Novel substrates for nitric oxide synthases.

Article abstract of DOI:10.1016/S0968-0896(02)00155-4

Enzymatic generation of nitric oxide (NO) by nitric oxide synthase (NOS) consists of two oxidation steps. The first step converts L-arginine to N(G)-hydroxy-L-arginine (NOHA), a key intermediate, and the second step converts NOHA to NO and L-citrulline. To fully probe the substrate specificity of the second enzymatic step, an extensive structural screening was carried out using a series of N-alkyl (and N-aryl) substituted-N'-hydroxyguanidines (1-14). Among the eleven N-alkyl-N'-hydroxyguanidines evaluated, N-n-propyl (2), N-iso-propyl (3), N-n-butyl (4), N-s-butyl (5), N-iso-butyl (6), N-pentyl (8) and N-iso-pentyl (9) derivatives were efficiently oxidized by the three isoenzymes of NOS (nNOS, iNOS and eNOS) to generate NO. N-Butyl-N'-hydroxyguanidine (4) was the best substrate for iNOS (K(m)=33 microM) and N-iso-propyl-N'-hydroxyguanidine (3) was the best substrate for nNOS (K(m)=56 microM). When the alkyl substituents were too small (such as ethyl 1) or too large (such as hexyl 10 and cyclohexyl 11), the activity decreased significantly. This suggests that the van der Waals interaction between the alkyl group and the hydrophobic cavity in the NOS active site contributes significantly to the relative reactivity of compounds 3-11. Moreover, five N-aryl-N'-hydroxyguanidines were found to be good substrates for iNOS, but not substrates for eNOS and nNOS. N-phenyl-N'-hydroxyguanidine was the best substrate among them (K(m)=243 microM). This work demonstrates that N-alkyl substituted hydroxyguanidine compounds are novel NOS substrates which 'short-circuit' the first oxidation step of NOS, and N-aryl substituted hydroxyguanidine compounds are isoform selective NOS substrate.

Full text of DOI:10.1016/S0968-0896(02)00155-4

Bioorganic & Medicinal Chemistry 10 (2002) 3049–3055
Novel Substrates for Nitric Oxide Synthases
Ming Xian,a Noriko Fujiwara,b Zhong Wen,a Tingwei Cai,a Satoshi Kazuma,b
a
b
Adam J.Janczuk, Xiaoping Tang,a Vladislav V.Telyatnikov,
Yingxin Zhang,a Xinchao Chen,a Yasuhide Miyamoto,b
Naoyuki Taniguchib,* and Peng George Wanga,*
aDepartment of Chemistry, Wayne State University, Detroit, MI 48202, USA
bDepartment of Biochemistry, Osaka University Medical School, Osaka 565-0871, Japan
Received 20 December 2001; accepted 30 January 2002
Abstract—Enzymatic generation of nitric oxide (NO) by nitric oxide synthase (NOS) consists of two oxidation steps.The first step
converts l-arginine to NG-hydroxy-l-arginine (NOHA), a key intermediate, and the second step converts NOHA to NO and
l-citrulline.To fully probe the substrate specificity of the second enzymatic step, an extensive structural screening was carried out
using a series of N-alkyl (and N-aryl) substituted-N0-hydroxyguanidines (1–14).Among the eleven N-alkyl-N0-hydroxyguanidines
evaluated, N-n-propyl (2), N-iso-propyl (3), N-n-butyl (4), N-s-butyl (5), N-iso-butyl (6), N-pentyl (8) and N-iso-pentyl (9) deri-
vatives were efficiently oxidized by the three isoenzymes of NOS (nNOS, iNOS and eNOS) to generate NO. N-Butyl-N0-hydroxy-
guanidine (4) was the best substrate for iNOS (Km=33 ꢀM) and N-iso-propyl-N0-hydroxyguanidine (3) was the best substrate for
nNOS (Km=56 ꢀM).When the alkyl substituents were too small (such as ethyl 1) or too large (such as hexyl 10 and cyclohexyl 11),
the activity decreased significantly.This suggests that the van der Waals interaction between the alkyl group and the hydrophobic
cavity in the NOS active site contributes significantly to the relative reactivity of compounds 3–11.Moreover, five N-aryl-
N0-hydroxyguanidines were found to be good substrates for iNOS, but not substrates for eNOS and nNOS. N-phenyl-N0-hydroxy-
guanidine was the best substrate among them (Km=243 ꢀM).This work demonstrates that N-alkyl substituted hydroxyguanidine
compounds are novel NOS substrates which ‘short-circuit’ the first oxidation step of NOS, and N-aryl substituted hydroxy-
guanidine compounds are isoform selective NOS substrate. # 2002 Published by Elsevier Science Ltd.
Introduction
inhibitors of NOS has been a subject of intense inter-
est.7,8 Among hundreds of candidates that have been
tested, arginine competitors seem to be promising tar-
gets and numerous studies have been performed to
determine the influence of substrate modifications on
the interaction with the NOS active site.However,
although it has been reported that the C¼N–OH func-
tional group of various N-hydroxyguanidines including
NOHA, N-hydroxydebrisoquine and even some
N-hydroxyguanidine containing drugs could be oxida-
tively cleaved by other enzymes such as cytochromes
P450 and horseradish peroxidase with the formation of
corresponding ureas and nitrogen oxides including
NO,9ꢀ11 only a few compounds have been clearly shown
to be NOS substrates.12ꢀ15 Besides l-arginine and
NOHA, homo-l-arginine, No-hydroxy-homo-l-arginine,
and several l-arginine derivatives have been reported to
be oxidized into corresponding ureas and NO by NOSs.
But most other arginine derivatives such as d-arginine,
l-arginine methyl ester, N-acetyl-l-arginine and agmatine
Nitric oxide (NO) plays an important role in numerous
physiological and pathological processes.1,2 The bio-
chemical production of NO starts from the initial oxi-
dation of l-arginine to produce NG-hydroxy-l-arginine
(NOHA), followed by a second oxidation forming NO
and l-citrulline.3,4 This conversion is catalyzed by three
distinct mammalian nitric oxide synthases (NOS) [i.e.,
endothelial NOS (eNOS), neuronal NOS (nNOS), and
inducible NOS (iNOS)].5,6
Since NO has been implicated in a wide variety of dis-
ease states, inhibitors and substrates of NOS could have
great therapeutic potential in the treatment of these
diseases.So far, identification of potent and selective
*Corresponding author.Te:l. +1-313-993-6759; fax: +1-313-577-
2554; e-mail: pwang@chem.wayne.edu
0968-0896/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0968-0896(02)00155-4

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M. Xian et al. / Bioorg. Med. Chem. 10 (2002) 3049–3055
Scheme 1.
most substrates of NOSs are arginine or NOHA deri-
vatives, it also suggests that the a-amino acid portions
of those substrates play an important role in the cat-
alysis.16ꢀ18 However, since some simple guanidines and
isothioureas such as aminoguanidine, S-ethyl-iso-
thiourea, and N-phenyl-S-methyl-isothiourea are strong
NOS inhibitors through binding at the active site,19ꢀ23 it
appears that the a-amino acid moiety of arginine can be
removed without detrimental consequences, while the
integrity of the guanidine function must be partially
retained.It is logical then to assume that simple com-
pounds bearing guanidine or hydroxyguanidine func-
tions, not derived from l-Arg or NOHA, can interact
with NOS active site and possibly serve as substrates of
NOS.
.
Scheme 2. (a) BrCN, Et3N, CH2Cl2; (b) NH2OH HCl, K2CO3, EtOH.
Table 1. NO formation (nmol/min/mg protein) from oxidation of
compounds 1–17 in the presence of NOSa
Compd
iNOS
nNOS
eNOS
NOHA
l-Arg
NHG
1
2
3
4
5
6
7
120 (100%)
55 (46%)
0 (0%)
595 (100%)
477 (79%)
0 (0%)
71 (100%)
27 (38%)
0 (0%)
4 (6%)
17 (24%)
27 (38%)
14 (20%)
3 (4%)
7 (10%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
3 (3%)
6 (1%)
18 (15%)
38 (32%)
50 (42%)
14 (12%)
6 (5%)
418 (70%)
450 (76%)
380 (64%)
107 (18%)
14 (2%)
0 (0%)
185 (31%)
39 (6%)
24 (4%)
19 (3%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
In an effort to better understand the structural factors
that are important for the substrates of NOS and to find
a new type of enzymatic NO donors, we have synthe-
sized a series of compounds bearing an N-hydro-
xyguanidine functional group and studied their
oxidation by recombinant NOSs.Herein we report that
several N-alkyl-N0-hydroxyguanidines can be efficiently
oxidized by NOS to generate NO.Most interestingly,
some N-aryl-N0-hydroxyguanidines have been found to
be selective substrates for iNOS.
0 (0%)
8
9
10
38 (32%)
29 (24%)
3 (3%)
11
4 (3%)
12a
12b
12c
12d
12e
13
14a–e
15a–c
16
39 (32%)
35 (29%)
29 (24%)
16 (13%)
18 (15%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
Results and Discussion
17
aThe initial rate of NO synthesis was determined at 37 ꢁC using spectro-
photometric oxyhemoglobin assay for NO.The concentration of substrate
was 0.5 mM. The rates were also expressed as a percentage of those found
for NOHA.
Starting from the corresponding amines, 22 N-sub-
stituted hydroxyguanidine derivatives (1–14) (Scheme 1)
were synthesized using a general procedure previously
reported24 with some modification (Scheme 2) (see the
experimental section for detail).These compounds were
were found not to be substrates. No-methyl-l-arginine is
even a widely used NOS inhibitor.
fully characteristized by H, 13C NMR, and high-reso-
1
lution mass spectroscopy.Activity of these N-hydroxy-
guanidine compounds as substrates of NOS was
evaluated by hemoglobin assay.25 For our evaluation,
NOHA was used as a control indicating 100% activity.
The results were summarized in Table 1.
The very limited number of substrates for NOSs sug-
gests that highly specific structural features are required
for NO generation from NOSs.Furthermore, because

M. Xian et al. / Bioorg. Med. Chem. 10 (2002) 3049–3055
3051
As expected, most of the synthetic hydroxyguanidine
derivatives were not as active as l-Arg or NOHA.
Without the a-amino acid moiety that allowed l-Arg or
NOHA to be specifically recognized by NOS, these
synthetic compounds may fail to interact most favor-
ably with the corresponding binding site of NOS.26,27
However, the results listed in Table 1 indicated that the
activity of these compounds was related to the structure
of substituents.Without any substituents, N-hydro-
xyguanidine (NHG) itself was not a substrate.23 When a
small alkyl group (such as ethyl, 1) was attached to
hydroxyguanidine, only low reactivity was observed.
Surprisingly, one additional methylene group, n-propyl
substitution in 2, significantly enhanced the activity,
especially for nNOS (up to 70% of NOHA).As the size
of the alkyl chain increased to isopropyl or n-butyl
group, 3 and 4 turned out to be good substrates.The
Km values of 3 was 77 mM for iNOS and Km=56 mM for
nNOS, whereas the Km values of 4 was 33 mM for iNOS
and 67 mM for nNOS.However, if the alkyl substituents
became too long or too bulky (such as n-hexyl in 10 and
cyclohexyl in 11), the activity decreased significantly.It
was interesting to note that n-propyl in 2 permitted very
good NOS activity but one more methyl group at its
terminal carbon (6) significantly diminished its reactiv-
ity.A similar effect could also be found for n-butyl
substituent (between 4 and 9).Compared to its isopro-
pyl counterpart (3), N-tert-butyl-N0-hydroxyguanidine
(7) was not the substrate for all three NOSs.
of 7 can be related to the bulky t-butyl group near the
guanidino functionality.Steric hindrance between the
t-butyl group and heme ring would disfavor the oxi-
dation reaction by disrupting the orientation of guani-
dino group in the heme active site.This result indicates
that the interaction between hydrophobic chains on
substrates and the hydrophobic cavity in the enzyme
active site could play a crucial role in determining the
affinity of substrates.However, for
N-hydroxy-
guanidines bearing an a-amino acid function, their
binding site should be different from N-alkyl hydroxy-
guanidines.For example, NOHA and homo-NOHA
bind well with NOS and serve as good substrates;
whereas nor-NOHA, which is one methylene group
shorter than NOHA, is not a NOS substrate.Moali et
al.suggested that the strong binding between a-amino
acid group and NOS makes it too short to position the
N-hydroxyguanidine function in nor-NOHA close to
the reaction center.18 Overall, our experiments clearly
demonstrated that, in the absence of the a-amino acid
moiety, a favorable interaction between the hydro-
phobic patch of NOS and a suitable alkyl group of
substrates contributed significantly to the binding.
Since N-isopropyl-N0-hydroxyguanidine (3) and N-n-butyl-
N0-hydroxyguanidine (4) are excellent substrates for
NOS, their corresponding derivatives, isopropyl-
guanidine (16) and butylguanidine (17) have also been
tested in our experiment.Unfortunately, these two
compounds can not generate NO by NOSs even at high
concentration (10 mM).This suggests that the two steps
in the NO biosynthesis are distinct processes.The first
oxidation step seems to require an exact ‘natural’ sub-
strate such as l-arginine.Such substrate specificity can
not be compromised by replacing l-arginine with simple
N-substituted guanidine compounds.In contrast, the
second oxidation step of NOS reaction is much more
compromising.Some N-substituted hydroxyguanidines
can be oxidized to produce NO in this step.
The above results were consistent with a recent structure
study,28 which revealed the importance of nonpolar van
der Waals interactions between inhibitor and NOS.As
shown in crystal structures of NOS oxygenase
domains,27ꢀ32 NOHA is anchored at the catalytic site
through H-bonds between N-hydroxyguanidino moiety
and the conserved amino acid residues (Glu and Trp).A
small hydrophobic cavity formed by three conserved
amino acid residues (Pro, Phe, and Val) is located in
close proximity to the catalytic site and is connected to
the open substrate access channel.Specifically, accord-
ing to the crystal structure study of substrate-bound
NOS by Poulos’ laboratory (University of California,
Irvine, USA), N-alkyl-N0-hydroxyguanidine differs from
NOHA in the orientation of N-substituent group (the
results will be published elsewhere).Although the
absence of H-bonding components in the N-substituent
(e.g., amino and carboxylate groups in NOHA) leads to
a distinct orientation of the N-hydroxyl group, yet sub-
strates like 4 still retain fairly good NOS reactivity.This
is because the hydroxyguanidino moiety is still anchored
by multiple H-bonds and is orientated in a manner
similar to NOHA at NOS active site, allowing efficient
oxidation of hydroxyguanidino moiety by super-
oxoiron(III) heme intermediate.19 Such orientation also
allows an N-alkyl group of suitable size to be accom-
modated in the hydrophobic cavity via van der Waals
interaction, including both the n-butyl chain in 4 and
the i-propyl group in 3, whereas such interaction would
diminish for shorter alkyl chains (for NHG and 1).On
the other hand, the limited space in this cavity would
hardly allow either long (in 10), bulky (in 11), or term-
inally branched alkyl chains (in 9).The poor reactivity
Next a series of N-aryl-N0-hydroxyguanidines (12a–e)
were investigated.Most interestingly, it was found that
these compounds were selective substrates for iNOS.
Previously 12d was shown to be a substrate for iNOS by
Mansuy and co-workers.12 In our study, N-phenyl-
N0-hydroxyguanidine (12a) was the best substrate in this
series (Km=243 mM).The rate of NO formation, when
using 500 mM 12a, was 39 nmol/min/mg protein, as high
as 70% of l-Arg and 32% of NOHA.The NO forma-
tion rates of other substrates (12b–e) were in the range
of 16–35 nmol/min/mg protein.When used in the assay
for nNOS and eNOS, none of them produced detectable
NO even at high concentrations (10 mM).This is in
sharp contrast to that of their aliphatic counterparts
(3–10), which exhibited good activity but hardly any
isoform selectivity.These N-aryl-N0-hydroxyguanidine
compounds are the first reported isoform selective NOS
substrates so far.Given the high active-site conservation
and high structural similarity among NOS isoforms, this
observation deserves further mechanistic consideration.
Further study on the effects of incubation conditions on
the formation of nitrite from these substrates indicated

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M. Xian et al. / Bioorg. Med. Chem. 10 (2002) 3049–3055
that the characteristics of this reaction were very similar
to those of the NOS-dependent oxidation of the endo-
genous NOHA.The reaction required the presence of
both the active enzyme and other cofactors, and could
be strongly inhibited by classical NOS inhibitors such as
N-nitro-l-arginine (NNA) and N-methyl-l-arginine
(NMA) (data not shown).It indicated that the oxi-
dation of these substrates should occur in the active site
of NOS and the mechanism was similar to NOHA.
significant not only for understanding the NOS
mechanism, but also in using such compound as iso-
form-specific probe in biomedical experiments.
Experimental
Chemistry
General. All reagents were used as purchased from
commercial suppliers without further processing.The
NMR data were recorded on a Mercury-400 or
-500 MHz spectrometer.MS spectra were obtained from
a Kratos MS 80 spectrometer using electrospray ioni-
zation mode (ESI) or electronic ionization (EI).Silica
gel F254 plates (Merck) and Silica Gel 60 (70–230 mesh)
were used in analytical thin-layer chromatography
(TLC) and column chromatography, respectively.
Since 12a–e were not substrates of nNOS and eNOS, we
further examined them as inhibitors of these two
enzymes.As shown in Table 2, 12a and 12d displayed
the most efficient concentration-dependent inhibition of
NOS-mediated nitrite formation in the presence of
l-Arg (0.1 mM). Other compounds partially inhibited
nitrite formation only at higher concentrations.The
weak inhibition of these compounds suggested that their
binding affinity to nNOS and eNOS was relatively weak
compared with l-Arg.
General procedure for synthesis of N-hydroxyguanidines
To further explore the structural effect on the activity,
some structure-related compounds (13–15) were pre-
pared and assayed.When the phenyl ring was replaced
by a naphthalene ring (13) or by a benzyl group (14), the
activity of substrate was destroyed completely.This
increase in size might over-grow the ‘specific’ phenyl-
binding pocket in iNOS.As for N-alkyl substituted
compounds, when the hydroxyguanidine group was
changed to a guanidine group (15), none of them acted
as a substrate of NOSs.
Synthesis of corresponding cyanamide. A solution of
corresponding amine (10 mmol) and anhydrous Et3N
(1.512 g, 15.1 mmol) in dry CH2Cl2 (30 mL) was cooled
to 0 ꢁC under argon atmosphere.To the solution was
added BrCN (3.5 mL 3.0 M in CH2Cl2, 10.5 mmol)
dropwise.After stirring 10 h at rt, the mixture was fil-
trated through a short silicon gel column.The filtrate
was concentrated.The crude product was purified by
flash chromatography in 55–85% yield.
N-Substituted-N0-hydroxyguanidines. A mixture of cyan-
amide (0.6 mmol), hydroxyamine hydrochloride (43 mg,
0.61 mmol) and anhydrous K2CO3 (173 mg, 1.21 mmol)
in anhydrous EtOH (5 mL) was stirred at rt for 5 h.
Then the mixture was filtrated through Celite.After
removing the solvent by rota-vap, the crude product
was purified with flash chromatography to give
N-hydroxyguanidine (40–78%).
In summary, an extensive structural screening of
N-hydroxyguanidine compounds revealed that relatively
simple exogenous compounds, not bearing an amino
acid function, could be oxidized by NOS in a manner
similar to NOHA, with a significant rate of NO forma-
tion.The structure–activity relationship showed that a
potent NOS substrate shared at least two character-
istics: (1) an N-hydroxyguanidine functional group,
capable of anchoring the substrate in the NOS active
site and furnishing the second step of the NOS reaction;
(2) a suitable hydrophobic chain that interacts favorably
with the hydrophobic cavity in NOS active site.Cur-
rently, l-arginine supplementation has been studied in a
variety of clinical situation where the increase of NO
production is desired.33,34 For example, l-arginine
coated endovascular stents have been tested in control-
ling restoration after balloon angioplasty.35 Findings in
the present study indicated simple N-substituted
hydroxyguanidine might also be used as NO donor
supplementation.Moreover, to our best knowledge,
N-aryl-N0-hydroxyguanidines (12a–e) are the first series
of isoform selective substrates for NOS.This finding is
N-Ethyl-N0-hydroxyguanidine 1. 1H NMR (500 MHz,
CD3OD) d 3.27 (q, J=7.0 Hz, 2H), 1.21 (t, J=7.0 Hz,
3H); 13C NMR (125 MHz, CD3OD) d 159.0, 36.2, 13.4.
HRMS calcd for C3H9N3O 103.0746, found 103.0741.
N-n-Propyl-N0-hydroxyguanidine
2.
1H
NMR
(400 MHz, CD3OD) d 3.16 (t, J=7.6 Hz, 2H), 1.57–1.65
(m, 2H), 0.97 (t, J=7.6 Hz, 3H); 13C NMR (100 MHz,
CD3OD) d 158.6, 42.7, 22.0, 10.1. HRMS calcd for
C4H11N3O 117.0902, found 117.0899.
0
1
N-i-Propyl-N -hydroxyguanidine 3. H NMR (400 MHz,
CD3OD) d 3.70–3.78 (m, 1H), 1.23 (d, J=6.4 Hz, 6H);
13C NMR (100 MHz, CD3OD) d 158.4, 43.9, 21.4.
HRMS calcd for C4H11N3O 117.0902, found
117.0901.
Table 2. IC50 (mM) of compounds 12a–e to nNOS and eNOS
Compd
nNOS
eNOS
N-n-Butyl-N0-hydroxyguanidine 4. H NMR (500 MHz,
1
CD3OD) d 3.00 (t, J=7.0 Hz, 2H), 1.45–1.53 (m, 2H),
1.34–1.38 (m, 2H), 0.993 (t, J=7.5 Hz, 3H); 13C NMR
(125 MHz, CD3OD) d 157.9, 40.7, 31.7, 19.9, 13.0.
HRMS calcd for C5H13N3O 131.1059, found
131.1060.
12a
12b
12c
12d
12e
900
3500
4500
1500
2600
1500
>10,000
>10,000
2000
5600

M. Xian et al. / Bioorg. Med. Chem. 10 (2002) 3049–3055
3053
N-s-Butyl-N0-hydroxyguanidine 5. H NMR (400 MHz,
N-(4-Chloro)-phenyl-N0-hydroxyguanidine 12d. H NMR
(500 MHz, CD3OD) d 7.19 (d, J=8.5 Hz, 2H), 7.14 (d,
J=8.5 Hz, 2H); 13C NMR (125 MHz, CD3OD) d 153.7,
140.4, 128.4, 125.3, 119.4. HRMS calcd for C7H8ClN3O
185.0356, found 185.0355.
1
1
CD3OD) d 3.52 (m, 1H), 1.56 (m, 2H), 1.20 (d,
J=6.4 Hz, 2H), 0.95 (t, J=7.2 Hz, 3H); 13C NMR
(100 MHz, CD3OD) d 159.95, 50.80, 30.40, 20.53,
10.67. HRMS calcd for C5H13N3O 131.1059, found
131.1059.
N-(4-Bromo)-phenyl-N0-hydroxyguanidine 12e. H NMR
1
N-iso-Butyl-N0-hydroxyguanidine
6.
1H
NMR
(400 MHz, CD3OD) d 7.28 (d, J=9.0 Hz, 2H), 7.14 (d,
J=9.0 Hz, 2H); 13C NMR (100 MHz, CD3OD) d 153.5,
141.0, 131.4, 119.6, 112.3; HRMS calcd for C7H8BrN3O
228.9851, found 228.9854.
(500 MHz, CD3OD) d 2.86 (t, J=6.0 Hz, 2H), 1.79 (m,
1H), 0.94 (d, J=6.9 Hz, 6H); 13C NMR (125 MHz,
CD3OD) d 159.20, 49.74, 29.51, 20.48. HRMS calcd for
C5H13N3O 131.1059, found 131.1060.
N-2-Naphthyl-N0-hydroxyguanidine 13. 1H NMR
(400MHz, CD3OD) d 7.64–7.78 (m, 4H), 7.26–7.42 (m,
3H); 13C NMR (100MHz, CD3OD) d 153.3, 144.4, 134.6,
130.5, 128.6, 127.4, 126.8, 126.2, 123.9, 120.4, 117.0,
HRMS calcd for C11H11N3O 201.0902, found 201.0899.
N-t-Butyl-N0-hydroxyguanidine 7. H NMR (500 MHz,
1
CD3OD) d 1.40 (s, 9H); 13C NMR (125 MHz, CD3OD)
d 159.35, 53.27, 29.17. HRMS calcd for C5H13N3O
131.1059, found 131.1062.
N-n-Pentyl-N0-hydroxyguanidine 8. 1H NMR (500 MHz,
CD3OD) d 3.17 (t, J=7.0 Hz, 2H), 1.56–1.61 (m, 2H),
1.32–1.39 (m, 4H), 0.931 (t, J=6.5 Hz, 3H); 13C NMR
(125 MHz, CD3OD) d 159.3, 41.1, 28.7, 28.5, 22.2,
13.1. HRMS calcd for C6H15N3O 145.1215, found
145.1217.
N-Benzyl-N0-hydroxyguanidine
14a.
1H
NMR
(400 MHz, CD3OD) d 7.38–7.24 (m, 5H), 4.45 (s, 2H);
13C NMR (100 MHz, CD3OD) d 159.2, 136.6, 128.7,
127.8, 127.1, 44.4; HRMS calcd for C8H11N3O
165.0902, found 165.0908.
N-(4-Methoxyl)-benzyl-N0-hydroxyguanidine 14b. 1H
NMR (400 MHz, CD3OD) d 7.25 (d, J=9.0 Hz, 2H),
6.92 (d, J=9.0 Hz, 2H), 4.35 (s, 2H), 3.78 (s, 3H); 13C
NMR (100 MHz, CD3OD) d 159.8, 159.2, 128.5, 128.3,
114.0, 54.3, 44.0. HRMS calcd for C9H13O2N3
195.1008, found 195.1010.
0
1
N-Isoamyl-N -hydroxyguanidine 9. H NMR (500 MHz,
CD3OD) d 3.02 (q, J=7.5 Hz, 2H), 1.65 (m, 1H), 1.40
(q, J=7.0 Hz, 2H), 0.92 (d, J=6.5 Hz, 6H); 13C NMR
(125 MHz, CD3OD) d 159.10, 40.44, 39.65, 26.89, 22.88.
HRMS calcd for C6H15N3O 145.1215, found 145.1216.
N-Hexyl-N0-hydroxyguanidine 10. H NMR (400 MHz,
N-(4-Methyl)-benzyl-N0-hydroxyguanidine 14c. H NMR
1
1
CD3OD) d 3.01 (t, J=7.6 Hz, 2H), 1.50–1.54 (m, 2H),
1.30–1.37 (m, 6H), 0.91 (t, J=6.4 Hz, 3H); 13C NMR
(100 MHz, CD3OD) d 159.2, 42.2, 32.8, 30.6, 27.7, 23.7,
14.4. HRMS calcd for C7H17N3O 159.1372, found
159.1367.
(400 MHz, CD3OD) d 7.19 (d, J=8.0 Hz, 2H), 7.11 (d,
J=8.0 Hz, 2H), 4.13 (s, 2H), 2.29 (s, 3H); 13C NMR
(100 MHz, CD3OD) d 157.8, 136.6, 136.4, 128.9, 127.3,
44.7, 20.0; EIMS 180.2 (M++1).HRMS calcd for
C9H13N3O (M+ꢀO) 163.1100, found 163.1107.
N-Cyclohexyl-N0-hydroxyguanidine 11. 1H NMR
(400 MHz, CD3OD) d 3.34–3.40 (m, 1H), 1.63–1.92 (m,
5H), 1.16–1.40 (m, 5H); 13C NMR (100 MHz, CD3OD)
d 158.2, 50.8, 32.4, 25.0, 24.6. HRMS calcd for
C7H15N3O 157.1215, found 157.1209.
N-(4-Chloro)-benzyl-N0-hydroxyguanidine 14d. 1H NMR
(400 MHz, CD3OD) d 7.37(d, 2H, J=8 Hz), 7.33 (d, 2H,
J=8 Hz), 4.93 (s, 2H); 13C NMR (100 MHz, CD3OD) d
154.5, 138.6, 128.8, 128.3, 118.6, 44.0; HRMS calcd for
C8H10ON3Cl 199.0512, found 199.0513.
N-Phenyl-N0-hydroxyguanidine 12a. 1H NMR (400 MHz,
CD3OD) d 7.30c7.20 (m, 4H), 7.03–6.98 (m, 1H); 13C
NMR (100 MHz, CD3OD) d 155.4, 139.4, 129.0, 122.9,
120.4. HRMS calcd for C7H9N3O 151.0746, found
151.0747.
N-(4-Nitro)-benzyl-N0-hydroxyguanidine 14e. 1H NMR
(400 MHz, CD3OD) d 8.20 (d, J=9.0 Hz, 2H), 7.59 (d,
J=9.0 Hz, 2H), 4.99 (s, 2H); d 8.20 (d, J=9.0 Hz, 2H),
7.59 (d, J=9.0 Hz, 2H), 4.99 (s, 2H); 13C NMR
(100 MHz, CD3OD) d 158.6, 147.4, 145.8, 127.9, 123.5,
43.8; EIMS 211.0 (M++1).HRMS calcd for
C8H9N3O3 (M+ꢀNH) 195.0644, found 195.0643.
N-Phenylguanidine 15a. 1H NMR (400 MHz, CD3OD) d
7.45–7.52 (m, 2H), 7.32–7.38 (m, 1H), 7.25–7.32 (m,
2H); 13C NMR (100 MHz, CD3OD) d 152.1, 134.9,
129.9, 127.6, 125.6. HRMS calcd for C7H9N3 135.0796,
found 135.0801.
N-(4-Methoxyl)-phenyl-N0-hydroxyguanidine 12b. 1H
NMR (500 MHz, CD3OD) d 7.12 (d, J=9.0 Hz, 2H),
6.80 (d, J=9.0 Hz, 2H), 3.73 (s, 3H); 13C NMR
(125 MHz, CD3OD) d 159.5, 158.6, 127.5, 126.8, 115.0,
55.0. HRMS calcd for C8H11N3O2 181.0851, found
181.0851.
N-(4-Methyl)-phenyl-N0-hydroxyguanidine
12c.
1H
NMR (400 MHz, CD3OD) d 7.09 (d, J=8.0 Hz, 2H),
7.02 (d, J=8.0 Hz, 2H), 2.24 (s, 3H); 13C NMR
(100 MHz, CD3OD) d 154.6, 138.5, 131.2, 129.2, 119.3,
19.5. HRMS calcd for C8H11N3O 165.0902, found
165.0903.
N-(4-Methoxyl)-phenylguanidine
15b.
1H
NMR
(500 MHz, CD3OD) d 7.21 (d, J=9.0 Hz, 2H), 7.01 (d,
J=9.0 Hz, 2H), 3.83 (s, 3H); 13C NMR (125 MHz,
CD3OD) d 159.8, 157.6, 127.8, 127.0, 115.0, 55.1.
HRMS calcd for C8H11N3O 165.0902, found 165.0904.

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M. Xian et al. / Bioorg. Med. Chem. 10 (2002) 3049–3055
1
N-(4-Methyl)-phenylguanidine 15c. H NMR (500 MHz,
CD3OD) d 7.23 (d, J=8.0 Hz, 2H), 7.16 (d, J=8.0 Hz,
2H), 2.34 (s, 3H); 13C NMR (125 MHz, CD3OD) d
156.9, 137.9, 132.0, 130.5, 125.6, 19.9. HRMS calcd for
C8H11N3 149.0953, found 149.0951.
Assay of the nitric oxide synthase catalyzed NO gener-
ation from N-hydroxyguanidines. The initial rate of NO
synthesis was determined at 37 ꢁC using the classical
spectrophotometric oxyhemoglobin assay for NO.
Briefly, 20–30 mL aliquots containing NOS, 5 mM BH4,
and 2–5 mM DTT were added to a prewarmed cuvette
that contained 50 mM HEPES (pH 7.4), supplemented
with 15 mM oxyhemoglobin, 100 units/mL SOD,
100 units/mL catalase, 200 mM NADPH, 4 mM FAD,
4 mM FMN, 5 mM BH4, and 0.5 mM substrate at the
desired concentration, to give a final volume of 0.9 mL.
In the case of nNOS and eNOS, 1 mM CaCl2 and 10 mg/
mL CaM were present.For iNOS, 1 mM magnesium
acetate was added.The reference cuvette had the same
composition except that 50 mM HEPES, 5 mM BH4,
and 2–5 mM DTT were added instead of NOS-contain-
ing solutions.The NO-mediated conversion of oxy-
hemoglobin to methemoglobin was monitored over time
as an increase in absorbance at 401 nm and quantitated
Isopropylguanidine (16) and butylguanidine (17) were
prepared as previous report.36,37
Biochemistry
General. Dithiothreitol, NADPH, FAD, FMN, l-argi-
nine, NG-hydroxy-l-arginine (NOHA), hydroxy-
guanidine (NHG), manganese superoxide dismutase
(SOD), N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic
acid (HEPES), and (6R)-5,6,7,8-tetrahydrobiopterin
were purchased from Sigma.UV–vis spectra were
recorded on a HP 8453 spectrophotometer.
using an extinction coefficient of 38 mMꢀ1 cmꢀ1
.
Production and purification of human eNOS
Culture of Spodoptera frugiperda (Sf21) cells and manipu-
lations of the baculovirus were performed according to the
procedures described by Piwnica-Worms.38 Sf21 cells were
maintained in Grace’s medium containing 3.3 mg/mL
yeastolate, 3.3 mg/mL lactoalbumin hydrolysate, and
50 mg/mL gentamycin supplemented with 10% heat-inac-
tivated fetal bovine serum (GIBCO/BRL) at 27 ꢁC.
Acknowledgements
This work is supported by National Institute of Health
Grant GM54074 (P.G.W). We thank Dr. H. Li and
Professor T.L.Poulos (University of California at
Irvine) for their valuable discussions. P.G.W. also
thanks a summer support from Osaka University which
made the collaboration possible.
For the overproduction of human eNOS by the baculo-
virus/insect cells system, human eNOS cDNA cloned
previously39 was ligated into a baculovirus transfer vec-
tor pVL1392 (Invitrogen) at EcoRI site and cotrans-
fected with Baculogold (Pharmingen) into Sf21 cells
using lipofectin (GIBCO/BRL).2 ꢂ 108 Sf21 cells were
infected with baculovirus carrying human eNOS cDNA
and were incubated for 3 days at 27 ꢁC.Purification of
the eNOS proteins which were overproduced in Sf21
cells was carried out essentially as described previously40
with some modifications.Briefly, the Sf21 cells were
homogenated in buffer A (50 mM Tris–HCl, 5 mM
CHAPS, 1 mM DTT, 0.1 mM EDTA, 10% glycerol and
protein inhibitor complex (Complete EDTA free, Roche).
The homogenate was slowly stirred for 30min at 4 ꢁC and
then centrifuged at 15,000g for 30min.4 M NaCl was
added into the supernatant to a concentration of 0.1 M,
and mixed with 2 mL of 20,50-ADP-sepharose.The slurry
was stirred slowly for 1 h at 4 ꢁC and subsequently poured
into a column.The column was washed with buffer A, and
buffer A containing 0.5 M NaCl, and then with buffer A
alone.The human eNOS was eluted with buffer A con-
taining 2 mM NADPH.The elute was concentrated using
a Centricon YM30 microconcentrator (Amicon).The
purity of the eNOS was assessed with SDS-PAGE.
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