NG-aminoguanidines from primary amines and the preparation of nitric oxide synthase inhibitors

Article abstract of DOI:10.1021/jm701119v

A concise, general, and high-yielding method for the preparation of N ^G-aminoguanidines from primary amines is reported. Using available and readily prepared materials, primary amines are converted to protected N ^G-aminoguanidines in a one-pot procedure. The method has been successfully applied to a number of examples including the syntheses of four nitric oxide synthase (NOS) inhibitors. The inhibitors prepared were investigated as competitive inhibitors and as mechanistic inactivators of the inducible isoform of NOS (iNOS). In addition, one of the four inhibitors prepared, N^G-amino-N^G-2,2,2-trifluoroethyl-L-arginine 19, displays the unique ability to both inhibit NO formation and prevent NADPH consumption by iNOS without irreversible inactivation of the enzyme.

Full text of DOI:10.1021/jm701119v

924
J. Med. Chem. 2008, 51, 924–931
NG-Aminoguanidines from Primary Amines and the Preparation of Nitric Oxide Synthase
Inhibitors
Nathaniel I. Martin,† William T. Beeson, Joshua J. Woodward, and Michael A. Marletta*
Departments of Chemistry, Molecular and Cellular Biology, and DiVision of Physical Sciences, Lawrence Berkeley National Laboratory,
UniVersity of California, Berkeley, Berkeley, California 94720-3220
ReceiVed September 9, 2007
A concise, general, and high-yielding method for the preparation of NG-aminoguanidines from primary
amines is reported. Using available and readily prepared materials, primary amines are converted to protected
NG-aminoguanidines in a one-pot procedure. The method has been successfully applied to a number of
examples including the syntheses of four nitric oxide synthase (NOS) inhibitors. The inhibitors prepared
were investigated as competitive inhibitors and as mechanistic inactivators of the inducible isoform of NOS
(iNOS). In addition, one of the four inhibitors prepared, NG-amino-NG-2,2,2-trifluoroethyl-L-arginine 19,
displays the unique ability to both inhibit NO formation and prevent NADPH consumption by iNOS without
irreversible inactivation of the enzyme.
Scheme 1. Reaction Catalyzed by NOS
Introduction
NG-Aminoguanidines have been of medicinal interest for
some time and have been previously described as anticancer,
antibacterial, and antiviral agents.1,2 This class of compounds
also display dopamine ꢀ-oxidase inhibition and antihypertensive
properties.3 Substituted aminoguanidines have been shown to
inhibit the advanced nonenzymatic glycosylation of proteins4,5
and arylaminoguanidines have been described as a novel class
of 5-HT2aA receptor antagonists.6 Recent work has also
suggested that NG-aminoguanidine functionalities can be utilized
in the design of potent inhibitors for trypsin-like serine
proteases.7,8 Our interest in these compounds stems from our
continuing work with the nitric oxide synthases (NOSsa , EC
1.14.13.39) and previous reports that certain NG-ami-
noguanidines inhibit NOS activity.9,10 The NOSs are a family
of enzymes that catalyze the conversion of L-arginine to
L-citrulline and nitric oxide (NO) via the intermediate NG-
hydroxy-L-arginine (NHA; Scheme 1).11,12 NOSs are active as
homodimers, with each subunit containing a C-terminal reduc-
tase domain (with binding sites for NADPH, FAD, and FMN)
and an N-terminal oxygenase domain containing the heme
prosthetic group. The substrate, L-arginine, and a redox cofactor,
(6R)-5,6,7,8 tetrahydro-L-biopterin (H4B), both bind near the
heme center in the oxygenase domain.13
orders, and inflammation.15 For this reason, compounds capable
of inhibiting the NOSs, including NG-aminoguanidines, remain
of interest.
Despite the widespread interest in NG-aminoguanidines, few
general methods have been developed by which these func-
tionalities can be conveniently and safely constructed under mild
conditions. Traditional approaches involve the use of toxic
reagents and harsh reaction conditions and have generally relied
upon the addition of hydrazines to cyanimides (prepared by
CNBr treatment of the starting amine)16,17 or to thioureas
requiring preactivation with HgCl2, PbO, or alkyl iodides.18,19
Recently, Katritzky and co-workers described an alternate route
to NG-aminoguanidines via treatment of di(benzotriazol-1-
yl)methanimine with the appropriate amines and hydrazines in
refluxing toluene.20
As mentioned above, NG-aminoguanidine containing com-
pounds have also been described as inhibitors of NOS.9 NG-
Amino-L-arginine is a well-known irreversible inhibitor of NOS
and exhibits mechanism-based, time-dependent inactivation of
the enzyme.21 The nature of this inactivation has been previously
investigated and has been shown to be the result of a covalent
modification of the enzyme. Based on mass spectrometric
evidence, Osawa and co-workers have proposed a modification
pathway whereby the inhibitor is converted to a reactive
intermediate by the action of NOS itself.22 This reactive species
then covalently cross-links with the heme prosthetic group,
resulting in a catalytically inactive form of the enzyme (Scheme
2). Whether this heme-modification pathway is a general
mechanism by which NG-aminoguanidines inhibit NOS is
NO regulates numerous physiological processes, including
neurotransmission, smooth muscle relaxation, platelet reactivity,
and the cytotoxic activity of immune cells.14 Overproduction
of NO has been linked to the pathogenesis of a number of
disease states, including septic shock, neurodegenerative dis-
* To whom correspondence should be addressed. Departments of
Chemistry and Molecular and Cellular Biology, and Division of Physical
Sciences, Lawrence Berkeley National Laboratory, University of California,
Berkeley, 570 Stanley Hall, Berkeley, CA 94720-3220. Tel.: (510) 666-
2763. Fax: (510) 666-2765. E-mail: marletta@berkeley.edu.
†
Current address: Department of Medicinal Chemistry and Chemical
Biology, Utrecht University, F.A.F.C. Wentgebouw, Room Z615, Sorbon-
nelaan 16, 3584 CA Utrecht, The Netherlands.
a
Abbreviations: NOS, nitric oxide synthase; iNOS, inducible NOS;
iNOSheme, heme domain of inducible NOS; H4B, (6R)-5,6,7,8-tetrahydro-
L-biopterin; NADPH, nicotinamide adenine dinucleotide phosphate; FAD,
flavin adenine dinucleotide; FMN, flavin monomucleotide; CbZNCS,
N-benzyloxycarbonyl isothiocyanate; OxyMb, oxymyoglobin; DTT, dithio-
threitol; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid.
10.1021/jm701119v CCC: $40.75
2008 American Chemical Society
Published on Web 01/26/2008

NG-Aminoguanidines from Primary Amines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 925
Table 1. Cbz-Protected NG-Aminoguanidines Prepared
Scheme 2. Proposed Inactivation of NOS by
NG-Amino-L-arginine22
unknown and presented an additional motivation for our
investigation.
Results and Discussion
An initial objective in our investigation was the development
of a convenient and general approach for the synthesis of NG-
aminoguanidines. While the addition of nucleophiles to thioureas
has been previously described in this regard,18,19 the lack of
suitable protecting groups along with the use of toxic reagents
and harsh coupling conditions has somewhat limited the
generality of these approaches. We recently described the
preparation and use of N-benzyloxycarbonyl isothiocyanate
(CbzNCS) in the conversion of amines to protected NG-
hydroxyguanidines via thiourea intermediates. The thiourea
intermediates need not be isolated and are readily converted to
protected NG-hydroxyguanidines in a high-yielding, one-pot
procedure.23 We have found that a similar approach, whereby
Cbz-protected thioureas are activated with N-(3-dimethylami-
nopropyl)-N-ethylcarbodiimide hydrochloride (EDCI) and treated
with benzyl carbazate, is of equal generality in the synthesis of
NG-aminoguanidines (Scheme 3).
a Isolated yield. b An equivalent of NEt3 added to the amine hydrochloride
prior to treatment with CbzNCS.
secondary amines are required, the approach of Katritzy and
co-workers presents a viable alternative.20
After establishing the validity of this approach for the
preparation of the NG-aminoguanidine core, the methodology
was further applied to the preparation of four L-arginine
analogues to be investigated as inhibitors of iNOS. The four
L-arginine analogues were designed to probe the role of both
electron-rich and electron-poor substituents located at either of
the two amino positions originally deriving from the hydrazine
used in constructing the NG-aminoguanidine center. Benzyl
carbazate as well as the commercially available 2,2,2-trifluo-
roethyl- and monomethyl-substituted hydrazines were used as
starting materials to yield the four analogues each deriving from
the same thiourea precursor 8. The orthogonally protected
L-ornithine species 7 was first prepared by standard means and
converted to 8 by TFA deprotection, followed by treatment with
CbzNCS (Scheme 4).
Scheme 3. One-Pot Preparation of NG-Aminoguanidines
The strategy illustrated in Scheme 3 was successfully applied
to a variety of amines. The expected Cbz-protected NG-
aminoguanidine products were all rapidly formed at room
temperature and in good yields (Table 1). A practical advantage
of this approach is that the NG-hydroxyguanidine moiety is
installed in a fully Cbz-protected form, allowing for purification
by standard chromatographic techniques prior to deprotection.
While previously reported syntheses of NG-aminoguanidines
have provided the target molecules directly, the lack of
protecting groups can complicate purification owing to the high
polarity of such compounds. In addition, NG-aminoguanidines
are stable to reductive conditions, allowing for global removal
of the Cbz groups by standard hydrogenation to directly yield
the fully deprotected products (see below for further examples
and description of deprotection conditions). It should be noted,
however, that it is not possible to convert secondary amines to
the corresponding NG-aminoguanidines using this procedure.
The EDCI-mediated coupling likely occurs via the formation
of a transient carbodiimide24 not attainable with secondary
amines. In cases where NG-aminoguanidines derived from
Scheme 4. Preparation of Thiourea 8
The required hydrazines were then Cbz-protected using
standard carbamate chemistry (Scheme 5). In choosing the
position for the Cbz protecting group on the hydrazine, it is
possible to dictate the substitution pattern in the final NG-
aminoguanidine product. Compound 9 was prepared by direct
treatment of methylhydrazine with CbzCl leading to carbamate

926 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Martin et al.
Scheme 5. Preparation of Cbz-Protected Methyl and
2,2,2-Trifluoroethylhydrazines
substrate(s) in identical fashion to the full-length enzyme and
is catalytically competent when provided with an exogenous
source of electrons such as sodium dithionite.25 Addition of
16-19 to H4B-bound iNOSheme induced a low- to high-spin state
conversion at the heme center resulting from loss of water (FeIII-
aqua f FeIII-unligated) upon binding. This transition leads to
a decrease in absorbance at ∼418 nm and an increase in
absorbance at ∼394 nm in the UV–visible spectrum. The
magnitude of spectral change, ∆∆Abs (where ∆∆Abs )
∆Abs394 - ∆Abs418), is dependent on the concentration of the
added analogue. Spectral Kd values for each compound are then
determined by fitting the titration data to the saturation binding
equation: ∆∆Abs ) (∆∆Absmax × [compound])/(Kd + [com-
pound]). Table 2 summarizes the spectral binding assay results
for compounds 16-19 (graphic depictions of the titration
experiments for all four analogs provided in Supporting
Information).
protection at the more nucleophilic (methylated) nitrogen. When
methylhydrazine was first treated with Boc2O followed by
CbzCl, the Cbz group was installed at the less nucleophilic
nitrogen. Acid treatment to remove the Boc carbamate then
yielded compound 10. Compound 11 was obtained by treating
2,2,2-trifluoroethylhydrazine with CbzCl. In this case, the
electron withdrawing effect of the trifluoromethyl group led to
preferential protection of the less-substituted nitrogen.
Table 2. Spectral Kd Values for 16–19
a
compound
spectral Kd (µM)
L-arginineb
7.0 ( 0.7 (5.8 ( 1.6)
1.3 ( 0.2
NG-amino-L-arginine (16)
NG-methylamino-L-arginine (17)
NG-amino-NG-methyl-L-arginine (18)
29.0 ( 2.5
10.0 ( 0.5
NG-amino-NG-2,2,2-trifluoroethyl-L-arginine (19) 40.4 ( 3.8
The four Cbz-protected NG-amino-L-arginine analogues
12-15 were then prepared by treatment of thiourea 8 with
EDCI, NEt3, and the appropriate Cbz-protected hydrazine
(Scheme 6). Deprotection of 12-15 was accomplished by
hydrogenation over Pd/C to provide the amino acid products
16-19 as the acetate salts after lyophilization (with the
exception of compound 18, which was isolated as the free base
after lyophilization. as evidenced by NMR; Scheme 6).
a Spectral Kd values reported for 16–19 are averages obtained from
triplicate analysis. b Previously reported value given in parentheses.25
Next, compounds 16–19 were examined as inhibitors for full-
length iNOS. NOS activity was measured using a spectral assay
making use of the NO-induced oxidation of oxymyoglobin to
metmyoglobin observable at 405 nm. Table 3 lists the Ki values
determined for compounds 16–19 (with graphic depictions of
the iNOS inhibition assays for all four analogs provided in
Supporting Information).
Scheme 6. Preparation of Amino Acid Products 16–19
Table 3. Ki Values Determined for 16–19
compound
Kia (µM)
NG-amino-L-arginine (16)b
2.2 ( 0.5 (1.7 ( 0.6)
16.4 ( 5.0
NG-methylamino-L-arginine (17)
NG-amino-NG-methyl-L-arginine (18)
34.1 ( 7.2
NG-amino-NG-2,2,2-trifluoroethyl-L-arginine (19) 10.5 ( 5.7
a Values reported are averages obtained from triplicate analysis. b Previously
reported value given in parentheses.26
Compounds 16–19 were next investigated as time-dependent
inactivators of iNOS. Each compound was used at a concentra-
tion 10-fold higher than the measured Ki and allowed to react
with iNOS under turnover conditions (NADPH, H4B, O2). Time
points were obtained by quenching aliquots from each reaction
mixture into a concentrated L-arginine solution (final concentra-
tion of L-arginine 100-fold higher than compound tested). The
oxymyoglobin assay was then used to measure residual iNOS
activity as a function of preincubation time with 16–19. Of the
four compounds tested, only NG-amino-L-arginine 16 (as ex-
pected21) and NG-amino-NG-methyl-L-arginine 18 displayed an
ability to irreversibly inactivate iNOS in a time-dependent
manner. Kinactivation values were determined by plotting residual
iNOS activity as a function of preincubation time (Figure 1).
The Kinactivation value measured for NG-amino-L-arginine 16 was
0.18 ( 0.01 min-1 (compared to a previously reported value21
of 0.26 min-1). Compound 18 inactivates iNOS at a higher rate
NOS binding affinities for the four NG-amino-L-arginine
analogues were determined using a previously described spectral
binding assay.25 For this analysis, a truncated construct of iNOS
(iNOSheme, comprised of amino acids 1–490) was used. This
construct has been previously shown to bind heme, H4B, and
with a measured Kinactivation value of 0.39 ( 0.02 min-1
.
As mentioned above, Osawa and co-workers have shown that
NG-amino-L-arginine inactivates NOS by covalent modification
of the heme cofactor.22 To investigate whether or not a heme

NG-Aminoguanidines from Primary Amines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 927
Figure 1. Time-dependent inactivation of iNOS by 18.
modification mechanism was responsible for the iNOS inactiva-
tion observed with 18, a mass spectral analysis was used. iNOS
was reacted with either NG-amino-L-arginine or compound 18
in the presence of NADPH for 30 min after which the heme
content of the samples was assessed by LC-MS. While treatment
of iNOS with NG-amino-L-arginine led to formation of the
expected adduct22 (MW ) 775.3), no modified heme species
were detected in iNOS samples treated with 18. This suggests
that heme modification is not an inactivation pathway common
to all NG-aminoguanidine-based inhibitors of NOS. The irreversible
iNOS inactivation caused by compound 18 is possibly due to
modification of an amino acid(s) in the enzyme active site.
Compounds 17 and 19 were shown not to be time-dependent
inactivators of iNOS and were further examined for an ability
to inhibit iNOS-mediated NADPH oxidation (Figure 2). The
rate at which NADPH is oxidized by NOS is regulated by a
number of factors27 and in the absence of L-arginine the enzyme
consumes NADPH (Figure 2, basal activity) to generate reactive
oxygen species like superoxide and peroxide.27,28 While incuba-
tion with compound 17 led to no significant difference versus
the basal rate (no L-arginine present) of iNOS-mediated NADPH
consumption, compound 19 severely inhibited NADPH oxidase
activity.
Figure 3. Extracted spectral intermediates observed for heme transi-
tions from stopped-flow UV–visible spectroscopic analysis. Oxidation
of iNOSheme containing H2B in the presence of (A) L-arginine or (B)
compound 19.
To further elucidate the nature of the inhibition of iNOS-
mediated NADPH consumption by compound 19, stopped-flow
rapid-scanning UV/visible spectroscopic analyses were per-
formed. As previously described,29,30 the introduction of oxygen
to FeII-iNOSheme (reconstituted with redox-inactive (6R)-7,8-
dihydro-L-biopterin (H2B) and premixed with L-arginine) results
in a rapid conversion of the ferrous protein to the ferrous-oxy
complex, followed by the appearance of high-spin ferric protein
due to the dissociation of superoxide from the heme. Under
our experimental conditions, oxygen binding occurs at a rate
that was not resolvable, resulting in no observable ferrous
starting protein (Figure 3A). The data fit well to an A f B
kinetic model in which dissociation of superoxide is clearly
observed as a transition of an intermediate with a Soret
absorbance at 428 nm (FeII-O2) to a species at 396 nm (FeIII)
with a kobs ) 0.13 ( 0.02 s-1 (published value29 0.16 ( 0.02
s-1). Figure 3B illustrates the results obtained when oxygenated
buffer was mixed with H2B reconstituted FeII-iNOSheme pre-
mixed with compound 19. Again the data fit well to an A f B
Figure 2. Effect of 17 and 19 on iNOS-mediated NADPH consump-
tion. Compound 17 has little impact on the rate of NADPH consumption
by iNOS (similar to basal oxidation rate). Compound 19 causes a
significant decrease in the observed rate of NADPH oxidation (<5%
of the rate observed with L-arginine).

928 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Martin et al.
kinetic model, however, the protein showed a transition from
an intermediate with a Soret absorbance at 414 nm (FeII) to a
species at 396 nm (FeIII), with a kobs ) 0.0267 ( 0.0006. Unlike
the sample containing L-arginine, there was no observable build
up of a ferrous-oxy species when iNOSheme was premixed with
compound 19. The overall rate of iNOSheme oxidation when
premixed with 19 is much slower (∼20%) than when premixed
with L-arginine. The absence of a detectable ferrous-oxy species
with 19 suggests that the rate of oxidation under these conditions
is limited by the ability of oxygen to bind in the iNOS active
site. With L-arginine, oxygen binding occurs rapidly (no ferrous-
unligated iNOSheme detected) after which the rate-limiting step
is dissociation of superoxide from the heme. With compound
19, however, oxygen binding is likely the rate-limiting step with
superoxide dissociation occurring at a higher relative rate,
preventing accumulation of the ferrous-oxy species. A possible
explanation for these results is that when compound 19 binds
to iNOS the NG-amino or -2,2,2-trifluoroethyl group may be
positioned in the active site in such a way so as to effectively
interfere with oxygen binding due to steric crowding. This
inhibition of iNOS-mediated NADPH consumption by 19 is
reminiscent of another NOS inhibitor with similar properties,
NG-nitro-L-arginine (L-NNA).31 Crytallographic studies have
shown that when bound to the NOS active site, the nitro group
of L-NNA is specifically located above the heme cofactor, likely
preventing productive oxygen binding.32 Like compound 19,
NG-nitro-L-arginine inhibits NO production by competing for
the L-arginine binding site while also limiting the production
of reactive oxygen species by shutting off NADPH consumption.
These properties are unique and likely important in designing
therapeutically useful NOS inhibitors that can prevent both the
formation of NO and the formation of potentially damaging
reactive oxygen species.
isoform selective inhibition of NOS by compound 19 as well
as crystallographic investigations into its mode of binding at
the enzyme active site are warranted.
Experimental Section
All reagents employed were of American Chemical Society
(ACS) grade or finer and were used without further purification
unless otherwise stated. CbzNCS was prepared as described in the
literature.23 Melting points are uncorrected and were determined
on a Büchi oil immersion apparatus using open capillary tubes. 1H
NMR, 13C NMR, and 19F NMR spectra were obtained on a Bruker
AVQ-400 spectrometer. High-resolution FAB and ESI mass spectra
were obtained using VG ZAB2-EQ and Q-Tof Premier (Waters)
instruments, respectively. Microanalyses were obtained using a
Perkin-Elmer 2400 Series II combustion analyzer. Optical rotations
1
were recorded at 20 °C using a Perkin Elmer 241 polarimeter. H
and 13C NMR spectra for compounds 16–19 are provided in
Supporting Information.
(Z)-Benzyl
2-((Benzylamino)(benzyloxycarbonylamino)
methylene)hydrazinecarboxylate (1). Benzylamine (0.5 mmol,
54 mg, 55 µL) was dissolved in CH2Cl2 (5.0 mL) and treated with
CbzNCS (∼1.0 mL of a 0.5 M solution in CH2Cl2) at room
temperature until consumption of the amine was complete (typically
within minutes), as evidenced by ninhydrin visualized TLC analysis.
The mixture, now containing the thiourea intermediate, was then
treated with NEt3 (1.1 equiv, 0.55 mmol, 56 mg, 77 µL), EDCI
(1.1 equiv, 0.55 mmol, 105 mg), and benzyl carbazate (1.1 equiv,
0.55 mmol, 91 mg). After one hour, TLC analysis (2:1 hexane/
ethyl acetate) indicated formation of the desired product (Rf ) 0.35)
with residual thiourea (Rf ) 0.65) still present. An additional 1.1
equiv of each reagent was added, and after stirring for another hour,
the thiourea was completely consumed. The mixture was then
washed with 0.1 M HCl (2 × 5 mL), H2O (2 × 5 mL) and brine
(2 × 5 mL). The organic layer was dried over Na2SO4 followed
by evaporation under vacuum. The residue was applied to a silica
column and eluted with 2:1f1:1 hexane/ethyl acetate, yielding the
desired product as a white solid (210 mg, 97%). Mp 116–118 °C;
1H NMR (CDCl3, 400 MHz) δ 7.34–7.22 (m, 15H), 5.08 (s, 2H),
5.05 (s, 2H), 4.89 (s, 2H); 13C NMR (CDCl3, 100 MHz) δ 156.4,
137.3, 135.3, 128.7, 128.6, 128.4, 128.2, 127.6, 127.55, 68.0, 67.5,
45.0; HRMS (FAB, M + H) calcd for C24H25N4O4, 433.1876;
found, 433.1867; Anal. (C24H24N4O4) C, H, N.
From the results obtained, it is apparent that the electon-rich/
poor nature, as well as the location of substituents on the NG-
aminoguanidine center in the L-arginine analogues studied,
dictates the nature and mechanism of NOS inhibition observed.
While the four compounds investigated share the L-arginine
amino acid scaffold, different inhibitory activities are observed
with each species. Further study, employing a broader range of
substituted NG-aminoguanidines (now readily available using
the synthetic methodology described here), is likely to provide
a basis upon which the different inhibitory behaviors observed
with this class of NOS inhibitors may be rationalized.
(Z)-Benzyl 2-((Benzyloxycarbonylamino)(cyclopentylamino)
methylene)hydrazinecarboxylate (2). Compound 2 was prepared
as described above for compound 1. Product obtained as a white
solid (95%). Mp 118–120 °C; 1H NMR (CDCl3, 400 MHz) δ
7.38–7.27 (m, 10H), 5.10 (s, 2H), 5.09 (s, 2H), 4.13 (m, 1H),
1.95–1.90 (m, 2H), 1.61–1.53 (m, 4H), 1.35–1.26 (m, 2H); 13C
NMR (CDCl3, 100 MHz) δ 159.8, 156.6, 137.0, 135.5, 128.5, 128.4,
128.1, 127.9, 67.8, 67.1, 52.4, 33.0, 23.5; HRMS (FAB, M + H)
calcd for C22H27N4O4, 411.2032; found, 411.2040; Anal.
(C22H26N4O4) C, H, N.
Conclusion
In summary, we have developed a high-yielding methodology
for the preparation of NG-aminoguanidines that avoids the use
of toxic reagents and harsh reaction conditions. This method
was successfully applied to a number of examples, including
the synthesis of four L-arginine-based inhibitors of iNOS. The
mode of inhibition by these compounds was investigated and
shown to be different depending upon the nature of the
substituents present on the NG-aminoguanidine moiety. While
covalent modification of the heme cofactor plays a role in the
time-dependent inactivation of iNOS by NG-amino-L-arginine
16, this is not a general mechanism for all NG-aminoguanidines.
Compound 18 was shown to irreversibly inactivate iNOS by a
different mechanism and future efforts will be aimed at
establishing the nature of this inactivation. Furthermore, one
of the four compounds prepared, NG-amino-NG-2,2,2-trifluoro-
ethyl-L-arginine 19, was shown to be a competitive inhibitor of
iNOS that also inhibits enzymatic NADPH consumption by
interfering with oxygen binding. Further investigations into the
(Z)-Benzyl 2-((Benzyloxycarbonylamino)(tert-butylamino)
methylene)hydrazinecarboxylate (3). Compound 3 was prepared
as described above for compound 1. Product obtained as a sticky
1
foam (94%). H NMR (CDCl3, 400 MHz) δ 7.38–7.27 (m, 10H),
5.14–5.12 (m, 4H), 1.39 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ
156.6, 135.6, 128.5, 128.43, 128.36, 128.1, 128.0, 67.8, 67.6, 52.1,
29.0; HRMS (FAB, M + H) calcd for C21H27N4O4, 399.2032;
found, 399.2039; Anal. (C21H26N4O4) C, H, N.
(Z)-Benzyl 2-((Benzyloxycarbonylamino)(4-bromophenylamino)
methylene)hydrazinecarboxylate (4). Compound 4 was prepared
as described above for compound 1. Product obtained as a white
solid (91%). Mp 131–133 °C; 1H NMR (CDCl3, 400 MHz) δ
7.39–7.27 (m, 14H), 5.12 (s, 2H), 5.11 (s, 2H); 13C NMR (CDCl3,
100 MHz) δ 156.6, 136.1, 135.5, 132.1, 128.85, 128.76, 128.60,
128.57, 128.42, 128.38, 128.2, 124.1, 117.7, 67.94, 67.88; HRMS
(FAB, M + H) calcd for C23H22N4O4Br, 497.0824; found, 497.0821;
Anal. (C23H21N4O4Br) C, H, N.

NG-Aminoguanidines from Primary Amines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 929
(Z)-Benzyl 2-((Benzyloxycarbonylamino)(3,5-dimethylphenyl-
amino)methylene) hydrazinecarboxylate (5). Compound 5 was
prepared as described above for compound 1. Product obtained as
a white solid (96%). Mp 127–129 °C; 1H NMR (CDCl3, 400 MHz)
δ 7.38–7.27 (m, 10H), 6.93 (s, 2 H), 6.81 (s, 1H), 5.12 (s, 2H),
5.11 (s, 2H), 2.27 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 156.7,
139.2, 136.2, 135.7, 128.51, 128.46, 128.24, 128.19, 128.1, 128.0,
67.7, 67.5, 21.3; HRMS (FAB, M + H) calcd for C25H27N4O4,
447.2032; found, 447.2043; Anal. (C25H26N4O4) C, H, N.
oil. The HCl salt was prepared by dissolving the residue in methanol
(5 mL) followed by dropwise addition of 1.25 M HCl in methanol
(10 mL). The methanol was then removed under vacuum, and the
product was obtained as white needles by recrystallization from
1
isopropyl alcohol/diethyl ether (1.7 g, 78%). Mp 166–168 °C; H
NMR (DMSO-d6, 400 MHz) δ 7.43–7.28 (m, 5H), 5.17 (s, 2H),
3.15 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ 155.4, 136.0,
128.9, 128.7, 128.3, 68.4, 36.2; HRMS (FAB, M + H) calcd for
C9H13N2O2, 181.0977; found, 181.0976; Anal. (C9H13N2O2Cl) C,
H, N.
(S)-Benzyl
4-Benzyl-5-oxo-1H-imidazole-1,2(4H,5H)-
diyldicarbamate (6). Compound 6 was prepared as described
above for compound 1. Product obtained as a clear glass (89%).
1H NMR (CDCl3, 400 MHz) δ 7.52–7.04 (m, 15H), 5.14–4.99 (m,
4H), 4.27 (m, 1H), 3.31 (m, 1H), 2.88 (m, 1H); 13C NMR (CDCl3,
100 MHz) δ 170.1, 163.0, 160.3, 154.7, 136.3, 135.5, 134.9, 129.5,
129.3, 128.8, 128.6, 128.4, 128.0, 68.6, 67.9, 58.4, 38.0; HRMS
(FAB, M + H) calcd for C26H25N4O5, 473.1825; found, 473.1819;
Anal. (C26H24N4O5) C, H, N.
N-Benzyloxycarbonyl-2-methylhydrazine
Hydrochloride
(10). At room temperature, methyl hydrazine (26.0 mmol, 1.2 g,
1.4 mL) and Boc2O (19.8 mmol, 4.3 g) were combined in methanol
(25 mL). After stirring for 30 min, the solvent was removed under
vacuum, and the residue was dissolved in a mixture of CH2Cl2 (20
mL) and 1 M NaOH (20 mL). While stirring rapidly, benzyl
chloroformate (20.0 mmol, 3.4 g, 2.8 mL) was added. After stirring
for one hour, the layers were separated and the organic layer was
washed with 1 M HCl (2 × 15 mL), H2O (2 × 15 mL), and brine
(2 × 15 mL) and dried over Na2SO4. The solvent was removed
under vacuum and the residue was dissolved in methanol (40 mL),
chilled on ice, and treated with acetyl chloride (10 mL). After
stirring for 20 min, the mixture was concentrated under vacuum
and the product was obtained as white needles by recrystallization
N-δ-tert-Butoxycarbonyl-N-r-benzyloxycarbonyl-L-
ornithine Benzyl Ester (7). N-R-Cbz-L-ornithine (5.0 g, 19.0
mmol) was stirred in methanol (30 mL) and treated with a 10%
NEt3 solution in methanol (30 mL) followed by Boc2O (8.2 g, 38.0
mmol). The mixture was warmed to 40 °C and stirred for one hour,
at which point TLC indicated consumption of starting material. The
methanol was removed under vacuum, and the residue was treated
with 20 mL of dilute HCl (pH 2.5) on ice, followed by extraction
with ethyl actetate (4 × 50 mL). The combined ethyl acetate layers
were combined, washed with brine, and dried over Na2SO4, and
the solvent was removed to yield the Boc-protected intermediate
as a pale yellow oil, used without further purification. This material
was then dissolved in acetonitrile (175 mL), treated with diisopro-
pylamine (1.05 equiv, 20.0 mmol, 2.7 g, 3.6 mL), followed by
benzyl bromide (1.05 equiv, 20.0 mmol, 3.7g, 2.6 mL), and left to
stir at room temperature for 14 h. The solvent was next removed
under vacuum and the residue was redissolved in ethyl acetate (250
mL), washed with water (4 × 100 mL) and brine (2 × 100 mL),
and dried over Na2SO4. Following solvent removal, recrystallization
from ethyl acetate/hexanes yielded 7 as a white solid (7.89 g, 91%
over 2 steps). Mp 107–109 °C; 1H NMR (CDCl3, 400 MHz) δ
7.42–7.28 (m, 10H), 5.44 (br d, J ) 7.6 Hz, 1H), 5.29–5.11 (m,
4H), 4.52 (br s, 1H), 4.44 (m, 1H), 3.08 (m, 2H), 1.88 (m, 1H),
1.67 (m, 1H), 1.56–1.40 (m, 11H); 13C NMR (CDCl3, 100 MHz)
δ 172.1, 155.9, 136.2, 135.2, 128.6, 128.5, 128.3, 128.2, 128.1,
79.2, 67.2, 67.0, 53.6, 39.9, 29.9, 28.4, 25.9; HRMS (FAB, M +
H) calcd for C25H33N2O6, 457.2339; found, 457.2336; Anal.
(C25H32N2O6) C, H, N.
1
from isopropyl alcohol (3.3 g, 77%). Mp 175–177 °C; H NMR
(DMSO-d6, 400 MHz) δ 7.39–7.28 (m, 5H), 5.15 (s, 2H), 2.70 (s,
3H); 13C NMR (DMSO-d6, 100 MHz) δ 155.2, 136.0, 128.9, 128.7,
128.5, 67.6, 35.9; HRMS (FAB, M + H) calcd for C9H13N2O2,
181.0977; found, 181.0981; Anal. (C9H13N2O2Cl) C, H, N.
N-Benzyloxycarbonyl-2-(2,2,2-trifluoroethyl)
Hydrazine
(11). On ice, 2,2,2-trifluoroethyl hydrazine (10.0 mmol, 1.63 g of
a 70% by weight solution in water) was dissolved in a mixture of
CH2Cl2 (15 mL) and 1 M NaOH (15 mL) and treated with benzyl
chloroformate (10.0 mmol, 1.7 g, 1.4 mL). The mixture was stirred
rapidly at room temperature for 6 h, after which the layers were
separated. The aqueous layer was extracted with an additional 20
mL of CH2Cl2 and the combined organic layers were dried over
Na2SO4 and evaporated to yield a white solid. Compound 11 was
obtained as the free base by recrystallization from ethyl acetate/
hexanes (1.91 g, 77%). Mp 79–81 °C; 1H NMR (CDCl3, 400 MHz)
δ 7.45–7.30 (m, 5H), 6.73 (br s, 1 H), 5.16 (s, 2H), 4.38 (br s,
1H), 3.43 (m, 2H); 13C NMR (CDCl3, 100 MHz) δ 157.2, 135.6,
128.6, 128.5, 128.2, 124.8 (q, JCF ) 278.0 Hz), 67.5, 52.7 (q, JCF
) 30.3 Hz); 19F NMR (CDCl3, 376.5 MHz) δ -70.9 (t, JFH ) 9.1
Hz); HRMS (FAB, M + H) calcd for C10H12N2O2F3, 249.0851;
found, 249.0856; Anal. (C10H12N2O2F3) C, H, N.
N-Uriedo-benzyloxycarbonyl-N-r-benzyloxycarbonyl-L-
thiocitrulline Benzyl Ester (8). Compound 7 (5.0 g, 11.0 mmol)
was treated with 1:1 TFA/CH2Cl2 (80 mL) and stirred at room
temperature for 1 h. The mixture was then evaporated under vacuum
and the residue was dissolved in CH2Cl2 (100 mL) and treated with
NEt3 to neutralize excess TFA. CbzNCS was next added dropwise
(as a 0.5 M solution in CH2Cl2) until the amine intermediate was
consumed. Following solvent removal, the crude material was
applied to a silica gel column (2:1 hexanes/ethyl acetate) and 8
NG-Benzyloxycarbonylamino-NG′-benzyloxycarbonyl-N-r-
benzyloxycarbonyl-L-arginine Benzyl Ester (12). Thiourea 8
(0.5 mmol, 275 mg) was dissolved in CH2Cl2 (5.0 mL) and treated
with NEt3 (1.1 equiv, 0.55 mmol, 56 mg, 77 µL), EDCI (1.1 equiv,
0.55 mmol, 105 mg), and benzyl carbazate (1.1 equiv, 0.55 mmol,
91 mg). After one hour, TLC analysis (3:1 hexane/ethyl acetate)
indicated formation of the desired product (Rf ) 0.30) with residual
thiourea (Rf ) 0.6) still present. An additional 1.1 equiv of each
reagent was added, and after stirring for another hour, the thiourea
was completely consumed. The mixture was then washed with 0.1
M HCl (2 × 5 mL), H2O (2 × 5 mL), and brine (2 × 5 mL). The
organic layer was dried over Na2SO4, followed by evaporation under
vacuum. The residue was applied to a silica column and eluted
with 3:2 hexane/ethyl acetate, yielding the desired product as an
1
isolated as a colorless oil 9 (4.9 g, 82%). H NMR (CDCl3, 400
MHz) δ 9.63 (br s, 1H), 8.21 (br s, 1H), 7.42–7.28 (m, 15H), 5.44
(br d, J ) 8.0 Hz, 1H), 5.22–5.08 (m, 6H), 4.47 (m, 1H), 3.63 (m,
2H), 1.95–1.58 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 179.1,
171.9, 155.9, 152.5, 136.1, 135.1, 134.5, 128.9, 128.8, 128.7, 128.5,
128.4, 128.3, 128.2, 128.1, 68.2, 67.4, 67.1, 53.5, 44.9, 30.0, 24.1;
HRMS (FAB, M + H) calcd for C29H32N3O6S, 550.2012; found,
550.2002; Anal. (C29H31N3O6S) C, H, N.
1
oil (310 mg, 91%). H NMR (CDCl3, 400 MHz) δ 10.15 (br s,
1H), 7.42–7.21 (m, 20H), 5.95–5.56 (br m, 2H), 5.20–5.00 (m, 8H),
4.35 (m, 1H), 3.25 (m, 2H), 1.90–1.37 (m, 4H); 13C NMR (CDCl3,
100 MHz) δ 172.1, 163.5, 160.8, 156.2, 137.2, 136.1, 135.3, 135.2,
128.62, 128.58, 128.5, 128.3, 128.2, 128.1, 127.7, 68.0, 67.2, 67.0,
53.6, 40.1, 29.5, 25.2; HRMS (FAB, M + H) calcd for C37H40N5O8,
682.2877; found, 682.2872; Anal. (C37H39N5O8) C, H, N.
N-Benzyloxycarbonyl-1-methylhydrazine
Hydrochloride
(9). At room temperature, methyl hydrazine (10.0 mmol, 0.46 g,
0.53 mL) was dissolved in a mixture of CH2Cl2 (15 mL) and 1 M
NaOH (15 mL) and treated with benzyl chloroformate (10.0 mmol,
1.7 g, 1.4 mL). The mixture was stirred rapidly for one hour, after
which the layers were separated. The aqueous layer was extracted
with an additional 20 mL of CH2Cl2, and the combined organic
layers were dried over Na2SO4 and evaporated to yield a colorless
NG-Benzyloxycarbonylmethylamino-NG′-benzyloxycarbonyl-N-r-
benzyloxycarbonyl-L-arginine Benzyl Ester (13). Compound
13 was prepared and purified as described above for 12 using Cbz-
protected methyl hydrazine 9 in place of benzyl carbazate. Product

930 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Martin et al.
1
isolated as an oil (313 mg, 90%). H NMR (CDCl3, 400 MHz) δ
39.6, 26.9, 23.9; [R]D ) +28.5° (c 0.7, H2O); HRMS (FAB, M +
10.40 (br s, 1H), 7.44–7.24 (m, 20H), 5.61 (br m, 1H), 5.20–5.03
(m, 8H), 4.38 (m, 1H), 3.35 (br m, 2H), 3.12 (s, 3H), 1.93–1.41
(m, 4H); 13C NMR (CDCl3, 100 MHz) δ 172.0, 163.6, 159.8, 156.4,
156.0, 153.0, 137.3, 136.1, 135.5, 135.1, 128.64, 128.57, 128.5,
128.4, 128.3, 128.2, 128.1, 128.0, 127.7, 68.4, 67.3, 67.0, 53.4,
40.1, 38.4, 29.7, 25.3; HRMS (FAB, M + H) calcd for C38H42N5O8,
696.3033; found, 696.3036; Anal. (C38H41N5O8) C, H, N.
H) calcd for C7H18N5O2, 204.1461; found, 204.1464.
NG-Amino-NG-2,2,2-trifluoroethyl-L-arginine Acetate (19).
Compound 19 was prepared from 15 as described above for
16. The product was lyophilized to yield the amino acid mono-
acetate salt as a clear glass (95%). 1H NMR (D2O, 400 MHz)
δ 4.28 (q, JHF ) 8.6 Hz, 2H), 3.70 (t, J ) 6.1 Hz, 1H), 3.27 (t, J
) 7.0 Hz, 3H), 1.87–1.77 (m, 2H), 1.75–1.54 (m, 2H); 13C NMR
NG-Benzyloxycarbonylamino-NG-methyl-NG′-benzyloxycarbonyl-N-r-
benzyloxycarbonyl-L-arginine Benzyl Ester (14). Compound
14 was prepared as described above for 12 using Cbz-protected
methyl hydrazine 10 in place of benzyl carbazate. Chromatographic
purification of the product required a much more polar solvent
system (1:1 hexane/ethyl acetate f 5% methanol in chloroform)
(D2O, 100 MHz) δ 180.1 (AcOH), 174.3, 157.5, 123.9 (q, JCF
)
280.5 Hz), 54.2, 52.7 (q, JCF ) 33.2 Hz), 41.1, 27.5, 23.7,
22.5 (AcOH); 19F-NMR (D2O, 376.5 MHz) δ -69.6 (t, JFH ) 8.4
Hz); [R]D ) +20.7° (c 0.9, H2O); HRMS (FAB, M + H)
calcd for C8H17N5O2F3, 272.1334; found, 272.1333; Anal.
(C8H16N5O2F3 ·1.4AcOH·0.2H2O) C, H, N.
1
after which 14 was isolated as an oil (278 mg, 80%). H NMR
Spectral Kd Titrations. iNOSheme was expressed and purified
as previously described.25 In a typical assay, H4B-bound iNOSheme
(500 µL of 0.5–1 µM) was titrated with the compound of choice,
working at concentrations appropriate to detect the spectral transi-
tion from 418 nm (FeIII-aqua) to 394 nm (FeIII-unligated). Absor-
bance spectra were corrected for the dilution due to the volume of
titrant added. As described in the text, absorbance changes (∆∆Abs
) ∆Abs394 - ∆Abs418) were plotted as a function of compound
concentration and the Kd was determined by fitting the data by a
nonlinear least-squares fit to the saturation binding equation: ∆∆Abs
) (∆∆Absmax × [compound])/(Kd + [compound]) (KaleidaGraph,
Abelbeck Software). All analyses were performed in triplicate and
the average value ( the standard deviation reported.
iNOS Inhibition Assays. A modified, high-throughput oxymyo-
globin assay34 using a 96-well plate reader was utilized to assess
the inhibition exhibited by compounds 16–19 with full-length iNOS.
Described here is the Ki determination for 19. Assay reactions were
prepared containing L-arginine (0, 10, 20, 50, 100, 200, 400, and
1000 µM), 19 (30, 60, 90, and 120 µM), 12 µM H4B, 150 µM
DTT, 8.5 µM oxymyoglobin, and 15 nM iNOS in 100 mM HEPES
(pH 7.5). The NOS reaction was initiated by addition of NADPH
(final concentration 100 µM) to each well using a multichannel
pipet. NO production was monitored by measuring the increase in
absorbance at 405 nm, corresponding to the NO-mediated conver-
sion of oxymyoglobin to metmyoglobin. The data derived from
the Michaelis–Menten plots of iNOS inhibition by 19 was used to
generate an apparent Km versus [19] replot from which the Ki for
19 was determined to be 10.5 ( 5.7 µM. Each analysis was
performed in triplicate and the average reported along with the
associated error in each fit.
Time-Dependent Inactivation of iNOS. An assay using a 96-
well plate reader was utilized to assess the time-dependent
inactivation of iNOS exhibited by compounds 16–19. Described
here is the Kinactivation determination for 18. Inactivation was initiated
by adding 18 (300 µM) to a solution containing 100 mM HEPES
(pH 7.5), 150 nM iNOS, 27 µM H4B, 345 µM DTT, 12 µM oxyMb,
and 100 µM NADPH. Inactivation of iNOS was terminated at
different time points (0–5.0 min) by transferring 30 µL of the
inactivation mixture to 270 µL of a reaction solution containing
100 mM HEPES (pH 7.5), 1.0 mM L-arginine, 27 µM H4B, 345
µM DTT, 12 µM oxyMb, and 100 µM NADPH. NO production
was monitored at 405 nm, as described for the previous kinetic
assays.
Inhibition of iNOS-Mediated NADPH Oxidation. Compounds
17 and 19 were investigated for their ability to prevent iNOS-
mediated NADPH oxidation. A solution containing 100 mM HEPES
pH 7.5, 1.0 mM inhibitor, 80 nM iNOS, 2.6 µM H4B, 30 µM DTT,
and 100 µM NADPH (final volume 450 µL) was added to a quartz
cuvette. The conversion of NADPH to NADP+ was monitored
spectrally at 340 nm using a Cary 3E UV–visible spectrophotometer
for 5.0 min.
(CDCl3, 400 MHz) δ 8.01 (br s, 1H), 7.43–7.22 (m, 20H), 5.62 (br
m, 1H), 5.19–5.03 (m, 8H), 4.33 (m, 1H), 3.24–3.01 (m, 5H),
1.85–1.36 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 172.0, 160.2,
156.2, 155.1, 136.7, 136.1, 135.3, 135.2, 128.6, 128.53, 128.50,
128.46, 128.42, 128.37, 128.3, 128.5, 128.08, 128.0, 68.0, 67.6,
67.2, 67.0, 53.6, 43.5, 39.9, 29.3, 25.3; HRMS (FAB, M + H)
calcd for C38H42N5O8, 696.3033; found, 696.3029; Anal.
(C38H41N5O8) C, H, N.
NG-Benzyloxycarbonyl-NG-2,2,2-trifluoroethylamino-NG′
-
benzyloxycarbonyl-N-r-benzyloxycarbonyl-L-arginine Benzyl
Ester (15). Compound 15 was prepared and purified as described
above for 12 using Cbz-protected 2,2,2-trifluoromethyl hydrazine
11 in place of benzyl carbazate. The product was isolated as an oil
1
(330 mg, 87%). H NMR (CDCl3, 400 MHz) δ 8.06 (br s, 1H),
7.45–7.20 (m, 20H), 5.58 (br m, 1H), 5.20–4.99 (m, 8H), 4.13 (m,
1H), 3.49 (br m, 2H), 3.28–2.95 (br m, 2H), 1.85–1.37 (m, 4H);
13C NMR (CDCl3, 100 MHz) δ 172.0, 161.7, 156.4, 154.4, 154.0,
136.9, 136.0, 135.1, 135.0, 128.7, 128.6, 128.5, 128.4, 128.34,
128.26, 128.21, 128.15, 128.1, 127.9, 124.2 (q, JCF ) 279.7 Hz),
68.2, 67.4, 67.3, 53.2, 50.3 (br m), 43.9, 29.9, 25.1; 19F NMR
(CDCl3, 376.5 MHz) δ -69.2; HRMS (FAB, M + H) calcd for
C39H41N5O8F3, 764.2907; found, 764.2904; Anal. (C39H40N5O8F3)
C, H, N.
NG-Amino-L-arginine Acetate (16). Compound 12 (200 mg,
0.29 mmol) was dissolved in 10 mL of 1:1 methanol/H2O (1%
acetic acid), and the system was purged with N2(g) prior to addition
of 10% Pd/C (100 mg). Hydrogen gas was delivered with a balloon
to the stirring reaction mixture throughout the course of the reaction
(5 h, room temperature). Following reaction completion, the catalyst
was removed by filtering through Celite and washed with several
volumes of MeOH/H2O (1% acetic acid). The filtrate was concen-
trated under vacuum and lyophilized to yield the amino acid
1
monoacetate salt as a clear glass (72 mg, 98%). H NMR (D2O,
400 MHz) δ 3.69 (t, J ) 6.1 Hz, 1H), 3.19 (t, J ) 6.9 Hz, 2H),
1.86–1.78 (m, 2H), 1.73–1.50 (m, 2H); 13C NMR (D2O, 100 MHz)
δ 180.6 (AcOH), 174.4, 157.9, 54.3, 40.1, 27.5, 24.0, 22.7 (AcOH);
[R]D ) +15.3° (c 0.8, H2O), lit.33 +7.4° (c 0.46, MeOH); HRMS
(FAB, M + H) calcd for C6H16N5O2, 190.1304; found, 190.1303;
Anal. (C6H15N5O2 ·1.4AcOH·0.3H2O) C, H, N.
NG-Methylamino-L-arginine Acetate (17). Compound 17 was
prepared from 13 as described above for 16. The product was
lyophilized to yield the amino acid monoacetate salt as a clear glass
(99%). 1H NMR (D2O, 400 MHz) δ 3.67 (t, J ) 6.1 Hz, 1H), 3.17
(t, J ) 6.9 Hz, 2H), 2.45 (s, 3H), 1.85–1.77 (m, 2H), 1.70–1.48
(m, 2H); 13C NMR (D2O, 100 MHz) δ 180.5 (AcOH), 174.3, 156.9,
54.3, 40.1, 37.6, 27.5, 24.0, 22.8 (AcOH); [R]D ) +9.6° (c 0.8,
H2O); HRMS (FAB, M + H) calcd for C7H18N5O2, 204.1461;
found, 204.1460; Anal. (C7H17N5O2 ·1.1AcOH·0.5H2O) C, H, N.
NG-Amino-NG-methyl-L-arginine (18). Compound 18 was
prepared from 14 as described above for 16. The product was
lyophilized to yield the amino acid with only trace acetic acid
(evidenced by NMR) as a clear glass (94%). The lyophilized
material is extremely hygroscopic, preventing satisfactory C, H, N
Stopped-Flow UV–Visible Spectroscopic Analysis. iNOSheme
oxidation was measured on a Hi-Tech Scientific SF-61 stopped-
flow spectrophotometer equipped with a Neslab RTE-100 temper-
ature controller set at 4 °C. Directly before use, iNOSheme was
desalted into 100 mM HEPES (pH 7.5), followed by addition of
H2B (400 µM) and DTT (5 mM). The sample was monitored
1
analysis. H NMR (D2O, 400 MHz) δ 4.05 (t, J ) 6.2 Hz, 1H),
3.25 (t, J ) 6.8 Hz, 2H), 3.17 (s, 3H), 2.01–1.86 (m, 2H), 1.81–1.58
(m, 2H); 13C NMR (D2O, 100 MHz) δ 171.9, 157.2, 52.6, 40.7,

NG-Aminoguanidines from Primary Amines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 931
(13) Roman, L. J.; Martasek, P.; Masters, B. S. Intrinsic and extrinsic
modulation of nitric oxide synthase activity. Chem. ReV. 2002, 102,
1179–1190.
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Structure, function, and inhibition. Biochem. J. 2001, 357, 593–615.
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spectrally until no further change was observed, indicating complete
reconstitution with the H2B. The protein was transferred into a Coy
anaerobic glovebag and allowed to sit at 4 °C for >1 h to equilibrate
the reconstituted protein in an O2-free environment. An aliquot of
the protein (200 µL) was then reduced with sodium dithionite (50
µM) and mixed with either L-arginine or compound 19 (10 mM)
and 100 mM HEPES (pH 7.5) to give a final volume of 1.0 mL.
The protein was rapidly mixed with an equal volume of a solution
of O2-saturated buffer (2.2 mM). The system had been previously
scrubbed of oxygen with a solution of 500 µM sodium dithionite
in 100 mM HEPES (pH 7.5). After mixing, the sample was
monitored spectrally from 350–650 nm. Final solution concentra-
tions consisted of iNOSheme (2–4 µM), H2B (50–100 µM), DTT
(50–125 µM), L-arginine or compound 19 (5 mM), and O2 (1.1
mM). All data was fit with Specfit global analysis software from
Spectrum Software Associates (Chapel Hill, NC, USA).
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by diaminoguanidine and NG-amino-L-arginine. Arch. Biochem. Bio-
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Acknowledgment. This work was supported by the Aldo
DeBenedictis Fund, the Natural Sciences and Engineering
Council of Canada, and the Alberta Heritage Foundation for
Medical Research (postdoctoral fellowships to N.I.M.). Nathaniel
Fernhoff and Michelle Chang are gratefully acknowledged for
providing full-length iNOS.
(22) Vuletich, J. L.; Lowe, E. R.; Jianmongkol, S.; Kamada, Y.; Kent,
U. M.; Bender, A. T.; Demady, D. R.; Hollenberg, P. F.; Osawa, Y.
Alteration of the heme prosthetic group of neuronal nitric-oxide
synthase during inactivation by NG-amino-L-arginine in vitro and in
vivo. Mol. Pharmacol. 2002, 62, 110–118.
Supporting Information Available: 1H and 13C NMR spectra
for compounds 16–19 and complete results of the spectral binding
and inhibition assays for compounds 16–19. This material is
available free of charge via the Internet at http://pubs.acs.org.
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from primary amines. Org. Lett. 2006, 8, 4035–4038.
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efficient method for the preparation of guanidines. Tetrahedron Lett.
1992, 33, 5933.
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