Hydroxyethylene isosteres of selective neuronal nitric oxide synthase inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.06.038

Nitric oxide (NO) is an important second messenger molecule for blood pressure homeostasis, as a neurotransmitter, and in the immune defense system. Excessive NO can lead to neurodegeneration and connective tissue damage. Three different isozymes of the e

Full text of DOI:10.1016/j.bmc.2007.06.038

Bioorganic & Medicinal Chemistry 15 (2007) 6096–6108
Hydroxyethylene isosteres of selective neuronal
nitric oxide synthase inhibitors
a
b,
Erik P. Erdal, Pavel Martasek, Linda J. Romanb,à and Richard B. Silvermana,
*
´
aDepartment of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology, and the Center for Drug
Discovery and Chemical Biology, Northwestern University, Evanston, IL 60208-3113, USA
bDepartment of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78384-7760, USA
Received 30 March 2007; revised 13 June 2007; accepted 15 June 2007
Available online 23 June 2007
Abstract—Nitric oxide (NO) is an important second messenger molecule for blood pressure homeostasis, as a neurotransmitter, and
in the immune defense system. Excessive NO can lead to neurodegeneration and connective tissue damage. Three different isozymes
of the enzyme nitric oxide synthase regulate NO production in endothelial (eNOS), neuronal (nNOS), and macrophage (iNOS) cells.
Whereas creating a lower level of NO in some cells could be beneficial, it also could be detrimental to the protective effects that NO
has on other cells. Therefore, it is essential that therapeutic NOS inhibitors be made that are subtype selective. Previously, we
reported a series of nitroarginine-containing dipeptide amides as potent and selective nNOS inhibitors. Here we synthesize pepti-
domimetic hydroxyethylene isosteres of these dipeptide amides for potential increased bioavailability. None of the compounds is
as potent or selective as the dipeptide amides, but they exhibit good inhibition and selectivity. When the terminal amino group
was converted to a hydroxyl group, potency and selectivity greatly diminished, supporting the importance of the terminal amino
group for binding.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
lication of smooth muscle cells.3 Pharmacological inhi-
bition of eNOS in animal models was shown to cause
vasoconstriction, hypertension, and enhanced platelet
activation.4 ‘Knockout’ mice are more prone to ath-
erogenesis and developing aneurysms.5 These inhibi-
tion experiments strongly support the importance of
NO production from the endothelial isoform. The iso-
form originally identified in neuronal cells (nNOS),
also constitutive, produces NO that is known to be in-
volved in neurotransmission and is important for
brain development and learning,6 modification of pain
perception,7 and long-term potentiation.8 The induc-
ible form of the enzyme (iNOS) is expressed in
macrophages (white blood cells) as an immune re-
sponse.9 The NO produced from iNOS acts as a
cytotoxic agent against bacterial endotoxins, pro-
inflammatory cytokines, protozoa, fungi, and
viruses.10,11
Nitric oxide (NO), an important biomolecule with a
wide array of functions, is a cell-signaling agent that
is involved in the cardiovascular, gastrointestinal, gen-
itourinary, respiratory, and nervous systems.1 NO is
known to be involved in important processes, such
as neuronal transmission, cytoprotection, and platelet
aggregation. The regulation of NO biosynthesis is
the responsibility of the heme-containing metalloen-
zyme nitric oxide synthase (NOS) (EC 1.14.13.39).2
NOS exists in three distinct isoforms: the constitu-
tively expressed endothelial isoform (eNOS) controls
blood pressure by the regulation of smooth muscle
relaxation and is involved in the inhibition of platelet
and white blood cell adhesion and to suppress the rep-
Keywords: Nitric oxide; Nitric oxide synthase; Enzyme inhibitor;
nNOS inhibitor; Hydroxyethylene isostere.
Because of its wide range of function, nitric oxide has
gained much interest in the field of medicinal chemis-
try. The overproduction of NO has been implicated in
pathophysiological changes in virtually every organ
system linking it to a large variety of disease states.
Excess generation of NO from nNOS has been linked
*
Corresponding author. Tel.: +1 847 491 5653; fax: +1 847 491
7713; e-mail: Agman@chem.northwestern.edu
Developed the eNOS overexpression system in E. coli and the
purification of eNOS.
à
Developed the overexpression system for nNOS in E. coli and the
purification of the nNOS.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.038

E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
6097
to the ischemia and neurodegeneration resulting from
stroke,12 migraine headache,13 Parkinson’s disease,14
Alzheimer’s disease,15 amyotrophic lateral sclerosis,16
and Huntington’s disease.17 Enhanced NO derived
from iNOS has been related to arthritis,18 colitis,19
septic shock,20 inflammatory bowel disease,21 and
asthma.22,23 Since overproduction has been linked to
the variety of disease states discussed above, it would
be beneficial to attenuate the generation of NO di-
rectly related to a specific condition. Whereas creating
a lower level of NO in some cells could be beneficial,
it also could be detrimental to the protective effects
that NO has on other cells. Therefore, it is essential
that therapeutic NOS inhibitors be made that are sub-
type selective. Selectivity is especially needed over
eNOS because of its importance in the fundamental
physiology of blood pressure homeostasis.
taken to potentially increase bioavailability. Here we ex-
plore the utilization of a hydroxyethylene isostere of 2 as
a bioisosteric replacement for the amide bond.34
2. Results and discussion
2.1. Design rationale
The reason the hydroxyethylene isostere is appealing is
3-fold. First, the peptide bond is replaced by an alcohol
functional group, which is not hydrolyzable, thereby
preventing proteolysis. On the basis of X-ray crystallo-
graphy, the peptide bond nitrogen appears to be involved
in a hydrogen bond interaction with the propionate
chain of the heme porphyrin through a bridged water
molecule.28 The alcohol moiety of the hydroxyethylene
isostere also should be able to undergo this interaction.
Furthermore, the hydroxyl group might displace the
structural water molecule and lead to a direct interaction
with the heme propionate group, possibly resulting in
higher potency. The loss of the peptide carbonyl should
not have a detrimental effect on potency or selectivity
because the 2 series with the carbonyl group reduced
(i.e., 3) is more potent and selective than the correspond-
ing 2 series.35 Furthermore, an X-ray crystal structure of
3 (n = 1) bound to nNOS exhibited a similar interaction
between the reduced amide nitrogen and the enzyme as
was observed with the peptide bond nitrogen of the
dipeptide inhibitors.28 In fact, the reduced amide nitro-
gen may be close enough to interact directly with the
propionate chain of the heme porphyrin. These two con-
clusions produced the hypothesis that the hydroxyethyl-
ene isosteres could potentially produce more potent and
selective inhibitors than the corresponding amides.
Crystal structure studies have shown that the active sites
of eNOS and iNOS are nearly identical.24,25 However,
the height above the heme cofactor differs among the
isoforms creating a difference in active site size that de-
creases in the order nNOS > iNOS > eNOS.26 Along
with the difference in size, there appear to be subtle, al-
beit relatively minor, structural differences among the
substrate binding sites of the three isozymes.27–30 These
slight disparities present avenues that may be exploited
to successfully develop isoform-specific NOS inhibitors
with broad therapeutic potential. Many amino acids,
as well as nonamino acid analogues, are known to be
selective nNOS inhibitors.31
Prior to the publication of the NOS crystal structures,
we synthesized a library of 152 dipeptide amides con-
taining L- and D-NG-nitroarginine (NNA) and a vari-
ety of amino acids.32 Several analogues were relatively
potent and selective nNOS inhibitors; L-NNA-L-Dbu-
NH2 (1) had a Ki of 130 nM with 1538-fold selectivity
for nNOS over eNOS and 192-fold selectivity over
iNOS. To simplify the structure, it was found that
excision of the carboxamide group did not have a ma-
jor impact on the potency or selectivity (2, n = 1–3);
the propanediamine analogue (2, n = 2), which corre-
sponds to 1, had a Ki of 460 nM with nNOS/eNOS
and nNOS/iNOS selectivities of 463 and 256,
respectively.33
HN
NHNO2
NH
H
N
NH2
H2N
n
3
Four different chiral hydroxyethylene isosteric ana-
logues of 3, in addition to their corresponding alcohols
in case simple hydrogen bonding interactions were
important, were synthesized (4–11). Their syntheses
and biological evaluation as inhibitors of NOS isozymes
are reported here.
HN
NHNO2
NH
HN
NHNO2
NH
H
H
N
NH2
N
2.2. Chemistry
NH2
H2N
H2N
n
O
CONH2
O
The synthetic plan was to prepare intermediates con-
taining the terminal hydroxyl group, then convert them
into amino groups. The synthetic route designed to ob-
tain the terminal 3-hydroxypropyl analogues (4 and 5) is
shown in Scheme 1. Boc-Orn(Z)-OH (12) was used as
the starting material. An Arndt-Eistert homologation
was employed to obtain the Weinreb amide (13), fol-
lowed by reduction to the aldehyde (14) using lithium
aluminum hydride.34,36,37 The crucial step in the synthesis
1
2
Although dipeptides, such as L-NNA-L-Dbu-NH2, pro-
duce exceptional biological activity, they are not desir-
able as drug candidates. The peptide bond can be
hydrolyzed, so it can easily be metabolized in the gut
prior to delivery to the desired target enzyme. Because
of this, peptide-based compounds tend to exhibit poor
bioavailability. A peptidomimetic approach can be

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E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
O2NHN NH
NH
O2NHN
NH
NH
OH
O2NHN
NH
NH
O2NHN
NH
NH
OH
OH
OH
OH
OH
H2N
OH
H2N
OH
H2N
H2N
6
7
4
5
O2NHN
NH
O2NHN
NH
O2NHN
NH
O2NHN
NH
NH
OH
NH
OH
NH
OH
NH
OH
NH2
NH2
H2N
H2N
H2N
NH2
H2N
NH2
8
9
10
11
was the subsequent Grignard addition to this aldehyde.
The addition of allylmagnesium bromide successfully
produced the secondary alcohol adducts (15a,b).38 The
addition was conducted without any stereocontrol, but
the two diastereomers were separated by column chro-
matography. To determine the absolute stereochemistry
of each alcohol, a number of methods were considered.
Since the chiral center of the ornithine compound was
fixed in the L-configuration, cyclization would allow
the two chiral centers to be compared using 1D-NOESY
experiments (see Supplementary Material). Sodium
hydride-induced condensation of the alcohol with the
carbonyl of the Boc-protecting group produced the
corresponding perhydro-1,3-oxazin-2-ones (19a,b),
which allowed stereochemical identification of each dia-
stereomer (Scheme 2). The anti-diastereomer has the two
chiral protons in parallel axial positions; the relevant
peaks are multiplets at 3.4 and 4.2 ppm (Fig. 1). Irradi-
ation of the peak at 3.4 ppm produced a strong NOE at
4.2 ppm, and irradiation at 4.2 ppm produced a strong
NOE at 3.4 ppm. These data are consistent with two
1,3-diaxial protons, as in the anti-diastereomer. The
syn-diastereomer has one proton axial (3.4 ppm) and
one equatorial (4.3 ppm), which should not exhibit a
NOE. When the peak at 3.4 ppm was irradiated, the
peak at 4.3 ppm was not affected by the a-proton. Sim-
ilarly, when the peak at 4.3 ppm was irradiated, no NOE
was observed at 3.4 ppm. These results indicate that the
compound that produced these spectra is the syn-
diastereomer.
Cbz
NH
OH
Cbz
NH
Cbz
NH
Cbz
NH
BocHN
BocHN
BrMg
ZnCl2
NMM, ClCO2iBu, CH2N2
H
LiAlH4
Cbz
NH
OH
, Et3N, PhCO2Ag
O
N
H3C NOCH3
O
H
OH
CH3
OCH3
BocHN
BocHN
BocHN
O
13
14
12
15a,b
O2NHN
NH
O2NHN
NH
Cbz
NH
OH
NH
NH2
OH
NH
OH
OH
OH
TFA/CH2Cl2
NH
NHNO2
OH
H3CS
BocHN
O2NHN
OH
BocHN
BocHN
OH
BocHN
BocHN
H2N
H2
10% Pd/C
4
Cbz
NH
OH
NH
9-BBN
ii
O2NHN
NH2
OH
NH
NH
NH
OH
OH
OH
OH
OH
BocHN
17a,b
OH
16a,b
H2N
18a,b
5
NH
NH
HNO2
H2SO4
H2SO4
H3CS
NHNO2
H3CS
NH2
2
ii
i
Scheme 1.

E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
6099
Cbz
NH
Cbz
Cbz
NH
NH
NaH
OH
HN
HN
DMF
BocHN
O
O
O
O
15a,b
19a,b
Scheme 2.
The original plan was to convert the terminal hydroxyl
group of 4–7 to a protected amine by employing a Mits-
unobu reaction with N-Boc ethyl oxamate. Unfortu-
nately, the secondary alcohol attacked the Mitsunobu
intermediate to form a cyclic ether; therefore, a protec-
tion scheme was needed to prevent this nucleophilic at-
tack. Scheme 4 shows the synthesis of the terminal
aminopropyl analogues 8 and 9. Starting from Grignard
adducts 15a,b, the tri-protected adducts (23a,b) were
afforded.42 The terminal alcohols (24a,b) were obtained
using the same hydroboration–oxidation method pre-
sented in Scheme 1.38 Using a previously established
method,43 the terminal alcohol was converted to Boc-
protected amines 26a,b. Removal of the Cbz-protecting
group and the three benzyl groups necessitated harsher
hydrogenation conditions than normally needed for
benzyl deprotection. Palladium hydroxide on carbon
at 60 ꢁC successfully afforded the unprotected ornithine
analogues (27a,b). Nitroguanidination followed by re-
moval of the Boc-protecting groups yielded the first set
of terminal amine compounds (8 and 9).39
Cbz
HN
Cbz
HN
N
O
H
4.3 ppm
N
O
H
O
3.4 ppm
O
H
3.4 ppm
H
4.2 ppm
anti-diastereomer
syn-diastereomer
Figure 1. Two diastereomers isolated from the NaH cyclization of
15a,b.
The synthetic route was completed for each isomer sepa-
rately to obtain both diastereomers of the final product.
Employing a hydroboration–oxidation reaction with 9-
BBN produced the terminal alcohols (16a,b).39 Deprotec-
tion of the Cbz-protecting group by hydrogenation in the
presence of 10% palladium on carbon produced the free
amines (17a,b), and addition of N-nitromethylthioguani-
dine afforded the desired analogues (18a,b).40 Removal of
the Boc-protecting group using trifluoroacetic acid com-
pleted the synthesis of 4 and 5.
The synthetic route for the final set of terminal amine
compounds (10 and 11) is presented in Scheme 5. Start-
ing from Grignard adducts 15a,b, ozonolysis and reduc-
tion produced the terminal alcohols (20a,b).40 The
secondary alcohol did not need protection because a
four-membered ring cyclization was not favored, so
Mitsunobu conditions, followed by LiOH hydrolysis,
successfully afforded Boc-protected amines 30a,b.42
Deprotection of the Cbz group, followed by nitroguani-
dination and Boc deprotection, yielded the final two
compounds (10 and 11).39
The synthetic route designed to obtain the terminal
hydroxyethyl analogues (6 and 7) is given in Scheme 3.
This method only differs from the scheme to develop
the first two compounds at one of the steps. Originally,
vinylmagnesium bromide was substituted to provide the
compounds one carbon shorter, but this addition was
not successful. As an alternative, ozonolysis of the allyl
Grignard addition products (15a,b) followed by reduc-
tion with sodium borohydride produced the desired
alcohols (20a,b).41 Deprotection and nitroguanidination
as discussed above completed the synthesis of 6 and 7.39
2.3. Enzyme inhibition studies
The eight synthetic targets were tested against all three
isoforms of NOS to determine potency and selectivity.
O2NHN
NH
O2NHN
NH
NH
Cbz
NH
OH
Cbz
NH
OH
NH
OH
NH2
OH
OH
BocHN
NH
OH
TFA/CH2Cl2
BocHN
OH
H2N
OH
BocHN
OH
BocHN
1) O3/CH3OH
6
H3CS NHNO2
H2
10% Pd/C
Cbz
NH
OH
Cbz
NH
OH
NH2
OH
O2NHN
ii
NH
O2NHN
BocHN
NH
2) NaBH4
NH
OH
NH
OH
OH
BocHN
BocHN
OH
BocHN
15a,b
21a,b
20a,b
OH
H2N
OH
7
22a,b
Scheme 3.

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E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
Cbz
NH
OH
Cbz
N
Cbz
Cbz
N
N
Bn
OBn
Bn
Bn
Boc
N
OBn
OBn
O
O
OH
BocHN
BocN
Bn
BocN
Bn
BocN
Bn
OEt
OEt
O
N-Boc Ethyl Oxamate
DIAD, PPh3
Cbz
NH
OH
Cbz
N
Cbz
N
Cbz
N
NaH
BnBr, Bu4N+I-
9-BBN
Bn
Bn
Bn
Boc
N
OBn
OBn
OBn
OH
BocHN
BocN
Bn
BocN
Bn
BocN
Bn
15a,b
O
23a,b
24a,b
25a,b
O2NHN
NH
Cbz
O2NHN
NH
NH2
OH
N
Bn
NH
OBn
NH
OH
NH
NHBoc
NHBoc
NHBoc
H2
BocHN
BocHN
OH
BocN
Bn
NHBoc
TFA/CH2Cl2
BocHN
NHNO2
H3CS
NH2
LiOH
H2N
NH2
OH
Cbz
ii
Pd(OH)2/C
EtOH 60
8
O2NHN
NH
N
Bn
OBn
O2NHN
C
°
NH
NH
NHBoc
NH
OH
BocN
Bn
27a,b
OH
NHBoc
BocHN
26a,b
NH2
H2N
28a,b
9
Scheme 4.
Cbz
NH
OH
Cbz
NH
OH
Cbz
NH2
OH
NH
OH
O
CO2Et
LiOH
NHBoc
BocHN
N
NHBoc
BocHN
BocHN
BocHN
OH
BocHN
Boc
Cbz
NH
OH
H2
10% Pd/C
Cbz
NH
OH
Cbz
NH
OH
N-Boc Ethyl Oxamate
DIAD, PPh3
NH2
OH
O
CO2Et
NHBoc
BocHN
NH
N
NHBoc
BocHN
OH
BocHN
Boc
30a,b
31a,b
20a,b
29a,b
O2NHN
O2NHN
NH
NH
NH
OH
OH
NH
NHNO2
NHBoc
NH2
BocHN
O2NHN
H2N
H3CS
10
TFA/CH2Cl2
O2NHN
NH
NH
ii
NH
NH
OH
OH
NHBoc
NH2
BocHN
H2N
11
32a,b
Scheme 5.
The isoforms used are recombinant enzymes, which
were overexpressed in Escherichia coli from different
sources: murine macrophage iNOS, rat brain nNOS,
and bovine eNOS. The biological activities for the termi-
nal alcohol compounds (4–7) are given in Table 1.
Table 1. Inhibition of NOS isozymes by 4–7
Compound
IC50 (lM)
Selectivity
nNOS iNOS eNOS eNOS/nNOS iNOS/nNOS
4
5
6
7
255
490
106
168
524 2582 10.1
266 879 1.8
331 1094 10.2
1293 1662 9.9
2.1
0.54
3.1
7.7
The potency results are reported as IC50 values, which
are the inhibitor concentrations that cause 50% loss of
enzyme activity. The selectivities that the inhibitors have

E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
Table 2. Inhibition of NOS isozymes by 8–11
6101
was observed, but the results were not consistent across
the two sets of aminoalkyl compounds. Between the two
aminoethyl side chain inhibitors, the syn-diastereomer
(10) proved to be more potent, whereas the anti-diaste-
reomer (9) of the aminopropyl compounds was the more
potent isomer. In contrast, 9 showed higher potency
than 11. Since the position of the terminal amino group
appears to be so crucial for enzyme binding, the resul-
tant position of the terminal amino group ultimately will
determine inhibitor potency. With the terminal amino
group in the favored position, the hydroxyl groups of
the syn-aminoethyl diastereomer (10) and the anti-
aminopropyl diastereomer (9) are in preferred positions,
whereas the hydroxyl groups of the anti-aminoethyl dia-
stereomer (11) and the syn-aminopropyl diastereomer
(8) are in less optimal positions.
Compound
IC50 (lM)
Selectivity
nNOS
23.5 (2.8)a 361 1395
iNOS eNOS eNOS/nNOS iNOS/nNOS
8
9
59
15
33
6.8 (0.83)a 313 1529 225
2.4 (0.29)a 257 1141 475
9.4 (1.1)a 167 1342 143
10
11
107
18
a Data in parentheses represent Ki values.
for nNOS over the other two isoforms are also pre-
sented. This series of compounds showed poor potency
against nNOS, with 3 providing the best inhibition,
IC50 = 106 lM. Some selectivity toward nNOS was ob-
served, but not significant enough to explore further.
It was proposed that the terminal hydroxyl compounds
might produce good potency because of a potential
hydrogen bond interaction with the structural water
molecule. These results provide strong evidence that
the enzyme has an affinity for a positively charged ami-
no side chain, not just a hydrogen-bond donor group.
Previous inhibitor docking experiments suggest that a
potential long-range electrostatic interaction is lost
when incorporating a hydroxyl group at the side chain
position.44
3. Summary
A series of chiral hydroxyethylene isosteres of Nx-nitro-
L-arginine amides were synthesized and shown to exhibit
potent and selective nNOS inhibition, but not as potent
or selective as the parent compounds. However,
hydroxyethylene isosteres have been shown in the litera-
ture to be more bioavailable than the corresponding
parent peptide analogues.46
The amino side chain-containing inhibitors (8–11) pro-
duced better results (Table 2). All four of these com-
pounds are much more potent inhibitors of nNOS.
Compounds 9 and 10 exhibited sub-micromolar Ki val-
ues, 830 and 290 nM, respectively. Compound 10 is
4. Experimental
4.1. 6-(Benzyloxycarbonylamino)-(3S)-(tert-butyloxycar-
bonylamino)-(N-methoxy-N-methyl)hexamide (13)
31
comparable in potency to L-NNA-L-Dbu-NH2 and
the reduced amide peptidomimetic compounds.34 The
chain length of 10 corresponds to the optimal chain
length in the reduced amide series (3, n = 1), suggesting
a similar binding geometry for the two series of com-
pounds. The aminoalkyl hydroxyethylene compounds
exhibited good selectivity over eNOS, but only moderate
selectivity over iNOS, although the selectivity of the
hydroxyethylene isosteres toward nNOS is significantly
lower than that of the previous inhibitors in this class.
The potency indicates that this isostere successfully
binds to nNOS, but we had thought that the hydroxyl
group would displace a structural water molecule and
bind directly to the heme cofactor, resulting in higher
potency. This increased inhibition was not observed,
suggesting that the molecules either are not binding as
anticipated or the proposed binding does not lead to
greater potency. Recently, we found that the N-hydroxyl
analogue of 3 (n = 1; OH on the secondary NH group),
which computer modeling predicted also could displace
the structural water molecule and bind directly to the
heme side chain was shown by X-ray crystallography
to do that, yet the Ki value was not changed from the
parent compound.45 However, other important interac-
tions were moderated, thereby compensating for the di-
rect interaction with the enzyme. Apparently, a similar
result occurs with the hydroxyethylene isosteric
molecules.
To a flame-dried 250 mL flask charged with argon was
added N,O-dimethyl hydroxylamine hydrochloride
(1.60 g, 16.4 mmol) in dry THF (20 mL). Triethylamine
(3.04 mL, 26.8 mmol) was added dropwise at room tem-
perature, and the mixture was allowed to stir overnight.
The resulting white precipitated salt was filtered and
washed with dry THF.
To
a
solution of Boc-L-Orn(Z)-OH (12, 4.0 g,
10.9 mmol) in ethylene glycol dimethyl ether (DME)
(10 mL), cooled to ꢀ20 ꢁC and under an argon atmo-
sphere, was added with vigorous stirring 4-meth-
ylmorpholine (1.27 mL, 11.4 mmol) followed by
isobutyl chloroformate (1.56 mL, 12.0 mmol). The
resulting white precipitate was removed by filtration
and washed with DME. The filtrate was added to dia-
zomethane in ether, generated using an Aldrich Diazald
Kit (ꢁ16 mmol). The solution was allowed to stir for 1–
2 h at 0 ꢁC and concentrated in vacuo. The resulting res-
idue was dissolved in dry THF (ꢁ50 mL). To this solu-
tion was added the filtrate resulting from the previous
reaction followed by silver benzoate (0.51 g, 2.20 mmol)
in triethylamine (5.8 mL), and the reaction mixture was
allowed to stir overnight. The above reaction was per-
formed in duplicate, and the solutions were combined
and worked up as one reaction. The resulting dark
brown solution was concentrated in vacuo, and the res-
idue was dissolved in ethyl acetate. The insoluble mate-
rial was removed by filtration through a pad of Celite.
The two diastereomers of each compound are expected
to bind differently to the NOS isozymes. A difference

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E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
The filtrate was washed with saturated sodium bicar-
bonate (3· 100 mL), water (100 mL), 1 M potassium
bisulfite (2· 50 mL), brine, dried over anhydrous magne-
sium sulfate, and concentrated in vacuo. The resulting
clear, yellow oil was purified by flash chromatography
(ethyl acetate/hexane 2:1) to afford a clear, colorless oil
drous magnesium sulfate, and concentrated in vacuo.
The resulting cloudy oil was purified by flash chromatog-
raphy (hexane/ethyl acetate 4:1) to afford two separate
diastereomers as clear, colorless oils (15a, S,S-isomer
[1.0 g, 44%] and 15b, S,R-isomer [0.72 g, 32%]).
1
(6.15 g, 67%). H NMR (400 MHz, CDCl3) d 7.36 (m,
Compound 15a: 1H NMR (400 MHz, CDCl3) d 7.30 (m,
5H), 5.79 (m, 1H), 5.25 (m, 1H), 5.05 (s, 2H), 5.05 (m,
2H), 4.81 (m, 1H), 3.96 (m, 1H), 3.62 (m, 1H), 3.11
(m, 2H), 2.18 (m, 2H) 1.22–1.56 (m, 4H), 1.40 (s, 9H).
MS (ES) (m/z): M+H+ Calcd for C22H34N2O5, 406.5.
Found: 407.5.
5H), 5.38 (m, 1H), 5.09 (s, 2H), 4.99 (br s, 1H), 3.90
(m, 1H), 3.68 (s, 3H), 3.20 (m, 2H), 3.17 (s, 3H), 2.70
(m, 1H), 2.59 (m, 1H) 1.28–1.66 (m, 4H), 1.40 (s, 9H).
MS (ESI) (m/z): 424.2 (M+1).
4.2. 6-(Benzyloxycarbonylamino)-(3S)-(tert-butyloxycar-
bonylamino)hexanal (14)
Compound 15b: 1H NMR (400 MHz, CDCl3) d 7.31 (m,
5H), 5.76 (m, 1H), 5.35 (m, 1H), 5.08 (m, 2H), 5.03 (s,
2H), 4.84 (m, 1H), 3.66 (m, 2H), 3.13 (m, 2H), 2.19
(m, 2H), 1.25–1.55 (m, 4H), 1.39 (s, 9H). MS (ESI)
(m/z): 407.3 (M+1).
A 250 mL three-necked round bottom flask was
equipped with a thermometer, flame dried, and charged
with argon and a 1 M solution of lithium aluminum hy-
dride in THF (29 mL, 29 mmol). To this solution was
added
a
solution of Weinreb amide 13 (6.15 g,
The following procedures were completed using both iso-
mers, but one example procedure is given for each.
14.5 mmol) in THF (30 mL) dropwise by cannula while
keeping the temperature below ꢀ60 ꢁC. After addition
was complete the dry ice bath was removed, and the
solution was allowed to stir for 30 min or until all start-
ing material was consumed. The dry ice bath was re-
placed, and the reaction was cautiously quenched with
saturated potassium bisulfate (10 mL). The dry ice bath
was removed once again, and the solution was allowed
to warm to room temperature for 2 h, resulting in a
clear, yellow solution with a white precipitate. The pre-
cipitate was removed by filtration through a pad of Cel-
ite, and the filtrate was concentrated in vacuo. The
resulting residue was dissolved in ethyl acetate and
washed with cold 1 N HCl (3· 50 mL), saturated sodium
bicarbonate (2· 50 mL), brine, dried over anhydrous
magnesium sulfate, and concentrated in vacuo. The
resulting yellow oil was purified by flash chromatogra-
phy (hexane/ethyl acetate 3:1) to afford a clear, colorless
oil that crystallized under vacuum (2.05 g, 38%). 1H
NMR (400 MHz, CDCl3) d 9.61 (s, 1H), 7.26 (m, 5H),
5.41 (m, 1H), 5.05 (m, 1H), 5.00 (s, 2H), 3.93 (m, 1H),
3.08 (m, 2H), 2.43 (m, 2H), 1.15–1.55 (m, 4H), 1.34 (s,
9H). MS (ESI) (m/z): 365.5 (M+1).
4.4. N-[(4S)-(tert-Butyloxycarbonylamino)-(6R,9)-
dihydroxynonyl-N-(benzyloxycarbonyl)]amine (16b)
A sample of allyl alcohol 15b (0.12 g, 0.29 mmol) was
added to a 50 mL round bottom flask, dried overnight
using a vacuum pump, and charged with argon. To this
sample was added a 0.5 M solution of 9-BBN in THF
(5.8 mL, 2.9 mmol) at room temperature, and the reac-
tion mixture was allowed to stir overnight. The mixture
was then cooled to 0 ꢁC, and 2 M potassium hydroxide
(5 mL, 10 mmol) was added, followed by 30% hydrogen
peroxide (5 mL). After being stirred for 5 min, the solu-
tion was diluted with ethyl acetate (25 mL), washed with
water (3· 50 mL) and brine, dried over anhydrous magne-
sium sulfate, and concentrated in vacuo. The cloudy oil
was purified by flash chromatography (methylene chlo-
ride/methanol 50:1) to afford a clear, colorless oil
(35 mg, 29%). This procedure was repeated for the other
isomer.
1
R-isomer: H NMR (400 MHz, CDCl3) d 7.31 (m, 5H),
5.19 (m, 1H), 5.10 (m, 1H), 5.04 (s, 2H), 4.78 (m, 1H),
3.62 (m, 2H), 3.34 (m, 1H), 3.18 (m, 2H), 1.65 (m,
2H), 1.26–1.60 (m, 8H), 1.40 (s, 9H). MS (ESI) (m/z):
425.0 (M+1).
4.3. N-[(4S)-(tert-Butyloxycarbonylamino)-(6S and 6R)-
hydroxynon-8-enyl-N-(benzyloxycarbonyl)]amine (15a,b)
A three-necked 250 mL round bottom flask was
equipped with a thermometer, flame dried, and charged
with argon. To a stirred 1 M solution of zinc chloride
in ether (16 mL, 16 mmol) was added THF (40 mL).
The solution was cooled to 0 ꢁC, and a 1 M solution of
allylmagnesium bromide was added (24 mL, 24 mmol).
After being stirred for 30 min, the suspension was cooled
to ꢀ30 ꢁC, and a solution of aldehyde 14 (2.05 g,
5.6 mmol) in THF (30 mL) was added slowly, keeping
the internal temperature below ꢀ20 ꢁC. The reaction
mixture was stirred and allowed to warm to room tem-
perature overnight. The mixture was then cooled to
ꢀ30 ꢁC, cautiously quenched with 5% citric acid
(100 mL), and diluted with ethyl acetate (200 mL). The
resulting organic layer was washed with water (50 mL),
saturated sodium bicarbonate (50 mL), dried over anhy-
1
S-isomer: H NMR (400 MHz, CDCl3) d 7.35 (m, 5H),
6.52 (br s, 1H), 5.08 (s, 2H), 4.99 (m, 1H), 3.84 (m, 2H),
3.71 (m, 2H), 3.20 (m, 2H), 1.64 (m, 2H), 1.34–1.58 (m,
8H), 1.41 (s, 9H). MS (ESI) (m/z): 425.0 (M+1).
4.5. N-[(4S)-(tert-Butyloxycarbonylamino)-(6R,9)-
dihydroxynonyl]amine (17b)
To a 25 mL round bottom flask was added Cbz-pro-
tected alcohol 16b (0.035 g, 0.082 mmol), which was dis-
solved in methanol (10 mL). To this solution was added
10% Pd/C (10 mg), and the suspension was exposed to
hydrogen gas overnight. The suspension was then fil-
tered through a pad of Celite and concentrated in vacuo
to afford a yellow oil. The crude yellow oil was used in

E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
6103
the next reaction without purification. This procedure
was repeated for the other isomer.
with water (3–5 times) and brine, dried over magnesium
sulfate, and concentrated in vacuo. The resulting yellow
oil was purified by flash chromatography (hexane/ethyl
acetate 2:1) to afford a clear, colorless oil (0.024 g,
45%). This procedure was repeated for the other isomer.
4.6. N-[(4S)-(tert-Butyloxycarbonylamino)-(6R,9)-
(dihydroxynonyl)]nitroguanidine (18b)
1
To a solution of crude amine 17b (0.23 g, 0.082 mmol) in
absoluteethanol (5 mL) was addedN-nitromethylthiogua-
nidine47 (0.012 g, 0.091 mmol). The solution was heated at
40 ꢁC for 5 min and allowed to stir overnight at room tem-
perature. The reaction mixture was concentrated in vacuo
and purified by flash chromatography (ethyl acetate/hex-
ane 7:1) to afford a clear, colorless oil (0.025 g, 81%). This
procedure was repeated for the other isomer.
R-isomer: H NMR (500 MHz, CDCl3) d 7.36 (m, 5H),
6.14 (br s, 1H), 5.81 (m, 1H), 5.14 (m, 2H), 5.10 (s, 2H),
5.03 (m, 1H), 4.26 (m, 1H), 3.46 (m, 1H), 3.22 (m, 2H),
2.48(m, 1H),2.37(m, 1H),1.95(m, 1H),1.27–1.72(m, 6H).
1
S-isomer: H NMR (500 MHz, CDCl3) d 7.35 (m, 5H),
6.85 (br s, 1H), 5.80 (m, 1H), 5.13 (m, 2H), 5.10 (s, 2H),
4.35 (m, 1H), 3.47 (m, 1H), 3.21 (m, 2H), 2.50 (m, 1H),
2.36 (m, 1H), 1.82 (m, 1H), 1.22–1.76 (m, 6H).
R-isomer: 1H NMR (400 MHz, CD3OD) d 3.72 (m, 1H),
3.56 (t, J = 8.0 Hz, 2H), 3.30 (m, 1H), 3.24 (m, 2H), 1.84
(m, 2H), 1.32–1.70 (m, 8H), 1.44 (s, 9H). MS (ESI) (m/
z): 378.3 (M+1).
4.9. N-[(4S)-(tert-Butyloxycarbonylamino)-(6R,8)-
dihydroxyoctyl-N-(benzyloxycarbonyl)]amine (20b)
To a 100 mL three-necked round bottom flask was
added a solution of allyl alcohol 15b (0.30 g, 0.74 mmol)
in methanol (10 mL). Ozone was bubbled through the
solution at a constant rate for 1–2 h or until all starting
material was consumed. The reaction was then worked
up by the addition of sodium borohydride (0.28 g,
7.4 mmol). After being stirred for several hours, the mix-
ture was concentrated in vacuo, and the resulting resi-
due was dissolved in ethyl acetate. The solution was
then washed with saturated ammonium chloride, brine,
dried over anhydrous magnesium sulfate, and concen-
trated in vacuo to afford a cloudy oil. The oil was puri-
fied by flash chromatography (methylene chloride/
methanol 50:1) to afford a clear, colorless oil (0.15 g,
49%). This procedure was repeated for the other isomer.
S-isomer: 1H NMR (400 MHz, CD3OD) d 3.61 (m, 1H),
3.56 (t, J = 8.0 Hz, 2H), 3.31 (m, 1H), 3.23 (m, 2H),
1.34–1.70 (m, 10H), 1.42 (s, 9H). MS (ESI) (m/z):
378.3 (M+1).
4.7. N-[(4S)-Amino-(6S,9)-dihydroxynonyl]nitroguanidine
(4) and N-(4S-amino-(6R,9)-dihydroxynonyl)nitroguani-
dine (5)
Compound 18a (0.012 g, 0.032 mmol) or 18b (0.025 g,
0.066 mmol) was dissolved in 50% TFA in methylene
chloride (2 mL) and allowed to stir for 3 h at room tem-
perature. The mixture was concentrated in vacuo, and
the resulting yellow oil was dissolved in ethanol. Con-
centrated HCl was added dropwise until the solution be-
came cloudy, and the solution was concentrated once
again. The resulting yellow oil was purified by HPLC
to afford a white solid that became sticky when exposed
to air (0.010 g, 99% for 4; 0.02 g, 99% for 5).
1
R-isomer: H NMR (400 MHz, CDCl3) d 7.35 (m, 5H),
5.23 (br s, 1H), 5.07 (s, 2H), 4.92 (br s, 1H), 4.30 (m,
1H), 4.19 (m, 2H), 3.24 (m, 4H), 2.14 (m, 2H), 1.96
(m, 1H), 1.25–1.76 (m, 6H), 1.46 (s, 9H). MS (ESI)
(m/z): 433.3 (M+Na+).
Compound 4: 1H NMR (500 MHz, D2O) d 3.69 (m,
1H), 3.46 (m, 2H), 3.32 (m, 1H), 3.20 (m, 2H), 1.30–
1.79 (m, 10H). 13C NMR (100 MHz, D2O) d 159.0,
70.1, 61.6, 51.0, 40.7, 38.2, 33.6, 29.7, 27.4. HRMS
1
S-isomer: H NMR (400 MHz, CDCl3) d 7.35 (m, 5H),
5.28 (s, 2H), 4.98 (m, 1H), 4.64 (m, 1H), 4.56 (m, 1H),
3.78 (m, 2H), 3.31 (m, 1H), 3.17 (m, 2H), 1.19–1.78
(m, 8H), 1.43 (s, 9H). MS (ESI) (m/z): 433.3 (M+Na+).
(ES) (m/z): M+H+ Calcd for C10H24N5O4, 278.1828.
24
D
Found: 278.1818. ½aꢂ ꢀ2.50. (syn-isomer).
4.10. N-[(4S)-(tert-Butyloxycarbonylamino)-(6R,8)-
dihydroxyoctyl]amine (21b)
Compound 5: 1H NMR (500 MHz, D2O) d 3.72 (m,
1H), 3.46 (m, 2H), 3.35 (m, 1H), 3.19 (m, 2H), 1.32–
1.72 (m, 10H). 13C NMR (100 MHz, D2O) d 159.0,
67.5, 61.6, 51.9, 51.5, 40.6, 37.6, 33.2, 29.3, 27.5. HRMS
To solution of 20b (0.050 g, 0.12 mmol) in methanol
(10 mL) was added Pd/C (0.050 g). The suspension was
exposed to hydrogen gas and allowed to stir overnight.
The suspension was then filtered through a pad of Celite
and concentrated in vacuo to afford a yellow oil. The
crude yellow oil was used in the next reaction without
purification. This procedure was repeated for the other
isomer.
(ES) (m/z): M+H+ Calcd for C10H24N5O4, 278.1828.
24
D
Found: 278.1838. ½aꢂ +2.34. (anti-isomer).
4.8. (6R)-Allyl-(4S)-3-(benzyloxycarbonylamino)propyl-
(perhydro-1,3-oxazin-2-one) (19b)
To a solution of allyl alcohol 15b (0.065 g, 0.16 mmol) in
dry DMF (2 mL) was added NaH (60% in mineral oil,
10 mg, 0.23 mmol). The reaction mixture was allowed
to stir overnight, cooled to 0 ꢁC, slowly quenched with
saturated ammonium chloride (10 mL), and diluted with
ethyl acetate (20 mL). The organic layer was washed
4.11. N-[(4S)-(tert-Butyloxycarbonylamino)-(6R,8)-
(dihydroxyoctyl)]nitroguanidine (22b)
To a solution of crude alcohol 21b in absolute ethanol
(5 mL) was added N-nitromethylthioguanidine (16 mg,

6104
E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
0.12 mmol). The solution was heated at 40 ꢁC for 5 min
and allowed to stir overnight at room temperature. The
reaction mixture was concentrated in vacuo and purified
by flash chromatography (ethyl acetate/hexane 7:1) to
afford a clear, colorless oil (20 mg, 46%). This procedure
was repeated for the other isomer.
oil containing a white solid. The mixture was dissolved
in methylene chloride and was purified by flash chroma-
tography (hexane/ethyl acetate 6:1) to afford a clear, col-
orless oil (0.46 g, 44%). This procedure was repeated for
the other isomer.
1
R-isomer: H NMR (400 MHz, CDCl3) d 7.05–7.45 (m,
R-isomer: 1H NMR (400 MHz, CD3OD) d 3.80 (m, 1H),
3.68 (m, 2H), 3.26 (m, 3H), 1.64 (m, 2H), 1.34–1.58 (m,
6H), 1.44 (s, 9H). MS (ESI) (m/z): 364.5 (M+1).
20H), 5.84 (m, 1H), 5.18 (s, 2H), 5.08 (m, 2H), 4.08–4.50
(m, 7H), 2.94–3.42 (m, 3H), 2.32 (m, 2H), 1.06–1.89 (m,
6H), 1.42 (s, 9H). MS (ESI) (m/z): 677.4 (M+1).
1
S-isomer: 1H NMR (400 MHz, CD3OD) d 3.78 (m, 1H),
3.67 (m, 2H), 3.29 (m, 2H), 3.23 (m, 1H) 1.63 (m, 2H),
1.30–1.55 (m, 6H), 1.43 (s, 9H). MS (ESI) (m/z): 364.5
(M+1).
S-isomer: H NMR (400 MHz, CDCl3) d 7.05–7.45 (m,
20H), 5.60 (m, 1H), 5.12 (s, 2H), 4.98 (m, 2H), 3.80–4.65
(m, 7H), 2.90–3.35 (m, 3H), 2.11 (m, 2H), 1.18–1.55 (m,
6H), 1.43 (s, 9H). MS (ESI) (m/z): 677.4 (M+1).
4.12. N-[(4S)-Amino-(6S,8)-dihydroxyoctyl]nitroguani-
dine (6) and N-(4S-amino-(6R,8)-dihydroxyoctyl)nitro-
guanidine (7)
4.14. N-[(4S)-[N-Benzyl-N-tert-butyloxycarbonyl]amino-
(6R)-benzyloxy-9-hydroxynonyl-{N-benzyl-N-(benzyl-
oxycarbonyl)}]amine (24b)
Compound 22a (0.016 g, 0.044 mmol) or 22b (0.021 g,
0.058 mmol) was dissolved in 50% TFA in methylene
chloride (2 mL) and was allowed to stir for 3 h at room
temperature. The mixture was concentrated in vacuo,
and the resulting yellow oil was dissolved in ethanol.
Concentrated HCl was added dropwise until the solu-
tion became cloudy, and the solution was concentrated
once again. The resulting yellow oil was purified by
HPLC to afford a white solid that became sticky when
exposed to air (0.015 g, 99% for 6; 0.017 g, 99% for 7).
To a 100 mL round bottom flask, flame-dried and
charged with argon, was added tribenzyl compound
23b (0.46 g, 0.68 mmol). The oil was then dissolved in
a 0.5 M solution of 9-BBN in THF (13.6 mL, 6.8 mmol)
and was allowed to stir at room temperature overnight.
The flask was cooled to 0 ꢁC, and 2.0 M aqueous KOH
(10 mL, 20 mmol) was added followed by 30% hydrogen
peroxide (10 mL). After being stirred for 5 min, the solu-
tion was diluted with ethyl acetate (50 mL). The result-
ing organic layer was washed with water (5· 50 mL)
and brine, and concentrated in vacuo to afford a white,
cloudy oil. The crude product was purified by flash chro-
matography (methylene chloride/methanol 40:1). Some
impurities remained, so the crude material was purified
by flash chromatography a second time (hexane/ethyl
acetate 3:1) to afford a soap-like white oil (0.39 g,
82%). This procedure was repeated for the other isomer.
Compound 6: 1H NMR (500 MHz, D2O) d 3.82 (q,
J = 7.9 Hz, 1H), 3.55 (t, J = 7.1 Hz, 2H), 3.36 (m, 1H),
3.17 (m, 2H), 1.53–1.70 (m, 8H). 13C NMR (100 MHz,
D2O) d 159.0, 64.7, 58.3, 49.0, 40.6, 38.8, 37.9, 29.4,
23.5. HRMS (ESI) (m/z): M+H+ Calcd for
24
D
C9H22N5O4, 264.1672. Found: 264.1663. ½aꢂ +1.85
(syn-isomer).
1
R-isomer: H NMR (400 MHz, CDCl3) d 6.88–7.50 (m,
1
Compound 7: H NMR (500 MHz, D2O) d 3.81 (m,
20H), 5.15 (s, 2H), 4.39 (s, 2H), 4.39 (m, 1H), 4.21 (s,
2H), 4.15 (s, 2H), 3.56 (m, 2H), 2.92–3.33 (m, 3H),
1.86 (m, 2H), 1.05–1.72 (m, 8H), 1.44 (s, 9H). MS
(ESI) (m/z): 717.9 (M+Na+).
1H), 3.55 (t, J = 6.7 Hz, 2H), 3.32 (m, 1H), 3.16 (m,
2H), 1.45–1.78 (m, 8H). 13C NMR (100 MHz, D2O) d
159.0, 67.3, 58.1, 50.9, 40.7, 39.3, 38.4, 29.7, 23.4.
HRMS (ESI) (m/z): M+H+ Calcd for C9H22N5O4,
24
D
1
264.1672. Found: 264.1682. ½aꢂ ꢀ6.61 (anti-isomer).
S-isomer: H NMR (400 MHz, CDCl3) d 6.95–7.58 (m,
20H), 5.14 (s, 2H), 3.80–4.66 (m, 7H), 3.53 (m, 2H),
2.80–3.33 (m, 3H), 1.10–1.85 (m, 10H), 1.44 (s, 9H).
MS (ESI) (m/z): 717.9 (M+Na+).
4.13. N-[(4S)-[N-Benzyl-N-tert-butyloxycarbonyl]amino-
(6R)-benzyloxynon-8-enyl-[N-benzyl-N-(benzyloxycar-
bonyl)]amine (23b)
4.15. N-[(4S)-[N-Benzyl-N-(tert-butyloxycarbonyl]amino-
(6R)-benzyloxy-9-{N-tert-butyloxycarbonyl)-N-[2-eth-
oxy-(oxoacetyl)amino}nonyl-{N-benzyl-N-(benzyloxy-
carbonyl)}]amine (25b)
To a 100 mL round bottom flask, flame-dried and
charged with argon, was added allyl alcohol 15b
(0.63 g, 1.55 mmol). The oil was dissolved in anhydrous
DMF (10 mL) and treated with 60% sodium hydride in
mineral oil (0.62 g, 15.5 mmol). The suspension was
cooled to 0 ꢁC followed by addition of tetrabutylammo-
nium iodide (1.91 g, 5.17 mmol) and benzyl bromide
(0.61 mL, 5.17 mmol). The mixture was allowed to
warm to room temperature overnight, cooled to 0 ꢁC,
and cautiously quenched with saturated ammonium
chloride. The solution was extracted with methylene
chloride (3· 50 mL), and the combined organic layers
were concentrated in vacuo to afford a mixture of a clear
A two-necked 100 mL round bottom flask was equipped
with a water-jacketed condenser, flame-dried, and
placed under an argon atmosphere. Triphenylphosphine
(0.16 g, 0.62 mmol) and N-Boc-ethyl oxamate (0.14 g,
0.62 mmol) were added followed by a solution of
tribenzyl alcohol 24b (0.39 g, 0.56 mmol) in dry THF
(10 mL). The solution was cooled to 0 ꢁC, and diisopro-
pyl azodicarboxylate (0.12 mL, 0.62 mmol) was added
dropwise. The reaction mixture was then heated to

E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
6105
60 ꢁC and was allowed to stir overnight. The resulting
light yellow liquid was concentrated in vacuo and puri-
fied by flash chromatography (hexane/ethyl acetate 7:1)
to afford a clear, colorless oil (0.24 g, 48%). This proce-
dure was repeated for the other isomer.
ture was concentrated in vacuo and purified by flash
chromatography (ethyl acetate/hexane 7:1) to afford a
clear, colorless oil (0.20 g, 33%). This procedure was re-
peated for the other isomer.
R-isomer: 1H NMR (400 MHz, CDCl3) d 8.59 (br s, 1H),
7.67 (m, 2H), 4.94 (m, 2H), 3.65 (m, 2H), 3.39 (m, 1H),
3.23 (m, 1H), 3.05 (m, 2H), 1.32–1.70 (m, 10H), 1.38 (s,
9H), 1.36 (s, 9H). MS (ESI) (m/z): 477.4 (M+1).
1
R-isomer: H NMR (400 MHz, CDCl3) d 7.04–7.49 (m,
20H), 5.16 (s, 2H), 3.97–4.50 (m, 9H), 3.65 (m, 2H),
2.93–3.34 (m, 3H), 1.82 (m, 2H), 1.22–1.72 (m, 9H),
1.51 (s, 9H), 1.48 (s, 9H). MS (ESI) (m/z): 917.3
(M+Na+).
S-isomer: 1H NMR (400 MHz, CDCl3) d 8.60 (br s, 1H),
7.57 (m, 2H), 5.02 (br s, 1H), 4.79 (br s, 1H) 3.63 (m,
2H), 3.38 (m, 1H), 3.24 (m, 1H), 3.07 (m, 2H), 1.26–
1.74 (m, 10H), 1.39 (s, 18H). MS (ESI) (m/z): 477.4
(M+1).
1
S-isomer: H NMR (400 MHz, CDCl3) d 6.96–7.72 (m,
20H), 5.12 (s, 2H), 4.95 (m, 2H) 3.78–4.71 (m, 9H),
2.88–3.36 (m, 3H), 0.64–1.82 (m, 11H), 1.24 (s, 9H),
1.23 (s, 9H). MS (ESI) (m/z): 917.3 (M+Na+).
4.19. N-[(4S,9)-Diamino-(6S)-hydroxynonyl]nitroguani-
dine (8) and N-[(4S,9)-diamino-(6R)-hydroxynonyl]nitro-
guanidine (9)
4.16. N-[(4S)-[N-Benzyl-N-tert-Butyloxycarbonyl]amino-
(6R)-benzyloxy-9-(N-tert-butyloxycarbonylamino)nonyl-
{N-benzyl-N-(benzyloxycarbonyl)}]amine (26b)
Compound 28a (0.035 g, 0.073 mmol) or 28b (0.050 g,
0.10 mmol) was dissolved in 50% TFA in methylene
chloride and was allowed to stir for 3 h at room temper-
ature. The mixture was concentrated in vacuo, and the
resulting yellow oil was dissolved in ethanol. Concen-
trated HCl was added dropwise until the solution be-
came cloudy, and the solution was concentrated once
again. The resulting yellow oil was purified by HPLC
to afford a white solid that became sticky when exposed
to air (0.026 g, 99% for 8; 0.036 g, 99% for 9).
To a solution of oxamate 25b (0.50 g, 0.56 mmol) in
THF (4 mL) a solution of lithium hydroxide (0.14 g,
6.0 mmol) in water (3 mL) was added at room tempera-
ture. After 3 h the reaction mixture was diluted with
water and extracted with methylene chloride. The or-
ganic layer was dried over anhydrous sodium sulfate
and concentrated in vacuo. The resulting cloudy oil
was purified by flash chromatography (hexane/ethyl ace-
tate 1:1) to afford a clear, colorless oil (0.40 g, 90%). This
procedure was repeated for the other isomer.
1
Compound 8: H NMR (500 MHz, D2O) d 3.72 (m,
1
R-isomer: H NMR (400 MHz, CDCl3) d 7.05–7.50 (m,
1H), 3.35 (m, 1H), 3.17 (m, 2H), 2.86 (m, 2H), 1.34–
1.71 (m, 10H). 13C NMR (100 MHz, D2O) d 159.0,
67.0, 51.9, 51.5, 40.6, 39.4, 37.7, 33.6, 29.3, 23.2. HRMS
20H), 5.18 (m, 2H), 4.94 (m, 1H), 3.94–4.64 (m, 9H),
2.92–3.34 (m, 5H), 1.92 (m, 2H), 1.12–1.72 (m, 8H),
1.44 (s, 18H). MS (ESI) (m/z): 794.6 (M+1).
(ES) (m/z): M+H+ Calcd for C10H24N6O3, 277.1988.
24
D
Found: 277.1997. ½aꢂ +3.85 (syn-isomer).
1
S-isomer: H NMR (400 MHz, CDCl3) d 6.90–7.62 (m,
1
20H), 5.15 (m, 2H), 5.04 (m, 1H), 4.10–4.78 (m, 9H),
2.68–3.41 (m, 5H), 1.08–1.66 (m, 10H), 1.42 (s, 18H).
MS (ESI) (m/z): 794.6 (M+1).
Compound 9: H NMR (500 MHz, D2O) d 3.68 (m,
1H), 3.31 (m, 1H), 3.16 (m, 2H), 2.85 (m, 2H), 1.32–
1.76 (m, 10H). 13C NMR (100 MHz, D2O) d 159.0,
69.5, 50.8, 40.7, 39.4, 38.2, 34.0, 29.6, 23.0. HRMS
4.17. (4S)-[N-(tert-Butyloxycarbonylamino)-(6R)-hydro-
xy-9-(N-tert-butyloxycarbonylamino)nonylamine (27b)
(ES) (m/z): M+H+ Calcd for C10H24N6O3, 277.1988.
24
D
Found: 277.2000. ½aꢂ +1.23 (anti-isomer).
To solution of 26b (1.01 g, 1.27 mmol) in ethanol
(50 mL) was added Pd(OH)2/C (0.25 g). The suspension
was exposed to hydrogen gas, heated to 60 ꢁC, and al-
lowed to stir overnight. The suspension was then filtered
through a pad of Celite and concentrated in vacuo to af-
ford a yellow oil. The crude yellow oil was used in the
next reaction without purification. This procedure was
repeated for the other isomer.
4.20. N-[(4S)-(tert-Butyloxycarbonylamino)-(6R)-hydro-
xy-8-{N-tert-butyloxycarbonyl-N-[2-ethoxy-(oxoace-
tyl)amino}octyl-N-(benzyloxycarbonyl)]amine (29b)
A two-necked 100 mL round bottom flask was equipped
with a water-jacketed condenser, flame-dried, and
placed under an argon atmosphere. Triphenylphosphine
(0.070 g, 0.26 mmol) and N-Boc-ethyl oxamate (0.059 g,
0.26 mmol) were added followed by a solution of the
Cbz-protected ethyl alcohol 20b (0.10 g, 0.24 mmol) in
dry THF (5 mL). The solution was cooled to 0 ꢁC, and
diisopropyl azodicarboxylate (0.053 mL, 0.26 mmol)
was added dropwise. The reaction mixture was then
heated to 60 ꢁC and was allowed to stir overnight. The
resulting light yellow liquid was concentrated in vacuo
and purified by flash chromatography (hexane/ethyl ace-
tate 5:1) to afford a clear colorless oil (0.050 g, 31%).
This procedure was repeated for the other isomer.
4.18. N-{(4S)-[tert-Butyloxycarbonylamino]-(6R)-hydro-
xy-9-(N-tert-butyloxycarbonylamino)nonyl}nitroguani-
dine (28b)
To a solution of crude amine 27b in absolute ethanol
(20 mL) were added N-nitromethylthioguanidine
(0.17 g, 1.27 mmol) and triethylamine (1 mL). The solu-
tion was heated at 40 ꢁC for 5 min and was allowed to
stir overnight at room temperature. The reaction mix-

6106
E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
1
R-isomer: H NMR (400 MHz, CDCl3) d 7.37 (m, 5H),
5.10 (s, 2H), 4.93 (m, 1H), 4.62 (m, 1H), 4.32 (q,
J = 8.0 Hz, 2H), 3.79 (m, 2H), 3.65 (m, 2H), 3.21 (m,
3H), 1.72 (m, 2H), 1.39–1.60 (m, 6H), 1.51 (s, 9H),
1.42 (s, 9H), 1.35 (t, J = 7.9 Hz, 3H). MS (ESI) (m/z):
632.5 (M+Na+).
hexane 7:1) to afford a clear, colorless oil (15 mg,
32%). This procedure was repeated for the other isomer.
R-isomer: 1H NMR (400 MHz, CD3OD) d 3.65 (m, 1H),
3.11–3.38 (m, 5H), 1.60 (m, 2H), 1.25–1.50 (m, 6H), 1.43
(m, 18H). MS (ES) (m/z): 463.3 (M+1).
1
S-isomer: H NMR (400 MHz, CDCl3) d 7.34 (m, 5H),
S-isomer: 1H NMR (400 MHz, CD3OD) d 3.66 (m, 2H),
3.24 (m, 2H), 3.14 (m, 2H), 1.63 (m, 2H), 1.40–1.50 (m,
6H), 1.43 (m, 9H), 1.42 (m, 9H). MS (ES) (m/z): 463.3
(M+1).
5.07 (s, 2H), 4.84 (m, 1H), 4.53 (m, 1H), 4.33 (q,
J = 8.0 Hz, 2H), 3.79 (m, 3H), 3.60 (m, 2H), 3.19 (m,
2H), 1.66 (m, 2H), 1.38–1.58 (m, 6H), 1.51 (s, 9H),
1.43 (s, 9H), 1.35 (t, J = 8.0 Hz, 3H). MS (ESI) (m/z):
632.5 (M+Na+).
4.24. N-[(4S,8)-Diamino-(6S)-hydroxyoctyl]nitroguani-
dine (10) and N-[(4S,8)-diamino-(6R)-hydroxyoctyl]nitro-
guanidine (11)
4.21. N-[(4S)-(tert-Butyloxycarbonylamino)-(6R)-hydro-
xy-8-{N-(tert-butyloxycarbonyl)amino}octyl-N-(benzyl-
oxycarbonyl)]amine (30b)
Compound 32a (0.010 g, 0.022 mmol) or 32b (0.015 g,
0.032 mmol) was dissolved in 50% TFA in methylene
chloride (2 mL) and allowed to stir for 3 h at room tem-
perature. The mixture was concentrated in vacuo, and
the resulting yellow oil was dissolved in ethanol. Con-
centrated HCl was added dropwise until the solution be-
came cloudy, and the solution was concentrated once
again. The resulting yellow oil was purified by HPLC
to afford a white solid that became sticky when exposed
to air (0.007 g, 95% for 10; 0.010 g, 93% for 11).
To a solution of oxamate 29b (0.05 g, 0.08 mmol) in
THF (4 mL), a solution of lithium hydroxide (0.14 g,
6 mmol) in water (3 mL) was added at room tempera-
ture. After 3 h the reaction mixture was diluted with
water and extracted with methylene chloride. The or-
ganic layer was dried over anhydrous sodium sulfate
and concentrated in vacuo. The resulting cloudy oil
was purified by flash chromatography (hexane/ethyl ace-
tate 1:1) to afford a clear, colorless oil (0.04 g, 98%). This
procedure was repeated for the other isomer.
Compound 10: 1H NMR (500 MHz, D2O) d 3.82 (m, 1H),
3.36(m, 1H),3.15(m, 2H),2.97(m,2H), 1.52–1.77(m, 8H).
13C NMR (100 MHz, D2O) d 159.0, 65.6, 51.9, 51.5, 40.6,
1
R-isomer: H NMR (400 MHz, CDCl3) d 7.35 (m, 5H),
5.08 (s, 2H), 4.95 (m, 2H), 4.68 (m, 1H), 3.78 (m, 1H),
3.63 (m, 2H), 3.43 (m, 1H), 3.19 (m, 2H), 3.10 (m,
1H), 1.16–1.73 (m, 8H), 1.43 (s, 18H). MS (ESI) (m/z):
510.3 (M+1).
38.0, 36.9, 34.0, 30.1. HRMS (ES) (m/z): M+H+ Calcd for
24
D
C9H23N6O3, 263.1832. Found: 263.1837. ½aꢂ +6.45 (syn-
isomer).
1
Compound 11: H NMR (500 MHz, D2O) d 3.80 (m,
1
S-isomer: H NMR (400 MHz, CDCl3) d 7.34 (m, 5H),
1H), 3.34 (m, 1H), 3.19 (m, 2H), 2.97 (m, 2H), 1.42–
1.82 (m, 8H). 13C NMR (100 MHz, D2O) d 159.0,
65.6, 51.9, 51.5, 40.5, 38.0, 36.8, 34.0, 29.4. HRMS
5.07 (s, 2H), 4.92 (m, 2H), 4.70 (m, 1H), 3.75 (m, 1H),
3.67 (m, 2H), 3.37 (m, 1H), 3.18 (m, 3H), 1.16–1.72
(m, 8H), 1.43 (s, 9H), 1.42 (s, 9H). MS (ESI) (m/z):
510.3 (M+1).
(ES) (m/z): M+H+ Calcd for C9H23N6O3, 263.1832.
24
D
Found: 263.1842. ½aꢂ +2.53 (anti-isomer).
4.22. N-[(4S)-(tert-Butyloxycarbonylamino)-(6R)-hydro-
xy-8-{N-(tert-butyloxycarbonyl)amino}octylamine (31b)
4.25. Enzyme assay
The NOS isoforms used were recombinant enzymes over-
expressed in E. coli. Murine macrophage iNOS,48 rat
nNOS,49 and bovine eNOS50 were overexpressed and iso-
lated as reported. Interspecies similarity of each isozyme
is quite high (80–94% identical);51 therefore, the source
of the NOS used in various studies has been thought not
to be important. The formation of nitric oxide was mon-
itored using a hemoglobin capture assay as described pre-
viously.52 Briefly, a solution of nNOS or eNOS containing
10 lM L-arginine, 1.6 mM CaCl2, 11.6 lg/mL calmodu-
lin, 100 lM DTT, 100 lM NADPH, 6.5 lM H4B,
3 mM oxyhemoglobin, and varying concentrations of
inhibitor in 100 mM Hepes (pH 7.4) was monitored at
30 ꢁC. For the determination of inhibition of iNOS, no
additional Ca2+ or calmodulin was added. The assay
was initiated by the addition of enzyme, and the absorp-
tion of UV light at 400 nm was recorded over 1 min. As
NO was evolved and coordinated to the hemoglobin,
the absorption at 400 nm increased, producing a value
for the enzyme velocity under these conditions.
To a solution of 30b (0.050 g, 0.10 mmol) in methanol
(10 mL) was added Pd/C (0.050 g). The suspension was
exposed to hydrogen gas and allowed to stir overnight.
The suspension was then filtered through a pad of Celite
and concentrated in vacuo to afford a yellow oil. The
crude yellow oil was used in the next reaction without
purification. This procedure was repeated for the other
isomer.
4.23. N-[(4S)-(tert-Butyloxycarbonylamino)-(6R)-hydro-
xy-8-{N-(tert-butyloxycarbonyl)amino}octyl]nitroguani-
dine (32b)
To a solution of crude amine 31b in absolute ethanol
(5 mL) was added N-nitromethylthioguanidine (13 mg,
0.10 mmol). The solution was heated at 40 ꢁC for
5 min and allowed to stir overnight at room tempera-
ture. The reaction mixture was concentrated in vacuo
and purified by flash chromatography (ethyl acetate/

E. P. Erdal et al. / Bioorg. Med. Chem. 15 (2007) 6096–6108
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4.26. Enzyme inhibition studies
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A value for the initial rate was obtained when no inhib-
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then determined in the presence of varying concentra-
tions of inhibitor, until a concentration of inhibitor that
reduced the enzyme velocity to half its initial value
(v/v0 ꢁ 0.5) was discovered. Concentrations of inhibitor
above and below this value were tested and a graph of
v/v0 versus inhibitor concentration ([I]) was plotted.
Extrapolation of this graph allowed the determination
of an IC50 value. The Ki was estimated from the IC50
using the equations below. The Km values used were:
1.3 lM (nNOS), 8.3 lM (iNOS), and 1.7 lM (eNOS).
16. Urushitani, M.; Shimohama, S. Amyotrop. Lateral Scler.
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We are grateful to the National Institutes of Health
(GM 49725 to R.B.S., and GM52419 and HL30050 to
Prof. Bettie Sue Masters, in whose laboratory P.M.
and L.J.R. work) for financial support of this work.
Supplementary data
NOESY spectra for determination of absolute diastereo-
meric structures of 19a,b. HPLC traces with two different
eluents to demonstrate purity of 4–11. Supplementary
data associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2007.06.038.
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