Aminolysis of an N-diazeniumdiolated amidine as an approach to diazeniumdiolated ammonia

Article abstract of DOI:10.1021/jo500551n

Recent theoretical studies have suggested that the parent diazeniumdiolate ion, H₂N-N(O)=NO^- ( diazeniumdiolated ammonia ), might be stable enough to be isolated and that it could potentially serve as a uniquely advantageous prodrug form of bioactive nitroxyl (HNO). Here, we report on an attempt to isolate its O²-benzylated derivative by aminolysis of the C=N bond in PhC(NH₂)=N-N(O)=NOBn. The reaction proved remarkably sluggish in comparison to aminolysis of unsubstituted benzamidine, and the desired product could not be isolated, apparently because of base sensitivity of the NH₂ group. Consistent with this interpretation, O-benzylhydroxylamine and N₂O were recovered from the reaction mixture in high yields, along with N,N′-dibutylbenzamidine. Theoretical calculations rationalize the observed slow aminolysis by demonstrating that the diazeniumdiolate group greatly suppresses the electrophilicity of the adjacent C=N carbon center, rendering attack at that position endothermic. The data provide significant insights into the challenges inherent to the pursuit of diazeniumdiolated ammonia.

Full text of DOI:10.1021/jo500551n

Article
pubs.acs.org/joc
Aminolysis of an N‑Diazeniumdiolated Amidine as an Approach to
Diazeniumdiolated Ammonia
Debanjan Biswas,† Joseph A. Hrabie,† Joseph E. Saavedra,‡ Zhao Cao,‡ Larry K. Keefer,† Joseph Ivanic,§
and Ryan J. Holland*,†
‡
§
†Drug Design Section, Chemical Biology Laboratory, and Basic Science Program and Advanced Biomedical Computing Center,
Information Systems Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick,
Maryland 21702, United States
S
* Supporting Information
ABSTRACT: Recent theoretical studies have suggested that the
parent diazeniumdiolate ion, H2N−N(O)NO− (“diazeniumdio-
lated ammonia”), might be stable enough to be isolated and that it
could potentially serve as a uniquely advantageous prodrug form of
bioactive nitroxyl (HNO). Here, we report on an attempt to isolate
its O2-benzylated derivative by aminolysis of the CN bond in
PhC(NH2)N−N(O)NOBn. The reaction proved remarkably
sluggish in comparison to aminolysis of unsubstituted benzamidine,
and the desired product could not be isolated, apparently because
of base sensitivity of the NH2 group. Consistent with this
interpretation, O-benzylhydroxylamine and N2O were recovered
from the reaction mixture in high yields, along with N,N′-dibutylbenzamidine. Theoretical calculations rationalize the observed
slow aminolysis by demonstrating that the diazeniumdiolate group greatly suppresses the electrophilicity of the adjacent CN
carbon center, rendering attack at that position endothermic. The data provide significant insights into the challenges inherent to
the pursuit of diazeniumdiolated ammonia.
recently, primary amine diazeniumdiolates have been shown to
hydrolyze according to two mechanisms running in parallel, one
mimicking the NO-forming pathway of eq 1 and the other
initiated by N-protonation at a different site to generate similar
amounts of a second nitrogenous bioeffector of current intense
interest, nitroxyl (HNO).3 The divergent primary amine
pathways are shown in eq 2.
INTRODUCTION
■
Reaction of nitric oxide (NO) with a variety of aliphatic amines
generates diazeniumdiolate ions [R1R2N−N(O)NO−].1,2
Secondary amine derivatives (R1 ≠ H ≠ R2) have become
important tools in studying the chemistry and biology of NO by
virtue of their tunable rates and extents of hydrolysis to
regenerate the starting materials, as shown in eq 1.2 More
If replacing one N-alkyl group of the “pure” NO donor shown
in eq 1 with a proton converts it to a mixed NO/HNO prodrug,
one might wonder whether a “pure” HNO donor might result if
both alkyl groups were removed, producing the parent
diazeniumdiolate structure, “diazeniumdiolated ammonia” (eq
3). Indeed, recent calculations have indicated that the species
would produce NO only very slowly, suggesting that HNO
release would take over as the default hydrolysis pathway,
potentially producing HNO with nontoxic dinitrogen and water
as the only byproducts (eq 3).4
Here we describe our initial attempts to prepare a derivative of
diazeniumdiolated ammonia for studies of its fundamental
physicochemical properties. The data provide important
information about its reactivity.
Received: March 7, 2014
Published: April 25, 2014
© 2014 American Chemical Society
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RESULTS AND DISCUSSION
■
Synthetic Approach. Unlike secondary and primary amines,
ammonia has shown no tendency to react directly with NO.
Placing a solution of ammonia in methanol or tetrahydrofuran in
contact with 3 atm of NO led to no observable gas uptake,
precipitation, or development of an ultraviolet peak at the
characteristic diazeniumdiolate wavelength of 250 nm.
Even if such a direct NO/NH3 reaction were to occur, the
product might be too reactive to isolate. While secondary amine
diazeniumdiolate salts are generally stable in the absence of
protonation sources, their primary amine counterparts are highly
pH-sensitive and prone to rapid decomposition.3 Primary amine
diazeniumdiolate ions can be stabilized by O-derivatization,5
however, so we decided to pursue O-benzylated derivative H2N−
N(O)NOBn (1) as our initial synthetic target.
As starting material, we chose known compound 2, already
available from a previous study.6 Early attempts at hydrolyzing 2
under either basic or acidic conditions were unsuccessful, so we
resorted to aminolysis by n-butylamine as a possible means of
breaking the CN bond to free the desired ammonia derivative.
This reaction was chosen on the basis of a previous report7 on the
aminolysis of benzamidine with n-butylamine to produce N-
butylbenzamidine and N,N′-dibutylbenzamidine, which were
fully characterized. Our expectation was that 2 would follow a
similar reaction course to produce 1 in addition to the alkylated
benzamidines.
Aminolysis of 2. Accordingly, 2 was dissolved in n-
butylamine to make a 3.7 mM solution and heated at 70 °C.
The time course of the reaction was monitored by LC−MS. After
1 h, significant amounts of one expected product, N-
butylbenzamidine 3 (along with its further aminolysis product
N,N′-dibutylbenzamidine 4), were seen, but there was no trace of
the desired product 1. After 30 h, the starting material was almost
gone, the same two butylated amidines were the major products,
and a tiny amount of mono-N-butylated 2 (structure 5 in Scheme
1) was detected, again without any evidence of the presence of 1.
To probe the origins of the significant signal associated with
the void volume in these chromatograms, a different chromato-
graphic system was used to identify the remaining products and
establish a material balance. Again, no trace of 1 was seen.
Instead, a near-quantitative yield of O-benzylhydroxylamine (6)
Figure 1. Time course of reaction of 2 with butylamine. Total ion
chromatograms show the rates of starting material (2) consumption and
product (3, 4, and 5) formation. Time points were taken at 1, 10, and 30
h for panels A, B, and C, respectively. Product 6 (not shown in these
chromatograms) was identified in a separate chromatographic treatment
of the same reaction.
was found by LC−MS, along with a high recovery of nitrous
oxide as determined by gas chromatography (Figure S1,
Supporting Information). The overall transformation is shown
in Scheme 1, while its time course is shown in Figure 1.
Products were identified as follows: 3, m/z obsd = 177.1382
m/z, calcd = 177.1386; 4, m/z obsd = 233.2018, m/z calcd =
233.2012; 5, m/z obsd = 327.1815, m/z calcd = 327.1816.
Product 6, m/z = 124.0764, separated with distinct chromato-
graphic conditions, was identified and quantified by LC−MS
comparison to an authentic standard, m/z = 124.0761. The
structures of product 3 and product 4 were confirmed via
Scheme 1. Aminolysis of 3.7 mM 2 in n-Butylamine Solution at 70 °C
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1
purification and H NMR (Figures S2 and S3, Supporting
Information, respectively).
Mechanistic Considerations. While various mechanisms
can be conceived, we rationalize the above results as follows. The
first step is postulated to be attack by the amine at the CN
carbon of 2 to generate tetrahedral intermediate 7, shown in
Scheme 2. Of its three C−N bonds, cleavage of the NH2 group of
Scheme 2. Pathways Leading to Decomposition of
Tetrahedral Intermediate 7
7 to form 5 was very slow, with only a small yield being observed
after many hours. Cleavage to expel the butylamine substituent
would simply regenerate starting material 2. The fastest
nondegenerate reaction was loss of the diazeniumdiolated
nitrogen to release 3 and 1, with the latter undergoing a series
of deprotonation/tautomerization steps as represented in
Scheme 1, ultimately leading to nitrous oxide and hydroxylamine
6 in good yields.
Kinetic Comparisons. To probe the effect of N-
diazeniumdiolating an amidine group on its susceptibility to
nucleophilic attack at the CN carbon, we reacted benzamidine
itself with butylamine under the conditions used in the
aminolysis of 2. The results are shown in Figure 2. Benzamidine
quickly gave way to 3, which was then converted to 4. The time
courses of reacting benzamidine vs 2 with butylamine are
compared in Figure 3. It is apparent that the presence of the
diazeniumdiolate substituent has a pronounced dampening effect
on the electrophilicity of the imine carbon, since a significant
quantity of 2 remains even after a 30-h reaction.
Figure 2. Reaction of 3.7 mM benzamidine with n-butylamine solvent at
70 °C. Total ion chromatograms showing the rates of starting material
(benzamidine) consumption and product (3 and 4) formation. Time
points were taken at 1, 3, and 18 h (panels A, B, and C, respectively).
Computational Chemistry Study Comparing Aminol-
ysis Rates of 2 and Benzamidine. In an effort to explain the
unexpectedly sluggish aminolysis rate of 2, we performed a
computational chemistry study comparing rate-determining
aminolysis reaction energetics of 2 and benzamidine. The
theoretical methodology we have used has been shown to
provide very reliable predictions of chemical properties for these
types of systems when compared against experimental
observations.4,8 For facility we have used methylamine (instead
of n-butylamine) and the O2-methyl (instead of benzyl)
derivatives of 2 (2a) and 1 (1a), as we expect that the results
will be commensurate. Lowest energy structures were located for
all species occurring in reactions 4 and 5, with the only structural
constraint being a Z conformation for the N(O)NO fragment in
2a and 1a.
Figure 3. Relative rates of aminolysis of 2 (red line) vs benzamidine
(blue) in n-butylamine at 70 °C. Apparently, the presence of the
diazeniumdiolate group on a benzamidine nitrogen greatly diminishes
the electrophilicity of the carbon atom of the adjacent CN double
bond.
2a + MeNH2 → 1a + PhC(NMe)NH2
(4)
PhC(NH)NH2 + MeNH2
simply attributed to the endothermicity of the first reaction step
while the exothermicity of benzamidine aminolysis gives rise to a
comparably faster rate of reaction. Experimental confirmation of
these predicted free energy changes should be possible in
principle; however, the essentially immediate decomposition of 1
via release of N2O would make such a determination for 2
difficult. In fact, it may be that this quick degradation helps drive
→ PhC(NMe)NH2 + NH3
(5)
The resultant computed changes in free energies, ΔG310(H2O
solvent), for the aminolysis reactions of 2a and benzamidine are
3.78 and −5.45 kcal/mol, respectively. Therefore, the slow
aminolysis rate of 2 (assuming a similar behavior to 2a) can be
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Figure 4. Optimized MP2/ADZ [PCM] structures of 2a (A) and benzamidine (B) together with selected bond lengths (Å) and Mulliken charges (red)
where the value of −0.06 corresponds to the O2-methyldiazenium diolate group (sum over all of its atoms).
the aminolysis of 2 forward, albeit very slowly; i.e., the reaction is
akin to a slow leak.
CONCLUSION
■
Our results show that the presence of a diazeniumdiolated amine
group attached to an imine nitrogen of an amidine dramatically
slows the rate-determining step of its aminolysis, namely
nucleophilic attack at the imine carbon, while also playing a
dominant role in the subsequent product-determining step.
Thus, presumed tetrahedral intermediate 7 formed on attack of
butylamine on the CN carbon of 2 eliminated the elements of
1 far more rapidly than it expelled ammonia. Theoretical
calculations confirmed that the presence of a diazeniumdiolate
group increases the electron density at the imine carbon of 2a,
rendering the initial nucleophilic attack endothermic.
The results also point to base sensitivity on the part of the
desired O2-protected diazeniumdiolate 1, whose NH2 group’s
acidity allows the two protons to be ionized or to tautomerize
readily to neighboring basic sites en route to complete removal.
Loss of N2O from the resulting intermediate leaves O-
benzylhydroxylamine in near-quantitative yield.
Although the observed slow aminolysis rate of 2 relative to that
of benzamidine can be rationalized via reaction energetics, it is
also instructive to investigate the electrophilicity of the imine
carbon center in the two systems. Figure 4 shows the optimized
structures of 2a and benzamidine together with selected bond
lengths and Mulliken atom charges.
When comparing 2a against benzamidine we find that the
imine CN bond length in the former (1.323 Å) is noticeably
longer than that of the latter (1.304 Å) while the formally
designated single C−N bond length of 2a (1.352 Å) is shorter
than its benzamidine counterpart (1.382 Å). Furthermore, the
C−NH2 group is more nearly planar in 2a as indicated by the
dihedral angle between the two planes defined by atoms H−N−
C and H′−N−C (θ = 159°) which is noticeably larger than that
for benzamidine (θ = 139°). The results suggest that in 2/2a
there is some resonance of the type shown in Scheme 3 that
While the desired diazeniumdiolated ammonia derivative 1
was neither isolated from the above-described experiments nor
directly observed as an intermediate, our data strongly suggest
that it was generated in essentially theoretical yield, only to be
rapidly destroyed through a series of prototropic and base-
induced reactions to form N2O and O-benzylhydroxylamine 6,
both of which were also recovered in high yields.
Scheme 3. Resonance of the π Bond Occurring in 2/2a
As for attempts to isolate diazeniumdiolated ammonia
derivatives for studies of their physicochemical properties and
possible pharmacological utility, future work will seek to avoid
the basic conditions that proved problematic in our aminolysis
reactions, focusing instead on neutral and acidic conditions in an
effort to minimize the influence of the prototropic equilibria that
lead to fragmentation of the H2N−N(O)NOR.
results in delocalization of the π bond. The 60:40 ratio of the two
resonance forms was crudely estimated by comparing the C−N
bond length differences between benzamidine and 2a.
Indeed, the computed Mulliken atomic charges support this
notion whereby the imine nitrogen in 2a has a more negative
charge (−0.73) than in benzamidine (−0.53). Additionally, the
imine carbon is less positive in 2a (+0.18) than in benzamidine
(+0.29).
The comparative bond lengths and charges indicate that
nucleophilic attack on the imine double bond is more difficult for
2/2a than benzamidine since (i) the π electrons are delocalized
over three centers in 2/2a and (ii) the electrophilicity of the 2/2a
imine carbon is subdued, relative to benzamidine, due to its lower
positive charge. Therefore, we must conclude that the
diazeniumiolate group acts to greatly suppress the reactivity of
the imine CN group.
EXPERIMENTAL SECTION
General Methods. Compound 2 was prepared as previously
described.6 All other compounds used here including authentic
compound 6 were obtained from commercial sources.
■
Aminolysis Conditions. In their respective reactions either 7 mg of
compound 2 or 3 mg of benzamidine was dissolved in 7 mL of neat
butylamine, for a final concentration of 3.7 mM, in a tightly sealed
reaction vessel and heated to 70 °C. At each reported time point the
reaction was allowed to cool to room temperature before aliquots were
removed for dilution and analysis. Each aliquot was diluted 100-fold in
acetonitrile and analyzed by LC−MS/MS. All compounds were
separated and identified with an HPLC coupled with a Quadrupole
Time-of-Flight (Q-TOF) mass spectrometer. Separations of all
compounds, with the exception of compound 6, were performed on a
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reversed-phase C18 column, 3 μm, 2.1 × 150 mm, at a flow rate of 0.2
mL/min under H2O/acetonitrile/0.1% formic acid gradient conditions.
Separation of compound 6 was performed on a polar end-capped C18
column, 3-μm, 2.1 × 150 mm, at a flow rate of 0.2 mL/min under H2O/
acetonitrile gradient conditions. Positive ions were generated by a dual
electrospray source with a 150 V fragmenter voltage.
Time Course of N2O Evolution. Reactions were run according to
the conditions presented above with the following exceptions. Increased
quantities of compound 2 (14 mg) and butylamine (14 mL) were used.
Each reaction was run under a constant stream of N2, which flowed out
of a condensing column and into a gas chromatography system with an
electron capture detector. A high surface area carbon molecular sieve
column (2 m × 2.0 mm i.d., 80/100 mesh) was used with helium as
carrier gas. The GC operation conditions were as follows: injector and
detector temperatures were at 250 °C, oven temperature was
programmed from 90 to 200 °C at 20 °C/min and held at 200 °C for
1.1 min. Helium flow was 30 mL/min, and nitrogen was used as makeup
gas at 2 mL/min.
Computational Chemistry. Computations were performed using
the second-order Møller−Plesset perturbation theory (MP2)9−11
method together with the correlation-consistent aug-cc-pVDZ (ADZ)
basis set.12,13 Water solvent effects were modeled by use of the polarized
continuum model (PCM),14−16 whereby the density of tesserae on the
cavity surface was set to 240. Geometries were optimized using analytical
gradients,12 and Hessians were computed seminumerically with analytic
gradients so that Gibbs free energies at 310 K could be determined.
Molecular structures were illustrated using MacMolPlt.17 All
computations were performed using the GAMESS package.18
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ASSOCIATED CONTENT
■
S
* Supporting Information
Time course of N2O generation in the aminolysis of 2; selected
NMR spectra; Cartesian coordinates and energies for all the
computed structures. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: hollandrj@mail.nih.gov.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project has been funded with federal funds from the
National Cancer Institute, National Institutes of Health, under
Contract No. HHSN261200800001E, and by the Intramural
Research Program of the NIH, National Cancer Institute, Center
for Cancer Research. We thank Dr. Sergey Tarasov and Ms.
Marzena A. Dyba of the Biophysics Resource in the Structural
Biophysics Laboratory, NCI-Frederick, for assistance with the
high-resolution mass spectrometry studies.
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