O2-functionalized methylamine diazeniumdiolates: Evidence for e ←2 Z equilibration in an acyclic system

Article abstract of DOI:10.1021/jo3020837

Diazeniumdiolates that have the structure RHNa-N(O)=NOR′ are of interest as prodrug (caged) forms of the bioeffectors nitric oxide (NO) and nitroxyl (HNO). Previous work has focused on examples possessing α-branched R groups, with isopropylamine (IPA)/NO (R = isopropyl) being the smallest examined to date. To probe the effect of minimizing the alkyl-group size on the chemistry of IPA/NO, we prepared the corresponding methylamine derivative as a sodium salt that was highly unstable but could be trapped in very low overall yield as the stable O²-benzyl derivative. To prepare enough for efficient characterization, we devised an alternate synthesis involving a novel N-dealkylation route. CH₃HNa-N(O)=NOBn, synthesized in high yield and crystallized as the Z isomer as determined by X-ray crystallography, was observed to exist as a 11:1 mixture of two isomeric forms in dynamic equilibrium in solution. Similar results were seen for the O ²-ethyl derivative, whose two equilibrium constituents were partially separated by HPLC to reveal essentially identical UV and mass spectra, indicating them to be Z and E isomers of CH₃HNa-N(O)=NOEt. The results could lead the way to a fuller understanding of the chemistry of the acyclic (E)-diazeniumdiolates.

Full text of DOI:10.1021/jo3020837

Article
pubs.acs.org/joc
O2‑Functionalized Methylamine Diazeniumdiolates: Evidence for E ⇄
Z Equilibration in an Acyclic System
Debanjan Biswas,*,† Ryan J. Holland,† Jeffrey R. Deschamps,‡ Zhao Cao,§ Larry K. Keefer,†
and Joseph E. Saavedra*,§
†Drug Design Section, Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702,
United States
§Basic Science Program, SAIC-Frederick Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United
States
‡Center for Biomolecular Science and Engineering, Naval Research Laboratory, Washington, District of Columbia 20375, United
States
S
* Supporting Information
ABSTRACT: Diazeniumdiolates that have the structure RHN−
N(O)NOR′ are of interest as prodrug (caged) forms of the
bioeffectors nitric oxide (NO) and nitroxyl (HNO). Previous work has
focused on examples possessing α-branched R groups, with isopropyl-
amine (IPA)/NO (R = isopropyl) being the smallest examined to date.
To probe the effect of minimizing the alkyl-group size on the chemistry
of IPA/NO, we prepared the corresponding methylamine derivative as
a sodium salt that was highly unstable but could be trapped in very low
overall yield as the stable O2-benzyl derivative. To prepare enough for
efficient characterization, we devised an alternate synthesis involving a
novel N-dealkylation route. CH3HN−N(O)NOBn, synthesized in high yield and crystallized as the Z isomer as determined by
X-ray crystallography, was observed to exist as a 11:1 mixture of two isomeric forms in dynamic equilibrium in solution. Similar
results were seen for the O2-ethyl derivative, whose two equilibrium constituents were partially separated by HPLC to reveal
essentially identical UV and mass spectra, indicating them to be Z and E isomers of CH3HN−N(O)NOEt. The results could
lead the way to a fuller understanding of the chemistry of the acyclic (E)-diazeniumdiolates.
groups might achieve that objective. Here we provide evidence
that replacing the isopropyl group of anion 1 in Scheme 1 with
a methyl group (R3 = benzyl, ethyl) provides just such an
equilibrium mixture of isomers.
INTRODUCTION
■
We are pursuing an increasingly refined understanding of the
fundamental physicochemical properties of the N-bound
diazeniumdiolates,1 which have the structure R1R2N−N(O)
NOR3, as a basis for the rational design of therapeutically useful
prodrugs of nitric oxide2 (NO) and nitroxyl3 (HNO) as well as
novel tools for probing the chemical biology of these important
bioeffectors.4 One aspect that to date has been difficult to
elucidate fully concerns the factors that control the cis/trans
stereochemistry of the oxygens in the −N(O)NOR3 group.
Arulsamy and co-workers very recently addressed this issue as it
relates to compounds in which the diazeniumdiolate nitrogen is
attached to carbon,5,6 but to date we know of only one report in
which an E configuration has been confirmed in a nitrogen-
bound diazeniumdiolate; this special case (R1 = isopropyl, R2 =
H, R3 = 2-bromoethyl)7 in which base-induced removal of the
N−H proton led to an anion whose E isomer was trapped by an
intramolecular alkylation process to form cyclic product 28 (see
Scheme 1). In an effort to obtain a macroscopically observable
pair of acyclic Z and E isomers in sufficient equilibrium
concentrations that the E form’s properties can be directly
characterized and compared with those of the Z isomer, we
hypothesized that minimizing steric interactions among the R
RESULTS AND DISCUSSION
■
Direct Synthesis of MA/NO. Preparation of primary-amine
diazeniumdiolates has been an enduring challenge on account
of their low thermodynamic stabilities.9 Initial attempts toward
the synthesis of methylamine analog 3 (see Scheme 2) involved
the reaction of NO with a solution of methylamine in ether/
acetonitrile precooled to −80 °C. Filtration of the resulting
methylammonium salt (obtained in very low yield) and
subsequent treatment with sodium methoxide afforded a
white solid in ∼3% overall yield (Scheme 2). All of the
manipulations were performed at −80 °C, and the solids were
found to be thermally unstable, often decomposing explosively
at ambient temperature. Nevertheless, the powder survived
long enough under cold conditions to allow the anion in 3 to
be trapped by reaction with benzyl bromide at −80 °C to
Received: October 1, 2012
© XXXX American Chemical Society
A
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Scheme 1. Synthesis of a Crystallographically Confirmed (E)-Diazeniumdiolate, 2
material obtained in the low-temperature benzylation of the
Scheme 2. Reaction of Methylamine with NO
MA/NO anion in 3.
1
Isomer Equilibration in 4. Careful examination of the H
NMR spectral pattern of 4 in CDCl3 revealed the presence of a
pair of doublets at 2.98 and 3.05 ppm in a 11:1 ratio along with
two overlapped singlets at 5.18 ppm, indicating the existence of
this O2-benzyl MA/NO analogue as a mixture of two isomeric
forms in solution. Similarly, the proton-coupled 13C NMR
spectrum of 4 also exhibited two sets of six peaks, further
supporting the existence of two isomeric forms of this analogue.
Recrystallization of the product mixture from diethyl ether at 4
°C followed by X-ray crystallographic analysis revealed it to
exist in the Z conformation (Z-4) (Figure 1A). We believe the
minor isomer to be the E conformation (E-4), although it could
not be isolated by recrystallization. Low-temperature NMR
analysis of a freshly prepared solution of recrystallized Z-4 in
CDCl3 at −20 °C revealed the Z:E isomeric ratio of 4 to be
∼66:1, indicating that the crystalline material contained no
more than 1.5% E-4 (Figure 1B). At room temperature, the
sample was found to equilibrate to a 11:1 Z-4:E-4 mixture,
similar to the purified product mixture, indicating that the two
isomers exist in a dynamic equilibrium in solution at room
temperature (see the Supporting Information).
produce the O2-benzyl derivative 4 in trace quantities.
Evidently, however, a more convenient alternative synthetic
route for the preparation of caged MA/NO prodrugs was
desired for systematic studies of the chemistry of MA/NO.
Dehydrohalogenation-Mediated Preparation of O2-
Protected MA/NO. Accordingly, we devised the dealkylation
route outlined in Scheme 3 for the preparation of derivatized
Scheme 3. N-Dealkylation Route to O2-Benzyl-Protected
MA/NO
LC/MS Identification and Partial Chromatographic
Separation of Z and E Isomers. With the same synthetic
protocol, O2-ethyl-protected MA/NO (13) was also prepared
in high yield starting from 10 via the formation of the
corresponding N-bromoethyl (11) and N-vinyl (12) inter-
mediates (Scheme 4). NMR analysis of this product was
consistent with that of the O2-benzyl analogue and suggested
the formation of 13 as an 11:1 mixture of two isomers,
presumably also having the Z and E conformations. Injection of
a purified mixture of these two isomers on an HPLC column
allowed a reasonable separation of the two peaks, which were
fused at the baseline (Figure 2A). Isolation of either form
followed by reinjection resulted in a chromatogram containing
both peaks with an integration ratio matching that of the
original mixture (Figure 2B,C). These results were confirmed
using HRMS detection. The measured m/z values for the [M +
H]+ ions of the two peaks were both 120.07692, which is in
excellent agreement (<1.5 ppm) with the calculated value for
the molecular formula C3H9N3O2, m/z 120.07675 (Figure 3).
The m/z value for the sodium adduct was extracted and plotted
as a function of time. This analysis showed that the taller peak
readily forms a sodium adduct, while such an adduct was not
detected for the smaller peak (see the Supporting Information).
These data are consistent with the view that 13 exists as a
mixture of Z and E isomers, with the Z isomer’s cis oxygens
being uniquely favorably oriented for bidentate coordination to
the alkali metal ion (see the Supporting Information). This
isomerization is presumably mediated through a proton shift
from the amine nitrogen (N3) to the diazeniumdiolate nitrogen
(N2) and/or direct ionization of the N3−H bond, thus
rendering largely single-bond character to the N1−N2 bond;
this allows free rotation about this bond, as indicated by
MA/NO analogues starting with the sodium salt of N-
diazeniumdiolated 2-(methylamino)ethanol (6),10 which was
used as a synthon. Preparation of the O2-benzyl-protected
diazeniumdiolate 7 and subsequent bromination with NBS/
Ph3P afforded the corresponding N-bromoethyl analogue 8.
Recently, we reported the use of 2,8,9-trimethyl-2,5,8,9-
tetraaza-1-phosphabicyclo[3.3.3]undecane (Verkade’s super
base) for dehydrohalogenation of diazeniumdiolate-containing
compounds under mild conditions.11 Reaction of 8 with this
base in acetonitrile was facile, but the desired N-vinyl derivative
9 was found to be unstable and could not be isolated. Acidic
hydrolysis of the in situ-generated 9 with p-toluenesulfonic acid
followed by purification of the crude reaction mixture afforded
a 69% isolated yield of O2-benzyl-protected MA/NO 4, whose
physicochemical properties were identical to those of the
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1
Figure 1. (A) Molecular structure and numbering scheme for the crystals of Z-4 (displacement ellipsoids are shown at the 50% level). (B) 1D H
NMR spectra of recrystallized Z-4 in CDCl3 at −20 °C, indicating that Z-4 and E-4 exist in a 66:1 ratio in solution.
theoretical calculations reported previously.8,12 (See Figure 1A
for the numbering scheme).
single peak was found for 13 at λmax = 241 nm at all pH from 1
to 11. As the pH was increased above 11, the peak intensity
decreased, and a new peak due to the ionization of the NH
proton emerged at λmax = 278 nm. Reacidification of the
alkaline solution restored the 241 nm peak at its original
intensity.
The pKa of the process was measured by adjusting the pH in
small increments between 11.0 and 12.0 until the 241 and 278
nm peaks were each at half-maximal intensity (Table 1). As the
extinction coefficients for the un-ionized and anionic forms of
13 were found to be 11.0 and 10.0 mM−1 cm−1, respectively,
UV Spectroscopic Determination of pKa. The potential
acidity of the NH bond in a purified mixture of Z-13 and E-13
was examined by UV spectroscopy using buffers of different
pH, following the similar procedure reported previously for the
analysis of O2-methyl IPA/NO.12 The extinction coefficients
were determined by dissolving a known amount of 13 in a
given quantity of ethanol, diluting with a large excess of 0.01 M
phosphate buffer for runs at pH 7.4−12, and then recording the
spectrum within 1−2 s of dilution at room temperature. A
C
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Scheme 4. Preparation of a Mixture of O2-Ethylated MA/NO
Isomers
broadening of the N-methyl and O-methylene signals was
observed for both isomers at pD 10.5 (Figure 4C). The
observed spectrum at pD 13.0 indicated ionization of the acidic
primary-amine proton to form the corresponding charge-
delocalized monoanionic species having a line-broadened
singlet and quartet for the N-methyl and O-methylene groups,
respectively (Figure 4D). Kinetic data for the exchange between
the anion and its conjugate acid were determined from the
signal broadening of the peak centered at 2.95 ppm using
previously described saturation-transfer and dynamic NMR
spectroscopic methods.8,14 The rate constant for exchange,
∼1.2 × 103 s−1, is similar to that of a structurally analogous
primary-amine diazeniumdiolate that was shown to have an
acidic NH bond.12 The interconversion barrier was found to be
17.4 kcal/mol, in close agreement with the results of previously
reported theoretical calculations for a similar molecule.8
Reacidification of this solution to pD 3.5 using DCl revealed
a re-equilibration with a Z/E ratio and spectral patterns
identical to those of the parent mixture at pD 7.5. Analysis of
this sample after 14 h at room temperature indicated no
significant change in the isomer ratio.
CONCLUSIONS
■
Our results reveal important similarities as well as differences
between the well-characterized iPrHN−N(O)NOR deriva-
tives and their methylamine analogues. While the sodium salt of
the former (R = Na+) can be stored indefinitely without
incident,15 salts of its analogue MA/NO have to date defied
isolation in pure form, even when the MA/NO reaction was
carried out at cryogenic temperatures. Both the IPA/NO and
MA/NO anions can be O-alkylated to produce stable
derivatives, but because the instability of MA/NO has
precluded isolation of the salts in reasonable yield, we had to
devise an indirect dealkylative synthesis in order to produce
enough MeHN−N(O)NOR (R = benzyl, ethyl) for efficient
characterization. The O-alkylated IPA/NO and MA/NO
derivatives were very similar to each other in their UV
properties, the pKa’s for ionization of their N−H bonds, and
the rate constants for protonation/deprotonation at pHs near
the pKa values. Additionally, there was evidence that the O-
alkylation products of both IPA/NO and MA/NO can
isomerize from the favored Z conformation (cis oxygens) to
the E form, but with one critical difference: the E isomer of
IPA/NO had to be trapped in an intramolecular cyclization
reaction to confirm its existence, while that of MeHN−N(O)
NOR proved to be directly observable as a constituent of the
equilibrium mixture. The isomers of the MA/NO derivatives
were partially separable. It may be that conditions can be found
that will permit a future MA/NO derivative to be isolated as a
pure E form, facilitating further insights into the chemistry (and
possibly the biology) of diazeniumdiolates having this thus far
little-studied configuration.
Figure 2. HPLC traces of a mixture of the two isomers of 13 observed
at 240 nm. (A) Chromatogram of the mixture of isomers purified by
silica gel chromatography. Both peaks have a λmax at 240 nm. (B)
Chromatogram obtained when the shaded portion of the first peak was
collected at 0 °C and immediately injected into the HPLC column,
showing re-equilibration. (C) Chromatogram obtained similarly to
(B), showing re-equilibration upon collection of the shaded portion of
the second peak.
the pH was adjusted so that the apparent extinction coefficients
were ∼5.5 and ∼5.0 mM−1 cm−1, respectively. Half-maximal
absorption intensities for both forms were observed at a pH of
∼11.70, and this pH value was taken as the pKa for 13.
Kinetic Studies. Ionization of the N−H bond in a mixture
of these two isomers at room temperature in D2O was also
examined by 1H NMR spectroscopy. Samples were run in D2O
adjusted to a pD value13 of either ∼8.5, ∼10.5, or ∼13.0 by
adding NaOD to solutions in D2O. Prominent exchange line
EXPERIMENTAL SECTION
■
Instrumentation. All reactions were performed under an inert
atmosphere. All chemicals were purchased from commercial sources
and used without further purification. UV spectra were recorded on a
diode array spectrophotometer. NMR spectra were collected with a
400 MHz spectrometer using appropriate deuterated solvents;
chemical shifts are reported in parts per million (ppm) downfield
from tetramethylsilane. The analytical HPLC/MS analyses were
performed using systems with a photodiode array UV−vis detector
D
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Figure 3. HRMS spectra for the two peaks of compound 13 in the extracted-ion chromatogram presented in the Supporting Information. Peak 1 has
[M + H]+ at m/z 120.07682. Peak 2 has [M + H]+ at m/z 120.07692 and [M + Na]+ at m/z 142.05889. The ion at m/z 121.051 is due to purine,
which was added as an internal reference to obtain high-resolution spectra of our analytes. All other ions are attributable to background interference.
and an accurate-mass quadrupole time-of-flight (Q-TOF) mass
spectrometer.
constant stirring. A solution of benzyl bromide (3.89 g, 22.8 mmol) in
5 mL of anhydrous DMF was slowly added. The reaction mixture was
then allowed to warm to room temperature, stirred for an additional
24 h, quenched with H2O, and extracted with ether (3 × 20 mL). The
combined organic layers were washed with water and dried over
anhydrous sodium sulfate. After concentration under reduced pressure,
the crude product was purified on a silica gel column (15:85 hexanes/
ethyl acetate) to afford 2.62 g (61% yield) of 7 as a hygroscopic, pale-
Reaction of Methylamine with NO. The equipment used for
conducting reactions with NO gas under anaerobic conditions has
been described previously.16,17 Nitric oxide was obtained in ultrahigh-
purity grade and allowed to stand in a ballast tank at a pressure of ∼5
atm over potassium hydroxide pellets for a minimum of several hours
before use. A 2 M solution of methylamine in THF (20 mL) was
diluted with 20 mL of diethyl ether and 10 mL of acetonitrile. The
resulting solution was placed in a standard thick-walled glass
hydrogenation bottle, cooled to −80 °C, flushed with nitrogen,
charged with 50 psi NO, and stirred for 2 h. After completion of the
reaction, ∼500 mg (∼10% yield) of the methylammonium salt of MA/
1
yellow oil. H NMR (CDCl3, 400 MHz): δ 7.40−7.32 (m, 5H), 5.22
(s, 2H), 3.66−3.62 (m, 2H), 3.34 (t, J = 5.2 Hz, 2H), 2.97 (s, 3H),
2.36 (br s, 1H). 13C NMR (CDCl3, 100 MHz): δ 135.5, 128.6, 128.5,
128.5, 75.7, 59.4, 57.1, 42.0. UV (MeOH) λmax (ε): 244 nm (10.0
mM−1 cm−1). Anal. Calcd for C10H15N3O3·0.3H2O: C, 52.07; H, 6.82;
N, 18.22. Found: C, 52.09; H, 6.66; N, 18.08.
NO was collected by filtration using a precooled Buchner funnel.
̈
While this product was still moist and cold, it was promptly suspended
in ether, cooled to −80 °C, and treated with 750 μL of 4.6 M sodium
methoxide in methanol; care was taken to use less than 1 equiv of
sodium methoxide as estimated from the mass of the crude
methylammonium salt. The resulting product was filtered while cold
to afford ∼140 mg (∼3% yield) of 3 as a white powder, which was
quickly stored under nitrogen at −20 °C. UV (0.01 M NaOH) λmax
(ε): 249 nm (8.3 mM−1 cm−1). (Caution! Compound 3 and its
methylammonium analogue are unstable and decompose, often
explosively, at ambient temperature.)
O2-Benzyl [N-(2-Bromoethyl)-N-methylamino]diazen-1-ium-
1,2-diolate (8). Compound 7 (1.37 g, 6.08 mmol) was dissolved in
15 mL of dichloromethane and cooled to 0 °C with constant stirring.
Triphenylphosphine (1.91 g, 7.30 mmol) was dissolved in 5 mL of
dichloromethane and added to the reaction mixture, which was then
stirred for 15 min. NBS (1.29 g, 7.30 mmol) was added in portions to
the reaction mixture over a period of 10 min, after which the mixture
was allowed to warm to room temperature and stirred overnight.
Solvent was removed on a rotary evaporator, and the crude product
was purified on a silica gel column (80:20 hexanes/ethyl acetate) to
1
O2-Benzyl [N-(2-Hydroxyethyl)-N-methylamino]diazen-1-
ium-1,2-diolate (7). Compound 610 (3.00 g, 19.1 mmol) was
dissolved in 15 mL of anhydrous DMF and cooled to 0 °C with
afford 1.20 g (69% yield) of 8 as a dark-yellow oil. H NMR (CDCl3,
400 MHz): δ 7.40−7.34 (m, 5H), 5.23 (s, 2H), 3.60 (t, J = 6.8 Hz,
2H), 3.35 (t, J = 6.8 Hz, 2H), 3.02 (s, 3H). 13C NMR (CDCl3, 100
E
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Isomeric Mixture of O2-Benzyl (N-Methylamino)diazen-1-
ium-1,2-diolate (Z-4 and E-4). Compound 8 (0.50 g, 1.7 mmol) was
dissolved in 4 mL of anhydrous acetonitrile and slowly added to a
stirring solution of 2,8,9-trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo-
[3.3.3]undecane (0.41 g, 1.91 mmol) in 3 mL of anhydrous
acetonitrile precooled to 0 °C. Subsequently, the reaction mixture
was warmed to room temperature and stirred for another 2 h while the
reaction was monitored by TLC. After the completion of the reaction,
the reaction mixture was recooled to 0 °C, and a solution of p-
toluenesulfonic acid (0.33 g, 1.74 mmol) in 1 mL of H2O was added.
The resulting mixture was stirred for 1 h, concentrated under reduced
pressure, and extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were washed with saturated brine solution and dried
over anhydrous sodium sulfate. After concentration under reduced
pressure, the crude product was purified on a silica gel column (40:60
hexanes/ethyl acetate) to afford 0.22 g (69% yield) of an 11:1 mixture
of the Z and E isomers of 4 as a white solid. Mp 48−50 °C. 1H NMR
of Z-4 (CDCl3, 400 MHz): δ 7.40−7.10 (m, 5H), 6.37−6.33 (m, 1H),
5.18 (s, 2H), 2.98 (d, J = 5.6 Hz, 3H). 13C NMR of Z-4 (CDCl3, 100
MHz): δ 135.7, 128.6, 128.6, 128.5, 75.4, 34.3. UV (MeOH) λmax (ε):
245 nm (12.1 mM−1 cm−1). Anal. Calcd for C8H11N3O2: C, 53.03; H,
6.12; N, 23.19. Found: C, 53.06; H, 6.02; N, 23.14.
Table 1. UV Absorbances of the Un-ionized and
Monoanionic Forms of 13 at 241 and 278 nm as Functions
of pH
absorbance (AU)
entry
pH
7.40
241 nm
278 nm
a
1
2
0.94
0
11.00
0.88
0.72
0.52
0.48
0.47
0.42
0.29
0.23
0.17
0
0.35
0.36
0.39
0.41
0.42
0.44
0.51
0.68
0.80
3
11.20
4
11.40
5
11.60
6
11.70 (pKa)
11.75
7
O2-Ethyl [N-(2-Hydroxyethyl)-N-methylamino]diazen-1-ium-
1,2-diolate (10). Compound 610 (3.00 g, 19.1 mmol) was dissolved
with constant stirring in a suspension of 1.00 g of finely powdered
potassium carbonate in 20 mL of precooled anhydrous methanol. To
this mixture, a solution of diethyl sulfate (4.11 g, 28.7 mmol) in 10 mL
of anhydrous methanol was slowly added. The reaction mixture was
allowed to warm to room temperature, stirred for an additional 10 h,
and filtered. The methanol was removed on a rotary evaporator, and
the residue was extracted with ethyl acetate (3 × 25 mL). The
combined organic layers were washed with saturated brine solution
and dried over anhydrous sodium sulfate. After concentration under
reduced pressure, the crude product was purified on a silica gel column
(20:80 hexanes/ethyl acetate) to afford 1.71 g (55% yield) of 10 as a
hygroscopic, pale-yellow oil. 1H NMR (CDCl3, 400 MHz): δ 4.17 (q, J
= 7.2 Hz, 2H), 3.72−3.57 (m, 2H), 3.26 (t, J = 5.2 Hz, 2H), 2.89 (s,
3H), 2.71 (br s, 1H), 1.25 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 100
MHz): δ 69.7, 59.3, 57.0, 42.0, 14.2. UV (MeOH) λmax (ε): 242 nm
(11.7 mM−1 cm−1). Anal. Calcd for C5H13N3O3·0.2H2O: C, 36.01; H,
8.10; N, 25.20. Found: C, 35.77; H, 7.82; N, 24.95.
8
11.80
9
11.90
10
12.00
b
11
14.00
0.85
a
b
ε = 11.0 mM−1 cm−1. ε = 10.0 mM−1 cm−1.
O2-Ethyl [N-(2-Bromoethyl)-N-methylamino]diazen-1-ium-
1,2-diolate (11). Compound 10 (3.00 g, 18.4 mmol) was dissolved
in 15 mL of dichloromethane and cooled to 0 °C with constant
stirring. Triphenylphosphine (9.64 g, 36.8 mmol) was dissolved in 5
mL of dichloromethane and added to the reaction mixture, which was
stirred for 15 min. NBS (6.51 g, 36.80 mmol) was added in portions to
the reaction mixture over a period of 10 min, after which the mixture
was allowed to warm to room temperature and stirred overnight.
Solvent was removed on a rotary evaporator, and the crude product
was purified on a silica gel column (80:20 hexanes/ethyl acetate) to
afford 2.75 g (74% yield) of 11 as a yellow oil. 1H NMR (CDCl3, 400
MHz): δ 4.20 (q, J = 7.2 Hz, 2H), 3.53 (t, J = 7.2 Hz, 2H), 3.35 (t, J =
6.8 Hz, 2H), 2.95 (s, 3H), 1.28 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3,
100 MHz): δ 69.8, 55.8, 42.0, 27.3, 14.3. UV (EtOH) λmax (ε): 244 nm
(10.6 mM−1 cm−1). Anal. Calcd for C5H12N3O2Br: C, 26.56; H, 5.35;
N, 18.59. Found: C, 26.81; H, 5.43; N, 18.25.
1
Isomeric Mixture of O2-Ethyl (N-Methylamino)diazen-1-ium-
1,2-diolate (Z-13 and E-13). Compound 11 (0.50 g, 2.21 mmol)
was dissolved in 4 mL of anhydrous acetonitrile and slowly added to a
stirring solution of 2,8,9-trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo-
[3.3.3]undecane (0.57 g, 2.65 mmol) in 3 mL of anhydrous
acetonitrile precooled to 0 °C. Subsequently, the reaction mixture
was warmed to room temperature and stirred for another 2 h while the
reaction was monitored by TLC. After the completion of the reaction,
the reaction mixture was recooled to 0 °C, and a solution of p-
toluenesulfonic acid (0.42 g, 2.21 mmol) in 1 mL of H2O was added.
The resulting mixture was stirred for 1 h, concentrated under reduced
pressure, and extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were washed with saturated brine solution and dried
Figure 4. H NMR spectra of the mixture of Z-13 and E-13 at 400
MHz revealing the slow exchange process accompanying ionization of
the N−H bond at room temperature. Spectra A−D were run in D2O
adjusted to measured pD values of ∼7.5, ∼8.5, ∼10.5, and ∼13.0,
respectively, by addition of NaOD. Spectrum E was run after the pD of
the sample used in D was adjusted back to 3.5 from 13.0 by addition of
DCl. Spectrum F shows the status of the pD 3.5 solution in D2O run
after 14 h.
MHz): δ 135.6, 128.7, 128.7, 128.6, 75.8, 55.9, 41.8, 27.3. UV (EtOH)
λmax (ε): 246 nm (9.17 mM−1 cm−1). Anal. Calcd for C10H14N3O2Br:
C, 41.68; H, 4.90; N, 14.58. Found: C, 41.44; H, 4.98; N, 14.29.
F
dx.doi.org/10.1021/jo3020837 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
over anhydrous sodium sulfate. After concentration under reduced
pressure, the crude product was purified on a silica gel column (40:60
hexanes/ethyl acetate) to afford 0.17 g (65% yield) of an 11:1 mixture
601−608. (b) Miranda, K. M. Coord. Chem. Rev. 2005, 249, 433−455.
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2009, 47, 1318−1324. (d) Paolocci, N.; Jackson, M. I.; Lopez, B. E.;
Miranda, K. M.; Tocchetti, C. G.; Wink, D. A.; Hobbs, A. J.; Fukuto, J.
M. Pharmacol. Ther. 2007, 113, 442−458.
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Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 708−713. (b) Espey, M. G.;
Miranda, K. M.; Thomas, D. D.; Xavier, S.; Citrin, D.; Vitek, M. P.;
Wink, D. A. Ann. N.Y. Acad. Sci. 2002, 962, 195−206.
(5) Arulsamy, N.; Bohle, D. S.; Holman, C. L.; Perepichka, I. J. Org.
Chem. 2012, 77, 7313−7318.
1
of the Z and E isomers of 13 as a pale-yellow oil. H NMR of Z-13
(CDCl3, 400 MHz): δ 4.25 (q, J = 8.4 Hz, 2H), 3.02 (d, J = 5.6 Hz,
3H), 1.37 (t, J = 7.2 Hz, 3H). 13C NMR of Z-13 (CDCl3, 100 MHz):
δ 69.4, 34.3, 14.3. UV (MeOH) λmax (ε): 245 nm (11.1 mM−1 cm−1).
HRMS (ESI) m/z: calcd for C3H9N3O2 [M + H]+, 120.07675; found,
120.07692; Δ = 1.4 ppm. Anal. Calcd for C3H9N3O2·0.076EtOAc: C,
31.54; H, 7.70; N, 33.40. Found: C, 31.14; H, 7.65; N, 33.00.
HPLC Analysis. HPLC separations of the mixture of the two
isomeric forms of 13 (Figure 2) were performed using an LC
instrument connected to a photodiode array UV−vis detector. A
reversed-phase C18 column (150 mm × 4.6 mm) with a particle size
of 5 μm and a guard column of the same material, both at 25 °C, were
used for the stationary phase. The mobile phase used for the
separation was composed of an isocratic solution of 30% acetonitrile in
water containing 0.1% formic acid. The eluate was monitored at a
wavelength of 250 nm. The fractions were collected manually at room
temperature and immediately cooled to 0 °C and preserved at that
temperature until further analysis.
(6) (a) Arulsamy, N.; Bohle, D. S.; Perepichka, I. Can. J. Chem. 2007,
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(7) (a) Drago, R. S.; Karstetter, B. R. J. Am. Chem. Soc. 1961, 83,
1819−1822. (b) Miranda, K. M.; Katori, T.; Torres de Holding, C. L.;
Thomas, L.; Ridnour, L. A.; McLendon, W. J.; Cologna, S. M.; Dutton,
A. S.; Champion, H. C.; Mancardi, D.; Tocchetti, C. G.; Saavedra, J. E.;
Keefer, L. K.; Houk, K. N.; Fukuto, J. M.; Kass, D. A.; Paolocci, N.;
Wink, D. A. J. Med. Chem. 2005, 48, 8220−8228. (c) Andrei, D.;
Salmon, D. J.; Donzelli, S.; Wahab, A.; Klose, J. R.; Citro, M. L.;
Saavedra, J. E.; Wink, D. A.; Miranda, K. M.; Keefer, L. K. J. Am. Chem.
Soc. 2010, 132, 16526−16532.
ASSOCIATED CONTENT
* Supporting Information
■
(8) Wang, Y.-N.; Bohle, D. S.; Bonifant, C. L.; Chmurny, G. N.;
Collins, J. R.; Davies, K. M.; Deschamps, J.; Flippen-Anderson, J. L.;
Keefer, L. K.; Klose, J. R.; Saavedra, J. E.; Waterhouse, D. J.; Ivanic, J. J.
Am. Chem. Soc. 2005, 127, 5388−5395.
S
Full 1H and 13C NMR spectra for new compounds,
crystallographic data for compound Z-4 (CIF), details of the
dynamic NMR experiment, pKa studies, and chromatographic
isomer separation. This material is available free of charge via
the Internet at http://pubs.acs.org.
(9) Dutton, A. S.; Suhrada, C. P.; Miranda, K. M.; Wink, D. A.;
Fukuto, J. M.; Houk, K. N. Inorg. Chem. 2006, 45, 2448−2456.
́
(10) Velazquez, C. A.; Chen, Q.; Citro, M. L.; Keefer, L. K.; Knaus, E.
E. J. Med. Chem. 2008, 51, 1954−1961.
AUTHOR INFORMATION
Corresponding Author
*Email: biswasd@mail.nih.gov; saavedjo@mail.nih.gov
(11) Hong, S. Y.; Saavedra, J. E.; Keefer, L. K.; Chakrapani, H.
Tetrahedron Lett. 2009, 50, 2069−2071.
■
(12) Saavedra, J. E.; Bohle, D. S.; Smith, K. N.; George, C.;
Deschamps, J. R.; Parrish, D.; Ivanic, J.; Wang, Y.; Citro, M. L.; Keefer,
L. K. J. Am. Chem. Soc. 2004, 126, 12880−12888.
Notes
The authors declare no competing financial interest.
(13) The pD value of each solution was taken as pD = pH + 0.4. See:
Keefer, L. K.; Hrabie, J. A.; Hilton, B. D.; Wilbur, D. J. Am. Chem. Soc.
1988, 110, 7459−7462 and ref 28 therein.
ACKNOWLEDGMENTS
■
(14) Sandstrom, J. Dynamic NMR Spectroscopy; Academic Press:
We thank Mr. John Klose (LPAT-ATP, SAIC-Frederick Inc.,
Frederick National Laboratory for Cancer Research) for
assistance in NMR determination of the exchange rate constant
and Dr. Sergey Tarasov and Ms. Marzena A. Dyba (Biophysics
Resource in the Structural Biophysics Laboratory, Frederick
National Laboratory for Cancer Research) for assistance with
HRMS. This project was funded by the National Cancer
Institute (NCI), National Institutes of Health (NIH), under
Contract HHSN261200800001E and by the Intramural
Research Program of the NIH, NCI, Center for Cancer
Research. Crystallographic studies were supported by the
National Institute on Drug Abuse (NIDA) under Contract Y1-
DA1101 and by the Naval Research Laboratory.
̈
London, 1982; p 78.
(15) Salmon, D. J.; Torres de Holding, C. L.; Thomas, L.; Peterson,
K. V.; Goodman, G. P.; Saavedra, J. E.; Srinivasan, A.; Davies, K. M.;
Keefer, L. K.; Miranda, K. M. Inorg. Chem. 2011, 50, 3262−3270.
(16) Hrabie, J. A.; Klose, J. R.; Wink, D. A.; Keefer, L. K. J. Org.
Chem. 1993, 58, 1472−1478.
(17) Keefer, L. K.; Nims, R. W.; Davies, K. M.; Wink, D. A. Methods
Enzymol. 1996, 268, 281.
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