O-Alkylation of Cupferron: Aiming at the Design and Synthesis of Controlled Nitric Oxide Releasing Agents

Article abstract of DOI:10.1021/jo000157+

O-Alkylation of N-nitroso-N-phenylhydroxylamine ammonium salt (cupferron) was studied for the synthesis of novel nitric oxide (NO) releasing agents. The alkylation occurred regioselectively at the terminal oxygen, leading to a single product N-(alkyloxy)-N′-phenyldiimide N′-oxide as indicated by NMR and X-ray analysis. The O-alkyl derivatives exhibited significantly improved stability compared to their parent compound, cupferron. It was demonstrated that the cupferron O-alkyl derivatives could function as photoreleasing NO donor compounds. N-(N″-acetylphenylalanylmethylenyloxy)-N′-phenyldiimide N′-oxide), which linked the cupferron portion with an amino acid via an acetal moiety, was synthesized as an model NO prodrug where controlled NO release would occur either by increasing pH or by a protease-catalyzed hydrolysis.

Full text of DOI:10.1021/jo000157+

J . Org. Chem. 2000, 65, 4333-4337
4333
O-Alk yla tion of Cu p fer r on : Aim in g a t th e Design a n d Syn th esis of
Con tr olled Nitr ic Oxid e Relea sin g Agen ts
Yongchun Hou, Wenhua Xie, Adam J . J anczuk, and Peng George Wang*
Department of Chemistry, Wayne State University, Detroit, Michigan 48202
pwang@chem.wayne.edu
Received February 4, 2000
O-Alkylation of N-nitroso-N-phenylhydroxylamine ammonium salt (cupferron) was studied for the
synthesis of novel nitric oxide (NO) releasing agents. The alkylation occurred regioselectively at
the terminal oxygen, leading to a single product N-(alkyloxy)-N′-phenyldiimide N′-oxide as indicated
by NMR and X-ray analysis. The O-alkyl derivatives exhibited significantly improved stability
compared to their parent compound, cupferron. It was demonstrated that the cupferron O-alkyl
derivatives could function as photoreleasing NO donor compounds. N-(N′′-acetylphenylalanylmeth-
ylenyloxy)-N′-phenyldiimide N′-oxide), which linked the cupferron portion with an amino acid via
an acetal moiety, was synthesized as an model NO prodrug where controlled NO release would
occur either by increasing pH or by a protease-catalyzed hydrolysis.
Sch em e 1. Cu p fer r on a n d Its Su bstitu ted
In tr od u ction
Der iva tives Ca n Relea se NO via Oxid a tion ,
Th er m a l Activa tion , a n d P h otoa ctiva tion
Nitric oxide (NO) has been identified as a major
mediator in physiological processes.1-4 Besides the three
“classical” NO-mediated functions, which are vasodilatory
and antiplatelet effects,5,6 macrophage-induced cytotox-
icity,7,8 and neurotransmission,9,10 new and more detailed
biological roles of NO are being uncovered.11,12 NO is
synthesized in vivo from arginine mediated by nitric
oxide synthase (NOS).13,14 Administration of NO-donating
compounds, such as glycerin trinitrate (GTN), has been
practiced in cardiovascular medicine for over a century.15
Most NO donors release NO either by spontaneous
decomposition or by metabolic transformation. One of the
major problems associated with the current NO donors
is the indiscriminate release of NO. Therefore, the
development of donors that can deliver NO at specific
time and space to evoke the desired biological function
is of great current interest.16-18
Cupferron, an analogue of diazeniumdiolate, is com-
monly used in industry as a metal chelator19 and a
polymerization inhibitor.20 It is also a donor that can
release NO upon enzymatic,21 electrochemical, 22,23 as well
as chemical oxidation.24 Hwu et al. also discovered that
cupferron can thermally and photochemically decompose
to azoxy compounds and release NO (Scheme 1).25
* To whom correspondence should be addressed. Tel: (313) 993-
6759. Fax: (313) 577-5831.
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1991, 43, 109.
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1993, Dec 20, 26.
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(4) Bredt, D. S.; Snyder, S. H. Annu. Rev. Biochem. 1994, 63, 175.
(5) Furchgott, R. F.; Zawadzki, J . V. Nature 1980, 288, 373.
(6) Feelisch, M.; Poel, M. T.; Zamora, R.; Deussen, A.; Moncada, S.
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(9) Hibbs, J ., J r.; Taintor, R. R.; Vavrin, Z. Science 1987, 235, 473.
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Paediatr. 1999, 88, 1147.
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L.; Facchinetti, F.; Garfield, R. E. Am. J . Obstet. Gynecol. 1999, 181,
981.
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Keefer, L. K. J . Org. Chem. 1992, 57, 6134. (b) Saavedra, J . E.; Billiar,
T. R.; Williams, D. L.; Kim, Y.-M.; Watkins, S. C.; Keefer, L. K. J . Med.
Chem. 1997, 40, 1947.
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(19) Paneli, M.; Ouguenoune, H.; David, F.; Bolyos, A. Anal. Chim.
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(22) (a) Lawless, J . G.; Hawley, M. D. Anal. Chem. 1968, 40(6), 948.
(b) Cupferron, Wako Pure Chemical Industries, Ltd., 1995.
(23) (a) McGill, A. D.; Yang, Y.-F.; Echegoyen, L.; Wang, P. G.
Methods Enzymology, Nitric Oxide, Part C: Biological and Antioxidant
Activities; Packer, L., Ed. 1999, 301, 235. (b). McGill, A. D.; Zhang,
W.; Wittbrodt, J .; Wang, J .-Q.; Schlegel, H. B.; Wang, P. G. Bioorg.
Med. Chem. 1999, in press.
(24) (a) Balaban, A. T.; Garfield, R. E.; Lesko, M. J .; Seitz, W. A.
Org. Prep. Proced. Int. 1998, 30, 439. (b) Garfield, R. E.; Balaban, A.
T.; Seitz, W. A.; Klein, D. J .; Lesko, M. U. S. Pat. 5,698,738 (Dec 16,
1997); Int. Pat. Appl. PCT/US/96/0694 (May 15, 1996). (c). Balaban,
A. T.; Negoita, N.; Pascaru, I. Rev. Roum. Chim. 1971, 16, 721. (d).
Taylor, D. K.; Bytheway, I.; Barton, D. H. R.; Bayse, C. A.; Hall, M. B.
J . Org. Chem. 1995, 60, 443.
(13) Nathan, C.; Xie, Q.-W. Cell 1994, 78, 915.
(14) Marletta, M. A. Cell 1994, 78, 927.
(15) Bauer, J . A.; Booth, B. P.; Fung, H.-L. In Nitric Oxide Donors:
Biochemical Pharmacology and Therapeutics, Nitric Oxide, Biochem-
istry, Molecular Biology, and Therapeutic Implications; Ignarro, L.,
Murad, F., Eds.; Academic Press: San Diego, 1995; pp 361-381.
(16) (a). Hou, Y.-C.; J anczuk, A. J .; Wang, P. G. Curr. Pharm. Design
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10.1021/jo000157+ CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/07/2000

4334 J . Org. Chem., Vol. 65, No. 14, 2000
Hou et al.
Ta ble 1. O-Alk yla tion Der iva tives of Cu p fer r on a
RX
PhN(O)NOR
yield (%)
MeI
EtI
n-PrI
CH2dCH2CH2Br
HOCH2CH2CH2Br
CH3OCH2Br
CH3SCH2Cl
PhSCH2Cl
C6H5CH2Br
Me
Et
n-Pr
CH2dCH2CH2
HOCH2CH2CH2
CH3OCH2
CH3SCH2
PhSCH2
C6H5CH2
1
2
3
4
5
6
7
8
9
55
49
40
31
23
35
16
17
41
36
41
44
p-CH3O-C6H5CH2Cl
p-NO2-C6H5CH2Br
o-NO2-C6H5CH2Br
p-CH3O-C6H5CH2
p-NO2-C6H5CH2
o-NO2-C6H5CH2
10
11
12
a
Ratio of RX to Cupferron: 3:1; isolated yield %.
F igu r e 1. Stability of cupferron and its O-alkyl derivatives
at 25 °C: (9) cupferron, (]) N-(o-nitrobenzyl)oxy)-N′-phenyl-
diimide N′-oxide (12), (2) N-methoxy-N′-phenyldiimide N′-
oxide (1), and (b) N-(N′′-acetylphenylalanylmethylenyloxy)-
N′-phenyldiimide N′-oxide (14). The NO donors were dissolved
at 6.0 mM in a mixture of CH3CN and phosphate buffer (10
mM, pH 7.0, with 1.0 mM EDTA) (4/1 (v/v)). The solutions were
deoxygenated with argon.
Cupferron has a NONO moiety attached directly to a
carbon instead of a nitrogen atom. The advantage of this
type of donor is that, after the release of NO, the
byproducts are noncarcinogenic.24 Balaban and co-
workers24a synthesized a series of ortho-substituted
derivatives of cupferron. They found that these deriva-
tives were good donors both in vitro and in vivo. These
derivatives showed a faster decomposition rate than
cupferron due to the ortho substitution, which prevents
the NONO moiety from becoming planar.24b Recently, our
group has developed a series of para-substituted cupfer-
ron derivatives.23 These compounds evolve NO and a
nitrosobenzene derivative by a spontaneous dissociation
mechanism after undergoing a one-electron oxidation.
These para-substituted cupferron derivatives constitute
a series of redox-sensitive NO donors. However, the
instability of cupferron or its ortho- and para-substituted
derivatives can be a liability in the pharmaceutical realm
where targeted delivery is crucial to the success of NO
donor drug efforts. To provide better donor compounds
for the controlled release of NO, we designed and
synthesized a series of O-alkyl derivatives of cupferron.
We found that the spontaneous NO-release rate of these
O-alkyl derivatives is significantly slower than that of
cupferron. In addition, these derivatives could be used
as a new type of photoreleasing NO donor, as well as NO
prodrugs that release NO chemically and enzymatically.
analogue of cupferron, only one type of alkylation prod-
uct, N-(alkyloxy)-N′-phenyldiimide N′-oxide, was isolated
from the reaction. Thus, the alkylation was highly
regioselective. For example, in compound 10, the only
product isolated from the reaction of p-methoxybenzyl
chloride, the benzyl group is attached to the terminal
oxygen rather than the interior oxygen. The products
1
were characterized by H NMR, 13C NMR, and MS and
confirmed by X-ray analysis in certain cases.
The O-alkylation reactions of cupferron gave reason-
able yields in most cases. The highest yields were
obtained with active alkyl halides (e.g., methyl iodide).
The reaction yield was slightly higher than that of the
O-alkylation of diazeniumdiolate.17a We have explored
different reaction conditions to increase the reaction
yield. Raising the reaction temperature and increasing
the reaction time only resulted in the formation of more
byproduct azoybenzene,25 which was the decomposition
product from cupferron. An increase in yield, however,
was observed by increasing the ratio of alkyl halide to
cupferron.
Resu lts a n d Discu ssion
Th e Sta bility of O-Alk yl Cu p fer r on Der iva tives.
Cupferron and its substituted derivatives are unstable
and decompose to release NO (Scheme 1).25 Using a
commercial NO detector, the stability of the O-alkyl
derivatives was directly monitored and compared to the
parent compound, cupferron. It was found that, in
general, O-alkyl derivatives exhibited remarkable stabil-
ity compared to cupferron (Figure 1). On the other hand,
the stability of each O-alkyl derivative differs slightly in
a very small range. As shown in Figure 2, when the
O-alkyl group was O-nitrobenzyl, no detectable decom-
position was observed at all.
Th e Alk yla tion Rea ction . O-Alkyl derivatives of
cupferron can be prepared by adding an excess amount
of an alkyl halide to a solution of cupferron in DMF at 0
°C (Scheme 2). Upon completion of the reaction, as moni-
tored by TLC, the mixture is diluted with water, ex-
tracted with methylene chloride, and dried with Na2SO4.
The product is then further purified by silica gel chro-
matography using ethyl acetate-hexane as an eluent.
Sch em e 2. Syn th esis of O-Alk yl Der iva tives of
Cu p fer r on
P h otoch em ica l Relea se of NO fr om O-Alk yl De-
r iva tives of Cu p fer r on . With the purpose of developing
NO donors for controlled release, we carried out some
preliminary studies on photochemical behaviors of the
O-alkyl cupferron derivatives. It was found that while
the alkyl derivatives were remarkably stable at room
temperature without light they readily released NO upon
A series of O-alkyl derivatives of cupferron were
synthesized (Table 1). Similar to diazenumdiolate,17 an
(26) Chertnova, L. F.; Marchenko, G. A.; Kovalenko, V. I.; Struchkov,
Yu. T. Bull. Acad. Sci. USSR, Div. Chem. Sci. (Engl. Transl.) 1988,
37, 2266.
(25) Hwu, J . R.; Yau, C. S.; Tsay, S.-C.; Ho, T.-I. Tetrahedron. Lett.
1997, 38, 9001.

O-Alkylation of Cupferron
J . Org. Chem., Vol. 65, No. 14, 2000 4335
Sch em e 3. Aceta l-Lin k ed O-Alk yl Der iva tives a s NO P r od r u gs
released NO, which in turn converted the oxyHb to
methemoglobin (metHb). Positive conversion was indi-
cated by a characteristic peak shift from 413 nm (oxyHb)
to 406 nm (metHb) in the UV spectra.27 N-(O-Nitroben-
zyloxy)-N′-phenyldiimide N′-oxide (entry 12 in Table 1)
showed the best NO- releasing ability under UV irradia-
tion. This property of the O-alkyl cupferrons provides a
unique feature for this class of compounds in realizing
photocontrolled NO-release.
O-Alk yl Der iva tives a s NO P r od r u gs. It has been
our intention to develop novel NO prodrugs that possess
targeting effects and can deliver NO in vivo at the desired
time to the desired region. Due to its poor stability,
cupferron itself cannot function as a prodrug. We have
demonstrated that, by O-alkylation, the stability of
cupferron can be remarkably improved. This proved to
be crucial for the development of cupferron-based pro-
drugs. To demonstrate that O-alkyl derivatives of cup-
ferron could be designed to act as NO prodrugs, we
designed and synthesized a model compound N-(N′′-
acetylphenylalanylmethylenyloxy)-N′-phenyldiimide N′-
oxide (14). As illustrated in Scheme 3, our strategy was
to conjugate a ligand (e.g., a peptide) with an O-alkyl
derivative of cupferron via an acetal linkage. The binding
portion can be then specifically recognized by a protease
and hydrolyzed at the ester bond to generate an unstable
NO releasing intermediate. By varying the binding
ligand, NO can be specifically delivered and released. The
synthesis of compound 14 is illustrated in Scheme 4.
F igu r e 2. Stability of different O-alkyl derivatives: (]) N-(o-
nitrobenzyl)oxy)-N′-phenyldiimide N′-oxide (12), (2) N-meth-
oxy-N′-phenyldiimide N′-oxide (1), and (b) N-(N′′-acetylphen-
ylalanyl-methylenyloxy)-N′-phenyldiimide N′-oxide (14). The
NO donors were dissolved at 6.0 mM in a mixture of CH3CN
and phosphate buffer (10 mM, pH 7.0, with 1.0 mM EDTA)
(4/1 (v/v)). The solutions were deoxygenated with argon.
Sch em e 4. Syn th esis of
N-(N′′-Acetylp h en yla la n ylm eth ylen yloxy)-
N′-p h en yld iim id e N′-oxid e (14)
F igu r e 3. Photochemical NO release from O-alkyl derivatives
of cupferron: (9) N-(o-nitrobenzyl)oxy)-N′-phenyldiimide N′-
oxide (12), (b) N-(p-nitrobenzyl)oxy)-N′-phenyldiimide N′-oxide
(11), and (2) N-methoxy-N′-phenyldiimide N′-oxide (1). The
NO donors were dissolved at a concentration of 6.0 mM in CH3-
CN. The solutions were deoxygenated with argon and irradi-
ated with an UV lamp of 254 nm in the dark.
UV irradiation (λmax ) 254 nm). As shown in Figure 3,
after a 2-min initiation period, NO release occurred
rapidly. The NO release was confirmed using an NO
detector and as well as by an oxyhemoglobin (oxyHb)
assay. In the oxyhemoglobin assay, irradiation at 254 nm

4336 J . Org. Chem., Vol. 65, No. 14, 2000
Hou et al.
been shown that O-alkyl cupferron derivatives can func-
tion as photoreleasing NO donor agents. They can also
be used for the design and synthesis of novel NO
prodrugs, which expect to find wide applications in
biomedical and clinic research.
Exp er im en ta l Section
All the chemicals were purchased from commercial suppliers
and used without further purification. Melting points were
measured on a digital apparatus without correction.
Syn th esis of O-Alk yl Der iva tives of Cu p fer r on . All
reactions were carried out under argon in dark. In general,
cupferron was dissolved in DMF and cooled to 0 °C. Alkyl
halides were then injected through a septum and stirred
overnight. TLC was used to monitor the reaction progress. The
reaction mixture was diluted with water, extracted with
methylene chloride and dried over Na2SO4. After evaporation
of the solvent in vacuo, the product was purified by chroma-
tography using ethyl acetate-hexane as an eluent.
N-Meth oxy-N′-p h en yld iim id e N′-oxid e (1). Synthesized
from the reaction of cupferron with MeI: mp 39.5 °C (lit.28
mp 38-40 °C); UV λmax ) 259 nm, 1.8 × 102 M-1 cm-1); 1H
NMR (CDCl3, 300 MHz) δ 4.00 (s, 3H), 7.26 (m, 3H), 7.75 (d,
2H, J ) 7.8 Hz); 13C NMR (CDCl3, 75 MHz) δ 61.62, 120.86,
128.80, 131.11, 142.86. MS m/z (EI, relative intensity) 77 (100),
152 (M+, 54); HRMS (EI) calcd for C7H8N2O2 (M+) 152.0586,
found 152.0590 (M+).
F igu r e 4. Chemical release of NO from N-(N′′-acetylphenyl-
alanylmethylenyloxy)-N′-phenyldiimide N′-oxide (14). The com-
pound was dissolved at 6.0 mM in a mixture of CH3CN and
phosphate buffer (10 mM with 1.0 mM EDTA) (1/1 (v/v)). Top
line, pH, 11.0; Bottom line, pH 8.0.
N-Eth oxy-N′-p h en yld iim id e N′-Oxid e (2). Synthesized
from the reaction of cupferron with EtI: mp 41.3 °C; 1H NMR
(CDCl3, 300 MHz) δ 1.44 (t, 3H, J ) 6.0 Hz), 4.49 (q, 2H, J )
6.0 Hz), 7.40-7.49 (m, 3H), 7.93-7.98 (m, 2H); 13C NMR
(CDCl3, 75 MHz) δ 14.56, 70.66, 121.11, 128.90, 131.11, 143.29;
MS m/z (EI, relative intensity) 77 (100), 166 (M+, 21); HRMS
(EI) calcd for C8H10N2O2 (M+) 166.0742, found 166.0743 (M+).
N-n -P r op oxy-N′-p h en yld iim id e N′-Oxid e (3). Synthe-
sized from the reaction of cupferron with n-PrI: 1H NMR
(CDCl3, 500 MHz) δ 1.02 (t, 3H, J ) 7.5 Hz), 1.83-1.90 (m,
2H), 4.39 (t, 2H, J ) 7.0 Hz), 7.43-7.51 (m, 3H), 7.95-7.97
(m, 2H); 13C NMR (CDCl3, 125 MHz) δ 10.83, 23.09, 77.19,
121.84, 129.64, 131.83, 143.99; MS m/z (EI, relative intensity)
108 (100), 180 (M+, 20); HRMS (EI) calcd for C9H12N2O2 (M+)
180.0899, found 180.0903 (M+).
F igu r e 5. Enzymatic release of NO from N-(N′′-acetylphenyl-
alanylmethylenyloxy)-N′-phenyldiimide N′-oxide (14, b). The
lower line is the control experiment without the addition of
the enzyme (2). The compound was dissolved at 1.0 mM in a
mixture of CH3CN and phosphate buffer (10 mM, pH 7.0, with
1.0 mM EDTA) (10% (v/v)). R-Chymotryptsin (EC 3.4.21.1) was
then added to a final concentration of 0.2 mg/mL.
N-(Allyoxy)-N′-p h en yld iim id e N′-Oxid e (4). Synthesized
from the reaction of cupferron with allyl bromide:. 1H NMR
(CDCl3, 300 MHz) δ 4.89-4.92 (m, 3H), 5.31-5.46 (m, 2H),
6.00-6.14 (m, 1H), 7.45-7.53 (m, 3H), 7.94-7.98 (m, 2H); 13
C
Cupferron was first allowed to react with chloromethyl
methyl sulfide; after treatment with sulfuryl chloride,
compound 13 was obtained in situ. N-Acetylphenylala-
nine was then neutralized by Cs2CO3, and reacted with
compound 13 to afford the model compound 14.
Compound 14 was essentially stable at neutral pH.
Only very slow NO-release was observed. However, as
shown in Figure 4, upon increasing the pH to 8 or above,
it quickly hydrolyzes and releases NO. To test the initial
design, compound 14 was treated with R-chymotrypsin
(EC 3.4.21.1), a protease from bovine pancreas. As
expected, NO-release was significantly accelerated upon
addition of the enzyme (Figure 5). The results from this
model study proved the feasibility of our cupferron-based
prodrug design. Further studies are in progress focusing
on the design of different binding ligands.
NMR (CDCl3, 75 MHz) δ 75.36, 119.98, 121.17, 128.93, 131.23,
132.05, 143.27;. MS m/z (EI, relative intensity) 41 (100), 178
(M+, 3); HRMS (EI) calcd for C9H10N2O2 (M+) 178.0742, found
178.0742 (M+).
N-(3-Hyd r oxyp r op oxy)-N′-p h en yld iim id e N′-Oxid e (5).
Synthesized from the reaction of cupferron with 3-bromo-1-
propanol: 1H NMR (CDCl3, 500 MHz) δ 2.04-2.08 (m, 2H),
2.84 (s, 1H), 3.79 (t, 2H, J ) 5.9 Hz), 4.57 (t, 2H, J ) 6.1 Hz),
7.40-7.48 (m, 3H), 7.92 (d, 2H, J ) 7.5 Hz); 13C NMR (CDCl3,
125 MHz) δ 31.75, 58.96, 72.16, 121.06, 128.96, 131.25, 143.06.
MS m/z (EI, relative intensity) 108 (100), 180 (M - 17, 16);
MS m/z (CI, relative intensity) 197 (M + H+, 87); HRMS (EI)
calcd for C9H12N2O3 (M - 17) 180.0898, found 180.0897 (M -
17).
N-((Meth oxym eth yl)oxy)-N′-p h en yld iim id e N′-Oxid e
(6). Synthesized from the reaction of cupferron with bromo-
methyl methyl ether: 1H NMR (CDCl3, 400 MHz) δ 3.58 (s,
3H), 5.45 (s, 2H), 7.47-7.57 (m, 3H), 8.02-8.05 (m, 2H); 13C
NMR (CDCl3, 100 MHz) δ 58.48, 99.71, 122.43, 130.12, 132.65,
144.33; MS m/z (EI, relative intensity) 77 (100), 183 (M + H+,
0.3); MS m/z (CI, relative intensity) 183 (M + H+, 100); HRMS
(EI) calcd for C8H10N2O3 (M - 30) 152.0712, found 152.0713
(M - 30).
In summary, we have demonstrated that O-alkylation
of cupferron, which occurred regioselectively at the
terminal oxygen, led to a single type of product, N-
(alkyloxy)-N′-phenyldiimide N′-oxide. The O-alkyl deriva-
tives exhibited remarkably improved stability. It has
N-(Meth ylth iom eth yloxy)-N′-p h en yld iim id e N′-Oxid e
(7). Synthesized from the reaction of cupferron with chloro-
(27) Feelish, M.; Kubitzek, D.; Werringloger, J . In The Oxyhemo-
globin Assay, Methods in Nitric oxide Research; Feelisch, M., Stamler,
J . S., Eds.; 1996, Wiley: New York, 1996; p 455.
(28) Stevens, T. E. J . Org. Chem. 1964, 29, 311.

O-Alkylation of Cupferron
J . Org. Chem., Vol. 65, No. 14, 2000 4337
methyl methyl sulfide: mp 40.1 °C; 1H NMR (CDCl3, 500 MHz)
δ 2.32 (s, 3H), 5.46 (s, 2H), 7.44-7.51 (m, 3H), 7.96-7.98 (m,
2H); 13C NMR (CDCl3, 125 MHz) δ 16.12, 79.99, 121.93, 129.74,
132.20, 143.93; MS m/z (EI, relative intensity) 61 (100), 168
(M - 30, 5); MS m/z (CI, relative intensity) 61 (100), 199 (M
+ H+, 6); HRMS (EI) calcd for C8H10N2O3 (M - 30) 168.0483,
found 168.0486 (M - 30).
N-(P h en ylth iom eth yloxy)-N′-p h en yld iim id e N′-Oxid e
(8). Synthesized from the reaction of cupferron with chloro-
methyl phenyl sulfide: mp 42-43 °C; 1H NMR (CDCl3, 500
MHz) δ 5.76 (s, 2H), 7.27-7.36 (m, 3H), 7.43-7.47 (m, 2H),
7.50-7.57 (m, 3H), 7.88-7.90 (m, 2H); 13C NMR (CDCl3, 125
MHz) δ 122.94, 128.52, 129.67, 129.92, 132.20, 132.25, 134.79,
143.92; MS m/z (EI, relative intensity) 77 (100), 230 (M - 30,
6); MS m/z (CI, relative intensity) 123 (100), 230 (M - 30, 6);
HRMS (EI) calcd for C8H10N2O3 (M - 30) 230.0640, found
230.0640 (M - 30).
N-(Ben zyloxy)-N′-p h en yld iim id e N′-Oxid e (9). Synthe-
sized from the reaction of cupferron with benzyl bromide: mp
80.2-80.8 °C (lit.29 mp 81-82 °C); 1H NMR (CDCl3, 300 MHz)
δ 5.44 (s, 2H), 7.35-7.51 (m, 8H), 7.92-7.95 (m, 2H); 13C NMR
(CDCl3, 75 MHz) δ 76.46, 121.20, 128.62, 128.73, 128.92,
131.23; MS m/z (EI, relative intensity) 91 (100), 198 (M - 30,
7); MS (FAB, M + H+, 229); HRMS (EI) calcd for C13H12N2O2
(EI, M - 30) 198.0919, found 198.0922 (M - 30).
gradually warm to room temperature. Upon completion and
removal of the solvent and the excess sufuryl chloride, N-
(choloromethyloxy)-N′-phenyldiimide N′-oxide 13 was obtained
as a yellowish liquid without further purification.
N-Acetylphenylalanine (248 mg, 1.20 mmol) and CsCO3 (391
mg, 1.20 mmol) were combined in 5 mL of anhydrous DMF.
The resultant suspension was stirred for 30 min at room
temperature. Compound 13 was dissolved in 3 mL of DMF
and cooled to 0 °C. The compound 13 was then added to the
suspension via syringe. The mixture was stirred overnight at
room temperature. After completion, the mixture was diluted
with 30 mL of water, extracted with CH2Cl2, and dried with
anhydrous Na2SO4. The desired product 14 was isolated as a
white powder after column chromatography (hexanes/acetyl
acetate, 3:2) (158 mg, 0.43 mmol, yield, 43%): 1H NMR (CDCl3,
500 MHz) δ 1.97 (s, 3H), 3.09-3.17 (m, 2H), 4.93-4.97 (m,
1H), 5.91-5.92 (d, 1H, J ) 7.0), 5.96-5.97 (d, 1H, NH, J )
8.0), 6.12-6.14 (d, 1H, J ) 7.5), 7.06-7.20 (m, 5H), 7.47-7.58
(m, 3H), 7.96-7.98 (m, 2H); 13C NMR (CDCl3, 125 MHz) δ
23.29 (CH3), 37.65, 53.17, 88.54, 121.61, 127.46, 128.87, 129.39,
129.47, 130.58, 132.21, 135.46, 143.24, 169.97, 170.60; MS m/z
(EI, relative intensity) 120 (100), 357 (M+); HRMS (EI) calcd
for C18H19N3O5 (M+) 357.1325, found 357.1327 (M+).
Sta bility Tests. Cupferron was dissolved in a 10 mM
phosphate buffer (pH, 7.0; EDTA, 1.0 mM). O-Alkyl derivatives
were dissolved in CH3CN and diluted with a 10 mM phosphate
buffer (pH, 7.0; EDTA, 1.0 mM). Solutions were deoxygenated
with argon. NO generation was monitored and measured using
a commercial ISO-NO Mark II isolated nitric oxide probe30 in
the dark.
P h otoch em ica l NO Relea se. O-Alkyl derivatives, dis-
solved in CH3CN and deoxygenated with argon, were irradi-
ated at a wavelength of 254 nm with a UV lamp (Spectroline,
Model ENF-240C) at a fixed position. NO evolution was
monitored and measured using the NO probe in the dark.
Ch em ica l NO Relea se fr om N-(N′′-Acetylp h en yla la -
n ylm et h ylen yloxy)-N′-p h en yld iim id e N′-Oxid e. Com-
pound 14 (10 mg) was dissolved in 3 mL of CH3CN and diluted
with 1 mL of a 10 mM phosphate buffer (pH 7.0, EDTA 1 mM).
The solution was stirred and deoxygenated with argon. The
pH was adjusted using a 0.1 M aqueous solution of KOH. NO
evolution was monitored and measured using the NO probe
in the dark.
N-(p-Meth oxyben zyloxy)-N′-p h en yld iim id e N′-Oxid e
(10). Synthesized from the reaction of cupferron with 4-meth-
1
oxybenzyl chloride: mp 84.4-84.8 °C; H NMR (CDCl3, 400
MHz) δ 3.80 (s, 3H), 5.37 (s, 2H), 6.89-6.93 (m, 2H), 7.38-
7.50 (m, 5H), 7.92-7.95 (m, 2H); 13C NMR (CDCl3, 100 MHz)
δ 55.25, 76.31, 113.99, 121.18, 127.50, 128.89, 130.65, 131.14,
144.30, 160.05; MS m/z (EI, relative intensity) 121 (100), 228
(M-30, 0.4); HRMS (EI) calcd for C14H14N2O3 (EI, M - 30)
228.1026, found 228.1024 (M - 30).
N-(p-Nitr oben zyloxy)-N′-p h en yld iim id e N′-Oxid e (11).
Synthesized from the reaction of cupferron with 4-nitrobenzyl
bromide: mp 101.2-101.5 °C; UV λmax 269 nm (CHCl3, ꢀ 2.2
1
× 104 M-1 cm-1); H NMR (CDCl3, 300 MHz) δ 5.52 (s, 2H),
7.42-7.55 (m, 3H), 7.59-7.62 (m, 2H), 7.89-7.94 (m, 2H),
8.22-8.25 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ 74.60, 121.14,
123.88, 128.78, 129.05, 131.59, 142.67; MS m/z (EI, relative
intensity) 77 (100), 243 (M - 30, 16); HRMS (EI) calcd for
C
13H11N3O4 (EI, M+) 273.0750, found 273.0749 (M+).
En zym a tic NO Relea se fr om N-(N′′-Acetylp h en yla la -
n ylm eth ylen yloxy)-N′-p h en yld iim id e N′-Oxid e. The com-
pound was dissolved at 1.0 mM in a 10% (v/v) mixed solvent
of CH3CN and phosphate buffer (10 mM, pH 7.0, with 1.0 mM
EDTA). R-Chymotryptsin (EC 3.4.21.1) was then added to a
final concentration of 0.2 mg/mL. NO release was monitored
and measured using the NO probe in the dark.
N-(o-Nitr oben zyloxy)-N′-p h en yld iim id e N′-Oxid e (12).
Synthesized from the reaction of cupferron with 2-nitrobenzyl
bromide: mp 96.3-97.1 °C; UV λmax 261 nm (CHCl3, ꢀ 2.8 ×
104 M-1 cm-1); 1H NMR (CDCl3, 300 MHz) δ 5.92 (s, 2H), 7.44-
7.56 (m, 4H), 7.66-7.75 (m, 2H), 7.95-7.98 (m, 2H), 8.17-
8.20 (m, 1H); 13C NMR (CDCl3, 75 MHz) δ 72.53, 121.17,
125.06, 128.64, 128.83, 129.04, 131.56, 134.18; MS m/z (EI,
relative intensity) 136 (100), 243 (M - 30, 5); MS (FAB) 274
(M + H+); HRMS (EI) calcd for C13H11N3O4 (M - 30) 243.0770,
found 243.0768 (M - 30).
Ack n ow led gm en t. This work was supported by a
research grant from the NIH (GM54074).
Syn th esis of N-(N′′-Acetylph en ylalan ylm eth ylen yloxy)-
N′-p h en yld iim id e N′-Oxid e (14). Sulfuryl chloride (84 µL,
1.04 mmol), dissolved in 3 mL of CH2Cl2 (anhydrous), was
added dropwise to a solution of N-(methylthiomethyloxy)-N′-
phenyldiimide N′-oxide (204 mg, 1.03 mmol) in 3 mL of CH2-
Cl2 (anhydrous) at 0 °C. The stirring mixture was allowed to
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR for
compounds 1-12 and 14 and complete crystal structure
parameters for compound 10. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O000157+
(29) Yandovskii, V. N.; Misharev, A. D.; Tselinskii, I. V.; Traore, U.
J . Org. Chem. USSR (Engl. Transl.) 1978, 14, 2308.
(30) Both manufactured by World Precision Instruments, Inc.,
Sarasota, FL.