Conformationally-restricted arginine analogues as alternative substrates and inhibitors of nitric oxide synthases

Article abstract of DOI:10.1016/S0968-0896(99)00029-2

Conformationally restricted arginine analogues (1-5) were synthesized and found to be alternative substrates or inhibitors of the three isozymes of nitric oxide synthase (NOS). A comparison of k(cat)/K(m) values shows that (E)-3,4-didehydro-d,l-arginine (

Full text of DOI:10.1016/S0968-0896(99)00029-2

Bioorganic & Medicinal Chemistry 7 (1999) 1097±1104
Conformationally-restricted Arginine Analogues as Alternative
Substrates and Inhibitors of Nitric Oxide Synthases
Younghee Lee,a,y Michael A. Marletta,b,{ Pavel Martasek,c,x Linda J. Roman,c,x
Bettie Sue Siler Mastersc,x and Richard B. Silvermana,*
aDepartment of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston,
IL 60208-3113, USA
bInterdepartmental Program in Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
cDepartment of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78284-7760, USA
Received 1 October 1998; accepted 13 November 1998
AbstractÐConformationally restricted arginine analogues (1±5) were synthesized and found to be alternative substrates or inhibi-
tors of the three isozymes of nitric oxide synthase (NOS). A comparison of kcat/Km values shows that (E)-3,4-didehydro-d,l-argi-
nine (1) is a much better substrate than the corresponding (Z)-isomer (2) and 3-guanidino-d,l-phenylglycine (3), although none is as
good a substrate as is arginine; 5-keto-d,l-arginine (4) is not a substrate, but is an inhibitor of the three isozymes. Therefore, it
appears that arginine binds to all of the NOS isozymes in an extended (E-like) conformation. None of the compounds exhibits time-
dependent inhibition of NOS, but they are competitive reversible inhibitors. Based on the earlier report that No-propyl-l-arginine is
a highly selective nNOS inhibitor (Zhang, H. Q.; Fast, W.; Marletta, M.; Martasek, P.; Silverman, R. B. J. Med. Chem. 1997, 40,
3869), (E)-No-propyl-3,4-didehydro-d,l-arginine (5) was synthesized, but it was shown to be weakly potent and only a mildly
selective inhibitor of NOS. Imposing conformational rigidity on an arginine backbone does not appear to be a favorable approach
for selective NOS inhibition. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
Many di€erent inhibitors of NOS are known.2 Substrate
modi®cation is a well-tested strategy for the discovery of
inhibitors of an enzyme, and this approach has been
successful in the discovery of early inhibitors of NOS.
These inhibitors include a variety of l-arginine deriva-
tives in which the terminal guanidine group of the argi-
nine is modi®ed to give methylarginine3 and
nitroarginine.4 Non-amino acid NOS inhibitors also
have been reported, and these include isothioureas,5,6
amidines,7,8 guanidines,9 imidazole,10 indazoles,11 and
2-iminohomopiperidinium salts.12 Although some selec-
tive inhibitors of the enzyme have been described, the
selectivity is modest in most cases. However, a recent
report by the GlaxoWellcome group showed that
N-[3-(aminomethyl)benzyl]acetamidine is a potent inhi-
bitor of NOS with a selectivity of 5000 for iNOS versus
eNOS.13 We have found that No-propyl-l-arginine is
about 3000-fold selective for nNOS over iNOS and
about 150-fold selective for nNOS versus eNOS.14
These ®ndings constitute the largest selectivities
reported for the inhibition of NOS and suggest that
the design of selective inhibitors of the enzyme is
achievable.
Nitric oxide synthase (NOS, EC 1.14.13.39) is a family
of three heme-containing enzymes that catalyzes the
conversion of l-arginine to l-citrulline and nitric oxide.
The three isozymes are expressed di€erently and play
di€erent physiological roles; the endothelial enzyme
(eNOS) is involved in the regulation of smooth muscle
relaxation and blood pressure, in neuronal tissue
(nNOS) NO serves as an important intracellular second
messenger involved in neurotransmission, and in mac-
rophage cells an inducible form of the enzyme (iNOS) is
produced by in¯ammatory stimuli.1 The uncontrolled
production of NO has been implicated in numerous
disease states, therefore, the design of isoform-speci®c
NOS inhibitors to regulate NO synthesis in speci®c cells
has potential therapeutic bene®ts.1
Key words: Arginine analogues; nitric oxide synthase; con-
formationally-restricted; enzyme inhibition.
*Corresponding author. Tel.: 847-491-5653; fax: 847-491-7713; e-mail:
yagman@chem.nwu.edu
Designed and carried out all of the experiments in this study.
Provided the recombinant Escherichia coli cells expressed with mur-
{
ine macrophage iNOS used in this study.
x
Until the three-dimensional structure of the oxygenase
domain of iNOS was determined by X-ray crystallography,
Provided the recombinant eNOS and the Escherichia coli cells
expressed with bovine neuronal nNOS used in this study.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00029-2

1098
Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1097±1104
Results and Discussion
little information of the active site of any of the iso-
zymes of the enzyme was available.15 A novel approach
to probe the active site topologies of the three NOS
isozymes using various aryldiazenes suggested that the
sizes of the active sites of these enzymes decrease in the
order of nNOS>iNOS>eNOS, although they are very
similar in their global topology.16 The fact that most of
the l-arginine-derived inhibitors have modest isoform
selectivity supports this result.
Syntheses of conformationally-restricted analogues
(E)-3,4-Didehydro-d,l-arginine (1) was synthesized by
the treatment of (E)-2-acetamido-5-amino-3-pentenoic
acid (6a)18 with 1H-pyrazole-1-carboxamidine19 in aqu-
eous Na2CO3 followed by deprotection of the N-acetyl
group in HCl (Scheme 1). (Z)-3,4-Didehydro-d,l-argi-
nine (2) was prepared from (Z)-3,4-didehydroornithine
(6b)18 employing the same guanylation reaction as
described for the synthesis of 1 (Scheme 1). m-Guanidi-
no-d,l-phenylglycine (3) was prepared by the route
shown in Scheme 2. N-Boc protection of 3-nitrophenyl-
glycine (8)20 followed by catalytic hydrogenation of the
nitro group provided N-Boc protected 3-aminophe-
nylglycine (10). Guanylation of 10 with 1H-pyrazole-1-
carboxamidine in the presence of diisopropylethylamine
at elevated temperature, and subsequent N-Boc depro-
tection, a€orded 3. 5-Keto-l-arginine (4) was prepared
by the reaction of l-glutamic acid 5-methyl ester with
guanidine. (E)-N-Propyl-3,4-didehydro-d,l-arginine (5)
was prepared by guanylation of 6a with 1H-pyrazole-N-
propyl-1-carboxamidine in aqueous Na2CO3. 3-Ureido-
d,l-phenylglycine (13), the potential oxidation product
of 3, was synthesized by conversion of N-Boc-(m-ami-
nophenyl)glycine (10) to the corresponding cyanamide
derivative (12), followed by simultaneous hydrolysis and
deprotection (Scheme 3).
We envisioned that some conformationally-restricted
arginine analogues, however, may be alternative sub-
strates and/or inhibitors of the isozymes of NOS,
thereby allowing us to determine the preferred orienta-
tion of the natural substrate in each of the isozymes. If a
di€erence in the preferred geometry exists, this would be
a key component in the design of selective inhibitors of
the NOS isozymes. On that basis we designed ®ve con-
formationally-restricted arginine analogues, (E)-(1)- and
(Z)-3,4-didehydro-d,l-arginine (2), m-guanidino-d,l-
phenylglycine (3), and 5-keto-d,l-arginine (4), as
potential alternative substrates or inhibitors. Com-
pounds 1±3 were both substrates and inhibitors of all
three isozymes of NOS, and 4 and 5 were only inhibi-
tors; little selectivity (425-fold) was displayed by any of
these compounds. While this work was in progress, a
di€erent set of conformationally-restricted arginine
analogues was reported that had no better than a 12-
fold selectivity of eNOS and nNOS over iNOS.17
Inhibition of NOS isoforms by the conformationally-
restricted analogues
Table 1 shows the substrate and inhibitory activities of
compounds 1±5. Compounds 1±3 are both substrates
and inhibitors of all three NOS isozymes. Under initial
velocity conditions with the (E)-didehydro arginine
analogue 1, the maximal rate of NO formation by
nNOS and eNOS is observed at 5 and 10 mM, respec-
tively, and the rate of NO formation is approximately
30% that of when 10 mM l-arginine was used. The Km
values with nNOS (1.5 mM) and eNOS (1 mM) are
lower than the respective Km values for l-arginine (2.7
and 1.1 mM, respectively) with those isozymes; the kcat
/
Km values for 1 are only slightly lower than those with
l-arginine. In the case of iNOS, the maximal rate of
NO formation, which was observed at a 300 mM con-
centration, corresponds to only 4% of that when l-
arginine was used. The Km value for 1 with iNOS was
much higher (35 mM) than those for the other two iso-
forms and for l-arginine, and the kcat/Km value is an
Scheme 1.

Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1097±1104
1099
Scheme 2.
Scheme 3.
Table 1. Kinetic constants for substrate and inhibitor activity of l-arginine and compounds 1±5
1
1
1
Compound
Km
Ki
kcat/Km (s 1 mM
)
Km
Ki
kcat/Km (s 1 mM
)
Km
Ki
kcat/Km (s 1 mM
)
l-Arginine
2.7 mM
1.5 mM 25 mM
5 mM
40 mM
180
110
9.5 mM
35 mM
20 mM 13 mM
110
13
1.1 mM
73
26
1
2
3
4
5
45 mM
1.0 mM 50 mM
15 mM 10 mM
25 mM 50 mM
3 mM
3
3
4
6.5 mM
2.0 mM
770 mM
640 mM
1.8Â10
6.9Â10
8.0Â10
2
3
2
1.8Â10
20 mM
8 mM
900 mM
9 mM
4.4Â10
7.9Â10
2.5 mM
order of magnitude lower than that for l-arginine. On
the basis of Km alone, the selectivities of 1 for nNOS and
eNOS over iNOS are 23- and 35-fold, respectively
(Table 1); however, on the basis of kcat/Km values the
selectivities are a much more modest 8- and 4-fold,
respectively. As a competitive inhibitor, 1 shows little or
no selectivity.
favor of iNOS (4- and 9-fold, respectively), when kcat
Km values are compared.
/
To further rigidify 1 in an attempt to increase the bind-
ing e€ectiveness, analogue 3 was synthesized, which can
be drawn in a conformer with the same geometry as 1,
but has additional atoms from the aromatic ring. How-
ever, this compound is a much poorer substrate than 1;
its Km and kcat/Km values are 3±4 orders of magnitude
greater than those for 1 (Table 1), suggesting a possible
steric hindrance to its binding at the arginine binding
site by the aromatic ring or that 1 and 3 bind in di€erent
geometries. Interestingly, the Ki values for 3 with nNOS,
iNOS, and eNOS are much lower (by 3±4 orders of
magnitude) than the corresponding Km values. This
suggests that nonproductive binding is important and
that this alternative substrate can bind to the active site,
but poorly elicit oxidation.
The Z-isomer (2) was found to be a much poorer sub-
strate for all three isoforms of NOS than was 1, having
Km values of 3±4 orders of magnitude greater than 1 and
kcat/Km values 3±5 orders of magnitude lower than those
for 1 (Table 1)! Although there are many conformers
that 1 and 2 can assume, these results suggest that the
binding orientation of the 2±5 carbon atoms of arginine
in the active site is better mimicked by an extended
geometry rather than a folded one. The maximal velo-
cities with 2 were 12±17% that for l-arginine with the
respective isoforms, and substrate selectivities for nNOS
and eNOS over iNOS were 4- and 1.3-fold, respectively,
if only Km is considered, but the selectivities reverse in
While monitoring turnover with 3, the NO formation
was found to diminish with time. Compound 3 was

1100
Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1097±1104
shown not to be a time-dependent inhibitor, so product
inhibition was suspected. As monitored by HPLC, the
major product (other than NO) formed was shown to be
3-ureido-d,l-phenylglycine (13), the expected oxidation
product. However, 13 was found to be a poor inhibitor
of nNOS; at a concentration of 5 mM (the highest con-
centration allowable because of its low solubility), less
than 10% of enzyme activity was inhibited. Compound
13 also was not a time-dependent inhibitor of nNOS at
1 mM concentration for 1 h. Possibly, other products,
formed in much smaller concentrations, are responsible
for the inhibitory activity of 3 or the release of 13 from
the enzyme is slow.
recently reported to be NOS inhibitors by the Glaxo-
Wellcome group.17
The arginine analogue 15 (R=NH2), in which the
amino acid group is ortho to that of the guanidine
group, inhibits all three human NOS isoforms with Ki
values for iNOS, eNOS, and nNOS of 2.60, 0.25, and
0.37 mM, respectively. Arginine analogue 16 (R=NH2)
also inhibits all isoforms of NOS, but the inhibitory
potencies are lower (100, 46, and 44 mM, respectively)
than those of 15. On the basis of these results, it was
proposed that arginine based inhibitors preferentially
bind to the active sites of all of the NOS isoforms in an
orientation where the arginine alkyl chain is in a `folded
conformation.' This hypothesis appears to be di€erent
from what we proposed above using 1±3, in which the
`extended' E-arginine analogue is a better substrate than
the `folded' Z-isomer. However, this is really just
semantics, because 15 can be drawn in a conformer that
is identical to that of 1 (Fig. 1). On the other hand, 3
cannot be drawn in a conformer that is identical to that
of 15, but it can be drawn in a conformer that is similar
to it (Fig. 2). This may account for our observation that
3 is a poor substrate but a comparable inhibitor to 1; it
may be bound appropriately for inhibition, but not for
oxygenation. However, with regard to selectivities for the
NOS isozymes (Table 2), 1 is more similar to 15 than 3 is
to 15. Structure 16, however, is not really an analogue of
arginine, but rather, is an analogue of homoarginine
5-Ketoarginine (4) also was investigated as a potential
conformationally-restricted analogue of arginine
because of the potential intramolecular hydrogen bond-
ing properties of the ketone carbonyl with the terminal
NH2 group. This compound was not a substrate at all
for any of the isozymes of NOS, and showed only weak
inhibitory properties (Table 1). In addition to its possi-
ble cyclic structure, as a result of hydrogen bonding, the
carbonyl group will strongly decrease the pKa of the
guanidino group. In fact, l-canavadine (14), with an
oxygen in place of the carbonyl, has a guanidino pKa of
7, and it is not a substrate for NOS either.21
Two conformationally-restricted series of arginine ana-
logues (15 and 16, R=NH2, NHCH3, and CH3) were
Figure 1. Comparison of speci®c conformations of 1 and 15.
Figure 2. Comparison of speci®c conformations of 3 and 15.

Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1097±1104
1101
Experimental
Table 2. Comparison of the selectivities of NOS isozyme inhibition
by conformationally-rigid analogues
Materials
Compound
Selectivity nNOS/eNOSa
Selectivity nNOS/iNOSa
NADPH, HEPES, DTT, calmodulin, and human fer-
rous hemoglobin were purchased from Sigma Chemical
Co. Tetrahydrobiopterin (H4B) was obtained from B.
Schircks Laboratories (Jona, Switzerland) or from
Alexis Biochemicals (San Diego, CA). Acids, bases, and
conventional organic solvents were purchased from
Fisher. All chemicals were purchased from Aldrich
unless otherwise stated. TLC plates (silica gel 60-F254,
250 mM), and silica gel 60 (230±400 mesh) were pur-
chased from VWR Scienti®c.
1
3
15b
16b
17c
18c
2
25
0.68
1
155
33
1.8
4
7
22
50
10
aRatio of the inverse of the Ki values (i.e. a ratio >1 means that nNOS
is a more potent inhibitor).
bData taken from ref 17.
cData taken from ref 22.
(there is an extra carbon in 16 relative to arginine),
whereas 1±3 are all analogues of arginine. Therefore, 1
and 3 are not similar in structure to 16, although the
low selectivities of 1 for the NOS isozymes are similar to
those of 16; 3 is much more selective than either 1 or 16.
Better structural comparisons are made between 3 and
Analytical methods
Optical spectra and enzyme assays were recorded on
either a Perkin±Elmer Lambda 10 UV±vis spectro-
photometer. NMR spectra were recorded on a Varian
Gemini-300 300-MHz spectrometer (75 MHz for 13C
spectra). Chemical shifts are reported as d values in
parts per million down®eld from Me4Si as the internal
standard in CDCl3, unless stated otherwise. HPLC was
performed on a Beckman System Gold instrument
(Model 125P solvent module and Model 166 detector).
Samples were analyzed by elution from an Alltech
Econosil C18 10 m (4.6Â250 mm) column. Mass spectra
were recorded on a VG Instruments VG70-250SE high-
resolution spectrometer. Combustion analyses were
performed by the Department of Geological Sciences at
Northwestern University. Melting points were obtained
with a Fisher±Johns melting point apparatus and are
uncorrected.
N-[3-(aminomethyl)benzyl]acetamidine13
(17)
and
between 16 and N-[3-(aminoethyl)benzyl]acetamidine22
(18). On the basis of selectivities (Table 2), 3 appears to
be closer to 18 than to 17. However, 3 is a guanidine
and 17 and 18 are amidines, and that could be very
important to the binding interactions. The fact that the
selectivities of 16 are not similar to either 17 or 18 may
indicate that they bind in di€erent conformations than
those depicted in the structures drawn. Relatively minor
structural changes can have major consequences with
regard to isozyme selectivities. For example, the dele-
tion of one methylene group from N-[3-(aminomethyl)
benzyl]acetamidine13 transforms the compound from an
iNOS-selective inhibitor into an nNOS-selective inhi-
bitor.22
(E)-3,4-Didehydro-D,L-arginine (1). A mixture of (E)-
2-acetoamido-5-amino-3-pentenoic acid18 (344 mg,
2.5 mmol), 1H-pyrazole-1-carboxamidine hydrochlo-
ride19 (366 mg, 2.5 mmol), and 1 M Na2CO3 (4 mL) was
stirred for 2 days at room temperature. Concentrated
NH4Cl (1 mL) was added, and the mixture was con-
centrated. The residue was loaded onto Dowex 50W-X8
(1Â2 cm), washed with H2O, then eluted with a gradient
of 0.5 to 1 N HCl. The combined fractions of HCl elu-
tion were concentrated, the residue was loaded onto
Dowex 1X2-100, washed with H2O, then eluted with
3% NH4OH. After evaporation of the combined frac-
tions containing product, the colorless foam (265 mg,
62%) of Na-acetyl (E)-3,4-didehydro-d,l-arginine (7a)
was used in the next reaction without further puri®ca-
tion; 1H NMR (D2O) d 1.88 (s, 3H), 3.68 (d,
J=4.61 Hz, 1H), 4.57 (m, 1H), 5.57 (dt, J=16.00,
4.61 Hz, 1H), 5.65 (dd, J=16.00, 5.62 Hz, 1H).
Since 1 is a very good substrate, we attempted to con-
vert it into a potent and selective inactivator of the iso-
forms of NOS by No-substitution. Earlier, we found
that No-propyl-l-arginine is a potent and selective inhi-
bitor of nNOS.14 However, (E)-N-propyl-3,4-didehy-
dro-d,l-arginine (5) is a poor, low selective inhibitor of
the nNOS isoform (Table 1). There, obviously, are sub-
tle binding interactions that are disturbed when sub-
stitution of 1 by a propyl group is made.
A solution of 7a (420 mg, 2.0 mmol; two preps were
combined) in 6 N HCl (7 mL) was re¯uxed for 2 h. After
concentration of the reaction mixture, the residue was
loaded onto Dowex 50W-X8 and eluted with a gradient
of 1±2.5 N HCl. The fractions containing product were
combined, concentrated, then lyophilized to a foamy
Conclusion
On the basis of the results reported here and those by
Shearer et al.17 conformational restriction appears to
prevent the molecules from assuming the appropriate
discriminatory binding orientations needed for high
selectivity of the isozymes of NOS.
1
solid (132 mg, 27%) of 1; H NMR (D2O) d 3.89 (d,
J=3.30 Hz, 2H), 4.64 (d, J=8.01 Hz, 1H), 5.77 (app dd,
J=15.72, 8.01 Hz, 1H), 6.05 (dt, J=15.72, 4.54 Hz, 1H);
13C NMR (75 MHz, D2O) 41.7, 54.1, 121.4, 134.2,

1102
Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1097±1104
.
156.9, 170.4. Anal. calcd for C6H12N4O2 2HCl: C, 29.40;
H, 5.76; N, 22.86. Found: C, 29.22; H, 6.08; N, 22.69.
2 N HCl. The fractions stained by ninhydrin treatment
were combined and concentrated. The resulting crude
solid was loaded on Dowex 50W-X8 and eluted with a
gradient of 1±2.5 N HCl. The fractions containing 3
were combined and concentrated to give m-guanidino-
d,l-phenylglycine dihydrochloride (280 mg, 20%) as a
colorless solid; 1H NMR (D2O) d 5.18 (s, 1H), 7.35±7.43
(3H), 7.50±7.54 (m, 1H); 13C NMR (D2O) 56.1, 125.6,
127.3, 127.5, 131.4, 133.3, 135.4, 156.2, 170.4; HRMS
(FAB, M+1) calcd for C9H13N4O2 209.1038; found
209.1072. Anal. calcd for C9H13N4O2 (0.2 H2O): C, 37.96;
H, 5.09; N, 19.67. Found: C, 37.65; H, 5.12; N, 19.71.
(Z)-3,4-Didehydro-D,L-arginine (2). A mixture of (Z)-
3,4-didehydro-d,l-ornithine dihydrochloride18 (120 mg,
0.59 mmol), 1H-pyrazole-1-carboxamidine hydrochlo-
ride19 (87 mg, 0.59 mmol), and 0.9 mL of 1.0 M Na2CO3
was stirred for 3 days at room temperature. Con-
centrated NH4Cl (1 mL) was added to the mixture, and
the solution was concentrated to dryness. The residue
was loaded onto a column of Dowex 50 (1Â2 cm), and
eluted with a gradient of 0.1±2 M HCl. The fractions
containing product were combined and concentrated to
give a sticky solid (66 mg, 46%), which was recrys-
tallized from a mixture of H2O:EtOH (5:95); mp 170±
172 C; H NMR (D2O) d 4.04 (dt, J=6.59, 2.05 Hz,
2H), 4.86 (dd, J=9.84, 0.88 Hz, 1H), 5.66 (ddt,
J=10.86, 9.84, 1.78 Hz, 1H), 5.96 (dtd, J=10.86, 6.59,
0.88 Hz, 1H); 13C NMR (D2O) 38.9, 50.5, 122.5, 133.6,
156.7, 170.0; Anal. calcd for C6H12N4O2(2HCl): C, 29.40;
H, 5.76; N, 22.86. Found: C, 29.67; H, 5.98; N, 22.70.
5-Keto arginine (4). To a solution of sodium ethoxide
(20 mmol) in 40mL of EtOH was added guanidine hydro-
chloride (1.91 g, 20 mmol) portionwise at room tem-
perature. After the mixture had been stirred for 1 h, the
solution was ®ltered, and the ®ltrate was transferred to
the ¯ask containing l-glutamic acid 5-methyl ester
(1.61 g, 10 mmol). The mixture was stirred overnight at
room temperature. The homogeneous solution was
concentrated under reduced pressure, and the residue
was loaded on Dowex 1X2-100 and eluted with water.
The combined fractions containing the product was
concentrated to give 4 as a white crystalline solid which
was recrystallized from a mixture of H2O:EtOH
ꢀ
1
Nꢀ-Boc-m-nitro-D,L-phenylglycine (9). To a stirred solu-
tion of di-tert-butyl dicarbonate (32 g, 0.146 mol) and
m-nitro-d,l-phenylglycine20 (8.26 g, 0.133 mol) in
dichloromethane (400 mL) was added triethylamine
(37 mL, 0.266 mol). After being stirred overnight, the
reaction mixture was concentrated under reduced pres-
sure, applied to a silica gel column (6Â25 cm), and
eluted with hexane:ethyl acetate (3:1). Fractions con-
taining the desired compound were combined and con-
centrated to give 9 as a pale yellow foamy solid (28.3 g,
72%); 1H NMR (CDCl3) d 1.19 (s, 9H), 5.27 (d,
J=4.35 Hz, 1H), 7.60 (app t, J=7.97 Hz, 1H), 7.80 (d,
J=7.41 Hz, 1H), 8.18 (d, J=8.25 Hz, 1H), 8.32 (bs 2H),
12.23 (bs, 1H); 13C NMR (CDCl3) 28.0 (3C) 58.3, 82.8,
122.4, 123.2, 129.7, 133.2, 140.6, 148.3, 157.0, 172.3;
HRMS (EI) calcd for C13H16N2O6 296.1009; found
296.1018.
(322 mg, 17%); mp 88±89 ꢀC; H NMR (D2O) d 1.87
1
(m, 1H), 2.26 (dd, J=8.78, 6.89 Hz, 2H), 2.35 (m, 1H),
4.03 (dd, J=9.03, 5.76 Hz, 1H); 13C NMR (D2O) 25.3,
29.6, 58.2, 157.9, 180.2, 181.5; HRMS (M+1) calcd for
C6H13N4O3 189.0988; found 189.1001. Anal. calcd for
C6H13N4O3: C, 38.29; H, 6.42; N, 29.82. found: C, 38.02;
H, 6.57; N, 29.82.
(E)-N!-Propyl-3,4-didehydro-D,L-arginine (5). A mix-
ture of (E)-2-acetoamido-5-amino-3-pentenoic acid18
(861 mg, 5 mmol), 1H-pyrazole-N-propyl-1-carbox-
amidine hydrochloride23 (940 mg, 5 mmol) in 1 N
Na2CO3 (10 mL) was stirred for 6 days at room tem-
perature. Concentrated NH4Cl (2 mL) was added, and
the mixture was concentrated, then the residue was loa-
ded on Dowex 50W-X8 (2.5Â6 cm) and washed with
water (200 mL). The amino acid was eluted with a HCl
gradient (0.5±2.5 N), and the eluate was concentrated to
give 5 as a colorless foam (210 mg, 18%); 1H NMR
(D2O) d 0.86 (t, J=7.38 Hz, 3H), 1.53 (m, 2H), 3.12 (t,
J=7.00 Hz, 2H), 3.91 (d, J=4.11 Hz, 2H), 4.6 (d,
J=7.95 Hz, 1H), 5.75 (ddt, J=15.69, 8.10, 1.65 Hz, 1H),
6.05 (dt, J=15.69, 4.41 Hz, 1H); 13C NMR (D2O) 10.5,
21.6, 41.7, 43.0, 54.0, 120.9, 134.6, 155.7, 170.2; HRMS
(FAB, M+1) calcd for C9H19N4O2 215.1508; found
215.1512. Anal. calcd for C9H19N4O2 (0.3 H2O): C,
37.64; H, 7.09; N, 19.14. found: C, 37.46; H, 7.17; N, 19.12.
Nꢀ-Boc-m-amino-D,L-phenylglycine (10). A mixture of 9
(9.24 g, 0.081 mol) and 10% Pd/C (1.5 g) in EtOH
(400 mL) was ¯ushed with hydrogen. After being stirred
vigorously under a hydrogen atmosphere (1 atm) with
frequent re®ll of hydrogen for 7 h, the reaction mixture
was ®ltered through Celite and concentrated to a foamy
solid which was recrystallized from chloroform to give
10 as a pale yellow solid (15 g, 70%); mp 148±149 ꢀC; 1H
NMR (CDCl3) d 1.34 (s, 9H), 4.88 (d, J=8.07 Hz, 1H),
6.53±6.59 (2H), 7.01 (app t, J=7.61 Hz, 1H), 7.27 (d,
J=8.31 Hz, 1H); HRMS (EI) calcd for C13H18N2O4
266.1267; found 266.1271.
m-Guanidino-D,L-phenylglycine (3). A mixture of 10
(1.33 g, 5 mmol), 1H-pyrazole-1-carboxamidine hydro-
chloride19 (733 mg, 5 mmol), and diisopropylethylamine
(870 mL) in DMF (2 mL) was stirred overnight at
140 ꢀC. DMF was removed under reduced pressure, and
the crude mixture was stirred in tri¯uoroacetic
acid:CH2Cl2 (1:2, 20mL) for 2 h at room temperature.
After evaporation of the volatile material, the mixture was
treated with 2 N NH4OH (3mL), loaded on the Dowex
1X2-100, washed with water (100 mL), then eluted with
Nꢀ-Boc-m-cyanamido-D,L-phenylglycine (12). To a mix-
ture of 10 (700 mg, 2.63 mmol) and sodium acetate
(220 mg, 2.63 mmol) stirred in MeOH (40 mL) at 0 ꢀC
was added cyanogen bromide (276 mg, 2.63 mmol) por-
tionwise. After being stirred for 3 h at room temperature
the volatile materials were removed under reduced
pressure, and the residue was dissolved in ethyl acetate
(50 mL) and extracted with 0.5 N HCl (30 mL). The
organic layer was concentrated, and the crude mixture

Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1097±1104
1103
was puri®ed by ¯ash silica gel chromatography using
ethyl acetate as eluant to give 12 as a white foam
(420 mg, 55%); H NMR (DMSO-d6) d 1.38 (s, 9H),
(GM49725), M.A.M. (CA50414) and B.S.S.M.
(GM52419) and to the Robert A. Welch Foundation
(AQ-1192) for ®nancial support to B.S.S.M.
1
5.01 (d, J=7.74 Hz, 1H), 6.85 (dd, J=7.98, 1.41 Hz,
1H), 6.97 (s, 1H), 7.03 (d, J=7.88 Hz, 1H), 7.29 (t,
J=7.83 Hz, 1H), 7.51 (d, J=7.89 Hz, 1H); 13C NMR
(DMSO-d6) 28.1 (3C), 57.4, 111.9, 114.1, 114.4, 122.0,
129.7, 138.6, 139.2, 155.1, 171.9; HRMS (FAB, M + 1)
calcd for C14H18N3O4 292.1297; found 292.1303.
References and Notes
1. Kerwin, J. F.; Heller, M. Med. Res. Rev. 1994, 14, 23.
2. Marletta, M. A. J. Med. Chem. 1994, 37, 1899.
3. Olken, N. M.; Marletta, M. A. Biochemistry 1993, 32, 9677.
4. Fur®ne, E. S.; Harmon, M. F.; Paith, J. E.; Garvey, E. P.
Biochemistry 1993, 32, 8215.
m-Ureido-D,L-phenylglycine
(13).
Compound
12
(260 mg, 0.89 mmol) in 1 N HCl (20 mL) was heated
under re¯ux for 10 min. After evaporation of the sol-
vent, the residue was loaded on Dowex 50W-X8,
washed with water (40 mL), then eluted with 1 N
NH4OH (150 mL). The fractions stained by ninhydrin
treatment were combined and concentrated to yield 13
as a colorless solid (133 mg, 82%), which was recrys-
tallized from water; ESMS (M+1) 210.0; 1H NMR
(D2O) d 4.71 (s, 1H), 7.13 (m, 1H), 7.27 7.40 (3H); 13C
NMR (D2O) 58.3, 120.8, 122.4, 123.5, 130.2, 135.0,
138.7, 159.3, 173.1; HRMS (FAB, M+1) calcd for
C9H12N3O3 210.0878; found 210.0819. Anal. calcd for
C9H12N3O3: C, 51.67; H, 5.30; N, 20.09. found: C, 51.64;
H, 5.57; N, 20.09.
5. Garvey, E. P.; Oplinger, J. A.; Tanoury, G. J.; Sherman, P.
A.; Fowler, M.; Marshall, S.; Harmon, M. F.; Paith, J. E.;
Fur®ne, E. S. J. Biol. Chem. 1997, 269, 26669.
6. Shearer, B. G.; Lee, S.; Oplinger, J. A.; Frick, L. W.; Gar-
vey, E. P.; Fur®ne, E. S. J. Med. Chem. 1997, 40, 1901.
7. (a) Moore, W. M.; Webber, R. K.; Jerome, G. M.; Tjoeng,
F. S.; Misko, T. P.; Currie M. G. J. Med. Chem. 1994, 37,
3886. (b) Moore, W. M.; Webber, R. K.; Fok, K. F., Jerome,
G. M.; Kornmeier, C. M.; Tjoeng, F. S.; Currie, M. G. Bioorg.
Med. Chem. 1996, 4, 1559.
8. (a) Moore, W. M.; Webber, R. K.; Fok, K. F.; Jerome, G.
M.; Connor, J. R.; Manning, P. T.; Wyatt, P. S.; Misko, T. P.;
Tjoeng, F. S.; Currie, M. G. J. Med. Chem. 1996, 39, 699. (b)
Webber, R. K.; Metz, S.; Moore, W. M.; Connor, J. R.; Cur-
rie, M. G.; Fok, K. F.; Hagen, T. J.; Hansen, D. W., Jr.; Jer-
ome, G. M.; Manning, P. T.; Pitzele, B. S.; Toth, M. V.;
Trivedi, M.; Zupec, M. E.; Tjoeng, F. S. J. Med. Chem. 1998,
41, 96.
Enzymes
9. (a) Wol€, D. J.; Lubeskie, A. Arch. Biochem. Biophys. 1995,
316, 290. (b) Fast, W.; Hu€, M. E.; Silverman, R. B. Bioorg.
Med. Chem. Lett. 1997, 7, 1449.
10. Wol€, D. J.; Gribin, B. J. Arch. Biochem. Biophys. 1994,
311, 293.
All of the enzymes used were recombinant enzymes
overexpressed in Escherichia coli. The murine macro-
phage iNOS was expressed24 and isolated25 as reported;
the bovine endothelial eNOS was prepared as previously
described,26 and the rat neuronal nNOS was expressed
and puri®ed as described.27
11. Wol€, D. J.; Gribin, B. J. Arch. Biochem. Biophys. 1994,
311, 300.
12. Hansen, D. W., Jr.; Peterson, K. B.; Trivedi, M.; Kramer,
S. W.; Webber, R. K.; Tjoeng, F. S.; Moore, W. M.; Herone,
G. M.; Kornmeier, C. M.; Manning, P. T.; Connor, J. R.;
Misko, T. P.; Currie, M. G.; Pitzele, B. S. J. Med. Chem. 1998,
41, 1361.
13. Garvey, E. P.; Oplinger, J. A.; Fur®ne, E. S.; Ki€, R. J.;
Laszlo, F.; Whittle, B. J. R.; Knowles, R. G. J. Biol. Chem.
1997, 272, 4959.
14. Zhang, H. Q.; Fast, W.; Marletta, M.; Martasek, P.; Sil-
verman, R. B. J. Med. Chem., 1997, 40, 3869.
15. Crane, B. R.; Arvai, A. S.; Gachhui, R.; Wu, C.; Ghosh,
D. K.; Getzo€, E. D.; Stuehr, D. J.; Tainer, J. A. Science 1997,
278, 425.
Initial velocity measurements via the hemoglobin assay
The generation of nitric oxide by NOS was measured
using the hemoglobin capture assay,28 which utilizes
the rapid oxidation of oxyhemoglobin by NO to
produce methemoglobin, which is detected spectro-
photometrically at 401 nm (e=19,700 M 1 cm 1) on a
Perkin±Elmer Lamda 10 UV±vis spectrophotometer. A
typical assay mixture for nNOS and eNOS contained
10mM l-arginine, 1.6 mM CaCl2, 11.6mg/mL calmodu-
lin, 100 mM NADPH, 6.5 mM BH4, and 3 mM oxyhe-
moglobin in 100 mM HEPES bu€er (pH 7.5). The
reaction mixture for iNOS contained 10mM of l-arginine,
100 mM NADPH, 6.5 mM BH4, and 3 mM oxyhemoglobin
in 100 mM HEPES bu€er (pH 7.5). All assays were in a
®nal volume of 600 mL and were initiated with enzyme.
16. Gerber, N. C.; Rodriguez-Crespo, I.; Nishida, C. R.; Ortiz
de Montellano, P. R. J. Biol. Chem. 1997, 272, 6285.
17. Shearer, B. G.; Lee, S.; Franzmann, K. W.; White, H. A.
R.; Sanders, D. C. J.; Ki€, R. J.; Garvey, E. P.; Fur®ne, E. S.
Bioorg. Med. Chem. Lett. 1997, 7, 1763.
18. Havlicek, L.; Hanus, J.; Sedmera, P.; Nemecek, J. Collect.
Czech. Chem. Commun. 1990, 55, 2074.
19. Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. J. Org.
Chem. 1992, 57, 2497.
Substrate activity/reversible inhibition measurements
20. Friis, P.; Kjñr, A. Acta Chem. Scand. 1963, 17, 2391.
21. McCall, T. B.; Boughton-Smith, N. K.; Palmer, M. J.;
Whittle, B. J. R.; Moncata, S. Biochem. J. 1989, 261, 293.
22. Collins, J. L.; Shearer, B. G.; Oplinger, J. A.; Lee, S.;
Garvey, E. P.; Salter, M.; Du€y, C.; Burnette, T. C.; Fur®ne,
E. S. J. Med. Chem. 1998, 41, 2858.
Substrate activity was measured as described above
using varying concentrations of the alternative substrate
(1±3) instead of l-arginine. Ki values were determined
by the method of Dixon.29
23. This compound was prepared as follows: To a stirred
solution of n-propylamine (8.2 mL, 100 mmol) in diethyl ether
(150 mL) at 0 ꢀC was added cyanogen bromide (5.2 g,
50 mmol) in diethyl ether (30 mL) over a period of 10 min. The
Acknowledgements
The authors are grateful to the National Institutes of
Health for ®nancial support of this research to R. B. S.

1104
Y. Lee et al. / Bioorg. Med. Chem. 7 (1999) 1097±1104
mixture was stirred further for 30 min at room temperature,
then the precipitated solid was ®ltered o€, and the ®ltrate was
concentrated under reduced pressure to a€ord N-propylcyan-
amide as an oil which was used directly for the next step. To
this oil in dioxane (20 mL) were added pyrazole (2.7 g,
40 mmol) and 4 N HCl in dioxane (10 mL, 40 mmol), and the
mixture was stirred overnight at room temperature. The pre-
cipitated white crystals were ®ltered, washed with cold diox-
ane, and dried to a constant weight (6.5 g, 69%); mp 154±
24. Rusche, K. M.; Spiering, M. M.; Marletta, M. A., manu-
script submitted.
25. Hevel, J. M.; White, K. A.; Marletta, M. A. J. Biol. Chem.
1991, 266, 22789.
26. Martasek, P.; Liu, Q.; Liu, J.; Roman, L. J.; Gross, S. S.;
Sessa, W. C.; Masters, B. S. S. Biochem. Biophys. Res. Com-
mun. 1996, 219, 359.
27. Roman, L. J.; Sheta, E. A.; Martasek, P.; Gross, S. S.;
Liu, Q.; Masters, B. S. S. Proc. Natl. Acad. Sci. USA 1995, 92,
8428.
156 ꢀC; H NMR (D2O) d 0.96 (t, J=7.41 Hz, 3H), 1.72 (m,
1
2H), 3.43 (t, J=6.90 Hz, 2H), 6.66 (m, 1H), 7.91 (s, 1H), 8. 26
(d, J=2.94 Hz, 1H); 13C NMR (D2O) 10.6, 20.9, 44.5, 111.7,
130.5, 146.0, 150.7.
28. Hevel, J. M.; Marletta, M. A. Methods Enzymol. 1994,
233, 250.
29. Dixon, M. Biochem. J. 1953, 55, 170.