Aromatic reduced amide bond peptidomimetics as selective inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1021/jm0202932

Nitric oxide synthase inhibitors could act as important therapies for disorders arising from overstimulation or overexpression of individual nitric oxide synthase (NOS) isoforms. But preservation of physiologically important nitric oxide functions require the use of isoform-selective inhibitors. Recently we reported reduced amide bond pseudodipeptide analogues as potent and selective neuronal nitric oxide synthase (nNOS) inhibitors (Hah, J.-M.; Roman, L. J.; Martasek, P.; Silverman, R. B. J. Med. Chem. 2001, 44, 2667-2670). To increase the lipophilicity a series of aromatic, reduced amide bond analogues (6-25) were designed and synthesized as potential selective nNOS inhibitors. The hypothesized large increase in isoform selectivity of nNOS over inducible NOS was not obtained in this series. However, the high potency with nNOS as well as high selectivity of nNOS over endothelial NOS was retained in some of these compounds (15, 17, 21), as well as good selectivity over inducible NOS. The most potent nNOS inhibitor among these compounds is N-(4S)-{4-amino-5-[2-(2-aminoethyl)-phenylamino]-pentyl}-N′- nitroguanidine (17) (K_i = 50 nM), which also shows the highest selectivity over eNOS (greater than 2100-fold) and 70-fold selectivity over iNOS. Further modification of compound 17 should lead to even more potent and selective nNOS inhibitors.

Full text of DOI:10.1021/jm0202932

J . Med. Chem. 2003, 46, 1661-1669
1661
Ar om a tic Red u ced Am id e Bon d P ep tid om im etics a s Selective In h ibitor s of
Neu r on a l Nitr ic Oxid e Syn th a se
J ung-Mi Hah,‡,§ Pavel Marta´sek,†, Linda J . Roman,†,| and Richard B. Silverman*,‡
Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University,
Evanston, Illinois 60208-3113, and Department of Biochemistry, The University of Texas Health Science Center,
San Antonio, Texas 78284-7760
Received J uly 8, 2002
Nitric oxide synthase inhibitors could act as important therapies for disorders arising from
overstimulation or overexpression of individual nitric oxide synthase (NOS) isoforms. But
preservation of physiologically important nitric oxide functions require the use of isoform-
selective inhibitors. Recently we reported reduced amide bond pseudodipeptide analogues as
potent and selective neuronal nitric oxide synthase (nNOS) inhibitors (Hah, J .-M.; Roman, L.
J .; Martasek, P.; Silverman, R. B. J . Med. Chem. 2001, 44, 2667-2670). To increase the
lipophilicity a series of aromatic, reduced amide bond analogues (6-25) were designed and
synthesized as potential selective nNOS inhibitors. The hypothesized large increase in isoform
selectivity of nNOS over inducible NOS was not obtained in this series. However, the high
potency with nNOS as well as high selectivity of nNOS over endothelial NOS was retained in
some of these compounds (15, 17, 21), as well as good selectivity over inducible NOS. The
most potent nNOS inhibitor among these compounds is N-(4S)-{4-amino-5-[2-(2-aminoethyl)-
phenylamino]-pentyl}-N′-nitroguanidine (17) (Ki ) 50 nM), which also shows the highest
selectivity over eNOS (greater than 2100-fold) and 70-fold selectivity over iNOS. Further
modification of compound 17 should lead to even more potent and selective nNOS inhibitors.
In tr od u ction
Additionally, the nNOS-derived nitric oxide or peroxy-
nitrite (a reaction of nitric oxide with superoxide anion)9
exacerbates acute ischemic injury. Animal studies and
early clinical trials suggest that nitric oxide synthase
inhibitors could be therapeutic in many of these disor-
ders,10 but preservation of physiologically important
nitric oxide functions would require the use of isoform-
selective inhibitors. Because eNOS plays a key role in
maintaining normal blood pressure, most investigators
believe therapeutically useful NOS inhibitors must not
inhibit eNOS.11
Nitric oxide (NO)1 is involved in a number of physi-
ological functions, acting as an inter- and intracellular
signaling agent. A family of enzymes, the nitric oxide
synthases (NOS, EC 1.14.13.39), catalyzes the stepwise
oxidation of L-arginine to L-citrulline and nitric oxide.
Three distinct NOS isoforms have been isolated and
characterized, each associated with different physiologi-
cal functions: blood-vessel dilation (endothelial NOS or
eNOS),2 neuronal signal transmission (neuronal NOS
or nNOS),3 immune response such as cytotoxicity against
pathogens and tumors (inducible NOS or iNOS).4
Overstimulation or overexpression of individual nitric
oxide synthase isoforms plays a role in a wide range of
disorders including septic shock, arthritis, diabetes,
ischemia-reperfusion injury, pain, and various neuro-
degenerative diseases.5,6 High levels of sustained nitric
oxide generated by nNOS activation as a result of
disrupted calcium homeostasis are associated with
numerous neurodegenerative disorders such as ischemic
brain, Parkinson’s disease, and Huntington’s disease.7
Studies with nNOS knockout mice have indicated that
nitric oxide produced by nNOS during neuronal injury
is associated with glutamate toxicity in the brain.8
Our aim is to develop nNOS-selective inhibitors. In
our labs libraries of nitroarginine-containing dipep-
tides,12 dipeptide esters,12 and dipeptide amides13 were
synthesized as possible candidates for nNOS-selective
inhibition based on two observations. First, L-nitroargi-
nine itself is a potent nNOS inhibitor (Ki ) 15 nM),14
and it has about a 250-fold selectivity over iNOS, but
only a 2.5-fold selectivity over eNOS. Second, L-arginine-
containing dipeptides are good substrates for the various
isoforms of NOS,15,16 suggesting that the active site of
NOS is flexible enough to accommodate larger molecules
than arginine. Indeed, crystal structures of the oxyge-
nase domains of eNOS17 and iNOS18 showed that there
is a large opening, which allows the diffusion of both
the substrate and the product (L-arginine and L-citrul-
line). Furthermore, there are significant structural
differences between isoforms just outside of the substrate-
binding pocket. From these two aspects, it was hypoth-
esized that dipeptides containing nitroarginine could fit
into the active site of NOS and at the same time utilize
the structural differences to achieve isoform selectivity.
Twelve nitroarginine (L or D)-containing dipeptides and
dipeptide esters were synthesized in the standard way,12
* Address correspondence to this author at the Department of
Chemistry. Professor Richard B. Silverman. Phone: (847) 491-5653.
Fax: (847) 491-7713. E-mail: Agman@chem.northwestern.edu.
‡ Department of Chemistry and Department of Biochemistry, Mo-
lecular Biology, and Cell Biology, Northwestern University.
† Department of Biochemistry, The University of Texas Health
Science Center.
§ Carried out all of the experiments in this study.
Developed the eNOS overexpression system in E. coli and isolated
and purified the eNOS.
| Developed the overexpression system for nNOS in E. coli and the
purification of the enzyme.
10.1021/jm0202932 CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/29/2003

1662 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Hah et al.
Ch a r t 1. Dipeptides Containing Nitroarginine as
Selective Inhibitors of nNOS
and 152 dipeptide amides were prepared using a solid-
phase synthesis method.13 They were tested against
each isoform of NOS. Among these libraries of com-
pounds, several dipeptides were found to be potent and
nNOS-selective inhibitors (Chart 1). The dipeptide
amides containing amino acids with a nitrogen-contain-
ing side chain, such as Lys, Orn, and Dbu, were
relatively potent inhibitors of nNOS. They also have
great selectivity over eNOS (1, 3 in Chart 1), implying
that the terminal amine group is significant for selectiv-
ity over eNOS.13 Among the dipeptide esters, D-Phe-D-
ArgNO2-OMe (2) showed excellent selectivity for nNOS
over iNOS (1800-fold), although the potency is weak,
and the selectivity for nNOS over eNOS is minimal.
Even though there are several exceptions, when
L-ArgNO2 is at the N-terminus of the dipeptide inhibitors,
an L-amino acid is also favored at the C-terminus. But
when ArgNO2 is at the C-terminus, the amino acid was
more selective as the D-isomer. From these observations,
a retro-inverso dipeptide model was proposed.13 In this
case, the nitroarginine residue binds to the same
binding site, regardless of its position in the dipeptide
(Figure 1). For example, D-ArgNO2 of 4 may flip over 180°
to assume an L-ArgNO2 configuration (like 3) at the
N-terminus for binding (Figure 1). Molecular modeling
and energy minimization of the retro-inverso dipeptide
amides 3 and 4 demonstrate perfect overlap; further-
more, recent ENDOR spectroscopic results19 confirmed
that the dipeptides bind to holo-nNOS similarly from
the point of view of the nitroguanidino functionality.
If the retro-inverso binding model holds for D-Phe-D-
ArgNO2-OMe (2) at the active site of nNOS, then it can
be expected to bind shown in Figure 2. The important
terminal nitrogen interaction of 1 (or 3 or 4) in the active
site is lost; instead, the phenyl ring moiety replaces the
C-terminal residue active site. This can explain the
weaker potency and minimal selectivity for nNOS over
eNOS of 2. However, the replacement of the phenyl ring
could also attribute to the high selectivity of nNOS over
iNOS (1800-fold); it is not as effective as the amine
group moiety at the nNOS active site, but much more
unfavorable in the active site of iNOS.
F igu r e 1. A proposed model for binding of the retro-inverso
dipeptide amide 3 and 4 at the active site of nNOS, taken from
ref 10.
F igu r e 2. A proposed model for binding of the retro-inverso
dipeptide amide 2 at the active site of nNOS.
is from the aromatic ring moiety), it was thought that
great nNOS-selective inhibitors could be achieved by
combining the aromatic ring and the terminal nitrogen
moieties into a dipeptide inhibitor. This also would
increase the lipophilicity of the molecule for enhanced
absorption properties. Finally, the successful surrogate
of the amide bond, namely, the reduced-amide bond,20
could be incorporated. Based on all of these observa-
tions, two series of aromatic, reduced amide bond
peptidomimetic compounds as possible selective inhibi-
tors of nNOS were designed (Charts 2 and 3).
Ch em istr y
All of the desired compounds (5-25, Charts 2 and 3)
were prepared by reductive amination, using N-Boc-L-
nitroargininal (26)21 as the key intermediate, which was
reductively coupled to various aromatic diamines. Com-
pounds 8-13 were synthesized according to Scheme 1.
N-Boc-L-nitroargininal (26) was coupled with (2-, 3-, or
4-aminomethyl)anilines (27-29), and [2-,3-, or 4-(2-
aminoethyl)]anilines (30-32), respectively. The reduc-
tive amination was carried out in good yields without
protection of the aniline group because alkylamines are
From this hypothesis (selectivity for nNOS over eNOS
is from the terminal nitrogen, and selectivity over iNOS

Inhibitors of Neuronal Nitric Oxide Synthase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1663
Sch em e 2. Syntheses of 30 and 31
Ch a r t 2. Phenyl Reduced Amide Bond Peptidomimetics
Sch em e 3. Synthesis of the Substituted Phenyl,
Reduced Amide Bond Analogues (6-7, and 14-19)
Ch a r t 3. Pyridinyl Reduced Amide Bond
Peptidomimetics
Sch em e 1. Synthesis of the Substituted Phenyl,
Reduced Amide Bond Analogues (8-13)
with 0.05% acetic acid, flow rate ) 1 mL/min) gave one
peak for 13, 17, and 25. It is not clear if the enantiomers
were not separated by these procedures or if there is
only one isomer formed, but the racemization of ni-
troargininal is known to be limited (only 9% at room
temperature after 22 h.22 The specific rotations for all
three of these compounds were greater than zero, so, if
the stereochemistry of the compounds was compro-
mised, it was not to the degree of racemization.
Most of the corresponding aromatic diamines were
commercially available, but 2- and 3-(2-aminoethyl)-
aniline (30, 31) were synthesized (Scheme 2). The
starting materials were the corresponding (nitrophenyl)-
acetonitriles, which were sequentially reduced by BH3-
THF23 complex and tin chloride hydrate24 to give 30 and
31.
much more reactive than aniline. Because of the relative
acidity of the R-proton of N-Boc-L-nitroargininal, it is
reasonable that reductive amination could lead to
racemization. Chiral chromatography was carried out
to determine if loss of chirality occurred. Chiral normal-
phase HPLC of the free base (Daicel ChiralPak AD,
ChiralPakAS, ChiralPak OD; solvents system: hexanes/
isopropyl alcohol (30-80%) with 0.5% diethylamine,
flow rate ) 1 mL/min), reverse-phase HPLC with
o-phthaldialdehyde, N-acetyl-L-cysteine derivatization
(Alltech, Econosil C18; solvent system: 80% methanol
+ 20% 50 mM sodium acetate pH 9, flow rate ) 1 mL/
min), and reverse-phase chiral HPLC of the salts (Regis
ChirosilSCA; solvent system: water/methanol (40-70%)
The aniline amino group of the (aminoalkyl)anilines
(27-32) were coupled with N-Boc-L-nitroargininal
(Scheme 3). In this case, protection of the alkylamine
moieties preceded the reductive amination (except 36
and 39 were commercially available).25 Using 2 equiv

1664 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Hah et al.
Sch em e 4. Synthesis ofthe Pyridyl, Reduced-amide
Bond Analogues (20-25)
F igu r e 3. Inhibition of nNOS by 17. A: Dixon plot for the
inhibition of nNOS by 17. B: Cornish-Bowden plot of com-
petitive inhibition.
of (aminoalkyl)aniline was sufficient to attain mono-
protection, while 8 equiv of the (aminoalkyl)aniline was
necessary for the monoprotection of R,ω-dialkylamines.
Resu lts a n d Discu ssion
As shown in the Table 1, all of the aromatic, reduced
amide bond analogues were found to be nNOS-selective
inhibitors. When compared to the lead compound,
RedAm-ethyl (50), the potencies on nNOS are decreased
(except 17), but most of the compounds showed still
higher potency than D-Phe-D-ArgNO2-OMe (2). However,
the potencies with iNOS also were decreased from those
of 50, except for 14 and 17, but not as low as 2, giving
good nNOS/iNOS selectivities. Disappointingly, the
Ki values with iNOS are closer to that of 50 or L-
ArgNO2-L-Dbu-NH2 (1) than to that of D-Phe-D-
ArgNO2-OMe (2), indicating that the desired unfavorable
interaction of the aromatic moiety in the iNOS active
site did not occur in this series, and the nNOS/iNOS
selectivities were not as high as expected.
The first two benzenediamine compounds (6, 7) were
found to be poorer inhibitors of nNOS and iNOS than
RedAm-ethyl (50), while having little difference toward
eNOS inhibition. This results in the dramatic decrease
of isoform selectivity of nNOS over eNOS (from 2577-
fold to 36- and 51-fold). This implies the amino residue
of the C-terminal phenyl ring is not long enough for an
effective interaction with the enzyme.
The members of the (aminomethyl)aniline series
(8-10) were also poor inhibitors of nNOS and iNOS.
The rigid C-terminal aniline structure does not seem
to be favored for interaction with the enzyme in the
nNOS active site. This is clear in the case of the ortho-
isomers (8 and 14), which showed almost the same
potency with all of the isozymes, thereby resulting in
the loss of isoform selectivity. However, when the
(aminomethyl)aniline moiety was attached at the ani-
lineamino group (but with the same length, 14-16), a
favorable result was obtained for the meta-isomer (15),
indicating that a flexible amino residue is better for
selective interaction with nNOS and that spatial geom-
etry seems to be very important. Compound 15 showed
almost the same potency and selectivity as RedAm-ethyl
(50) implying that the spatial geometry in the enzyme
active site is similar.
Among these target molecules, 5 (Chart 2) could not
be made using this same synthetic method. The reduc-
tive coupling of mono-Boc-protected benzene-1,2-di-
amine (34) with N-Boc-L-nitroargininal was not suc-
cessful; the bulky Boc-group was thought to be the
reason. However, it turned out that the low reactivity
of benzene-1,2-diamine, not the steric factor, was the
cause, because reductive aminations with either free
benzene-1,2-diamine or monomethylcarbamoyl-protect-
ed benzene-1,2-diamine proved to be fruitless. Further-
more, N-Boc-L-nitroargininal is also somewhat unreac-
tive, because it exists in two forms in solution: cyclized
hemiaminal and aldehyde.12 To accomplish this reac-
tion, different reaction conditions, other than reductive
amination, are needed. However, further investigation
of this reaction was postponed until enzyme tests could
show whether this family of compounds was potent
enough to warrant synthesis of this analogue.
Scheme 4 shows the synthetic route for the pyridyl,
reduced amide bond analogues. All of the aminoalkyl
pyridines (44-49) were commercially available, and in
each case the reductive amination with N-Boc-L-ni-
troargininal worked well.
All products from the coupling reactions were purified
by column chromatography using various solvent sys-
tems, and the Boc group was deprotected in 30% TFA/
methylene chloride.
En zym ology. All of the aromatic, reduced amide
analogues are competitive inhibitors of the three iso-
forms of NOS. Representative plots for the competitive
inhibition of nNOS by 17 are shown in Figure 3. Dixon
analysis (Figure 3A)26 and the method of Cornish and
Bowden (Figure 3B)27 were used to determine the type
of inhibition.
The Ki data for the aromatic, reduced amide bond
analogues (6-25) are given in Table 1 along with the
data for the D-Phe-D-ArgNO2-OMe (2) and the reduced
amide ethyl analogue (50).20

Inhibitors of Neuronal Nitric Oxide Synthase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1665
Ta ble 1. NOS Inhibition by the Aromatic, Reduced Amide Analoguesa
Ki (µM)b
selectivityc
compound
nNOS
iNOS
eNOS
iNOS/nNOS
eNOS/nNOS
D-Phe-D-ArgNO2-OMe (2)d
RedAm-Ethyl (50)
2
3600
39
5
314
78.6
116
86.1
185
303
30.1
47.6
170
39.8
194
409
105
100
414
443
523
589
123
275
193
1800
320
127
149
4.25
278
204
135
122
458
5.28
381
245
70.2
118
217
53
2.5
2577
36
51
4.24
90
189
22.1
35
230
7.17
924
299
2121
45.2
249
153
951
370
254
133
56
0.12
2.20
2.28
20.3
2.06
1.60
1.36
1.36
0.74
5.55
0.21
1.37
0.05
2.21
1.66
2.90
0.55
1.59
2.21
2.06
0.76
6 (A, n ) 0, m ) 0, meta)
7 (A, n ) 0, m ) 0, para)
8 (A, n ) 1, m ) 0, ortho)
9 (A, n ) 1, m ) 0, meta)
10 (A, n ) 1, m ) 0, para)
11 (A, n ) 2, m ) 0, ortho)
12 (A, n ) 2, m ) 0, meta)
13 (A, n ) 2, m ) 0, para)
14 (A, n ) 0, m ) 1, ortho)
15 (A, n ) 0, m ) 1, meta)
16 (A, n ) 0, m ) 1, para)
17 (A, n ) 0, m ) 2, ortho)
18 (A, n ) 0, m ) 2, meta)
19 (A, n ) 0, m ) 2, para)
20 (B, n ) 1, ortho)
279
339
86.3
572
326
184
166
339
29.3
80
335
3.51
260
360
154
141
392
72.6
178
395
21 (B, n ) 1, meta)
256
247
33
86
520
22 (B, n ) 1, para)
23 (B, n ) 2, ortho)
24 (B, n ) 2, meta)
25 (B, n ) 2, para)
a
The Ki values are calculated from the measured IC50 values. The enzymes used are recombinant rat nNOS, recombinant murine
b
iNOS, and recombinant bovine eNOS. The Ki values represent at least duplicate measurements; standard deviations of (8-12% were
observed. c The ratio of Ki (eNOS or iNOS) to Ki (nNOS); all are nNOS selective. Data taken from ref 10.
d
were recorded on a Varian Inova 500-MHz or Varian Mercury
400-MHz NMR spectrometer. Chemical shifts are reported as
δ values in parts per million downfield from TMS (δ 0.0) as
the internal standard in CDCl3. For samples run in D2O, the
HOD resonance was arbitrarily set at 4.80 ppm, and the CD3-
OH resonance was set at 4.87 ppm in CD3OD. An Orion
Research model 701 pH meter with a general combination
electrode was used for pH measurements. Electrospray mass
spectra were obtained on a Micromass Quattro II spectrometer.
Elemental analyses were obtained from Oneida Research
Services, Inc., Whiteboro, NY. Thin-layer chromatography was
carried out on E. Merck precoated silica gel 60 F254 plates.
Amino acids were visualized with a ninhydrin spray reagent
or a UV/vis lamp. E. Merck silica gel 60 (230-400 mesh) was
used for flash column chromatography.
The inhibitory potency of the (aminoethyl)aniline
series 11-13 and 17-19 also varied depending on the
location of the rigid phenyl ring. It appears that the
para-isomer is preferred when the phenyl ring is at the
C-terminus (13), but the ortho-isomer is much more
preferred when the flexible alkylamine residue is at the
C-terminus (17) in the same length of compounds.
Compound 17 was the best nNOS inhibitor (Ki ) 50 nM)
of all of the compounds this series, and the isoform
selectivity over eNOS was also excellent (>2000-fold);
however, the selectivity over iNOS is only 70-fold.
The variation of the inhibitory potencies with nNOS
as a result of a small structural change was also found
in the pyridinyl series (20-25). They are generally weak
inhibitors of nNOS; 21 was the best of these compounds,
with good isoform selectivity over iNOS and eNOS.
In conclusion, the hypothesized great increase in
isoform selectivity of nNOS over iNOS was not obtained
in the aromatic, reduced amide bond peptidomimetic
series. However, the high potency with nNOS as well
as high selectivity of nNOS over eNOS was retained in
some of these compounds (15, 17, 21), as well as good
selectivity over iNOS. Further modification of compound
17, which was the best nNOS-inhibitor in these series
of compounds, and the most potent nNOS inhibitor of
all of the dipeptide or dipeptidomimetic compounds so
far reported, should be an effective way to develop more
potent and selective nNOS-inhibitors.
High-performance liquid chromatography was performed on
a Beckman System Gold (Model 125P solvent module and
Model 166 detector). In case of analytical HPLC, samples were
analyzed by elution from a Hypersil ODS C18 column (Agilent,
5 µm, 4.0 × 250 mm), with a flow rate of 1 mL/min. The mobile
phase was a gradient from 100% solvent A (0.1% TFA in water)
and 0% solvent B (0.1% of TFA in CH3CN) to 60% solvent A
and 40% solvent B over 5 min, then to 30% solvent A and 70%
solvent B over 20 min and to 100% solvent A over 10 min.
Sample elution was detected by absorbance at 254 nm. For
the purification of final products, a Whatman Partsil C18 semi-
prep HPLC column (9.4 × 125 mm) was used. Samples were
eluted using a gradient of 100% solvent A (0.1% TFA in water)
to 70% of solvent B (0.1% of TFA in CH3CN) over 30 min at a
flow rate of 4 mL/min.
Rea gen ts a n d Ma ter ia ls. Amino acids were purchased
from Advanced ChemTech, Inc. NADPH, calmodulin, and
human ferrous hemoglobin were obtained from Sigma Chemi-
cal Co. Tetrahydrobiopterin (H4B) was purchased from Alexis
Biochemicals. HEPES, DTT, and conventional organic solvents
were purchased from Fisher Scientific. 2-, 3-, and 4-(amino-
Exp er im en ta l Section
Gen er a l Meth od s. NOS assays were recorded on a Perkin-
Elmer Lambda 10 UV/vis spectrophotometer. 1H NMR spectra

1666 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Hah et al.
methyl)aniline were purchased from TCI, America. All other
chemicals were purchased from Aldrich, unless otherwise
stated.
1.66 mmol) and (4-aminomethyl)aniline (284 µL, 2.49 mmol).
The residue was purified by flash column chromatography on
silica gel (CH2Cl2:MeOH ) 3:2) to afford NR-(tert-butoxycar-
bonyl)-(4S)-[4-amino-5-(2-aminobenzylamino)pentyl]-N′-ni-
troguanidine (410 mg, 62%) as a white solid. An aliquot of 304
mg was treated with 30% TFA in CH2Cl2 to give 10 (204 mg,
89%, brown solid): 1H NMR (500 MHz, D2O) δ 7.43 (d, J )
8.5 Hz, 2H), 7.28 (d, J ) 8.5 Hz, 2H), 4.16 (s, 2H), 3.52 (q, J )
5.5 Hz, 1H), 3.26 (d, J ) 5.5 Hz, 2H), 3.10 (brs, 2H), 1.45-
1.73 (m, 4H). HRMS (ES) (m/z): M + H+ calcd for C13H23N7O2
310.1991, found 310.1992.
N -(4S )-{[4-Am in o -5-(2-a m in o p h e n y l)e t h y la m in o ]-
p en tyl}-N′-n itr ogu a n id in e (11). This compound was pre-
pared as described above using NR-(tert-butoxycarbonyl)-L-
nitroargininal (400 mg, 1.32 mmol) and 2-(2-aminoethyl)-
aniline (361 mg, 2.65 mmol). The purification by flash column
chromatography on silica gel (CH2Cl2:MeOH ) 1:1) gave
NR-(tert-butoxycarbonyl)-(4S)-{[4-amino-5-(2-aminophenyl)eth-
ylamino]pentyl}-N′-nitroguanidine (415 mg, 74%). Removal of
the Boc-group yielded 282 mg of 11 (89%, brown solid): 1H
NMR (500 MHz, D2O) δ 7.26 (m,4H), 3.53 (m, 1H), 3.26 (m,
4H), 3.12 (brs, 2H), 2.95 (t, J ) 6.8 MHz, 2H), 1.36-1.80 (m,
4H). HRMS (ES) (m/z): M + H+ calcd for C14H26N7O2 324.2148,
found 324.2151
N -(4S )-{[4-Am in o -5-(3-a m in o p h e n y l)e t h y la m in o ]-
p en tyl}-N′-n itr ogu a n id in e (12). This compound was pre-
pared as described above using NR-(tert-butoxycarbonyl)-L-
nitroargininal (400 mg, 1.32 mmol) and 3-(2-aminoethyl)-
aniline (361 mg, 2.65 mmol). The purification by flash column
chromatography on silica gel (CH2Cl2:MeOH ) 1:1) gave
NR-(tert-butoxycarbonyl)-(4S)-[4-amino-5-(3-amino-phenyl)-eth-
ylamino)pentyl]-N′-nitroguanidine (250 mg, 45%). Removal of
the Boc-group yielded 189 mg of 12 (99%, brown solid): 1H
NMR (400 MHz, D2O) δ 7.30 (d, J ) 7.2 Hz, 1H), 7.21 (d, J )
7.2 Hz, 1H), 7.11 (m, 2H), 3.49 (m, 1H), 3.20 (m, 4H), 3.11
(brs, 2H), 2.90 (t, J ) 7.2 MHz, 2H), 1.33-1.77 (m, 4H). HRMS
(ES) (m/z): M + H+ calcd for C14H26N7O2 324.2148, found
324.2147.
N -(4S )-{[4-Am in o -5-(4-a m in o p h e n y l)e t h y la m in o ]-
p en tyl}-N′-n itr ogu a n id in e (13). This compound was pre-
pared as described above using NR-(tert-butoxycarbonyl)-L-
nitroargininal (500 mg, 1.66 mmol) and 4-(2-aminoethyl)-
aniline (338 µL, 2.49 mmol). The purification by flash column
chromatography on silica gel (CH2Cl2:MeOH ) 2:1) gave
NR-(tert-butoxycarbonyl)-(4S)-[4-amino-5-(4-aminophenyl)eth-
ylamino)pentyl]-N′-nitroguanidine (380 mg, 54%). Removal of
the Boc-group yielded 218 mg of 13 (95%, yellow solid): 1H
NMR (500 MHz, D2O) δ 7.28 (d, J ) 8 Hz, 2H), 7.21 (d, J ) 8
Hz, 2H), 3.53 (m, 1H), 3.24 (m, 4H), 3.15 (brs, 2H), 2.94 (t, J
) 7.5 MHz, 2H), 1.43-1.77 (m, 4H). HRMS (ES) (m/z): M +
H+ calcd for C14H26N7O2 324.2148, found 324.2150. Anal.Calcd
Ch em ica l Syn th esis. Gen er a l P r oced u r e for th e Re-
d u ctive Am in a tion of 6-25. To a solution of NR-(tert-
butoxycarbonyl)-L-nitroargininal21 (1 equiv) in dry methanol
were added aromatic diamine (1.5 equiv) and 3 Å molecular
sieves, and the mixture was stirred at room temperature. After
being stirred for 1 h, the reaction mixture was treated with
sodium triacetoxyborohydride (2 equiv) and was stirred over-
night. The reaction mixture was filtered, and the filtrate was
concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel to afford the N-Boc
product as a solid, which was treated with 30% TFA in CH2-
Cl2 for 2 h. Excess trifluoroacetic acid and solvent were
removed by evaporation. The residue was dissolved in a small
amount of water, which was washed with ether and lyophi-
lized.
N-(4S)-{[4-Am in o-5-(3-a m in o)p h en yla m in o]p en tyl}-N′-
n itr ogu a n id in e (6). This compound was prepared as de-
scribed above using NR-(tert-butoxycarbonyl)-L-nitroargininal
(500 mg, 1.66 mmol) and (3-aminophenyl)carbamic acid tert-
butyl ester (514.9 mg, 2.49 mmol). The purification by flash
column chromatography on silica gel (EtOAc:hexane ) 3:1)
gave
NR-(tert-butoxycarbonyl)-(4S)-{[4-amino-5-(3-amino)-
phenylamino]pentyl}-N′-nitroguanidine (300 mg, 37%). Re-
moval of the Boc-group yielded 170 mg of 6 (95%, purple
solid): 1H NMR (500 MHz, D2O) δ 7.11 (t, J ) 8 Hz, 1H), 6.60
(d, J ) 8 Hz, 1H), 6.52 (d, J ) 8 Hz, 1H), 6.47 (s, 1H), 3.31 (m,
1H), 3.22 (m, 2H), 3.10 (brs, 2H), 1.41-1.73 (m, 4H). HRMS
(ES) (m/z): M + H+ calcd for C12H22N7O2 296.1835, found
296.1846.
N-(4S)-{[4-Am in o-5-(4-a m in o)p h en yla m in o]-p en t yl}-
N′-n itr ogu a n id in e (7). This compound was prepared as
described above using NR-(tert-butoxycarbonyl)-L-nitroargini-
nal (500 mg, 1.66 mmol) and (4-aminophenyl)carbamic acid
tert-butyl ester (515 mg, 2.49 mmol). The purification by flash
column chromatography on silica gel (EtOAc:hexane ) 2:1)
gave
NR-(tert-butoxycarbonyl)-(4S)-{[4-amino-5-(4-amino)-
phenylamino]pentyl}-N′-nitroguanidine (400 mg, 49%). Re-
moval of Boc-group from 360 mg yielded 197 mg of 7 (92%,
orange solid): 1H NMR (500 MHz, D2O) δ 7.04 (d, J ) 8 Hz,
2H), 6.65 (d, J ) 8 Hz, 2H), 3.34 (m, 1H), 3.30 (m, 2H), 3.14
(brs, 2H), 1.40-1.81 (m, 4H). HRMS (ES) (m/z): M + H+ calcd
for C12H22N7O2 296.1835, found 296.1848.
N-(4S)-[4-Am in o-5-(2-a m in oben zyla m in o)p en tyl]-N′-n i-
tr ogu a n id in e (8). This compound was prepared as described
above using NR-(tert-butoxycarbonyl)-L-nitroargininal (359 mg,
1.19 mmol) and (2-aminomethyl)aniline (204 µL, 2.49 mmol).
The residue was purified by flash column chromatography on
silica gel (CH2Cl2:MeOH ) 3:2) to afford NR-(tert-butoxycar-
bonyl)-(4S)-{4-amino-5-[(2-amino)benzylamino]pentyl}-N′-ni-
troguanidine (270 mg, 56%) as a white solid. Removal of the
Boc-group (30% TFA) gave a yellow foam (204 mg, 95% yield):
1H NMR (400 MHz, D2O) δ 7.57 (m, 2H), 7.46 (d, J ) 3.6 Hz,
1H), 7.29 (m, 1H), 3.44 (m, 1H), 3.31 (m, 2H), 3.17 (m, 2H),
1.46-1.90 (m, 4H). HRMS (ES) (m/z): M + H+ calcd for
for
C14H25N7O2‚3TFA‚H2O: C, 35.15,. H, 4.42,. N,14.35;
Found: C, 35.65, H, 4.25, N, 14.30. [R]24 +2.96 (c ) 1.15,
D
MeOH).
N -(4S )-{4-Am in o-5-[(2-a m in om e t h yl)p h e n yla m in o]-
p en tyl}-N′-n itr ogu a n id in e (14). This compound was pre-
pared as described above using NR-(tert-butoxycarbonyl)-L-
nitroargininal (200 mg, 0.73 mmol) and (2-aminobenzyl)-
carbamic acid tert-butyl ester (324 mg, 1.46 mmol). The
purification by flash column chromatography on silica gel
(EtOAc:hexane ) 2:1) gave NR-(tert-butoxycarbonyl)-(4S)-{4-
amino-5-[(3-aminomethyl)phenylamino]pentyl}-N′-nitroguani-
dine (160 mg, 43%). Removal of the Boc-group yielded 92.3
mg of 14 (95%, orange solid): 1H NMR (400 MHz, D2O) δ 7.55
(m, 2H), 7.46 (m, 1H), 7.27 (m, 1H), 3.63 (m, 1H), 3.31 (m,
2H), 3.23 (m, 4H), 1.40-1.88 (m, 4H). HRMS (ES) (m/z): M +
H+ calcd for C13H23N7O2 310.1991, found 310.1931.
N -(4S )-{4-Am in o-5-[(3-a m in om e t h yl)p h e n yla m in o]-
p en tyl}-N′-n itr ogu a n id in e (15). This compound was pre-
pared as described above using NR-(tert-butoxycarbonyl)-L-
nitroargininal (458.5 mg, 1.52 mmol) and (3-aminobenzyl)-
carbamic acid tert-butyl ester (675 mg, 3.04 mmol). The
purification by flash column chromatography on silica gel
(EtOAc:hexane ) 1:1) gave NR-(tert-butoxycarbonyl)-(4S)-{4-
C
13H23N7O2 310.1991, found 310.1920.
N-(4S)-[4-Am in o-5-(3-a m in oben zyla m in o)p en tyl]-N′-n i-
tr ogu a n id in e (9). This compound was prepared as described
above using NR-(tert-butoxycarbonyl)-L-nitroargininal (500 mg,
1.66 mmol) and (3-aminomethyl)aniline (284 µL, 2.49 mmol).
The residue was purified by flash column chromatography on
silica gel (CH2Cl2:MeOH ) 3:2) to afford NR-(tert-butoxycar-
bonyl)-(4S)-[4-amino-5-(2-aminobenzylamino)pentyl]-N′-ni-
troguanidine (400 mg, 58.9%) as a white solid. Removal of Boc-
group gave a light brown foam of 9 (278 mg, 92% yield): 1H
NMR (500 MHz, D2O) δ 7.23 (m, 2H), 7.18 (brs, 1H), 7.13 (d,
J ) 3 Hz, 1H), 4.01 (s, 2H), 3.39 (q, J ) 6 Hz, 1H), 3.12 (d, J
) 5.5 Hz, 2H), 2.93 (brs, 2H), 1.25-1.65 (m, 4H). HRMS (ES)
(m/z): M + H+ calcd for C13H23N7O2 310.1991, found 310.2008.
N-(4S)-[4-Am in o-5-(4-a m in oben zyla m in o)p en tyl]-N′-n i-
tr ogu a n id in e (10). This compound was prepared as described
above using NR-(tert-butoxycarbonyl)-L-nitroargininal (500 mg,

Inhibitors of Neuronal Nitric Oxide Synthase
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1667
amino-5-[(3-aminomethyl)phenylamino]pentyl}-N′-nitroguani-
dine (320 mg, 41%). Removal of the Boc-group yielded 120 mg
of 15 (62%, brown solid): 1H NMR (400 MHz, D2O) δ 7.18 (t,
J ) 7.5 Hz, 1H), 6.79 (m, 3H), 3.91 (s, 2H), 3.55 (m, 1H), 3.37
(m, 2H), 3.09 (m, 2H), 1.36-1.82 (m, 4H). HRMS (ES) (m/z):
M + H+ calcd for C13H23N7O2 310.1991, found 310.1992.
Anal.Calcd for C13H23N7O2‚3TFA‚2H2O: C, 33.19,‚ H, 4.40,. N,-
14.26; Found: C, 33.62, H, 4.38, N, 14.19.
N -(4S )-{4-Am in o-5-[(4-a m in om e t h yl)p h e n yla m in o]-
p en tyl}-N′-n itr ogu a n id in e (16). This compound was pre-
pared as described above using NR-(tert-butoxycarbonyl)-L-
nitroargininal (500 mg, 1.66 mmol) and (4-aminobenzyl)-
carbamic acid tert-butyl ester (569 mg, 2.49 mmol). The
purification by flash column chromatography on silica gel
(EtOAc:hexane ) 2:1) gave NR-(tert-butoxycarbonyl)-(4S)-{4-
amino-5-[(4-aminomethyl)phenylamino]pentyl}-N′-nitroguani-
dine (390 mg, 46%). Removal of the Boc-group yielded 234 mg
of 16 (99%, pale yellow solid): 1H NMR (400 MHz, D2O) δ 7.12
(d, J ) 8 Hz, 2H), 6.65 (d, J ) 8 Hz, 2H), 3.89 (s, 2H), 3.36 (m,
1H), 3.29 (m, 2H), 3.14 (brs, 2H), 1.36-1.62 (m, 4H). HRMS
(ES) (m/z): M + H+ calcd for C13H23N7O2 310.1991, found
310.1992.
yellow solid. Removal of the Boc-group gave 20 as a pale yellow
foam (251 mg, 99%, brown solid): 1H NMR (500 MHz, D2O) δ
8.64 (d, J ) 5.5 Hz, 1H), 8.37 (t, J ) 8 Hz, 1H), 7.91 (d, J ) 8
Hz, 1H), 7.83 (t, J ) 6.5 Hz, 1H), 4.51 (s, 2H), 3.57 (m, 1H),
3.37 (m, 2H), 3.12 (brs, 2H), 1.42-1.80 (m, 4H). HRMS (ES)
(m/z): M + H+ calcd for C12H22N7O2 296.1385, found 296.1837.
N-(4S)-{4-Am in o-5-[(pyr idin -3-yl)m eth yl]am in o]pen tyl}-
N′-n itr ogu a n id in e (21). This compound was prepared as
described above using NR-(tert-butoxycarbonyl)-L-nitroargini-
nal (400 mg, 1.32 mmol) and 1-(pyridin-3-yl)methylamine (202
µL, 1.98 mmol). The residue was purified by flash column
chromatography on silica gel (EtOAc:MeOH ) 1:1) to afford
NR-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(pyridin-3-ylmethy-
l)amino]pentyl}-N′-nitroguanidine (300 mg, 58%) as a yellow
solid. Removal of the Boc-group gave 21 as a greenish yellow
foam (217 mg, 97%): 1H NMR (500 MHz, D2O) δ 8.78 (brs,
1H), 8.68 (d, J ) 5.5 Hz, 1H), 8.56 (d, J ) 7.5 Hz, 1H), 7.96 (t,
J ) 7.5 Hz, 1H), 4.39 (s, 2H), 3.55 (m, 1H), 3.36 (m, 2H), 3.08
(brs, 2H), 1.39-1.82 (m, 4H). HRMS (ES) (m/z): M + H+ calcd
for C12H22N7O2 296.1835, found 296.1839. Anal.Calcd for
C
12H21N7O2‚3TFA‚2H2O: C, 32.10, H, 4.19, N,14.56; Found:
C, 31.62, H, 4.16, N, 14.47.
N -(4S )-{4-Am in o-5-[2-(2-a m in oe t h yl)p h e n yla m in o]-
p en tyl}-N′-n itr ogu a n id in e (17). This compound was pre-
pared as described above using NR-(tert-butoxycarbonyl)-L-
nitroargininal (463.5 mg, 1.53 mmol) and [2-(2-aminophenyl)-
ethyl]carbamic acid tert-butyl ester (544 mg, 2.30 mmol). The
purification by flash column chromatography on silica gel
(EtOAc:hexane ) 2:1) gave NR-(tert-butoxycarbonyl)-(4S)-{4-
amino-5-[2-(2-aminoethyl)phenylamino]pentyl}-N′-nitroguani-
dine (300 mg, 38%). Removal of the Boc-group yielded 176 mg
of 17 (95%, brown solid): 1H NMR (400 MHz, D2O) δ 7.06 (t,
J ) 6.8 Hz, 1H), 6.94 (d, J ) 6.8 Hz, 1H), 6.60 (m, 2H), 3.37
(m, 1H), 3.26 (m, 2H), 3.10 (brs, 2H), 3.04 (t, J ) 7.2 Hz, 2H),
2.72 (t, J ) 7.2 Hz, 2H), 1.38-1.78 (m, 4H). HRMS (ES) (m/z):
M + H+ calcd for C14H26N7O2 324.2148, found 324.2137.
Anal.Calcd for C14H25N7O2‚3TFA‚H2O: C, 35.15, H, 4.42, N,-
N-(4S)-{4-Am in o-5-[(pyr idin -4-yl)m eth yl]am in o]pen tyl}-
N′-n itr ogu a n id in e (22). This compound was prepared as
described above using NR-(tert-butoxycarbonyl)-L-nitroargini-
nal (420 mg, 1.39 mmol) and 1-(pyridin-4-yl)methylamine (211
µL, 2.09 mmol). The residue was purified by flash column
chromatography on silica gel (EtOAc:MeOH ) 5:2) to afford
NR-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(pyridin-4-ylmeth-
yl)aminopentyl}-N′-nitroguanidine (419 mg, 76%) as a yellow
solid. Removal of the Boc-group gave 22 as a orange yellow
foam (310 mg, 99%): 1H NMR (500 MHz, D2O) δ 8.72 (d, J )
6 Hz, 2H), 8.01 (d, J ) 6 Hz, 2H), 4.50 (s, 2H), 3.62 (m, 1H),
3.43 (m, 2H), 3.15 (brs, 2H), 1.46-1.83 (m, 4H). HRMS (ES)
(m/z): M + H+ calcd for C12H22N7O2 296.1835, found 296.1848.
N-(4S)-{4-Am in o-5-[(2-pyr idin -2-yl)eth yl]am in open tyl}-
N′-n itr ogu a n id in e (23). This compound was prepared as
described above using NR-(tert-butoxycarbonyl)-L-nitroargini-
nal (415 mg, 1.37 mmol), 2-(pyridin-2-yl)ethylamine (190 µL,
1.51 mmol). The residue was purified by flash column chro-
matography on silica gel (EtOAc:MeOH ) 1:1) to afford NR-
(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(2-pyridin-2-yl)ethyl]-
aminopentyl}-N′-nitroguanidine (400 mg, 71%) as a yellow
solid. Removal of the Boc-group gave 23 as a light brown foam
(296 mg, 98%): 1H NMR (500 MHz, D2O) δ 8.38 (d, J ) 5.5
Hz, 1H), 8.21 (t, J ) 8 Hz, 1H), 7.66 (d, J ) 8 Hz, 1H), 7.63
(m, 1H), 3.41 (m, 1H), 3.30 (m, 2H), 3.24 (m, 2H), 3.16 (m,
2H), 2.95 (brs, 2H), 1.26-1.62 (m, 4H). HRMS (ES) (m/z): M
+ H+ calcd for C13H24N7O2 310.1991, found 310.1995.
N-(4S)-{4-Am in o-5-[(2-pyr idin -3-yl)eth yl]am in open tyl}-
N′-n itr ogu a n id in e (24). This compound was prepared as
described above using NR-(tert-butoxycarbonyl)-L-nitroargini-
nal (460 mg, 1.52 mmol), 2-(pyridin-3-yl)ethylamine dihydro-
bromide salt (509 mg, 1.82 mmol). The residue was purified
by flash column chromatography on silica gel (EtOAc:MeOH
) 1:1) to afford NR-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(2-
pyridin-3-yl)ethyl]aminopentyl}-N′-nitroguanidine (404 mg,
65%) as a yellow solid. Removal of the Boc-group gave 24 as a
pale yellow foam (290 mg, 95%): 1H NMR (500 MHz, D2O) δ
8.49 (brs, 1H), 8.45 (d, J ) 4.5 Hz, 1H), 8.30 (d, J ) 6.5 Hz,
1H), 7.77 (ddd, J ) 4.5, 6 Hz, 1H), 3.46 (m, 1H), 3.25 (m, 2H),
3.20 (m, 2H), 3.07 (m, 2H), 3.02 (brs, 2H), 1.35-1.71 (m, 4H).
HRMS (ES) (m/z): M + H+ calcd for C13H24N7O2 310.1991,
found 310.1995.
N-(4S)-{4-Am in o-5-[(2-pyr idin -4-yl)eth ylam in o]pen tyl}-
N′-n itr ogu a n id in e (25). This compound was prepared as
described above using NR-(tert-butoxycarbonyl)-L-nitroargini-
nal (400 mg, 1.32 mmol), 2-(pyridin-4-yl)ethylamine (249 µL,
1.98 mmol). The residue was purified by flash column chro-
matography on silica gel (EtOAc:MeOH ) 1:1) to afford
NR-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(2-pyridin-4-yl)eth-
ylamino]pentyl}-N′-nitroguanidine (400 mg, 74%) as a yellow
solid. Removal of the Boc-group gave 25 as a white foam (287
mg, 95%): 1H NMR (500 MHz, D2O) δ 8.45 (d, J ) 6 Hz, 1H),
14.35; Found: C, 35.59,‚ H, 4.32, ‚N, 14.41. [R]24 +2.70 (c )
D
0.89, MeOH).
N -(4S )-{4-Am in o-5-[3-(2-a m in oe t h yl)p h e n yla m in o]-
p en tyl}-N′-n itr ogu a n id in e (18). This compound was pre-
pared as described above using NR-(tert-butoxycarbonyl)-L-
nitroargininal (433 mg, 1.43 mmol) and [3-(2-aminophenyl)-
ethyl]carbamic acid tert-butyl ester (508 mg, 2.15 mmol). The
purification by flash column chromatography on silica gel
(EtOAc:hexane ) 2:1) gave NR-(tert-butoxycarbonyl)-(4S)-{4-
amino-5-[3-(2-aminoethyl)phenylamino]pentyl}-N′-nitroguani-
dine (314 mg, 42%). Removal of the Boc-group yielded 174 mg
of 18 (90%, brown solid): 1H NMR (400 MHz, D2O) δ 7.07 (t,
J ) 7.6 Hz, 1H), 6.59 (m, 3H), 3.55 (m, 1H), 3.45 (m, 2H), 3.31
(m, 2H), 3.09 (m, 4H), 1.34-1.74 (m, 4H). HRMS (ES) (m/z):
M + H+ calcd for C14H26N7O2 324.2148, found 324.2137.
N -(4S )-{4-Am in o-5-[4-(2-a m in oe t h yl)p h e n yla m in o]-
p en tyl}-N′-n itr ogu a n id in e (19). This compound was pre-
pared as described above using NR-(tert-butoxycarbonyl)-L-
nitroargininal (477 mg, 1.58 mmol) and [4-(2-amino-phenyl)-
ethyl]carbamic acid tert-butyl ester (560 mg, 2.37 mmol). The
purification by flash column chromatography on silica gel
(EtOAc:hexane ) 2:1) gave NR-(tert-butoxycarbonyl)-(4S)-{4-
amino-5-[4-(2-aminoethyl)phenylamino]pentyl}-N′-nitroguani-
dine (300 mg, 36%). Removal of the Boc-group yielded 176 mg
of 19 (95%, light brown solid): 1H NMR (500 MHz, D2O) δ
7.01 (d, J ) 8 Hz, 2H), 6.64 (d, J ) 8 Hz, 2H), 3.60 (m, 1H),
3.49 (m, 1H), 3.29(m, 3H), 3.15 (m, 2H), 2.72 (t, J ) 7.5 Hz,
2H), 1.42-1.81 (m, 4H). HRMS (ES) (m/z): M + H+ calcd for
C
14H26N7O2 324.2148, found 324.2154.
N-(4S)-{4-Am in o-5-[(pyr idin -2-yl)m eth yl]am in open tyl}-
N′-n itr ogu a n id in e (20). This compound was prepared as
described above using NR-(tert-butoxycarbonyl)-L-nitroargini-
nal (400 mg, 1.32 mmol) and 1-(pyridin-2-yl)methylamine
(204.8 µL, 1.98 mmol). The residue was purified by flash
column chromatography on silica gel (EtOAc:MeOH ) 1:1) to
afford NR-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(pyridin-2-yl-
methyl)amino]pentyl}-N′-nitroguanidine (340 mg, 65%) as a

1668 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9
Hah et al.
7.73 (d, J ) 6 Hz, 2H), 3.49 (q, J ) 6 Hz, 1H), 3.30 (m, 2H),
3.21 (m, 1H), 3.15 (t, J ) 7.5 Hz, 2H), 3.03 (brs, 2H), 1.33-
1.76 (m, 4H). HRMS (ES) (m/z): M + H+ calcd for C13H24N7O2
Hz, 1H), 6.63 (t, J ) 7.2 Hz, 1H), 3.18 (t, J ) 8 Hz, 2H), 2.67
(t, J ) 8 Hz, 2H), 1.43 (s, 9H). HRMS (EI) m/z (M+) calcd for
C
13H20N2O2 236.1524, found 236.1537.
310.1991, found 310.1994. [R]24 +5.30 (c ) 0.88, MeOH).
[(3-Am in op h en yl)eth yl]ca r ba m ic Acid ter t-Bu tyl Ester
D
(42). This compound was prepared as described above using
di-tert-butyl dicarbonate (401 mg, 1.84 mmol) and 3-(2-ami-
noethyl)aniline (401 mg, 1.84 mmol). The residue was purified
by flash column chromatography on silica gel (EtOAc:hexane
) 2:1) to afford 508 mg of product (quantitative yield, yellow
2-(2-Am in oeth yl)a n ilin e (30). To a solution of (2-nitro-
phenyl)acetonitrile (3 g, 18.5 mmol) was added dropwise a 1
M BH3-THF solution (125 mL) at 0 °C. After 4 h of stirring
at 25 °C, 6 N HCl solution (125 mL) was added to the reaction
mixture at 0 °C. After evaporation of the organic solvent in
vacuo, the aqueous phase was basified with a 4 N NaOH
solution to pH 10. The product was extracted with EtOAc, and
the organic phase was dried over MgSO4 and concentrated in
vacuo. A brown liquid (32, 2 g, 10.8 mmol) was isolated.
Without further purification, it was mixed with SnCl2‚2H2O
(13.57 g, 60.2 mmol) and absolute ethanol (20 mL). The
suspension was heated at 70 °C under nitrogen. After being
stirred for 30 min, the starting material disappeared, the
solution was allowed to cool, then poured into ice (100 g). The
pH was made slightly basic (pH 8) by the addition of a 5%
aqueous NaHCO3 solution, and the resulting basic mixture was
stirred for 1 h. The precipitate was extracted with ethyl acetate
(100 mL × 3), then the extract was washed with water (50
mL × 3) and dried over MgSO4. The product was obtained in
a yield of 52% (1.3 g, brown liquid, over two steps) after
evaporation of the solvent: 1H NMR (400 MHz, CD3OD) δ 6.97
(t, J ) 7.2 Hz, 2H), 6.72 (d, J ) 7.2 Hz, 2H), 6.64 (t, J ) 7.2
Hz, 1H), 2.82 (t, J ) 7.2 Hz, 2H), 2.66 (t, J ) 7.2 Hz, 2H).
HRMS (EI) m/z (M+) calcd for C8H12N2 136.1000, found
136.1002.
1
solid): H NMR (400 MHz, CD3OD) δ 7.00 (t, J ) 8 Hz, 1H),
6.58 (m, 3H), 3.21 (t, J ) 7.6 Hz, 2H), 3.63 (t, J ) 7.6 Hz, 2H),
1.42 (s, 9H). HRMS (EI) m/z (M+) calcd for C13H20N2O2
236.1524, found 236.1508.
[(4-Am in op h en yl)eth yl]ca r ba m ic Acid ter t-Bu tyl Ester
(43). This compound was prepared as described above using
di-tert-butyl dicarbonate (1 g, 7.34 mmol) and 4-(2-aminoethyl)-
aniline (0.8 g, 3.67 mmol). The product was obtained in a yield
of 98% (850 mg, pale yellow solid): 1H NMR (400 MHz, CDCl3)
δ 6.96 (d, J ) 8.4 Hz, 2H), 6.62 (d, J ) 8.4 Hz, 2H), 3.29 (t, J
) 6.4 Hz, 2H), 2.66 (t, J ) 6.4 Hz, 2H), 1.43 (s, 9H). HRMS
(EI) m/z (M+) calcd for C13H20N2O2 236.1524, found 236.1555.
En zym e a n d Assa y. All of the NOS isoforms used are
recombinant enzymes overexpressed in E. coli from different
sources; there is very high sequence identity for the isoforms
from different sources. The murine macrophage iNOS was
expressed and isolated according to the procedure of Hevel et
al.28 The rat nNOS was expressed29 and purified as described.30
The bovine eNOS was isolated as reported.31 Nitric oxide
formation from NOS was monitored by the hemoglobin capture
assay as described at 30 °C.32 A typical assay mixture for
nNOS contained 3-15 µM l-arginine, 1.6 mM CaCl2, 11.6 µg/
mL calmodulin, 100 µM DTT, 100 µM NADPH, 6.5 µM BH4,
and 3 mM oxyhemoglobin in 100 mM Hepes (pH 7.5). The
reaction mixture for the iNOS assay included 10 µM L-
arginine, 100 µM DTT, 100 µM NADPH, 6.5 µM BH4, and 3
mM oxyhemoglobin in 100 mM Hepes (pH 7.5). The eNOS
assay mixture contained 3-25 µM l-arginine, 10 µM CaCl2, 1
µg/mL calmodulin, 100 µM DTT, 100 µM NADPH, 5 µM BH4,
and 80 µM oxyhemoglobin in 50 mM Hepes (pH 7.5). All assays
were in a final volume of 600 µL and were initiated by enzyme.
Nitric oxide reacts with oxyHb to yield methemoglobin which
was detected at 401 nm (ꢀ ) 19700 M-1 cm-1) on a Perkin-
Elmer Lambda 10 UV/vis spectrophotometer.
Deter m in a tion of Ki Va lu es. The reversible inhibition of
NOS by reduced-amide bond analogues was studied under
initial rate conditions with the hemoglobin assay as described
above. The type of competitive inhibition of nNOS was
determined from Dixon plots33 with various L-arginine and
inhibitor concentrations. The apparent Ki values were obtained
by measuring percent inhibition in the presence of 10 µM
L-arginine with at least three concentrations of inhibitor. The
parameters of the following inhibition equation33 were fitted
to the initial velocity data: % inhibition ) 100[I]/{[I] + Ki (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.
3-(2-Am in oeth yl)a n ilin e (31). This compound was pre-
pared as described above using (3-nitrophenyl)acetonitrile (2.2
g, 13.6 mmol). Evaporation of the solvent gave the product in
1
a yield of 81% (1.5 g, brown liquid, over two steps): H NMR
(400 MHz, CD3OD) δ 7.02 (t, J ) 7.2 Hz, 1H), 6.59 (m, 2H),
6.56 (m, 1H), 2.84 (t, J ) 7.2 Hz, 2H), 2.64 (t, J ) 7.2 Hz, 2H).
HRMS (EI) m/z (M+) calcd for C8H12N2 136.1000, found
136.0980.
(3-Am in op h en yl)ca r ba m ic Acid ter t-Bu tyl Ester (36).
A solution of di-tert-butyl dicarbonate (2 g, 9.16 mmol, 1 equiv)
in dioxane (25 mL) was added over a period of 30 min to a
solution of 1,3-phenylenediamine (2 g, 18.5 mmol, 2 equiv) in
dioxane (25 mL). The mixture was allowed to stir for 22 h,
and the solvent was removed using a rotary evaporator. The
residue was purified by flash column chromatography on silica
gel (EtOAc:hexane ) 2:1) to afford 1.7 g of product (89% based
on dicarbonate) as a peach color solid: 1H NMR (500 MHz,
CDCl3) δ 7.11 (t, J ) 8 Hz, 1H), 6.60 (d, J ) 8 Hz, 1H), 6.52 (t,
J ) 8 Hz, 1H), 6.47 (s, 1H), 4.79 (s, 1H), 1.44 (s, 9H). HRMS
(EI) m/z (M+) calcd for C11H16N2O2 208.1211, found 208.1201.
(2-Am in oben zyl)ca r ba m ic Acid ter t-Bu tyl Ester (38).
This compound was prepared as described above using di-tert-
butyl dicarbonate (2 g, 9.16 mmol) and 2-aminomethylaniline
(2 g, 18.5 mmol). The residue was purified by flash column
chromatography on silica gel (EtOAc:hexane ) 2:1) to afford
1.5 g of product (79% based on dicarbonate) as a light yellow
1
solid: H NMR (400 MHz, CDCl3) δ 7.10 (t, J ) 7.6 Hz, 1H),
Ack n ow led gm en t. We are grateful to the National
Institutes of Health for financial support of this research
to R.B.S. (GM49725) and Bettie Sue Siler Masters
(GM52419) and to the Robert A. Welch Foundation (AQ-
1192) for financial support to B.S.S.M, in whose lab P.M.
and L.J .R. work. Sincere thanks go to Prof. Michael A.
Marletta (University of California, Berkeley) for the E.
coli cells which express murine macrophage iNOS, to
Yaoqiu Zhu (Northwestern University) and Ted Szcz-
erba (Regis Technologies, Inc.) for chiral HPLC work
on the final compounds, and to Dr. Wenxin Gu for
obtaining the optical rotation data.
7.02 (d, J ) 6.8 Hz, 1H), 6.67 (m, 2H), 4.80 (s, 1H), 1.44 (s,
9H). HRMS (EI) m/z (M+) calcd for C12H18N2O2 222.1369, found
222.1357.
(3-Am in oben zyl)ca r ba m ic Acid ter t-Bu tyl Ester (39).
This compound was prepared as described above using di-tert-
butyl dicarbonate (0.89 g, 4.07 mmol) and 3-aminomethyla-
niline (1 g, 8.18 mmol). The residue was purified by flash
column chromatography on silica gel (EtOAc:hexane ) 2:1) to
afford 0.95 g of brown liquid (quantitative yield): 1H NMR (400
MHz, CDCl3) δ 7.08 (t, J ) 7.6 Hz, 1H), 6.64 (d, J ) 7.6 Hz,
1H), 6.58 (m, 2H), 4.91 (s, 2H), 1.45 (s, 9H). HRMS (EI) m/z
(M+) calcd for C12H18N2O2 222.1369, found 222.1357.
[(2-Am in op h en yl)eth yl]ca r ba m ic Acid ter t-Bu tyl Ester
(41). This compound was prepared as described above using
di-tert-butyl dicarbonate (0.3 g, 2.2 mmol) and 2-(2-amino-
ethyl)aniline (481 mg, 2.2 mmol). The product was obtained
as a pale yellow solid in a quantitative yield (544 mg): 1H NMR
(400 MHz, CD3OD) δ 6.97 (t, J ) 7.2 Hz, 2H), 6.71 (d, J ) 8
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