The secondary amine/nitric oxide complex ion R2N[N(O)NO]- as nucleophile and leaving group in SNAr reactions

Article abstract of DOI:10.1021/jo0016529

Ions of structure R₂N[N(O)NO]^- and their alkylation products have seen increasing use as nitric oxide (NO)-generating agents for biomedical research applications. Here we show that such diazeniumdiolate anions can readily displace halide from a variety of electrophilic aza- or nitroaromatic substrates to form O²-arylated derivatives of structure R₂N-N(O)=N-OAr. The site of arylation and the cis arrangement of the oxygens were confirmed by X-ray crystallography. Displacement by various nucleophiles showed R₂N[N(O)NO]^- to be a reasonably good leaving group, with rate constants for displacement by hydroxide, methoxide, and isopropylamine that were between those of chloride and fluoride in the S_NAr reactions we surveyed. The Meisenheimer intermediate could be spectrally observed. These O²-aryl diazeniumdiolates proved capable of reacting with the nucleophilic sulfur of the HIV-1 p7 nucleocapsid protein's zinc finger assembly to eject the zinc, disrupting a structural motif critical to viral replication and suggesting possible utility in the drug discovery realm.

Full text of DOI:10.1021/jo0016529

3090
J . Org. Chem. 2001, 66, 3090-3098
Th e Secon d a r y Am in e/Nitr ic Oxid e Com p lex Ion R2N[N(O)NO]- a s
Nu cleop h ile a n d Lea vin g Gr ou p in SNAr Rea ction s
J oseph E. Saavedra,*,† Aloka Srinivasan,‡ Challice L. Bonifant,‡ J ingxi Chu,‡
Anna P. Shanklin,‡ J udith L. Flippen-Anderson,§ William G. Rice,† J im A. Turpin,|
Keith M. Davies, and Larry K. Keefer†
Intramural Research Support Program, SAIC Frederick, National Cancer Institute at Frederick,
Frederick, Maryland 21702, Chemistry Section, Laboratory of Comparative Carcinogenesis,
National Cancer Institute at Frederick, Frederick, Maryland 21702, Laboratory for the Structure of
Matter, Naval Research Laboratory, Washington, District of Columbia 20375, Infectious Disease
Research, Southern Research Institute, Frederick, Maryland 21702, and Department of Chemistry,
George Mason University, Fairfax, Virginia 22030
saavj@mail.ncifcrf.gov
Received November 22, 2000
Ions of structure R2N[N(O)NO]- and their alkylation products have seen increasing use as nitric
oxide (NO)-generating agents for biomedical research applications. Here we show that such
diazeniumdiolate anions can readily displace halide from a variety of electrophilic aza- or
nitroaromatic substrates to form O2-arylated derivatives of structure R2N-N(O)dN-OAr. The site
of arylation and the cis arrangement of the oxygens were confirmed by X-ray crystallography.
Displacement by various nucleophiles showed R2N[N(O)NO]- to be a reasonably good leaving group,
with rate constants for displacement by hydroxide, methoxide, and isopropylamine that were
between those of chloride and fluoride in the SNAr reactions we surveyed. The Meisenheimer
intermediate could be spectrally observed. These O2-aryl diazeniumdiolates proved capable of
reacting with the nucleophilic sulfur of the HIV-1 p7 nucleocapsid protein’s zinc finger assembly
to eject the zinc, disrupting a structural motif critical to viral replication and suggesting possible
utility in the drug discovery realm.
In tr od u ction
introduce a series of such compounds below, compare the
reactivity of 1 with that of chloride as a leaving group in
SNAr reactions, and report on the ability of the O2-aryl
diazeniumdiolates12 to degrade a protein’s zinc finger
assembly by electrophilically attacking the constituent
thiolate ligands.
Diazeniumdiolate anions (1) are of current interest for
their ability to generate bioregulatory nitric oxide (NO)
spontaneously in physiological fluids, as in eq 1.1-4 They
can be alkylated at the terminal oxygen to produce
charge-neutral derivatives5 that are proving useful as
potential prodrugs, molecules that are stable in aqueous
media but that are capable of generating nitric oxide
beneficially when the protecting group is hydrolytically
or enzymatically removed in the target cell.6-11
Resu lts a n d Discu ssion
Dia zen iu m d iola te Ion s a s Nu cleop h iles. We began
by studying the reactivity of various electron-deficient
aryl halides with the DiEthylAmine/NO adduct, DEA/
NO (2; see Scheme 1). In general, aryl rings with two or
more activating moieties reacted smoothly to give the
anticipated products. Thus, not only could 2,4-dinitro-
phenyl derivative 3a be prepared in good yield, but so
too could the two analogues in which one or the other of
the nitro groups is replaced by trifluoromethyl (3b and
3c). Analogous nitropyridyl compounds (3d and 3e)
We are interested in exploring the potential utility of
the corresponding aryl derivatives. To this end, we
† SAIC Frederick.
‡ National Cancer Institute.
§ Naval Research Laboratory.
(9) Saavedra, J . E.; Mooradian, D. L.; Mowery, K. A.; Schoenfisch,
M. H.; Citro, M. L.; Davies, K. M.; Meyerhoff, M. E.; Keefer, L. K.
Bioorg. Med. Chem. Lett. 2000, 10, 751-753.
(10) Mowery, K. A.; Schoenfisch, M. H.; Saavedra, J . E.; Keefer, L.
K.; Meyerhoff, M. E. Biomaterials 2000, 21, 9-21.
(11) Saavedra, J . E.; Shami, P. J .; Wang, L. Y.; Davies, K. M.; Booth,
M. N.; Citro, M. L.; Keefer, L. K. J . Med. Chem. 2000, 43, 261-269.
(12) We refer to the compounds of structure R2NN(O)dNOAr studied
herein as "O2-arylated diazeniumdiolates", rather than using strict
Chemical Abstracts or International Union of Pure and Applied
Chemistry nomenclature systems, to emphasize their status as deriva-
tives of the 1-(N,N-dialkylamino)diazen-1-ium-1,2-diolate ion. Ration-
ale for maintaining the “diazeniumdiolate” root for all such ions having
potential biomedical relevance is discussed in detail in Keefer, L. K.;
Flippen-Anderson, J . L.; George, C.; Shanklin, A. P.; Dunams, T. M.;
Christodoulou, D.; Saavedra, J . E.; Sagan, E. S.; Bohle, D. S. Nitric
Oxide: Biol. Chem., in press.
| Southern Research Institute.
George Mason University.
(1) Hanson, S. R.; Hutsell, T. C.; Keefer, L. K.; Mooradian, D. L.;
Smith, D. J . Adv. Pharmacol. (San Diego) 1995, 34, 383-398.
(2) Keefer, L. K.; Nims, R. W.; Davies, K. M.; Wink, D. A. Methods
Enzymol. 1996, 268, 281-293.
(3) Keefer, L. K. CHEMTECH 1998, 28, 30-35.
(4) Keefer, L. K. Pharm. News 2000, 7, 27-32.
(5) Saavedra, J . E.; Dunams, T. M.; Flippen-Anderson, J . L.; Keefer,
L. K. J . Org. Chem. 1992, 57, 6134-6138.
(6) 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.
(7) Saavedra, J . E.; Booth, M. N.; Hrabie, J . A.; Davies, K. M.;
Keefer, L. K. J . Org. Chem. 1999, 64, 5124-5131.
(8) Hrabie, J . A.; Saavedra, J . E.; Roller, P. P.; Southan, G. J .;
Keefer, L. K. Bioconjugate Chem. 1999, 10, 838-842.
10.1021/jo0016529 CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/11/2001

R2N[N(O)NO]- Ion as Nucleophile and Leaving Group
J . Org. Chem., Vol. 66, No. 9, 2001 3091
Sch em e 1. Scop e of Electr op h ilic
Dia zen iu m d iola tion Rea ction . Rea ction s of 2 w ith
Va r iou s Ar om a tic Electr op h iles
Sch em e 2. Scop e of Electr op h ilic
Dia zen iu m d iola tion Rea ction (p a r t 2). Rea ction s of
1-F lu or o-2,4-d in itr oben zen e w ith Va r iou s
R2N[N(O)NO]- Ion s
readily formed, but the otherwise unsubstituted halo-
pyridines have thus far not been successfully linked to
diazeniumdiolates. However, a single nitro group in the
para position was sufficiently activating that DEA/NO
could displace halide to produce 3f, consistent with the
view that the nitro group is a more potent inducer of SNAr
reactivity than a heteroaromatic nitrogen.13,14 Two ni-
trogens in the ring, on the other hand, could under
appropriate circumstances allow for facile displacement
of halide; an interesting case in point is the reaction of
DEA/NO with 2,4-dichloropyrimidine, in which the 4-po-
sition was readily substituted by displacing the halide
to produce 3g but the 2-position could not be substituted
by the anion of 2 in dimethyl sulfoxide solution even
when silver ion was added to the medium. This result is
consistent with the known reactivities of the 2 and 4
positions in 2,4-dichloropyrimidine toward other nucleo-
philes.15
Sch em e 3. Ad d ition a l O2-Ar yl Dia zen iu m d iola tes
P r ep a r ed for Th is In vestiga tion
Similar results were seen when the diazeniumdiolate
ions formed by reacting nitric oxide with a variety of
other secondary amines were in turn reacted with
1-fluoro-2,4-dinitrobenzene, as expected. These reactions
are summarized in Scheme 2, with the structures of six
additional compounds synthesized for this investigation
shown in Scheme 3.
Interestingly, the ortho and para nitro groups of 1,3-
difluoro-4,6-dinitrobenzene (11; see Scheme 4) activated
both fluorines for displacement by diazeniumdiolate ions.
Thus exposure to excess DEA/NO converted the difluoride
to symmetrically substituted 12 (Scheme 4), but reaction
of the difluoride first with N-methylaniline to produce
13 followed by exposure to the dimethylamino analogue
of 2 (DMA/NO) produced 14. This order of addition was
necessary because reaction of monodiazeniumdiolate 15
with N-methylaniline led to extensive displacement of the
diazeniumdiolate ion as well as of fluoride. Similarly,
N-methylaniline displaced the diazeniumdiolate ion from
14 as well. These reactions are shown in Scheme 4.
Th e Dia zen iu m d iola te Ion a s a Lea vin g Gr ou p .
These latter reactions demonstrated that the diazen-
iumdiolate ion can serve as a leaving group as well as a
nucleophile in SNAr reactions. To probe this behavior
further, we studied the reaction of 3a with hydroxide,
(13) March, J . Advanced Organic Chemistry: Reactions, Mechanisms,
and Structure, 4th ed.; Wiley: New York, 1992; pp 649-651.
(14) Terrier, F. In Nucleophilic Aromatic Displacement. The Influ-
ence of the Nitro Group; VCH Publishers: New York, 1991.
(15) Shepherd, R. G.; Fedrick, J . L. Adv. Heterocycl. Chem. 1965, 4,
145-423.

3092 J . Org. Chem., Vol. 66, No. 9, 2001
Saavedra et al.
Ta ble 1. Ra te a n d Activa tion P a r a m eter s for Rea ction
Sch em e 4. Am in a tion ver su s Dia zen iu m d iola tion
of 1,3-Diflu or o-4,6-d in itr oben zen e (11)
a
of 3a w ith OH-, CH3O-, a n d i-P r NH2
k2 at 25 °C
(M-1 s-1
∆Hq
∆Sq
nucleophile solvent
)
(kJ mol-1
)
(J mol-1 K-1
)
OH-
H2O
MeOH
7.4 × 10-3
45.2
57.5
59.9
-134
-70.2
-65.2
MeO-
i-PrNH2
110 × 10-3
Me2SO 80 × 10-3
a
Rate constants k at 25 °C were calculated from the corre-
sponding activation parameters.
F igu r e 1. Ultraviolet spectral changes observed after adding
sodium methoxide at a concentration of 1.75 mM to a metha-
nolic solution of 96 µM 3a at 37 °C. Spectra were recorded 10,
60, 120, 240, 480, and 3000 s after mixing.
methoxide, and isopropylamine (Table 1). While no
displacement of the diazeniumdiolate ion was detectable
when 3a was placed in water at around pH 7, raising
the pH led to release of 2,4-dinitrophenoxide and dia-
zeniumdiolate ions (eq 2A). When the reaction of 3a with
hydroxide was studied kinetically by monitoring loss of
substrate by HPLC while maintaining constant ionic
strength with sodium perchlorate, it was found to be first-
order in substrate and hydroxide ion, with a second-order
rate constant, 7 × 10-3 M-1 s-1 at 25 °C, that compares
with values of 1.2 × 10-1 and 1.2 × 10-4 M-1 s-1 reported
for the reaction of hydroxide with 1-fluoro- and 1-chloro-
2,4-dinitrobenzene, respectively.14
product spectrum that was consistent with the presence
of 2,4-dinitroanisole (λmax ) 292 nm, ꢀ ) 10.5 mM-1 cm-1
)
and DEA/NO (λmax ) 250 nm, ꢀ ) 8.0 mM-1 cm-1) in the
solution. When the rate of disappearance of 3a was
followed by HPLC, the measured second-order rate
constant (0.11 M-1 s-1; see Table 1) was found to be ca.
four times as large as that (0.03 M-1 s-1) reported16 for
1-chloro-2,4-dinitrobenzene.
Reactions of dinitrohalobenzenes with anionic nucleo-
philes in hydroxylic solvents are generally assumed to
follow an SNAr reaction mechanism involving formation
of a σ, or Meisenheimer, complex resulting from attack
of the nucleophile on the ipso carbon. In polar aprotic
solvents, such as dimethyl sulfoxide, enhanced stability
has been found for such complexes, which are character-
ized by strong absorption bands in the visible region. In
some cases, stable Meisenheimer complexes have been
identified.14,17 Direct spectral evidence for an intermedi-
ate Meisenheimer complex was obtained during the
reaction of sodium methoxide with 3a in dimethyl sulf-
oxide, a transformation illustrated in Scheme 5. Strong
visible absorbance [λmax 504 and 346 nm (ꢀ 26 and 15
mM-1 cm-1, respectively)] appeared within seconds of
mixing 3a with excess methoxide and slowly decayed,
ultimately yielding a solution showing UV absorbance
consistent with the expected reaction products (2,4-
dinitroanisole and the free diazeniumdiolate, DEA/NO),
with the latter species reacting slowly to generate amine
and nitric oxide. The spectral changes documenting this
The reaction of 3a with methoxide (eq 2B) was exam-
ined both in methanol and in dimethyl sulfoxide. Ultra-
violet spectral changes observed for the reaction of 0.2
mM substrate with excess sodium methoxide in methanol
(Figure 1) showed the characteristic 3a absorption maxi-
mum (λmax ) 302 nm, ꢀ ) 16 mM-1 cm-1) shifting to 292
nm (ꢀ ) 12 mM-1 cm-1) and a second maximum appear-
ing at 250 nm (ꢀ ) 16 mM-1 cm-1), leading to a final
(16) Bartoli, G.; Todesco, P. E. Acc. Chem. Res. 1977, 10, 125-132.
(17) Orvik, J . A.; Bunnett, J . F. J . Am. Chem. Soc. 1970, 92, 2417-
2427.

R2N[N(O)NO]- Ion as Nucleophile and Leaving Group
J . Org. Chem., Vol. 66, No. 9, 2001 3093
Sch em e 5. In ter m ed ia cy of a Meisen h eim er
Com p lex in th e Rea ction of 3a w ith Meth oxid e Ion
in Dim eth yl Su lfoxid e
F igu r e 2. Electronic spectra of: (a) 3a at a concentration of
35 µM in dimethyl sulfoxide; (b-e) same as a but 0.3, 60, 145,
and 267 min, respectively, after adding excess (44 mM) sodium
methoxide at 37 °C to generate the transient Meisenheimer
intermediate, as in Scheme 5.
mechanism of SNAr reactivity are shown in Figure 2. A
transient visible coloration was also observed on mixing
3a with sodium methoxide in methanol; while this was
not evident among the spectral changes of Figure 1, it
nevertheless suggests that loss of the diazeniumdiolate
anion from the Meisenheimer complex was also rate
limiting in that solvent.
the R2N nitrogen (N3) was slightly pyramidal, with N3
being from 0.27 to 0.37 Å out of the plane formed by N1
and the two R groups. Crystal data and structure
refinement parameters are summarized in Table 2.
An tivir a l Activity of th e O2-Ar yl Dia zen iu m d io-
la tes. The results for the N- and O-nucleophiles pre-
sented above suggested that the O2-aryl diazenium-
diolates might be selectively activated for nitric oxide
release by reaction with cellular thiolate groups. Known
to be potent nucleophiles, these RS- species retain their
high reactivity even when coordinated to metal centers,
such as those in the zinc finger motifs of many proteins.18
The human immunodeficiency virus (HIV-1) synthesizes
a nucleocapsid protein (NCp7) containing two copies of
a specific zinc finger motif without which packaging of
viral genomic RNA and infectivity are blocked.19-23
Several classes of electrophilic agent have been shown
to bind these sulfurs covalently, ejecting the zinc and
inhibiting viral replication.24 In fact, 1-chloro-2,4-di-
nitrobenzene has itself shown promise as a treatment for
early HIV disease in human patients.25
When isopropylamine was employed as a nucleophile
in dimethyl sulfoxide, its reaction with 3a (eq 2C)
proceeded cleanly to products with no evidence of base
catalysis. Spectral changes between 200 and 450 nm
showed a decrease in the 3a absorbance (λmax ) 310 nm,
ꢀ ) 16 mM-1 cm-1) accompanied by formation of a
product with a strong visible maximum at 376 nm and a
shoulder at 432 nm. An isosbestic point, observed at 345
nm, was maintained for the duration of the reaction
(Figure A of Supporting Information). The values of the
second-order rate constant, measured under pseudo first-
order conditions with the isopropylamine concentration
(0.059-0.118) in large excess over that of 3a (94 µM),
were found to be independent of the excess amine
concentration and the wavelengths used (310, 370, and
432 nm) to monitor the reaction. The value obtained
spectrally was also in good agreement with that meas-
ured by HPLC in experiments having [isopropylamine]
) 12-29 mM. The reaction of 3a with isopropylamine
occurred five times more rapidly than the reaction of
isopropylamine with 1-chloro-2,4-dinitrobenzene meas-
ured in this laboratory.
X-r a y Cr ysta llogr a p h ic Stu d ies. The regio- and
stereochemistry of these ArO2N2dN1(O1)N3R2 systems
were in keeping with those of the alkyl derivatives. The
structure of 3a (see Supporting Information for full
structural details) shows the electrophilic moiety to be
attached to the O2-oxygen, with the two diazeniumdiolate
oxygens being in the typical cis arrangement. This was
true even with the sterically more demanding substitu-
tion pattern of 14. The O1-N1-N2-O2 torsion angles
were -2.5° and -3.9° for the two molecules in 3a and
-0.6° in 14. The substitution in 14 also caused the para
nitro group to be rotated out of the plane of the six-
membered ring. In 3a only one ortho nitro group was
rotated out of the plane of the six-membered ring, due
in this case to intermolecular packing forces rather than
intramolecular interactions as in 14. In both 3a and 14,
To determine whether the O2-arylated diazenium-
diolates are sufficiently electrophilic to react with the
NCp7 thiolate moiety, the protein was dissolved in 10
(18) Roehm, P. C.; Berg, J . M. J . Am. Chem. Soc. 1998, 120, 13083-
13087.
(19) Rice, W. G.; Supko, J . G.; Malspeis, L.; Buckheit, R. W., J r.;
Clanton, D.; Bu, M.; Graham, L.; Schaeffer, C. A.; Turpin, J . A.;
Domagala, J .; Gogliotti, R.; Bader, J . P.; Halliday, S. M.; Coren, L.;
Sowder, R. C., II; Arthur, L. O.; Henderson, L. E. Science (Washington,
D. C.) 1995, 270, 1194-1197.
(20) Turpin, J . A.; Terpening, S. J .; Schaeffer, C. A.; Yu, G.; Glover,
C. J .; Felsted, R. L.; Sausville, E. A.; Rice, W. G. J . Virol. 1996, 70,
6180-6189.
(21) Rice, W. G.; Turpin, J . A. Med. Virol. 1996, 6, 187-199.
(22) McDonnell, N. B.; De Guzman, R. N.; Rice, W. G.; Turpin, J .
A.; Summers, M. F. J . Med. Chem. 1997, 40, 1969-1976.
(23) Cushman, M.; Golebiewski, W. M.; Graham, L.; Turpin, J . A.;
Rice, W. G.; Fliakas-Boltz, V.; Buckheit, R. W., J r. J . Med. Chem. 1996,
39, 3217-3227.
(24) Rice, W. G.; Turpin, J . A.; Schaeffer, C. A.; Graham, L.; Clanton,
D.; Buckheit, R. W., J r.; Zaharevitz, D.; Summers, M. F.; Wallqvist,
A.; Covell, D. G. J . Med. Chem. 1996, 39, 3606-3616.
(25) Stricker, R. B.; Zhu, Y. S.; Elswood, B. F.; Dumlao, C.; Van Elk,
J .; Berger, T. G.; Tappero, J .; Epstein, W. L.; Kiprov, D. D. Immunol.
Lett. 1993, 36, 1-6.

3094 J . Org. Chem., Vol. 66, No. 9, 2001
Saavedra et al.
Ta ble 2. Cr ysta l Da ta a n d Str u ctu r e Refin em en t P a r a m eter s
3a
14
empirical formula
formula weight
temperature (K)
wavelength (Å)
crystal system
C
10H13N5O6
C15H16N6O6
376.34
293(2)
1.54178
monoclinic
P21/n
299.25
293(2)
1.54178
triclinic
P1h
space group
unit cell dimensions
a ) 7.3410(10) Å, R ) 92.52(1)°
b ) 7.748(2) Å, â ) 93.54(1)°
c ) 24.434(4) Å, γ ) 99.98(2)°
1364.0(5)
a ) 13.152(3) Å, R ) 90°
b ) 7.3661(15) Å, â ) 98.33(2)°
c ) 17.821(4) Å, γ ) 90°
volume (Å3)
1708.3(6)
Z
4
4
density (calculated) (Mg/m3)
absorption coefficient (mm-1
F(000)
1.457
1.055
624
1.463
0.99
784
)
crystal size (mm3)
θ range for data collection
index ranges
0.66 × 0.60 × 0.40
0.28 × 0.24 × 0.04
5.81 to 56.28%
1.81 to 22.88%
-7 e h e 7, -8 e k e 8, -26 e l e 0
3802
-1 e h e 14, -2 e k e 8, -19 e l e19
3300
reflections collected
independent/observed reflections
completeness to theta ) 22.88°
absorption correction
3580/2964 [R(int) ) 0.0172]
2339/1538 [R(int) ) 0.0212]
99.7%
100.0%
face corrected
face corrected
max. and min. transmission
refinement method
0.960 and 0.925
full-matrix least-squares on F2
3580/0/380
0.962 and 0.802
full-matrix least-squares on F2
2339/0/244
data/restraints/parameters
goodness-of-fit on F2
1.102
1.035
final R indices [I >20 (I)]
R indices (all data)
largest diff. peak and hole
R1 ) 0.0592, wR2 ) 0.1408
R1 ) 0.0739, wR2 ) 0.1596
0.327 and -0.241 eÅ3
R1 ) 0.0560, wR2 ) 0.1282
R1 ) 0.0943, wR2 ) 0.1474
0.264 and -0.253 e Å3
mM phosphate buffer at 22 °C and pH 7.0 and mixed
with a selection of these novel compounds; evidence of
zinc loss was followed by fluorescence,24 as shown in
Figure C of Supporting Information. Time-dependent loss
of fluorescence due to the Trp37 residue of the protein’s
C-terminal zinc finger was seen for all four O2-aryl
diazeniumdiolates studied, with the observed decreases
of 27-45% reflecting significant loss of bound zinc, as
predicted. Upon the basis of this reactivity, we next
assessed the compounds’ ability to inhibit HIV-1Rf rep-
lication in the XTT cytoprotection assay. In this screen,
T-cells of the lymphoblastoid line CEM-SS are infected
with the laboratory-adapted cytopathic HIV strain Rf and
antiviral activity is measured as cytoprotection. While
none of the 19 compounds tested provided consistent
inhibition of HIV-1 replication, three of them did show
measurable activity in at least one antiviral determina-
tion (IC50 values of: 2.7 µM for 4b in two of four
determinations; 47 µM for 6 in one of four experiments;
and 16 µM for 8 in one of three determinations). The data
suggest that these compounds do interact with NCp7, but
that there are other factors affecting their ability to
inhibit viral replication. One such factor is cytotoxicity;
3b, 4f, and 12 were all toxic to the lymphoblastic CEM-
SS cells at submicromolar concentrations, suggesting
reactivity against a cell-based target. As a reference, the
positive control 3′-azido-3′-deoxythymidine (AZT), a nu-
cleoside reverse transcriptase inhibitor, inhibited virus
replication with an IC50 between 1 and 9 nM and was
nontoxic at 1 µM. Cytotoxicity values for the aryl dia-
zeniumdiolates evaluated in this assay are summarized
in Table B of Supporting Information. Despite their lack
of consistent activity in the anti-HIV screen, the ability
of these compounds to disrupt the NCp7 protein’s zinc
finger as predicted from the physicochemical data sug-
gests that this family of compounds may be useful
starting points for drug design activities. Efforts to exploit
their reactivity with other nucleophilic biomolecules for
therapeutic benefit are currently underway.
Exp er im en ta l Section
Compound 2 was prepared and characterized as previously
described,26 as were the analogous piperidine,27 pyrrolidine,6
and substituted piperazine7 salts. Unless otherwise indicated,
nuclear magnetic resonance (NMR) spectra were recorded in
chloroform-d with a Varian Unity Plus or Varian XL-200 NMR
spectrometer and ultraviolet (UV) data were collected on a
Hewlett-Packard Model 8451A diode array spectrophotometer.
Elemental analyses were performed by Atlantic Microlab, Inc.
NO was purchased from Matheson Gas Products (Montgom-
eryville, PA). Other reagents were obtained from Aldrich
Chemical Co. (Milwaukee, WI). Melting points were deter-
mined on a Thomas-Hoover capillary melting point apparatus
and are not corrected.
Sod iu m 1-(N,N-Dim et h yla m in o)d ia zen -1-iu m -1,2-d i-
ola te (DMA/NO). A 40% aqueous solution (25 mL, 0.198 mol)
of dimethylamine was cooled to 0 °C. To this cold solution was
added 8 g (0.2 mol) of sodium hydroxide. After stirring under
nitrogen until the pellets had dissolved, the ice-bath was
removed, 250 mL of dioxane was added, and the two-phase
mixture was charged with 40 psi of nitric oxide. After 48 h of
stirring, the pressure was released, and the mixture containing
a white precipitate was poured into 2 L of ether. The product
was collected by filtration, washed with copious amounts of
ether, and dried under vacuum to give 25 g of product: 1H
NMR (D2O) δ 2.75 (s); 13C NMR (0.1 M NaOD) δ 45.93; UV
(0.1 M NaOH) λmax (ꢀ) 250 nm (7.6 mM-1 cm-1). All DMA/NO
preparations contain significant amounts of inorganic salts,
and thus a useful elemental analysis was not obtained.
To prove the authenticity of the compound, 1.95 g (0.015
mol) was dissolved in 20 mL of methanol, cooled to 0 °C,
treated with 1.5 mL (0.016 mol) of dimethyl sulfate, and kept
overnight at 25 °C. The product was concentrated on a rotary
evaporator, extracted with dichloromethane, washed with
water, dried over sodium sulfate, filtered, and evaporated. A
vacuum distillation at 50-51 °C at approximately 1 mmHg
gave O2-methyl 1-(N,N-dimethylamino)diazen-1-ium-1,2-di-
olate as a colorless liquid: 1H NMR δ 3.0 (s, 6 H), 4.01 (s, 3
(26) 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.
(27) Drago, R. S.; Karstetter, B. R. J . Am. Chem. Soc. 1961, 83,
1819-1822.

R2N[N(O)NO]- Ion as Nucleophile and Leaving Group
J . Org. Chem., Vol. 66, No. 9, 2001 3095
H); 13C NMR δ 42.89, 60.85; UV (water) λmax (ꢀ) 234 nm (6.8
mM-1 cm-1). Anal. Calcd for C3H9N3O2: C, 30.25; H, 7.62; N,
35.27. Found: C, 30.32, H, 7.63, N, 35.18.
with water, extracted with ether, dried over sodium sulfate,
and evaporated. The product is either recrystallized or purified
by flash chromatography.
Sod iu m 1-(N-Ben zyl-N-m eth yla m in o)d ia zen -1-iu m -1,2-
d iola te. A solution of 50 mL (0.39 mol) of N-benzyl-N-
methylamine in 30 mL of methanol was placed in a Parr bottle.
The solution was treated with 83 mL (0.38 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 ether. The product was dried under vacuum to
give 14.7 g of product: mp 150-152 °C; UV (0.01 M NaOH)
Meth od C: A solution of 1.76 mmol of the activated bromo-,
fluoro-, or chloroarene reagent in 3 mL of dimethyl sulfoxide
is added to a slurry (1.76 mmol) of the diazeniumdiolate in 3
mL of THF at room temperature under nitrogen. The mixture
is stirred for 72 h. The resulting homogeneous solution is
treated with 100 mL of water. The precipitate is extracted with
ether. The organic layer is dried and evaporated, whereupon
the product is purified as described in method A.
O2-(2,4-Din itr op h en yl) 1-(N,N-Dieth yla m in o)d ia zen -1-
iu m -1,2-d iola te (3a ). This compound was prepared by method
A to give 1.3 g of a red oil which crystallized on standing.
Recrystallization from ethanol gave yellow-orange needles: mp
76-77 °C; 1H NMR δ 1.25 (t, 6 H), 3.58 (q, 4 H), 7.68 (d, 1 H),
8.44 (m, 2 H), 8.89 (m, 1 H); 13C NMR δ 11.51, 46.92, 117.53,
122.17, 129.13, 142.05, 154.22; UV (ethanol) λmax (ꢀ) 218 nm
(17.4 mM-1 cm-1) and 302 nm (15.6 mM-1 cm-1). Anal. Calcd
for C10H13N5O6: C, 40.13; H, 4.35; N, 23.41. Found: C, 40.21;
H, 4.43, N, 23.37.
Rea ction of 3a w ith Nu cleop h iles. A solution of 85 mg
(0.28 mmol) of 3a in 1 mL of ether was cooled to -4 °C and
treated with 1 mL of diethylamine. The solution was kept at
-4 °C for 1 h, giving a precipitate. The solid was collected by
filtration. The filtrate was concentrated and analyzed by NMR;
the residue was identical to an authentic sample of 2,4-dinitro-
N,N-diethylaniline. The precipitate was washed with petro-
leum ether and dried under nitrogen to give 5.4 mg of product
having λmax 250 nm (6.5 mM-1 cm-1); 1H NMR (D2O) δ 0.96 (t,
6 H), 1.28 (t, 6 H), 2.94 (q, 4 H), 3.08 (q, 4 H). This product
proved to be identical to an authentic sample of the diethyl-
ammonium salt of anion 2 prepared as previously described.
A solution of 16 mg (0.064 mmol) of 3a in 1 mL of ether
was treated with 29 mL of 25% sodium methoxide in methanol
(0.14 mmol) and allowed to stand at -4 °C for 2 h. The solid
precipitate was collected by filtration, washed with ether, and
dried under vacuum to give 4 mg of a solid identical to an
authentic sample of 2 (sodium salt).
Kin et ic Mea su r em en t s of t h e R ea ct ion of 3a w it h
Nu cleop h iles. Kinetic data on reactions of 3a with hydroxide,
methoxide, and isopropylamine were obtained by HPLC under
pseudo first-order conditions with the nucleophile concentra-
tion in g10-fold excess of the diazeniumdiolate substrate. In
a typical experiment involving methoxide, the reaction was
initiated by treating 10 mL of a 23 mM solution of sodium
methoxide in methanol, in a thermostated water bath, with
10 µL of a 0.95 M solution of 3a in the same solvent. Aliquots
of 100 µL each were withdrawn at timed intervals and
quenched by addition to 0.10 M hydrogen chloride in methanol.
The concentration of 3a in the resulting solution was deter-
mined by HPLC using a Phenomenex Luna column with 60%
aqueous acetonitrile as mobile phase. Pseudo first-order rate
constants, kobs, were obtained from the linear plots of log[sub-
strate] versus time for runs having methoxide concentrations
of 13-23 mM. Second-order rate constants (k2 ) kobs/[MeO-])
were independent of the excess methoxide concentrations
employed.
Similar conditions were used to follow the reaction of 1 mM
3a with 12-29 mM isopropylamine in dimethyl sulfoxide
except that the mobile phase was 65% aqueous methanol. The
reaction of 64-69 µM 3a with 6.2-6.5 mM hydroxide in 1%
aqueous dioxane was followed by HPLC using similar condi-
tions except that the mobile phase was 60:40 acetonitrile:0.5%
aqueous trifluoroacetic acid. Values for the activation param-
eters were calculated from linear fit of the data [ln(k/T versus
1/T] to the Eyring absolute rate equation.
1
λmax (ꢀ) 252 nm (10.0 mM-1 cm-1); H NMR (0.01 M NaOD in
D2O) δ 2.82 (s, 3 H); 4.06 (s, 2 H); 7.37-7.38 (m, 5 H); 13C
NMR (0.01 M NaOD in D2O) δ 138.1, 132.5, 131.3, 130.9, 61.88,
44.61. Anal. Calcd for C8H10N3O2Na‚H2O: C, 43.44; H, 5.47;
N, 19.00; Na, 10.39. Found: C, 43.45; H, 5.53; N, 18.84; Na,
10.67.
Sod iu m 1-[4-Ca r b oxa m id o)p ip er id in -1-yl]d ia zen -1-
iu m -1,2-d iola te. This compound was prepared from 12.8 g
(0.1 mol) of isonipecotamide and nitric oxide as described
above. A yield of 13 g of product was obtained: 1H NMR (D2O)
δ 1.87 (m, 2 H), 2.03 (m, 2 H), 2.44 (m, 1 H), 3.08 (m, 2 H),
3.23 (m, 2 H); 13C NMR (0.1 M NaOD) δ 30.29, 43.46, 54.24,
183.44; UV (0.1 M NaOH) λmax (ꢀ) 250 nm (8.2 mM-1 cm-1).
The sample contained approximately 7% starting isonipecot-
amide and was used without further purification in arylation
reactions, all of which gave acceptable elemental analyses.
A portion of this material was methylated with dimethyl
sulfate as described above for the dimethylamine analogue to
give O2-methyl 1-[(4-carboxamido)piperidin-1-yl]diazen-1-ium-
1,2-diolate: mp 155-157 °C; UV (ethanol) λmax (ꢀ) 241 nm (7.1
mM-1 cm-1); 1H NMR δ 2.06 (m, 4 H); 2.44 (m, 1 H); 3.08 (m,
4 H); 3.68 (s, 3 H), 6.21 (b, 2 H); 13C NMR δ 27.53, 41.29, 51.01,
61.08, 175.69. Anal. Calcd for C7H14N4O3‚2/3 H2O: C, 39.25;
H, 7.21; N, 26.15. Found: C, 39.41; H, 6.93; N, 25.95.
Sod iu m 1-[(3-Ca r b oxa m id o)p ip er id in -1-yl]d ia zen -1-
iu m -1,2-d iola te. A solution of 10 g (0.078 mol) of nipecotamide
in 25% methanolic sodium methoxide was diluted and charged
with nitric oxide as described for its isomer above to give 9.04
g (55%) of a white powder: 1H NMR (0.1 M NaOD) δ 1.52 (m,
1 H), 1.76 (m, 1 H), 1.94 (m, 2 H), 2.77 (m, 1 H), 3.10 (m, 3 H),
3.29 (m, 1 H), 3.34 (s, 1.6 H); 13C NMR (0.1 M NaOD) δ 25.97,
44.88, 55.12, 56.71, 181.73; UV (10 mM NaOH) λmax (ꢀ) 250
nm (7.2 mM-1 cm-1).
A portion of the sample was methylated in methanol as
described for the dimethylamine analogue to give O2-methyl
1-[(3-carboxamido)piperidin-1-yl]diazen-1-ium-1,2-diolate for
characterization: mp 140-142 °C; UV (ethanol) λmax (ꢀ) 240
1
nm (7.1 mM-1 cm-1); H NMR δ 2.01 (m, 4 H); 2.29 (m, 2 H);
3.02 (sextet, 2 H); 3.89 (m, 2 H), 4.02 (s, 3 H); 5.52 (b, 2 H);
13C NMR δ 22.84, 26.74, 41.68, 51.90, 61.09, 174.77. Anal.
Calcd for C7H14N4O3: C, 41.58; H, 6.98; N, 27.71. Found: C,
41.69; H, 6.92; N, 27.57.
Gen er a l P r oced u r es for th e Ar yla tion of Dia zen iu m -
d iola tes
Meth od A: solution of 11 mmol of a diazeniumdiolate anion
in 20 mL of 5% aqueous sodium bicarbonate is cooled to 0 °C
under nitrogen. A solution containing 10 mmol of the activated
fluoro- or chloroarene in 10 mL of tert-butyl alcohol is added
slowly. A precipitate normally forms upon addition. The
mixture is allowed to warm gradually to room temperature
and then stirred overnight. The product is extracted with
dichloromethane and washed subsequently first with cold
dilute hydrochloric acid and then with sodium bicarbonate
solution. The organic layer is dried over sodium sulfate, filtered
through a layer of magnesium sulfate, and evaporated under
vacuum to give the crude product. Purification is carried out
by recrystallization, flash chromatography, or preparative
HPLC.
Spectral changes observed during the reactions of 3a were
obtained using a Hewlett-Packard 8452A Diode Array UV-
visible spectrophotometer with the nucleophile (methoxide or
isopropylamine) in large excess. In a typical experiment, the
reaction (in methanol or dimethyl sulfoxide) was initiated by
adding 10- to 50-µL aliquots of nucleophile stock solutions to
1.0 mL of 3a contained in the same solvent in a thermostated
Met h od B: To a solution of 7.5 mmol of the diazenium-
diolate in 10 mL of dimethyl sulfoxide at 0 °C under a steady
stream of nitrogen is added dropwise 6 mmol of the activated
aryl compound. The reaction is allowed to warm to room
temperature and stirred for 24 to 72 h. Reaction is quenched

3096 J . Org. Chem., Vol. 66, No. 9, 2001
Saavedra et al.
cuvette. First-order rate constants were calculated from the
spectral changes using the Hewlett-Packard kinetics software.
O2-(2-Tr iflu or om eth yl-4-n itr op h en yl) 1-(N,N-Dieth yl-
a m in o)d ia zen -1-iu m -1,2-d iola te (3b). A solution of 859 mg
(5.54 mmol) of 2 in dimethyl sulfoxide was treated with 1.05
g (5.02 mmol) of 2-fluoro-5-nitrobenzotrifluoride according to
method B to give 1.45 g of a pale yellow crystallized product.
This was recrystallized from ether-petroleum ether: mp 67-
68 °C; 1H NMR δ 1.24 (t, 6 H), 3.52 (q, 4 H), 7.58 (d, 1 H), 8.44
(m, 1 H), 8.57 (d, 1 H); 13C NMR δ 11.53, 47.16, 115.88, 118.25,
119.95, 123.45, 128.90, 142.45, 158.44; UV (ethanol) λmax (ꢀ)
302 nm (14.4 mM-1 cm-1). Anal. Calcd for C11H13N4O4F3: C,
41.00; H, 4.07; N, 17.39. Found: C, 40.93; H, 4.11; N, 17.26.
O2-[2-Nitr o-4-(tr iflu or om eth yl)p h en yl] 1-(N,N-Dieth yl-
a m in o)d ia zen -1-iu m -1,2-d iola te (3c). Method A was fol-
lowed in the preparation of this compound. Purification of the
product was carried out on a KP-Sil column fitted to a Flash
40 apparatus, eluted with 2:1 cyclohexane:ethyl acetate, to give
183 mg (38%) of an oil: 1H NMR δ 1.23 (t, 6 H), 3.50 (q, 4 H),
7.66 (d, 1 H), 7.84 (d, 1 H), 8.28 (s, 1 H); 13C NMR δ 11.48,
47.20, 118.29, 123.64, 131.13, 137.89, 152.16; UV (ethanol) λmax
(ꢀ) 244 nm (8.26 mM-1 cm-1). Anal. Calcd for C11H13N4O4F3:
C, 41.00; H, 4.07; N, 17.39. Found: C, 41.28; H, 4.16; N, 17.26.
O2-(5-Nitr op yr id -2-yl) 1-(N,N-Dieth yla m in o)d ia zen -1-
iu m -1,2-d iola te (3d ). This reaction was carried out as de-
scribed in method A. The yellow solid was recrystallized from
ether-petroleum ether to give a 73% yield of pure material:
mp 77-78 °C; 1H NMR δ 1.24 (t, 6 H), 3.53 (q, 4 H), 7.21 (dd,
1 H), 8.52 (dd, 1 H), 9.17 (dd, 1 H); 13C NMR δ 11.42, 47.21,
109.25, 134.99, 141.15, 145.10, 165.21; UV (ethanol) λmax (ꢀ)
300 nm (12.9 mM-1 cm-1). Anal. Calcd for C9H13N5O4: C,
42.35; H, 5.13; N, 27.44. Found: C, 42.46; H, 5.14; N, 27.53.
O2-(3-Nitr op yr id -2-yl) 1-(N,N-Dieth yla m in o)d ia zen -1-
iu m -1,2-d iola te (3e). This compound was prepared as de-
scribed in method A to give a yellow oil. The crude product
mM-1 cm-1) and 304 nm (23.4 mM-1 cm-1). Anal. Calcd for
C8H9N5O6: C, 35.43; H, 3.34; N, 25.82. Found: C, 35.56; H,
3.37; N, 25.66.
O2-(2,4-Din itr oph en yl) 1-(P yr r olidin -1-yl)diazen -1-iu m -
1,2-d iola t e (4b). Method A was followed to give a product
which was recrystallized from ethanol: mp 94-95 °C; 1H NMR
δ 2.04 (m, 4 H), 3.35 (m, 4 H), 6.90 (d, 1 H), 8.20 (dd, 1 H),
8.67 (d, 1 H); 13C NMR δ 23.44, 50.53, 117.17, 122.17, 129.10,
136.88, 141.69, 154.53; UV (ethanol) λmax (ꢀ) 300 nm (11.4
mM-1 cm-1). Anal. Calcd for C10H11N5O6: C, 40.41; H, 3.73;
N, 23.56. Found: C, 40.36; H, 3.77; N, 23.42.
O2-(2,4-Din itr op h en yl) 1-(P ip er id in -1-yl)d ia zen -1-iu m -
1,2-d iola te (4c). Method A gave a product from sodium
1-(piperidin-1-yl)diazen-1-ium-1,2-diolate27 that was purified
by flash chromatography using a KP-Sil column and a Flash
40 system with dichloromethane as the eluant: mp 111-112
1
°C; H NMR (500 MHz) δ 1.60 (m, 2 H), 1.82 (m, 4 H), 3.63
(m, 4 H), 7.68 (d, J ) 9.3 Hz, 1 H), 8.45 (d, J 1 ) 2.7 Hz, J 2
)
9.3 Hz, 1 H), 8.87 (d, J ) 2.7 Hz, 1 H); 13C NMR δ 23.15, 24.31,
51.62, 117.54, 122.08, 129.10, 137.04, 142.01, 154.10; UV
(ethanol) λmax (ꢀ) 216 nm (14.6 mM-1 cm-1) and 300 nm (14.7
mM-1 cm-1). Anal. Calcd for C11H13N5O6: C, 42.45; H, 4.21;
N, 22.50. Found: C, 42.53; H, 4.25; N, 22.50.
O2-(2,4-Din itr op h en yl) 1-(4-Ca r boxa m id op ip er id in -1-
yl)d ia zen -1-iu m -1,2-d iola te (4d ). Method A was followed in
this preparation. The crude product was recrystallized from
1:1 methanol:dichloromethane to give an 85% yield of pale
yellow crystals: mp 165-166 °C; 1H NMR (500 MHz, DMSO-
d6) δ 1.71 (m, 2 H), 1.90 (m, 2 H), 2.32 (m, 1 H), 3.20 (m, 2 H),
4.18 (m, 2 H), 6.89 (br s, 1 H), 7.36 (br s, 1 H), 7.92 (d, J ) 9.3
Hz, 1 H), 8.54 (d, J 1 ) 2.8 Hz, J 2 ) 9.3 Hz, 1 H), 8.87 (d, J )
2.8 Hz, 1 H); 13C NMR (DMSO-d6) δ 26.63, 39.86, 49.46, 118.01,
121.75, 129.74, 136.81, 142.01, 152.86, 175.33; UV (ethanol)
λmax (ꢀ) 302 nm (7.5 mM-1 cm-1). Anal. Calcd for C12H14N6O7:
C, 40.68; H, 3.98; N, 23.72. Found: C, 40.61; H, 3.99, N, 23.70.
O2-(2,4-Din itr op h en yl) 1-(3-Ca r boxa m id op ip er id -1-yl)-
d ia zen -1-iu m -1,2-d iola te (4e). This compound was prepared
as described in method A. A 45% yield of product was obtained
after recrystallization from 1:1 methanol:dichloromethane: mp
was purified on
a KP-Sil column fitted to a Flash 40
apparatus, eluted with 5:1 dichloromethane:ethyl acetate, to
give a 52% yield of an oil: 1H NMR δ 1.25 (t, 6 H), 3.55 (q, 4
H), 7.26 (m, 1 H), 8.48 (m, 2 H); 13C NMR δ 11.44, 47.06,
119.62, 122.94, 135.63, 152.06, 152.39; UV (ethanol) λmax (ꢀ)
262 nm (15.4 mM-1 cm-1). Anal. Calcd for C9H13N5O4: C,
42.35; H, 5.13; N, 27.44. Found: C, 42.59; H, 5.20; N, 27.20.
O2-(4-Nitr op h en yl) 1-(N,N-Dieth yla m in o)d ia zen -1-iu m -
1,2-d iola te (3f). Method B was used in the preparation of this
compound. It was obtained in 31% yield after recrystallization
1
149-150 °C; H NMR (300 MHz, DMSO-d6) δ 1.56 (m, 2 H),
1.86 (m, 2 H), 2.55 (m, 1 H), 3.15 (m, 2 H), 4.17 (m, 2 H), 6.99
(br s, 1 H), 7.44 (br s, 1 H), 7.90 (d, J ) 9.3 Hz, 1 H), 8.56 (d,
J 1 ) 2.6 Hz, J 2 ) 9.3 Hz, 1 H), 8.87 (d, J ) 2.8 Hz, 1 H); 13C
NMR (DMSO-d6) δ 22.60, 26.51, 40.40, 50.10, 51.99, 118.08,
121.83, 129.86, 136.89, 142.07, 152.88, 173.78; UV (ethanol)
from ether-petroleum ether: mp 81-82 °C; H NMR δ 1.25
λ
max (ꢀ) 222 nm (6.9 mM-1 cm-1) and 302 nm (8.2 mM-1 cm-1).
1
(t, 6 H), 3.43 (q, 4 H), 7.36 (d, 2 H), 8.28 (d, 2 H); 13C NMR δ
11.45, 47.58, 115.12, 125.77, 143.62, 160.87; UV (ethanol) λmax
(ꢀ) 306 nm (13.7 mM-1 cm-1). Anal. Calcd for C10H14N4O4: C,
47.24; H, 5.55; N, 22.04. Found: C, 47.16; H, 5.51; N, 21.95.
O2-(2-Ch lor op yr im id in -4-yl) 1-(N,N-Dieth yla m in o)d ia -
zen -1-iu m -1,2-d iola te (3g). A solution of 600 mg (4 mmol)
of 2,4-dichloropyrimidine in 2 mL of dimethyl sulfoxide and 5
mL of tetrahydrofuran was added via syringe to a slurry of
678 mg (4.37 mmol) of 2 in 15 mL of tetrahydrofuran at room
temperature under nitrogen, and the resulting mixture was
stirred for 72 h. The mixture was shaken with 100 mL of ether.
The organic layer was washed with water, dried over sodium
sulfate, filtered through a layer of magnesium sulfate, and
evaporated to give 679 mg of an oil which crystallized in the
freezer. This material was recrystallized from ether-petro-
Anal. Calcd for C12H14N6O7: C, 40.68; H, 3.98; N, 23.72.
Found: C, 40.50; H, 3.98; N, 23.61.
O2-(2,4-Din itr op h en yl) 1-(4-P h en ylp ip er a zin -1-yl)d ia -
zen -1-iu m -1,2-d iola te (4f). Sodium 1-(4-phenylpiperazin-1-
yl)diazen-1-ium-1,2-diolate (732 mg; 3 mmol) was allowed to
react with 372 mg (3 mmol) of 1-fluoro-2,4-dinitrobenzene as
described in method A. Recrystallization from methanol gave
1
a pure product in 90% yield: mp 144-145 °C; H NMR (500
MHz) δ 3.42 (m, 4 H), 3.84 (m, 4 H), 6.96 (m, 3 H), 7.31 (m, 2
H), 7.69 (d, J ) 9.2 Hz, 1 H), 8.76 (d, J 1 ) 2.7 Hz, J 2 ) 9.2 Hz,
1 H), 8.88 (d, J ) 2.7 Hz, 1 H); 13C NMR δ 48.28, 50.55, 116.89,
117.62, 121.16, 122.15, 129.10, 129.35, 137.21, 142.28, 149.97,
153.82; UV (ethanol) λmax (ꢀ) 246 nm (9.3 mM-1 cm-1) and 298
nm (6.6 mM-1 cm-1). Anal. Calcd for C16H16N6O6‚1/4H2O: C,
48.92; H, 4.23; N, 21.39. Found: C, 48.78; H, 4.04; N, 21.46.
O2-(2,4-Din itr op h en yl) 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 te (4g). Method A was used to
prepare 4g in 90% yield after recrystallization from ethanol:
1
leum ether: mp 37-38 °C; H NMR δ 1.25 (t, 6 H), 3.56 (q, 4
H), 7.00 (d, 1 H), 8.50 (d, 1 H); 13C NMR δ 11.35, 46.90, 104.68,
160.61, 160.71, 169.23; UV (ethanol) λmax (ꢀ) 268 nm (9.3 mM-1
cm-1). Anal. Calcd for C18H12N5O2Cl: C, 39.11; H, 4.92; N,
28.51; Cl, 14.43. Found: C, 38.96; H, 4.96; N, 28.35; Cl, 14.60.
Attempts to displace the second chlorine by addition of
excess 2 resulted in no reaction.
1
mp 149-150 °C; H NMR (500 MHz, DMSO-d6) δ 3.73 (m, 4
H), 4.00 (m, 4 H), 6.71 (t, J ) 4.8 Hz, 1 H), 7.95 (d, J ) 9.3 Hz,
1 H), 8.41 (d, J ) 4.8 Hz, 2 H), 8.54 (d, J 1 ) 2.8 Hz, J 2 ) 9.3
Hz, 1 H), 8.86 (d, J ) 2.8 Hz, 1 H); 13C NMR (DMSO-d6) δ
41.74, 49.66, 110.87, 118.04, 121.69, 129.64, 136.73, 142.01,
152.82, 157.98, 160.83; UV (ethanol) λmax (ꢀ) 242 nm (21.1
mM-1 cm-1) and 300 nm (8.5 mM-1 cm-1). Anal. Calcd for
O2-(2,4-Din itr op h en yl) 1-(N,N-Dim eth yla m in o)d ia zen -
1-iu m -1,2-d iola te (4a ). This compound was prepared accord-
ing to method B. The crude product was recrystallized from
methanol to give a 33% yield of 4a : mp 146-147 °C; 1H NMR
(DMSO-d6) δ 3.26 (s, 6 H), 7.88 (d, 1 H), 8.54 (dd, 1 H), 8.85
(d, 1 H); 13C NMR (DMSO-d6) δ 41.17, 117.68, 121.68, 129.66,
136.66, 141.73, 153.14; UV (methanol) λmax (ꢀ) 220 nm (23.8
C
14H14N8O6: C, 43.08; H, 3.62; N, 28.71. Found: C, 43.04; H,
3.62; N, 28.84.
O2-(2,4-Din itr oph en yl) 1-[(4-Eth oxycar bon yl)piper azin -
1-yl]d ia zen -1-iu m -1,2-d iola te (4h ). The product, prepared

R2N[N(O)NO]- Ion as Nucleophile and Leaving Group
J . Org. Chem., Vol. 66, No. 9, 2001 3097
λmax (ꢀ) 250 nm (22.9 mM-1 cm-1). Anal. Calcd for C17H16
N3O4F3: C, 49.64; H, 3.92; N, 17.03. Found: C, 49.64; H, 3.95;
N, 16.95.
as in method A, was recrystallized from ethanol:dichloro-
methane: mp 140-141 °C; 1H NMR δ 1.32 (t, 3 H), 3.63 (m, 4
H), 3.74 (m, 4 H), 4.19 (q, 2 H), 7.66 (d, 1 H), 8.48 (q, 1 H),
8.88 (d, 1 H); 13C NMR δ 14.60, 42.18, 50.55, 62.07, 113.23,
117.72, 122.19, 129.07, 142.48, 153.69, 154.99; UV (ethanol)
λmax (ꢀ) 300 nm (12 mM-1 cm-1). Anal. Calcd for C13H16N6O8:
C, 40.61; H, 4.20; N, 21.87. Found: C, 40.74; H, 4.13; N, 21.98.
O2-(2,4-Din it r op h en yl) 1-(N-Ben zyl-N-m et h yla m in o)-
d ia zen -1-iu m -1,2-d iola te (4i). This material was prepared
in 49% yield as described in method A: mp 86-87 °C; 1H NMR
(500 MHz) δ 3.25 (s, 3 H), 4.76 (s, 2 H), 7.36 (m, 6 H), 8.35
(dd, J 1 ) 2.7 Hz, J 2 ) 9.3 Hz, 1 H), 8.84 (d, J ) 2.8 Hz, 1 H);
13C NMR δ 39.50, 57.63, 117.41, 122.01, 128.48, 128.57, 128.91,
129.05, 134.03, 136.87, 141.91, 154.06; UV (ethanol) λmax (ꢀ)
302 nm (11.0 mM-1 cm-1). Anal. Calcd for C14H13N5O6: C,
48.42; H, 3.77; N, 20.17. Found: C, 48.34; H, 3.80; N, 19.97.
-
O2-(5-Nitr op yr id -2-yl) 1-(4-P h en ylp ip er a zin -1-yl)d ia -
zen -1-iu m -1,2-d iola te (10). A tetrahydrofuran solution of
2-bromo-5-nitropyridine was added to sodium 1-(4-phenyl-
piperazin-1-yl)diazen-1-ium-1,2-diolate as described in method
B. The product was recrystallized from acetonitrile to give 248
1
mg (36%) of light yellow crystals: mp 175-176 °C; H NMR
(500 MHz) δ 3.42 (m, 4 H), 3.84 (m, 4 H), 6.96 (m, 3 H), 7.21
(d, J ) 9.0 Hz, 1 H), 7.31 (m, 2 H), 8.53 (d, J 1 ) 2.8 Hz, J 2
)
9.0 Hz, 1 H), 9.17 (d, J ) 2.8 Hz, 1 H); 13C NMR δ 48.30, 50.72,
109.39, 116.84, 121.04, 129.34, 135.06, 141.27, 145.04, 150.07,
164.89; UV (ethanol) λmax (ꢀ) 246 nm (5.2 mM-1 cm-1) and 296
nm (4.9 mM-1 cm-1). Anal. Calcd for C15H16N6O4: C, 52.33;
H, 4.65; N, 24.42. Found: C, 52.09; H, 4.65; N, 24.28.
1,5-Bis{[1-(N,N-dieth ylam in o)diazen -1-iu m -1,2-diolato]-
O2}-2,4-d in itr oben zen e (12). This reaction was carried out
in tetrahydrofuran:dimethyl sulfoxide according to method C.
The product was recrystallized from ethanol: mp 128-129 °C;
1H NMR δ 1.26 (t, 12 H), 3.59 (q, 8 H), 7.60 (s, 1 H), 8.89 (s, 1
H); 13C NMR δ 11.54, 46.76, 104.52, 125.57, 131.22, 154.51;
UV (ethanol) λmax (ꢀ) 294 nm (11.4 mM-1 cm-1). Anal. Calcd
for C14H22N8O8: C, 39.07; H, 5.15; N, 26.04. Found: C, 39.05;
H, 5.20; N, 25.95.
O2-(2-Ch lor op yr im id in -4-yl) 1-(P yr r olid in -1-yl)d ia zen -
1-iu m -1,2-d iola te (5). Method B gave a pure product that was
1
collected by filtration: mp 150-151 °C; H NMR δ 2.00 (t, 4
H), 3.77 (t, 4 H), 6.99 (d, 1 H), 8.49 (d, 1 H); 13C NMR δ 23.36,
50.49, 104.56, 160.43, 160.58, 169.35; UV (ethanol) λmax (ꢀ) 274
nm (18.5 mM-1 cm-1). Anal. Calcd for C8H10ClN5O2: C, 39.44;
H, 4.14; N, 28.74. Found: C, 39.51; H, 4.24; N, 28.87.
O2-(5-Nitr op yr id -2-yl) 1-(P yr r olid in -1-yl)d ia zen -1-iu m -
1,2-d iola te (6). To a slurry of 459 mg (3 mmol) of sodium
1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate in 4 mL of dimethyl
sulfoxide at 4 °C was added a solution of 406 mg (2 mmol) of
2-bromo-5-nitropyridine in 6 mL of tetrahydrofuran. The
reaction mixture was stirred overnight under nitrogen at room
temperature. The mixture was treated with water and ex-
tracted with ether. The organic solution was washed with
water, dried over sodium sulfate, filtered through a layer of
magnesium sulfate, and concentrated on a rotary evaporator.
The product was recrystallized from methanol: mp 162-163
°C; 1H NMR (500 MHz) δ 2.10 (m, 4 H), 3.80 (m, 4 H), 7.17 (d,
J ) 9.1 Hz, 1 H), 8.50 (d, J 1 ) 2.7 Hz, J 2 ) 9.1 Hz, 1 H), 9.14
(d, J ) 2.7 Hz, 1 H); 13C NMR δ 23.34, 50.57, 109.02, 134.84,
140.80, 145.03, 165.46; UV (ethanol) λmax (ꢀ) 302 nm (11.2
mM-1 cm-1). Anal. Calcd for C9H11N5O4: C, 42.69; H, 4.35; N,
27.67. Found: C, 42.72; H, 4.37; N, 27.51.
O2-(2-Ch lor op yr im id in -4-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 (7). Prepared accord-
ing to method C, this compound had mp 136-137 °C; 1H NMR
δ 1.29 (t, 3 H), 3.67 (m, 4 H), 3.71(m, 4 H), 4.18 (q, 2 H), 6.99
(d, 1 H), 8.52 (d, 1 H); 13C NMR δ 14.59, 42.14, 50.55, 61.98,
104.80, 155.04, 160.73, 168.79; UV (ethanol) λmax (ꢀ) 268 nm
(11.7 mM-1 cm-1). This compound undergoes nucleophilic
substitution with methoxide displacing the chlorine at C-2 and
the diazeniumdiolate at C-4 to give 2,4-dimethoxypyrimidine.
Anal. Calcd for C11H15N6O4Cl: C, 39.95; H, 4.57; N, 25.41; Cl,
10.72. Found: C, 40.07; H, 4.44; N, 25.32; Cl, 10.80.
N-Meth yl-N-p h en yl-2,4-d in itr o-5-flu or oa n ilin e (13). To
a solution of 3.15 g (0.0154 mol) of 1,5-difluoro-2,4-dinitroben-
zene in 15 mL of tetrahydrofuran was added 2.1 g (0.02 mol)
of anhydrous sodium carbonate. To the slurry was added a
solution of 1.67 mL (0.02 mol) of N-methylaniline in 10 mL of
tetrahydrofuran. This was done dropwise over a 20-min period.
After 24 h, the tetrahydrofuran was removed on a rotary
evaporator, the residue was extracted with dichloromethane,
and the organic layer was filtered into a separatory funnel.
The solution was washed with water, dried over sodium
sulfate, filtered through a layer of magnesium sulfate, and
evaporated to give 4.46 g of an orange solid. The product was
crystallized from ethanol: mp 158-159 °C; 1H NMR δ 3.45
(s, 3 H), 6.88 (d, 1 H), 7.12 (m, 2 H), 7.28 (m, 1 H), 7.42 (m, 2
H), 8.58 (d, 1 H); 13C NMR δ 42.99, 107.99, 108.33, 123.74,
126.50, 127.07, 130.27, 145.67, 156.03, 159.87; UV (ethanol)
λ
max (ꢀ) 248 nm (12 mM-1 cm-1) and 369 nm (13.2 mM-1 cm-1).
Anal. Calcd for C13H10N3O4F: C, 53.62; H, 3.46; N, 14.43.
Found: C, 53.73; H, 3.57; N, 14.26.
O2-[2,4-Din it r o-5-(N-m et h yl-N-p h en yla m in o)p h en yl]
1-(N,N-Dim eth ylam in o)diazen -1-iu m -1,2-diolate (14). This
compound was prepared using a slight modification of method
A. The arylating agent, 13, was dissolved in dioxane rather
than in tert-butyl alcohol. After 15 h the dioxane and water
were removed on a rotary evaporator, and the residue was
extracted with dichloromethane. The solution was washed with
water, dried over magnesium sulfate, and evaporated to give
2.7 g of a red-orange solid. The crude product was purified on
a 4-cm by 15-cm KP-Sil column of a Flash 40 system and
eluted with dichloromethane: mp 163-164 °C; 1H NMR δ 3.18
(s, 6 H), 3.45 (s, 3 H), 7.08 (s, 1 H), 7.15 (m, 3 H), 7.38 (m, 2
H), 8.63 (s, 1 H); 13C NMR δ 41.85, 42.51, 107.79, 113.22,
123.23, 126.18, 126.72, 128.63, 130.04, 134.20, 146.17, 147.82;
UV (DMSO) λmax (ꢀ) 258 nm (33.2 mM-1 cm-1) and 386 nm
(15.6 mM-1 cm-1). Anal. Calcd for C15H16N6O6: C, 47.84; H,
4.29; N, 22.33. Found: C, 47.70; H, 4.34; N, 22.04.
X-r a y Stu d y. Diffraction data for both 3a and 14 were
collected on a Bruker P4 automated diffractometer with a
graphite monochromator on the incident beam. All measure-
ments were performed using the θ/2 θ scan technique with a
varying scan speed depending upon the intensity of a reflec-
tion. Crystal and experimental data are summarized in Table
2. The data sets were corrected for Lorentz and polarization
effects and for absorption using the face-indexed algorithm in
program XPREP.28 The structures were solved by direct
methods and refined by full matrix least-squares on F2 values
O2-(5-Nitr op yr id -2-yl) 1-(N,N-Dim eth yla m in o)d ia zen -
1-iu m -1,2-d iola te (8). This reaction was carried out in tet-
rahydrofuran:dimethyl sulfoxide as described in Method C.
The product was crystallized from methanol to give a 56% yield
1
of 8: mp 180-181 °C; H NMR δ 3.24 (s, 6 H), 7.20 (m, 1 H),
8.51 (m, 1 H), 9.15 (b, 1 H); 13C NMR δ 41.33, 109.02, 136.11,
141.15, 144.84, 164.97; UV (ethanol) λmax (ꢀ) 218 nm (11 mM-1
cm-1) and 302 nm (13.6 mM-1 cm-1). Anal. Calcd for
C7H9N5O4: C, 37.00; H, 3.96; N, 30.84. Found: C, 37.10; H,
4.00; N, 30.92.
O2-[2-Nit r o-(4-t r iflu or om et h yl)p h en yl] 1-(4-P h en yl-
p ip er a zin -1-yl)d ia zen -1-iu m -1,2-d iola te (9). A solution of
4-fluoro-3-nitrobenzotrifluoride in tert-butyl alcohol was added
to a cold solution of sodium 1-(4-phenylpiperazin-1-yl)diazen-
1-ium-1,2-diolate as described in method A. The product was
purified by preparative HPLC using an Altima-C18 column
with a mobile phase of 20/80 water/acetonitrile: mp 136-137
1
°C; H NMR (500 MHz) δ 3.40 (m, 4 H), 3.78 (m, 4 H), 6.96
(m, 3 H), 7.31 (m, 2 H), 7.64 (d, J ) 8.7 Hz, 1 H), 7.85 (d, J 1
)
0.6 Hz, J 2 ) 2.3 Hz, J 3 ) 8.8 Hz, 1 H), 8.26 (d, J ) 1.9 Hz, 1
H); 13C NMR δ 48.32, 50.69, 116.90, 118.36, 121.11, 123.67,
129.35, 131.09, 131.12, 137.99, 150.07, 151.91; UV (ethanol)
(28) XPREP. 1997. Madison, WI, Bruker Analytical Instruments.
Ref Type: Computer Program

3098 J . Org. Chem., Vol. 66, No. 9, 2001
Saavedra et al.
using all the independent data. Non-hydrogen atoms were
refined anisotropically and hydrogen atoms were refined using
a riding model in which the C-H distances are fixed at 0.96
Å, and the hydrogen coordinates are reset after each cycle of
least-squares. The structure solution, refinement, and prepa-
ration of publication plots and tables were accomplished using
the SHEXLTL set of computer programs.29
using a mitochondrion-activated dye to assess cell viability.
Absorbance at 450 nM of 2,3-bis(2-methoxy-4-nitro-5-sulfo-
phenyl)-[5-(phenylamino)carbonyl]-2H-tetrazolium hydroxide
dye reduction following activation with phenazine metho-
sulfate was taken as an index of cell survival at 6 days post-
infection. The IC50 (concentration resulting in 50% reduction
in virus replication) and TC50 (concentration resulting in 50%
loss of cell viability in cells without virus) values were
calculated using linear regression analysis. AZT was included
as a positive control for all antiviral determinations.
Zin c Ejection Assa y. Fluorescence measurement of the
Trp37 residue in the C-terminal zinc finger of recombinant
HIV-1 NCp7 protein (kindly provided by L. O. Arthur of the
AIDS Vaccine Program of NCI at Frederick, Frederick, MD)
was performed as previously described.19 Briefly, a solution
of 20 µg/mL of NCp7 in 10 mM sodium phosphate buffer (pH
7.0) was treated with the indicated compounds for various
lengths of time at concentrations of 25 µM each. Aliquots were
removed at the indicated times and diluted 10-fold in 10 mM
sodium phosphate buffer (pH 7.0). Fluorescence intensity was
measured with a Shimadzu RF5000 spectrofluorimeter at
excitation and emission wavelengths of 280 and 352 nm,
respectively. The disulfide corresonding to 4-[N-(2-mercapto)-
benzoyl]aminobenzene sulfonamide19 was used as a positive
control for zinc ejection.
Ack n ow led gm en t. We thank Larry Arthur for
providing the NCp7 protein and Mary McGuire for
determining the NMR spectra. This project was funded
in part by the National Cancer Institute under contract
NO1-CO-56000 and by the Office of Naval Research.
Su p p or tin g In for m a tion Ava ila ble: Spectral changes
accompanying the reaction of 3a with isopropylamine, rate and
activation parameters for reaction of 3a with nucleophiles,
results from the zinc-ejection assay, toxicity data from the anti-
HIV screen, and full crystal structure reports for 3a and 14.
This material is available free of charge via the Internet at
http://pubs.acs.org.
An tivir a l Assa y. Antiviral activity was measured by
inhibition of virus cytopathic effects using HIV-1Rf infection
of CEM-SS lymphoblastoid T-cells.30 Virally induced cyto-
pathogenicity and cell survival (cytoprotection) were measured
J O0016529
(30) Buckheit, R. W., J r.; Fliakas-Boltz, V.; Decker, W. D.; Roberson,
J . L.; Stup, T. L.; Pyle, C. A.; White, E. L.; McMahon, J . B.; Currens,
M. J .; Boyd, M. R.; Bader, J . P. Antiviral Res. 1995, 26, 117-132.
(29) Sheldrick, G. M. SHELXTL. [v5.1]. 1997. Madison, WI, Bruker
Analytical Instruments. Ref Type: Computer Program