Hydrolytic reactivity trends among potential prodrugs of the O 2-glycosylated diazeniumdiolate family. Targeting nitric oxide to macrophages for antileishmanial activity

Article abstract of DOI:10.1021/jm8000482

Glycosylated diazeniumdiolates of structure R₂NN(O)=NO-R′ (R′ = a saccharide residue) are potential prodrugs of the nitric oxide (NO)-releasing but acid-sensitive R₂NN(O)=NO- ion. Moreover, cleaving the acid-stable glycosides under alkaline conditions provides a convenient protecting group strategy for diazeniumdiolate ions. Here, we report comparative hydrolysis rate data for five representative glycosylated diazeniumdiolates at pH 14, 7.4, and 3.8-4.6 as background for further developing both the protecting group application and the ability to target NO pharmacologically to macrophages harboring intracellular pathogens. Confirming the potential in the latter application, adding R₂NN(O)=NO-GlCNAc (where R₂N = diethylamino or pyrrolidin-1-yl and GlcNAc = N-acetylglucosamin-1-yl) to cultures of infected mouse macrophages that were deficient in inducible NO synthase caused rapid death of the intracellular protozoan parasite Leishmania major with no host cell toxicity.

Full text of DOI:10.1021/jm8000482

J. Med. Chem. 2008, 51, 3961–3970
3961
Hydrolytic Reactivity Trends among Potential Prodrugs of the O2-Glycosylated
Diazeniumdiolate Family. Targeting Nitric Oxide to Macrophages for Antileishmanial Activity
Carlos A. Valdez,† Joseph E. Saavedra,‡ Brett M. Showalter,† Keith M. Davies,§ Thomas C. Wilde,† Michael L. Citro,‡
Joseph J. Barchi, Jr.,| Jeffrey R. Deschamps, Damon Parrish, Stefan El-Gayar,#,× Ulrike Schleicher,#,¶ Christian Bogdan,#,¶
and Larry K. Keefer*,†
Chemistry Section, Laboratory of ComparatiVe Carcinogenesis, National Cancer Institute at Frederick, Frederick, Maryland 21702, Basic
Research Program, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland 21702, Department of Chemistry, George
Mason UniVersity, Fairfax, Virginia 22030, Laboratory of Medicinal Chemistry, National Cancer Institute at Frederick, Frederick, Maryland
21702, Laboratory for the Structure of Matter, NaVal Research Laboratory, Washington, D.C. 20375, Mikrobiologisches Institut-Klinische
Mikrobiologie, Immunologie und Hygiene, UniVersita¨tsklinikum Erlangen, Wasserturmstrasse 3-5, D-91054 Erlangen, Germany, and Abteilung
Mikrobiologie und Hygiene, Institut fu¨r Medizinische Mikrobiologie und Hygiene, UniVersita¨tsklinikum Freiburg, D-79104 Freiburg, Germany
ReceiVed January 17, 2008
Glycosylated diazeniumdiolates of structure R2NN(O)dNO-R′ (R′ ) a saccharide residue) are potential
prodrugs of the nitric oxide (NO)-releasing but acid-sensitive R2NN(O)dNO- ion. Moreover, cleaving the
acid-stable glycosides under alkaline conditions provides a convenient protecting group strategy for
diazeniumdiolate ions. Here, we report comparative hydrolysis rate data for five representative glycosylated
diazeniumdiolates at pH 14, 7.4, and 3.8-4.6 as background for further developing both the protecting
group application and the ability to target NO pharmacologically to macrophages harboring intracellular
pathogens. Confirming the potential in the latter application, adding R2NN(O)dNO-GlcNAc (where R2N
) diethylamino or pyrrolidin-l-yl and GlcNAc ) N-acetylglucosamin-l-yl) to cultures of infected mouse
macrophages that were deficient in inducible NO synthase caused rapid death of the intracellular protozoan
parasite Leishmania major with no host cell toxicity.
Introduction
been induced to express inducible NO synthase (iNOS).6 As
prospective prodrugs of NO, we have derivatized a series of
monosaccharides reportedly recognized and transported by the
MR (including N-acetylglucosamine as well as mannose)7 by
substituting them at the anomeric position with diazeniumdiolate
ions 1 that in their free form spontaneously release molecular
NO at physiological pH levels. Glycosylating ions of structure
1 as in eq 1 has been proposed by Wu et al. as a route to
prodrugs capable of delivering bioactive NO for pharmacological
benefit.8 For this strategy to be successful in the present
connection, the glycosylated diazeniumdiolate must be hydro-
lytically stable enough in the systemic circulation to reach the
physiological site of need but sufficiently labile under conditions
unique to the target site (e.g., the acidic [pH 4-5] interior of a
macrophage’s phagolysosome) to free the aglycone for proton-
induced generation of NO, as depicted in eq 2.
Macrophages and the nitric oxide (NOa) they produce on
stimulation by appropriate cytokine combinations are key players
in a mammalian host’s defense against microbial pathogens1
and cancer cells,2–4 but they can fail in this role, allowing disease
to progress.
With the hypothesis that pharmacologic supplementation of
the immune cells’ NO output might restore their host-protective
effects, we are exploring the possibility that prodrugs exploiting
the mannose receptor (MR) of macrophages5 might be actively
imported and then selectively activated in the intracellular milieu
for NO release and consequent therapeutic benefit.
As a test system, we have chosen Leishmania major (L.
major), a protozoan parasite that is known to be susceptible to
NO’s toxic effects but that is able to survive and proliferate in
highly lytic phagolysosomes of macrophages that have not yet
* To whom correspondence should be addressed. Phone: 301-846-1467.
Fax: 301-846-5946. E-mail: keefer@ncifcrf.gov.
†
Laboratory of Comparative Carcinogenesis, National Cancer Institute
at Frederick.
‡
SAIC-Frederick.
George Mason University.
Laboratory of Medicinal Chemistry, National Cancer Institute at
§
|
Frederick.
Naval Research Laboratory.
Universita¨tsklinikum Erlangen.
Present address: Abteilung fu¨r Pa¨diatrische Ophthalmologie, Strabis-
#
×
mologie and Ophthalmogenetik, Universita¨tsklinikum Regensburg, Franz-
Josef-Strauss-Allee 11, D-93503 Regensburg, Germany.
Glycosylation as in eq 1 has also been introduced as a
convenient protecting group strategy for ions of structure 1.9
Here too, success in a given application depends on a knowledge
of the glycoside’s hydrolytic reactivity. Best results will be
obtained if the glycoside is adequately stable through all the
needed transformations at neutral or acidic pH but easily cleaved
at high pH to generate the base-stable 1 ion.
¶
Universita¨tsklinikum Freiburg. Present address: Universita¨tsklinikum
Erlangen.
a Abbreviations: NO, nitric oxide; L. major, Leishmania major; MR,
mannose receptor; PBS, phosphate buffered saline; iNOS, inducible nitric
oxide synthase; IFN, interferon; TNF, tumor necrosis factor; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GlcNAc, N-acetyl-
glucosamine; DMF, N,N-dimethylformamide; NAGase, ꢀ-N-acetylglu-
cosaminidase.
10.1021/jm8000482 CCC: $40.75
2008 American Chemical Society
Published on Web 06/06/2008

3962 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Valdez et al.
Scheme 1. Ionic Diazeniumdiolates Used as Starting Materials
for Preparations Described in This Work
Scheme 2. General Synthesis of Glycosylated Diazeniumdiolates
Described in This Work
Here we report the physicochemical characterization of a
small library of these O2-glycosylated diazeniumdiolates as
protected starting materials for future syntheses as well as for
pharmacological testing, focusing especially on the rates of their
hydrolytic cleavage at three different pH ranges. We then
provide preliminary data suggesting their potential utility for
delivering therapeutic levels of NO to intracellular pathogens
resident in infected macrophages.
anomeric carbon of glucose protected them under a variety of
conditions that would otherwise lead to their decomposition but
that brief exposure to concentrated aqueous alkali could free
the aglycone, regenerating the intact diazeniumdiolate ion under
basic conditions that prevented the ion’s acid-catalyzed dissocia-
tion.9 The reaction was first order in hydroxide ion with a rate
constant kOH of 5.3 × 10-3 M-1 s-1 for the prototypical glycoside
–
Results
3a. Indeed, 3a has proven to be the most sensitive to base-induced
hydrolysis of the five glycosides studied here. The half-lives,
determined by following the first order decrease of the diazeni-
umdiolate chromophore’s ultraviolet absorbance as a function of
time and summarized in Table 1, show that fucosyl derivative 7a
hydrolyzed half as fast as 3a in 1 M sodium hydroxide, with
GlcNAc analogue 9a being slower than 2-deoxy derivative 11a
by a similar factor under these conditions (half-life of 51 min versus
half-life of 23 min, respectively). Mannose derivative 5a was most
resistant of the five with a half-life of 298 min.
The results suggest that ꢀ-D-glucosyl is an advantageous
saccharide to use as a protecting group for diazeniumdiolate
ions. The glucose derivatives could generally be prepared with
ease (not always the case in our experience with the other
glycosides), and they hydrolyzed in base to regenerate the free
diazeniumdiolate ions more quickly than the others as well.
Differing Order of Hydrolytic Reactivity at pH 7.4 and
Below. In contrast to their relatively sluggish rates of cleavage
in 1 M base, 9a and 11a were the most easily hydrolyzed of
Synthesis. A variety of ions 1 (Scheme 1) were converted to
the corresponding peracetylated glycosides by reacting them
with the appropriate anomerically activated monosaccharide (i.e.,
glycosyl halide) (Scheme 2). Although initially silver acetate
was commonly added to reaction mixtures comprising peracety-
lated 1-halopyranoses and sodium salts 1, it was eventually
discovered that this was in most cases unnecessary. Following
installation of the diazeniumdiolate group at C-1, the resulting
peracetylated, O2-glycosylated products were treated with
catalytic amounts of sodium methoxide in methanol to cleave
the ester groupings, as detailed in eqs 3–7. The R stereochem-
istry of mannosyl derivative 4a was confirmed by X-ray
crystallography, as shown in Figure 1. The R vs ꢀ configurations
of all other compounds were shown by proton NMR to be as
assigned in eqs 3–7.
Rates of Base-Induced Deglycosylation: Dependence on
Identity of the Saccharide. It was previously shown that
attaching acid- and heat-sensitive diazeniumdiolate ions to the

Targeting NO to Macrophages
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3963
Figure 1. Structure of 4a as determined by X-ray diffraction analysis. Displacement ellipsoids are at the 40% probability level. Some H-atoms are
omitted for clarity.
Table 1. Half-Lives of Hydrolysis at 37 °C for Glycosides of Structure
Et2NN(O)dNOR in 1.0 M NaOH, in 0.1 M Phosphate at pH 7.4, or in
0.05 M Citrate at pH 3.8-4.6
half-life
pH 14
(min)
pH 7.4
(day)
pH 4.6
(day)
R
ꢀ-D-glucosyl (3a)
2.1
4.0
23
51
298
>10
>10
0.5
0.5
>10
>10a
>10a
1.2b
ꢀ-L-fucosyl (7a)
ꢀ-(2-deoxy)-D-glucosyl (11a)
ꢀ-(N-acetyl-D-glucosaminyl) (9a)
R-D-mannosyl (5a)
NO and thence to nitrite in 10-20% recovery during a 24 h
incubation. By contrast, mannose derivative 5a generated little
or no nitrite under the same conditions.
Surprisingly, hydrolysis of pyrrolidine derivative 9b in PBS
was more than an order of magnitude slower than that of 9a
(Figure 2). Differing only in the fact that the two CH3 groups
of 9a are joined to form the five-membered ring of 9b, we had
anticipated that they would be very similar in rate. However,
the half-lives of 9b in 0.1 M phosphate at pH 7.4 or in 0.05 M
citrate at pH 4.6 were both >10 days at 37 °C.
Hydrolysis of 0.25-1 mM 9b was considerably faster when
incubated in RPMI cell culture medium containing 2.5% fetal
calf serum instead of PBS, as shown in Figure 2. Importantly,
the rates of nitrite release from 9b were increased even further
when the serum-containing medium was used to culture mouse
macrophages deficient in inducible NO synthase (iNOS-/-). In
the experiments summarized in Figure 2, the cells increased
the nitrite yield 4-fold in medium initially containing 0.25 mM
9b. Similarly, the macrophages approximately doubled the rate
of nitrite accumulation in a parallel incubation of 0.25 mM 9a.
These serum- and macrophage-induced catalytic effects were
further explored in additional experiments with 9b. As shown
in Figure 3, the rate of nitrite formation in cell culture medium
proved quite dependent on serum concentration, with the
macrophage-induced acceleration being clearly evident in
medium containing 0%, 0.1%, or 1% fetal bovine serum but
not in the presence of 5% or, with some batches of serum, 2.5%.
Infecting the macrophages with L. major did not significantly
affect the rate of nitrite formation (data not shown).
0.7a
>10a
a pH 4.6. b pH 3.8.
Table 2. Half-Lives of Tetrahydropyranyl Derivative 12 at Various pH
Levels at 37 °C
pH
t1/2 (min)
14.0 (1.0 M NaOH)
2.5
2.7
3.5
2.9
3.5
3.1
3.2
13.0 (0.10 M NaOH)
12.1 (0.0125 M NaOH)
10.0 (0.05 M glycine/NaOH)
8.1 (0.10 M phosphate)
7.4 (0.10 M phosphate)
4.0 (0.05 M citrate)
the five 1a derivatives in pH 7.4 phosphate buffer, with half-
lives estimated as 0.5 days (Table 1). The other three glycosides
included in this study hydrolyzed so slowly that their rates could
not be monitored with confidence by following the changes in
their absorbance maxima with time.
Rates of hydrolysis at pH 3.8-4.6 were little different from
those at pH 7.4, with 9a and 11a displaying half-lives of 0.7
and 1.2 days and the other three showing values of >10 days
(Table 1). Interestingly, tetrahydropyranyl derivative 12, pro-
duced by reacting 1a with dihydropyran as in eq 8, hydrolyzed
within minutes at all pH values studied (Table 2).
The relatively high susceptibility of GlcNAc derivative 9a
to hydrolysis at pH 7.4 was confirmed by following the
accumulation of the nitric oxide auto-oxidation product, nitrite
ion, in both phosphate-buffered saline (PBS) and cell culture
medium containing 2.5% fetal calf serum. As shown in Figure
2, there was little difference between the PBS and culture
medium results, with each one engendering cleavage of 9a to
Catalysis of Hydrolysis by Glycosidases. The above results
suggested that the macrophages may harbor factors capable of
accelerating the conversion of glycosides 9a and especially 9b
to NO. As one possibility, Wu, et al. have reported on the rapid

3964 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Valdez et al.
Table 3. Rates and Extents of NO Generation on Hydrolysis of 9a and
9b under Catalysis by N-Acetylglucosaminidases from Both Jack Bean
(JB) and Human Placenta (HP)a
moles produced per mole of glycoside
-
-
compd enzyme
[NO]
[NO2
]
[NO + NO2
]
k(s-1
)
9a
9a
9b
9b
JB
HP
JB
1.26
1.73
1.72
1.69
0.05
0.24
0.12
0.36
1.31
1.97
1.84
2.05
6.2 × 10-4
4.1 × 10-4
3.1 × 10-4
3.6 × 10-4
HP
a All reactions were run at 37 °C in 0.1 M phosphate, pH 7.4, containing
0.05 mM diethylenetriaminepentaacetic acid. Jack bean (JB) enzyme was
present at 0.11 units/mL, while its human placenta (HP) counterpart was
added at 0.044 units/mL. Substrates were added at the following initial
concentrations: 9a, 0.10 mM; 9b, 0.16 mM; 5a, 0.09 mM. NO production
was followed by chemiluminescence detection until no more was evolved.
Nitrite formed during hydrolysis was measured in the remaining solution
by the colorimetric Griess assay. Rate constants were cleanly first order, as
determined by ultraviolet spectrophotometry; wavelengths followed were
227 nm for 9a and 5a but 256 nm for 9b. Absorbance changes in the 5a
incubations were negligible under the conditions employed. The enzymes
used were confirmed to be fully active by means of control incubations
with the manufacturer’s recommended substrate, 1-(p-nitrophenyl)-2-deoxy-
2-(N-acetyl)-ꢀ-D-glucosamine.
Figure 2. Nitrite yields as a reflection of NO generation on incubating
5a, 9a, and 9b in PBS, in cell culture medium containing 2.5% fetal
calf serum, or in the serum-containing medium while being used to
culture macrophages for 24 h at 37 °C. When the glycoside solutions
were assayed immediately after they were dissolved, no nitrite was
detected using the Griess test. Data shown are mean values of triplicates.
Standard deviations were <5%. Shown is one of two comparable
experiments.
Figure 4. Chemiluminescence trace showing the time course of NO
release from 0.39 µM 9a at 37 °C in 50 mM citrate buffer (pH 5.0)
with 1.6 µg/mL of jack bean ꢀ-N-acetylglucosaminidase.
ꢀ-D-glucosamine. The time course of NO release in a typical
hydrolysis is illustrated in Figure 4. Negligible NO generation
was seen in a parallel incubation of mannose derivative 5a,
which was not expected to be hydrolyzed by an N-acetylglu-
cosaminidase.
Antileishmanial Activity of 9a and 9b. We next tested the
hypothesis that the infected macrophages’ ability to accelerate
the hydrolysis of 9a and 9b to NO might help them defeat their
intracellular L. major amastigotes. Previous studies have shown
that intracellular L. major amastigotes are killed by cytokine-
activated macrophages in an iNOS-dependent manner.10–13 For
the analysis of the NO donors we utilized macrophages from
iNOS-deficient mice, which allowed us to exclude any contribu-
tion by iNOS-derived endogenous NO. As shown in Figure 5,
unstimulated macrophages allowed replication of the intracel-
lular parasites during the 72 h observation period. Because of
the absence of iNOS, stimulation of the macrophages with
interferon (IFN)-γ plus tumor necrosis factor (TNF) had only a
very weak antileishmanial effect, as seen before.14 In contrast,
addition of 9a or 9b led to a highly significant decrease in the
number of intracellular parasites, with 9a being more potent
than 9b. Non-NO-releasing N-acetyl-ꢀ-D-glucosaminyl deriva-
tive 13 did not have any effect on the number of intracellular
Leishmania (data not shown).
Figure 3. Generation of NO after parallel incubations of 9b at
concentrations of (A) 250 and (B) 125 µM under different conditions.
Substance 9b was incubated in phosphate-buffered saline (PBS)
alone; RPMI cell culture medium without (w/o) or with addition of
0.1%, 1%, 2.5%, and 5% fetal calf serum (FCS) in the absence or
presence of peritoneal exudate macrophages from iNOS-deficient
mice (PE-MF); cultured in serum-free or serum-containing (0.1%,
1%, 2.5%, or 5% FCS) RPMI. Supernatants were harvested after
24 h, and cumulative NO release was estimated by determining its
auto-oxidation product, nitrite ion, using the Griess assay. Data
shown are mean values of triplicates. Standard deviations were <5%.
The results confirm that the macrophages are capable of metabolizing
9b to NO and that the accelerating effect of serum on the hydrolysis
of 9b is concentration-dependent. Shown are results from one of
two identically designed experiments, which yielded compar-
able results.
enzymatic cleavage of 5b and certain other diazeniumdiolated
carbohydrates by the corresponding glycosidases.8 To determine
whether N-acetylglucosamine derivatives 9a and 9b are similarly
susceptible, we incubated them with N-acetylglucosaminidases
isolated from jack bean and from human placenta. As sum-
marized in Table 3, both enzymes hydrolyzed both substrates
with rates and efficiencies that were comparable to those of the
reference compound, 1-(p-nitrophenyl)-2-deoxy-2-(N-acetyl)-
Importantly, in the concentrations tested, neither of the NO
donors caused any toxicity to the macrophage host cells as
determined microscopically by trypan blue staining of dead cells
or by measuring the mitochondrial activity of the total cell
monolayer using the MTT assay (see Experimental Section).

Targeting NO to Macrophages
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3965
to the screening results; accordingly, stimulation of the cells
with cytokines, a procedure that in normal cells resoundingly
activates wild type macrophages to produce iNOS-derived
antimicrobial NO in abundance, had little effect on the parasites.
Recognizing that the MR serves as a receptor not only for
mannose and fucose but also for certain other saccharides such
as N-acetylglucosamine, we added glycosides 5a, 5b, 7a, 7c,
9a, and 9b at concentrations of 125 or 250 µM to culture
medium containing the infected macrophages. Neither of the
fucose derivatives had any perceivable effect on the parasite
burden of these cells, and only one of the mannosides (5a)
showed any activity, that being of borderline significance.
However, both of the N-acetylglucosamine (GlcNAc) derivatives
(9) were decidedly antileishmanial (Figure 5).
Figure 5. Effect of two glycosylated diazeniumdiolates on the
intracellular proliferation of L. major in primary mouse macrophages.
Peritoneal exudate macrophages from iNOS-deficient mice were
cultured in medium alone or stimulated with IFN-γ plus TNF for 16 h
prior to a 4 h infection period with L. major amastigotes (parasite/
macrophage ratio of 5:1). Thereafter, the macrophages remained
unstimulated (NS), were stimulated with IFN-γ plus TNF, or were
exposed to glycosylated diazeniumdiolates (9a or 9b) at the indicated
concentrations. The mean ((SD) number of intracellular parasites per
100 macrophages (immediately after infection [0 h], after 48 and 72 h),
and the amount of nitrite in the culture medium (accumulated between
24 and 48 h and between 48 and 72 h) are given: (/) p < 0.01, (//) p
< 0.001, significantly different compared to unstimulated macrophages.
The numbers above the bars represent the mean value of nitrite (units
of micromolar) detected in the culture supernatants after the final
accumulation period (48-72 h of stimulation). Nitrite ion was devoid
of antileishmanial activity under these conditions.
The data are consistent with the interpretation that the cultured
macrophages were able to effect cleavage of the glycosidic
bonds of prodrugs 9a and 9b, with the resulting spontaneous
release of NO significantly reducing the parasite burden with
no observable toxicity to the host cells.
Significance. The results suggest that N-acetylglucosamine
derivatives 9a and 9b may be promising lead compounds for
the development of antileishmanial agents. They were designed
with the hypothesis that they might enter the infected macroph-
ages via the cells’ mannose receptor and be cleaved therein to
concentrate intracellular NO for antileishmanial activity. While
none of the analogous fucosides and mannosides was more than
marginally active, if at all, the results for 9a and 9b were
consistent with that hypothesis. The findings for 9b were
particularly illuminating. Its hydrolysis to generate NO in cell
culture medium was greatly accelerated in the presence of
macrophages, as reflected in an increased rate of accumulation
of nitrite (the auto-oxidation product of NO) that correlated
suggestively with antileishmanial activity.
Thus, these glycosylated diazeniumdiolates exert potent anti-
parasitic effects against intracellular L. major at concentrations
that do not cause overt damage to the host cells.
Very importantly, the iNOS-knockout macrophages harboring
the affected parasites were well spread and quite healthy, more
so than their wild type counterparts that were stimulated to kill
resident L. major by up-regulation of iNOS using interferon-
γ/tumor necrosis factor treatment. We conclude that NO donors
9a and 9b exert their antileishmanial activity by cell-mediated
activation to one or more metabolites (presumably including
NO) that directly harm the parasites and not to any nonspecific
toxicity toward the host cells.
Discussion
Parasites of the genus Leishmania are among a variety of
pathogens that thrive intracellularly in the macrophage despite
their demonstrated sensitivity to the cytostatic and microbicidal
action of NO, a major component of this cell type’s normal
immunological armamentarium.15 It has been suggested that the
parasites’ ability to survive in the normally hostile interior of
phagolysosomes of activated macrophages is due at least in part
to impairment of endogenous NO production by the phagocy-
tized parasites.16–18
With the hypothesis that pharmacological supplementation
of the macrophages’ NO reserves might restore the immune
system’s capacity to contain and eliminate these pathogens, we
are exploring ways of delivering exogenously derived NO to
the intracellular compartments of infected macrophages. The
strategy described here is aimed at exploiting the so-called
mannose receptor [MR, formerly also termed mannose/fucose
receptor (MFR)] that decorates the surfaces of macrophages and
certain other cells of the immune system. If these receptors can
be harnessed to import glycosylated, diazeniumdiolated NO
prodrugs into the cell, one might rely on intracellular glycosi-
dases and/or the acidic environment in the phagolysosome (pH
4-5.5) to free the sequestered NO and kill the pathogens.
The infectious agents in our test system were L. major
amastigotes. As host cells, we used primary peritoneal mac-
rophages from iNOS-knockout mice to minimize the possibility
that endogenously produced NO might contribute significantly
Future work with these lead compounds will focus on the
additional hypothesis that divalent or polyvalent analogues might
have exponentially increased potency. We are also examining
the mechanistic origins of the often surprising differences in
hydrolysis rate among the test agents studied here. Preliminary
indications suggest that the >20-fold increase in hydrolysis rate
for acyclic 9a in pH 7.4 phosphate buffer relative to its more
constrained heterocyclic analogue 9b may be a consequence of
electronic interaction between the pyrrolidino nitrogen and the
-N(O)dNO- group to which it is attached. An X-ray crystal-
lographic study is currently underway aimed at further exploring
this hypothesis.
The chemical data reported here may also aid in the synthesis
of future prodrug candidates as well as in the design of drugs
with NO release profiles needed for specific pharmacological
effects. In particular, the results support the utility of glucosy-
lation as a protecting group strategy for diazeniumdiolate ions
1 to provide derivatives that remain stable through synthetic
transformations at neutral-to-acidic pH and/or at elevated

3966 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Valdez et al.
3.06-3.26 (m, 4 H), 3.41-3.49 (m, 5 H), 3.63-3.69 (m, 1 H),
4.59 (t, 1 H, J ) 5.9 Hz), 4.85 (d, 1 H, J ) 7.8 H), 5.00 (d, 1 H,
temperatures but that can be efficiently removed at high pH
levels, conditions that stabilize the newly formed diazenium-
diolate ions.
J ) 5.2 Hz), 5.09 (d, 1 H, J ) 4.3), 5.36 (d, 1 H, J ) 5.0 Hz); 13
C
NMR δ 22.32, 50.59, 60.59, 69.34, 71.57, 76.60, 77.33, 103.17.
Anal. (C10H19N3O7 ·H2O) C, H, N.
O2-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl) 1-(N,N-Dim-
ethylamino)diazen-1-ium-1,2-diolate (2c) (CV-30). To 3.81 g
(0.03 mol) of 1c were added catalytic amounts of silver acetate
and 12 g (0.029 mol) of acetobromoglucose as described in the
general procedure. A foam (1.5 g, 12%) was isolated: UV
(methanol) λmax (ε) 228 nm (8.3 mM-1 cm-1); 1H NMR (300 MHz,
CDCl3) δ 2.02 (s, 3 H), 2.037 (s, 3 H), 2.04 (s, 3 H), 2.09 (s, 3 H),
3.08 (s, 6 H), 3.82-3.88 (m, 1 H), 4.08-4.19 (m, 1 H), 4.23-4.31
(m, 1 H), 5.12-5.37 (m, 1 H); 13C NMR δ 20.34, 20.48, 42.09,
61.51, 67.67, 69.00, 72.35, 72.62, 100.12, 168.82, 169.16, 169.93,
170.41. Anal. (C16H25N3O11) C, H, N.
Experimental Section
NO was purchased from Matheson Gas Products (Montgomer-
yville, PA). 2,3,4,6-Tetraacetyl-1-R-glucopyranosyl bromide (ac-
etobromoglucose) was purchased from Fluka Chemical Corp.
(Milwaukee, WI). Sodium salts 1a,19 1b,20 1c,21 1e,22 and 1f22
were prepared as previously described, as were 2a,9 3a,9 R-D-
acetobromomannose,23 R-D-acetochloro-(N-acetylglucosamine),24
and R-L-acetobromofucose.25 Unless otherwise indicated, all other
chemicals were purchased from Aldrich Chemical Co. (Milwaukee,
WI). Nuclear magnetic resonance (NMR) spectra were collected
with a 400 MHz NMR spectrometer. Chemical shifts (δ) are
reported in parts per million downfield from tetramethylsilane or
3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt. Elemental
analyses were performed by Atlantic Microlab, Inc. (Norcross, GA)
or Midwest Microlab, LLC (Indianapolis, IN).
Sodium 1-(Piperidin-1-y1)diazen-1-ium-1,2-diolate (1d) (JS-
11-105). A solution of 100 mL of freshly distilled piperidine in
100 mL of ether was placed in a Parr bottle, purged with nitrogen,
and cooled to -80 °C. The cold solution was charged with 60 psi
of nitric oxide. A solid mass formed within 30 min, but the mixture
was allowed to stand for an additional 3 h. The pressure was
released; the cold material was collected by filtration and washed
with ether. The piperidinium salt was treated with 20 mL of 10 M
NaOH and 10 mL of methanol for cation exchange, and the resulting
pasty mass was flooded with 500 mL of ether. The diazeniumdiolate
1d was collected by filtration and dried under vacuum to give 18 g
of product: UV (0.01 M NaOD) λmax (ε) 249 nm (8.15 mM-1
cm-1); 1H NMR (10 mM NaOD) δ 1.46-1.52 (m, 2 H), 1.72-1.78
(m, 4 H), 3.06 (t, 4 H, J ) 5.6 Hz); 13C NMR δ 25.31, 27.39,
55.80.
O2-(ꢀ-D-Glucopyranosyl) 1-(N,N-Dimethylamino)diazen-1-
ium-1,2-diolate (3c) (CV-36). Compound 2c was deacylated to
give a 66% yield of 2a as a white foam: UV (methanol) λmax (ε)
224 nm (4.3 mM-1 cm-1); 1H NMR (300 MHz, DMSO-d6) δ 2.96
(s, 6 H), 3.09-3.30 (m, 4 H), 3.39-3.48 (m, 2 H), 3.67 (d, 1 H, J
) 11.2 Hz,), 4.61 (br, 1 H), 4.90 (d, 1 H, J ) 7.8 Hz), 5.03-5.11
(br, 2 H), 5.40 (br, 1 H); 13C NMR δ 42.19, 60.63, 69.38, 71.62,
76.62, 77.45, 103.42. Anal. (C8H17N3O7 ·H2O) C, H, N.
O2-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl) 1-(Piperidin-
1-yl)diazen-1-ium-1,2-diolate (2d) (CV-16). To 5.0 g (0.03 mol)
of 1d were added catalytic amounts of silver acetate and 12 g (0.029
mol) of acetobromoglucose as described in the general procedure.
A white solid (0.512 g, ∼4%) was isolated: mp 112-113 °C; UV
(methanol) λmax (ε) 230 nm (6.9 mM-1 cm-1); 1H NMR (CDCl3)
δ 1.48-1.56 (m, 2 H), 1.72-1.82 (m, 4 H), 2.02 (s, 3 H), 2.03 (s,
3 H), 2.04 (s, 3 H), 2.09 (s, 3 H), 3.39-3.52 (m, 4 H), 3.78-3.84
(m, 1 H), 4.14-4.19 (m, 1 H), 4.24-4.32 (m, 1 H), 5.12-5.38
(m, 4 H); 13C NMR δ 20.53, 20.69, 23.34, 24.60, 52.24, 61.68,
67.82, 69.21, 72.58, 72.82, 100.34, 168.99, 169.30, 170.15, 170.60.
Anal. (C19H29N3O11) C, H, N.
General Procedure for the Preparation of O2-Glycosylated
Diazeniumdiolates. A 0.5 M solution of the appropriate peracety-
lated glycosyl halide in tetrahydrofuran was slurried with solid
sodium bicarbonate to remove any traces of acid, filtered, injected
dropwise into a 0.5 M solution of the appropriate diazeniumdiolate
salt (1) in dimethyl sulfoxide, and stirred for 24-78 h at room
temperature. The reaction mixture was poured over ice-water (100
mL) 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 deriva-
tives were purified by recrystallization, while the glassy mannose,
fucose, and GlcNAc adducts required column chromatography.
Deacylation was carried out according to established procedures
prescribed by Wolfram and Thompson26 by adding 10 mol % of a
25% solution of sodium methoxide in methanol to 0.1 M solutions
of the peracetylated/glycosylated diazeniumdiolates in methanol.
Reactions were monitored by thin layer chromatography on 2.5
cm × 7.5 cm silica gel plates developed using various solvents.
Spots were visualized by ultraviolet quenching.
O2-(ꢀ-D-Glucopyranosyl) 1-(Piperidin-1-yl)diazen-1-ium-1,2-
diolate (3d) (CV-21). Compound 2d was deacylated to give a
quantitative yield of 3d as a white foam: UV (methanol) λmax (ε)
1
230 nm (7.7 mM-1 cm-1); H NMR (CDCl3) δ 1.43-1.55 (m, 2
H), 1.66-1.78, (m, 4 H), 3.30-3.46 (m, 5 H), 3.57-3.78 (m, 3
H), 3.79-4.00 (m 3 H), 4.95-5.14 (m, 2 H), 5.24-5.53 (m, 2 H);
13C NMR δ 23.31, 24.64, 52.28, 61.16, 69.01, 71.40, 76.05, 76.25,
103.32. Anal. (C11H21N3O7 ·0.5H2O) C, H, N.
O2-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl) 1-[(4-Ethoxy-
carbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (2e) (CV-06).
To 1.2 g (0.005 mol) of 1e were added catalytic amounts of silver
acetate and 1.83 g (0.0044 mol) of acetobromoglucose. A colorless
syrup (1.33 g, 55%) was isolated: UV (ethanol) λmax (ε) 228 nm
1
(7.2 mM-1 cm-1); H NMR (CDCl3) δ 1.28 (t, 3 H, J ) 7.1),
2.02 (s, 3H), 2.04 (s, 3 H), 2.04 (s, 3 H), 2.09 (s, 3 H), 3.44-3.48
(m, 4 H), 3.65-3.68 (m, 4 H), 3.80-3.86 (m, 1 H), 4.16 (q, 2 H,
J ) 7.2), 4.11-4.19 (m, 1 H), 4.25-4.30 (m, 1 H), 5.11-5.37 (m,
4 H); 13C NMR δ 14.52, 20.46, 20.49, 20.63, 42.21, 50.71, 61.58,
61.76, 67.71, 69.06, 72.62, 100.33, 154.99, 168.92, 169.24, 170.02,
170.48. Anal. (C21H32N4O13) C, H, N.
O2-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl) 1-(Pyrrolidin-
1-yl)diazen-1-ium-1,2-diolate (2b) (CV-05). The general procedure
above was employed as follows. To 4.48 g (0.029 mol) of 1b were
added catalytic amounts of silver acetate and 12 g (0.029 mol) of
acetobromoglucose. A solid (9.2 g, 69%) was isolated: mp 146-147
°C; UV (H2O) λmax (ε) 258 nm (7.9 mM-1 cm-1); 1H NMR
(CDCl3) δ 1.94-1.98 (m, 4 H), 2.02 (s, 3 H), 2.04 (s, 6 H), 2.09
(s, 3 H), 3.58-3.62 (m, 4 H), 3.78-3.84 (m, 1 H), 4.14-4.19 (m,
1 H), 4.25-4.30 (m, 1 H), 5.12-5.19 (m, 2 H), 5.26 (t, 1 H, J )
9.2 Hz), 5.33-5.39 (m, 1 H); 13C NMR δ 20.54, 20.60, 20.70,
23.01, 50.60, 61.74, 69.14, 72.50, 72.91, 100.27, 169.05, 169.33,
170.17, 170.63. Anal. (C18H27N3O11) C, H, N.
O2-(ꢀ-D-Glucopyranosyl) 1-[(4-Ethoxycarbonyl)piperazin-1-
yl]diazen-1-ium-1,2-diolate (3e) (CV-11). Compound 2e was
deacylated to give an 86% yield of 3e as a white foam after column
chromatography (silica gel, 9:1 CH2Cl2/MeOH): UV (methanol)
1
λmax (ε) 232 nm (8.8 mM-1 cm-1); H NMR (DMSO-d6) δ 1.19
(t, 3 H, J ) 7.1 Hz), 3.08-3.14 (m, 1 H), 3.21-3.25 (m, 3 H),
3.32-3.35 (m, 5 H), 3.42-3.55 (m, 5 H), 3.65-3.68 (m, 1 H),
4.06 (q, 2 H, J ) 7.1 Hz), 4.52-4.68 (m, 1 H), 4.90-4.93 (m, 1
H), 5.04 (br, 1 H), 5.42 (br, 1 H); 13C NMR δ 14.52, 42.05, 50.34,
54.92, 60.58, 61.10, 69.31, 71.54, 77.51, 103.53, 154.49. Anal.
(C13H24N4O9 ·0.75H2O) H, N, C: calcd, 39.19; found, 39.67.
O2-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl) 1-[(4-tert-Bu-
toxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (2f) (CV-
42). To 1.0 g (0.0037 mol) of 1f were added catalytic amounts of
silver acetate and 1.73 g (0.0042 mol) of acetobromoglucose. A
O2-(ꢀ-D-Glucopyranosyl) 1-(Pyrrolidin-1-yl)diazen-1-ium-1,2-
diolate (3b) (CV-10). Compound 2b was deacylated to give a 79%
yield of 3b as a white foam: UV (methanol) λmax (ε) 252 nm (7.2
mM-1 cm-1); 1H NMR (DMSO-d6) δ 1.83-1.92 (m, 4 H),

Targeting NO to Macrophages
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3967
white solid (1.17 g, 55%) was isolated: mp 122-125 °C; UV
(methanol) λmax (ε) 232 nm (9.3 mM-1 cm-1); 1H NMR (CDCl3)
δ 1.47 (s, 9 H), 2.02 (s, 3 H), 2.04 (s, 3 H), 2.04 (s, 3 H), 2.09 (s,
3 H), 3.43-3.46 (m, 4 H), 3.59-3.63 (m, 4 H), 3.79-3.89 (m, 1
H), 4.14-4.19 (m, 1 H), 4.25-4.30 (m, 1 H), 5.11-5.38 (m, 4 H);
13C NMR δ 20.52, 20.56, 20.70, 28.31, 50.84, 61.64, 67.78, 69.12,
72.69, 80.50, 100.40, 154.17, 168.99, 169.29, 170.10, 170.56. Anal.
(C23H36N4O13) C, H, N.
increasing to 3:2 to yield 4.1 g of O2-(2,3,4,6-tetra-O-acetyl-R-D-
mannopyranosyl) 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate, 4b.
The product, without further purification, was deacylated as
described above to give crude 5b. The product was chromato-
graphed on silica gel using a gradient of 95%-8% dichloromethane
1
in methanol: UV (H2O) λmax (ε) 254 nm (7.8 mM-1 cm-1); H
NMR (D2O) δ 2.00-2.01 (m, 4 H), 3.46-3.52 (m, 1 H), 3.56-3.63
(m, 4 H), 3.65-3.82 (m, 3 H), 3.93-3.97 (m, 1 H), 4.23 (dd, 1 H,
J ) 0.9 Hz, J ) 2.9 Hz), 5.38 (d, 1 H, J ) 0.9 Hz); 13C NMR δ
25.34, 53.84, 63.55, 69.10, 72.23, 75.36, 79.73, 103.02.
O2-(ꢀ-D-Glucopyranosyl) 1-[(4-tert-Butoxycarbonyl)piperazin-
1-yl]diazen-1-ium-1,2-diolate (3f) (CV-167). Compound 3e was
deacylated as described above to give a 63% yield of 3f as a white
foam: UV (methanol) λmax (ε) 232 nm (8.2 mM-1 cm-1); 1H NMR
(CDCl3) δ 1.46 (s, 9 H), 2.70 (br, 1 H), 3.32-3.51 (m, 4 H),
3.51-3.79 (m, 7 H), 3.86 (br, 3 H), 4.93-5.12 (m, 2 H), 5.25-5.47
(m, 2 H); 13C NMR δ 28.34, 42.19, 42.36, 50.92, 61.01, 68.84,
71.39, 76.00, 76.28, 80.48, 103.42, 154.22. Anal. (C15H28N4O9) C,
H, N.
O2-(2,3,4-Tri-O-acetyl-ꢀ-L-fucopyranosyl) 1-(N,N-Diethylami-
no)diazen-1-ium-1,2-diolate (6a) (JS-33-181 Triacetate). This
compound was prepared as described in general methods but
without using silver ion. Purification of 1.8 g of crude product was
carried out by column chromatography on 80 g of silica gel and
the product was eluted with 5:1 dichloromethane/ethyl acetate to
give 986 mg of a clear glass (43%): UV (methanol) λmax (ε) 226
1
nm (7.0 mM-1 cm-1); H NMR (CDCl3) δ 1.11 (t, 6 H, J ) 7.2
O2-(2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl) 1-(N,N-Di-
ethylamino)diazen-1-ium-1,2-diolate (4a) (JS-37-137). A solution
of 5 g (0.012 mol) of acetobromomannose in tetrahydrofuran (50
mL) was added to 7.75 g (0.05 mol) of 1a in 30 mL of DMSO and
250 mg of silver acetate. After being stirred for 24 h, the reaction
mixture was treated with 100 mL of ether, cooled in an ice bath to
avoid possible hydrolysis on adding water, placed in a separatory
funnel, and washed with cold water. The ether solution was dried
over sodium sulfate, filtered through a layer of magnesium sulfate,
and concentrated under vacuum to give 1.7 g of an amber resin.
The product was purified on column chromatography (silica gel)
using 1:1 hexane/ethyl acetate as the eluant. A fraction containing
about 25 mg of product was concentrated, and the isolated resin
was crystallized on standing while covered with ether/petroleum
ether. The rest of the fractions containing 814 mg of product could
be isolated only as a foam. Because of the difficulty in totally
purifying the tetraacetylmannose adduct, it is recommended that
the deacylation step proceed without thorough purification. How-
ever, sufficient material was isolated for characterization: mp
99-100 °C; UV (H2O) λmax (ε) 228 nm (8.4 mM-1 cm-1); 1H
NMR (CDCl3) δ 1.12 (t, 6 H), 2.00 (s, 3 H), 2.05 (s, 3 H), 2.07 (s,
3 H), 2.19 (s, 3 H), 3.15-3.30 (m, 4 H), 4.04-4.08 (m, 1 H),
4.10-4.14 (m, 4 H), 4.26-4.31 (m, 1 H), 5.31-5.36 (m, 1 H),
5.41-5.44 (dd, 1 H, J ) 3.51 Hz, J ) 9.86 Hz), 5.49-5.51 (dd, 1
H, J ) 1.85 Hz, J ) 3.51 Hz), 5.58 (d, 1 H, J ) 1.75 Hz); 13C
NMR δ 11.53, 20.68, 20.70, 20.76, 20.89, 48.49, 62.55, 66.15,
69.94, 68.52, 68.72, 92.21, 169.79, 169.99, 170.16, 170.79. Anal.
(C18H29N3O11) C, H, N.
Hz), 1.23 (d, 3 H, J ) 6.4 Hz), 1.99 (s, 3 H), 2.03 (s, 3 H), 2.17
(s, 3 H), 3.19 (q, 4 H, J ) 7.2 Hz), 4.10-4.14 (m, 1 H), 5.05-5.30
(m, 3 H), 5.44-5.53 (m, 1 H). This compound was used without
further purification or characterization in the procedure described
in the next paragraph below.
O2-(ꢀ-L-Fucopyranosyl) 1-(N,N-Diethylamino)diazen-1-ium-
1,2-diolate (7a) (JS-33-182). A solution of 920 mg (2.21 mmol)
of the above glass in methanol was deacylated as described for
other sugar analogues, yielding 532 mg (86%) of pure 7a as a white
foam: UV (H2O) λmax (ε) 228 nm (6.3 mM-1 cm-1); 1H NMR
(methanol-d4) δ 1.01 (t, 6 H, J ) 7.1 Hz), 1.29 (d, 3 H, J ) 6.4
Hz), 3.16 (q, 4 H, J ) 7.1 Hz), 3.68-3.76 (m, 3 H), 3.81 (b, 1 H),
3.98-4.08 (m, 1 H), 4.15 (b, 1 H), 5.01 (d, 1 H, J ) 8 Hz), 5.08
(b, 1 H); 13C NMR δ 11.45, 16.13, 48.19, 71.35, 71.42, 73.84,
107.71. Anal. (C10H21N3O5 ·H2O) C, H, N.
O2-(2,3,4-Tri-O-acetyl-ꢀ-L-fucopyranosyl) 1-(N,N-Dimethyl-
amino)diazen-1-ium-1,2-diolate (6c) (JS-33-164). Acetobromo-
fucose (2.66 g, 0.0075 mol) in tetrahydrofuran was added to 1.52 g
(12 mmol) of 1c without silver acetate to give 2.74 g of crude
product. Purification on column chromatography (silica gel) using
5:1 CH2Cl2/EtOAc as the eluant provided 2.2 g of a white glass:
UV (methanol) λmax (ε) 231 nm (7.9 mM-1 cm-1); 1H NMR
(CDCl3) δ 1.25 (d, 3 H, J ) 6.5 Hz), 2.02 (s, 3 H), 2.08 (s, 3 H),
2.18 (s, 3 H), 3.07 (s, 6 H), 3.9-4.35 (m, 1 H), 5.07-5.10 (dd, 1
H, J ) 3.4, 10.4 Hz), 5.26-5.27 (dd, 1 H, J ) 0.9, 8.4 Hz),
5.47-5.52 (dd, 1 H, J ) 8.4, 10.4 Hz), 5.15 (d, 1 H, J ) 8.4 Hz);
13C NMR δ 16.01, 20.58, 20.61, 20.71, 42.42, 66.94, 69.90, 70.08,
71.46, 100.97, 169.13, 170.14, 170.56. Anal. (C14H23N3O9 ·1/4 H2O)
C, H, N.
O2-(r-D-Mannopyranosyl) 1-(N, N-Diethylamino)diazen-1-
ium-1,2-diolate (5a) (CV-123). A solution of 624 mg of 4a in 10
mL of methanol was treated with 0.15 mL of 25% methanolic
sodium methoxide and stirred at 25 °C for 30 min. Dowex-50W-
H+ resin (250 mg) was added to the methanolic solution to give
390 mg of a white foam. Purification was carried out on a 4 cm ×
7 cm KP-Sil column fitted into a Flash 40 chromatography system
and the product was eluted with 9:1 dichloromethane/methanol to
give 208 mg of foamy 5a: UV (methanol) λmax (ε) 228 nm (8.3
O2-(ꢀ-L-Fucopyranosyl) 1-(N,N-Dimethylamino)diazen-1-ium-
1,2-diolate (7c) (JS-33-177). Deacylation in methanol with catalytic
amounts of sodium methoxide was carried out as described above.
A white glass was isolated: UV (methanol) λmax (ε) 235 nm (9.6
mM-1 cm-1); 1H NMR (CDCl3) δ 1.36 (d, 3 H, J ) 6.4 Hz), 2.99
(s, 6 H), 3.70-3.79 (m, 3 H), 3.96-4.10 (m, 1 H), 4.96 (d, 1 H, J
) 8.2 Hz); 13C NMR δ 11.45,16.13, 48.19, 71.35, 71.42, 73.84,
107.71. Anal. (C8H17N3O6 ·0.67H2O·0.33CH3OH) C, H, N.
O2-(3,4,6-Tri-O-acetyl-ꢀ-D-N-acetylglucosaminyl) 1-(N,N-Di-
ethylamino)diazen-1-ium-1,2-diolate (8a) (CV-144). To 1.2 g (9.3
mmol) of 1a in 3:1 dimethylsulfoxide/tetrahydrofuran was added
2-acetamido-2-deoxy-R-D-glucopyranosyl chloride 3,4,6-triacetate
(3.3 g, 9.0 mmol) and 47 mg of silver acetate. A white foam was
isolated (3.0 g, 70%): UV (methanol) λmax (ε) 226 nm (7.2 mM-1
1
mM-1 cm-1); H NMR (D2O) δ 1.06 (t, 6 H), 3.14-3.24 (m, 4
H), 3.60-3.64 (m, 1 H), 3.74-3.91 (m, 2 H), 4.19-4.21 (dd, 1 H,
J ) 1.76 Hz, J ) 3.42 Hz), 5.73 (d, 1 H, J ) 1.75 Hz); 13C NMR
(D2O) δ 13.31, 51.70, 63.37, 68.93, 70.95, 73.07, 77.52, 104.86.
Anal. (C10H21N3O7 ·0.5 H2O) C, H, N.
O2-(r-D-Mannopyranosyl) 1-(Pyrrolidin-1-yl)diazen-1-ium-
1,2-diolate (5b) (CV-2-09). To a slurry of 4.29 (28 mmol) of 1b
in 30 mL of dimethyl sulfoxide and 10 mL of tetrahydrofuran was
added 140 mg of silver acetate. A solution of 10 g (24.3 mmol) of
acetobromomannose in 10 mL of tetrahydrofuran was injected
gradually into the slurry under a blanket of argon. After being stirred
at room temperature overnight, the reaction mixture was poured
over ice-water and extracted with ether. The organic layer was
washed with 5% sodium bicarbonate solution followed by aqueous
sodium chloride. The solution was dried over sodium sulfate,
filtered, and evaporated to give a brown syrup. This was chromato-
graphed on silica gel using ethyl acetate/hexane in a 7:6 ratio
1
cm-1); H NMR (CDCl3) δ 1.12 (t, 6 H, J ) 7.1 Hz), 1.92 (s, 3
H), 2.03 (s, 3 H), 2.04 (s, 3 H), 2.06 (s, 3 H), 3.27 (q, 4 H, J ) 7.1
Hz), 3.83-3.89 (m, 1 H), 3.93-4.02 (m, 1 H), 4.12-4.28 (m, 2
H), 5.06-5.12 (m, 1 H), 5.54-5.60 (m, 1 H), 5.68 (d, 1 H, J )
8.8 Hz), 6.22 (d, 1 H, J ) 8.2 Hz); 13C NMR 11.35, 20.58, 20.60,
20.64, 23.23, 47.62, 53.42, 61.91, 68.36, 71.65, 72.35, 99.76,
169.40, 170.39, 170.42, 170.58. Anal. (C18H30N4O10) C, H, N.
O2-(N-Acetyl-ꢀ-D-glucosaminyl) 1-(N,N-Diethylamino)diazen-
1-ium-1,2-diolate (9a) (CV-159). A solution of 309 mg (0.67
mmol) of 8a in methanol was deacylated, yielding 141 mg (63%

3968 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Valdez et al.
yield) of a white foam: UV (H2O) λmax (ε) 228 nm (7.6 mM-1
cm-1); 1H NMR (D2O) δ 1.05 (t, 6 H, J ) 7.2 Hz), 2.02 (s, 3 H),
3.19 (q, 4 H, J ) 7.1 Hz), 3.46-3.69 (m, 4 H), 3.77-3.96 (m, 3
H), 4.03 (t, 1 H, J ) 9.0 Hz), 5.37 (d, 1 H, J ) 8.9 Hz); 13C NMR
δ 13.29, 24.91, 51.56, 56.63, 63.19, 72.07, 76.48, 79.70, 104.57,
177.32. Anal. (C12H24N4O7) C, H, N.
glass: UV (methanol) λmax (ε) 229 nm (8.2 mM-1 cm-1); 1H NMR
(CDCl3) δ1.11 (t, 6 H, J ) 7.13 Hz), 1.84-1.93 (m, 1 H),
2.39-2.48 (m, 1 H), 3.93-3.95 (m, 5 H), 5.34 (dd, 1 H, J ) 2.2,
10.2 Hz); 1H NMR (D2O) δ 1.07 (t, 6 H, J ) 7.1 Hz), 1.74-1.82
(m, 1 H), 2.45 (ddd, 1 H, J ) 2.22, 5.04, 12.3 Hz), 3.20 (q, 4 H,
J ) 7.2 Hz), 3.37-3.43 (m, 1 H), 3.50-3.54 (m, 1 H), 3.76-3.85
(m, 2 H), 3.93 (dd, 1 H, J ) 2.3, 7.17 Hz), 5.55 (dd, 1 H, J )
2.22, 9.94 Hz); 13C NMR (CDCl3) δ 11.49, 36.21, 48.29, 61.64,
71.09, 71.01, 76.18, 100.25: 13C NMR (D2O) δ 13.33, 38.69, 51.64,
63.39, 72.86, 73.09, 79.60, 103.28. The chemical shifts proved to
be solvent-dependent. Anal. (C10H21N3O6 ·0.2H2O·0.2C6H12O5) C,
H, N.
O2-(3,4,6-Tri-O-acetyl-ꢀ-D-N-acetylglucosaminyl) 1-(Pyrroli-
din-1-yl)diazen-1-ium-1,2-diolate (8b) (CV-154). This compound
was prepared in 73% yield by reacting 1b with acetochloroglu-
cosamine in the presence of 25 mg of silver acetate. Purification
was carried out on a silica gel column by eluting with 7:3 ethyl
acetate/hexane increasing to 100% ethyl acetate: mp 159-161 °C;
UV (methanol) λmax (ε) 254 nm (7.7 mM-1 cm-1); 1H NMR
(CDCl3) δ 1.93 (s, 3 H), 1.96-2.00 (m, 4 H), 2.03 (s, 3 H), 2.04
(s, 3 H), 2.07 (s, 3 H), 3.60-3.65 (m, 4 H), 3.83-3.89 (m, 1 H),
3.96-4.31 (m, 3 H), 5.06 (t, 1 H, J ) 13.3 Hz), 5.59 (t, 1 H, J )
O2-Tetrahydropyran-2-yl 1-(N,N-Diethylamino)diazen-1-ium-
1,2-diolate (12) (JS-45-97). A solution of 360 mg (3.02 mmol) of
2-chlorotetrahydropyran (prepared by the method of Viola et al.)28
in 5 mL of tetrahydrofuran was cooled to 0 °C under nitrogen. A
solution of 468 mg (3 mmol) of 1a in 5 mL of THF, 1 mL of
DMF, and 660 mg (3 mmol) of 15-crown-5 was added slowly to
the cold solution and stirred for 1 h, at which time TLC indicated
that the reaction was complete. The volatile solvents were removed
on a rotary evaporator, and the residual oil was extracted with ether
that was quickly washed with cold, dilute sodium bicarbonate
solution, dried over sodium sulfate, filtered, and evaporated to give
662 mg of a brown oil. Flash chromatography on a 13.5 cm × 3.3
cm silica gel column eluted with 5:1 dichloromethane/ethyl acetate
gave 358 mg (55%) of JS-45-97 as a colorless liquid: UV (ethanol)
9.4 Hz), 5.68 (d, 1 H, J ) 8.9 Hz), 6.75 (d, 1 H, J ) 8.4 Hz); 13
C
NMR δ 20.58, 20.66, 22.96, 23.29, 53.13, 61.90, 68.35, 71.80,
72.26, 99.63, 169.42, 170.34, 170.59, 170.64. Anal. (C18H28N4O10)
C, H, N.
O2-(ꢀ-D-N-Acetylglucosaminyl) 1-(Pyrrolidin-1-yl)diazen-1-
ium-1,2-diolate (9b) (CV-166). A solution of 1 g (2.17 mmol) of
8b in 13 mL of methanol was treated with 25% sodium methoxide
in methanol as described above. The hydrolyzed product was
chromatographed on silica gel and eluted with 9:1 dichloromethane/
ethyl acetate to give 660 mg (91%) of 9b as a white foam: UV
(methanol) λmax (ε) 254 nm (7.5 mM-1 cm-1); 1H NMR (D2O) δ
1.96-2.0 (m, 4 H), 2.03 (s, 3 H), 3.54-3.62 (m, 4 H), 3.62-3.96
(m, 4 H), 4.04 (t, 1 H, J ) 9.9 Hz), 5.25 (d, 1 H, J ) 7 Hz); 13C
NMR δ 24.93, 25.39, 53.75, 56.38, 63.24, 72.17, 76.57, 79.61,
103.91, 177.38. Anal. (C12H22N4O6 ·0.5H2O) C, H, N.
1
λmax (ε) 233 nm (6.2 mM-1 cm-1); H NMR (CDCl3) δ 1.11 (t,
6H, J ) 7.12 Hz), 1.58-1.99 (m, 6H), 3.4 (q, 4H, J ) 7.1 Hz),
3.56-3.68 (m, 1H), 3.86-3.92 (m, 1H), 5.51 (t, 1H, J ) 2.83 Hz);
13C NMR (CDCl3) δ 11.53, 18.36, 24.82, 28.03, 48.59, 62.07,
100.15. Anal. (C9H19N3O3 ·0.1C5H10O2) C, N. H: calcd, 8.85; found,
9.27. (Note: C5H10O2 is 2-hydroxytetrahydropyran.)
O2-(3,4,6-Tri-O-acetyl-2-deoxy-ꢀ-D-glucopyranosyl) 1-(N,N-
Diethylamino)diazen-1-ium-1,2-diolate (10a) (JS-45-76). 1,3,4,6-
Tetraacetyl-2-deoxy-ꢀ-D-glucose (1.59 g, 4.8 mmol), prepared from
2-deoxyglucose with pyridine and acetic anhydride using the method
of Wolfrom and Thompson,26 was brominated in 25 mL of
dichloromethane with 24 mL of 30% HBr/acetic acid according to
a literature procedure27 to give 1.16 g (68%) of 3,4,6-tri-O-acetyl-
2-deoxy-R-D-glucopyranosyl bromide. A solution of 950 mg (2.69
mmol) of this compound in 10 mL of tetrahydrofuran was cooled
to 0 °C and stirred under nitrogen. In a separate container were
placed 551 mg (3.6 mmol) of 1a, 10 mL of tetrahydrofuran, and 1
mL of N,N-dimethylformamide (DMF). The resulting slurry was
treated with 792 mg (3.6 mmol) of 15-crown-5, resulting in a near-
homogeneous solution that was slowly added to the bromosugar
and stirred at 0-4 °C. The reaction was complete within an hour
as indicated by TLC on silica gel. The reaction flask was placed
on a rotary evaporator and concentrated to an oil (1.6 g) containing
the product, impurities, and crown ether as well as DMF. The oil
was flash-chromatographed on a 3.3 cm × 29 cm prepacked silica
gel column and eluted with 5:1 dichloromethane/ethyl acetate to
give 640 mg (59%) of 10a as a colorless glass that was difficult to
separate from the 3,4,6-tri-O-acetyl-2-deoxy-ꢀ-D-glucose. The
analytical sample contained the latter compound and 10a in a 1:3
ratio: UV (methanol) λmax (ε) 227 nm (8.2 mM-1 cm-1); 1H NMR
(CDCl3) δ 1.15 (t, 6 H, J ) 7.13 Hz), 2.05 (s, 3 H), 2.06 (s, 3 H),
2.06 (s, 3 H), 1.99-2.10 (m, 1 H), 2.49 (ddd, 1 H, J ) 2.32, 4.69,
12.49 Hz), 4.27 (dd, 1 H, J ) 4.9, 12.2 Hz), 5.01-5.12 (m, 2 H),
5.36 (dd, 1 H, J ) 2.34, 9.96 Hz); 13C NMR (CDCl3) δ 11.53,
20.69, 20.86, 33.42, 48.51, 62.19, 68.35, 69.90, 72.53, 99.25,
169.63, 170.19, 170.65 (note: only 13 out of 14 carbons are
detected). Anal. (C16H27N3O9 ·0.33C12H18O9) C, H, N.
N-(ꢀ-D-N′-Acetylglucosaminyloxy)succinamate O-Methyl Es-
ter (13) (BMS-3-80). This non-NO-releasing control compound
was prepared as previously described. The compound’s NMR
spectrum matched that reported by Cao et al.29
Rates of Hydrolysis. Rate constants for hydrolysis of 3a, 5a,
7a, 9a, and 11a measured at pH 7.4 and below under protection
from light were determined by measuring absorption spectra
(200-400 nm) of l mL aliquots taken daily from 40 mL reaction
solutions maintained in sealed bottles thermostated at 37 °C for up
to 11 days. First-order rate constants were obtained from the slope
of plots of ln(At - Ainf) versus time, where A is absorbance at time
t at λmax and where Ainf is the constant absorbance value at the end
of the reaction. For reactions that had not gone to completion,
infinity absorbance values were estimated from the exponential At
versus time decay curves that were obtained at g80% of reaction.
Faster reactions occurring in 1.0 M NaOH solutions were
followed directly in the thermostated cell compartment of the HP
8453 diode-array spectrophotometer, and rate constants were
obtained through the instrument’s kinetics software.
Rates of NO release in cell culture medium containing fetal calf
serum in the presence and absence of macrophages were estimated
by following the accumulation of its auto-oxidation product, nitrite
ion, using the colorimetric Griess reagent assay as described.30
Glycosidase-Induced Hydrolysis. The specific activity for
hydrolysis of PNP-NAG (p-nitrophenyl-N-acetyl-D-glucosaminide)
by ꢀ-N-acetylglucosaminidase (NAGase) was confirmed at 25 °C
as recommended in the manufacturer’s protocol by monitoring UV
absorbance changes at 345 nm. Activities for enzymes from both
jack bean and human placenta were determined at their pH rate
maxima, 5.0 and 4.25, respectively. Chemiluminescence detection
and quantification of NO evolving from the reactions were
conducted using a Sievers 280i nitric oxide analyzer (Boulder, CO)
essentially as described previously.31 The NO release profile was
followed at 37 °C, and the resulting curve was integrated to quantify
the amount of NO released per mole of compound. Details of the
enzymatic hydrolysis studies on 5a, 9a, and 9b are described in
Figure 4 and Table 3.
O2-[2-Deoxy-ꢀ-D-glucopyranosyl] 1-(N,N-Diethylamino)dia-
zen-1-ium-1,2-diolate (11a) (JS-45-112). To a solution of 208 mg
(0.51 mmol) of 10a in 5 mL of methanol was added 5 µL (0.023
mmol) of 25% methanolic sodium methoxide. The resulting solution
was stirred at room temperature for 2 h whereupon 500 mg of
prewashed Dowex 50W-H+ resin was added. After stirring for an
additional 5 min, the mixture was filtered and evaporated in vacuo.
The residue was taken up in dichloromethane and filtered through
a short alumina column to give 130 mg of JS-45-112 as a colorless

Targeting NO to Macrophages
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3969
X-ray Crystallographic Characterization of Mannose Deri-
vative 4a. Single-crystal X-ray diffraction data on compound 4a
were collected at 103 K using Mo KR radiation and a Bruker
SMART 1000 CCD area detector. A 0.38 × 0.21 × 0.20 mm3
crystal was prepared for data collection coating with high viscosity
microscope oil (Paratone-N, Hampton Research). The oil-coated
crystal was mounted on a glass rod and transferred immediately to
the cold stream (-170 °C) on the diffractometer. The crystal was
orthorhombic in space group P212121 with unit cell dimensions a
) 8.919(3) Å, b ) 11.086(3) Å, and c ) 23.857(7) Å. Corrections
were applied for Lorentz, polarization, and absorption effects. Data
were 99.7% complete to 28.28° θ (approximately 0.75 Å). The
structure was solved by direct methods and refined by full-matrix
least-squares on F2 values using the programs found in the
SHELXTL suite (Bruker, SHELXTL version 6.10, 2000, Bruker
AXS Inc., Madison, WI). Parameters refined included atomic
coordinates and anisotropic thermal parameters for all non-hydrogen
atoms. Hydrogen atoms on carbons were included using a riding
model [coordinate shifts of C applied to H atoms] with C-H
distance set at 0.96 Å. The absolute configuration was set on the
basis of the known configuration of the sugar. Atomic coordinates
for compound 4a have been deposited with the Cambridge
Crystallographic Data Centre (deposition number 260728). Copies
of the data can be obtained, free of charge, on application to CCDC,
12 Union Road, Cambridge, CB2 1EZ, UK [fax, +44(0)-1223-
336033; e-mail, deposit@ccdc.cam.ac.uk].
Institute, Center for Cancer Research, by the German Research
Foundation (Grants Bo 996/3-1 and 3/2 and SFB 620 A9), and
by the National Cancer Institute under Contract NO1-CO-12400.
We gratefully acknowledge NIDA Contract Y1-DA6002 for
support of the X-ray work.
Supporting Information Available: Complete crystal structure
report for 4a and results from elemental analysis. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Fang, F. C., Ed. Nitric Oxide and Infection; Kluwer Academic/Plenum
Publishers: New York, 1999.
(2) Klimp, A. H.; deVries, E. G. E.; Scherphof, G. L.; Daemen, T. A
potential role of macrophage activation in the treatment of cancer.
Crit. ReV. Oncol. Hematol. 2002, 44, 143–161.
(3) Malkinson, A. M. Role of inflammation in mouse lung tumorigenesis:
a review. Exp. Lung Res. 2005, 31, 57–82.
(4) Xu, W.; Liu, L. Z.; Loizidou, M.; Ahmed, M.; Charles, I. G. The role
of nitric oxide in cancer. Cell Res. 2002, 12, 311–320.
(5) Martinez-Pomares, L.; Linehan, S. A.; Taylor, P. R.; Gordon, S.
Binding properties of the mannose receptor. Immunobiology 2001, 204,
527–535.
(6) Bogdan, C.; Ro¨llinghoff, M.; Diefenbach, A. Nitric Oxide in Leish-
maniasis. In Nitric Oxide and Infection; Fang, F. C., Ed.; Kluwer
Academic/Plenum Publishers: New York, 1999; pp 361-377.
(7) Taylor, M. E.; Drickamer, K. Structural requirments for high affinity
binding of complex ligands by the macrophage mannose receptor.
J. Biol. Chem. 1993, 268, 399–404.
Leishmania Parasites, Macrophages, and Assay for Leish-
manicidal Activity. L. major amastigotes were prepared from the
skin lesions of BALB/c mice that had been infected subcutaneously
withpromastigotesoftheL.majorstrainMHOM/IL/81/FE/BNI.14,32,33
Thioglycolate-elicited peritoneal exudate macrophages were
prepared from C57BL/6 iNOS-deficient mice34 (Jackson Labora-
tories, Bar Harbor, ME) 4 days after ip injection of 2 mL of 4%
Brewer’s thioglycolate broth.14,35 The macrophages were seeded
into eight-well LabTek Tissue Culture Permanox Chambers (Nalge
Nunc International, Naperville, IL) at 3 × 105 macrophages/well
and cultured in RPMI-1640 medium (supplemented with 2 mM
glutamine, 10 mM HEPES, 13 mM NaHCO3, 50 µM 2-mercap-
toethanol, 100 µg/mL penicillin, 100 µg/mL streptomycin, and 2.5%
or 5% fetal bovine serum [lot 77H3398; Sigma, Deisenhofen,
Germany]). After an adherence phase of 2-3 h, nonadherent cells
were washed off and the macrophages were either cultured in
medium alone or stimulated with interferon (IFN)-γ (obtained from
Dr. G. Adolf at the Ernst Boehringer Institut, Vienna, Austria; 20
ng/mL) and tumor necrosis factor (TNF, 10 ng/mL) for 16 h.
Thereafter, the monolayers were infected with amastigotes of L.
major at a parasite/macrophage ratio of 5:1 for 4 h (with the
remaining extracellular Leishmania washed off thereafter). For the
following 72 h, the macrophages were cultured in medium alone
(no stimulus), stimulated with IFN-γ plus TNF, or incubated with
glycosylated diazeniumdiolates at the indicated concentrations. After
24 and 48 h, the culture medium was replaced with fresh medium
containing the respective stimuli. Immediately after the 4 h pulse
infection period (time point 0 h after the infection) as well as 48
and 72 h after infection, the percentage of infected macrophages
and the number of intracellular parasites per infected macrophage
were determined microscopically using the Diff-Quick staining
procedure (Dade Behring, Marburg, Germany). The results were
used to calculate the number of parasites per 100 macrophages in
culture. All given values are mean values ((SD) and based on the
evaluation of at least 4 × 100 macrophages for the infection rate
and 3 × 60 macrophages for the number of parasites per cell.
The viability of the macrophage monolayers was verified by
microscopic analysis using trypan blue staining of dead cells as
well as by the ability of viable macrophages to transform yellow
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
into violet formazan (this photometric assay was performed in
triplicate using an ELISA reader as described36,37).
(8) Wu, X.; Tang, X.; Xian, M.; Wang, P. G. Glycosylated diazenium-
diolates: a novel class of enzyme-activated nitric oxide donors.
Tetrahedron Lett. 2001, 42, 3779–3782.
(9) Showalter, B. M.; Reynolds, M. M.; Valdez, C. A.; Saavedra, J. E.;
Davies, K. M.; Klose, J. R.; Chmurny, G. N.; Citro, M. L.; Barchi,
J. J., Jr.; Merz, S. I.; Meyerhoff, M. E.; Keefer, L. K. Diazeniumdiolate
ions as leaving groups in anomeric displacement reactions: a protection-
deprotection strategy for ionic diazeniumdiolates. J. Am. Chem. Soc.
2005, 127, 14188–14189.
(10) Liew, F. Y.; Li, Y.; Millott, S. Tumor necrosis factor-R synergizes
with IFN-γ in mediating killing of Leishmania major through the
induction of nitric oxide. J. Immunol. 1990, 145, 4306–4310.
(11) Green, S. J.; Crawford, R. M.; Hockmeyer, J. T.; Meltzer, M. S.; Nacy,
C. A. Leishmania major amastigotes initiate the L-arginine-dependent
killing mechanism in IFN-γ-stimulated macrophages by induction of
tumor necrosis factor-R. J. Immunol. 1990, 145, 4290–4297.
(12) Green, S. J.; Meltzer, M. S.; Hibbs, J. B., Jr.; Nacy, C. A. Activated
macrophages destroy intracellular Leishmania major amastigotes by
an L-arginine-dependent killing mechanism. J. Immunol. 1990, 144,
278–283.
(13) Diefenbach, A.; Schindler, H.; Donhauser, N.; Lorenz, E.; Laskay,
T.; MacMicking, J.; Ro¨llinghoff, M.; Gresser, I.; Bogdan, C. Type 1
interferon (IFNR/ꢀ) and type 2 nitric oxide synthase regulate the innate
immune response to a protozoan parasite. Immunity 1998, 8, 77–87.
(14) Blos, M.; Schleicher, U.; Soares Rocha, F. J.; Meissner, U.; Ro¨lling-
hoff, M.; Bogdan, C. Organ-specific and stage-dependent control of
Leishmania major infection by inducible nitric oxide synthase and
phagocyte NADPH oxidase. Eur. J. Immunol. 2003, 33, 1224–1234.
(15) DeGroote, M. A.; Fang, F. C. Antimicrobial Properties of Nitric Oxide.
In Nitric Oxide and Infection; Fang, F. C., Ed.; Kluwer Academic/
Plenum Publishers: New York, 1999; pp 231-261.
(16) Proudfoot, L.; Nikolaev, A. V.; Feng, G.-J.; Wei, X.-Q.; Ferguson,
M. A. J.; Brimacombe, J. S.; Liew, F. Y. Regulation of the expression
of nitric oxide synthase and leishmanicidal activity by glycoconjugates
of Leishmania lipophosphoglycan in murine macrophages. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 10984–10989.
(17) Balestieri, F. M.; Queiroz, A. R. P.; Scavone, C.; Costa, V. M. A.;
Barral-Netto, M.; Abrahamsohn, I. A. Leishmania (L.) amazonensis-
induced inhibition of nitric oxide synthesis in host macrophages.
Microbes Infect. 2002, 4, 23–29.
(18) Ghosh, S.; Bhattacharyya, S.; Sirkar, M.; Sa, G. S.; Das, T.; Majumdar,
D.; Roy, S.; Majumdar, S. Leishmania donoVani suppresses activated
protein 1 and NF-κB activation in host macrophages via ceramide
generation: involvement of extracellular signal-regulated kinase. Infect.
Immun. 2002, 70, 6828–6838.
(19) Drago, R. S.; Paulik, F. E. The reaction of nitrogen(II) oxide with
diethylamine. J. Am. Chem. Soc. 1960, 82, 96–98.
(20) Saavedra, J. E.; Billiar, T. R.; Williams, D. L.; Kim, Y.-M.; Watkins,
S. C.; Keefer, L. K. Targeting nitric oxide (NO) delivery in vivo.
Design of a liver-selective NO donor prodrug that blocks tumor
necrosis factor-R-induced apoptosis and toxicity in the liver. J. Med.
Chem. 1997, 40, 1947–1954.
Acknowledgment. This project was funded in part by the
Intramural Research Program of the NIH, National Cancer

3970 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Valdez et al.
(21) Saavedra, J. E.; Srinivasan, A.; Bonifant, C. L.; Chu, J.; Shanklin,
A. P.; Flippen-Anderson, J. L.; Rice, W. G.; Turpin, J. A.; Davies,
K. M.; Keefer, L. K. The secondary amine/nitric oxide complex ion
R2N[N(O)NO]– as nucleophile and leaving group in SNAr reactions.
J. Org. Chem. 2001, 66, 3090–3098.
(22) Saavedra, J. E.; Booth, M. N.; Hrabie, J. A.; Davies, K. M.; Keefer,
L. K. Piperazine as a linker for incorporating the nitric oxide-releasing
diazeniumdiolate group into other biomedically relevant functional
molecules. J. Org. Chem. 1999, 64, 5124–5131.
(30) Vodovotz, Y.; Bogdan, C.; Paik, J.; Xie, Q. W.; Nathan, C. Mecha-
nisms of suppression of macrophage nitric oxide release by transform-
ing growth factor beta. J. Exp. Med. 1993, 178, 605–613.
(31) Keefer, L. K.; Nims, R. W.; Davies, K. M.; Wink, D. A. “NONOates”
(1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors:
convenient nitric oxide dosage forms. Methods Enzymol. 1996, 268,
281–293.
(32) Solbach, W.; Lohoff, M.; Streck, H.; Rohwer, P.; Ro¨llinghoff, M.
Kinetics of cell-mediated immunity developing during the course of
Leishmania major infection in “healer” and “non-healer” mice:
progressive impairment of response to and generation of interleukin-
2. Immunology 1987, 62, 485–492.
(23) Kartha, K. P. R.; Jennings, H. J. A simplified, one-pot preparation of
aceto bromo sugars from reducing sugars. J. Carbohydr. Chem. 1990,
9, 777–781.
(33) Stenger, S.; Solbach, W.; Ro¨llinghoff, M.; Bogdan, C. Cytokine
interactions in experimental cutaneous leishmaniasis. II. Endogenous
tumor necrosis factor-R production by macrophages is induced by the
synergistic action of interferon (IFN)-γ and interleukin (IL) 4 and
accounts for the antiparasitic effect mediated by IFN-γ and IL 4. Eur.
J. Immunol. 1991, 21, 1669–1675.
(34) Laubach, V. E.; Shesely, E. G.; Smithies, O.; Sherman, P. A. Mice
lacking inducible nitric oxide synthase are not resistant to lipopolysac-
charide-induced death. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10688–
10692.
(35) Schindler, H.; Lutz, M. B.; Ro¨llinghoff, M.; Bogdan, C. The production
of IFN-γ by IL-12/IL-18-activated macrophages requires STAT4
signaling and is inhibited by IL-4. J. Immunol. 2001, 166, 3075–3082.
(36) Mosmann, T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol.
Methods 1983, 65, 55–63.
(37) Bogdan, C.; Paik, J.; Vodovotz, Y.; Nathan, C. Contrasting mechanisms
for suppression of macrophage cytokine release by transforming growth
factor-beta and interleukin-10. J. Biol. Chem. 1992, 267, 23301–23308.
(24) Horton, D. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-R-D-glucopyra-
nosyl Chloride. In Organic Syntheses CollectiVe Volume V; Baum-
garten, H. E., Ed; John Wiley and Sons: New York, 1973; pp 1-5.
(25) Flowers, H. M.; Levy, A.; Sharon, N. Synthesis of 2-O-R-L-
fucopyranosyl-L-fucopyranose. Carbohydr. Res. 1967, 4, 189–195.
(26) Wolfrom, M. L.; Thompson, A. Deacetylation. In Methods in
Carbohydrate Chemistry; Whistler, R. L., Wolfrom, M. L., BeMiller,
J. N., Eds.; Academic Press Inc.: New York and London, 1963; pp
215-220.
(27) Huang, X.; Surry, C.; Hiebert, T.; Bennet, A. J. Hydrolysis of (2-
deoxy-ꢀ-D-glucopyranosyl)pyridinium salts. J. Am. Chem. Soc. 1995,
117, 10614–10621.
(28) Viola, A.; Collins, J. J.; Filipp, N.; Locke, J. S. Acetylenes as potential
antarafacial components in concerted reactions. Formation of pyrroles
from thermolyses of propargylamines, as a dihydrofuran from a
propargylic ether, and of an ethylidenepyrrolidine from a ꢀ-amino
acetylene. J. Org. Chem. 1993, 58, 5067–5075.
(29) Cao, S.; Tropper, F. D.; Roy, R. Stereoselective phase transfer
catalyzed syntheses of glycosyloxysuccinimides and their transforma-
tions into glycoprobes. Tetrahedron 1995, 51, 6679–6686.
JM8000482