Structure-based design and synthesis of Nω-nitro-L-arginine- containing peptidomimetics as selective inhibitors of neuronal nitric oxide synthase. Displacement of the heme structural water

Article abstract of DOI:10.1021/jm061305c

The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric oxide in the central nervous system, represents an attractive target for the treatment of various neurodegenerative disorders. X-ray crystal structur

Full text of DOI:10.1021/jm061305c

J. Med. Chem. 2007, 50, 2089-2099
2089
Structure-Based Design and Synthesis of Nω-Nitro-L-Arginine-Containing Peptidomimetics as
Selective Inhibitors of Neuronal Nitric Oxide Synthase. Displacement of the Heme Structural
Water
Jiwon Seo,† Jotato Igarashi,‡ Huiying Li,‡ Pavel Marta´sek,§,# Linda J. Roman,§,| Thomas L. Poulos,‡ and
Richard B. Silverman†,*
Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology, and the Center for Drug DiscoVery and Chemical
Biology, Northwestern UniVersity, EVanston, Illinois 60208-3113, Department of Biochemistry, The UniVersity of Texas Health Science Center,
San Antonio, Texas 78384-7760, and Departments of Molecular Biology and Biochemistry, Physiology and Biophysics, and Chemistry and
Program in Macromolecular Structure, UniVersity of California, IrVine, California 92697-3900
ReceiVed NoVember 9, 2006
The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric
oxide in the central nervous system, represents an attractive target for the treatment of various
neurodegenerative disorders. X-ray crystal structures of complexes of nNOS with two nNOS-selective
inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N′-nitroguanidine (1) and 4-N-(Nω-nitro-L-
argininyl)-trans-4-amino-L-proline amide (2), led to the discovery of a conserved structural water molecule
that was hydrogen bonded between the two heme propionates and the inhibitors (Figure 2). On the basis of
this observation, we hypothesized that by attaching a hydrogen bond donor group to the amide nitrogen of
2 or to the secondary amine nitrogen of 1, the inhibitor molecules could displace the structural water molecule
and obtain a direct interaction with the heme cofactor. To test this hypothesis, peptidomimetic analogues
3-5, which have either an N-hydroxyl (3 and 5) or N-amino (4) donor group, were designed and synthesized.
X-ray crystal structures of nNOS with inhibitors 3 and 5 bound verified that the N-hydroxyl group had,
indeed, displaced the structural water molecule and provided a direct interaction with the heme propionate
moiety (Figures 5 and 6). Surprisingly, in vitro activity assay results indicated that the addition of a hydroxyl
group (3) only increased the potency slightly against the neuronal isoform over the parent compound (1).
Rationalizations for the small increase in potency are consistent with other changes in the crystal structures.
Introduction
Nitric oxide (NO),1 a highly reactive free radical, is an
essential signaling molecule involved in various physiological
processes in humans. Generally, NO acts as a potent activator
of soluble guanylate cyclase (sGC), which catalyzes the conver-
sion of guanosine 5′-triphosphate (GTP) to an intramolecular
second messenger, guanosine 3′,5′-cyclic monophosphate (cG-
MP).2 This NO/cGMP signaling pathway is essential in many
physiological processes including vasodilation, neurotransmis-
sion, and platelet aggregation.3 NO also participates in responses
that are not mediated via sGC.4
Figure 1. Nω-Nitro-L-arginine-containing dipeptides and their deriva-
tives.
Nitric oxide is synthesized by a family of enzymes called
nitric oxide synthases (NOS, EC 1.14.13.39),5 which catalyze
the oxidation of L-arginine to L-citrulline and nitric oxide. Three
isozymes of NOS have been identified so far, each associated
with a distinct physiological function: neuronal signal transmis-
sion (neuronal NOS or nNOS),6 smooth muscle relaxation
(endothelial NOS or eNOS),7 and immune response (inducible
NOS or iNOS).8 The first two isoforms (nNOS and eNOS) are
constitutively expressed and intermittently produce small amounts
of NO. In contrast, the third isoform (iNOS) is inducible by
cytokines and produces large amounts of NO for both a
cytoprotective and cytotoxic effect. All three isozymes are
encoded by separate genes and, therefore, differently regulated.
Their activity depends on a number of cofactors: NADPH,
FAD, FMN, tetrahydrobiopterin (H4B), and heme. The activity
of the constitutive enzymes is regulated by phosphorylation and
by the Ca2+-binding protein calmodulin,9 while iNOS does not
depend on the intracellular calcium levels because iNOS carries
a permanently bound molecule of calmodulin.10
The three NOS isoforms share a similar structural architec-
ture.11 The C-terminus is a reductase domain, homologous to
cytochrome P450 reductase. NADPH, FAD, and FMN are
bound in this domain. The N-terminus is the oxygenase domain,
which contains binding sites for the essential cofactors; tet-
rahydrobiopterin, heme, and the substrate, L-Arg. These two
domains are linked by a calmodulin-binding motif and form
one monomer. Two of the monomers dimerize to form a
homodimer enzyme with the aid of a single intersubunit ZnS4
cluster, which stabilizes dimerization and H4B binding site
formation.12 Electrons are transferred from NADPH via FAD
* To whom correspondence should be addressed at the Department of
Chemistry. Phone: 1-847-491-5653. Fax: 1-847-491-7713. E-mail: Agman@
chem.northwestern.edu.
† Northwestern University.
§ The University of Texas Health Science Center.
‡ University of California.
# Developed the eNOS overexpression system in E. coli. Isolated and
purified the eNOS.
| Developed the nNOS overexpression system in E. coli.
10.1021/jm061305c CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/11/2007

2090 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Seo et al.
Scheme 1a
a Reagents and conditions: (a) N-Boc-ethylenediamine, HBTU, HOBt, DIEA, DMF/CH2Cl2, 78%; (b) BH3-THF, THF, -10 °C, 56%; (c) benzoyl peroxide,
Na2HPO4, Et2O/THF, 70 °C, 68%; (d) 0.3% NaOH, MeOH, ∼quant.; (e) 50% TFA, CH2Cl2, ∼quant.
Scheme 2a
Table 1. Dipeptide Amide Reduction.
entry reagent equiva
time (h)
temp (°C) yield of product (%)
1
2
3
4
5
6
7
8
9
LiAlH4
LiAlH4
LiAlH4
LiAlH4
AlH3
AlH3
BH3
BH3
BH3
BH3
BH3
2
2
overnight
overnight
2
overnight
overnight
overnight
72
overnight
2
30
r.t.
70
r.t.
r.t.
r.t.
70
r.t.
70
70
b
c
a Reagents and conditions: (a) 10, CHCl3, 97%; (b) 50% TFA, CH2Cl2,
∼quant.
10
10
10
10
2
2
5
10
10
traceb
c
17b
c
in vitro and in vivo. One limitation of L-NNA as a therapeutic
agent is its poor isoform selectivity.17 This weak selectivity is
commonly an issue for substrate arginine analogues such as
L-NNA and L-NMA (Nω-methyl-L-arginine) due to the lack of
distinctive residues among the different isozymes in the
substrate-binding pocket.18 To achieve a highly isoform-selective
inhibition, an inhibitor should possess a functional group that
can reach into the substrate-access channel remote from the
substrate-binding pocket, and the functional group can contribute
to increase isoform selectivity as well as binding affinity. We
have designed Nω-nitro-L-arginine-containing dipeptide amide
inhibitors for the purpose of retaining the advantages of L-NNA
and using the second amino acid to identify a region outside of
the substrate-binding pocket that would differentiate nNOS from
iNOS and eNOS.19 A series of dipeptidomimetic analogues were
synthesized and tested for in vitro activity, and 1 and 2 (Figure
1) were found to be superior to L-NNA, particularly, in terms
of their remarkable isoform selectivity.20 In a continued effort
to investigate the active site of the enzyme, further structure-
activity relationship studies along with X-ray crystallographic
studies have been performed.21,22 To enhance inhibitor binding
further, we present here the first example of a nNOS inhibitor
designed with the intention of displacing the heme structural
water molecule in the active site.
b
b
c
10
11
r.t.
-10 to 0
10
10
56d
a Molar equivalents of reagent for relative to starting material. b Starting
material returned. c Starting material decomposed. d 83% based on consumed
starting material.
and FMN to the heme of the other domain. This flow of
electrons during catalysis occurs from the reductase domain of
one monomer subunit to the oxygenase domain of the other
monomer, so an intact homodimer form of the enzyme is
essential for full enzyme activity.13
Overproduction of NO from neuronal isozyme has been
associated with harmful effects in the central nervous system.
Radical nitric oxide (NO•) reacts rapidly with superoxide (O2•
in aqueous media to form the highly reactive peroxynitrite anion
(ONOO-). Either nitric oxide or peroxynitrite causes damage
to neuronal cells and tissues and, therefore, produces neurode-
generation.14 Recently, Uehara et al. found that endogenous NO
caused S-nitrosylation on the cysteine residue of protein-
disulfide isomerase (PDI) and inhibited its activity as a
chaperone, indicating that nNOS was involved in the accumula-
tion of unfolded/misfolded proteins in the brain.15 Because
eNOS is important in maintaining normal blood pressure, nNOS
inhibitors must not inhibit eNOS. Thus, selective inhibition of
the neuronal isoform is essential if nNOS inhibitors are used
for therapeutic purposes.
-
)
Chemistry
Synthesis of Hydroxylamine 3. The synthesis of hydroxy-
lamine derivative 3 is depicted in Scheme 1. Secondary amine
7 was prepared via a peptide coupling reaction of Boc-L-Arg-
(NO2)-OH with N-Boc-ethylenediamine23 followed by amide
reduction of the resulting secondary amide (6). A widely
exploited protocol for the preparation of secondary amines is
Nitric oxide synthase inhibitors have been reviewed com-
prehensively.16 Among those inhibitors, Nω-nitro-L-arginine (L-
NNA) has been extensively studied for pharmacological effects

Inhibitors of Neuronal Nitric Oxide Synthase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2091
Scheme 3a
a Reagents and conditions: (a) DIAD, PPh3, THF, 85%; (b) MeOH, NaN3, 40 °C, 96%; (c) Boc-NH-O-Boc, DEAD, PPh3, THF, 45%; (d) 50% TFA,
CH2Cl2, 98%; (e) Fmoc-L-Arg(NO2)-Cl, Collidine, THF, 74%; (f) (i) 20% piperidine, DMF; (ii) Boc2O, NaHCO3, dioxane, 83% for two steps; (g) 5% LiOH,
i
THF, 94%; (h) butyl chloroformate, NMM, NH4OH, THF, 65%; (i) 50% TFA, CH2Cl2, ∼quant.
Table 2. Heme Structural Water Molecules Found in Rat Brain nNOS
PDB i.d.a
molecule
heme structural waterb
1zvl
1lzx
1om4
1k2r
1vag
1k2t
1qwc
1lzz
1mmw
1mmv
L-Arg
HOH 183
HOH 66
HOH 81
HOH 82
NAc
NG-hydroxy-L-Arg
L-Arg
L-NNA
AR-R1747734
S-ethyl-N-phenylisothiourea
HOH 318
1400W35
NAc
N-isopropyl-N′-hydroxyguanidine
vinyl-L-NIO
HOH 83
HOH 53
HOH 256
NG-propyl-L-Arg
a Protein Data Bank: http://www.rcsb.org/pdb. b Residue number in pdb
file. c Structural water molecule was displaced by the inhibitor.
using borane. The low-temperature conditions suppressed the
side reactions but were adequately effective for secondary amide
reduction. At low-temperature we isolated 7 in a 56% yield (83%
yield based on the consumed starting material).
Because the oxidation of secondary amines can give N-oxides,
which rearrange to the corresponding hydroxylamines, mCPBA
was first employed for the direct preparation of hydroxylamine
9 from amine 7.28 However, we found hydroxylamine 9 was
very susceptible to further oxidation with mCPBA.29 As
evidenced by many side product spots on the TLC plate and M
- 1, M, and M + 1 peaks in the mass spectrum, we concluded
that the mCPBA oxidation reaction was complicated by over-
oxidation. Varying the mCPBA ratio and the reaction temper-
ature did not help circumvent the problem. Therefore, we
changed the oxidant to a more controllable reagent, namely,
benzoyl peroxide. To trap the byproduct benzoic acid and
suppress the N-acylation side reaction, an auxiliary base (Na2-
HPO4) was added to the reaction mixture,30 and the oxidation
reaction proceeded smoothly to provide 8. A mild sodium
methoxide solution cleaved the O-benzoyl group successfully,
and deprotection of the Boc groups gave 3.
Synthesis of Hydrazine 4. The synthesis of 4 started from
intermediate 7 (Scheme 2). An electrophilic N-Boc transfer
reaction31 using the commercially available reagent N-Boc-3-
(4-cyanophenyl)oxaziridine (10) provided 11 in an excellent
yield. Subsequent deprotection of the Boc groups provided 4.
Synthesis of Hydroxamate 5. Hydroxamate derivative 5 was
synthesized by the synthetic route shown in Scheme 3. The
synthesis of an intermediate, hydroxylamine 15, started from
commercially available Fmoc protected trans-4-hydroxy-L-
proline. An intramolecular Mitsunobu reaction gave 12, and
sodium azide-catalyzed methanolysis provided 13 in an 82%
Figure 2. X-ray crystal structure of the active site heme and inhibitor
(top: 1, bottom: 2). PDB id of top and bottom structure is 1p6i and
1p6j, respectively. Numbers are in angstroms.
reductive amination.24 This method was applied both to the
solution- and solid-phase syntheses; however, there is a risk of
racemization because the proton on the R-carbon becomes acidic
during the intermediate iminium ion formation. Alternatively,
amides can be converted to thioamides using Lawesson’s reagent
and then subsequent reductive desulfurization provides the
corresponding reduced amide.25 A third way to synthesize
secondary amines is by a direct reduction of an amide to an
amine. This approach has been employed less frequently,
probably because of the lack of selectivity that the hydride
reagents have for various functionalities.26 For the synthesis of
7, we tried the direct reduction method using various conditions
(Table 1). LAH, alane, and borane were used as hydride-donor
reducing agents. Entries 1-10 show that all three reagents have
poor selectivity for the reduction of the amide relative to other
functionalities (carbamate and N-nitro) in 6. The milder reagents
showed slightly better selectivity (LiAlH4 < BH3 < AlH3).
Attempts to obtain a higher yield by raising the reaction
temperature resulted in side reactions; usually, the hydride
reacted with the carbamates. However, maximum selectivity was
achieved when a low reaction temperature (-10 to 0 °C) was
used (entry 11). This method was originally reported by Roeske
et al.27 for the selective reduction of an amide from an ester

2092 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Seo et al.
Figure 3. Superimposition of the docked conformations of 5 (purple) and X-ray crystal structure of 2 bound to nNOS (colored by element with
carbons in gray, nitrogens in blue, and oxygens in red; pdb id: 1p6j). AutoDock calculations of 5 bound to nNOS active site were performed (A)
with or (B) without the structural water molecule.
anhydrous conditions described above were used. Also, because
of its short half-life, it was best to use 15 directly after Boc-
deprotection. Prior to basic hydrolysis of the methyl ester, the
Fmoc protecting groups were switched to Boc groups (17). The
methyl ester was then converted to amide 18 by hydrolysis and
amidation reactions. Subsequent deprotection of the Boc groups
provided 5.
Results and Discussion
Inhibitor Design. X-ray crystal structures of the complexes
of nNOS with inhibitors 1 and 2 bound reveal the presence of
a structural water molecule that is hydrogen bonded to the two
heme propionates of the enzyme and to the inhibitor NH moiety
(Figure 2). An analogous water molecule is frequently found
in the active site of nNOS complexed with the substrate L-Arg,
the catalytic intermediate Nω-hydroxy-L-Arg, or with various
inhibitors bound (Table 2).
Generally, distances from the water molecule to the heme
propionates are between 2.5 and 3.5 Å, and the binding angles
are about 110°. This angle is larger than the H-O-H angle
(104.5°) but is close to tetrahedral geometry (109°). As is the
case of ice, the H-O-H angle of water in the ordered state is
close to tetrahedral; the angle increases about 5° from gaseous
water to solid water.36 Therefore, the tetrahedral binding angle
of the structural water molecule supports the fact that the water
molecule is in a highly ordered state, not in bulk aqueous
medium. A structural water molecule seems to be highly
conserved in heme-containing enzymes when the heme propi-
onates are open to the access of bulk solvent, as is the case
with nitric oxide synthases.
Structural water molecules in enzyme active sites are of
interest to medicinal chemists because they modify the active
site geometry and contribute to the binding affinity between a
ligand and a protein. In addition, the presence of structural water
molecules in enzyme-inhibitor binding can provide a possible
new direction for the design of more potent inhibitors, which
was successfully demonstrated by Lam et al. in their develop-
ment of a HIV protease inhibitor.37
Our nNOS inhibitor design is based on the following
considerations: (1) by extending a hydrogen bond donor group
from the inhibitor NH moiety, displacement of the structural
water molecule may occur, which would be entropically
favorable;38 (2) direct interaction of the inhibitor and the heme
cofactor may occur, which may enhance the binding affinity of
the inhibitors. On the basis of these considerations, peptidomi-
metic modifications of compounds 1 and 2 were made. We
chose R ) OH and NH2 as the hydrogen bond donor modifica-
tion groups on 1 and 2 (3-5, Figure 1). These moieties are
intended to mimic the hydrogen-bonding feature of the structural
water molecule and/or provide direct binding of the inhibitor
to the heme cofactor.
Figure 4. The omit Fo - Fc electron density maps contoured at 3.0σ
for (A) 3 bound in the nNOS active site, (B) 5 in nNOS, and (C) 3 in
eNOS. The simulated annealing protocol in CNS with starting tem-
perature at 1000 K was used for the calculation.
yield for two steps.32 The N,O-diBoc-protected hydroxylamine
was then incorporated into 13 using Mitsunobu conditions to
give 14; subsequent deprotection provided hydroxylamine 15.
The hydroxylamine was coupled to Fmoc-Arg(NO2)-Cl to
provide hydroxamate 16 in a 74% yield.33 Successful hydrox-
amate coupling was achieved when the low temperature and

Inhibitors of Neuronal Nitric Oxide Synthase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2093
Figure 5. X-ray crystal structures of the nNOS active site with inhibitors 1 (A) and 3 (B) bound (units are Å). (A)-1: Side view of 1; (A)-2: Top
view of 1; (B)-1: Side view of 3; (B)-2: Top view of 3.
Molecular Modeling. Initially, we carried out a computer
modeling study to determine if the displacement of the structural
water molecule is feasible. AutoDock 3.0.539 was used to predict
the binding mode of designed compound 5 in the nNOS active
site. For the docking simulation, the active site binding pocket
was defined using the crystal structure of 1 bound to nNOS
(pdb id: 1p6i) as described by Ji et al.18 Two different nNOS
active sites were employed for the modeling, one with and one
without the heme structural water molecule. Binding conforma-
tions of 5 obtained by AutoDock calculations are depicted in
Figure 3.
When 5 was docked into the nNOS active site with the
structural water molecule, the N-OH moiety of 5 was placed
away from the position of the water molecule; therefore, the
proline side chain was deviated from the conformation of 2
(Figure 3A). In the case of the nNOS active site without the
structural water molecule, the N-OH moiety was directed toward
the position of the water molecule, and the docked conformation
of 5 and the crystal structure of 2 showed similar conformations
(Figure 3B). Root-mean-square deviation (rmsd) values of the
docked conformation of 5 from the crystal structure of 2 were
(A) 0.37 Å and (B) 0.15 Å. These results led us to conclude
that in the minimal binding energy mode between 5 and the
nNOS active site, the incorporated N-OH moiety tends to be
placed at the position of the structural water molecule. Therefore,
displacement of the structural water should be feasible.
X-Ray Crystallographic Analysis and Biological Assay.
X-ray crystal structures of 3 and 5 complexed with nNOS and
3 complexed with eNOS were obtained to 2.00, 2.15, and 1.95
Å resolution, respectively (Figures 4-7). Clearly, the structural
water molecule (Water 1 in Figures 5-7) was displaced by the
water molecule mimic, the N-OH moiety, and the N-hydroxyl
group provided a direct hydrogen bonding interaction with the
heme propionate moiety. Therefore, the computer modeling was
successful in predicting the desired effect of incorporation of
an N-hydroxyl group into the inhibitors. Figure 7 depicts
overlapped X-ray crystal structures of the nNOS active sites
bound with the inhibitors: (A) 1 and 3 in nNOS, (B) 2 and 5
in nNOS, and (C) 1 and 3 in eNOS. For nNOS, no striking
change in the inhibitor/active site residue interactions is
observed, and the overall binding conformations of 3 and 5 are
quite similar to parent compounds 1 and 2, respectively.
Compounds 1-5 and L-NNA were tested in vitro against the
three isoforms of NOS using the hemoglobin capture assay (see
Experimental Section): nitric oxide produced by NOS from
L-arginine transforms oxyhemoglobin (Fe2+) to methemoglobin
(Fe3+), which is monitored at the maximum absorbance (401
nm); the assay results are shown in Table 3. Much to our surprise
the N-hydroxylated inhibitors 3 and 5 were only marginally
more potent than the parent compounds 1 and 2. The N-aminated
compound (4) showed more than a 3-fold decrease in potency
against nNOS. The activity difference between 3 and 4 can be
explained by the fact that the N-hydroxyl group is a strong
proton donor providing short contacts with carbonyl oxygens
whereas the N-amino group is a much weaker proton donor.40
In general, N-hydroxyl modifications (3 and 5) showed little
effect on iNOS binding but tighter binding against eNOS (more
than 2-fold), thereby decreasing the nNOS/eNOS selectivity.
Similarly, the N-amino compound (4) exhibited increased
binding affinity against eNOS but decreased the affinity against
iNOS.
Why should compounds that attain direct interactions with
the enzyme not produce much more potent binding interactions?
The explanation can be found in other changes in the crystal

2094 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Seo et al.
Figure 6. X-ray crystal structures of the nNOS active site with inhibitors 2 (A) and 5 (B) bound (units in Å). (A)-1: Side view of 2; (A)-2: Top
view of 2; (B)-1: Side view of 5; (B)-2: Top view of 5.
structures upon binding of 3 and 5. For compound 3 the new
interactions between the N-hydroxyl group and the heme
propionate causes the inhibitor molecules to move slightly
farther from Glu592, weakening somewhat the key hydrogen
bonding interaction between the R-amino group of the inhibitors
and Glu592.18,22 The distances from the R-amino group to
Glu592 are 2.7 Å for 1 and 2.9 Å for 3 ((A)-1 and (B)-1 in
Figure 5). The N-OH and heme interaction causes the R-amino
groups of 3 to move away from Glu592 by 0.2 Å. As a result,
the hydrogen-bonding distance from the R-amino group of 3 to
another structural water (water 3 in between Tyr588 and
Asp597) has also increased (comparison between (A)-1 and
(B)-1 of Figure 5). Although there is no direct experimental
relationship between hydrogen-bond lengths and hydrogen-bond
strengths,41 the bond length difference of approximately 0.25
Å can be significant enough to switch a moderate hydrogen
bond (2.65-2.75 Å) to a weak hydrogen bond (2.75-3.00 Å).42
In addition, the decreased basicity of the hydroxylamino nitrogen
in 3 compared to the basicity of the secondary amino group in
1 possibly resulted in a weaker interaction of the nitrogen with
the heme propionate.
Unlike 3, compound 5 contains a bulkier and conformation-
ally restricted proline amide side chain. The direct hydrogen
bond from N-OH to the heme propionate (pyrrole A) pulls the
proline amide tail of 5 closer to the heme compared to 2, which
results in a shorter hydrogen bond distance from the inhibitor
amide nitrogen to the second heme propionate (pyrrole D). This
causes a shift of the proline secondary amine away from another
structural water molecule (Water 2) located between the heme
propionate (pyrrole A) and the carbonyl oxygen (O-4) of tetra-
hydrobiopterin ((A)-2 and (B)-2 in Figure 6). When compounds
2 and 5 are compared, their binding interactions involving the
nitroguanidino and R-amino groups are similar, and differences
are confined to the proline amide tail. However, these do not
result in any significant differences in inhibitor-protein interac-
tions that might result in tighter binding. This may explain why
the binding affinity of 5 is almost identical to that of 2.
The differences between 3 and 5 deserve further discussion.
The hydroxamate linkage of 5 results in restricted rotation while
the ethylamino tail in 3 is small and more flexible. The restricted
and bulky proline amide tail in 5 makes more contacts with the
heme and the surrounding protein than does 3. These extensive
inhibitor-enzyme interactions prevent 5 from undergoing major
conformational changes relative to the parent compound 2. In
contrast, 3 is more flexible and thus is able to adapt to the
addition of the N-OH group, which is even more obvious in
the eNOS structures.
In a similar behavior to 1,22 compound 3 binds to wild type
eNOS in a different conformation from its binding mode seen
in wild type nNOS. Figure 7C indicates that the R-amino group
of 3 gains a direct interaction with the heme propionates. This
is the likely structural basis for the increased affinity to eNOS
in comparison with 1 (Table 3). The common feature for 1 and
3 bound in eNOS is that their R-amino groups stay away from
the Glu363 side chain. In contrast, 1 and 3 bound in nNOS
have the R-amino group directly hydrogen bonded to Glu592
(Figure 5). As discussed previously22 this isoform-specific bind-
ing mode of 1 and 3 originates from one amino acid substitution,
Asn368 in eNOS and Asp597 in nNOS, in the carboxylate
binding pocket of the substrate binding site. It is interesting to
see that the addition of the N-OH group in 3 allows the R-amino
group to interact directly with the heme propionate, which was
not the case for 1. This makes 3 a better inhibitor for eNOS but
a less isoform-selective inhibitor compared to 1 (Table 3).

Inhibitors of Neuronal Nitric Oxide Synthase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2095
was not observed, unlike that observed by Lam et al. for cyclic
urea inhibitors of HIV protease.37 In this case the inhibitors had
increased potency for multiple reasons: (1) displacement of a
structural water molecule; (2) conversion of a flexible and linear
inhibitor into a rigid cyclic urea structure with restricted
conformation; (3) a nearly perfect geometry obtained after
structural modifications. For compound 3, where the overall
hydrogen binding conformation is relatively conserved, a small
increase in binding affinity was obtained in nNOS. A larger
gain in binding affinity is obtained in eNOS as a result of direct
interaction between the R-amino group of 3 and the heme
propionate. However, in 5 the energy gain from the structural
water displacement did not outweigh the loss of critical
hydrogen-bonding energy. This indicates that displacement of
a structural water molecule itself is not sufficient for a dramatic
increase in binding affinity; increased binding requires a highly
conserved conformation without disruption of pre-existing
hydrogen bonds. Careful structural design of inhibitors is
necessary beyond the goal of displacement of a structural water
molecule.43 Another reason that there is not much of a change
is related to the effects of desolvation. Interchange of an
inhibitor-solvent hydrogen bond in the free inhibitor for an
inhibitor-protein hydrogen bond may not lead to much gain in
terms of binding energy unless the inhibitor-protein interaction
is ideal in its properties, such as geometry and distance.
Experimental Section
General Methods. Fisher silica gel 60 (230-400 mesh) was
used for flash column chromatography with >10% methanol eluent
system; otherwise, Sorbent Technologies silica gel 60 (200-400
mesh) was used. Thin-layer chromatography was carried out on E.
Merck precoated silica gel 60 F254 plates. Compounds were
visualized with a ninhydrin spray reagent or a UV/vis lamp.
Combustion analyses were performed by Atlantic Microlab, Inc.,
Norcross, GA. High-resolution mass spectroscopy analyses were
carried out at the University of Illinois, Urbana-Champaign using
a Micromass Quattro spectrometer. Electrospray mass spectra were
obtained on a Micromass Quattro II spectrometer. 1H NMR spectra
were recorded on a Varian Mercury 400 or Varian Inova 500 NMR
spectrometer. Chemical shifts were reported as δ values in parts
per million downfield from TMS (δ 0.0) as the internal standard
in CDCl3. Melting points were measured on a Buchi melting point
B-540 apparatus and are uncorrected. An Orion Research model
701 pH meter with a general combination electrode was used for
pH measurements. NOS assays were recorded on a Perkin-Elmer
Lambda 10 UV/vis spectrophotometer.
Compounds 1-5 (50 µL injection of a solution of 50 mM
sample) were purified by HPLC using a semipreparative column
[Phenomenex, Luna, 250 × 10 mm, C18(2)] with a precolumn
[Phenomenex, Luna 50 × 10 mm, C18(2)] at a flow rate of 4 mL/
min. Sample elution was detected by absorbance at 254 nm. The
mobile phase was as follows: (A, water + 0.1% TFA; B, CH3CN
+ 0.1% TFA) 10 min using 1% B, then a gradient to 10% B over
10 min, and then a gradient to 1% B over 5 min. The HPLC was
performed on a Beckman System Gold chromatograph (Model 125P
solvent module and Model 166 detector). Fractions containing the
pure product were concentrated in Vacuo, and the residue was
lyophilized.
Figure 7. Overlapped X-ray crystal structures of the NOS active site
complexed with inhibitors: (A) nNOS (1, gray; 3, orange); (B) nNOS
(2, gray; 5, orange); (C) eNOS (1, gray; 3, orange).
Table 3. NOS Inhibition by the N-OH and N-NH2 Derivatives and
Their Parent Compoundsa
Ki (µM)
selectivityd
compound
nNOSb
iNOSc eNOSc
4.28 0.72
18 191
n/i
n/e
1.2
1124
218
608
184
113
l-NNA
0.61 ( 0.005
0.17 ( 0.011
0.33 ( 0.019
0.12 ( 0.017
0.56 ( 0.024
0.31 ( 0.014
7
106
52
133
66
1
2
3
4
5
17
16
37
16
72
73
103
35
52
a The enzyme used for the Ki determinations are bovine brain nNOS,
recombinant murine iNOS, and recombinant bovine eNOS. See Experi-
mental Section. b Ki ( SEM. Results are given as a mean of more than or
equal to two independent experiments. c The Ki values represent single
measurements with five data points and correlation coefficients (r2) 0.959-
0.996. d The ratio of Ki (iNOS or eNOS) to Ki (nNOS).
Reagents and Materials. All reagents were purchased from
Aldrich, Advanced ChemTech, and Nova Biochem. They were used
without further purification unless stated otherwise. NADPH, cal-
modulin, and human ferrous hemoglobin were obtained from Sigma-
Aldrich Co. Tetrahydrobiopterin (H4B) was purchased from Alexis
Biochemicals. HEPES, DTT, and conventional organic solvents
were purchased from Fisher Scientific. Tetrahydrofuran was distilled
under nitrogen from sodium/benzophenone. Diethyl azodicarboxy-
late (DEAD) was purchased from Lancaster Synthesis, Inc., Pelham,
NH. N-Boc-3-(4-Cyanophenyl)oxaziridine was purchased from
Acros Organics, NJ (Product number: AC29709-5000).
Conclusions
Compound 3 showed the best potency and selectivity among
the N-OH and N-NH2 derivatives. However, a dramatic en-
hancement in binding affinity as a result of displacement of a
structural water molecule and direct interaction with the enzyme

2096 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Seo et al.
NR-(tert-Butoxycarbonyl)-L-nitroarginine 2-(N-tert-butoxycar-
bonylaminoethyl)amide (6). To a solution of Boc-L-Arg(NO2)-
OH (0.75 g, 2.34 mmol), HBTU (0.89 g, 2.34 mmol), and HOBt
(0.32 g, 2.34 mmol) in dry DMF (5 mL) and dry CH2Cl2 (5 mL)
was added N-Boc-ethylenediamine (0.25 g, 1.56 mmol) in dry DMF
(5 mL) dropwise at 0 °C. DIEA (0.30 mg, 2.34 mmol) in dry CH2-
Cl2 (5 mL) was added slowly, and stirring was continued for 2 h
at room temperature under argon. The solvent was removed under
reduced pressure, and the residue was treated with EtOAc (15 mL).
The organic mixture was washed successively with 5% NaHCO3,
water, 0.5 N HCl, and brine and dried over Na2SO4. The solution
was concentrated in vacuo, and the residue was purified by flash
column chromatography (EtOAc/MeOH ) 19:1, Rf ) 0.25) to
afford 6 (0.56 g, 78%) as a white solid; mp 98.6-99.6 °C. 1H NMR
(500 MHz, acetone-d6) δ 7.67 (s, 1H), 6.26 (s, 1H), 6.14 (s, 1H),
4.17 (s, 1H), 3.41-3.35 (m, 4H), 3.22 (s, 2H), 1.90 (s, 1H), 1.75
(br s, 3H), 1.42 (s, 18H). HRMS (ES) (m/z): M + H+ calcd for
C18H36N7O7 462.2676, found 462.2660.
(4S)-4-N-tert-Butoxycarbonylamino-5-[2-(N-tert-butoxycarbo-
nylaminoethyl)aminopentyl]-N′-nitroguanidine (7). Compound
6 (0.84 g, 1.8 mmol) was treated dropwise with a solution of 1.0
M BH3 (18 mL) in THF at -10 °C under argon. The reaction
temperature was allowed to rise to 0 °C. After being stirred at 0
°C for 10 h, the residual BH3 was quenched by cautious addition
of MeOH (30 mL) at 0 °C, and the mixture was stirred overnight
at room temperature. The solution was evaporated under reduced
pressure and treated three times with MeOH (50 mL), evaporating
to dryness after each addition, to remove boric acid as trimethyl
borate. The residue was purified by flash column chromatography
(CH2Cl2/MeOH/Et3N ) 12:1:0.1, Rf ) 0.3) to afford 7 (0.46 g,
56%) as a transparent oil. 1H NMR (500 MHz, CD3OD) δ 3.69 (s,
1H), 3.37-3.21 (m, 4H), 2.74-2.66 (m, 4H), 1.67 (br s, 2H), 1.61-
1.58 (m, 2H), 1.46 (s, 9H), 1.45 (s, 9H); 13C NMR (125 MHz,
CD3OD) δ 159.8, 157.5, 157.4, 79.1, 52.8, 49.5, 48.8, 40.9, 39.3,
30.4, 29.0, 27.6; HRMS (ES) (m/z): M + H+ calcd for C18H38N7O6
448.2884, found 448.2888.
(4S)-4-N-tert-Butoxycarbonylamino-5-{[2-(N-tert-butoxycar-
bonylaminoethyl)benzyoloxyamino]-pentyl}-N′-nitroguani-
dine (8). An oven-dried round-bottom flask equipped with a
magnetic stir bar and reflux condenser was charged with disodium
hydrogen phosphate (175 mg, 1.23 mmol), 7 (110 mg, 0.25 mmol),
and dry ether/THF (3 mL/3 mL). To this stirred suspension was
added dibenzoyl peroxide (121 mg, 0.50 mmol) in dry ether/THF
(2 mL/2 mL), and the reaction mixture was stirred at 70 °C for 24
h under argon atmosphere. The solvent was removed under reduced
pressure, and the residue was partitioned between EtOAc (5 mL)
and water (5 mL). The organic layer was washed twice with water
and dried over Na2SO4. The solution was concentrated in vacuo,
and the residue was purified by flash column chromatography
(hexane/EtOAc ) 1:4, Rf ) 0.4) to afford 8 (96 mg, 68%) as an
oil. 1H NMR (500 MHz, CDCl3) δ 7.98 (d, J ) 7.5 Hz, 2H), 7.60
(t, J ) 7.5 Hz, 1H), 7.46 (t, J ) 8.0 Hz, 2H), 5.56 (s, 1H), 5.19 (br
s, 1H), 3.77 (br s, 1H), 3.41 (br s, 1H), 3.21 (br s, 3H), 3.07 (s,
4H), 1.66 (br s, 4H), 1.43 (s, 9H), 1.37 (s, 9H); 13C NMR (125
MHz, CDCl3) δ 166.4, 159.6, 156.4, 134.0, 129.8, 128.9, 128.4,
80.3, 63.4, 60.0, 40.8, 37.8, 31.2, 29.7, 28.7, 28.6, 25.0; HRMS
(ES) (m/z): M + H+ calcd for C25H42N7O8 568.3095, found
568.3098.
1.45 (s, 18H); 13C NMR (125 MHz, CDCl3) δ 159.6, 157.8, 80.2,
63.9, 60.7, 48.3, 41.2, 38.1, 30.9, 29.0, 28.7, 25.3; HRMS (ES)
(m/z): M + H+ calcd for C18H38N7O7 464.2833, found 464.2821.
(4S)-N-{4-Amino-5-[(2-aminoethyl)-hydroxyamino]-pentyl}-
N′-nitroguanidine (3). Compound 9 (30 mg, 0.065 mmol) was
treated with trifluoroacetic acid/CH2Cl2 (5 mL/5 mL) at 0 °C under
nitrogen. The reaction temperature was then allowed to rise to room
temperature, and stirring continued for 45 min. Excess TFA and
solvent were removed by evaporation in vacuo. The residue was
repeatedly dissolved in 10 mL of CH2Cl2, and the solvents were
evaporated to remove traces of TFA. The residue was dissolved in
a small amount of water, and the solution was washed with CH2-
Cl2 and lyophilized to give a yellowish hygroscopic foam (26 mg,
96%). 1H NMR (500 MHz, D2O) δ 3.38 (s, 1H), 3.16 (s, 2H), 3.01-
3.12 (m, 2H), 2.80-2.90 (m, 3H), 2.69 (dd, J ) 10.0, 13.5, 1H),
1.58 (s, 4H); [R]24 +8.4 (c ) 0.50, MeOH); Anal. Calcd for
D
C8H21N7O3‚3.5HCl: C, H, N.
(4S)-4-N-tert-Butoxycarbonylamino-5-{[2-(N-tert-butoxycar-
bonylaminoethyl)-(N-tert-butoxycarbonylhydrazino)]pentyl}-N′-
nitroguanidine (11). A solution of 7 (16 mg, 0.036 mmol) in dry
CHCl3 (2 mL) was treated at 0 °C with a solution of N-Boc-3-(4-
cyanophenyl)oxaziridine (10, 13 mg, 0.054 mmol) in dry CHCl3
(2 mL). At the end of the addition, the cooling bath was removed
and stirring continued at room temperature overnight. After the
starting material was consumed, the mixture was concentrated in
vacuo, and the residue was purified by flash column chromatog-
raphy (hexane/EtOAc ) 1:4, Rf ) 0.35) to afford 11 (20 mg, 97%)
1
as a syrup. H NMR (500 MHz, CDCl3) δ 5.85 (s, 1H), 5.44 (s,
2H), 3.69 (s, 1H), 3.52 (s, 1H), 3.24 (s, 3H), 2.84 (s, 2H), 2.78 (s,
2H), 1.85 (s, 1H), 1.61-1.72 (m, 3H), 1.46 (s, 9H), 1.45 (s, 18H);
13C NMR (125 MHz, CDCl3) δ 159.6, 156.5, 80.1, 79.5, 62.9, 58.8,
47.8, 40.8, 38.0, 31.1, 28.7, 28.5, 25.1; HRMS (ES) (m/z): M +
H+ calcd for C23H47N8O8 563.3517, found 563.3518.
(4S)-N-{4-Amino-5-[(2-aminoethyl)-hydrazino]-pentyl}-N′-ni-
troguanidine (4). Using the procedure described for the preparation
of 3, compound 4 was obtained from 11 as a yellowish foam (32
1
mg, 97%); mp 59.8-61.2 °C. H NMR (500 MHz, D2O) δ 3.42
(s, 1H), 3.11-3.19 (m, 7H), 2.95 (dd, J ) 13.5, 11.0, 1H), 1.57 (s,
4H); Anal. Calcd for C8H22N8O2‚4.5HCl: C, H, N.
Compounds 12 to 18 were characterized for structural purity by
1H and 13C NMR spectrometry. The NMR spectra of those
compounds showed the characteristic presence of rotamer mixtures
originating from the restricted rotation about the tertiary amide bond
of the proline ring.44 A high-temperature 1H NMR experiment was
performed on 13 to see if the rotational energy barrier could be
overcome at a higher temperature. At ambient temperature, two
distinct peaks from the methyl ester were observed at δ 3.61 and
δ 3.55 having a ratio of 1.9 to 1.1, respectively. At 70 °C, the two
peaks merged to a singlet at δ 3.64 (500 MHz, DMSO).
N-Fmoc-4-Hydroxy-L-proline Lactone (12). To a stirred ice-
cold solution of Fmoc-Hyp-OH (3.0 g, 8.5 mmol) and triph-
enylphosphine (2.7 g, 10.2 mmol) in freshly distilled THF (200
mL) under nitrogen was added DIAD (2.1 g, 10.2 mmol) dropwise.
The reaction temperature was then allowed to rise to room
temperature and stirring continued for 2 h. The solvent was
evaporated under reduced pressure, and the residue was directly
purified by flash column chromatography (EtOAc/hexane ) 1:1,
Rf ) 0.35), to give 2.4 g (85%) of 12 as a white foamy solid; mp
(4S)-4-N-tert-Butoxycarbonylamino-5-{[2-(N-tert-butoxycar-
bonylaminoethyl)hydoxyamino]-pentyl}-N′-nitroguanidine (9).
Compound 8 (60 mg, 0.11 mmol) was treated with a solution of
0.3% NaOH in MeOH (3 mL), and the mixture was stirred at room
temperature for 4 h. After evaporation of the MeOH in vacuo, the
residue was partitioned between water (10 mL) and EtOAc (10
mL), and the aqueous phase was extracted with EtOAc (10 mL).
The combined organic layer was washed with brine (20 mL), dried
over Na2SO4, and evaporated. The residue was purified by flash
column chromatography (hexane/EtOAc ) 1:4, Rf ) 0.3) to afford
1
106.4-106.8 °C. H NMR (400 MHz, CDCl3) δ 7.78 (d, J ) 7
Hz, 2H), 7.64 (m, 2H), 7.42 (t, J ) 7.5 Hz, 2H), 7.34 (t, J ) 7.0
Hz, 2H), 5.14 (s, 1H), 4.67 (s, 1H), 4.39-4.50 (m, 2H), 4.27 (s,
1H), 3.43-3.58 (m, 2H), 2.27 (s, 1H), 2.06 (s, 1H); 13C NMR (125
MHz, CDCl3) δ 171.8, 154.5, 143.9, 141,7, 128.1, 127.4, 124.9,
120.3, 74.8, 67.3, 60.7, 47.4, 39.5, 35.3.
N-Fmoc-cis-4-Hydroxy-L-proline Methyl Ester (13). A solution
of 12 (2.0 g, 6.0 mmol) and sodium azide (1.1 g, 18.0 mmol) in
dry MeOH (50 mL) was stirred for 3 h at 40 °C under nitrogen.
The solvent was evaporated under reduced pressure, and the residue
was partitioned between water and EtOAc. The organic layer was
1
9 (50 mg, 98%) as an oil. H NMR (500 MHz, CDCl3) δ 7.11 (s,
1H), 5.38 (s, 1H), 4.99 (s, 1H), 3.86 (s, 1H), 3.43-3.45 (m, 2H),
3.25 (s, 2H), 2.65-2.77 (m, 4H), 1.71 (s, 2H), 1.58-1.64 (m, 2H),

Inhibitors of Neuronal Nitric Oxide Synthase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2097
washed with brine, dried over Na2SO4, and concentrated. The
compound was purified by flash column chromatography (EtOAc/
hexane ) 2:1, Rf ) 0.3), to give 13 (2.1 g, 96%) as a white solid;
mp 48.5-48.8 °C. 1H NMR (400 MHz, DMSO) δ 7.87 (t, J ) 8.0
Hz, 2H), 7.58-7.65 (m, 2H), 7.40 (d, J ) 6.8 Hz, 2H), 7.32 (d, J
) 6.0 Hz, 2H), 4.16-4.28 (m, 5H), 3.49-3.59 (m, 4H), 3.20 (m,
1H), 2.29 (s, 1H), 1.90 (m, 1H); 13C NMR (125 MHz, CD3OD) δ
172.8, 155.4, 143.9, 141.3, 127.6, 126.9, 124.7, 119.8, 69.6, 67.5,
58.0, 54.5, 51.8, 38.8, 37.9; MS (ESI, CH2Cl2) [M+H+] ) 368.2.
N-Fmoc-trans-4-(N,O-di-tert-Butoxycarbonyl-hydroxyamino)-
L-proline Methyl Ester (14). To a stirred ice-cold solution of the
13 (500 mg, 1.36 mmol), triphenylphosphine (393 mg, 1.50 mmol),
and Boc-HN-O-Boc (350 g, 1.50 mmol) in anhydrous THF (20
mL) under nitrogen was added diethyl azodicarboxylate (261 mg,
1.50 mmol) dropwise over 15 min. The reaction temperature was
allowed to rise to room temperature, and stirring continued for 30
min. The solvent was evaporated in vacuo, and the residue was
directly purified by flash column chromatography (EtOAc/hexane
) 1:3, Rf ) 0.35), to give 14 (332 mg, 42%) as a white foamy
d6) δ 172.73, 170.40, 160.19, 156.60, 154.65, 144.41, 144.24,
141.45, 127.95, 127.43, 127.36, 125.54, 125.32, 120.22, 67.58,
66.71, 59.98, 58.51, 57.94, 53.43, 52.10, 51.87, 49.93, 47.32, 47.22,
40.89, 40.72, 33.88, 32.94, 20.28; HRMS (ES) (m/z): M + H+
calcd for C42H44N7O10 806.3150, found 806.3156.
N-Boc-4-N-(NR-Boc-Nω-Nitro-L-argininyl)-trans-4-hydroxyami-
no-L-proline Methyl Ester (17). Piperidine (1 mL) was added to
a solution of 16 (63 mg, 0.078 mmol) in DMF (4 mL), and the
mixture was stirred at room temperature for 0.5 h (monitoring by
TLC). The volatile components were removed under reduced
pressure. To remove excess piperidine completely, the residue was
repeatedly dissolved in CH2Cl2, and the solvents were evaporated
several times. The residue was then dissolved in 1,4-dioxane (4
mL) and 10% aq NaHCO3 (3 mL), and the solution of Boc2O (51
mg, 0.23 mmol) in 1,4-dioxane (1 mL) was added dropwise at 0
°C. After being stirred at room temperature overnight, the reaction
mixture was partitioned between water (10 mL) and EtOAc (10
mL), and the aqueous layer was extracted with EtOAc (10 mL).
The combined organic layers were washed with brine (10 mL),
dried over Na2SO4, and evaporated. The residue was purified by
flash column chromatography (EtOAc/MeOH ) 19:1, Rf ) 0.3)
to afford 17 (36 mg, 83%) as a colorless foam. 1H NMR (500 MHz,
CDCl3) δ 5.18 (s, 1H), 4.78 (s, 1H), 4.40-4.49 (m, 1H), 3.72 (s,
3H), 3.67 (m, 1H), 3.55-3.61 (m, 2H), 3.30 (s, 1H), 2.54 (s, 1H),
2.26 (m, 1H), 1.83 (s, 1H), 1.70 (s, 2H), 1.55 (m, 1H), 1.38-1.42
(m, 18H); HRMS (ES) (m/z): M + H+ calcd for C22H40N7O10
562.2837, found 562.2839.
1
solid. H NMR (500 MHz, CDCl3) δ 7.71 (m, 2H), 7.54 (m, 2H),
7.35 (t, J ) 7.0 Hz, 2H), 7.27 (d, J ) 6.0 Hz, 2H), 4.98 (m, 1H),
4.21-4.51 (m, 4H), 3.82-3.94 (m, 1H), 3.57-3.72 (m, 4H), 2.36-
2.49 (m, 1H), 2.23 (s, 1H), 1.48 (s, 9H), 1.45 (s, 9H); 13C NMR
(125 MHz, CDCl3) δ 172.7, 154.8, 154.3, 152.5, 144.2, 141.5,
127.9, 127.3, 125.3, 120.2, 85.5, 83.5, 68.1, 60.5, 58.1, 56.3, 52.7,
47.3, 32.1, 28.3, 27.7; HRMS (ES) (m/z): M + H+ calcd for
C31H39N2O9 583.2656, found 583.2666.
N-Boc-4-N-(NR-Boc-Nω-Nitro-L-argininyl)-trans-4-hydroxyami-
no-L-proline Amide (18). A mixture of 17 (80 mg, 0.14 mmol) in
THF (5 mL) and 5% aq LiOH (5 mL) was stirred at room
temperature for 1 h. After evaporation of THF under reduced
pressure, the aqueous solution was washed twice with EtOAc (5
mL), acidified with 1 N HCl to pH 3, and extracted twice with
EtOAc (10 mL). The organic extract was washed with brine, dried
over Na2SO4, filtered, and evaporated. The residue was dried
overnight under high vacuum and dissolved in THF (5 mL) at
ambient temperature. The solution was cooled to -10 °C. To this
solution were added N-methylmorpholine (16 mg, 0.15 mmol) and
isobutyl chloroformate (21 mg, 0.15 mmol) successively, stirring
was continued for 30 min at -10 °C, and then 29% aqueous
ammonia (0.028 mL) was added. The mixture was stirred at
-10 °C for 30 min and at room temperature for 12 h. After addition
of water (10 mL), the solution was extracted twice with EtOAc
(10 mL), washed with brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by flash column chromatog-
raphy (EtOAc/MeOH ) 10:1, Rf ) 0.25) to afford 18 (47 mg, 61%
from 17) as a colorless foam. 1H NMR (500 MHz, CD3OD) δ 5.11
(m, 1H), 4.66 (s, 1H), 4.32 (m, 1H), 3.52-3.72 (m, 2H), 3.24 (s,
2H), 2.43 (m, 1H), 2.08 (m, 1H), 1.79 (s, 1H), 1.66 (s, 2H), 1.57
(m, 1H), 1.45 (s, 4H), 1.42 (s, 14H); HRMS (ES) (m/z): M + H+
calcd for C21H39N8O9 547.2840, found 547.2837.
N-Fmoc-trans-4-Hydroxyamino-L-proline Methyl Ester (15).
Compound 14 (300 mg, 0.52 mmol) was treated with trifluoroacetic
acid/CH2Cl2 (5 mL/5 mL) at 0 °C under nitrogen. The reaction
temperature was then allowed to rise to room temperature, and
stirring was continued for 45 min. Excess TFA and solvent were
removed by evaporation. The residue was repeatedly dissolved in
CH2Cl2 (10 mL), and the solvent was evaporated to remove traces
of TFA. Trituration of this brown oil with ether (10 mL) gave 15
(245 mg, 95%) as a yellowish solid, which was used in the next
reaction without further purification. TLC (EtOAc/MeOH, 19:1)
1
Rf ) 0.50; H NMR (400 MHz, CD3OD) δ 7.81 (s, 2H), 7.58-
7.64 (m, 2H), 7.40 (s, 2H), 7.32 (s, 2H), 4.48 (s, 2H), 4.37 (d, J )
8.8 Hz, 1H), 4.17-4.28 (m, 1H), 4.08-4.08 (m, 1H), 3.62-3.87
(m, 5H), 2.56 (s, 1H), 2.37 (s, 1H); 13C NMR (125 MHz, acetone-
d6) δ 172.6, 154.2, 144.3, 141.4, 128.0, 127.4, 125.5, 120.3, 67.7,
63.0, 58.7, 52.0, 49.0, 47.3, 32.6; HRMS (ES) (m/z): M + H+
calcd for C21H23N2O5 383.1607, found 383.1614.
N-Fmoc-4-N-(NR-Fmoc-Nω-Nitro-L-argininyl)-trans-4-hy-
droxyamino-L-proline Methyl Ester (16). A completely dissolved
solution of Fmoc-Arg(NO2)-OH (486 mg, 1.10 mmol) in freshly
distilled anhydrous THF (3 mL) was chilled in an ice-acetone bath
(-10 °C), and to this was added SOCl2 (262 mg, 2.20 mmol)
dropwise. The mixture was stirred under nitrogen for 1 h. Chilled
anhydrous ether (20 mL) was introduced to yield a syrupy
precipitate. Maintaining low temperature, the solvent was removed
under reduced pressure, and the residue was triturated with chilled
anhydrous ether (20 mL). A white syrupy solid was obtained after
evaporation under reduced pressure, and excess thionyl chloride
was removed. During the evaporations, an ice-cold solution of 15
(275 mg, 0.55 mmol) and 2,4,6-collidine (67 mg, 0.55 mmol) in
THF (5 mL) was prepared and added via cannula to a flask
containing the acid chloride. Stirring continued for 20 min at 0 °C
and then 20 min more at room temperature. The solvent was
removed under reduced pressure, and the residue was treated with
EtOAc (10 mL). The organic mixture was washed twice with 5%
NaHCO3, water, 0.5 N HCl, and brine and dried over Na2SO4. The
solution was concentrated in vacuo, and the residue was purified
by flash column chromatography (EtOAc/MeOH ) 19:1, Rf ) 0.45)
to afford 16 (330 mg, 74%) as a white foamy solid. 1H NMR (400
MHz, acetone-d6) δ 7.84 (d, J ) 5.6 Hz, 4H), 7.64 (m, 4H), 7.40
(d, J ) 7.2 Hz, 4H), 7.32 (d, J ) 5.2 Hz, 4H), 6.73 (m, 1H), 5.26
(m, 1H), 4.88 (s, 1H), 4.51 (d, J ) 5.6 Hz, 1H), 4.29 (m, 6H), 3.70
(m, 4H), 3.39 (s, 2H), 2.62 (m, 1H), 2.19 (d, J ) 6.4 Hz, 1H), 2.05
(q, J ) 2.5 Hz, 2H), 1.80 (m, 4H); 13C NMR (125 MHz, acetone-
4-N-(Nω-Nitro-L-argininyl)-trans-4-hydroxyamino-L-proline
Amide (5). Using the procedure described for the preparation of
3, compound 5 was obtained from 18 as a white hygroscopic foam
(17 mg, 97%). 1H NMR (500 MHz, D2O) δ 5.14 (m, 1H), 4.49 (t,
J ) 8.5 Hz, 1H), 4.40 (m, 1H), 3.39-3.63 (m, 2H), 3.17 (s, 2H),
2.42-2.47 (m, 1H), 2.28-2.34 (m, 1H), 1.82 (s, 2H), 1.58 (s, 2H);
HRMS (ES) (m/z): M + H+ calcd for C11H23N8O5 347.1791, found
347.1801; [R]24 +8.0 (c ) 0.20, MeOH); Anal. Calcd for
D
C11H21N8O5‚3TFA‚H2O: C, H, N.
Docking Analysis. Molecular modeling calculations were per-
formed using the software packages SYBYL 6.8 and AutoDock
3.0.5 running on a Silicon Graphics Octane 2 workstation. The
protein structure used in the docking study was prepared as
described previously.18 The 3-D structure of the ligand was built
in SYBYL 6.8 by modifying the molecular structure of 2, which
was extracted from the crystal structure (pdb id: 1p6j). Using the
SYBYL program, correct atom types were assigned assuming
physiological pH. Energy minimizations were performed following
both the addition of polar hydrogen atoms and partial atom charge
calculations by the Gasteiger-Marsilli method.45 The torsion and

2098 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Seo et al.
(2) (a) Waldman, S. A.; Murad, F. Cyclic GMP Synthesis and Function.
Pharmacol. ReV. 1987, 39, 163-196. (b) Hobbs, A. J. Soluble
Guanylate Cyclase: the Forgotten Sibling. Trends. Pharmacol. Sci.
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Physiology, Pathophysiology, and Pharmacology. Pharmacol. ReV.
1991, 43, 109-142. (b) Moncada, S.; Higgs, E. A. Molecular
Mechanisms and Therapeutic Strategies Related to Nitric Oxide.
FASEB J. 1995, 9, 1319-1330.
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rotatable bonds in the ligand were defined by AutoTors, an auxiliary
program of AutoDock 3.0.5, which also united the nonpolar
hydrogens and partial atomic charges to the bonded carbon atoms.
Parameters for the docking experiment were used as described in
detail previously.18 A flexible docking calculation yielded 100
docked conformations, and appropriate conformations were chosen
by visual comparison of the superimposition of the guanidino
moiety of the predicted conformations and that of 2.
Enzyme and Assay. All of the NOS isoforms used are
recombinant enzymes overexpressed in E. coli from different
sources. Murine macrophage iNOS,46 rat nNOS,47 and bovine
eNOS48 were expressed and isolated as reported. Nitric oxide
formation from NOS was monitored by the hemoglobin capture
assay at 30 °C as described previously.49 Briefly, a solution of nNOS
or eNOS contained 10 µM L-arginine, 1.6 mM CaCl2, 11.6 µg/mL
calmodulin, 100 µM DTT, 100 µM NADPH, 6.5 µM H4B, 3 mM
oxyhemoglobin, and specified inhibitor concentration in 100 mM
HEPES (pH 7.5) in 600 µL total volume; iNOS contained the same
concentrations of cofactors, except CaCl2 and calmodulin were not
added. The assay was initiated by addition of enzyme and was
monitored at 401 nm on a Perkin-Elmer Lambda 10 UV-vis
spectrophotometer.
Determination of Ki Values. The reversible inhibition of NOS
by peptidomimetic inhibitors was studied under initial rate condi-
tions using the hemoglobin assay as described above. The apparent
Ki values were obtained by measuring percent inhibition in the
presence of 10 µM L-arginine with at least four different concentra-
tions of inhibitor. Generally, the inhibitor concentrations of two
higher and two lower than 50% inhibition were used. The IC50
values were determined by linear (or logarithmic for a few cases)
regression analysis of the percent inhibition data. The apparent Ki
values were calculated from the IC50 values using the following
inhibition equation:50 % inhibition ) 100[I]/{[I] + Ki (1 + [S]/Km)}
or Ki ) IC50/(1 + [S]/Km). Km values for L-arginine were 1.3 µM
(nNOS), 8.3 µM (iNOS), and 1.7 µM (eNOS). The selectivity of
an inhibitor was defined as the ratio of the respective Ki values.
Crystal Preparation and Structure Determination. The pro-
cedures for preparing nNOS or eNOS crystals complexed with
inhibitors 3 and 5 are similar to what were reported previously.22
Cryogenic X-ray diffraction data were collected at either Stanford
Synchrotron Radiation Laboratory (Menlo Park, CA) or Advanced
Light Source (Berkeley, CA) using a Q315 CCD detector. Raw
data were integrated and scaled with HKL2000.51 The bound
inhibitors were revealed by difference Fourier synthesis using
CNS.52 Models of inhibitors were built in O53 and structures refined
with CNS. Reflections and coordinates of refined structures have
been deposited in the RCSB protein data bank. The data collection
and structure refinement statistics for the three new structures are
summarized in Table S2 of Supporting Information.
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Evolution: Structure Studies of a Cystein Thiolate-Liganded Heme
Protein that Hydroxylates L-Arginine to Produce NO as a Cellular
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Acknowledgment. The authors are grateful for financial
support from the National Institutes of Health, GM 49725 to
R.B.S., GM57353 to T.L.P., and GM52419 to Dr. Bettie Sue
Masters, in whose lab P.M. and L.J.R. work, and Grant No.
AQ1192 from The Robert A. Welch Foundation to B.S.M. We
also acknowledge the SSRL and ALS beamline staff for
assistance with X-ray data collection. J.S. thanks Dr. Haitao Ji
and Dr. Wenxin Gu for helpful discussions.
(14) (a) Vallance, P.; Leiper, J. Blocking NO Synthesis: How, Where
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2006, 441, 513-517.
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Supporting Information Available: Details of data collection,
structure refinement statistics of the three new structures; elemental
analysis data of compounds 3-5; recently reported amide reduction
method. This material is available free of charge via the Internet at
http://pubs.acs.org.
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