organic moiety and Q is an
aryl moiety. In this novel class of compounds an atom of
the aryl ring moiety Q is bonded to the 02-oxygen of the
N202- functional group. The diazeniumdiolates of Formula
(I) are stable with respect to the hydrolytic generation
of nitric oxide in neutral to acidic solutions.
Surprisingly, these novel compounds, or the resultant
product of these compounds after oxidative or
electrophilic activation, have proven capable of
generating nitric oxide in basic or nucleophilic
environments, in which the aryl moiety is separated from
the remainder of the diazeniumdiolate.
The present invention also provides 02-glycosylated
1-substituted diazen-1-ium-1,2-diolates and 02-substituted
1-((2-carboxylato)pyrrolidin-l-yljdiazen-1-ium-1,2-
diolates, both of which can be represented by the
formula:

CA 02814748 2013-05-02
N-O-R (Ia),
5 in which X and R are organic and/or inorganic moieties as
defined herein, although for 02-glycosylated
diazeniumdiolates, R must be a saccharide.
Further with -.espect to the 02-glycosylated 1-
substituted diazen-l-ium-1,2-diolates, the moiety X can
be any organic or inorganic group. Preferably, X
contains atoms other than carbon and hydrogen, and is
linked to the nitrogen of the diazeniumdiolate through an
atom other than carbon. Most preferably, X is an amino
group, and is linked to the nitrogen of the
diazeniumdiolate through a nitrogen atom.
With respect to the 02-substituted 1-11(2-
carboxylato)pyrrolidin-1-ylldiazen-1-ium-1,2-diolates, X
of Formula Ia can be
=
0
22
N 7\--171
such that the (1-(2-carboxylato)pyrrolidin-1-
yl]diazeniumdiolates can be structurally represented by
the formula:
0
¨C R22
N
0
N--0--R
wherein R22 is hydrogen, hydroxyl, OM, wherein M is a
cation, halo, or X1R23R24, wherein XI is oxygen, nitrogen
or sulfur and R23 and R241 are independently a substituted
or unsubstituted C1-24 alkyl, a substituted or
unsubstituted 03-24 cycloalkyl, a substituted or
unsubstituted C2-24 olefinic, a substituted or

CA 02814748 2013-05-02
6
unsubstituted aryl (such as acridine, anthracene,
benzene, benzofuran, benzothiophene, benzoxazole,
benzopyrazole, benzothiazole, carbazole, chlorophyll,
cinnoline, furan, imidazole, indole, isobenzofuran,
isoindole, isoxazole, isothiazole, isoquinoline,
naphthalene, oxazole, phenanthrene, phenanthridine,
phenothiazine, phenoxazine, phthalimide, phthalazine,
phthalocyanine, porphin, pteridine, purine, pyrazine,
pyrazole, pyridazine, pyridine, pyrimidine, pyrrocoline,
pyrrole, quinolizinium ion, quinoline, quinoxaline,
quinazoline, sydnone, tetrazole, thiazole, thiophene,
thyroxine, triazine, and triazole), or a heterocyclic
group, such as glycosyl, and the like, and when X1 is 0 or
= S, there is no R24 group. Alternatively, when X1 is
nitrogen, R23 and R24, together with X1, form a
heterocyclic ring, such as a heterocyclic ring selected
from the group consisting of:
R9
g>""10 N-- N¨

Re (C1-12)y
R25
N¨ (CH2CH2A)z---N
CH,CH,R26
,and
in which A is N, 0, or S, w is 1-12, y is 1 or 2, z is

CA 02814748 2013-05-02
7
1-5, R9, R9, R25, and R26 are hydrogen, a 01-8 straight
chain alkyl, a C3-8 branched chain alkyl, a 03-8
cycloalkyl, or an aryl. The aforementioned R23
and R24 groups can be unsubstituted or substituted as
appropriate. For example, the R23 and R24 groups can be
substituted as appropriate with acyloxy, acylthio,
hydroxyl, amino, carboxyl, mercapto, halo, amido,
sulfonyl, sulfoxy, sulfenyl, phosphono, phosphato, and
the like.
Further with respect to the 02-substituted 1-[(2-
carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates,
the moiety R of Formula Ia can be any organic or
inorganic moiety, which is covalently bound to the
terminal oxygen of the diazeniumdiolate as shown but
which is other than hydrogen and is a substituted or
unsubstituted 01-12 straight chain or C3-12 branched chain
alkyl, a substituted or unsubstituted C2-12 straight chain
or C3-12 branched chain olefinic, a substituted or
unsubstituted C1-12 acyl, sulfonyl, carboxamido, a glycosyl
group, an aryl group, or a group of the formula -(CH2)11-
ON=N(0)NR29R29, wherein n is an integer of 2-8, and R29 and
Rn are independently a C1-12 straight chain alkyl, a C3-12
branched chain alkyl, a C2-12 straight chain or C3-12
branched chain olefinic, or R29 and R", together with the
nitrogen atom to which they are bonded, form a
heterocyclic group, preferably a pyrrolidino, piperidino,
piperazino or morpholino group. The aforementioned R
groups can be unsubstituted or substituted. Preferred
substitutions include those made with hydroxy, halo,
acyloxy, alkoxy, acylthio, or benzyl.
In another aspect, the present invention comprises a
composition, including a pharmaceutical composition,
comprising a present inventive diazeniumdiolate. The
pharmaceutical composition preferably additionally
comprises a pharmaceutically acceptable carrier.

CA 02814748 2013-05-02
7a
In accordance with one aspect of the present
invention, there is provided an 02-glycosylated
1-substituted diazen-l-ium-1,2-diolate of Formula Ia:
X¨N1-1.- 0
II
N¨ 0 ¨R (Ia),
in which X is selected from the group consisting of
-03S¨, -0--, an amino, a polyamino, a C1-C24 aliphatic, a
C6-C30 aryl and a CE-C30 non-aromatic cyclic, and R is a
saccharide, which is attached to the 02 of the
diazeniumdiolate by the 2 position of a pyranose ring or a
furanose ring.
In accordance with another aspect of the present
invention, there is provided an 02-substituted 1-[(2-
carboxylato)pyrrolidin-l-yl]diazen-l-ium-1,2-diolate
derivative of formula II:
0
N R22
N--0--R
(II)
wherein
R is optionally substituted and is a C1-12 straight
chain alkyl, a C3-12 branched chain alkyl, a C2-12 straight
chain or a C3-12 branched chain olefinic, a C1-12 acyl,
sulfonyl, carboxamido, a glycosyl group, a C3-C30 aryl, or a
group of the formula -(CH2)n_0N=N(0)NR28R29, wherein n is an
integer of 2-8, and R28 and R29 are independently a C1-12
straight chain alkyl, a C3-12 branched chain alkyl, or a C2-12

CA 02814748 2013-05-02
7b
straight chain or a C3-12 branched chain olefinic, or R28 and
R29, together with the nitrogen atom to which they are
bonded, form a heterocyclic group selected from the group
consisting of a pyrrolidino, a piperidino, a piperazino and
a morpholino group; and
R22 is hydrogen, hydroxyl, OM, wherein M is a cation, a
halo, X1R23R24, wherein X1 is 0, N or S, and R23 and R24 are
independently a C1-24 alkyl, a C3-24 cycloalkyl, a C2-24
olefinic, a C3-C30 aryl, or a heterocyclic group, and, when
X' is 0 or S, there is no R24.
In accordance with yet another aspect of the present
invention, there is provided a compound having the formula:
0
'
R22
NI VCI
wherein R is a C1-12 straight chain alkyl, a C3-12
branched chain alkyl, a C2-12 straight chain or a C3_12
branched chain olefinic, a C1-12 acyl, sulfonyl, carboxamido,
a glycosyl group, a C1-C30 aryl group or a group of the
formula -(CH2)n_0N=N(0)NR28R29, wherein n is an integer of 2-
8, and R28 and R29 are independently a C1-12 straight chain
alkyl, a C3-12 branched chain alkyl, or a C2-12 straight chain
or a C3..12 branched chain olefinic, or R28 and R29, together
with the nitrogen atom to which they are bonded, form a
heterocyclic group selected from the group consisting of a
pyrrolidino, a piperidino, a piperazino and a morpholino
group; and R22 is hydrogen, hydroxyl, OM, wherein M is a
cation, a halo, X1R23R24, wherein X3- is 0, N or S, and R23 and

CA 02814748 2013-05-02
7c
R24 are independently a C1-24 alkyl, a C3-24 cycloalkyl, a C2-24
olefinic, a C3-C30 aryl, or a heterocyclic group, and, when
Xl is 0 or S. there is no R24.
In accordance with still another aspect of the present
invention, there is provided a use of an 02-substituted 1-
[(2-carboxylato)pyrrolidin-l-yl]diazen-1-ium-1,2-diolate
derivative of formula II:
0
II
C¨R
N-30-02 2
NI VC-I
D
N--0--R
(II)
wherein
R is a C1-12 straight chain alkyl, a C3-12 branched chain
alkyl, a C2-12 straight chain or a C3-12 branched chain
olefinic, a C1-12 acyl, sulfonyl, carboxamido, a glycosyl
group, a C3-C30 aryl, or a group of the formula -(CH2)n-
ON=N(0)NR28R29, wherein n is an integer of 2-8, and R28 and
R29 are independently a C1-12 straight chain alkyl, a C3-12
branched chain alkyl, or a C2-12 straight chain or a C3-12
branched chain olefinic, or R28 and R29, together with the
nitrogen atom to which they are bonded, form a heterocyclic
group selected from the group consisting of a pyrrolidino,
a piperidino, a piperazino and a morpholino group; and
R22 is hydrogen, hydroxyl, OM, wherein M is a cation, a
halo, X1R23R24, wherein X' is 0, N or S. and R23 and R24 are
independently a C1-24 alkyl, a C3-24 cycloalkyl, a C2-24
olefinic, a C3-C30 aryl, or a heterocyclic group, and, when
X1 is 0 or S, there is no R24, in the manufacture of a
medicament in the treatment or prevention of a biological
disorder treatable or preventable by nitric oxide.

CA 02814748 2013-05-02
8
In yet another aspect, the present invention
provides methods of using a compound in accordance with
the present invention.
In still another aspect, the present invention
provides a method of making 02-aryl diazeniumdiolates.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph of Trp37 fluorescence (RFU)
versus time (min), which depicts zinc ejection from HIV-1
nucleocapsid p7 protein by 02-aryl diazeniumdiolates. In
the graph, 0 represents the negative control, i.e., no
drug, 0 represents the positive control, i.e., 624151
(See Rice et al., Antimicrob. Agents Chemother. 41:
419426 (1997)), II represents the compound of Example 1
(LK1), = represents the compound of Example 8 (LK2),
represents the compound of Example 5 (LK3), = represents
the compound of Example 10 (LK4), and X represents the
compound of Example 11 (LK5).
Figure 2 is a graph of relative NO release rate
versus time (min), which depicts the catalysis of NO
release from DNP-PYRRO/NO by glutathione S-transferase
(GST).
DETAILED DESCRIPTION OF THE INVENTION
02-arylated diazeniumdiolates
The present invention provides an 02-aryl 1-
substituted diazeniumdiolate (i.e., 02-aryl 1-substituted
diazen-1-ium-1,2-diolate) having the formula:
X
fl
N-O-Q
(I)
wherein X is an organic or inorganic moiety and Q is an
aryl group.
In accordance with the invention, the 02-oxygen of
the N202- group is bonded directly to an atom of the ring
of the aryl group. Stated another way, there are no

ak 02814748 2013-05-02
9
spacer atoms (e.g., methylene) that separate the 02-oxygen
from the aryl ring. If the aryl group comprises a
bicyclic or polycyclic moiety and all rings of the aryl
group are not aromatic, then the linkage between the 02-
oxygen and the aryl group is through an atom that is part
of an aromatic ring. Further, the 02-oxygen can be linked
to any aromaticr.Eipg atom of the aryl group that is
capable of bonding to the 02-oxygen of the N202- group.
Atoms of the aromatic ring that are capable of bonding
with the 02-oxygen of the N202- group are typically carbon
and nitrogen, although there can be other linkages as
well. =
While not wishing to be bound to any particular
theory, it is presently believed that the bonding of the
02-oxygen with the atom of the aryl ring is accomplished
by bonding to an activated atom of the ring. Activation
can be accomplished through any suitable mechanism. In
this regard, a preferred mechanism of activating an aryl
ring is by reacting the diazeniumdiolate through an atom
of the aryl ring possessing a partial positive charge or,
more specifically, by displacing an amino substituent of
the ring structure.
In the first preferred reaction mechanism, the aryl
ring is substituted by a suitable electron-withdrawing
group(s), which can be part of the ring, as in Example
12, and a "leaving group" prior to reaction with the
diazeniumdiolate. It will be appreciated by those
skilled in the art that the electron-withdrawing group
and the leaving group can, in some instances, be the same
moiety. The leaving group is displaced by the
diazeniumdiolate to form the 02-aryl diazeniumdiolate of
the present invention. Suitable leaving groups include,
but are not limited to, F, Cl, Br, I, NO,, OSO2R, and
OSO3R, wherein R is an organic moiety, a metal center, or
the like, the composition of which is well understood by
those skilled in the art. By way of illustration and not
in limitation, suitable R groups include H, alkyl,

CA 02814748 2013-05-02
alkenyl, or aryl. This reaction mechanism is based on
the well known StAr mechanism; for example, see
Nucleophilic Aromatic Displacement: The Influence of the
Nitro Group, Francois Terrier, VCH Publishing, Inc., New
5 York, New York, pages 1-11 (1991). Preferably, these StAr
reactions are carried out in electron-deficient aromatic
rings comprising at least one electron-withdrawing group.
In the second preferred reaction mechanism, an aryl
reactant is substituted by a suitable amino group, which
10 allows direct derivatization (e.g., after diazotization
of the amino group) of the ring atom of the aryl group
that is bound to the displaced amino group. There is no
requirement for the atom of the aryl ring linked to the
02-oxygen to be activated after it has been incorporated
into the present inventive compound. However, if this
atom is activated after being incorporated into the
present inventive compound, then the diazeniumdiolate
moiety to which it is bound may be displaced through
further nucleophilic displacement (e.g., in a suitably
strong base). Alternatively, an oxidative or
electrophilic activation event can alter the present
inventive compound so that the aryl ring atom linked to
the 02-oxygen becomes activated, thereby rendering the
compound subject to further nucleophilic displacement, as
observed above.
Advantageously, the compounds of the present
invention have new and useful properties, which are not
possessed by other nitric oxide/nucleophile adducts
previously known in the art. In general, the compounds
of the present invention are stable at neutral or acidic
pH (i.e., at neutral or acidic pH, the compounds
indicated by Formula I do not generate NO). Another
advantageous property of the compounds of the present
invention is that the 02-aryl linkage is often susceptible
to cleavage by nucleophiles, including hydroxide ions.
When the typical 02-aryl diazeniumdiolate or the

ak 02814748 2013-05-02
11
oxidatively or electrophilically activated 02-aryl
diazeniumdiolate of the present invention is placed into
a basic or nucleophilic environment, the aryl linkage to
the 02-oxygen can be broken. The resulting
diazeniumdiolate ion spontaneously degrades via a
predictable, first order mechanism, giving rise to NO.
The resulting aryl group is substituted with a
nucleophile provided by the environment. If the
nucleophile provided by the environment is part of an
enzyme, that enzyme can be inactivated. The
susceptibility to nucleophilic attack of the 02-aryl
diazeniumdiolates also makes them particularly amenable
to designing prodrugs for targeting nitric oxide to
nucleophilic tissue components, body sites and
microenvironments in the body.
The compounds of the present invention are also
useful to identify and quantify individual thiols
(organic -SH containing compounds) when the thiols are
present in mixtures. For example, a sample suspected to
consist of C4-C8 straight-chain thiols can be analyzed by
dissolving the product of Example 1 in tetrahydrofuran or
another inert solvent, then mixing a molar excess of the
resulting solution with the sample to be assayed. After
the ensuing reaction is complete, an aliquot is subjected
to HPLC analysis using an ultraviolet detection system.
Peaks found in the resulting chromatogram can be
identified by comparing their retention times to those of
independently derivatized authentic standards of the
individual C4-C8 straight-chain thiols, and quantified by
transforming peak areas to concentrations via the
individual standard curves.
With respect to the 02-aryl diazeniumdiolates, "aryl
group" as used herein refers to any aromatic group,
regardless of whether it is part of a (homo)cyclic,
heterocylic, or polycyclic structure. The standard
understanding of "aromatic" is used herein (See, e.g.,
L.G. Wade, Jr., Organic Chemistry, 2d Edition, Prentice

CA 02814748 2013-05-02
12
Hall, Englewood Cliffs, New Jersey, 682-683 (1991)). The
aryl group, as used herein, can also have a wide variety
of substituent groups. Any suitable aryl substituent can
be used providing that the substituent does not destroy
the aromaticity of the aryl ring.
Turning to the aryl group Q of Formula I, Q is
intended to include all aryl groups that are (or can be
made) amenable to reaction with the 02-oxygen atom of a
diazeniumdiolate. The moiety Q thus includes homocyclic,
heterocyclic, and polycyclic aromatic structures as well
as derivatives thereof. Illustrative of the aryl groups
Q are acridine, anthracene, benzene, benzofuran,
benzothiophene, benzoxazole, benzopyrazole,
benzothiazole, carbazole, chlorophyll, cinnoline, furan,
imidazole, indole, isobenzofuran, isoindole, isoxazole,
isothiazole, isoquinoline, naphthalene, oxazole,
phenanthrene, phenanthridine, phenothiazine, phenoxazine,
phthalimide, phthalazine, phthalocyanine, porphin,
pteridine, purine, pyrazine, pyrazole, pyridazine,
pyridine, pyrimidine, pyrrocoline, pyrrole, quinolizinium
ion, quinoline, quinoxaline, quinazoline, sydnone,
tetrazole, thiazole, thiophene, thyroxine, triazine, and
triazole.
In keeping with the invention, each of these
aromatic compounds Q can be variably derivatized with the
=
numerous substituents well known in the art that are
capable of being substituted into an aromatic ring so
long as the aromaticity of the ring is maintained. For
example, the substituents of the aryl moiety, Q, can
include X[N(0)N0]-, wherein X is as defined hereinafter
and is the same as X of Formula I, halo, hydroxy,
alkylthio, arylthio, alkoxy, aryloxy, amino, mono- or di-
substituted amino, ammonio or substituted ammonio,
nitroso, cyano, sulfonato, mercapto, nitro, oxo, C1-C2
aliphatic, C3-C12 olefinic, C3-C24 cycloalkyl, C3-C24
heterocycloalkyl, benzyl, phenyl, substituted benzyl,
substituted phenyl, benzylcarbonyl, phenylcarbonyl,

CA 02814748 2013-05-02
13
saccharides, substituted benzylcarbonyl, substituted
phenylcarbonyl and phosphorus derivatives. Illustrative
phosphorus derivatives include phosphate and phosphono
moieties. Illustrative phosphate moieties include
(OH)2P(0)0- and substituted (OH)2P(0)0-moieties, wherein
one or more oxygen atoms can be independently replaced by
S or NR', wherejz R' is understood to be a C1-C10
containing aliphatic, cycloalkyl, or aryl group.
Illustrative C1-C24 aliphatic substituents comprise C1-C24
acyl, and
N-OH
Ii
R-C-
wherein R is hydrogen, substituted or unsubstituted CI-C23
aliphatic, substituted or unsubstituted C3-C2 cycloalkyl,
substituted or unsubstituted C3-C12 olefinic, benzyl,
phenyl, substituted benzyl or substituted phenyl,
and said substituted benzyl or substituted phenyl is
substituted with one to five substituents selected from
the group consisting of nitro, halo, hydroxy,
alkyl, C1-C24 alkoxy, amino, mono-C1-C24 alkylamino,
C24 alkylamino, cyano, phenyl and phenoxy.
Preferred saccharides include ribose, glucose,
deoxyribose, dextran, starch, glycogen, lactose, fucose,
galactose, fructose, glucosamine, galactosamine, heparin,
mannose, maltose, sucrose, sialic acid, and cellulose.
Other preferred saccharides are phosphorylated, 3,5-
cyclophosphorylated, and polyphosphorylated hexoses and
pentoses.
Examples of substituted aryl compounds of the
present invention that can be linked to the
diazeniumdiolate group comprise dinitrophenol (a
benzene), hypoxanthine (a purine), uridine (a
pyrimidine), vitamin Ks(a naphthalene) and ribosyl uridine
(a nucleoside).

CA 02814748 2013-05-02
14
In another particular embodiment of the present
invention, the aryl moiety is identical to or
structurally analogous to molecules, or substituents
thereof, normally found in living organisms. These
biologically relevant groups can be selected from
nucleotides, nucleosides, and nucleic acids, peptides,
including peptide hormones, non-peptide hormones,
vitamins and other enzyme cofactors such as porphyrins,
and others. Examples of biologically relevant aryl
groups are thyroxine, NAD (or NADH), chlorophyll,
hypoxanthine, uridine, and vitamin K5.
The following reaction schematics illustrate methods
of preparing the 02-aryl diazeniumdiolates of the present
invention. In these illustrative reactions, in general,
a solution of a diazeniumdiolate (X-[N202-]) in 5% aqueous
sodium bicarbonate (which is weakly basic) is cooled to
0 *C, preferably under a blanket of inert gas such as
nitrogen. A solution containing one equivalent of the
activated aromatic reagent in a solvent, such as t-butyl
alcohol, dimethyl sulfoxide, or N,N-dimethylformamide, is
then added slowly. While not being bound to any
particular theory, it is believed that polar non-protic
solvents are preferred. The reaction temperature is
raised slightly for less reactive aryl moieties, for
example, to ambient temperatures or higher. Generally, a
precipitate forms upon addition. The mixture is then
allowed to warm to room temperature gradually and stirred
overnight. The product may be extracted with a suitable
extraction agent, such as dichloromethane, and washed
subsequently with cold dilute hydrochloric acid and then
with sodium bicarbonate solution. The organic layer is
dried over a suitable drying agent, such as sodium
sulfate, filtered, preferably through a layer of
anhydrous magnesium sulfate, and evaporated under vacuum
to give the crude product. Usually, the product is
solid. Recrystallization from ethanol or other suitable
solvents is a preferred method of purifying the product.

CA 02814748 2013-05-02
It will be appreciated by one skilled in the art that
these conditions can be modified to suit the particular
application of the artisan. Accordingly, alternative
methods of preparation are also embraced.
5 Chlorinated quinoline and isoquinoline can be
reacted with a diazeniumdiolate such that the Cl
substituent is replaced by the 02-oxygen of a
diazeniumdiolate, as shown below:
X-N202- 0
00 _______________________________________ 0 0
10 Cl
N \O¨N=N¨X
Cl 0¨N=N--X
X-N202-
00 1.- 0 0
X-N202 ¨N=N¨X
00 _______________________________________ 0 0
0
X- N202

0 0 _______________________________________ 0N
Cl 0¨N=N ¨X
0
Additionally, quinazoline can be incorporated as shown:

CA 02814748 2013-05-02
16
0
Cl 0¨NN--X
N X-N202-
N
001 0
The phthalazines also are incorporated in accordance with
the present invention, as indicated:
X-N 0
N
Cl 0¨N=N¨X
0
Acridine can be incorporated as indicated:
0
Cl
X-N202-
0 0 0 0 0 0
\/\N./\/
Cinnoline can also be incorporated as indicated:
0
Cl 0¨N=N¨X
X-N202-
00 0 0 m
Quinoxaline can also be incorporated as indicated:

CA 02814748 2013-05-02
17
N....,, N,
X-N 0 - '/7-?'-
00 . 2200 0
t
NC1 -..õ..õ..õ----õ, ,õ----õ.
N 0--Nr-=N--X
Oxygen- and sulfur-containing heteroaromatics can
also be used as the aromatic reagent for 02-oxygen
substitution of tne diazeniumdiolate in accordance with
the present invention. For example, oxazole and
benzoxazole can be derivatized at the 2-position as
indicated:
______________________ N N 0
_
2
X-N2 0, _____
LI\ t
0 C 1 0 0 ¨N=N¨X
0
______________________ N
_
--...,..õõ.---... ..-L . X-N202 0
t
' ' \ ,õ.
0 CI 0 0 ¨N=N ¨X
Similarly, thiazole and benzothiazole can also be
derivatized at the 2-position.
______________ N - _____________ 0
X-N202
S 2 Cl S 0-N=N-X
./...^=,,
_________________________________________ N 0
0
)2 X-N202 0
3. t
S Cl S 0¨N=N¨X
A derivatized Vitamin K7 can also be prepared, as
indicated:

CA 02814748 2013-05-02
18
0
0
t
t
Br 0¨N--=-N¨X
0¨N=N¨X
,)! X-N202- .V.) [H]
NO2 NO2 NH2
The 02-diazeniumdiolated atom of the aryl ring in the
right-most (directly above) structure is not activated.
Therefore, the right-most structure is resistant to
nucleophilic attack, which would re-generate X-N202 ,
which, in turn, would spontaneously degrade to produce
NO. Therefore, the right-most structure must undergo
oxidative preactivation prior to nucleophilic attack in
order to generate NO. This oxidative preactivation
requirement would also be of advantage in targeting a
cell or organ type that is uniquely able to perform the
required oxidation, thereby limiting NO exposure to the
desired tissue while avoiding exposure at other NO-
sensitive portions of the anatomy.
Illustrative of the class of compounds requiring
electrophilic preactivation is the compound indicated
. below:
\+
X-N202-4- NU-K0 N-R
/
t Nu-
\ \
RX
X-N,02--(07 __________________________________ , X-N20T-(0 ______ //N¨R+
,
Nu- (nucleophile)
1 IH20
no reaction ---\
X-N.,0, + C) /N¨R

CA 02814748 2013-05-02
19
Triazines can likewise be the aromatic reagent that
forms the 02-aryl substituted diazeniumdiolates of the
present invention as shown below. The synthesis of such
compounds should enhance the potency of existing
triazine-derived herbicides.
0
0¨NN---X
Cl NN
X-N202- X ¨N=N-0
Cl Cl 0 0
Nucleic acids and the nitrogenous bases they
comprise (including ribosylated bases) can also be used
as the aromatic reagent to form the 02-aryl substituted
diazeniumdiolates of the present invention. This is
illustrated in Example 13.
Another interesting 02-arylated diazeniumdiolate is
the one shown as the product in the reaction below; it
can co-generate NO and allopurinol on hydrolysis.
N
I NN 0 X-N202 r.
NyJ
Cl O¨N=N¨X
0
Advantageously, allopurinol is already known to be
pharmaceutically useful. Thus, by converting known
pharmaceutically useful compounds containing a suitable .
aryl group to the 02-aryl diazeniumdiolates of the present

CA 02814748 2013-05-02
invention, the present invention allows existing drugs to
be enhanced by the release of NO.
Similarly, a derivative of a biopterin
diazeniumdiolate can be prepared from a substituted
5 pteridine, as indicated below.
0
4
Cl
N -X
CHOH-CHOH-CH3 CHOH-CHOH-CH3
X
HN H2N
An example of a suitable aryl substitution that
10 utilizes linkage through a heteroatom is shown in the
following scheme which can be effected by reaction with
BuONO or other suitable nitrosating agents.
N ______________________ N
BuONO
1
NH2 X- N2 _2 0
N\./
X
A structural analog of Bendazac, a well-known anti-
inflammatory agent, can be prepared as indicated:
/CH2Ph CH,Ph
/
CH2 Ph
N/
N 0
HOCH2COOH
Cl =
OCH2COOH
0-N=N-X
Benda zac

ak 02814748 2013-05-02
21
In accordance with the invention, any of the
compounds in the class of compounds defined as
diazeniumdiolates can be subjected to 02-aryl
substitution. Thus, for the compounds having Formula I,
X can be any organic or inorganic moiety. Preferably, X
contains atoms other than carbon and hydrogen, and is
linked to the nitrogen of the N202- group through an atom
other than carbon. Most preferably, X is an amine, and
is linked to the nitrogen of the N202- group through a
nitrogen atom. Suitable moieties of X also include, but
are not limited to, Cl-C24 aliphatic, aryl, and
nonaromatic cyclic. By "aliphatic" is meant acyclic
moieties containing carbon and hydrogen and optionally
containing nitrogen, oxygen, sulfur, phosphorus, and
halogens. By "aryl" is meant, as hereinabove, a moiety
containing at least one aromatic ring. Preferably, the
aryl moiety is a C3-C30-containing moiety. By nonaromatic
cyclic is meant a moiety containing at least one ring
structure and no aromatic rings. Preferably, the non-
aromatic cyclic moiety is a C3-C30-containing moiety.
The moiety .X of Formula I can be unsubstituted or
substituted with suitable additional moieties, such as,
for example, -[N(NO)0], halo, hydroxy, alkylthio, alkoxy,
aryloxy, amino, mono- or di- substituted amino, cyano,
sulfonato, mercapto, nitro, substituted or unsubstituted
C1-C12 aliphatic, substituted or unsubstituted C3-C8
cycloalkyl, substituted or unsubstituted C3-C8
heterocycloalkyl, substituted or unsubstituted C3-C12
olefinic, benzyl, phenyl, substituted benzyl, substituted
phenyl, benzylcarbonyl, phenylcarbonyl, saccharides,
substituted benzylcarbonyl, substituted phenylcarbonyl
and phosphorus derivatives. Illustrative phosphorus
derivatives include phosphato and phosphono moieties.
Illustrative phosphato moieties include (OH)2P(0)0- and
substituted (OH)2P(0)0- moieties, wherein one or more
oxygen atoms can be independently replaced by S or NR',

CA 02814748 2013-05-02
22
wherein R' is understood to be a C1-C8 -containing
aliphatic, cycloalkyl, or aryl group. Preferred C1 -C12
aliphatic substituents comprise Cl-C12 acyl, and
N-0 H
(III) ,
R-C-
wherein R is C1-C10 substituted or unsubstituted
aliphatic, C3-C11 olefinic, C3-C8 substituted or
unsubstituted cycloalkyl, benzyl, phenyl, substituted
benzyl or substituted phenyl, and said substituted benzyl
or substituted phenyl is substituted with one or two
substituents selected from the group consisting of halogen,
hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, mono-C1-C4
alkylamino, di-C1-C4alkylamino, phenyl and phenoxy.
Preferred saccharides and polysaccharides include ribose,
glucose, deoxyribose, dextran, starch, glycogen, lactose,
galactose, fructose, glucosamine, galactosamine, heparin,
mannose, maltose, sucrose, sialic acid, and cellulose.
Other preferred saccharides are phosphorylated, 3,5-
cyclophosphorylated, and polyphosphorylated pentoses and
hexoses.
In one embodiment of the invention, X is an inorganic
moiety as described in U.S. Pat. No. 5,212,204. Preferred
embodiments of Formula I, in which X is inorganic, are -03
S-- (sulfite) and -0- (oxide).
In another embodiment of the present invention, X is a
polyamine as defined in U.S. Pat. No. 5,155,137. Thus, the
polyamine substituted 02 -aryl diazeniumdiolates havethe
formula
Ri-NHCHA¨N¨RCHA¨N b-- RCH2)k¨N I -R3
d
N 0 R5 R4
R,2 I I
N-O-Q (II)
wherein Q is the same as the Q in Formula I and is
defined as above, b and d can be the same or different and
are zero or one, Rl, R2, R3, P4, and R5 are the same or

CA 02814748 2013-05-02
23
different and comprise hydrogen, substituted or
unsubstituted C3-C8 cycloalkyl, substituted or
unsubstituted C1-C12 straight or branched chain alkyl,
substituted or unsubstituted benzyl, substituted or
unsubstituted benzoyl, substituted or unsubstituted C3-C12
olefinic, phthaloyl, acetyl, trifluoroacetyl, p-toluyl,
t-butoxycarbonyl, or 2,2,2-tri-halo-t-butoxycarbonyl.
The values of i, j, and k in Formula II can be the same
or different and are integers from 2 to 12.
In a preferred embodiment of the present invention
the 02-aryl diazeniumdiolates are derived from the
compounds disclosed in U.S. Patent Nos. 5,039,705 (Keefer
et al.) and 4,954,526 (Keefer et al.) and, thus, have the
formula
R6R7N-N--o-
II
N-0--Q
wherein R6 and R7 can be the same or different and are
chosen from H, C1-C12 straight chain alkyl, C1-C12 alkoxy
or acyloxy substituted straight chain alkyl, C2-C12
hydroxy or halo substituted straight chain alkyl, C3-C12
branched chain alkyl, C3-C12 hydroxy, halo, alkoxy, or
acyloxy substituted branched chain alkyl, C2-C12 straight
chain olefinic and C3-C12 branched chain olefinic, which
are unsubstituted or which are substituted with hydroxy,
alkoxy, acyloxy, halo or benzyl, provided that both R6 and
R7 are not H; or R6 and R7, together with the nitrogen
atom to which they are bonded, form a heterocyclic ring
selected from the group consisting of:

CA 02814748 2013-05-02
24
R9
(CH2), N¨ N¨
(CH2CH2A)
(6E12) y and
H2CH2 _______________________________________________________________________
R8
wherein A is N, 0, or S, w is 1 to 12, y is 1 or 2, z
is 1 to 5, R8 is hydrogen, Cl-C8 straight chain alkyl, C3-C8
branched chain alkyl, C3-C8 cycloalkyl, unsubstituted or
substituted aryl, such as phenyl, tolyl or the like, and R9
is hydrogen, C1-C.8 straight chain alkyl or C3-C6 branched
chain alkyl. Exemplary aza crown groups are 1-aza-12-crown-
4, 1-aza-15-crown-5, and 1-aza-18-crown-6. Where A is
nitrogen, the nitrogen atom, itself, can be substituted, as
described, for example, in U.S. Patent No. 5,721,365.
Further examples include the 02-aryl substituted
diazeniumdiolates derived from the compounds disclosed in
U.S. Pat. No. 5,250,550 and, thus, have the formula
R10--N--(CH2)(--D
R" (IV),
wherein D is
\N¨N ---- 0 or ¨Nr¨\N¨N ¨0
/II \--/ II
N-O-Q N-O-Q
= and wherein R" and Ril are the same or different. The
substituents R1 and can be any suitable group, examples
of which include hydrogen, C3-C8 cycloalkyl, C1-C2 straight
or branched chain alkyl, benzyl, benzoyl, phthaloyl,
acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl, and
2,2,2-trihalo-t-butoxycarbonyl. In Formula IV, f is an
integer from 0 to 12.

ak 02814748 2013-05-02
Preferred 02-aryl substituted diazeniumdiolates also
include those of Example 14.
An alternative method of preparing 02-arylated
diazeniumdiolates is possible through adaptation of the
5 following literature reaction (Stevens, J.Org. Chem. 29:
311-315 (1964)).
0-
+ 0-
Me-N
Me-N/
OTs + Me0-
N OMe+
tosylate ion (0Ts-)
By substituting aryloxy anion Ar0- for the methoxide of
10 Stevens's reaction, it is possible to obtain 02-aryl
diazeniumdiolates of varied structure. Similarly, it is
possible to obtain derivatives corresponding to ArS-
species.
15 02-glycosylated diazeniumdiolates and 1-f(2-
carboxylato)pyrrolidin-1-yl] diazeniumdiolates
The present invention also provides two other new
classes of diazeniumdiolates, one class of which contains
a hydrolytically labile group (R), which, upon cleavage
20 to the free diazeniumdiolate (NO donor) X-NO=NO-, releases
an innocuous and possibly beneficial saccharide and
allows advantage to be taken of saccharide-based
receptor-mediated phenomena. The other class of
diazeniumdiolates provides, among others, prodrugs of the
25 salt disodium 1-[(2-carboxylato)pyrrolidin-1-yl]diazen-l-
ium-1,2-diolate (PROLI/NO), which is an ultrafast NO
donor of proven effectiveness as an antithrombotic agent
and a vasodilator but is inherently extremely difficult
to derivatize, due to its instability (Saavedra et al.,
J. Med. Chem. 31:4361-4365 (1996); and U.S. Patent No.
5,632,981 (Saavedra et al.)). The newly discovered
ability to generate prodrugs of the ultrafast NO donor
PROLI/NO allows the PROLI/NO prodrugs to move freely
through the circulatory system until they reach the
desired organ or cell type for metabolic removal of the

CA 02814748 2013-05-02
26
stabilizing 02-protecting group, thereby providing a rapid
release of NO at the specific or preferred site and
obviating the need for administration by infusion at a
controlled rate in the vicinity of the target tissue.
Additionally, the corresponding nitrosamine, N-
nitrosoproline, if formed in the biological medium, does
not pose a carci.nogenic threat, unlike other
nit rosamines.
Accordingly, the present invention provides 02-
glycosylated 1-substituted diazen-l-ium-1,2-diolates (02-
glycosylated diazeniumdiolates) and 02-substituted 1-((2-
carboxylato)pyrrolidin-1-ylldiazen-l-ium-1,2-diolates (1-
[(2-carboxylato)pyrrolidin-l-yl]diazeniumdiolates), both
of which can be represented by the formula:
X-N-*0
N-O-R (Ia).
In Formula Ia, X and R are organic and/or inorganic
moieties as defined herein.
02-GLYCOSYLATED DIAZENIUMDIOLATES
With respect to the 02-glycosylated
diazeniumdiolates, any of the compounds in the class of
compounds defined as diazeniumdiolates (see e.g., U.S.
Patent Nos. 5,039,705, 5,208,233, 5,155,137, 5,250,550,
5,389,675, 5,525,357, 5,405,919 and related patents and
patent applications) can be subjected to 02-glycosylation,
provided that the 02 of the diazeniumdiolate is available
for glycosylation. The moiety R of Formula Ia can be any
saccharide, which is attached to the 02 of the
diazeniumdiolate by the 2 position of a pyranose or
furanose ring. The saccharide can be functionalized.
Desirably, the sacaharide and its derivatives are
hydrolyzable at physiological pH. The saccharide can be
a monosaccharide, disaccharide, such as sucrose or
maltose, an oligosaccharide or a polysaccharide.
Preferred saccharides and polysaccharides include, among

CA 02814748 2013-05-02
27
others, ribose, glucose, deoxyribose, fucose, lactose,
galactose, fructose, glucosamine, galactosamine, mannose,
maltose, sucrose, and the many saccharide and
oligosaccharide units that serve as recognition sequences
in receptor-mediated cellular interactions. Other
preferred saccharides include those that are
phosphorylated, 3,5-cyclophosphorylated, and
polyphosphorylated pentoses and hexoses.
By way of illustration, the saccharide residue
(shown attached to the diazeniumdiolate for illustrative
purposes)can be an amino sugar, such as a glucosamine or
a substituted glucosamine having the structure:
13
R 0
0¨N¨X
IP
0__N
R
NHR
wherein Ru and Ru can be the same or different and are a
hydrogen, a C1-6 alkyl, an acyl, a phosphate, a sulfate, a
peptide or a protein. The saccharide residue can be, for
example, glucuronic acid or a derivative thereof:
0
11
0 O¨N¨X
HO ____________________________________
OH
wherein R14 is X1R15R16, wherein X1 is N, 0 or S and, when X1
is N, R15 and R16 are independently a hydrogen or a
substituted or an unsubstituted C1-24 alkyl, C3-29
cycloalkyl, C2-24 olefinic, aryl (such as acridine,
anthracene, benzene, benzofuran, benzothiophene,
benzoxazole, benzopyrazole, benzothiazole, carbazole,
chlorophyll, cinnoline, furan, imidazole, indole,

CA 02814748 2013-05-02
28
isobenzofuran, isoindole, isoxazole, isothiazole,
isoquinoline, naphthalene,oxazole, phenanthrene,
phenanthridine, phenothiazine, phenoxazine, phthalimide,
phthalazine, phthalocyanine, porphin, pteridine, purine,
pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,
pyrrocoline, pyrrole, quinolizinium ion, quinoline,
quinoxaline, quinazoline, sydnone, tetrazole, thiazole,
thiophene, thyroxine, triazine, and triazole), or
heterocyclic group, such as glycosyl and the like, and
when X' is 0 or S, there is no R" group.
Alternatively, when XI is nitrogen, R15 and R" form a
heterocyclic ring selected from the group consisting of:
R
N(CH2CH2A) _________________________________________________________
Kni2L,
N ¨ z
,
, and
- ______________________________________________________ CH2 CH2
R8
wherein A is N, 0, or S, w is 1-12, y is 1 or 2, z is
1-5. R8 is hydrogen, a C1.-6 straight chain alkyl, a C3..6
branched chain alkyl, a C3-6 cycloalkyl, an aryl (such as
phenyl, tolyl or the like), or carboxylato and derivatives
thereof as further described herein, and R9 is hydrogen, a
C1-6 straight chain alkyl or a C3-6 branched chain alkyl.
The aforementioned groups can be unsubstituted or
substituted as appropriate.
Exemplary aza crown groups (i.e., where A is N) are
1-aza-12-crown-4, 1-aza-15-crown-5, and 1-aza-18-crown-6.
Where A is nitrogen, the nitrogen atom, itself, can be
substituted, as described, for example, in U.S. patent
No. 5,721,365.
Further with respect to the 02-glycosylated
diazeniumdiolates, the moiety attached to the carbonyl
group through XI can be anything that does not interfere
with the cleavage to the diazeniumdiolate anion.

CA 02814748 2013-05-02
29
Further with respect to the 02-glycosylated
diazeniumdiolates, the moiety attached to the carbonyl
group through X1 can be anything that does not interfere
with the cleavage to the diazeniumdiolate anion.
Preferably, the moiety X contains atoms other than
carbon and hydrogen, and is linked to the nitrogen of the
N202- group through an atom other than carbon. Most
preferably, X is an amino group, and is linked to the
nitrogen of the N202- group through a nitrogen atom.
Suitable moieties of X include, but are not limited to,
C1-24 aliphatic, aryl and non-aromatic cyclic groups. By
"aliphatic" is meant an acyclic moiety containing carbon
and hydrogen and optionally containing nitrogen, oxygen,
sulfur, phosphorus or a halogen. By "aryl" is meant a
moiety containing at least one aromatic ring.
Preferably, the aryl moiety is a C8-30 moiety. By "non-
aromatic cyclic" is meant a moiety containing at least
one ring structure and no aromatic rings. Preferably,
the non-aromatic cyclic moiety is a C8-30 moiety. Further,
X can be unsubstituted or substituted with suitable
additional moieties, such as, for example, -[N(NO)0), a
halo, a hydroxy, an alkylthio, an alkoxy, an aryloxy, an
amino, a mono- or di- substituted amino, a cyano, a
sulfonato, a mercapto, a nitro, a substituted or
unsubstituted C1-12 aliphatic, a substituted or
unsubstituted C3-8 cycloalkyl, a substituted or
unsubstituted C3-C12 olefinic, a substituted or
unsubstituted C3-8 heterocycloalkyl, a benzyl, a phenyl, a
substituted benzyl, a substituted phenyl, a
benzylcarbonyl, a phenylcarbonyl, a saccharide, a
substituted benzylcarbonyl, a substituted phenylcarbonyl
and a phosphorus derivative. Illustrative phosphorus
derivatives include phosphato and phosphono moieties.
Illustrative phosphato moieties include (OH)2P(0)0- and
substituted (OH)2P(0)0- moieties, wherein one or more
oxygen atoms can be independently replaced by S or NR17,
wherein 07 is understood to be a Cl_s aliphatic, a

CA 02814748 2013-05-02
N-OH
1A II
R--C-
5 wherein RI8 is a C1-10 unsubstituted or substituted
aliphatic, a C3-13 unsubstituted or substituted cycloalkyl,
benzyl, phenyl, substituted benzyl or substituted phenyl.
When the benzyl or phenyl is substituted, preferably it
is substituted with one or two substituents selected from
10 the group consisting of halogen, hydroxy, a Ci..4 alkyl,
a C1-4 alkoxy, an amino, a mono-C1-4 alkylamino, a di-C1-4
alkylamino, phenyl and phenoxy.
In one embodiment of the invention, X in Formula Ia
is an inorganic moiety as described in U.S. Patent No.
15 5,212,204. Preferred embodiments of Formula Ia, in which
X is inorganic, are -03S- (sulfite) and -0- (oxide).
In another embodiment of the present invention, X in
Formula Ia is a polyamine as defined in U.S. Patent No.
5,250,550. Thus, the polyamine 02-glycosylated
20 diazeniumdiolates have the formula
R'¨ N¨ (CH2 ) ¨N¨( (CH2) N] ç¨[ (CF12) k-Nid-R5
i
R2 II R4
N--0Q
(II),
wherein Q is the same as the R in Formula Ia and is
defined as above, b and d can be the same or different and
are zero or one, RI, R2, R3, R4, and R5 are the same or
different and are hydrogen, substituted or unsubstituted
C3-8 cycloalkyl, substituted or unsubstituted C1-12 straight
or branched chain alkyl, substituted or unsubstituted
benzyl, substituted or unsubstituted benzoyl, substituted
or unsubstituted C3-C12 olefinic, phthaloyl, acetyl,
trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or 2,2,2-tri-
halo-t-butoxycarbonyl. The values of i, j, and k in

CA 02814748 2013-05-02
31
Formula II can be the same or different and are integers
from 2 to 12.
In a preferred embodiment of the present invention,
the diazeniumdiolates are derived from the compounds
disclosed in U.S. Patent Nos. 5,039,705 (Keefer et al.)
and 4,954,526 (Keefer et al.), and, thus, have the formula
R19R20N_N,_ 0
ii
N-O-R
(III),
wherein R is the same as the R in Formula Ia and is
defined as above, R19 and R2 are the same or different and
are hydrogen, a C1-12 straight chain alkyl, a C3-12 branched
chain alkyl, or a C2-12 straight or C3-12 branched chain
olefinic, provided that both R" and R2 are not hydrogen.
Any of the aforementioned substituents can be
unsubstituted or substituted with an alkoxy, an acyloxy,
an acylthio, a hydroxy, a halo or a benzyl group.
Alternatively, R19 and R20, together with the nitrogen
atom to which they are bonded, form a heterocyclic ring
selected from the group consisting of:
R9
N-(CH2CH2A)z---)
(CH2)w I N---
"(H
'>< 2)
R8 CH2CH2
"y
wherein A is N, 0, or S, w is 1-12, y is 1 or 2, z is 1-5,
R8 is hydrogen, a C1-6 straight chain alkyl, a C3-8 branched
chain alkyl, a C3-8 cycloalkyl, a substituted or an
unsubstituted aryl (such as phenyl, tolyl or the like), or
carboxylato and derivatives thereof as further described
herein, and R9 is hydrogen, a C1-6 straight chain alkyl or

ak 02814748 2013-05-02
32
a C3-6 branched chain alkyl. The aforementioned groups can
be unsubstituted or substituted as appropriate.
Exemplary aza crown groups (i.e., where A is N) are
1-aza-12-crown-4, 1-aza-15-crown-5, and 1-aza-18-crown-6.
Where A is nitrogen, the nitrogen atom, itself, can be=
substituted, as described, for example, in U.S. patent
No. 5,721,365.
Further examples include the 02-glycosylated
diazeniumdiolates derived from the compounds disclosed in
U.S. Patent No. 5,250,550, and, thus, have the formula
R10--N-(CH2)f-D
Rn
(IV),
wherein D is
/ ______________________________________________________
(: 0 ¨N
or \ __ /
N--OR II
N---OR
and wherein R21 is the same as the R in the saccharide of
Formula Ia and is defined as above, and R" and R", which
can be the same or different, can be any suitable group,
examples of which include hydrogen, a C3-6 cycloalkyl, a
C1-12 straight or branched chain alkyl, benzyl, benzoyl,
phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-
butoxycarbonyl and 2,2,2-trihalo-t-butoxycarbonyl. In
Formula IV, f is an integer from 0 to 12.
A preferred 02-glycosylated diazeniumdiolate is one
in which, with respect to Formula Ia, X is N(CH2CH2NH2)2
and R is fucose or mannose.
The above compounds can be prepared in accordance
with methods known to those of skill in the art.
Reagents for glycopyranosylation include acetobromo-a-
galactose and acetobromoglutosamine. Reagents for
glycofuranosylation include tribenzyl-a-arabinofuranosyl
bromide and bromoacetylxylose.
Oligosaccharides are commercially available from,
for example, Sigma Chemical Co. (St. Louis, MO) and

ak 02814748 2013-05-02
33
Carbomer Specialty Biochemicals and Polymers
(Westborough, MA). In addition, oligosaccharides can be
synthesized in accordance with well-established
procedures, including chemical and enzymatic preparation,
such as those described in Preparative Carbohydrate
Chemistry, Stephen Hanessian, ed., Marcel Dekker, New
York, NY (1997) and Polysaccharides in Medicinal
Applications, Severian Dumitriu, ed., Marcel Dekker, New
York, NY (1996).
A protected straight- or branched-chain
polysaccharide can be activated toward reaction with the
diazeniumdiolate ion by halogenation of the anomeric
terminus, followed by glycosylation of the
diazeniumdiolate. Activated disaccharides for generation
of 02-glycosylated diazeniumdiolates include acetobromo-
a-maltose and acetobromo-a-lactose.
02-Glycosylated diazeniumdiolates are useful where
molecular signalling and recognition processes, including
cell adhesion, involve carbohydrates. For example,
02-glycosylated diazeniumdiolates are believed to be
useful in the treatment of infection, such as that due to
a parasite (e.g., leishmania), a virus or a bacterium, as
well as inflammation and metastasis. In this regard, an
02-glycosylated diazeniumdiolate can be prepared so as to
be directed to a mannose-fucose receptor as exemplified
in Example 36. It is believed that the sugar residue, in
this instance mannose, protects the diazeniumdiolate.
The mannose binds to the mannose-fucose receptor on a
macrophage, and the 02-mannosylated diazeniumdiolate is
imported into the cell, where the sugar residue is
cleaved, and NO is released.
1-[(2-CARBOXYLATO)PYRROLIDIN-1-YLJDIAZENIUMDIOLATES
With respect to the 1-[(2-carboxylato)pyrrolidin-1-
yl]diazeniumdiolates, the moiety X of Formula ia can be

CA 02814748 2013-05-02
34
0
N
such that the 1-[(2-carboxylato)pyrrolidin-1-yl]diazen-
iumdiolates can be structurally represented by the
formula:
0
0
N-0--R
wherein R22 is hydrogen, hydroxyl, OM, wherein M is a
cation, halo, or X1R23R241, wherein x' is N, 0 or S and,
when x' is N, R23 and R24 are independently a substituted
or an unsubstituted C1-24 alkyl, 03-24 cycloalkYl,
C2-24 olefinic, aryl (such as acridine, anthracene,
benzene, benzofuran, benzothiophene, benzoxazole,
benzopyrazole, benzothiazole, carbazole, chlorophyll,
cinnoline, furan, imidazole, indole, isobenzofuran,
isoindole, isoxazole, isothiazole, isoquinoline,
naphthalene, oxazole, phenanthrene, phenanthridine,
phenothiazine, phenoxazine, phthalimide, phthalazine,
phthalocyanine, porphin, pteridine, purine, pyrazine,
pyrazole, pyridazine, pyridine, pyrimidine, pyrrocoline,
pyrrole, quinolizinium ion, quinoline, quinoxaline,
quinazoline, sydnone, tetrazole, thiazole, thiophene,
thyroxine, triazine, and triazole), or heterocyclic
group, such as glycosyl, and the like, and when X' is 0 or
S, there is no R24 group. Alternatively, when X' is
nitrogen, R23 and R24, together with the nitrogen to which
they are bonded, form a heterocyclic ring, such as a
heterocyclic ring selected from the group consisting of:

CA 02814748 2013-05-02
R9
7\
(CH2)õ N
Re
(CF12)y
R25
N¨ ( CH2CH2A)z---
NI/
/\N _R26
---n.
2
CH CH2 _______________________________________ //
, and
5
in which A is N, 0 or S, w is 1 to 12, y is 1 or 2, z is
1 to 5, Re, R9, R25 and R26 are hydrogen, a C1-8 straight
chain alkyl, a C3_8 branched chain alkyl, a C3-8
cycloalkyl, or an aryl. The aforementioned groups can be
10 unsubstituted or substituted as appropriate.
The R26 substituent on the nitrogen (N-4) can be a
hydrogen, a C1-8 alkyl group, an aryl group, or C(0)-YR",
wherein Y is sulfur or oxygen, or nitrogen and R" is
CH2OCH3, vinyl, a C1_9 straight chain alkyl, a C3...6 branched
15 chain alkyl, a C3-8 cycloalkyl, polyethylene glycol,
polysaccharide, or other polymer, a peptide, or a
protein. YR" can be an activating linker, such as a
hydroxy succinimidyl group, for linkage to proteins,
peptides, phospholipids, polysaccharides,
20 oligosaccharides, purines, pyrimidines, and biocompatible
polymers (i.e., polyethylene glycol, polylactides, and
polycaprolactone). YR" can be an activating moiety for
the carbonyl group, making the carbonyl group an
electrophilic site that reacts with nucleophilic
25 functionalities of oligopeptides, polyamines and
proteins. YR" can cause the carbonyl group to react with
many nucleophiles, and can react with a polymer, such as
polyethylene glycol, to form a polymer-bound compound.

CA 02814748 2013-05-02
36
Further with respect to the 1-[(2-
carboxylato)pyrrolidin-l-ylldiazeniumdiolates, the moiety
R of Formula ia can be any covalently bound organic or
inorganic moiety, which is other than hydrogen and is a
C1-12 straight chain or C3-12 branched chain alkyl, a C2-12
straight chain or C3-12 branched chain olefinic, a C1-12
acyl, sulfonyl, C3-,12 cycloalkyl, carboxamido, a glycosyl
group as described above, an aryl group as described
below, or a group of the formula -(CH2)n-ON=N(0)NR28R28,
wherein n is an integer of 2-8, and R28 and R28 are
independently a C1-12 straight chain alkyl, a C3-12 branched
chain alkyl, a C1_12 straight chain or C3-12 branched chain
olefinic, or R28 and R28, together with the nitrogen atom
to which they are bonded, form a heterocyclic group,
preferably a pyrrolidino, piperidino, piperazino or
morpholino group. The aforementioned R groups can be
unsubstituted or substituted as appropriate. Preferred
substitutions include those made with hydroxy, halo,
acyloxy, alkoxy, acylthio, or benzyl.
The above compounds can be prepared in accordance
with methods known to those of skill in the art. For
example, see Sanger, Biochem. J. 39: 507-515 (1945).
02-Substituted 1-[(2-carboxylato)pyrrolidin-1-
yl]diazeniumdiolates offer advantages over other
diazeniumdiolates in that they are more stable in aqueous
solution than the 02-unsubstituted anion and, in many
cases, they can be activated for NO release by enzymatic
action. Furthermore, if an N-nitroso derivative is
formed by net formal cleavage of the N-N double bond of
the 1-[(2-carboxylato)pyrrolin-1-yl)diazen-l-ium-1,2-
diolate, the N-nitroso compound is noncarcinogenic. Such
compounds are believed to be particularly useful in the
treatment of fulminant liver failure, malaria,
respiratory problems, impotence, and a variety of
cardiovascular/hematologic disorders.

CA 02814748 2013-05-02
37
Polymer Bound Diazeniumdiolates
Another particularly useful embodiment of the
present invention comprises 02-aryl diazeniumdiolates of
Formula I or 02-glycosylated diazeniumdiolates of Formula
Ia, wherein X is a polymer, or wherein any 02-aryl
diazeniumdiolate or 02-glycosylated diazeniumdiolate of
the present inveut_ion is incorporated into a polymeric
matrix. PROLI/NO also can be polymer bound--through R2
as well as R. Both of these embodiments result in the
N202 functional group being "bound to the polymer." By
"bound to a polymer," it is meant that the N202
-
functional group is associated with, part of,
incorporated with or contained within the polymeric
matrix physically or chemically.
Physical association or bonding of the N202
functional group to the polymer may be achieved by
coprecipitation of the polymer with a nitric oxide/
nucleophile complex as well as by covalent bonding of the
N202 group to the polymer. Chemical bonding of the N202
group to the polymer may be by, for example, covalent
bonding of the nucleophilic moiety of the nitric
oxide/nucleophile adduct to the polymer such that the
nucleophilic residue to which the NO2 group is attached
forms part of the polymer, itself, i.e., is in the
polymer backbone or is attached to pendant groups on the
polymer backbone. The manner in which the nitric oxide-
releasing NA functional group is associated with, part
of, or incorporated with or contained within, i.e.,
"bound" to the polymer is inconsequential to the present
invention and all means of association, incorporation and
bonding are contemplated herein.
Site-specific application of the polymer-bound
adduct composition enhances the selectivity of action of
the nitric-oxide releasing N202 functional group. If N20,-
functional groups attached to the polymer are necessarily
localized, then the effect of their nitric oxide release
will be concentrated in the tissues with which they are

CA 02814748 2013-05-02
38
in contact. If the polymer is soluble, selectivity of
action can still be arranged, for example, by linkage to
or derivatization of an antibody specific to the target
tissue. Similarly, linkage of N202 groups to small
peptides that mimic the recognition sequences of ligands
for important receptors provides localized nitric oxide
release, as would linkage to oligonucleotides capable of
site-specific interactions with target sequences in a
nucleic acid.
The 02-diazeniumdiolates of the present invention can
be derived from the materials disclosed in U.S. Patent
Nos. 5,525,357 (Keefer et al.) and 5,405,919 (Keefer et
al.), and in U.S. Patent No. 5,691,423 (Smith et al.).
Any of a wide variety of polymers can be
used in the context of the present invention. It is only
necessary that the polymer selected is biologically
acceptable. Illustrative of polymers suitable for use in
the present invention are polyolef ins, such as
polystyrene, polypropylene, polyethylene,
polytetrafluorethylene, polyvinyl chloride,
polyvinylidene difluoride, and polyethers such as
polyethylene glycol, polysaccharides such as dextran,
polyesters such as poly(lactide/glycolide), polyamides
such as nylon, polyurethanes, polyethyleneimines,
biopolymers such as peptides, proteins, oligonucleotides,
antibodies and nucleic acids, starburst dendrimers,
polysaccharides, and the like.
In this regard, a polymer containing a
diazeniumdiolate can be reacted with a saccharide, such
that the saccharide becomes bound to the N202- functional
group.
Formation of a diazeniumdiolate from a biopolymer
provides a biopolymer-bound diazeniumdiolate composition
that can be applied with specificity to a biological site
of interest. Site-specific application of the
biopolymer-bound diazeniumdiolate enhances the

CA 02814748 2013-05-02
39
selectivity of action of the nitric oxide-releasing
diazeniumdiolate, which occurs following the cleavage of
the 02-aryl or 02-glycosylated bond or the 0-R bond in
PROLI/NO (see pg. 33). As with the other polymers
disclosed above, if the diazeniumdiolate attached to the
biopolymer is localized because of the inherent
properties of the molecule, then the effect of its nitric
oxide release will be concentrated in the tissues with
which they are in contact. If the biopolymer is soluble,
selectivity of action can still be arranged, for example,
by attachment to or derivatization of an antibody
specific to the target tissue% Similarly, linkage of
diazeniumdiolate groups to small peptides that mimic the
recognition sequences of ligands for important receptors
provides localized nitric oxide release, as would linkage
to oligonucleotides capable of site-specific interactions
with target sequences in a nucleic acid. Other proteins,
peptides, polypeptides, nucleic acids and polysaccharides
can be similarly utilized. U.S. Patent No. 5,405,919
(Keefer et al.) and U.S. Patent No. 5,632,981 (Saavedra
et al.),
disclose similar compounds and manufactures
useful in the preparation of the diazeniumdiolates.
By way of illustration, an 02-arylated piperazine
diazeniumdiolate can be covalently attached to a
polypeptide containing the IKVAV recognition sequence,
which is important in tumor cell chemotaxis. Through
retention of both the capacity to regenerate NO as an
anti-adhesive agent and the affinity of the IKVAV
sequence for tumor cells and/or sites in the vascular and
lymphatic systems, where the tumor cells tend to attach,
metastasis can be reduced or even prevented. Further,
the aryl moiety can be chosen such that it provides
additional antitumor cell activity. Substitutions at the
N4 position of piperazine can be used to link the
glycosylated diazeniumdiolate to peptides, polypeptides,
proteins, polysaccharides and nucleotides.

CA 02814748 2013-05-02
It is contemplated that the diazeniumdiolates of the
present invention can be used to coat prostheses, stents,
and medical implants, such as breast implants, prior to
surgical connection to the body as a means of reducing
5 the risk of solid state carcinogenesis associated
therewith. Additionally, the prostheses and implants can
be manufactured using a diazeniumdiolate as an integral
component of the starting materials. Medical devices
incorporating a diazeniumdiolate provide an invaluable
10 two-pronged approach to the treatment of many biological
disorders, providing useful medical structures that also
advantageously provide local release of NO.
Compositions
15 As is well-known in the art, nitric oxide and
compounds comprising 1\1202- functional groups can have a
wide range of utilities, in part because of the
multifaceted role of nitric oxide in bioregulatory
processes. Accordingly, the present invention also
20 provides a composition, including a pharmaceutical
composition, comprising a present inventive
diazeniumdiolate. Preferably, the pharmaceutical
composition additionally comprises a pharmaceutically
acceptable carrier.
25 One skilled in the art will appreciate that suitable
methods of administering the diazeniumdiolate
compositions of the present invention to an animal, such
as a mammal, are available, and, although more than one
route can be used to administer a particular composition,
30 a particular route can provide a more immediate and more
effective reaction than another route. Pharmaceutically
acceptable carriers are also well-known to those who are
skilled in the art. The choice of carrier will be
determined, in part, both by the particular composition
35 and by the particular method used to administer the
composition. Accordingly, there is a wide variety of

CA 02814748 2013-05-02
41
suitable formulations of the pharmaceutical compositions
of the present invention.
Formulations suitable for oral administration can
consist of (a) liquid solutions, such as an effective
amount of the diazeniumdiolate dissolved in diluents,
such as water or saline, (b) capsules, sachets or-
tablets, each containing a predetermined ambunt of the
active ingredient, as solids or granules, (c) suspensions
in an appropriate liquid, and (d) suitable emulsions.
Tablet forms can include one or more of lactose,
mannitol, corn starch, potato starch, microcrystalline
cellulose, acacia, gelatin, colloidal silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, stearic
acid, and other excipients, colorants, diluents,
buffering agents, moistening agents, preservatives,
flavoring agents, and pharmacologically compatible
carriers. Lozenge forms can comprise the active
ingredient in a flavor, usually sucrose and acacia or
tragacanth, as well as pastilles comprising the active
ingredient in an inert base, such as gelatin and glycerin
or sucrose and acacia emulsions, gels, and the like
containing, in addition to the active ingredient, such
carriers as are known in the art.
The diazeniumdiolates of the present invention,
alone or in combination with other suitable components,
can be made into aerosol formulations to be administered
via inhalation. These aerosol formulations can be placed
into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations suitable for parenteral administration
include aqueous and non-aqueous solutions, isotonic
sterile injection solutions, which can contain
antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and

CA 02814748 2013-05-02
42
preservatives. The formulations can be presented in
unit-dose or multi-dose sealed containers, such as
ampules and vials, and can be stored in a freeze-
dried(lyophilized) condition requiring only the addition
of the sterile liquid carrier, for example, water, for
injections, immediately prior to use. Extemporaneous
injection solut+r,as and suspensions can be prepared from
sterile powders, granules, and tablets of the kind
previously described.
The dose administered to an animal, particularly a
human, in the context of the present invention should be
sufficient to effect a therapeutic response in the animal
over a reasonable time frame. The dose will be
determined by the strength of the particular compositions
employed (taking into consideration, at least, the rate
of NO evolution, the extent of NO evolution, and the
bioactivity of the decomposition products derived from
the diazeniumdiolates) and the condition of the
animal, as well as the body weight of the animal to be
treated. The size of the dose also will be determined by
the existence, nature, and extent of any adverse side
effects that might accompany the administration of a
particular composition. A suitable dosage for internal
administration is 0.01 to 100 mg/kg per day. A preferred
dosage is 0.01 to 35 mg/kg per day. A more preferred
dosage is 0.05 to 5 mg/kg per day. A suitable
concentration of 02-aryl diazeniumdiolates in
pharmaceutical compositions for topical administration is
0.05 to 15% (by weight). A preferred concentration is
from 0.02 to 5%. A more preferred concentration is from
0.1 to 3%.
Methods of Use
In view of the above, the present invention provides
methods of using a present inventive diazeniumdiolate.
In one embodiment, a method of treating an animal, such
as a mammal, with a biological disorder treatable with
nitric oxide, is provided. The method comprises

CA 02814748 2013-05-02
43
administering to the animal, e.g., the mammal, an amount
of an diazeniumdiolate in accordance with the present
invention sufficient to treat the biological disorder in
the animal. In this embodiment, "biological disorder"
can be any biological disorder, including a biological
disorder due to a genetic defect or infection with an
infectious agent, such as a virus, bacterium or parasite,
as long as the disorder is treatable with nitric oxide.
In another embodiment of a method of use, a method
is provided for treating an animal, such as a mammal, for
infection with, for example, a virus, a bacterium, or a
parasite (e.g., leishmania). The method comprises
administering to the animal, e.g., the mammal, an amount
of a diazeniumdiolate sufficient to treat the infection
in the animal.
In one aspect of this embodiment of the invention, a
method is provided for treating an animal, such as a
mammal, for infection with, for example, a virus, such as
a retrovirus, in particular HIV, more particularly HIV-1,
a bacterium, such as a Gram-positive bacterium, or a
parasite, such as Giardia, any one of which comprises a
zinc finger protein that can be inactivated by an 02-aryl
diazeniumdiolate. By "zinc finger protein" is meant a
protein comprising a short amino acid domain containing
cysteines alone or cysteine and histidine ligands, both
of which coordinate with zinc and interact with nucleic
acids (South and Summers, "Zinc Fingers," Chapter 7, In:
Adv. Inorg. Biochem. Ser. 8, pp. 199-248 (1990).
By "inactivated" is meant partial or complete loss of
activity of the zinc finger protein to be inactivated.
Such inactivation should not result in inactivation of
biologically important zinc finger proteins in the
animal, itself, to such an extent as to compromise unduly
the health and well-being of the animal_ The method
comprises administering to the animal, e.g., the mammal,

CA 02814748 2013-05-02
44
an amount of an 02-aryl diazeniumdiolate sufficient to
inactivate the zinc finger protein in said infectious
agent so as to treat the infection in the animal.
The above-described method also can be adapted as a
means of treating a plant, plant cell or tissue culture
thereof for infection with an infectious agent, such as a
virus, e.g., tobacco streak virus (TSV) or alfalfa mosaic
virus (AIMV) (South and Summers (1990), supra; and Sehnke
et al., Virology 168: 48 (1989)).
The methods described herein are useful against zinc
fingers comprising the motif C-X2-C-X4-H-X4-C (see, e.g.,
_Wain-Hobson et al., Cell 40(1): 9-17 (1985)), in which
"C" represents cysteine, "H" represents histidine, "X"
represents any amino acid, and the numbers "2" and "4"
represent the number of "X" amino acids. Such a motif is
characteristic of retroviruses, in particular the gag
protein of retroviruses. Accordingly, the methods herein
are useful against retroviruses, such as HIV, and, in
particular, HIV-1 (Rice et al., Nature Medicine 3(3):
341-345 (1997); and Rice et al., Reviews in Medical
Virology 6: 187-199 (1986)), which comprises nucleocapsid
p7 proteins (NCp7 proteins) that include two zinc binding
domains. Actual and/or potential zinc fingers also have
been identified in, among others, the gene products of
the EIA genomic region of adenoviruses, the large T
antigens from simian virus 40 (SV40) and polyoma viruses,
the UvrA protein in E. coil (Culp et al., PNAS USA 85:
6450 (1988)), murine leukemia virus (MuLV-F; Green et
al., PNAS USA 86: 4047 (1989)), and bacteriophage
proteins (Berg, Science 232: 484 (1986)), such as gene 32
protein (G32P) from bacteriophage T4 (Giedroc et al.,
Biochemistry 28: 2410 (1989)). Such proteins can be
isolated in accordance with methods known in the art (see
references cited in South and Summers (1990), supra), and
the 02-aryl diazeniumdiolates, which can inactivate such
zinc finger proteins, can be identified in accordance,

CA 02814748 2013-05-02
for example, with the zinc finger assay described herein
and in Rice et al., J. Med. Chem. 39: 3606-3616 (1996).
To the extent that steroid hormone receptors
comprise zinc fingers with motifs comprising 4 or 5
5 _cysteines, an 02-aryl diazeniumdiolate can be used to
modulate steroid hormone activity in an animal, such as a
mammal. Accordingly, the p-resent invention also provides
a method of modulating steroid hormone activity in an
animal, such as a mammal, which is in need of modulation
10 of steroid hormone activity and which comprises a steroid
hormone receptor protein comprising a zinc finger that
can be inactivated by an 02-aryl diazeniumdiolate. The
method comprises administering to the animal, e.g., the
mammal, an amount of an 02-aryl diazeniumdiolate
15 sufficient to inactivate the steroid hormone receptor
protein so as to modulate steroid hormone activity in the
animal.
In yet another embodiment, a method for treating an
animal, such as a mammal, for cancer and metastasis
20 thereof is provided. The method comprises administering
to the animal, e.g., the mammal, an amount of
diazeniumdiolate sufficient to prevent the growth or
metastasis of the cancer in the animal.
In one aspect of this embodiment, a method for
25 treating an animal, such as a mammal, for cancer is
provided, wherein the cancer is due, at least in part,
directly or indirectly, to the activity of a zinc finger
protein that can be inactivated by an 02-aryl
diazeniumdiolate. The method comprises administering to
30 the animal, e.g., the mammal, an amount of 02-aryl
diazeniumdiolate sufficient to inactivate the zinc finger
protein so as to treat the cancer in the animal (Rice et
al., PNAS 89: 7703-7707 (1992)), i.e., prevent the growth
or metastasis of the cancer in the animal.
35 In still yet another embodiment, a method is
provided for treating an animal, such as a mammal, for
cancer, wherein the cancer is resistant to treatment with

ak 02814748 2013-05-02
46
a chemotherapeutic agent (see, e.g., Kelley et al.,
Biochem. J. 304: 843-848 (1994)), in particular a DNA
damaging agent, such as an alkylating agent or an
oxidizing agent, due, for example, to the action of an
enzyme that adversely affects the activity of the
chemotherapeutic agent. The method comprises
administering to the animal, e.g., the mammal, an amount
of an 02-aryl diazeniumdiolate sufficient to render the
cancer in the animal susceptible to treatment with the
chemotherapeutic agent. Accordingly, such a method can
be used as an adjunct therapy to chemotherapy as needed.
For example, certain 02-aryl diazeniumdiolates can be
synthesized to fit into the active site of glutathione S-
transferase, specifically isoenzyme n (see, e.g., Ji et
al., Biochemistry 32(49): 12949-12954 (1993); and Ji et
al., Biochemistry 36: 9690-9702 (1997)). Accordingly,
inversible consumption or glutathione from the active
site of glutathione S-transferase-n with an 02-aryl
diazeniumdiolate could prevent the enzyme from
detoxifying a variety of xenobiotic compounds, such as
chemotherapeutic drugs, especially alkylating agents,
such as chlorambucil, melphalan and hepsulfam, and other
DNA-damaging agents, such as agents that induce
electrophilic attack or oxidization, by enzymatic
conjugation of the compound with glutathione (see, e.g.,
Morgan et al., Cancer Chemother. Pharmacol. 37: 363-370
(1996)). This method also has applicability to screening
drug-resistant cancer cell lines in vitro.
In another embodiment, a method is provided for
treating an inanimate object for the presence of a
potentially infectious virus, bacterium, or parasite.
The method comprises contacting the inanimate object with
an amount of a present inventive diazeniumdiolate
sufficient to reduce the presence of the potentially
infectious virus, bacterium or parasite. By "potentially
infectious" is meant the capability of infecting an
animal, such as a mammal.

ak 02814748 2013-05-02
47
In one aspect of this embodiment, a method is
provided for reducing on an inanimate object the presence
of a potentially infectious agent, such as a virus, a
bacterium, or a parasite, any one of which comprises a
zinc finger protein that can be inactivated by an 02-aryl
diazeniumdiolate. The method comprises contacting the
inanimate object.w-ith an amount of an 02-aryl
diazeniumdiolate sufficient to inactivate the zinc finger
protein so as to reduce the presence of the potentially
infectious agent, e.g., virus, bacterium or parasite, on
the inanimate object. By "potentially infectious" is
meant the capability of infecting an animal, such as a
mammal, directly or indirectly.
EXAMPLES
The following examples further illustrate the
present invention and, of course, should not be construed
as in any way limiting its scope. With respect to the
following examples, NO was obtained from Matheson Gas
Products (Montgomeryville, PA), 0- and a-glycosidases and
porcine liver esterase were obtained from Sigma Chemical
Co. (St. Louis, MO), polyurethane (TecofleIr was obtained
from Thermedics Inc. (Woburn, MA), and glucose and
mannose were obtained from Aldrich Chemical Co.
(Milwaukee, WI). Proton NMR spectra were recorded with a
300 MHz Varian Unity Plus or a Varian XL-200 NMR
spectrometer. Spectra were obtained in deuterochloroform
for covalent compounds and in D20 for salts. Chemical
shifts are reported in parts per million (ppm) downfield
from TMS. Low and high resolution mass spectral (MS)
measurements were carried out on a VG-Micromass Model
7070 spectrometer. Unless otherwise indicated, MS data
were collected in the electron impact mode with sample
introduction via direct probe. Ultraviolet ((JV) spectra
were run as solutions in water or 0.01 M NaOH on an HP
8451A Diode Array spectrophotometer. Glutathione S-
transferase kinetics were monitored by measuring the

ak 02814748 2013-05-02
48
change in UV absorbance at 380 nm with a Beckman DU 640
spectrophotometer. Chemiluminescence measurements were
done on a Thermal Energy Analyzer Model 502A instrument
(Thermedics, Inc., Woburn, MA). Elemental analyses were
performed by Atlantic Microlab Inc.
EXAMPLE 1
This Example illustrates the preparation of 02-(2,4-
dinitrophenyl) 1-(N,N-diethylamino)diazen-l-ium-1,2-
diolate.
A solution of 1.67 g (11 mmol) of sodium
diethylaminodiazeniumdiolate in 20 ml of 5% aqueous
sodium bicarbonate was cooled to 0 'C under nitrogen. A
solution of 1.3 ml (0.01 mol) of 2,4-dinitrofluorobenzene
in 10 ml of t-butyl alcohol was added slowly. A
precipitate formed upon addition. The mixture was
allowed to warm up to room temperature gradually, then
stirred overnight. The product was extracted with
dichloromethane and subsequently washed with cold dilute
hydrochloric acid followed by sodium bicarbonate
solution. The organic layer was dried over sodium
sulfate, filtered through a layer of magnesium sulfate,
and evaporated under vacuum to give 1.3 g of a red oil,
which crystallized on standing. Recrystallization from
ethanol gave yellow-orange needles: m.p. 76-7 'C; NMR 5
1.25 (t, 6H), 3.58 (q, 41-i), 7.68 (d, 1H), 8.44 (m, 2H),
8.89 (m, 1H); UV (ethanol) X.(E) 218 (17.4 mM-1 cm1-) and
302 (15.6 mM-1 cm-1) fin; MS, exact mass, calculated for
C10H13N506: (M+) 299.0865; measured M4 299.08658. Analysis,
C, H, N, calculated for: C10H13N506: C 40.13%, H 4.35%, N
23.41%. Found: C 40.21%, H 4.43%, N 23.37%.
EXAMPLE 2
This Example illustrates the regeneration of the
anionic diazeniumdiolate from its 02-aryl substituted form
(02-(2,4-dinitrophenyl) 1-(N,N-diethyamino)diazen-l-ium-
1,2-diolate).

ak 02814748 2013-05-02
49
A solution of 85 mg (0.28 mmol) of 02-(2,4-
dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-
diolate, prepared as in Example 1, in 1 ml of ether was
cooled to -4 'C and treated with 1 ml of diethylamine.
The solution was kept at -4 'C for 1 hr, giving a
precipitate. The solid was collected by filtration. The
filtrate was comentrated and analyzed by NMR; the
residue proved to be identical to an authentic sample of
2,4-dinitro-N,N-diethylaniline. The precipitate was
washed with petroleum ether and dried under N2 to give 5.4
mg of product having Xmax 250 nm; NMR (D20) ö 0.96 (t, 6H),
1.28(t, 6H), 2.94 (q, 4H), 3.08 (q, 4H). This product
proved to be identical to an authentic sample of
diethylammonium 1-(N,N-diethylamino)diazen-l-ium-1,2-
diolate.
EXAMPLE 3
This Example illustrates the chemical cleavage of
the 02-aryl bond of an 02-aryl diazeniumdiolate mediated
by sodium methoxide.
A solution of 16 mg (0.064 mmol) of 02-(2,4-
dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-
diolate in 1 ml of ether was treated with 29 1.11 of 25%
sodium methoxide in methanol (0.14 mmol) and allowed to
stand at -4 'C for 2 hr. The solid precipitate was
collected by filtration, washed with ether and dried
under vacuum to yield 4 mg of a solid identical to an
authentic sample of 1-(N,N-diethylamino)diazen-l-ium-1,2-
diolate sodium salt.
EXAMPLE 4
This Example illustrates the kinetics of reaction of
02-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-l-ium-
1,2-diolate with sodium methoxide in methanol. The
kinetics of this reaction show the rate of conversion of
02-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-l-ium-
1,2-diolate to 1-(N,N-diethylamino)diazen-l-ium-1,2-

ak 02814748 2013-05-02
diolate ion in alkaline or nucleophilic environments.
An excess of Na0Me was used in the reactions;
aliquots were collected at intervals and quenched with
0.1 N HC1 in methanol. The disappearance of 02-(2,4-
5 dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-
diolate, monitored by HPLC, was found to fit the first-
order rate equatimn. This was determined by plotting
log[02-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-l-
ium-1,2-diolate] vs. time to find }cab,' at four different
10 concentrations of Na0Me. Similarly, the second-order
rate constant (7.87 M' min') was determined by plotting
log }cobs vs. log[Na0Me].
= EXAMPLE 5
15 This Example illustrates the preparation of 02-(2,4-
dinitrophenyl) 1-(N-isopropylamino)diazen-l-ium-1,2-
diolate.
A solution of 84 mg (0.597 mmol) of sodium 1-(N-
isopropylamino)diazen-l-ium-1,2-diolate in 1 ml of 5%
20 sodium bicarbonate was cooled to 0 'C and 69 mg (0.55
mmol) of 2,4-dinitrofluorobenzene was added. The ice
bath was removed, the mixture was allowed to stir at room
temperature overnight, and then the mixture was extracted
with dichloromethane. The extract was dried over sodium
25 sulfate, filtered and evaporated in vacuo to give 86 mg
of a film, which crystallized on standing: m.p. 92-93 'C
NMR 8 1.39 (d, 6H), 3.99 (septet, 1H), 6.93 (d, 1 H),
8.27 (dd, 1H), 8.5 (b, 1H), 9.15 (d, 1H).
30 EXAMPLE 6
This Example illustrates the synthesis of
pyrrolidinium 1-[pyrrolidin-1-yl]diazen-l-ium-1,2-
diolate.
A solution of 36 g (0.507 mol) of pyrrolidine in 50
35 ml of ether and 25 ml of acetonitrile was placed in a 500
ml Parr bottle, degassed and charged with 40 psi of
nitric oxide. The reactor was cooled to -80 C. The

ak 02814748 2013-05-02
51
pressure was maintained at 40 psi. After 4 hr, the
pressure was released, and the crystalline product was
collected by filtration in a fritted glass funnel and
then washed with cold ether under an atmosphere of
nitrogen. The material was dried in a vacuum desiccator
at 1 mm Hg and 25 'C for 3 hr to give 23 g (45%) of white
needles: m.p. 68 70 'C. Analysis C,H,N: Calculated for
C8E1181\1402: C 47.51%, H 8.97%, N 27.70%; Found, C 47.62%, H
9.04%, N 27.46%.
The pyrrolidinium salt was converted to the more
stable sodium salt for subsequent 02-arylations by
treatment with 10 N NaOH to promote cation exchange. It
was then flooded with ether. The product was collected
by filtration.
EXAMPLE 7
This Example gives an alternate method of preparing
the sodium salt of the 1-(pyrrolidin-1-yl)diazen-1-
iuml,2-diolate presented in Example 6.
A solution of 28.2 g (0.397 mol) of pyrrolidine in
100 ml of acetonitrile and 100 ml of ether was mixed with
94 ml (0.4 mol) of 25% sodium methoxide in methanol. The
resulting solution was flushed with nitrogen then charged
with 40 psi of NO and stirred at room temperature for two
days forming a thick precipitate. (The precipitate had
begun to form within 1 hr of exposure to NO.) The
pressure was released and the product was collected by
filtration. The product was washed with ether and dried
under vacuum to give 32.1 g (54%) of a white powder: UV
(0.01 N NaOH) Xraax (E), 252 nm (8.84 mM-I t112 8.5
sec at 25 'C and 2.8 sec at 37 'C in pH 7.4 phosphate
buffer; NMR (D20) 6 1.91 (m, 4H) , 3.22 (m, 4H).
EXAMPLE 8
This Example illustrates the preparation of 02-(2,4-
dinitrophenyl) 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-
diolate.

ak 02814748 2013-05-02
52
A solution of 556 mg (3.63 mmol) of sodium
1(pyrrolidin-1-y1)diazen-1-ium-1,2-diolate in 10 ml of 5%
aqueous sodium bicarbonate was cooled to 0 C. A
solution of 456 1 (3.63 mmol) of 2,4-
dinitrofluorobenzene in 2 ml of t-butyl alcohol was added
and the resulting mixture was stirred at room temperature
overnight. The yellow-orange precipitate was collected
by filtration, washed with water, and dried to give 758
mg of product, which was recrystallized from ethanol:
m.p. 94-95 C; NMR, 5 2.04 (m, 4H), 3.35 (m, 4H), 6.90
(d, 1H), 8.20 (dd, 1H), 8.67 (d, IH); MS, m/z(%), 297 (M%
1), 220 (100), 237 (30), 190 (94), 180 (15), 162 (10),
149 (26), 130 (20), 100 (95), 70 (24), 63 (35), 56 (18).
Exact Mass: calculated for C10H11N506 (M+)297.0708;
measured 297.0709.
EXAMPLE 9
This Example illustrates the preparation of sodium
1-[(4-ethoxycarbonyl)piperazin-1-ylidiazen-1-ium-1,2-
diolate.
A solution of 20 g (0.126 mol) of N-
carboethoxypiperazine in 60 ml of methanol was placed in
a Parr bottle. The solution was treated with 27.4 ml
(0.126 mol) of 25% sodium methoxide in methanol; the
system was evacuated, charged with 40 psi of nitric oxide
and kept at 25 .0 for 48 hr. The white crystalline
product was collected by filtration and washed with cold
methanol as well as with copious amounts of ether. The
product was dried under vacuum to give a 14.5 g (48%)
yield of sodium 1-[(4-ethoxycarbonyl)piperazin-l-
yl]diazen-1-ium-1,2-diolate: m.p. 184-5.C; UV (0.01 N
NaOH) Ainax (E) 252 nm (10.4 mM-1 cm-1); NMR (D20) 5 1.25 (t,
3H), 2.15 (q, 2H) 3.11 (m, 4H), 3.68 (m, 4H). Anal
calcd. for C6H13N404Na: C 35.00%, H 5.42%, N 23.33%, Na
9.58%. Found: C 34.87%, H 5.53%, N 23.26%, Na 9.69%.
The half-life of this compound at pH 7 and 25 .0 was

ak 02814748 2013-05-02
53
assessed at 5 min. This measurement was based on the
loss of the 252 am chromophore in the ultraviolet
spectrum.
EXAMPLE 10
This Example illustrates the preparation of 02-(2,4-
dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-l-
yl]diazen-l-ium-1,2-diolate.
A solution of 1.073 g (0.0045 mol) of sodium 1-[(4-
ethoxycarbonyl)piperazin-1-yl]diazen-l-ium-1,2-diolate in
10 ml of 5% sodium bicarbonate was cooled at 0 .0 under
nitrogen. A partial solution of 0.89 ml (0.0044 mol) of
2,4-dinitrofluorobenzene in 10 ml of t-butyl alcohol was
added. A precipitate formed upon addition; the mixture
was allowed to stir at room temperature for 4 hr. The
product was extracted with dichloromethane. The extracts
were washed with water, dried over sodium sulfate and
filtered through a layer of anhydrous magnesium sulfate.
Evaporation of the solvent gave an orange glass which
crystallized on standing. The product was recrystallized
from ethanol:dichloromethane to give 1.3 g (76%) of
analytically pure material: m.p. 140-141 *C; NMR 8 1.32
(t, 3H), 3.63 (m, 4H), 3.74 (m, 4H), 4.19 (q, 2H), 7.66
(d, 1 H), 8. 48 (q, 1H) , 8.86 (d, 1 H); UV (H20) ?.max (c)
210 am (13.3 m11-1 cm-1), 300 am (12 mt4-1 cm-1). Anal calcd.
for Ci3Hi6N608: C 40.61%, H 4.20%, N 21.87%; Found: C
40.74%, H 4.13%, N 21.98%.
EXAMPLE 11
This Example illustrates the preparation of 02-(2-
chloropyrimidin-4-y1) 1-(N,N-diethylamino)diazen-l-ium-
1,2-diolate.
A solution of 600 mg (4 mmol) of 2,4-
dichloropyrimidine in 2 ml of dimethylsulfoxide and 5 ml
of tetrahydrofuran was added via syringe to a slurry of
678 mg (4.37 mmol) of sodium 1-(N,N-diethylamino)diazen-
1-ium-1,2-diolate in 5 ml of tetrahydrofuran at room

CA 02814748 2013-05-02
54
temperature under nitrogen and the resulting mixture was
stirred for 72 hr. Five (5) ml of ether was added to the
mixture. After washing with water, the organic layer was
dried over sodium sulfate, filtered through a layer of
magnesium sulfate, and evaporated to give 679 mg of an
oil which crystallized at -20 C. This material was
recrystallized fram ether-petroleum ether: m.p. 37-38 .C;
NMR 5 1.25 (t, 6H), 3.56 (q, 4H), 7.00 (d, 1H), 8.50
(d,1H); UV, Xmax (c) 268 nm (9.3 mM-1 cm(1). Analysis C, H,
N: Calculated for C8H12N502C1: 39.11%, H 4.92%, N 28.51%,
Cl 14.43%; Found: C 38.96%, H 4.96%, N 28.35%, Cl 14.60%.
EXAMPLE 12
This Example illustrates the preparation 02-(2-
chloropyrimidin-l-y1) 1-((4-ethoxycarbonyl)piperazin-l-
y1Jdiazen-l-ium-1,2-diolate.
A solution of 262 mg (1.76 mmol) of 2,4-
dichloropyrimidine in 3 ml of dimethylsulfoxide was added
to a slurry of 424 mg (1.76 mmol) of sodium 1-[(4-
ethoxycarbonyl)piperazin-l-ylldiazen-l-ium-1,2-diolate in
10 ml of tetrahydrofuran at room temperature under
nitrogen and stirred for 72 hr. The resulting
homogeneous solution was treated with 100 ml of water.
The precipitate was collected by filtration and dried
under vacuum to give 300 mg of product: m.p. 136-137 *C;
NMR 5 1.29 (t, 3H), 3.69 (m, 4H), 3.71 (m, 4H), 4.18 (q,
2 H) , 6. 99 (d, 1 H) , 8.52 (d, 1 H) ; (UV) 'max (e) 270
nm (4.1 mM-1 cm(1).
This compound undergoes nucleophilic substitution
with methoxide to displace the chlorine atom at the C2
position and the diazeniumdiolate at the C4 position to
give 2,4-dimethoxypyrimidine.
EXAMPLE 13
This Example describes the synthesis of the
following compounds:

CA 02814748 2013-05-02
Et2NN202 _________ ( 1 ) __ CF3 Et2NN202 0
COOH
NO2 NO2
1 2
5
N-
Et2NN202 ____________________________________________________ ())---i\TO2
Et2NN202 _________ 0)----NO2
NO2
10 3 4
Et2NN202
NO2
5
General synthesis of compounds 1 through 5: A 1 M
solution of sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-
diolate in dimethylsulfoxide was stirred at
5 'C under nitrogen. A 1 M solution containing 0.95
molar equivalents of the arylating agent in
tetrahydrofuran was injected through a septum. The
reaction mixture was allowed to warm up to room
temperature, stirred overnight, quenched with ice-water
and extracted with ether. The ether was washed with
water, dried over sodium sulfate, filtered through a
layer of maganesium sulfate and concentrated on a rotary
evaporator. The methods of purification varied with each
preparation and are described with the individual
compounds below. (Note: Compounds 1 through 5 are
selected products from 02-aryl compound libraries built

ak 02814748 2013-05-02
56
using solution phase synthetic methods in parallel
fashion). NMR spectra were run in CDC13.
02-(2-Nitro-4-trifluoromethylphenyl) 1-(N,N- .
diethylamino)diazen-1-ium-1,2-diolate, 1: Arylation was
carried out with 4-fluoro-3-nitrobenzotrifluoride.
Purification of th-e product was carried out on
preparative HPLC using a 1 inch C-18 column eluted with
20% aqueous acetonitrile with a solvent gradient to 50%
acetonitrile:50% water. A 42% yield of product was
obtained as an oil: NMR 5 1.23 (t,6H), 3.50 (q,4 H),
7.66 (d, 1 H), 7.82 (d, 1 H), 8.28 (s, 1 H).
02-(2-Nitro-4-carboxylatophenyl) 1-(N,N-
diethylamino)diazen-1-ium-1,2-diolate, 2:
4-Fluoro-3-nitrobenzoic acid was used in this
preparation. Purification of the product was carried out
on a Biotage Flash 40 system with a 4.0 x 15.0 cm KP-Sil
column. The system was eluted with 5:1
dichloromethane:ethyl acetate at 15 psi of air at a rate
of elution of 25 ml/min to give a 22% yield of product:
mp 115-6 'C; NMR 5 1.22 (t, 6 H), 3.33 -(q, 4 H), 7.06 (d,
1 H), 8.03 (dd, 1 H), 8.37 (m, 1 H).
02-(5-Nitropyrid-2-y1)1-(N,N-diethyl)diazen-1-ium-1,2-
diolate, 3: The product of reaction with 2-bromo-5-
nitropyridine was recrystallized from ether:ethanol to
give pure 3 in 62% yield: mp 77-8 .C; NMR .5 1.24 (t, 6
H), 3.53 (q, 4 H), 7.21 (dd, 1 H), 8.52 (dd, 1 H), 9.17
(dd, 1 H). Analysis C,H,N: Calculated for C9H13N504: C
42.35%, H 5.13%, N 27.44%, Found: C 42.46%, H 5.14%, N
27.52%.
02-(3,5-Dinitropyrid-2-y1)1-(N,N-diethyl)diazen-1-ium-1,2-
diolate, 4: Anylation was effected with 2-chloro-3,5-
dinitropyridine as described in the general procedure.
The crude product was recrystallized from ether:petroleum

CA 02814748 2013-05-02
57
ether to give 4 in 33% yield: mp 56-7 'C; NMR 5 1.28 (t 6
H), 3.57 (q,4 H), 8.81 (d, 1 H), 9.10 (d, 1 H).
02-(3-Nitropyrid-2-y1)1-(N,N-diethyl)diazen-1-ium-1,2-
diolate, 5: 2-Chloro-3-nitropyridine was used in this
reaction. The crude product was purified on a Flash 40
system using a 4.D x 7.0 cm KP-Sil column eluted with
100% dichloromethane to give a 52% yield of product as a
viscous oil: NMR 5 1.25 (t, 6 H), 3.55 (q, 4 H), 7.26
(m, 1 H), 8.48 (m, 2 H).
EXAMPLE 14
This Example illustrates the preparation of 02-vinyl
1-[(2-carboxylato)pyrrolidin-1-yljdiazen-1-ium-1,2-
diolate(V-PROLI/NO).
. To 3.56 g (9.2 mmol) of 02-(2-bromoethyl) 1-[(2-
carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate 2-
bromoethyl ester was added 10 ml of 10 N sodium hydroxide
solution.
The two-phase mixture was stirred at 25 *C,
whereupon the compound gradually dissolved in the aqueous
layer. After stirring overnight, the UV of the reaction
mixture exhibited an absorption maximum at 266 nm
(starting material absorbed at 252 rim), indicating the
formation of a vinyl group.
The solution was cooled to 0 *C and carefully
acidified to pH 4 by the slow addition of 10%
hydrochloric acid. Care must be taken to keep the
solution cold while acid is added. The acidic solution
was extracted with ethyl acetate, dried over sodium
sulfate and filtered through a layer of magnesium
sulfate. Evaporation of the solvent gave 1.4 g of an
oil. Purification was carried out on a Flash 40 System
(Biotage) using a 4.0 x 7.0 cm KP-Sil column and 2:1
ethyl acetate:cyclohexane as the eluant:ir (film) 3163,
2987, 1734, 1630, 1490 cm-1; NMR (CDC13) 8 2.06-2.3

CA 02814748 2013-05-02
58
(m,4H), 3.62 (m,2H), 4.47 (q,1H), 4.77 (ABq, 1H), 5.02
(ABq, 1H), 6.75 (q,1H); UV Amax (6) 266 nm(6.3 mM-1 cm-1);
MS,m/z(%)201 (M4,5), 176(10), 150(49), 145(27), 114(9),
99(45), 70(99.9), 69(57), 68(45).
EXAMPLE 15
This Example illustrates the regeneration of NO from
02-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-l-ium-
1,2-diolate in the presence, but not the absence, of
glutathione.
A solution containing 1 mM glutathione (GSH) in 10
mM phosphate buffer was degassed by purging with argon
for 10 min, whereupon a 3 ml aliquot was mixed with 3 1
of a dioxane solution that was 2 mM in 02-(2,4-
dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-
diolate. NO release was monitored by chemiluminescence
while the mixture was held at 37 C. After a brief lag
time, peak nitric oxide generation was observed at
approximately 15 minutes after the reaction was initiated
and continued at readily detectable levels for
approximately 100 minutes. Total NO generation during
the first 112 min was approximately 9 nmol. Assuming
that 2 nmol of NO is generated per mol of 02-(2,4-
dinitrophenyl) 1-(N,N-diethylamino)diazen-l-ium-1,2-
diolate, this 9 nmol corresponds to roughly 75% of the
theoretical yield.
When the reaction was repeated as above but with
exclusion of the GSH, no NO generation was observed. The
nucleophilic glutathione reacted with the 02-(2,4-
dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-
diolate to produce NO according to the equation shown
below.

ak 02814748 2013-05-02
59
0
N
NO2 O2
Or0\N,N
NEt2 + GSH
02N
01 SG
2N 0
0
Et 2 N
pH7.4
Et2INH + 2N0
This example is illustrative of the ability of some
of the 02-aryl diazeniumdiolate compounds of the present
invention to undergo nucleophilic substitution by
nucleophilic side-chains of amino acids such as cysteine,
which are often found in the active sites of enzymes.
The result of such nucleophilic substitution is the
generation of an aryl derivative of the displacing amino
acid residue and a diazeniumdiolate capable of producing
NO, through a predictable, first-order reaction.
02-(2,4-Dinitrophenyl) 1-(N,N-diethylamino)diazen-l-
ium-1,2-diolate and glutathione were also assayed in the
presence and absence of glutathione S-transferase.
Assays were conducted in a thermostated cell compartment
at 25 'C, using 0.1 M phosphate buffer at pH 7.4, with a
final volume of 3 ml. The concentration of the enzyme
was 0.7 g/ml, whereas that of glutathione was 1.4 mM.
The concentration of diazeniumdiolate was varied from 50-
100 M. Using the integrated form of the Henri-
Michaelis-Menten equation, K. was found to be 46.3 M and
Võ,,,, was found to be 0.89 M min-1.
EXAMPLE 16
This Example illustrates a route of synthesis which
is useful in the production of diazeniumdiolated
nucleotides, nucleosides, and nucleic acids and further

ak 02814748 2013-05-02
illustrates a route to synthesis of 02-aryl
diazeniumdiolates, which comprises converting an amino
group to a diazonium group, followed by reaction of the
diazonium group with a diazeniumdiolate.
5 2'-Deoxycytidine is reacted with nitric oxide in the
presence of a suitable 1-electron oxidant which results
in the conversiez .ef the amino group of the cytidine into
a diazonium group while reducing the oxidant and
producing hydroxide ion. The resulting diazotized (i.e.,
10 diazonium derivatized) pyrimidine is then reacted with 1-
(N,N-diethylamino)diazen-l-ium-1,2-diolate ion, as
described in the previous examples, to generate a
diazeniumdiolated 2'-deoxyuridine derivative. This
diazeniumdiolated 2'-deoxyuridine derivative can be
15 reacted with strong nucleophiles (e.g., hydroxide ions).
This will result in the regeneration of 1-(N,N-
diethylamino)diazen-l-ium-1,2-diolate ion plus 2'-
deoxyuridine. This regenerated 1-(N,N-
diethylamino)diazen-l-ium-1,2-diolate ion will generate
20 NO in a predictable, first-order reaction. This Example
demonstrates a basis for a mechanism that is suitable for
targeting nitric oxide to a particular site of a
mammalian body, so that the specificity of NO action can
be increased.
EXAMPLE 17
This example demonstrates the ability of an 02-aryl
diazeniumdiolate to inactivate a zinc finger protein by
zinc ejection.
Samples of recombinant nucleocapsid protein p7
(p7NC) from HIV-1 (L.O. Arthur, AIDS Vaccine Program,
NCI-FCRDC, Frederick, MD) were prepared at g/ml in 10 mM
sodium phosphate buffer (pH = 7.0) and treated with 25
mol of an 02-aryl diazeniumdiolate in a total volume of
1.0 ml. At various time intervals, as shown in Figure 1,
which is a graph of Trp37 fluorescence (RFU) versus time
(min), the samples were diluted 1/10 in 10 mM sodium

ak 02814748 2013-05-02
61
phosphate buffer (pH = 7.0) to prevent introduction of
any artifactual quenching effects and the fluorescence
intensity of the tryptophan residue (Trp37) in the C-
terminal zinc finger of p7NC in each sample was
determined as previously described (Rice et al., Int.
Antiviral News 3: 87-89 (1995)). The excitation and
emission wavelengths utilized with a Shimadzu RF5000
spectrofluorimeter were 280 and 351 nm, respectively.
The results are shown in Figure 1, in which 0 represents
the negative control, i.e., no drug, CI represents the
positive control, i.e., 642151 (see Rice et al. (1997),
supra), = represents the compound of Example 1 (LK1), =
represents the compound of Example 8 (LK2), ' represents
the compound of Example 5 (LK3), = represents the
compound of Example 10 (LK4), and x represents the
compound of Example 11 (LK5). The results indicate that
an 02-aryl diazeniumdiolate can eject zinc from a zinc
finger protein.
EXAMPLE 18
This example demonstrates the anti-HIV activity of
02-aryl diazeniumdiolates.
The tumor cell line of T4 lymphocytes designated
CEM-SS was grown in a synthetic medium with fetal bovine
serum (Rice et al., Advances in Pharmacol. 33: 389-438
(1995)). 02-aryl diazeniumdiolates were administered to
HIV-1-infected and uninfected CEM-SS cells at
concentrations ranging from 10-L5 to 10-7.0 M in accordance
with the XTT-based cell viability assay of the National
Cancer Institute (see, e.g., Rice et al. (1995), supra).
After exposure of CEM-SS cells to the compounds, the
percentage of T-cell viability was assessed. The
viability of HIV-1-infected CEM-SS cells, which were
contacted with a subtoxic concentration of any one of the
above-described 02-aryl diazeniumdiolates, was
substantially increased in comparison to untreated cells.

CA 02814748 2013-05-02
62
Compounds 1-3 from Example 13 were especially effective.
EXAMPLE 19
This example describes the preparation of disodium
1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-
diolate.
A solution of 10 g (0.087 mol) of L-proline in 39 ml
(0.18 mol) of 2596'sodium methoxide in methanol, 20 ml of
methanol and 40 ml of ether was degassed and exposed to
40 psi of nitric oxide for 20 hr. The pressure was
released and the solid residue was collected by
filtration, washed with ether and dried under vacuum to
give 17 g of a white solid: mp 250 00 (dec.); UV (0.01 N
NaOH) kmax (8) 252 rim (8.4 mM-1 cm-1); NMR (D20) 8 1.71 (m,
1H), 1.91 (m, 2H), 2.27 (m, 1H), 3.27-3.43 (m, 2H), 4.04
(m, 1H) (a methanol singlet at 3.34 is also observed); "C
NMR, 24.45 ppm, 30.97, 48.73 (methanol), 54.95, 67.70,
182.75.
Anal. C,H,N: Calculated for C5H7N304Na2-CH3OH, C
28.69%, H 4.41%, N 16.73%, Na 18.30%; Found C 28.65%, H
3.99%, N 16.74%, Na 18.04%.
EXAMPLE 20
This example describes the preparation of 02-methyl
1-[(2-carboxylato)pyrrolidin-1-yl)diazen-1-ium-1,2-
diolate methyl ester.
Disodium 1-[(2-carboxylato)pyrrolidin-l-ylldiazen-1-
ium-1,2-diolate (methanol solvate, FW 251; 6.8 g; 0.027
mol) was placed in a 300 ml 3-neck flask and cooled to
-20 C. Cold methanol (-20 00; 200 ml) was added to the
solid while stirring to give a homogeneous solution,
which was cooled further to -35 C. A solution of 9.5 ml
(0.1 mol) of dimethylsulfate in 25 ml of ether was added
dropwise over a 15 min period. The reaction mixture was
then allowed to warm to room temperature gradually and
stirred for an additional 4 hr. The progress of the
reaction was monitored on silica gel TLC using 10:1

CA 02814748 2013-05-02
63
dichloromethane:ethyl acetate as the eluant. The
reaction mixture was filtered, the methanol was removed
on a rotary evaporator, and the residue was extracted
with dichloromethane. The solution was washed with
aqueous sodium bicarbonate, dried over sodium sulfate and
filtered through a layer of magnesium sulfate.
Evaporation of the solvent gave an oil, which
crystallized on standing. Recrystallization from
ether:petroleum ether gave 945 mg (18%) of an
analytically pure sample: mp 62-63 C; UV (0.01 N NaOH),
?..max (E) 252 nm (6.79 mM-1 cm-1); NMR ö 2.05 (m, 3H), 2.30
(m, 1H) , 3.65 (m, IH), 3.75 (s, 3H), 3.83 (m, 1H), 3.96
(s, 3H), 4.55 (m, 1H); MS m/z (%) 203 (W, 6), 188 (20),
58 (35), 120 (22), 99 (100), 95 (34)1 69 (36), 59 (24);
exact mass calculated for C'H13N304 (M+) 203.0906, found
(W) 203.0906.
Anal. C,H,N: Calculated for C7H13N304, C 41.38%, H
6.45%, N 20.68%: Found C 41.48%, H 6.43%, N 20.59%.
EXAMPLE 21
This example describes the preparation of 02-(N,N-
dimethylsulfamoyl) 1-[(2-carboxylato)pyrrolidin-1-
yl]diazen-1-ium-1,2-diolate.
A solution of 1.08 ml (0.01 mol) of N,N-dimethyl-
sulfamoyl chloride in 5 ml of tetrahydrofuran was added
dropwise to a cold (0 C) solution of 1.57 g (0.0062' mol)
of disodium
1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-
diolate in 25 ml of 0.1 N NaOH in saline solution. The
reaction mixture was allowed to warm up to room
temperature and stirred overnight. The aqueous layer was
extracted with dichloromethane and the organic layer was
dried over anhydrous sodium sulfate. The aqueous layer
showed no significant UV absorption after extraction and,
thus, indicated that the extraction products were devoid
of diazeniumdiolate. The organic layer was filtered
through a layer of magnesium sulfate and the solvent was

ak 02814748 2013-05-02
64
removed on a rotary evaporator to give 989 mg of a pale
yellow oil, which was chromatographed on silica gel using
5:1 dichloromethane:ethyl acetate as the eluant. The
fractions containing the desired product were combined
and concentrated under vacuum to give a solid, which was
recrystallized from ether-petroleum ether: mp 97-98 C; UV
(0.01 N NaOH) (e) 266 nm (8.05 mM-1 cm-1); NMR 5 2.16
(m, 3H), 2.40 (m, 1H), 3.01 (s, 6H), 3.83 (m, 1H), 3.94
(m, 1H), 4.69 (q, 1H), 6.80 (b, 1H).
Anal. C,H,N,S: Calculated for C7H14N4S06, C 29.79%, H
5.00%, N 19.85%, S 11.36%; Found C 29.93%, H 5.09%, N
19.76%, S 11.27%.
= EXAMPLE 22
This example describes the preparation of 02-
methoxymethyl 1-((2-carboxylato)pyrrolidin-1-yl)diazen-1-
ium-1,2-diolate methoxymethyl ester.
A slurry of 485 mg (1.93 mmol) of disodium 1-((2-
carboxylato)pyrrolidin-1-yl)diazen-1-ium-1,2-diolate in
20 ml of anhydrous tetrahydrofuran was cooled to 0 C
under a nitrogen atmosphere. Triethylamine (0.5 ml) was
added to the cold solution followed by the slow addition
of 360 mg (4.45 mmol) of chloromethylmethyl ether and a
subsequent dropwise addition of 0.5 ml of methanol. The
solution was then stirred in the cold for 1.5 hr. The
reaction mixture was allowed to warm up to room
temperature and stirred under nitrogen for an additional
1.5 hr. The reaction was quenched with crushed ice,
whereupon the solvent was removed on a rotary evaporator
and the residue was extracted with dichloromethane. The
organic phase was washed with water, dried over sodium
sulfate, filtered through magnesium sulfate and
evaporated in vacuo to give 330 mg of a yellow oil, which
was purified on a silica gel column with 5:1
dichloromethane:ethyl acetate as the eluant: UV (H20) kmax
(e) 250 rim (8.58 mM-1 cm-1); NMR 8 2.09 (m, 3H), 2.35 (m,

CA 02814748 2013-05-02
IH), 3.48 (s, H), 3.71 (m, 2H), 3.90 (m, 1H), 4.61 (dd,
1H), 5.17 (ab q, 2H), 5.31 (ab q, 2H).
Anal. C,H,N: Calculated for C9H17N306: C 41.06%, H
6.51%, N 15.96 %: Found C 40.87%, H 6.53%, N 15.76%.
5
EXAMPLE 23
This example describe&the preparation of 02-(2-
bromoethyl) 1-[(2-carboxylato)pyrrolidin-1-ylidiazen-1-
ium-1,2-diolate 2-bromoethyl ester.
10 A solution of 20 ml (0.28 mol) of bromoethanol in 50
ml of dichloromethane was cooled to 0 C and 11.25 ml
(0.28 mol) of sulfuryl chloride in 50 ml of
dichloromethane was added dropwise to the solution. The
resulting solution was kept at 4 C for 72 hr. The
15 solution was washed with cold 10% NaOH until the washings
tested distinctly basic. The organic layer was dried
over sodium sulfate, filtered through a layer of
magnesium sulfate and concentrated on a rotary
evaporator. The resulting crude product (2-
20 bromoethoxysulfonyl chloride, BrCH2CH2OSO2C1) was vacuum-
distilled to give 35 g (56%) of a colorless oil: bp 73-75
C at 1.5 mmHg; NMR 5 3.64 (t, 2H), 4.752 (t, 2H); MS
miz (%) 221 (Mt, 1), 143 (10), 129 (25), 106 (100), 93
(62). Analysis C,H,N,S,X: Calculated for C2H4S03C1Br:
25 C 10.75%, H 1.80%, S 14.35%, total halogen as Br 71.52%
and as Cl 31.72%; Found: C 10.82% H 1.80%, S 14.35%;
total halogen as Br 71.63% and as Cl 31.78%.
Disodium 1-((2-carboxylato)pyrrolidin-1-ylidiazen-1-
ium-1,2-diolate (4.86 g; 0.0194 mol) was placed in a 100
30 ml round-bottom flask, together with 2.2 g of anhydrous
sodium carbonate. The flask was immersed in a dry ice-
acetonitrile bath (at -40 C) and 50 ml of cold
(-20 C) ethanol was added.. Then the mixture was stirred
and allowed to stabilize at -40 C under an atmosphere of
35 nitrogen. To the cold slurry was added, via a syringe,
9.45 g (0.0422 mol) of 2-bromoethoxysulfonyl chloride
over a period of 10 min. After stirring for 2 hr, the

CA 02814748 2013-05-02
66
reaction mixture was allowed to warm to 15 C and stirred
for an additional 2 hr. The reaction mixture was poured
into 250 ml of ice-water and extracted with
dichloromethane. The organic layer was washed with
aqueous sodium bisulfite solution, dried over sodium
sulfate and filtered through a layer of magnesium'
sulfate, whereupon the solvent was removed on a rotary
evaporator. The crude product was chromatographed on a
silica gel column using 1:1 cyclohexane:ethyl acetate as
the eluant to give 2.7 g (36%) of a pale yellow oil: NMR
5 2.11 (m, 3H), 2.35 (m, IH), 3.55 (m, 4H), 3.68 (m, 1H),
3.86 (m, 1H), 4.46 (m, 4H), 4.59 (m, 1H); UV (H20) ?ax (E)
252 nm (6.6 mM-1
EXAMPLE 24
This example describes the preparation of 02-[S-
acetyl-(2-mercaptoethyl)] 1-[(2-carboxylato)pyrrolidin-1-
yl]diazen-l-ium-1,2-diolate [S-acetyl-(2-mercaptoethyl))
ester.
1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (1.03 g;
0.0068 mol) was added to a solution of 1.33 g (0.0034
mol) of 02-(2-bromoethyl) 1-[(2-carboxylato)pyrrolidin-1-
yl]diazen-1-ium-1,2-diolate 2-bromoethyl ester in 35 ml
of tetrahydrofuran and the resulting solution was stirred
at room temperature under nitrogen. Two equivalents of
thiolacetic acid (0.479 ml, 0.0068 mol) were added and
the mixture was stirred at room temperature for 2 hr.
The mixture was filtered and the solid residue was washed
with ether. The filtrate was evaporated to dryness under
reduced pressure and the residue was extracted with
methylene chloride. The organic solution was
subsequently washed with ice-cold 5 N HC1, sodium
bicarbonate solution and water. The solution was dried
over sodium sulfate, filtered through a layer of
magnesium sulfate and evaporated in vacuo to give 710 mg
of a yellow oil. Chromatography was carried out on a
silica gel column eluted with 1:1 cyclohexane:ethyl

CA 02814748 2013-05-02
67
acetate: UV (H20) kmax (E) 232 rim (7.0 mM-1 cm-1); NMR 5
2.09 (m, 3H), 2.36 (m, IH), 2.38 (s, 6H), 3.09 (m, 4H),
3.78 (m, 2H), 4.27 (m, 4H), 4.55 (m, IH).
EXAMPLE 25
This example describes the determination of the
halflife of the compound produced in Example 24 in the
absence and presence of porcine liver esterase at 25 C
and pH 7.4.
A 0.009 M ethanolic stock solution of 02-[S-acetyl-
(2-mercaptoethyl)] 1-[(2-carboxylato)pyrrolidin-l-
yl]diazen-1-ium-1,2-diolate [S-acetyl-(2-mercapto-ethyl)]
ester was prepared. The decay of this compound was
monitored at 25 C as 1.5 x 10-4 M solutions in a 4 ml
quartz cuvette containing 3 ml of phosphate buffer (pH
7.4) and 50 ml of stock solution. The decay of the 232
nm chromophore was monitored on the ultraviolet
spectrophotometer. The halflife was estimated as 3.2 hr.
A second set of experiments was carried out using
the above parameters to measure the decay after addition
of 5 ml of porcine liver esterase suspension. The half-
life for the esterase reaction was 8 min at 25 C.
EXAMPLE 26
This example describes the preparation of a nitric
oxide-releasing polymer blend of 02-[S-acetyl-(2-mercapto-
ethyl)] 1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-
1,2-diolate [S-acetyl-(2-mercaptoethyl)] ester.
A solution of 50 mg (0.132 mmol) of 02-[S-acetyl-(2-
mercaptoethyl)] 1-[(2-carboxylato)pyrrolidin-l-yl]diazen-
1-ium-1,2-diolate (S-acetyl-(2-mercaptoethyl)] ester in 1
ml of tetrahydrofuran was dissolved in a solution of 498
mg of polyurethane in 10 ml of tetrahydrofuran. The
homogeneous lacquer was concentrated under a stream of
dry nitrogen followed by further drying under high vacuum
to give a solid, which contained 0.091 mg (0.24 mmol) of
02-[S-acetyl-(2-mercaptoethyl)] 1-[(2-

CA 02814748 2013-05-02
68
carboxylato)pyrrolidin-1-ylldiazen-1-ium-1,2-diolate [S-
acetyl-(2-mercaptoethyl)] ester per mg of polymer
composite. Rates of NO release were measured as a
function of time after immersing a 32 mg aliquot of the
diazeniumdiolate in 2 ml of phosphate buffer, pH 7.4, at
37 C, with a chemiluminescence detector. A set of
experiments was carried out in plain buffer, while.
another set was done in the presence of porcine liver
esterase. A very small amount of NO was released in the
absence of enzyme over a 200 hr period, while a
significant rate of NO production was observed when the
enzyme was present in the buffer. This indicates that as
the diazeniumdiolate oozes out of the polymer composite,
it is hydrolyzed by the enzyme with further cleavage to
NO.
EXAMPLE 27
This example describes the introduction of the
nitric oxide-releasing 02-[S-acetyl-(2-mercaptoethyl)] 1-
((2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate
[S-acetyl-(2-mercaptoethyl)] ester into P-cyclodextrin.
P-Cyclodextrin (228 mg, 0.201 mmol) was mixed with 2
ml of water and heated to 65 C to give a homogeneous
solution. To the warm solution was added 76 mg (0.201
mmol) of 02-[S-acetyl-(2-mercaptoethyl)] 1-[(2-
carboxylato)pyrrolidin-l-yl]diazen-l-ium-1,2-diolate [S-
acetyl-(2-mercaptoethyl)] ester. Upon mixing, a white
precipitate formed. The mixture was allowed to cool to
room temperature and the product was collected by
filtration, washed with water, and dried under vacuum to
give 170 mg of product. An aqueous solution containing
33 mg of the 02-[S-acetyl-(2-mercaptoethyl)] 1-[(2-
carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (S-
acetyl-(2-mercaptoethyl)) ester: P-cyclodextrin mixture
exhibited an absorbance maximum at .232 nm and a molar
absorptivity (c) of 10.8 mMcm-1. Rates of NO release
were measured as a function of time after immersing a 13

CA 02814748 2013-05-02
69
mg aliquot of the encapsulated material in 4 ml of
phosphate buffer, pH 7.4, at 37 C, with a
chemiluminescence detector. A set of experiments was
carried out in plain buffer, while another set was done
in the presence of porcine liver esterase. A very small
amount of NO was released in the absence of enzyme over
a 400 hr period while a significant rate of NO
production was observed when the enzyme was present in
the buffer.
EXAMPLE 28
This example describes a general procedure for the
preparation of 02-glycosylated diazeniumdiolates.
2,3,4,6-Tetraacetyl-a-D-glucopyranosyl bromide
(acetobromoglucose) was prepared as described in Redemann
et al., Org. Syn. Coll. Vol. III: 11-14 (1955).
2,3,4,6-Tetraacetyl-a-D-mannopyranosyl bromide
(acetobromomannose) was prepared as described in Levene
et al., J. Biol. Chem. 90: 247-250 (1931). Then, a
slurry of 1 eq of a diazeniumdiolate in
dimethylsulfoxide (DMSO) (0.5 mmol solid/ 1 ml of DMSO)
was stirred with 0.03 eq of silver oxide at room
temperature under nitrogen. A 0.5 M solution of 1.2 eq
of acetobromomannose or acetobromoglucose in DMSO was
injected dropwise and the mixture was stirred for three
days. The resulting homogeneous solution was poured into
100 ml of ice-water and extracted with ether. The ether
layer was washed with water, dried over sodium sulfate
and treated with charcoal. The solution was filtered
through magnesium sulfate, concentrated on a rotary
evaporator, and dried under vacuum. The glucose
derivatives were purified by recrystallization, while the
glassy mannose adducts required column chromatography.
EXAMPLE 29
This example describes the preparation of sodium 1-
(N,N-diethylamino)diazen-1-ium-1,2-diolate (-DEA/NO").

CA 02814748 2013-05-02
A solution of 119 g (1.63 mol) of diethylamine in
100 ml of 1:1 ether:acetonitrile was placed in a 500 ml
Parr bottle. The solution was degassed, charged with 40
psi of nitric oxide, and allowed to stand at room
5 temperature overnight. The pressure was released and the
crystalline product was collected by filtration and dried
under nitrogen tc give 13 g of diethylammonium 1-(N,N-
diethylamino)diazen-l-ium-1,2-diolate. The salt was
treated with 10 ml of 10 M sodium hydroxide solution and
10 the resulting paste was treated with 200 ml of ether to
give the sodium salt. The sodium salt ("DEA/NO") was
collected by vacuum filtration, washed with ether, and
dried under vacuum to give 7.1 g of product: UV (in 0.01
= N NaOH) krnax (E) 250 (6.88 mM-1cm-1); NMR (020) 8 0.96 (t, 3
15 H), 2.94 (q, 2 H); in DMSO-d6 8 0.84 (t, 3 H) and 2.75 (q,
2 H).
EXAMPLE 30
This example describes the preparation of 02-
20 (2,3,4,6-tetra-0-acetyl-a-D-glucopyranosyl) 1-(N,N-
diethylamino)diazen-l-ium-1,2-diolate.
DEA/NO (2.98 g; 0.019 mol) in DMS0 was reacted with
acetobromoglucose (6.9 g; 0.017 mol) as described in the
general procedure of Example 28. The product was
25 recrystallized from petroleum ether to give 5.7 g (72%)
108 mg of a crystalline solid: mp 107-108 C; UV krua. (E)
228 nm (6.92 mM-lcm-1); NMR 8 1.11 (t, 6 H, J= 7.11), 2.02
(s, 3 H), 2.03 (s, 3 H), 2.04 (s, 3 H), 2.07 (s, 3 H),
3.21 (q, 4 H, J= 7.12), 3.81 (m, 1 H), 4.20 (m, 2 H),
30 5.14 (m, 1 H), 5.33 (m, 3 H). Anal. Calcd for C18H29N3011:
C, 46.65; H, 6.31; N, 9.07. Found: C, 46.73; H, 6.26; N,
9.01.
EXAMPLE 31
35 This example describes the deacylation of 02-
(2,3,4,6-tetra-0-acetyl-a-D-glucopyranosyl) 1-(N,N-
diethylamino)diazen-1-ium-1,2-diolate(from Example 30).

CA 02814748 2013-05-02
71
A solution of 253 mg (0.55 mmol) of the above
compound in 5 ml of methanol was stirred with 10 1 of
25% methanolic sodium methoxide. The progress of the
reaction was monitored by TLC using 5:1 CH2C12:ethyl
acetate. The reaction was complete within 1 h at 25 'C.
TM
Dowex-50W-H* resin (1 g) was added to the stirring
methanolic solutliOn. The mixture was filtered to remove
the resin, and the methanolic solution was evaporated
under vacuum to give 122 mg (75%) of 02-glucopyranosyl 1-
(N,N-diethylamino)diazen-1-ium-1,2-diolate: UV A.max (E) 226
rim (6.4 mM-1 cm-1); NMR (CDC13) 8 1.08 (t,6H), 3.23 (9,4H),
5.59 (m,4H), 3.88 (m,2H), 5.29 (in, 1H)
Surprisingly, the deacetylated product cleaved to
the 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO)
anion, then to NO, only extremely slowly at pH 3, despite
its acetal-like structure. Even more surprisingly, the
cleavage proceeded extremely rapidly at pH 13.
EXAMPLE 32
This example describes the preparation of sodium 1-
[(1-ethoxycarbonyl)piperazin-4-ylldiazen-l-ium-1,2-
diolate.
A solution of 20 g (0.126 mol) of carboethoxy
piperazine in 60 ml of methanol was placed in a Parr
bottle. The solution was treated with 27.4 ml (0.126
mol) of 25% sodium methoxide in methanol. The system was
evacuated, charged with 40 psi of nitric oxide and kept
at 25 C for 48 hr. The white crystalline product was
collected by filtration and washed with cold methanol as
well as with copious amounts of ether. The product was
dried under vacuum to give 14.5 g (48% yield) of sodium
1-1(1-ethoxycarbonyl)piperazin-4-yl]diazen-l-ium-1,2-
diolate: mp: 184-185 C; UV (0.01 N NaOH) Amax (6) 252nm
(10 mM-I cm-1); NMR (020) 5 1.25 (t, 3 H), 3.11 (m, 2 H),
3.68(m, 2 H), 2.15 (g, 2 H). Anal calcd. for C6H13N404Na:
C 35.00%, H 5.42%, N 23.33%, Na 9.58%. Found: C 34.87%,
H 5.53%, N 23.26%, Na 9.69%. The half-life of this

CA 02814748 2013-05-02
72
compound at pH 7 and 25 C was estimated as 5 min. This
measurement was based on the loss of the 252 nm
chromophore in the ultraviolet spectrum.
EXAMPLE 33
This example describes the preparation of 02-
(glucopyranos-2,-y1) 1-[(1-ethoxycarbonyl)piperazin-4-
yl]diazen-l-ium-1,2-diolate tetraacetate ester.
Acetobromoglucose (2.055 g; 0.005 mol) and 1.11 g
(0.00466 mol) of sodium 1-[(1-ethoxycarbonyl)piperazin-4-
yl]diazen-l-ium-1,2-diolate were reacted as described
above to give 624 mg (25%) of 02-(glucopyranos-2-y1) 1-
[(1-ethoxycarbonyl)piperazin-4-yl]diazen-l-ium-1,2-
diolate tetraacetate ester: UV -1õ,, (E) 228 nm (7.20 mM-1
cm-1); NMR 5 1.26 (t, 3 H), 2.02 (s, 3H), 2.03 (s, 3H),
2.04 (s, 3 H), 2.09 (s, 3 H), 3.46 (m, 4H), 3.68 (m, 4
H), 3.82 (m, 1 H), 4.17 (q, 2 H), 4.25 (m, 3 H), 5.27 (m,
3 H).
EXAMPLE 34
This example describes the preparation of 02-
(mannopyranos-2-y1) 1-[(1-ethoxycarbonyl)piperazin-4-
yl)]diazen-l-ium-1,2-diolate tetraacetate.
Acetobromomannose (10.2 g ; 0.025 mol) and 5.28 g
(0.022 mol) of sodium 1-[(1-ethoxycarbonyl)piperazin-4-
yl]diazen-1-ium-1,2-diolate were reacted as described
above to give 6.4 g (53 %) of a glass: UV Xma. () 238 nm
(7.5 mM-1 cm-1); NMR 6 1.29 (t, 3 H), 2.01 (s, 3H), 2.05
(s, 3H), 2.11 (s, 3 H), 2.17 (s, 3 H), 3.13 (m, 1 H),
3.50 (m, 4 H), 3.78 (m, 5 H), 4.19 (q, 2 H), 4.27(m, 3
H), 5.28 (m, 3 H), 5.42 (m, 1H).

ak 02814748 2013-05-02
73
EXAMPLE 35
This example describes the preparation of an 02-
glycosylated diazeniumdiolate directed to a mannose-fucose
receptor.
Bis¨[2-(N-ethoxycarbonylamino)ethyl]amine: A three-neck
flask equipped with two dropping funnels was immersed in
an ice-water bath_ Diethylenetriamine (10.7 g, 0.104
mol) was placed in the cold flask and dissolved in 100 ml
of 95% ethanol. To the cold solution was added 10 ml
(0.205 mol) of ethylchloroformate, dropwise. A solution
of 10.6 g (0.1 mol) of sodium carbonate in 100 ml of
distilled water was added simultaneously with 10 ml
(0.205 mol) of ethylchloroformate. The reaction mixture
was allowed to stir at room temperature overnight. The
ethanol was removed on a rotary evaporator and the
aqueous portion was extracted with dichloromethane. The
organic layer was washed with water, then extracted with
5% hydrochloric acid. The organic layer containing the
neutral products was separated and set aside. The
aqueous layer was washed with dichloromethane and made
basic with sodium hydroxide. The product was extracted
into dichloromethane, dried over sodium sulfate, filtered
through magnesium sulfate and evaporated to give 4 g of a
colorless oil: NMR
(CDC13) 5 1.25 (t, 6H), 2.78 (m, 4H),
3.36 (m, 4H), 4.14 (q, 4H), 5,13 (b, 2H).
Sodium 1-(bis-(2-(N-ethoxycarbonylamino)ethyl)amino]
diazen-1-ium-1,2-diolate: A solution of 2.6 g (0.011
mol) of bis-[2-(N-ethoxycarbonylamino)ethyl]amine in 20
ml of ether and 5 ml of methanol was placed in a 50 ml
Parr bottle, treated with 2.4 ml (0.011 mol) of 25%
methanolic sodium methoxide, degassed, cooled to -80 C
and charged with 50 psi of nitric oxide. A thick
precipitate was observed after 3 hr of stirring. The
mixture was exposed to NO for 24 hr, the pressure was
released, and the product was collected by filtration.
The solid was washed with ether and dried under vacuum to

CA 02814748 2013-05-02
74
give 1.26 g (35%) of the diazeniumdiolate: mp 170-2 C;
UV imax (E) 252 nm (7.6 mM-1 cm-1); NMR 5 1.24 (t, 6H), 3.19
(m, 8H), 4.11 (q, 4H).
02-(Mannos-2-y1) 1-(bis-(2-(N-ethoxycarbonylamino)ethyll
amino]diazen-1-ium-1,2-diolate tetraacetate: A partial
solution of 251,--mg (0.763 mmol) of sodium 1-(bis-(2-(N-
ethoxycarbonylamino)ethyl}amino]diazen-l-ium-1,2-diolate
in 2 ml of dimethylsulfoxide (DMSO) was cooled to 0 C
under nitrogen. To this was added 10 mg (0.06 mmol) of
silver acetate, followed by the slow addition of 1 ml of
a 0.82 M solution of acetobromomannose in
tetrahydrofuran. The reaction mixture was allowed to
stir at room temperature for 48 hr, poured over ice-
water, and extracted with ether. The ether solution was
dried over sodium sulfate, filtered through a layer of
magnesium sulfate, and evaporated under vacuum to give
307 mg of an oil: UV A,õ 240 nm.
02-(Mannos-2-y1)1-(bis(2-aminoethyl)amino]diazen-l-ium-
1,2-diolate]:
A solution of 145 mg (0.23 mmol) of 02-(mannos-2-y1) 1-
(bis-(2-(N-ethoxycarbonylamino)ethyllaminoldiazen-l-ium-
1,2-diolate tetraacetate in a mixture of 0.2 ml of 10 N
NaOH, 2 ml of ethanol and 2 ml of water was heated at
reflux for 15 hr. The solution was concentrated under
vacuum and the remaining aqueous solution was extracted
with dichloromethane. The aqueous solution was
evaporated to dryness under vacuum. The residue was
taken up in methanol, put through a 10 g, 60 cc prepacked
C-18 column, and eluted with methanol. The fractions
exhibiting an absorption maximum at 236 nm were combined
and evaporated to give 32 mg of a white powder: NMR
(CD30D) 5 2.74 (t, 4H), 3.02 (t, 4H), 3.74 (m, 4H), 4.2
(m, 3H); UV kfm,,,, 238 nm.

ak 02814748 2013-05-02
EXAMPLE 36
This example describes the preparation of a
combinatorial library using disodium 1-(2-
carboxylato)pyrrolidin-1-y1 diazen-l-ium-1,2-diolate
5 (PROLI/NO) as starting material.
The piperazine trityl resin 1, available from
Calbiochem-Novabiachem Int'l. (San Diego, CA), is treated
with sulfuryl chloride to form the chlorosulfonamide 2.
Reaction of this resin with PROLI/NO gives compound 3.
10 The free carboxylic acid can be activated to 4 by
reaction with dicyclohexyl carbodiimide (DCC) and N-
hydroxysuccinimide. Nucleophilic addition of R"XH (X=0,
N, S) to the resin-bound diazeniumdiolate provides a
potentially large library of compounds, 5, substituted at
15 the carboxylato portion of the molecule. Base hydrolysis
of 5 frees the anionic diazeniumdiolate 6 from the resin.
This library, 6, may now be reacted with electrophiles
RflX to form new sets of compound having structure 7.

CA 02814748 2013-05-02
76
/ \ RC
HN N____1_41-e SO2CI,
- CISO2N N___I___
2
1
0)c0 0
C))10H
N N
I I \ )2(
N207 N------N- SO2- N ____ 1
I I
_______________________ . 00
3
- OCC 0
0c)___ N
N- 3G
R -XH
1
Hydroxy- 0
N==N
I I
Succinimide7- -0
0 0-S0 N \s2
4
_
0
0
0
o R31X (
0-)L'=XR3
_______ >k`XR30
I N
N
, - /--. N - 0
N20, S02-N I-
I
N202
NA 101
6 7
5
While this invention has been described with
emphasis upon preferred embodiments, it will be obvious
to those of ordinary skill in the art that the

CA 02814748 2013-05-02
77
scope of the claims should not be limited by the preferred
embodiments set forth in the examples, but should be given
the broadest interpretation consistent with the description
as a whole.
=