Piperazine as a linker for incorporating the nitric oxide-releasing diazeniumdiolate group into other biomedically relevant functional molecules

Article abstract of DOI:10.1021/jo9901539

Synthetic procedures have been devised to exploit the bifunctional amine piperazine (pip) as a linker capable of attaching the nitric oxide (NO)- releasing diazeniumdiolate functional group [N(O)NO]^- to a diverse selection of biomedically useful molecules. One of the amino groups bears the diazeniumdiolate, which may be substituted on oxygen as necessary to control its dissociation to NO, while the other is used to provide a site suitable for covalent bonding to the molecule requiring NO donor capability. N,N'- Disubstituted piperazines of the structure R-pip-N(O)=NOE were prepared either by using the nucleophilic character of the amino group or by converting it into an electrophilic moiety for reaction with nucleophilic centers in the molecules to be derivatized. Examples are reported in which E = CH₃ and the R groups are bound to the N'-nitrogen: via amide linkages to the carboxyl groups of the drug ibuprofen and the amino acid derivative N- acetylmethionine; through a urea grouping to the ε-amino group of a protected lysine; via a carbamate linkage to poly(ethylene glycol); and by replacing the NH₂ nitrogens of nicotinamide and adenosine. Synthesis of analogues in which E = vinyl has been facilitated by introduction of BrCH₂CH₂OSO₂Cl as a novel, efficient bromoethylating agent. Spontaneous NO releasers in the diazeniumdiolated piperazine series include both a fluorescent anion of half-life 5.5 min in which E = Na and R = dansyl and 'MOM-PIPERAZI/NO' (E = CH₃OCH₂, R = H), whose half-life for NO release was estimated as 17 days. The latter agent has made possible the conversion of poly(vinyl chloride) and phosphatidylethanolamine to NO-releasing derivatives. This chemistry should allow introduction of diazeniumdiolate groups into a wide variety of natural products, drugs, polymers, and other molecules whose activities could be beneficially combined with the ability to generate NO for biomedical applications.

Full text of DOI:10.1021/jo9901539

5124
J . Org. Chem. 1999, 64, 5124-5131
P ip er a zin e a s a Lin k er for In cor p or a tin g th e Nitr ic
Oxid e-Relea sin g Dia zen iu m d iola te Gr ou p in to Oth er Biom ed ica lly
Releva n t F u n ction a l Molecu les
J oseph E. Saavedra,*,† Melissa N. Booth,‡ J oseph A. Hrabie,§ Keith M. Davies, and
Larry K. Keefer‡
Intramural Research Support Program and Chemical Synthesis and Analysis Laboratory,
SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center,
Frederick, Maryland 21702, Chemistry Section, Laboratory of Comparative Carcinogenesis, National
Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, and
Department of Chemistry, George Mason University, Fairfax, Virginia 22030
Received J anuary 27, 1999
Synthetic procedures have been devised to exploit the bifunctional amine piperazine (pip) as a
linker capable of attaching the nitric oxide (NO)-releasing diazeniumdiolate functional group
[N(O)NO]- to a diverse selection of biomedically useful molecules. One of the amino groups bears
the diazeniumdiolate, which may be substituted on oxygen as necessary to control its dissociation
to NO, while the other is used to provide a site suitable for covalent bonding to the molecule requiring
NO donor capability. N,N′-Disubstituted piperazines of the structure R-pip-N(O)dNOE were
prepared either by using the nucleophilic character of the amino group or by converting it into an
electrophilic moiety for reaction with nucleophilic centers in the molecules to be derivatized.
Examples are reported in which E ) CH3 and the R groups are bound to the N′-nitrogen: via
amide linkages to the carboxyl groups of the drug ibuprofen and the amino acid derivative
N-acetylmethionine; through a urea grouping to the ꢀ-amino group of a protected lysine; via a
carbamate linkage to poly(ethylene glycol); and by replacing the NH2 nitrogens of nicotinamide
and adenosine. Synthesis of analogues in which E ) vinyl has been facilitated by introduction of
BrCH2CH2OSO2Cl as a novel, efficient bromoethylating agent. Spontaneous NO releasers in the
diazeniumdiolated piperazine series include both a fluorescent anion of half-life 5.5 min in which
E ) Na and R ) dansyl and “MOM-PIPERAZI/NO” (E ) CH3OCH2, R ) H), whose half-life for NO
release was estimated as 17 days. The latter agent has made possible the conversion of poly(vinyl
chloride) and phosphatidylethanolamine to NO-releasing derivatives. This chemistry should allow
introduction of diazeniumdiolate groups into a wide variety of natural products, drugs, polymers,
and other molecules whose activities could be beneficially combined with the ability to generate
NO for biomedical applications.
In tr od u ction
Drago7 introduced complexes of structure 1 formed by
the reaction of nucleophiles, including amines, with nitric
oxide; Hrabie et al.3 have extended the available library
of amine/NO complexes to the polyamine series 2. These
diazeniumdiolate-containing compounds are advanta-
geous agents for many studies requiring the controlled,
gradual release of nitric oxide, since they are capable of
regenerating nearly 2 equiv of NO upon dissolution in
neutral media.8 The half-lives, t1/2, of these complexes
range from 1.3 to 1200 min at 37 °C in pH 7.4 phosphate
buffer. The decomposition to NO is a spontaneous, first-
order reaction at constant pH (eq 1).9
Nitric oxide (NO) plays a critical role in a variety of
bioregulatory processes, including normal physiological
control of blood pressure, neurotransmission, and mac-
rophage-induced cytostasis and cytotoxicity.1 NO has
limited solubility in water, and it is unstable in the
presence of various oxidants. This makes it difficult to
introduce it as such into biological systems in a controlled
or specific fashion. Consequently, the development of
chemical agents that release nitric oxide (NO) is impor-
tant if we are to target its bioeffector roles to specific cell
types for pharmacological applications.2-6
† Intramural Research Support Program.
‡ Laboratory of Comparative Carcinogenesis.
§ Chemical Synthesis and Analysis Laboratory.
George Mason University.
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1991, 43, 109-142.
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L. K. J . Org. Chem. 1992, 57, 6134-6138.
(3) Hrabie, J . A.; Klose, J . R.; Wink, D. A.; Keefer, L. K. J . Org.
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(4) Taylor, D. K.; Bytheway, I.; Barton, D. H. R.; Bayse, C. A.; Hall,
M. B. J . Org. Chem. 1995, 60, 435-444.
(7) Drago, R. S. Adv. Chem. Ser. 1962, 36, 143-149.
(8) 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. 1991, 34, 3242-3247.
(9) Keefer, L. K.; Nims, R. W.; Davies, K. M.; Wink, D. A. Methods
Enzymol. 1996, 268, 281-293.
(5) Roy, B.; d’Hardemare, A. du M.; Fontecave, M. J . Org. Chem.
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(6) Makings, L. R.; Tsien, R. Y. J . Biol. Chem. 1994, 269, 6282-
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10.1021/jo9901539 CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/15/1999

Piperazine as a Diazeniumdiolate Group Linker
J . Org. Chem., Vol. 64, No. 14, 1999 5125
Sch em e 1. Syn th esis of An ion ic P ip er a zin e
Dia zen iu m d iola tes
We have been interested in reacting amine/NO complex
ions of structure 1 (X ) R2N) with alkylating agents (E+)
to provide stable, covalently bonded adducts 3 (eq 2).
Compounds of this type could serve as prodrugs that
cannot release nitric oxide until they are metabolically
reconverted to 1 by enzymes specific to the desired target
cell type. Anions of structure 1 (X ) R2N) proved reactive
toward a variety of electrophiles, such as alkyl halides,
sulfate esters and epoxides, as in eq 2,2 to give compounds
of type 3.
We recently reported the successful application of this
strategy to the design of a liver-selective NO donor, 4.10
Liver enzymes metabolize this compound to give a
diazeniumdiolate ion (1, X ) pyrrolidin-1-yl) that spon-
taneously and rapidly (t1/2 ) 3 s) decomposes to NO at
physiological pH. The administration of 4 to rats resulted
in the blocking of tumor necrosis factor-R-induced apo-
ptosis and hepatotoxicity, thus illustrating the potential
utility of O2-substituted diazeniumdiolates for targeting
NO delivery in vivo.
diazeniumdiolated piperazine to a desired substrate. The
examples described below illustrate the generality of this
approach.
Resu lts a n d Discu ssion
Syn th esis a n d NO Relea se Ch a r a cter istics of
An ion ic P ip er a zin e Dia zen iu m d iola tes. N-Substi-
tuted piperazines are easily prepared, with many being
commercially available. We have reacted a selection of
these (5) with nitric oxide gas at 4-6 atm of pressure in
methanolic sodium methoxide, as shown in Scheme 1.
The N4-substituent of 6 may be a base-labile protective
group as in 6a or b, or it can be acid sensitive such as
that in 6c. Commercially available N-arylpiperazines 5d
and e proved similarly amenable to diazeniumdiolation.
While we at first envisioned these products merely as
one step en route to our proposed linker reagents, they
proved to be useful NO-releasing agents themselves. To
address the need for a fluorescent diazeniumdiolate for
use in biological experiments, for example, 5f was
synthesized and converted to the diazeniumdiolate 6f.
This dansyl derivative has shown useful pharmacological
properties beyond its fluorescence. Local infusion of this
compound (“GLO/NO”) at the luminal surface of me-
chanically injured porcine carotid arteries led to signifi-
cant reductions of platelet adhesion at the injury site,
together with marked improvement in blood flow 24 h
after injury relative to control animals receiving no
drug.14 In view of this utility, we determined the NO-
release characteristics of each of the anionic materials
6a -f. All dissociated in physiological buffer with first-
order kinetics with half-lives of 2-6 min to generate
approximately 2 mol of NO per mole of dissociating 6.
These data are summarized in Table 1.
Besides this prodrug approach, we have shown that
polymers to which the [N(O)NO]- group is attached can
serve as localized sources of nitric oxide.11 For example,
cross-linked poly(ethylenimine) exposed to NO inhibited
the in vitro proliferation of rat aorta smooth muscle cells
and showed potent antiplatelet activity in arteriovenous
shunts placed in the baboon circulatory system.11
This paper introduces an alternate drug delivery
strategy in which the diazeniumdiolate group can be
bound via a linker moiety to a variety of substrates, thus
furnishing a convenient means of adding the capacity for
NO release to the substrate’s other useful functional
characteristics.
P ip er a zin e a s a Lin k er in Dia zen iu m d iola t ion
Rea ction s. Piperazine, a bifunctional base used medici-
nally for its anthelmintic properties, appeared to us to
be the ideal type of compound for these studies. Early
reports of the high-pressure reaction of piperazine with
NO show that various salts of diazeniumdiolated piper-
azines can be produced.7,12 Hrabie et al.13 took advantage
of the solubility properties of monodiazeniumdiolated
piperazine to isolate the anion as the sodium salt in its
pure form. We aim, with the assistance of protection and
deprotection techniques, to use one of piperazine’s two
nucleophilic sites for the reaction with nitric oxide while
the second nitrogen remains available for linking the
P r ep a r a tion of O2-Su bstitu ted P ip er a zin e Di-
a zen iu m d iola tes. Alkylation of diazeniumdiolates 6a -c
at their terminal oxygen, followed by removal of the
protecting group at N4 to free a secondary amino nitrogen
on the piperazine ring for subsequent reaction with
electrophilic centers, can provide molecules having useful
pharmacological properties. To illustrate this approach,
we have carried 6a through the transformations shown
in Scheme 2. The first step was an alkylation2 to attach
(10) 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.
(11) Smith, D. J .; Chakravarthy, D.; Pulfer, S.; Simmons, M. L.;
Hrabie, J . A.; Citro, M. L.; Saavedra, J . E.; Davies, K. M.; Hutsell, T.
C.; Mooradian, D. L.; Hanson, S. R.; Keefer, L. K. J . Med. Chem. 1996,
39, 1148-1156.
(12) Reilly, E. L. U.S. Patent 3,153,094, Oct 13, 1964; Chem. Abstr.
1965, 62, 5192g.
(13) Hrabie, J . A.; Saavedra, J . E.; Davies, K. M.; Keefer, L. K. Org.
Prep. Proced. Int. 1999, 31, 189-192.
(14) J eong, M. H.; Marks, D. S.; Keefer, L. K.; Hrabie, J . A.; Southan,
G. J .; Stewart, M. L.; Lerman, A.; Gregoire, J .; Staab, M. E.; Holmes,
D. R., J r.; Schwartz, R. S. Unpublished data.

5126 J . Org. Chem., Vol. 64, No. 14, 1999
Saavedra et al.
Sch em e 3. Syn th esis of O2-Vin yla ted P ip er a zin e
Dia zen iu m d iola tes
Ta ble 1. NO Relea se Ra tes of An ion ic P ip er a zin e
Dia zen iu m d iola tes (6)
dissociation kinetics in
pH 7.4 phosphate at 37 °C
obsd NO release per mole
compd
k (s-1
)
half-life (min) of diazeniumdiolate (mol)
6a
6b
6c
6d
6e
6f
4.08 × 10-3
5.69 × 10-3
4.39 × 10-3
6.14 × 10-3
5.74 × 10-3
2.05 × 10-3
2.8
2.0
2.6
1.9
2.0
5.6
1.92 ( 0.11
1.83 ( 0.06
1.85 ( 0.05
1.96 ( 0.02
2.36 ( 0.08
1.43 ( 0.17
Sch em e 2. Syn th esis of O2-Meth yla ted P ip er a zin e
Dia zen iu m d iola tes
molecules by taking advantage of the nucleophilic char-
acter of the free secondary amine in the piperazine ring,
in particular by linking it via amide formation with
carboxyl groups of bioactive molecules. The same nucleo-
philic nitrogen can combine with a reagent such as N,N′-
disuccinimidyl carbonate to give an activated carbam-
ate,16 9d , thus transforming the diazeniumdiolated
piperazine into an electrophile. Carbamate and urea
functions may now be established at the distal nitrogen
of the compound containing a diazeniumdiolate. Nucleo-
philic termini (N, O, or S) of amino acids, oligopeptides,
proteins, or synthetic polymers may now be used to
incorporate diazeniumdiolated piperazine moieties. For
example, coupling of 9d with NR-acetyllysine methyl
ester, a reaction in which the nucleophilic ꢀ-nitrogen
displaces the hydroxysuccinimide residue, formed the
derivative 9e.
Poly(ethylene glycol) (PEG) has been used for many
years as a covalent modifier of a variety of substrates in
order to make them suitable for biological applications.17
Attachment of a diazeniumdiolated piperazine to a PEG
derivative may be useful for altering the solubility and
pharmacokinetics18 of the NO donor. To demonstrate the
feasibility of this type of linkage, low molecular weight
poly(ethylene glycol) methyl ether-350 was reacted with
electrophile 9d in the presence of potassium tert-butoxide
to give oligomer-bound diazeniumdiolate 9f.
To illustrate the preparation of a diazeniumdiolated
nucleoside, 8 was reacted with 6-chloropurine riboside
to produce 9g in an adaptation of the procedure of
Iwamura et al.19 This strategy could serve as an approach
to diazeniumdiolation of oligonucleotides and nucleic
acids.
a methyl group to the terminal oxygen of the diazenium-
diolate function to produce 7. Removal of the ethoxycar-
bonyl group at N4 by basic hydrolysis gave 8, a useful
intermediate that was reacted with various electrophiles
to produce agents of structure 9.
Having noted the utility of incorporating NO-releasing
functions into nonsteroidal antiinflammatory drugs
(NSAIDs), a strategy that has in many cases allowed
introduction of potent antiinflammatories devoid of the
gastric toxicity often seen in many of these drugs,15 we
linked 8 to ibuprofen’s carboxyl group in a condensation
reaction to form amide 9a . If the methyl group proves to
be metabolically labile, such that NO is generated at or
near sensitive tissues in need of protection, 9a could be
an important addition to the list of NO-releasing NSAIDs.
Similarly, incorporation of the diazeniumdiolate group
into nicotinamide (vitamin B3) was accomplished by
aroylating 8 with nicotinoyl chloride to form 9b. To
demonstrate that a piperazine containing the O2-methyl
diazeniumdiolate group can be linked to an amino acid,
9c was synthesized.
Like the methyl group, an O2-vinyl substituent can
often be oxidatively removed by enzymes found in certain
organs, as was demonstrated through the introduction
of liver-selective NO donor 4.10 To extend this concept to
other molecules with functionality of pharmacological
relevance, we have synthesized 12 via the route shown
in Scheme 3. Introduction of the vinyl group was ac-
complished efficiently with the aid of a novel bromo-
ethylating agent, BrCH2CH2OSO2Cl. Compound 12 can
(16) Takeda, K.; Akagi, Y.; Saiki, A.; Tsukahara, T.; Ogura, H.
Tetrahedron Lett. 1983, 24, 4569-4572.
(17) Zalipsky, S. Bioconjugate Chem. 1995, 6, 150-165.
(18) Nathan, A.; Zalipsky, S.; Ertel, S. I.; Agathos, S. N.; Yarmush,
M. L.; Kohn, J . Bioconjugate Chem. 1993, 4, 54-62.
(19) Iwamura, H.; Ito, T.; Kumazawa, Z.; Ogawa, Y. Phytochemistry
1975, 14, 2317-2321.
Up to this point, we have discussed the introduction
of the diazeniumdiolate function into a variety of other
(15) Wallace, J . L.; Elliott, S. N.; Del Soldato, P.; McKnight, W.;
Sannicolo, F.; Cirino, G. Drug Dev. Res. 1997, 42, 144-149.

Piperazine as a Diazeniumdiolate Group Linker
J . Org. Chem., Vol. 64, No. 14, 1999 5127
Sch em e 4. Syn th esis of O2-Meth oxym eth yla ted
P ip er a zin e Dia zen iu m d iola tes
F igu r e 1. Observed rates of NO generation from 13b at initial
concentrations of 20-50 mM at various times after dissolution
in 0.1 M phosphate buffer at 37 °C. Individual results for three
separate incubations are shown. The pH remained at 7.4
throughout the incubations.
be used to incorporate O2-vinylated diazeniumdiolate
moieties into other molecules in the same way that 8 was
employed in synthesizing O2-methyl derivatives 9 (Scheme
2).
In contrast to the methyl derivatives shown in Scheme
2, introduction of O2-methoxymethyl substituents on the
terminal oxygen as acetals of the diazeniumdiolate
function should allow for two-stage hydrolytic release of
NO as well as for potential metabolic removal. The
preparations of six such O2-methoxymethyl derivatives
are shown in Scheme 4. To prepare phospholipid 13d ,
phosphatidyl ethanolamine was reacted with intermedi-
ate 13c to form a urea linkage. The immediate precursor
to 13c, 13b, was also reacted with ethylene sulfide to
produce 13e, an agent used to couple the diazeniumdi-
olate function to a protein in a previously reported
preliminary experiment.20 To determine whether an NO-
releasing poly(vinyl chloride) (PVC) derivative could be
prepared, solutions of PVC in tetrahydrofuran were
refluxed with 13b to produce diazeniumdiolated PVC of
structure 13f. The product’s elemental analytical data
and NMR spectrum indicated that 1-2% of the chloride
in the polymer had been replaced by 13b. This material,
when blended with various combinations of plasticizers
and ionophores, has proven useful as a thromboresistant
coating for intravascular biosensors and other surfaces
in contact with blood.21
NO Relea se Ch a r a cter istics of O2-Su bstitu ted
P ip er a zin e Dia zen iu m d iola tes. The prediction that an
O2-methoxymethylated diazeniumdiolate can serve as a
slow-release source of molecular NO was confirmed by
chemiluminescence analysis of the gaseous product swept
periodically from the incubation mixture after dissolution
of 13b at a concentration of 20-50 mM in 0.1 M
phosphate buffer (pH 7.4) at 37 °C. Production of molec-
ular NO proceeded rather smoothly for several weeks.
Rate data for three separate determinations are pre-
F igu r e 2. Time course of NO release observed on suspending
10 mg of diazeniumdiolated phospholipid 13d in 2 mL of 0.1
M phosphate buffer (pH 7.4) at 37 °C.
sented in Figure 1. Graphical analysis of these semilog
plots as previously described9 indicated that complete
dissociation of 13b generated 1.06 ( 0.59 mol of NO per
mol of 13b that was present at time zero. In addition,
N-nitrosopiperazine was detected when the reaction
mixture was examined by HPLC after most of the NO
had been released; the mechanistic origin of this product
is currently under investigation. The half-life for NO
release by 13b in pH 7.4 buffer at 37 °C was estimated
from the data of Figure 1 to be 17.2 ( 1.5 days [k ) (4.67
( 0.43) × 10-7 s-1].
A slow release of NO extending over several weeks was
also observed with phospholipid derivative 13d , as shown
in Figure 2. It was noted that 13d was incompletely
soluble at the concentration used (5 mg/mL). Despite this
fact, the two-phase mixture provided a remarkably
steady rate of NO generation over the first 3 weeks of
incubation. Integration of the data for Figure 2 indicated
that 3.1 ( 1.2 mol of NO was generated for each mol of
13d initially present. The half-life was estimated graphi-
cally to be 11.4 days.
In contrast to the spontaneous dissociation of the O2-
methoxymethyl diazeniumdiolates, the O2-methyl and
-vinyl derivatives, 8 and 12, respectively, produced no
observable chemiluminescence response at a detection
(20) Keefer, L. K.; Smith, D. J .; Pulfer, S.; Hanson, S. R.; Saavedra,
J . E.; Billiar, T. R.; Roller, P. P. In The Biology of Nitric Oxide, Part 5;
Moncada, S., Stamler, J ., Gross, S., Higgs, E. A., Eds.; Portland
Press: London, 1996; p 335.
(21) Mowery, K. A.; Schoenfisch, M. H.; Saavedra, J . E.; Keefer, L.
K.; Meyerhoff, M. E. Manuscript in preparation.

5128 J . Org. Chem., Vol. 64, No. 14, 1999
Saavedra et al.
needles: mp 199-200 °C; UV (0.01 M NaOH) λmax (ꢀ) 246 nm
(20.1 mM-1 cm-1) and 306 nm (1.7 mM-1cm-1); 1H NMR
(DMSO-d6) δ 2.5 (m, 4 H), 3.89 (m, 4 H), 6.66 (t, 1 H), 8.38 (d,
2 H); 13C NMR (0.1 M NaOD in D2O) δ 46.00, 54.08, 113.91,
161.25, 163.48. Anal. Calcd for C8H11N6O2Na: C, 39.03; H,
4.50; N, 34.13; Na, 9.34. Found: C, 38.94; H, 4.44; N, 34.10;
Na, 9.53.
Sod iu m 1-(4-P h en ylp ip er a zin -1-yl)d ia zen -1-iu m -1,2-d i-
ola te, 6e. Exposure of 15 g (0.093 mol) of 1-phenylpiperazine
to NO as described above gave 7.23 g (33%) of 6e as white
crystals: mp 215-216 °C; UV (0.01 M NaOH) λmax (ꢀ) 246 nm
(20.2 mM-1 cm-1); 1H NMR (0.1 M NaOD in D2O) δ 3.37 (m, 8
H), 7.16 (m, 3 H), 7.42 (m, 2 H); 13C NMR (0.1 M NaOD in
D2O) δ 52.01, 53.98, 120.80, 125.07, 132.39, 152.91. Anal. Calcd
for C10H13N4O2Na: C, 49.18; H, 5.37; N, 22.94; Na, 9.41.
Found: C, 48.94; H, 5.28; N, 22.81; Na, 9.63.
limit of 12 pmol of NO when solutions thereof were
incubated under the conditions used for 13b.
Con clu sion
The delivery of NO to a specific organ or cell type where
it is needed without affecting other parts of the anatomy
is a crucial goal in our research. We have successfully
utilized the chemistry of the dibasic amine piperazine to
introduce the diazeniumdiolate moiety, [N(O)NO-], into
a variety of substrates. The chemistry described in this
paper serves as a model for converting natural products,
drugs, and other important materials to NO-releasing
form.
Sodiu m 1-[4-(5-Dim eth ylam in o-1-n aph th alen esu lfon yl)-
p ip er a zin -1-yl]d ia zen -1-iu m -1,2-d iola te, 6f. A solution con-
taining 7.98 g (9.27 mmol) of piperazine and 5 g (18.5 mmol)
of dansyl chloride in 100 mL of toluene was heated at reflux
for 6 h. The product was isolated after the solution was washed
with 5% sodium hydroxide and then water and the organic
layer was dried over sodium sulfate. The solution was filtered
and concentrated on a rotary evaporator to give 5 g (84%) of
dansylpiperazine, 5f, as a yellow-green powder. To prepare the
diazeniumdiolate, a solution of 3.08 g (9.64 mmol) of 5f, 2.2
mL (9.64 mmol) of 25% sodium methoxide in methanol, and
25 mL of N,N-dimethylformamide was exposed to 5 atm of NO
gas for 2 days. After the solution was flushed with argon, 150
mL of ether was added, and 2 g (52%) of 6f was isolated by
filtration: mp 158-160 °C; UV (methanol) λmax (ꢀ) 252 nm (12.7
mM-1 cm-1) and 344 nm (3.0 mM-1 cm-1); the compound
fluoresced with an emission wavelength of 523 nm (excitation
wavelength of 342 nm); 1H NMR (D2O) δ 2.8 (s, 6 H), 3.15 (m,
4 H), 3.45 (m, 4 H), 7.5 (m, 3 H), 8.35 (m, 3 H); 13C NMR (CD3-
OD) δ 45.78, 45.99, 52.76, 116.62, 120.77, 124.35, 129.28,
131.41, 131.75, 132.01, 153.26, 164.87, 170.39. Anal. Calcd for
Exp er im en ta l Section
NO was purchased from Matheson Gas Products (Mont-
gomeryville, PA). Ultraviolet (UV) spectra were recorded in
water, except as otherwise indicated. Nuclear magnetic reso-
nance (NMR) spectra were collected with a 300-MHz NMR
spectrometer in deuteriochloroform (unless otherwise speci-
fied). Chemiluminescence measurements of nitric oxide were
performed with a Thermal Energy Analyzer model 502A or
610 (Thermedics, Inc., Woburn, MA). Elemental analyses were
done by Atlantic Microlab, Inc. (Norcross, GA). Sulfuryl
chloride and piperazine derivatives were obtained from Aldrich
Chemical Co. (Milwaukee, WI).
Sod iu m 1-(4-Eth oxyca r bon ylp ip er a zin -1-yl)d ia zen -1-
iu m -1,2-d iola te, 6a . A solution of 20 g (0.126 mol) of 1-ethoxy-
carbonylpiperazine 5a in 60 mL of methanol was placed in a
Parr bottle. The solution was treated with 27.4 mL (0.126 mol)
of 25% sodium methoxide in methanol. The system was
evacuated, charged with 40 psi of nitric oxide, and kept at 25
°C for 48 h. The white crystalline product was collected by
filtration and washed with cold methanol as well as with
copious amounts of ether. The product was dried under
vacuum to give a 14.5 g (48%) yield of 6a : mp 184-185 °C;
UV (0.01 M NaOH) λmax (ꢀ) 252 nm (10.4 mM-1 cm-1); 1H NMR
(0.1 M NaOD in D2O) δ 1.25 (t, 3 H), 3.11 (m, 2 H), 3.68 (m, 2
H), 4.15 (q, 2 H); 13C NMR (0.1 M NaOD in D2O) δ 16.62, 45.40,
54.10, 65.60, 159.71. Anal. Calcd for C7H13N4O4Na: C, 35.00;
H, 5.46; N, 23.33; Na, 9.57. Found: C, 34.87; H, 5.53; N, 23.26;
Na, 9.69.
C
16H20N5O4SNa: C, 47.87; H, 5.02; N, 17.45; S, 7.99. Found:
C, 47.94; H, 5.06; N, 17.32; S, 8.04.
O2-Meth yl 1-(4-Eth oxyca r bon ylp ip er a zin -1-yl)d ia zen -
1-iu m -1,2-d iola te, 7. To a solution of 10 g (0.042 mol) of
diolate 6a in 200 mL of methanol was added 10 g of anhydrous
potassium carbonate, and the resulting slurry was cooled in
an ice bath. Dimethyl sulfate (4.7 mL, 0.05 mol) was added
dropwise. The reaction mixture was stirred at 0 °C for 1 h and
then allowed to warm gradually to room temperature. After
an additional hour, the solution was concentrated on a rotary
evaporator; the residue was extracted with dichloromethane,
washed with water, dried over sodium sulfate, and filtered
Sod iu m 1-(4-Ben zyloxyca r bon ylp ip er a zin -1-yl)d ia zen -
1-iu m -1,2-d iola te, 6b. 1-Benzyloxycarbonylpiperazine (23.5
g, 107 mmol) in 6 mL (120 mmol) of 25% methanolic sodium
methoxide and 60 mL of methanol was exposed to NO as
described for the preparation of 6a : mp 180 °C dec; UV (0.01
through
a layer of magnesium sulfate. The solvent was
M NaOH) λmax (ꢀ) 252 nm (6.3 mM-1 cm-1); H NMR (0.1 M
1
evaporated under reduced pressure, and the residue was
chromatographed on silica gel. Elution with 2:1 dichlo-
romethane/ethyl acetate provided 5.7 g (56%) of 7 as an oil,
which crystallized on standing: mp 46 °C; UV λmax (ꢀ) 240 nm
(8.4 mM-1 cm-1); 1H NMR δ 3.38 (m, 4 H), 3.67 (m, 4 H), 4.03
(s, 3 H), 4.16 (q, 2 H); 13C NMR δ 14.52, 42.33, 51.08, 61.04,
61.72, 155.03. Anal. Calcd for C8H16N4O4: C, 41.38; H, 6.90;
N, 24.14. Found: C, 41.23; H, 6.82; N, 24.05.
O2-Meth yl 1-(P ip er a zin -1-yl)d ia zen -1-iu m -1,2-d iola te,
8. A mixture of 6.42 g (0.0276 mol) of 7, 200 mL of 10%
sodium hydroxide in ethanol, and 5 mL of water was heated
at reflux. After 45 min, no starting material remained in the
mixture, as assessed from qualitative thin-layer chromatog-
raphy. The solution was allowed to cool to room temperature
and evaporated to a viscous residue, which was extracted with
dichloromethane, dried over sodium sulfate, filtered, and
evaporated to give 3.8 g of a yellow oil. The product was
chromatographed on silica gel using a Flash 40 system eluted
with 9:2:0.1 dichloromethane/methanol/water to give 3.22 g
(73%) of 8 as a pale yellow oil: mp (picrate) 113-114 °C; UV
λmax (ꢀ) 234 nm (7.0 mM-1 cm-1); 1H NMR δ 3.03 (m, 4 H),
3.38 (m, 4 H), 4.06 (s, 3 H); 13C NMR δ 44.81, 52.53, 60.89.
Anal. Calcd for picrate salt C11H15N7O9‚0.5 C2H5OH: C, 35.00;
H, 4.28; N, 23.81. Found: C, 35.04; H, 4.43; N, 23.75.
NaOD in D2O) δ 3.12 (m, 4 H), 3.70 (m, 4 H), 5.18 (s, 2 H),
7.45 (s, 5 H); 13C NMR (0.1 M NaOD in D2O) δ 45.56, 54.13,
70.70, 130.68, 131.32, 131.66, 139.12, 159.31. Anal. Calcd for
C
12H15N4O4Na‚0.5 NaOH: C, 44.72; H, 4.85; N, 17.39; Na,
10.70. Found: C, 44.75; H, 4.78; N, 17.59; Na, 10.32.
Sodiu m 1-(4-ter t-Bu toxycar bon ylpiper azin -1-yl)diazen -
1-iu m -1,2-d iola te, 6c. This reaction was carried out as
described for the preparation of 6a . A solution of tert-butoxy-
carbonylpiperazine 5c (11.5 g; 62 mmol) and 15 mL (69 mmol)
of 25% methanolic sodium methoxide in 30 mL of methanol
was exposed to nitric oxide to give 7 g (42%) of 6c as a free-
flowing solid: mp 262-264 °C; UV (0.01 M NaOH) λmax (ꢀ) 252
nm (6.7 mM-1 cm-1); 1H NMR (0.1 M NaOD in D2O) δ 1.47 (s,
9 H), 3.11 (m 4 H), 3.65 (m, 4 H); 13C NMR (0.1 M NaOD in
D2O) δ 30.44, 45.44, 54.19, 85.05, 159.08. Anal. Calcd for
C9H17N4O4Na: C, 40.30; H, 6.39; N, 20.89; Na, 8.57. Found:
C, 40.09; H, 6.44; N, 20.64; Na, 8.69.
Sod iu m 1-[4-(P yr im id in -2-yl)p ip er a zin -1-yl]d ia zen -1-
iu m -1,2-d iola t e, 6d . A solution of 4 g (0.024 mol) of 1-(2-
pyrimidyl)piperazine, 5d , in 50 mL of methanol and 10 mL of
ether was placed in a Parr bottle with 5.2 mL of 25% sodium
methoxide in methanol. This was exposed to nitric oxide as
described above to give 1.7 g (25%) of 6d as large white

Piperazine as a Diazeniumdiolate Group Linker
J . Org. Chem., Vol. 64, No. 14, 1999 5129
O2-Meth yl 1-[4-(p-Isobu tyl-r-m eth ylp h en yla cetyl)p i-
p er a zin -1-yl]d ia zen -1-iu m -1,2-d iola te, 9a . A solution of 1.72
g (0.0083 mol) of ibuprofen in 5 mL of thionyl chloride was
stirred with a boiling stone at room temperature for 15 h.
Excess thionyl chloride was removed on a rotary evaporator,
and the residue was placed under high vacuum to remove
traces of the chlorinating agent. The resulting residue was
taken up in dichloromethane, washed with 5% sodium bicar-
bonate solution, dried over sodium sulfate, filtered, and
evaporated to give 1.76 g (94%) of the acid chloride as a
colorless oil. Without any further purification, 1.7 g (7.5 mmol)
of p-isobutyl-R-methylphenylacetyl chloride was added to a cold
solution of 8 in dichloromethane containing a slight molar
excess of triethylamine (1.11 mL; 8 mmol). The reaction
mixture was allowed to warm to room temperature, stirred
for 2 h, diluted with 20 mL of dichloromethane, washed with
5% aqueous hydrochloric acid followed by 10% sodium hydrox-
ide, dried, and evaporated to give 2.6 g of a yellow oil that
crystallized on standing. Recrystallization from ether/petro-
leum ether gave an analytically pure sample: mp 79-80 °C;
UV λmax (ꢀ) 224 nm (12.4 mM-1 cm-1), 234 (8.7 mM-1 cm-1);
1H NMR δ 0.88 (d, 6 H), 1.44 (d, 3 H), 1.84 (m, 1 H), 2.45 (d,
2 H), 3.36 (m, 4 H), 3.52 (m, 4 H), 3.83 (q, 1 H), 3.97 (s, 3 H),
7.11 (s, 4 H); 13C NMR δ 20.56, 22.29, 30.09, 40.42, 43.07,
43.87, 44.90, 61.01, 126.23, 129.79, 138.71, 140.48, 172.18.
Anal. Calcd for C18H28N4O4: C, 62.05; H, 8.10; N, 16.08.
Found: C, 61.80; H, 8.03; N, 16.04.
progress of the resulting reaction was monitored on silica gel
thin-layer chromatography using 1:1 dichloromethane/ethyl
acetate. After being stirred for 1 h, the solution was washed
with 1 M hydrochloric acid, followed by 5% aqueous sodium
hydroxide. The solution was dried as described previously and
evaporated under vacuum to give 2.44 g of a glassy substance.
Purification of the product was carried out on a Biotage Flash
40 system with a 4.0 × 15.0 cm KP-Sil column. The system
was eluted with 1:1 dichloromethane/ethyl acetate at 15 psi
of air at a rate of elution of 25 mL/min to give pure 9d : mp
104-105 °C; UV λmax (ꢀ) 234 nm (6.44 mM-1 cm-1); 1H NMR δ
2.85 (s, 4 H), 3.52 (m, 4 H), 3.74 (b, 2 H), 3.82 (b, 2 H), 4.04 (s,
3 H); 13C NMR δ 25.43, 43.46, 50.56, 61.19, 150.14, 169.42.
Anal. Calcd for C10H15N5O6: C, 39.87; H, 5.02; N, 23.25.
Found: C, 39.92; H, 4.97; N, 23.07.
Rea ction of 9d w ith Nr-Acetyl Lysin e Meth yl Ester To
F or m 9e. To a solution of 197 mg (0.654 mmol) of 9d in 10
mL of chloroform was added 167 mg (0.7 mmol) of NR-acetyl
lysine methyl ester hydrochloride followed by 278 µL (2 mmol)
of triethylamine. The resulting solution was heated at reflux
for 8 h, allowed to cool to room temperature, and washed with
water. The organic layer was dried over sodium sulfate,
filtered, and evaporated to give 160 mg of a yellow glass.
Purification was carried out on a Flash 40 apparatus using a
7.5 × 4 cm KP-Sil column and eluted with 10:1 dichlo-
romethane/methanol to give 39 mg of pure 9e: UV (ethanol)
1
λmax 240 nm (8.2 mM-1 cm-1); H NMR δ 1.36 (m, 2 H), 1.55
O2-Met h yl 1-[(4-Nicot in oyl)p ip er a zin -1-yl)d ia zen -1-
iu m -1,2-d iola te, 9b. To a solution of 306 mg (1.91 mmol) of 8
in 10 mL of dichloromethane was added 340 mg (1.91 mmol)
of nicotinyl chloride hydrochloride. The resulting mixture was
treated with 2 mL of triethylamine, stirred for 30 min, and
then washed with water. The organic layer was dried over
sodium sulfate, filtered through a layer of magnesium sulfate,
and evaporated. The residue was chromatographed on silica
gel using 1:1 dichloromethane/acetone as eluant. Evaporation
of the solvent gave 485 mg of a crystalline product: mp 85-
86 °C; UV λmax (ꢀ) 236 nm (10 mM-1 cm-1); 1H NMR δ 3.47 (m,
4 H), 3.83 (m, 4 H), 4.04 (s, 3 H), 7.29 (m, 2 H), 7.82 (m, 1 H),
8.72 (m, 1 H); 13C NMR δ 51.28, 61.22, 123.57, 130.68, 135.08,
147.93, 151.29, 151.50, 167.81. Anal. Calcd for C11H15N5O3: C,
49.81; H, 5.66; N, 26.42. Found: C, 50.01; H, 5.76; N 26.24.
O2-Meth yl 1-[4-(N-Acetyl-L-m eth ion yl)p ip er a zin -1-yl]-
d ia zen -1-iu m -1,2-d iola te, 9c. To a solution of 1.18 g (0.0062
mol) of N-acetyl-L-methionine in 20 mL of 2:1 dichloromethane/
acetonitrile was added a solution 1.44 g (0.007 mol) of
dicyclohexylcarbodiimide (DCC) in 10 mL of dichloromethane.
This was followed by the rapid introduction of 805 mg (0.007
mol) of N-hydroxysuccinimide in 6 mL of dichloromethane. The
resulting mixture was stirred at room temperature for 5 min,
and then 1 g (0.0062 mol) of 8 in 10 mL of dichloromethane
was added dropwise. The reaction mixture was stirred at 25
°C for 4 h. A few drops of glacial acetic acid were added to
decompose excess DCC. The mixture was filtered and washed
with dilute hydrochloric acid followed by dilute aqueous
sodium hydroxide. The organic layer was dried over sodium
sulfate, filtered through a layer of magnesium sulfate, and
evaporated in vacuo to give 2 g of a yellow oil. Purification of
the product was accomplished on a Flash 40 system using a 4
× 15-cm KP-Sil column eluted with 10:10:1 dichloromethane/
ethyl acetate/methanol to give 785 mg (38%) of a pale yellow
oil: UV λmax (ꢀ) 230 nm (8.7 mM-1 cm-1); 1H NMR δ 2.02 (s, 3
H), 2.07 (m, 2 H), 2.11 (s, 3 H), 3.34 (s, 1.5 H), 3.46 (m, 4 H),
3.83 (m, 4 H), 4.03 (s, 3 H), 5.15 (m, 1 H), 6.28 (b, 0.5 H), 6.35
(b, 0.5 H); 13C NMR δ 23.18, 33.81, 33.92, 40.74, 44.18, 47.48,
51.04 (CH3OH), 51.31, 61.21, 169.81, 170.14. Anal. Calcd for
(m, 2 H), 1.81 (m, 2 H), 2.04 (s, 3 H), 3.22 (m, 2 H), 3.40 (m, 4
H), 3.57 (m, 4 H), 3.75 (s, 3 H), 4.03 (s, 3 H), 5.0 (b, 1 H), 6.42
(d, 1 H); 13C NMR δ 22.89, 23.14, 29.02, 32.03, 42.28, 42.64,
51.06, 51.74, 52.42, 61.14, 157.49, 170.14, 172.98. Anal. Calcd
for C15H28N6O6‚H2O: C, 44.33; H, 7.44; N, 20.68. Found: C,
44.40; H, 7.12; N, 20.18.
Rea ction of 9d w ith P oly(eth ylen e glycol) Meth yl
Eth er To F or m 9f. A partial solution of 700 mg (2.0 mmol)
of poly(ethylene glycol) methyl ether (average MW 350) and
246 mg (2.2 mmol) of potassium tert-butoxide in 5 mL of
tetrahydrofuran and 1 mL of tert-butyl alcohol was heated at
reflux for 30 min. To the hot solution was added 700 mg (2.33
mmol) of 9d in 15 mL of tetrahydrofuran, and heating at reflux
was continued for 3 h. The reaction mixture was allowed to
cool to room temperature, diluted with 50 mL of dichlo-
romethane, and washed with 5% aqueous hydrochloric acid.
The solution was dried over sodium sulfate, filtered, and
evaporated to give 716 mg of 9f as a yellow oil. This material
was chromatographed on a Flash 40 system using a 4.0 × 15
cm KP-Sil column and 4:1 acetonitrile/tetrahydrofuran: UV
λ
max (ꢀ) 238 nm (6.4 mM-1 cm-1); 1H NMR δ 3.38 (s, 3 H), 3.41
(m, 4 H), 3.56 (m, 7 H), 3.65 (m, 25 H), 4.03 (s, 3 H). Anal.
Calcd for C21H42N4O11: C, 49.06; H, 7.86; N, 10.40. Found: C,
48.87; H, 7.96; N, 10.83.
O2-Meth yl 1-[4-[7-(â-Ribofu r a n osyl)p u r in -6-yl]p ip er -
a zin -1-yl]d ia zen -1-iu m -1,2-d iola te, 9g. To a solution of 275
mg (1.72 mmol) of 8 in 10 mL of ethanol and 0.250 mL (1.8
mmol) of triethylamine was added 460 mg (1.6 mmol) of
6-chloropurine riboside. The resulting slurry was heated at
reflux for a total of 3 h. A homogeneous solution resulted upon
heating. The solution was allowed to cool to room temperature
and concentrated on a rotary evaporator to give a solid residue.
The product was extracted with acetone, filtered, and evapo-
rated to give 689 mg of a white powder and then recrystallized
from ethanol: mp 195-197 °C; UV λmax (ꢀ) 218 nm (15.1 mM-1
cm-1), 278 (17.2 mM-1 cm-1); 1H NMR (DMSO-d6) δ 3.93 (s, 3
H), 4.1-4.58 (m, 8 H), 5.23 (m, 1 H), 5.32 (m, 1 H), 5.50 (d, 1
H), 5.93 (d, 1 H), 8.30 (s, 1 H), 8.48 (s, 1 H); 13C NMR (DMSO-
d6) δ 43.23, 50.58, 60.57, 61.40, 70.39, 73.55, 85.69, 87.74,
119.70, 139.23, 150.43, 151.72, 152.98. Anal. Calcd for
C
12H23N5O4S‚0.5 CH3OH: C, 42.97; H, 7.21; N, 20.04; S, 9.18.
Found: C, 43.30; H, 6.91; N, 20.08; S, 9.40.
C15H22N8O6: C, 43.90; H, 5.40; N, 27.30. Found: C, 43.78; H
O2-Meth yl 1-[4-(Su ccin im id -N-oxyca r bon yl)p ip er a zin -
1-yl]d ia zen -1-iu m -1,2-d iola te, 9d . A partial solution of 2.8
g (0.011 mol) of N,N′-disuccinimidyl carbonate in 15 mL of
dichloromethane was stirred in a round-bottom flask and
mixed with a solution of 1.76 g (0.011 mol) of 8 in 30 mL of
dichloromethane and 1.5 mL of triethylamine. The resulting
homogeneous solution was stirred at room temperature. The
5.38; N, 27.29.
2-Br om oeth ylsu lfu r yl Ch lor id e. A solution of 20 mL (0.28
mol) of bromoethanol in 50 mL of dichloromethane was cooled
to 0 °C, followed by the dropwise addition of 11.25 mL (0.28
mol) of sulfuryl chloride in 50 mL of dichloromethane. The
resulting solution was kept at 4 °C for 72 h. The solution was
washed with cold 10% sodium hydroxide until the washings

5130 J . Org. Chem., Vol. 64, No. 14, 1999
Saavedra et al.
tested distinctly basic. The organic layer was dried over sodium
sulfate, filtered through a layer of magnesium sulfate, and
concentrated on a rotary evaporator; the resulting crude
product was vacuum distilled to give 35 g (56%) of a colorless
buffered saline, 76 mM. Anal. Calcd for picrate C12H15N7O9:
C, 35.92; H, 3.77; N, 24.43. Found: C, 35.74; H, 3.73; N, 24.30.
O2-(Meth oxym eth yl) 1-[(4-Eth oxyca r bon yl)p ip er a zin -
1-yl]d ia zen -1-iu m -1,2-d iola te, 13a . A slurry of 4.4 g (0.018
mol) of 6a and 2 g of anhydrous sodium carbonate in 100 mL
of tetrahydrofuran was stirred under nitrogen at 0 °C. Through
a septum was added 1.5 mL (0.02 mmol) of methyl chloro-
methyl ether dropwise. Methanol, 1 mL, was added simulta-
neously with a syringe. The resulting mixture was allowed to
warm to room temperature and stirred overnight. The reaction
mixture was filtered and evaporated to dryness under reduced
pressure. The residue was extracted with dichloromethane,
dried over sodium sulfate, filtered through a layer of magne-
sium sulfate, and evaporated to give 4.74 g of an oil that
crystallized after several days on standing at ambient tem-
perature. The product was recrystallized from ether to give
pale yellow plates: mp 54-55 °C; UV λmax (ꢀ) 232 nm (7.3
1
oil: bp 73-75 °C at 1.5 mmHg; H NMR δ 3.64 (t, 2 H), 4.75
(t, 2 H); 13C NMR δ 25.39, 74.07. Anal. Calcd for C2H4SO3-
ClBr: C, 10.75; H, 1.80; S, 14.35; total halogen as Br, 71.52,
and Cl, 31.72. Found: C, 10.82; H, 1.80; S, 14.35; total halogen
as Br, 71.63, and Cl, 31.78.
O2-(2-Br om oeth yl) 1-(4-Eth oxycar bon ylpiper azin -1-yl)-
d ia zen -1-iu m -1,2-d iola te, 10a . To a cold (0 °C) slurry of 4.0
g (16.7 mmol) of 6a and 4 g of anhydrous sodium carbonate in
60 mL of methanol was added 3.75 g (16.8 mmol) of 2-bromo-
ethyl sulfuryl chloride in 5 mL of ether under nitrogen. The
reaction mixture was allowed to warm to 10 °C and stirred
for 2 h. The mixture was filtered, the filtrate was concentrated
on a rotary evaporator, and the residue was extracted with
dichloromethane. The solution was filtered through a layer of
magnesium sulfate and evaporated in vacuo to give a tan solid
that on recrystallization from petroleum ether/ether gave 540
mg (10%) of pale yellow plates: mp 83-84 °C; UV λmax (ꢀ) 240
1
mM-1 cm-1); H NMR δ 1.28 (t, 3 H), 3.46 (m, 4 H), 3.46 (s, 3
H), 3.65 (m, 4 H), 4.16 (q, 2 H), 5.22 (s, 2 H); 13C NMR δ 14.57,
42.37, 50.94, 57.08, 61.79, 97.92, 155.08. Anal. Calcd for
C9H18N4O5: C, 41.22; H, 6.87; N, 21.37. Found: C, 41.32; H,
6.95; N, 21.42.
1
nm (7.4 mM-1 cm-1); H NMR (methanol-d4) δ 1.28 (t, 3 H),
O2-Meth oxym eth yl 1-(P ip er a zin -1-yl)d ia zen -1-iu m -1,2-
d iola te, 13b. A solution of 3.51 g (13.4 mmol) of 13a in 100
mL of 10% ethanolic sodium hydroxide was heated at reflux
for 1 h. The reaction mixture was cooled to room temperature,
and the ethanol was removed on a rotary evaporator. The
residue was extracted with dichloromethane, filtered, and
extracted with aqueous hydrochloric acid. The aqueous layer
was washed with dichloromethane and then made basic with
aqueous sodium hydroxide. The product was extracted into
dichloromethane, dried over sodium sulfate, and filtered
through a layer of magnesium sulfate. Evaporation of the
solvent gave 2.1 g of product as a pale yellow oil. Purification
was carried out by Flash 40 chromatography using a 4 × 15
cm KP-Sil column and 10:1 dichloromethane/methanol as the
3.41 (m, 4 H), 3.57 (t, 2 H), 3.67 (m, 4 H), 4.16 (q, 2 H), 4.49 (t,
2 H); 13C NMR δ 14.57, 27.64, 42.35, 51.06, 61.82, 72.71,
155.06. Anal. Calcd for C9H17N4O4Br: C, 33.24; H, 5.27; N,
17.23; Br, 24.57. Found: C, 33.40; H, 5.19; N, 17.23; Br, 24.68.
O2-(2-Br om oeth yl) 1-(4-ter t-Bu toxyca r bon ylp ip er a zin -
1-yl)d ia zen -1-iu m -1,2-d iola te, 10b. A slurry of 6.9 g (26
mmol) of 6b, 25 g of anhydrous sodium carbonate, and 200
mL of methanol was cooled to 0 °C. A solution of 6 g (27 mmol)
of 2-bromoethyl sulfuryl chloride in 10 mL of ether was added
dropwise to the cold slurry under nitrogen. The reaction
mixture was allowed to warm to room temperature and stirred
overnight to give on workup 3.9 g of a brown solid. The crude
product was recrystallized from ether/petroleum ether to give
1 g of a white crystalline diazeniumdiolate: mp 86-87 °C; UV
1
eluent: UV λmax (ꢀ) 232 nm (8.2 mM-1 cm-1); H NMR δ 3.05
1
(methanol) λmax (ꢀ) 236 nm (8.9 mM-1 cm-1); H NMR δ 1.47
(m, 4 H), 3.46 (m, 4 H), 3.47 (s, 3 H), 5.22 (s, 2 H); 13C NMR δ
44.88, 52.41, 57.00, 97.78; maximum solubility in phosphate-
(t, 9 H), 3.34 (m, 4 H), 3.59 (m, 6 H), 4.49 (t, 2 H). Anal. Calcd
for C11H21N4O4Br: C, 37.41; H, 5.99; N, 15.86; Br, 22.62.
Found: C, 37.49; H, 6.03; N, 15.87; Br, 22.65.
buffered saline, 0.36 M. Anal. Calcd for picrate C12H17N7O10
:
C, 34.37; H, 4.09; N, 23.38. Found: C, 34.63; H, 4.13; N, 23.13.
O2-Meth oxym eth yl 1-[4-(Su ccin im id -N-oxyca r bon yl)-
p ip er a zin -1-yl]d ia zen -1-iu m -1,2-d iola t e, 13c. A partial
solution of 1.23 g (4.8 mmol) of N,N′-disuccinimidyl carbonate
in 15 mL of dichloromethane was added to a solution of 915
mg (4.8 mmol) of 13b in 15 mL of dichloromethane. To the
resulting homogeneous solution was added 0.7 mL (5 mmol)
of triethylamine. The reaction was complete on stirring at room
temperature within 1 h. The reaction mixture was evaporated
to dryness. The residue was taken up in dichloromethane and
washed first with cold, dilute hydrochloric acid and then with
aqueous bicarbonate. The organic layer was dried over sodium
sulfate, filtered through magnesium sulfate, and evaporated
to give 1.42 g of a glass. This compound was purified as
described for the methyl analogue 9d , giving a glass that
crystallized on standing: mp 123-125 °C; UV (ethanol) λmax
(ꢀ) 238 nm (7.69 mM-1 cm-1); 1H NMR δ 2.82 (s, 4 H), 3.49 (s,
3 H), 3.57 (m, 4 H), 3.81 (m, 4 H), 5.22 (s, 2 H); 13C NMR δ
25.43, 43.41, 50.38, 57.09, 98.00, 150.13, 169.41. Anal. Calcd
for C11H17N5O7: C, 39.88; H, 5.17; N, 21.14. Found: C, 40.16;
H, 5.23; N, 20.91.
Con ju ga tion of 13b w ith a P h osp h olip id To P r od u ce
13d . To a solution of 170 mg (0.513 mmol) of 13c in 10 mL of
chloroform was added 200 µL (1.4 mmol) of triethylamine and
346 mg (0.5 mmol) of dipalmitoyl DL-R-phosphatidyl ethanol-
amine. The partial solution was diluted with 10 mL of
chloroform to give a homogeneous solution. Heating was
continued for 8 h, whereupon the mixture was allowed to cool
to room temperature and concentrated on a rotary evaporator
to give 730 mg of a solid residue. Purification of 13b was
performed on a Flash 40 system using a 4 × 15 cm KP-Sil
column. The less polar impurities were eluted with 10:1
dichloromethane/methanol, while the remainder eluted in 1:1
dichloromethane/methanol to give 308 mg of product: mp
123-125 °C; UV (ethanol) λmax (ꢀ) 234 nm (8.4 mM-1 cm-1);
O2-Vin yl 1-(4-Eth oxyca r bon ylp ip er a zin -1-yl)d ia zen -1-
iu m -1,2-d iola te, 11a . To a solution of 917 mg (2.8 mmol) of
10a in 55 mL of tetrahydrofuran was added 1 g (25 mmol) of
powdered sodium hydroxide. The resulting slurry was heated
at reflux for 3 h, allowed to cool to room temperature, and
filtered. The solvent was evaporated, and the residue was
extracted with dichloromethane, dried over sodium sulfate,
filtered through magnesium sulfate, and concentrated under
vacuum to give 280 mg of an orange oil. This was purified on
a silica gel column with 1:1 ethyl acetate/cyclohexane as the
eluant to give 268 mg (39%) of 11a : UV (methanol) λmax (ꢀ)
1
260 nm (6.6 mM-1 cm-1); H NMR δ 1.28 (t, 3 H), 3.47 (m, 4
H), 3.68 (m, 4 H), 4.17 (q, 2 H), 4.44 (dd, 1 H), 4.869 (dd, 1 H),
6.82 (dd, 1 H); 13C NMR δ 14.58, 42.31, 50.89, 61.86, 92.67,
148.47, 155.06. Anal. Calcd for C9H16N4O4: C, 44.26; H, 6.60;
N, 22.94. Found: C, 44.18; H, 6.53; N, 22.87.
O2-Vin yl 1-(P ip er a zin -1-yl)d ia zen -1-iu m -1,2-d iola te, 12.
A solution of 400 mg (1.64 mmol) of 11a in 6 mL of 9% sodium
hydroxide in ethanol and 2 mL of water was heated at reflux
for 4 h. The reaction mixture was allowed to cool to room
temperature and concentrated under vacuum. The residue was
extracted with dichloromethane. The organic extract was in
turn extracted with cold 1 M hydrochloric acid, and the organic
layer was discarded. The aqueous layer was washed once with
dichloromethane, basified with 10% aqueous sodium hydrox-
ide, and extracted with dichloromethane. This organic extract
was dried over sodium sulfate, filtered through magnesium
sulfate, and concentrated under vacuum to give a pale yellow
oil. The crude product was chromatographed on silica gel with
20:1 acetonitrile/tetrahydrofuran as the eluant to give 200 mg
of 12: mp 133-135 °C (picrate); UV (methanol) λmax (ꢀ) 260
1
nm (8.7 mM-1 cm-1); H NMR δ 3.05 (m, 4 H), 3.46 (m, 4 H),
4.11 (dd, 1 H), 4.86 (dd, 1 H), 6.82 (dd, 1 H); 13C NMR δ 43.31,
50.99, 92.90, 148.44; maximum solubility in phosphate-

Piperazine as a Diazeniumdiolate Group Linker
J . Org. Chem., Vol. 64, No. 14, 1999 5131
1H NMR δ 0.89 (t, 6 H), 1.27 (s, 52 H), 1.58 (b, 8 H), 3.47 (m,
4 H), 3.60 (m, 4 H), 3.50 (s, 3 H), 3.93 (b, 4 H), 4.35 (m, 1 H),
5.23 (s, 2 H), 6.38 (b, 1 H). Anal. Calcd for C44H86N5O12P‚
2H2O: C, 55.97; H, 9.61; N, 7.42. Found: C, 56.33; H, 9.38; N,
6.89.
8451 UV-vis diode array spectrophotometer at 37 °C. Dis-
sociation of 6a -c was followed at the characteristic 252-nm
diazeniumdiolate maximum. With 6d and e, which displayed
more intense UV absorption and had maxima at 246 nm,
reactions were followed at that wavelength. Compound 6f was
solubilized for the kinetic runs by dissolving in methanol at a
concentration of 10 mM and then diluting 100-fold with pH
7.4 phosphate buffer to initiate the reaction.
For all except the pyrimidine-substituted derivative, absor-
bance-time (A-t) data followed good first-order kinetics with
ln(At - A∞) versus time plots, displaying good linearity over
3-4 half-lives. With the pyrimidine derivative, an initial drop
in absorbance from 2.23 to ca. 1.355 over the first 600 s was
followed by a slower decrease over longer reaction times (t >
1900 s). Using the 1.355 absorbance value as A∞, the initial
data also showed good first-order behavior (R2 ) 0.994 over
3.5 half-lives).
The rates for O2-substituted derivatives 13b and 13d were
measured by a chemiluminescence method developed previ-
ously.11 Briefly, solutions (or a suspension, in the case of
incompletely soluble 13d ) in 0.1 M phosphate, pH 7.4, were
incubated continuously at 37 °C except for short intervals
during which their contents were swept with inert gas into a
chemiluminescence detector for quantification of NO. After a
steady baseline was achieved, integration over several minutes
provided the NO generation rate during that interval. Plots
of these rates versus the times at the midpoints of the
corresponding interval yielded a curve from which the half-
life could be estimated.
Yields of NO were determined by integration of these rate
versus time plots with extrapolation to infinite time, as
previously described.9 For anionic diazeniumdiolates 6a -f,
infinite time was effectively reached within a single purge with
inert gas, allowing quantification by integration of the result-
ing chemiluminescence detector response versus time curve.
O2-Meth oxym eth yl 1-[4-(2-Mer ca p toeth yl)p ip er a zin -1-
yl]d ia zen -1-iu m -1,2-d iola te, 13e. A solution of 3.34 g (0.0176
mol) of 13b in 20 mL of dichloromethane and 5 mL of methanol
was treated with 6 g (0.1 mol) of ethylene sulfide. The resulting
solution was heated at reflux, under nitrogen, and the progress
of the reaction was monitored by thin-layer chromatography
(silica gel, 4:1 acetonitrile/tetrahydrofuran). The reaction
appeared to have gone to completion within 18 h; at this time,
the mixture was cooled to room temperature, filtered, and
evaporated to give 4.2 g of a turbid yellow oil. The oil was
chromatographed twice on a silica gel column and eluted with
4:1 acetonitrile/tetrahydrofuran to give 1.3 g of 13e: mp
(picrate) 45 °C; UV λmax (ꢀ) 232 nm (4.9 mM-1 cm-1); 1H NMR
δ 2.66-2.85 (m, 8 H), 3.49 (s, 3 H), 3.51 (m, 4 H), 5.22 (s, 2 H);
13C NMR δ 36.19, 50.84, 51.44, 56.99, 57.05, 97.81. Anal. Calcd
for the picrate C14H21N7O10S: C, 35.07; H, 4.42; N, 20.45; S,
6.69. Found: C, 35.23; H, 4.35; N, 20.16; S, 6.88.
Rea ction of 13b w ith P oly(vin yl ch lor id e) To P r od u ce
13f. Poly(vinyl chloride) (2.15 g, average molecular weight 62
kDa, Aldrich) was dissolved in 50 mL of tetrahydrofuran with
the aid of a vortex mixer. To this solution was added 197 mg
(1.04 mmol) of 13b in 5 mL of tetrahydrofuran. The resulting
solution was refluxed for 6 h and then cooled to room
temperature. The solvent was removed with a rotary evapora-
tor to give an amorphous solid that was washed with methanol
and then water to remove unreacted 13b. The polymer was
dried under vacuum to give 2.6 g of product: NMR (tetrahy-
drofuran-d8) δ 2.02-2.49 (m, 2 H), 2.93-3.01 (m, 0.03 H),
3.36-3.41 (m, 0.03 H), 3.38 (s, 0.023 H), 4.28-4.70 (m, 1 H),
5.10 (s, 0.015 H). Integration of the methylene and methyl
singlets at δ 5.10 and 3.32, respectively, relative to the
methylene and methinyl protons of the polymer backbone led
to an estimate of 1.3 mol percent incorporation of 13b into
the poly(vinyl chloride). Elemental analysis pointed to a
slightly higher incorporation, however. Anal. (Calcd on the
assumption that 2.3% of the Cl in the polymer is replaced by
13b and that tetrahydrofuran remains trapped in the polymer
matrix to the extent of 0.05 solvent molecules per monomer
unit): C, 40.29; H, 5.34; Cl, 49.88; N, 1.81. Found: C, 40.51;
H, 5.46; Cl, 50.27; N, 1.83.
Ack n ow led gm en t. We thank Michael Citro for
performing the chemiluminescence analyses and Mary
McGuire for obtaining the NMR spectra. This project
was funded in part by the National Cancer Institute
under contract NO1-CO-56000.
Su p p or tin g In for m a tion Ava ila ble: Individual least-
squares plots (assuming first-order kinetics) of the three
separate sets of NO release data found in Figure 1. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Mea su r em en t of Dissocia tion Kin etics a n d Yield s of
NO. Rate measurements for piperazine derivative 6a -f were
carried out spectrophotometrically by following the decrease
in the UV absorption maxima in 0.10 M phosphate buffer at
pH 7.4. All measurements were made using a Hewlett-Packard
J O9901539