Bis-diazeniumdiolates of dialkyldiamines: Enhanced nitric oxide loading of parent diamines

Article abstract of DOI:10.1021/ol050541z

(Chemical Equation Presented) The synthesis and characterization of a series of symmetric bis-dialkyldiamine-based diazeniumdiolates, RN[N(O)NO ^-Na⁺](CH₂)_xN[N(O)NO ^-Na⁺]R′, are reported. Preparation of corresponding intramolecular diazeniumdiolates of the form RN[N(O)NO]^-(CH ₂)_xNH₂⁺R′ with alkyl groups > (CH₂)₄CH₃ have been shown previously to lack stability. In contrast, sodium-stabilized bis-diazeniumdiolates of such lipophilic species can be readily formed when these same diamines are reacted with NO in basic media. The resulting compounds release 4 mol of NO per mole of original diamine. This approach enables the synthesis of more lipophilic NO donors than previously possible.

Full text of DOI:10.1021/ol050541z

ORGANIC
LETTERS
2005
Vol. 7, No. 14
2813-2816
Bis-diazeniumdiolates of
Dialkyldiamines: Enhanced Nitric Oxide
Loading of Parent Diamines
Melissa M. Reynolds, Zhengrong Zhou, Bong K. Oh, and Mark E. Meyerhoff*
The UniVersity of Michigan, Department of Chemistry, 930 North UniVersity AVenue,
Ann Arbor, Michigan 48109-1055
mmeyerho@umich.edu
Received March 11, 2005 (Revised Manuscript Received May 25, 2005)
ABSTRACT
-
+
-
+
The synthesis and characterization of a series of symmetric bis-dialkyldiamine-based diazeniumdiolates, RN[N(O)NO Na ](CH2)xN[N(O)NO Na ]R′,
are reported. Preparation of corresponding intramolecular diazeniumdiolates of the form RN[N(O)NO] (CH2)xNH2
-
+
R′ with alkyl groups > (CH2)4-
CH3 have been shown previously to lack stability. In contrast, sodium-stabilized bis-diazeniumdiolates of such lipophilic species can be
readily formed when these same diamines are reacted with NO in basic media. The resulting compounds release 4 mol of NO per mole of
original diamine. This approach enables the synthesis of more lipophilic NO donors than previously possible.
Nitric oxide (NO) is known to regulate vascular tone,1 kill
cancer cells,2 prevent the activation of platelets,3 and serve
as a neurotransmitter.4 As more is learned about the
biological roles of NO, a substantial body of research has
focused on synthesizing compounds that will store NO and
subsequently release this free-radical species under physi-
ological conditions.5
NO adducts) have been developed and can be classified
within three broad categories: intramolecularly stabilized
species (2), anionic (3), and protected (4) (Figure 1), all of
Among a host of organic NO donor species reported to
date,5,6 diazeniumdiolates have emerged as attractive can-
didates for direct use as pharmacological agents2,5-7 as well
as dopants within polymeric materials to create more
biocompatible polymers.8,9 Diazeniumdiolates (nucleophilic
(1) Maragos, C. M.; Morley, D.; Wink, D. A.; Dunams, T. M.; Saavedra,
J. E.; Hoffman, A.; Bove, A. A.; Isaac, L.; Hrabie, J. A.; Keefer, L. K. J.
Med. Chem. 1994, 34, 3242-3247.
(2) Wu, X. A.; Janczuk, J. J.; Cai, T.: Xian, M.; Wen, Z.; Wang, P. G.
Exp. Opin. Ther. Pat. 2002, 12, 819-826.
Figure 1. Structures of diamines and diazeniumdiolates where R,
R′, and R′′ are side groups, x is an integer, and M+ is a postively
charged species.
(3) Annich, G. M.; Meinhardt, J. P.; Mowery, K. A.; Ashton, B. A.;
Merz, S. I.; Hirshchl, R. B.; Meyerhoff, M. E.; Bartlett, R. H. Crit. Care
Med. 2000, 28, 915-920.
(4) Marletta, M. A.; Tayeh, M. A.; Hevel, J. M. Biofactors 1990, 2, 219-
225.
(5) Hrabie, J. A.; Keefer, L. K. Chem. ReV. 2002, 102, 1135-1154.
(6) Wang, P. G.; Xian, M.: Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk,
A. Chem. ReV. 2002, 102, 1091-1134.
which have been tested in vivo and shown to be biologically
active. For example, anionic diazeniumdiolates, such as the
diazeniumdiolate of proline (PROLI/NO), have been shown
(7) Saavedra, J. E.; Keefer, L. K. Chem. Br. 2000, 36, 30-33.
10.1021/ol050541z CCC: $30.25
© 2005 American Chemical Society
Published on Web 06/09/2005

to prevent cerebral spasms,10 and the protected adduct of
PYRRO/NO, V-PYRRO/NO, has been shown to be a liver-
selective NO donor.11 In addition, numerous zwitterionic
diazeniumdiolates such as (Z)-1-[N-methyl-N-[6-(N-methyl-
ammoniohexyl)amino]]diazen-1-ium-1,2-diolate (MAHMA/
NO) (see Figure 1, compound 2; where R,R′ ) CH3, x ) 6)
and the diazeniumdiolate of diethylene triamine (DETA/NO)
have been used to inhibit platelet aggregation, lower blood
pressure,12 and prevent overproliferation of cells in the blood
vessel wall after arterial injury.11,13
Scheme 1
Because of the recent success in using diamines as NO
storage and delivery agents, we sought to improve the NO-
loading efficiency of symmetrical parent diamines, especially
more lipophilic dialkyldiamines that could potentially be
doped into polymers to create NO releasing polymeric
materials that are thromboresistant owing to NO’s potent
inhibition of platelet function. In this work, we demonstrate
that NO can be reacted with symmetrical dialkyldiamine
compounds, including MAHMA as well as much more
lipophilic analogues (of the form RNH(CH2)6NHR), to
generate doubly loaded diazeniumdiolate adducts that can
release 4 mol of NO per mole of initial diamine species. In
addition, we show that this methodology is not limited to
diamines with a hexamethylene spacer by achieving similarly
enhanced NO loading with substrates of the form RNH-
(CH2)3NHR. Finally, we compare the initial rates of NO
release of these bis-adducts with their zwitterionic counter-
parts.
Hrabie et al. originally demonstrated that diamines of the
form RNH(CH2)xNHR′ can react with NO under medium-
pressure conditions to form diazeniumdiolates, with stabiliza-
tion believed to exist as a result of hydrogen bonding between
the terminal oxygen of the diazeniumdiolate moiety and the
hydrogen on the ammonium nitrogen (Scheme 1A).14 How-
ever, as the lipophilicity of these parent diamine compounds
increases, the ability to form stable diazeniumdiolates of this
type dramatically decreases.9 Indeed, the most lipophilic
intramolecular diamine diazeniumdiolates (2f,g) cannot be
isolated in the presence of air. Exposure to oxygen results
in immediate decomposition to the corresponding dialkyl-
hexamethylenediamine ammonium nitrite salt. The nitrite salt
decomposition product has also been observed by Drago and
co-workers for the anionic diazeniumdiolate of diethyl-
amine.15 However, longer air exposure times at room
temperature were required (i.e., days) before this decomposi-
tion was observed compared to 2f and 2g, which were found
to decompose within seconds.9,15
The reason for this lack of diazeniumdiolate stability with
increased side chain length is not yet known. However,
destabilization presumably occurs because the hydrogen bond
formed between the hydrogen on the ammonium nitrogen
and the oxygen on the diazeniumdiolate is significantly
weakened. This may be attributed to the flexibility of the
longer alkyl chains disrupting the H-bonding interaction.
Normally, this hydrogen bond stabilizes the zwitterionic
complexes.14 In view of this, we sought to eliminate this
dependency on the hydrogen bond stabilization and use
exogenous cations (sodium) to stabilize the corresponding
diazeniumdiolates of the dialkyldiamines. Beyond obtaining
stable diazeniumdiolates of the most lipophilic diamine
structures, the use of exogenous cations has the potential to
greatly enhance the NO storage capability of these species,
by enabling the formation of bis-diazeniumdiolate type
structures (see 5a-i).
(8) Smith, D. J.; Chakravarthy, D.; Pulfer, S.; Simmons, M. L.; Hrabie,
J. A.; Citro, M. L.; Saavedra, J. E.; Davies, K. M.; Husell, T. C.;
Moorandian, D. L.; Hanson, S. R.; Keefer, L. K. J. Med. Chem. 1996, 39,
1148-1156.
(9) Batchelor, M. M.; Reoma, S. L.; Fleser, P. S.; Nuthakki, V. K.;
Callahan, R. E.; Shanley, C. J.; Politis, J. K.; Elmore, J.; Merz, S. I.;
Meyerhoff, M. E. J. Med. Chem. 2003, 46, 5153-5161.
(10) Saavedra, J. E.; Southan, G. J.; Davies, K. M.; Lundell, A.; Markous,
C.; Hanson, S. R.; Adrie, C.; Hurford, W. E.; Zapol, W. M.; Keefer, L. K.
J. Med. Chem. 1996, 39, 4361-4365.
(11) Saavedra, J. E.; Billiar, T. R.; Williams, D. L.; Kim, Y. M.; Watkins,
S. C.; Keefer, L. K. J. Med. Chem. 1997, 40, 1947-1954.
(12) Diodati, J. G.; Quyyumi, A. A.; Keefer, L. K. J. CardioVasc.
Pharmacol. 1995, 25, 674-678.
(13) Mooradian, D. L.; Hutsell, T.; Keefer, L. K. J. CardioVasc.
Pharmacol. 1995, 25, 674-678.
(14) Hrabie, J. A.; Klose, J. R.; Wink, D. A.; Keefer, L. K. J. Org. Chem.
1993, 58, 1472-1476.
Incorporating sodium trimethylsilanolate salt during the
NO addition reaction with the dialkyldiamines allows air-
stable bis-diazeniumdiolates to be prepared (Scheme 1B).
For all diamines examined, the sodium-stabilized bis-
diazeniumdiolates (5a-i) were the only products observed.
The products can store twice as much NO in one molecule
and, more importantly, increasingly more lipophilic NO
donors can be synthesized that remain stable in the presence
(15) Ragsdale, R. O.; Karstetter, B. R.; Drago, R. S. Inorg. Chem. 1965,
4, 420-422.
2814
Org. Lett., Vol. 7, No. 14, 2005

of air. For example, an NO donor with a parent structure of
1g (with a log P > 12; P ) octanol/water partition
coefficient) can be synthesized and stored under ambient
conditions without significant loss of NO. Symmetrical
diamines with three methylenes between the amine nitrogens
can also be reacted with NO to yield the corresponding stable
bis-diazeniumdiolates (5h and 5i).
buffer). Note the decrease in the absorbance at 247 nm,
corresponding to the diazeniumdiolate absorption band. In
addition, the spectrum has an isobestic point, suggesting a
one-step decomposition pathway as previously reported for
diazeniumdiolates.16
NO Release. All sodium stabilized bis-diazeniumdiolates
are hydrolyzed at pH 7.4 to release essentially 4 mol of NO
per mole of diamine (see Table 1). These values were
determined via chemiluminescence, after adding a given
amount of the bis-diazeniumdiolate to PBS buffer. The NO
released was detected and integrated over time, until no
further release of NO was observed.
NMR Spectra. Spectroscopic data strongly suggests that
stable bis-diazeniumdiolates were formed. Compared to the
starting diamine, the NO-reacted species show symmetrical
1
downfield shifts in the H NMR of the methylene protons
adjacent to the NO-reacted nitrogens, with integrals corre-
sponding to eight protons around each nitrogen, indicative
of the proposed structures. In contrast, if a monodiazenium-
diolate were formed, two distinct proton resonances would
be observed, one from each of the methylene protons adjacent
to the diazeniumdiolated nitrogen and the other from the
nondiazeniumdiolated nitrogen (see the Supporting Informa-
tion).
Kinetics. The decomposition of N-based diazeniumdiolates
have been shown to be proton driven.16 The half-lives of
the anionic bis-diazeniumdiolates prepared in this work, at
pH 7.4 and 37 °C, are summarized in Table 1. The term
Table 1. Summary of Characteristics of the
UV-vis Spectra. In addition, the UV absorption λmax of
the complexes formed from reaction of the diamines with
NO in the presence of sodium trimethylsilanoate were found
to exist at 247 nm and had molar absorptivities (ꢀ) in the
range from 12000 to 17000 M-1 cm-1, twice that of the
corresponding zwitterionic complexes, where formation of
only one dizeniumdiolate is achieved.14,16
Microanaylsis. Further authenticity of the bis-diazenium-
diolates was confirmed by derivatizing representative com-
pounds (5a, 5b, 5d, 5h) via O2-methylation.11,17 Full char-
acterization, including combustion analyses and exact mass
measurements, were performed for the resulting products (6a,
6b, 6d, 6h) supporting the identification of the target bis-
diazeniumdiolate species (see the Supporting Information).
Decomposition. In general, discrete diazeniumdiolates
have been shown to decompose and release NO by a proton-
driven mechanism.16 To monitor the decomposition of the
bis-diazeniumdiolates under investigation with time at pH
7.4, UV-vis spectroscopy was used. Figure 2 shows
Bis-diazeniumdiolates
R
x
t1/2a (s)
ratiob of diamine/NO
5a
5b
5c
5d
5e
5f
5g
5h
5i
CH3
CH2CH3
6
6
6
6
6
6
6
3
3
59.8 ( 2.0
102.9 ( 5.4
107.8 ( 5.4
93.3 ( 2.3
102.6 ( 1.3
88.9 ( 4.8
50.4 ( 5.7
93.8 ( 9.1
155.0 ( 0.5
1:3.7
1:3.8
1:3.9
1:4.0
1:3.8
1:4.0
1:4.0
1:3.7
1:3.9
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)11CH3
CH3
CH(CH3)2
a “Apparent” t1/2 and t1/2 for diazeniumdiolates under investigation in
PBS buffer at 37 °C and pH 7.4. b Measurements are within e0.3 standard
deviation.
“apparent” half-life is used to describe those compounds that
have limited solubility in PBS buffer owing to their high
lipophilicity. Due to the insolubility of some of the diazeni-
umdiolates under investigation, chemiluminescence, rather
than UV absorbance, was used to monitor the NO loss with
time. This also makes it possible to differentiate between
the dissociation of the first diazeniumdiolate from the second
(i.e., half-life 1 and half-life 2).
The “apparent” half-lives for the bis-diazeniumdiolates
were found to be shorter than that of their corresponding
zwitterionic forms (see Tables 1 and ref 14). One plausible
explanation for the longer half-lives of the zwitterionic
species (2a-e) is that the more hydrophobic side groups of
the ring structure of such a structure impedes protons from
reacting with the amine to heterolytically cleave the N-N
bond. For the sodium-stabilized bis-diazeniumdiolates, the
parent diamine backbone is more linear and does not provide
shielding from the aqueous environment.
Beyond the differences in the half-lives of the bis-
diazeniumdiolates with their corresponding zwitterionic
Figure 2. UV-vis spectra of 5d as a function of time in PBS
buffer (pH 7.4) at 22 °C.
(16) Davies, K. M.; Wink, D. A.; Saavedra, J. E.; Keefer, L. K. J. Am.
Chem. Soc. 2001, 123, 5473-5491.
(17) Saavedra, J. E.; Bohle, D. S.; Smith, K. N.; George, C.; Deschamps,
J. R.; Parrish, D.; Ivanic, J.; Wang, Y. N.; Citro, M. L.; Keefer, L. K. J.
Am. Chem. Soc. 2004, 126, 12880-12887.
representative spectra of the decomposition of 5d as a
function of time under physiological conditions (i.e., PBS
Org. Lett., Vol. 7, No. 14, 2005
2815

counterparts, the kinetics of the bis-diazeniumdiolate de-
composition is more complex than that of the corresponding
zwitterions (see Table 1). For example, all zwitterionic
compounds (2a-e) follow first-order kinetics as do bis-
diazeniumdiolates 5a-d and 5h-i; however, the most
lipophilic bis-diazeniumdiolate compounds 5e-g do not. The
NO release curves for the more lipophilic bis-diazeniumdi-
olates can be described as biphasic where the rates of NO
release are greater initially then decrease as function of time
(see the Supporting Information). Attempts to fit either zero-
order, first-order, or second-order kinetics to the release
profiles was not possible at the R2 g 0.99 level. The detailed
kinetics of the decomposition of the bis-diazeniumdiolates
is currently under investigation; however, it seems likely that
the dissociation of one diazeniumdiolate group, yielding a
single secondary amine can alter the decomposition of the
second diazeniumdiolate group on the same molecule owing
to local pH changes around the structure (i.e., differing pKa
of the amines within the compound).
Finally, we examined the effect of the methylene spacer
length between the diamine nitrogens on the half-lives of
the bis-diazeniumdiolates. It has been previously reported
for the zwitterionic analogues that a decrease in the meth-
ylene spacer length leads to an increase in the half-lives of
the corresponding diazeniumdiolates under physiological
conditions. In the same manner, a decrease in the methylene
spacer between the diamine nitrogens also leads to an
increase in the half-lives of the bis-diazeniumdiolate species
(compare compounds 5a and 5h, Table 1). The rationale for
this enhance half-life as a function of methylene spacer length
for zwitterionic diazeniumdiolates is apparently due to the
stability of an 11-membered ring compared to an 8-mem-
bered ring.14 The reason for the enhanced stability for the
bis-diazeniumdiolates as a function of decreased methylene
spacer is not straightforward; however, it seems likely that
either the electron distribution of the compounds with the
shorter methylene spacer chain is more favorable or the
basicity of the nitrogen bearing the diazeniumdiolate is less
than that of the bis-compounds with a hexamethylene spacer,
leading to a longer half-life.
The potential advantages of the bis-diazeniumdiolates
described herein are numerous. Higher NO loading per parent
diamine increases the NO loading efficiency of diamine
compounds, thus providing twice the NO delivery capability
compared to their zwitterionic counterparts. More impor-
tantly, however, the use of an exogenous cation and base
provides the ability to synthesize far more lipophilic stable
diazeniumdiolates (i.e., didodecyldiamines) that could not
be synthesized by previous methods. Such NO donors are
attractive candidates to add to hydrophobic polymeric
materials used to prepare a wide variety of biomedical
devices (catheters, extracorporeal tubings, implantable sen-
sors, etc.). Indeed, use of these new bis-diazeniumdiolates
could aid in the manufacturing process (owing to better air
stability) as well as the in vivo thromboresistivity observed
with such polymers because of the enhanced levels of NO
that could be released from the surfaces of materials doped
with such compounds. Efforts to demonstrate the utility of
these new compounds in creating more thromboresistant
polymeric coatings are currently in progress within this
laboratory.
Acknowledgment. This work was supported through the
National Institutes of Health (Grant No. EB-000783).
Supporting Information Available: Experimental pro-
cedures and spectral data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL050541Z
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Org. Lett., Vol. 7, No. 14, 2005