Evaluation of 3-substituted arginine analogs as selective inhibitors of human nitric oxide synthase isozymes

Article abstract of DOI:10.1016/j.bmcl.2005.03.078

Nitric oxide (NO), a mediator of various physiological and pathophysiological processes, is synthesized by three isozymes of nitric oxide synthase (NOS). In developing candidate clinical drugs, it is very important not to inhibit endothelial NOS, because

Full text of DOI:10.1016/j.bmcl.2005.03.078

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2881–2885
Evaluation of 3-substituted arginine analogs as selective
inhibitors of human nitric oxide synthase isozymes
Ryosuke Ijuin, Naoki Umezawa, Shin-ichi Nagai and Tsunehiko Higuchi*
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
Received 2 March 2005; revised 20 March 2005; accepted 22 March 2005
Available online 25 April 2005
Abstract—Nitric oxide (NO), a mediator of various physiological and pathophysiological processes, is synthesized by three isozymes
of nitric oxide synthase (NOS). In developing candidate clinical drugs, it is very important not to inhibit endothelial NOS, because it
plays an important role in maintaining normal blood pressure and flow. Here, we describe the design, synthesis and human NOS-
inhibitory activities of S-methyl-L-isothiocitrulline-based 3-substituted arginine analogs. The 3R*-methyl compound 4, which has an
S-methyl isothiourea moiety, inhibited nNOS and iNOS, but not eNOS (IC50 >1 mM). However, the 3R*-methyl compound 7,
bearing a 5-iminoethyl moiety, did not inhibit any of the NOS isozymes, although L-N-iminoethylornithine (L-NIO) potently inhibi-
ted all three. A computational docking study was carried out to investigate the mechanism of the isozyme selectivity.
Ó 2005 Elsevier Ltd. All rights reserved.
Nitric oxide synthases (NOS)1 are a class of enzymes
found in mammals and other species that utilize L-argi-
nine to generate nitric oxide (NO). NO is an important
signaling molecule involved in a wide range of physio-
logical functions, as well as pathophysiological states.2
Three NOS isozymes have been identified in various
cells, that is, neuronal NOS (nNOS), inducible NOS
(iNOS), and endothelial NOS (eNOS). They all catalyze
the same two-step oxidation of L-arginine, using the
same cofactors, NADPH, FAD, FMN, hemin, and
BH4.3–5 Those NOS isozymes are associated with differ-
ent physiological functions: blood-vessel dilation
(eNOS),6 neuronal signal transmission (nNOS),3 and
immune response, such as cytotoxicity, against patho-
gens, and tumors (iNOS).7 NO overproduction by
NOS plays a role in a wide range of disorders, including
septic shock, arthritis, diabetes, ischemia-reperfusion in-
jury, pain, and various neurodegenerative diseases.8,9
However, in developing NOS inhibitors to treat these
disorders, it is very important to keep eNOS activity in-
tact, because of its role in maintaining normal blood
pressure and flow.10 Therefore, the design of isozyme-
selective NOS inhibitors to regulate NO synthesis in spe-
cific tissues is important. Structural analogs of L-argi-
nine, the natural substrate of NOS, have been shown
to inhibit the various forms of NOS, and nonamino acid
inhibitors of NOS have also been reported.11 However,
the first generation of arginine analog inhibitors had
low isozyme selectivity. For example, S-methyl-isothio-
citrulline (L-MIT 1)12 and N-iminoethyl-L-ornithine
(L-NIO 2) are potent inhibitors of all three NOS isozymes.
We attempted to design selective inhibitors based on
arginine, and found that S-methyl-L-isothiocitrullinyl-
L-phenylalanine (MILF 3),13,14 which has a phenylala-
nine moiety at the C-terminal of L-MIT, is an iNOS-
selective inhibitor (Fig. 1). Our results suggested that
the NOS isozymes have differences in the hydrophobic
region near the binding site of the C-terminal of the sub-
strate. To investigate the contribution of lipophilic sub-
stituents to the selectivity of NOS inhibition by arginine
Keywords: Nitric oxide; Nitric oxide synthase; Inhibitor; Arginine;
Docking study.
*
Corresponding author. Tel./fax: +81 52 836 3435; e-mail: higuchi@
phar.nagoya-cu.ac.jp
Figure 1. Structures of L-MIT, L-NIO, and MILF.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.078

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R. Ijuin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2881–2885
Figure 2. Design of 3-substituted arginine analogs.
analogs, we newly designed several 3-substituted argi-
nine analogs 4–7 (Fig. 2).
Here, we describe the synthesis of these 3-substituted
arginine analogs and the evaluation of their inhibitory
activity toward the three recombinant human NOS iso-
zymes. A docking study was conducted to throw light on
the effects of the substituents.
Our synthetic strategy was to use Claisen rearrangement
to form the amino acid backbone bearing an alkyl group
at the 3-position,15,16 followed by conversion of the
functional group. Protected glycine esters 9a–c were ob-
tained as precursors of Claisen rearrangement by con-
densation of the N-protected glycine 8 and the
corresponding 2,3-unsaturated alcohol. Since cis-buten-
1-ol was not commercially available, 2-butyn-1-ol was
used and the terminal alkyne was reduced with Lindlar
catalyst to the cis olefin for the preparation of 9c. The
3-substituted amino acids were generated in the Claisen
rearrangement with LHMDS and Al(O-i-Pr)3, affording
( )-N-Cbz-3-substituted 4,5-unsaturated amino acids
10a–c. In this rearrangement, the relative stereochemis-
try of the 3-substituent versus the 2-amino group was
controlled by the geometry of the substrate (Scheme
1). syn-Form amino acid derivatives were obtained from
trans substrates and anti-form from cis substrates.
Scheme 1. Reagents and conditions: (a) crotyl alcohol, EDCI, DMAP,
CH2Cl2, 0 °C, 95%; (b) cis-2-hexen-1-ol, EDCI, DMAP, CH2Cl2, 0 °C,
84%; (c) (i) 2-butyn-1-ol, EDCI, DMAP, CH2Cl2, 0 °C, (ii) Lindlar
cat., MeOH, H2, rt, 99%; (d) LHMDS, Al(O-i-Pr)3, THF, À78 °C, 10a
52%, 10b 52%, 10c 30%.
The carboxyl group was protected as the tert-butyl ester
11a–c. The terminal olefin of the resulting 11a–c was then
converted to the primary alcohol 12a–c by hydrobora-
tion followed by oxidation. Compound 12c was depro-
tected by catalytic reduction and then protected with a
Boc group to afford 13c. Hydroxyl groups on 12a–b
and 13c were converted to azide to afford 14a–c. Azide
compounds 14a–c were easily converted to the ornithine
derivatives 15a–c by catalytic reduction (Scheme 2).
Scheme 2. Reagents and conditions: (a) isobutene, H2SO4, CH2Cl2,
0 °C, 11a 72%, 11b 31%, 11c 98%; (b) (i) thexylborane, THF, 0 °C, (ii)
H2O2, pH 7.0, 12a 56%, 12b 78%, 12c 39%; (c) Pd/C, MeOH, (Boc)2O,
Et3N, 13c 53%; (d) (i) MsCl, pyridine, CH2Cl2, 0 °C, (ii) NaN3, DMF,
50 °C, 14a 67%, 14b 42%, 14c 9.1%; (e) for 14a: Lindlar cat., MeOH,
H2, rt; for 14b,c: Pd/C MeOH, H2, rt, 15a–c quant.
These 3-substituted ornithines 15a–c were treated with
thiophosgene, then NH3 in methanol, to give the thiou-
reas 16a–c, which were purified by HPLC. Treatment
with iodomethane in CH3CN converted 16a–c to S-
methyl isothiourea form, 17a–c. Deprotection afforded
the desired 3-substituted S-methyl-isothiourea-type argi-
nine analogs 4, 5, 6. To synthesize another type of argi-
nine analog, 15a was treated with ethyl acetimidate, and
subsequent deprotection gave compound 7 (Scheme 3).
NOS activity was measured by monitoring the conver-
sion of [14C]-L-Arg to [14C]-L-Cit.17 The IC50 values of
synthesized compounds were calculated from the
concentration dependence of the inhibitory activity
(Table 1). L-MIT 1 is a potent inhibitor of all the
Human NOS isozymes were expressed in Sf-9 (a Spodop-
tera frugiperda insect cell line). Recombinant human

R. Ijuin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2881–2885
2883
Scheme 3. Reagents and conditions: (a) (i) thiophosgene, CaCO3, CH2Cl2, H2O, rt, (ii) NH3, MeOH, 0 °C, 16a 77%, 16b 94%, 16c 54%; (b) MeI,
CH3CN, rt, 17a 80%, 17b 84%, 17c quant; (c) for 17a: HCl, THF, reflux; for 17b,c: (i) TFA, H2O, phenol, thioanisole, 1,2-ethanedithiol, rt; for 17c:
(ii) (À)ion exchange column, 4–6 quant; (d) (i) for 15a, ethyl acetimidate, K2CO3, MeOH, rt, 23%, (ii) HCl, THF, reflux, quant.
Table 1. IC50 values (lM) for inhibition of human NOS isozymesa
Entry
Compound
Substituent
5-Position
nNOSb
iNOSb
eNOSb
3-Position
1
2
3
4
5
6
7
1
H
S-Me-isothiourea
S-Me-isothiourea
S-Me-isothiourea
S-Me-isothiourea
S-Me-isothiourea
Iminoethyl
0.06
0.3
3.9
110
4.5
370
0.4
>1000f
>1000f
11
3c,d
H
36
38
( )-4e
( )-5e
( )-6e
2
(R*)-Me
(S*)-Me
(S*)-Pr
H
3.5
500
9
>1000f
>1000f
4
6
( )-7e
(R*)-Me
Iminoethyl
>1000f
>1000f
a Human NOS activity was measured by monitoring the conversion of [14C]-Arg to [14C]-L-Cit.
b Human NOS isozymes were expressed in Sf-9 cells.
c Data from Ref. 13.
d Dipeptide compound.
e The relative configuration at the 2-position of all compounds is S*.
f Not inhibited by 50% at concentrations up to 1 mM inhibitor.
NOS isozymes, with slight selectivity for nNOS, while
MILF 3 is an iNOS-selective inhibitor (entries 1 and
2). This difference between 1 and 3 could be explained
by the existence of a hydrophobic region in the iNOS ac-
tive site at a position corresponding to the C-terminal of
the ligand.
To study the binding mode of compounds 4 and 5 to the
active site, we calculated the minimum energy confor-
mations of 4 and 5 when they were docked into models
based on the crystal structures of rat nNOS (PDB code
1LZX) and human eNOS (PDB code 3NOS) using the
software package MACROMODEL 8.0, and we compared
the results with those obtained for L-MIT 1 (Fig. 3).
As shown in Figure 3a and b, there is a high degree of
similarity among the binding modes to nNOS of 1
(green), 4 (blue), and 5 (red). Next, we investigated the
binding modes to eNOS of 1, 4, and 5. In the case of
4, the carboxyl group cannot be overlapped with that
of 1 (Fig. 3c and d), owing to rotation of the alkyl chain
resulting from the presence of the 3R-methyl group at
the 3-position. On the other hand, the binding mode
of 5 is similar to that of 1. Thus, in the case of the nNOS
active site, the carboxyl groups and amino groups of 4
and 5 were superimposed in the binding pocket, while
such superimposition did not occur in the case of the
eNOS active site. This result is consistent with the differ-
ence of eNOS inhibitory activity between 4 and 5. It ap-
pears that a 3R-methyl group interferes with docking to
the eNOS active site and thus reduces eNOS-inhibitory
activity.
The 3R*-methyl derivative 4 had similar nNOS inhibi-
tory activity (IC50 = 38 lM) to MILF 3 (36 lM). How-
ever, the iNOS and eNOS inhibitory activities were very
low (iNOS 110 lM, eNOS >1000 lM) (entry 3). In con-
trast, the 3S*-methyl derivative 5 inhibited all the NOS
isozymes (nNOS 3.5 lM, iNOS 4.5 lM, eNOS 11 lM)
(entry 4). The 3S*-propyl derivative 6 had very weak
inhibitory activity for all of the NOS isozymes (nNOS
500 lM, iNOS 370 lM, eNOS >1000 lM) (entry 5).
Surprisingly, 7 had no inhibitory activity for any of
the NOS isozymes (entry 7). In contrast, L-NIO 2 po-
tently inhibited all of them (entry 6). Therefore, the 3R
substituent appears to have a critical negative effect on
substrate recognition. The 3R*-methyl derivative 4 was
slightly selective for nNOS over iNOS, and did not inhi-
bit eNOS, whereas the 3S*-methyl derivative 5 had
inhibitory activity for all of the NOS isozymes. This sug-
gests that R-configuration at the 3-position results in
lower potency than S-configuration (entries 3 and 4).
The difference between the 3R*-methyl compound and
3S*-methyl compound is remarkable. Comparing the
methyl 5 and propyl 6 substituents, it seems that the pro-
pyl group is too large to bind to the NOS L-arginine-
binding site (entries 4 and 5). In order to investigate sub-
stitution effects at the 3-position of L-MIT in detail, we
conducted docking calculations.
In summary, we have designed and synthesized L-MIT-
based compounds as 3-substituted arginine analogs in
order to find selective NOS inhibitors. The 3R*-methyl
compound 4 had no inhibitory effect on eNOS, though
it inhibited both nNOS and iNOS. Compound 7 did
not inhibit any of the NOS isozymes. The 3S*-methyl
compound 5 potently inhibited all of the isozymes. A
large substituent, such as the n-propyl group in 6,

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R. Ijuin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2881–2885
Figure 3. Superposition of the minimum energy conformations of 1 (green) and 4 (blue) and 5 (red) in the nNOS (a, b) and eNOS (c, d) active sites.
(a) nNOS protein is not shown for the sake of clarity. (b) View of the conformation of the inhibitors docked into the nNOS catalytic center. Residues
˚
within 5 A from the ligand are displayed as wire graphics and heme is displayed as ball and stick. (c) eNOS protein is not shown for the sake of
˚
clarity. (d) View of the conformation of the inhibitors docked into the eNOS catalytic center. Residues within 5 A from the ligand are displayed as
wire graphics and heme is displayed as ball and stick.
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Acknowledgements
We thank Dr. Jung-Min Park and Professor Tetsuo
Nagano of the University of Tokyo for providing hu-
man NOS baculovirus and for advice about the assay.
We also thank Dr. Takayoshi Suzuki and Professor
Naoki Miyata of this university for the docking study.
We are grateful to Uehara Memorial Foundation for
financial support.
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