Design, synthesis, and evaluation of new type of l-amino acids containing pyridine moiety as nitric oxide synthase inhibitor

Article abstract of DOI:10.1016/j.bmc.2006.01.020

New amino acids 7-12 were designed and synthesized as candidate inhibitors of human nitric oxide synthase (NOS). The 2-aminopyridine-containing l-amino acids 8 had potent inhibitory activity toward all of the human NOS isozymes. However, the regioisomers 9 and 10, and 2-methylpyridine-containing compound 11 had much lower inhibitory activity. Human NOS isozymes were also inhibited by 7, which lacks an amino group on the pyridine moiety. A computational docking study was carried out to investigate the mechanism of the inhibitory effect.

Full text of DOI:10.1016/j.bmc.2006.01.020

Bioorganic & Medicinal Chemistry 14 (2006) 3563–3570
Design, synthesis, and evaluation of new type of L-amino
acids containing pyridine moiety as nitric oxide synthase inhibitor
Ryosuke Ijuin, Naoki Umezawa and Tsunehiko Higuchi*
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
Received 14 November 2005; revised 6 January 2006; accepted 7 January 2006
Available online 8 February 2006
Abstract—New amino acids 7–12 were designed and synthesized as candidate inhibitors of human nitric oxide synthase (NOS). The
2-aminopyridine-containing L-amino acids 8 had potent inhibitory activity toward all of the human NOS isozymes. However, the
regioisomers 9 and 10, and 2-methylpyridine-containing compound 11 had much lower inhibitory activity. Human NOS isozymes
were also inhibited by 7, which lacks an amino group on the pyridine moiety. A computational docking study was carried out to
investigate the mechanism of the inhibitory effect.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Structural analogs of L-arginine, the natural substrate of
NOS, have been shown to inhibit the various forms of
NOS, and non-amino acid inhibitors of NOS have also
been reported.9 For example, S-methyl-isothiocitrulline
(L-MIT 1),10 N-iminoethyl-L-ornithine (L-NIO 2),11
and N-iminoethyl-L-lysine (L-NIL 3)12 are arginine ana-
logs with potent inhibitory activities toward all three
NOS isozymes, while 1400W (4)13 is a non-amino acid
type inhibitor that is iNOS-selective. We have previously
designed two types of NOS inhibitors based on the
working hypothesis that each NOS isozyme has a differ-
ent hydrophobic region at its L-arginine recognition site.
First, we designed a dipeptide-type NOS inhibitor
(5),14,15 which has a phenylalanine moiety at the C-ter-
minal of L-MIT, and found that it is an iNOS-selective
inhibitor. Next, we designed 6,16 which has an alkyl moi-
ety at the 3-position of L-MIT, we found that the 3-(R)-
methyl derivative 6 has no inhibitory effect on eNOS,
but inhibits both nNOS and iNOS, whereas the 3-(S)-
methyl derivative inhibits all the NOS isozymes
(Fig. 1). These results suggested that arginine analogs
with a hydrophobic substituent could be isozyme-selec-
tive inhibitors. To examine the contribution of hydro-
phobic substituents near the guanidino group of the
substrate, we have newly designed pyridine ring-contain-
ing arginine analogs (7–12) and investigated their struc-
ture–activity relationship (Fig. 2). These types of
aminopyridines should be more stable than those con-
taining an S-alkylated isothiourea moiety. Recently,
Connolly et al. reported 2-aminopyridines as iNOS-
selective inhibitors.17 Here, we describe the synthesis
of new 2-aminopyridine-type arginine analogs and the
Nitric oxide synthase (NOS) catalyzes the oxidation of a
guanidino nitrogen of L-arginine to nitric oxide (NO)
with concomitant formation of L-citrulline.1 NO is an
important signaling molecule involved in a wide range
of physiological functions, as well as pathophysiological
states.2–4 Three major isozymes of NOS have been iden-
tified. The endothelial isozyme (eNOS),5 which is consti-
tutive and tightly regulated by Ca2+ and calmodulin
(CaM), plays a key role in maintaining normal blood
pressure.6 The inducible isozyme (iNOS)7 is expressed
in response to endotoxin and inflammatory cytokines
in macrophages, in vascular endothelial and smooth
muscle cells, and in many other cell types. Because iNOS
is often expressed at high levels and is essentially unreg-
ulated once expressed, locally high, and therefore cyto-
toxic concentrations of NO can be generated.
Neuronal NOS (nNOS)8 as with eNOS, is constitutive
and tightly regulated by Ca2+/CaM. Although nNOS
normally generates low levels of NO for intercellular sig-
naling in the nervous system, overstimulation of nNOS
is implicated in ischemia-reperfusion injury following
stroke, migraine headache, pain, and several neurode-
generative disorders.
Keywords: Nitric oxide synthase; Inhibitor; Human; Pyridine; Picoline;
Design; Synthesis; IC50; Regioisomer; 2-Aminopyridine; Cysteine;
Arginine; Amino acid; Citrulline assay; Computational docking study;
Isozyme; Structure–activity relationship; Competitive; Nitric oxide.
*
Corresponding author. Tel./fax: +81 52 836 3435; e-mail: higuchi@
phar.nagoya-cu.ac.jp
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.020

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R. Ijuin et al. / Bioorg. Med. Chem. 14 (2006) 3563–3570
Figure 1. Structures of NOS inhibitors.
Figure 2. Designs of NOS inhibitors.
evaluation of their inhibitory activity toward the three
recombinant human NOS isozymes. A docking study
was conducted to throw light on the substituent.
a number of bromomethylated heteroarenes are readily
available.
2.2. Biological evaluation
2. Results and discussion
2.1. Chemistry
Human NOS isozymes were expressed in Sf-9
(a Spodoptera frugiperda insect cell line). Recombinant
human NOS activity was measured by monitoring the
conversion of [14C]-L-Arg to [14C]-L-Cit.21 The IC50
values of synthesized compounds were calculated from
the concentration dependence of the inhibitory activity
(Table 1). Compound 7, without an amino group on
the pyridine ring, inhibited all the human NOS isozymes
(IC50 values: nNOS 2 lM, iNOS 0.5 lM, and eNOS
4.8 lM), with slight selectivity for iNOS (entry 1). Com-
pound 8 had potent inhibitory activity toward each
NOS isozyme (IC50 values: nNOS 0.06 lM, iNOS
0.1 lM, and eNOS 0.02 lM), and potency was compa-
rable to that of L-MIT 1 (entries 2 and 7). The 2-amino-
pyridine group seems to provide higher affinity for NOS
than does a simple pyridine group. Compound 9, with
the amino acid moiety at the 4-position, had much
weaker inhibitory activity than 8 (entry 3). Moreover,
compound 10, substituted at the 5-position, showed
The first step in the synthesis was protection of 2-amino
picolines 13a–c. The protected picolines (14a–c) were
easily converted to bromomethyl derivatives 15a–c.18
These compounds were too unstable to isolate, so we
used them without further purification. 2-Picoline
(16d) and 2,6-lutidine (16e) were converted to bromo-
methyl derivatives 17d,e (Scheme 1). Protected cysteine
18 was obtained from L-cystine according to the litera-
ture.19,20 Reaction of 15a–c or 17d,e with 18 gave pyri-
dine ring-containing protected amino acids (19a–e).
Deprotection of 19a–e afforded the desired amino acids
(7–11) (Scheme 2). We also synthesized the sulfonyl
compound 20, by oxidation of 19d, and 12 was obtained
by deprotection of 20 (Scheme 3). The route allows
ready variation of the heteroaromatic groups because

R. Ijuin et al. / Bioorg. Med. Chem. 14 (2006) 3563–3570
3565
Scheme 1. Reagents: (a) Boc2O, DMAP, N,N-diisopropylethylamine, CH2Cl2, 14a 22%, 14b 33%, 14c 24%; (b) NBS, BPO, CCl4, 17d 28%, 17e 31%.
Table 2. Kinetic constants (lM) of obtained amino acids for human
NOS isozymesa
Entry
Compound
nNOSb
iNOSb
eNOSb
1
2
3
L-Arg (Km)c
1.5
16
4.0
2
2.0
3.7
0.5
7
8
1.5
9.5
a Ki values were analyzed by means of Dixon analysis. Measurement of
human NOS activity under initial velocity conditions is described in
Section 4.
b Human NOS isozymes were expressed in Sf-9 cells.
c Data from Ref. 15.
Scheme 2. Reagents and conditions: (a) 17, N,N-diisopropylethyl-
amine, CH2Cl2, rt, 16 h, 19a 35% in two steps, 19b 58% in two steps,
19c 53% in two steps, 19d 71%, (b) HCl–dioxane, rt, 12 h. All
compounds were obtained in quantitative yield.
dramatically decreased inhibitory activity (entry 4).
Compound 11, with a methyl group instead of an amino
group on the pyridine ring of 8, had low inhibitory activ-
ity toward each NOS isozyme (entry 5). This result indi-
cates that the amino group plays an important role in
recognition at the NOS active site. Moreover,
compound 12, with a sulfonyl linker, also had a low
inhibitory activity (entry 6). We also measured the kinet-
ic constant to reveal the manner of inhibition. Dixon
analysis22 for the inhibition by 7 and 8 was carried out
and the results indicated that they are competitive inhib-
itors of the three human NOS isozymes (Table 2 and
Scheme 3. Reagents and conditions: (a) m-CPBA, CH2Cl2, 32%; (b)
HCl–dioxane, rt, 12 h, quant.
Table 1. IC50 values (lM) for inhibition of human NOS isozymesa
Entry
Compound
nNOSb
iNOSb
eNOSb
4.8
0.02
42
1
2
7
8
2
0.06
90
0.5
0.1
50
3
9
4
10
11
12
1c
3d
5c
6e
390
500
100
0.06
61
120
140
45
400
260
200
5
6
7
0.3
5
0.4
8
138
>1000f
>1000f
9
10
36
38
3.9
110
a Human NOS activity was measured by monitoring the conversion of
[
14C]-L-Arg to [14C]-L-Cit.
b Human NOS isozymes were expressed in Sf-9 cells.
c Data from Ref. 15.
d Data from Ref. 12.
Figure 3. Dixon analysis of inhibition of human iNOS by 8.
Measurement of human iNOS activity under initial velocity conditions
is [14C]-L-arginine concentration was 4.2 or 8.3 lM.
e Data from Ref. 16.
f Not inhibited by 50% at concentrations up to 1 mM inhibitor.

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R. Ijuin et al. / Bioorg. Med. Chem. 14 (2006) 3563–3570
Fig. 3). We found that the direction of the amidino moi-
ety linked with the 2-aminopyridine in the amino acid
greatly affects the inhibitory activity. This result strongly
suggests that it is important to direct the amidino moiety
appropriately for binding to the carboxyl group of Glu
377 that should fix the guanidino group of L-arginine.
In order to investigate this hypothesis, we conducted
docking calculations.
Next, we calculated hydrogen-bonding interactions be-
tween iNOS residues and inhibitors 8 and 10 to investigate
the effects of the amino group on the pyridine ring (Figs.
5a anþd b, 6). In the case of 8, a hydrogen atom of
˚
oxygen atom of carboxylate was located at 2.11 A from
a hydrogen atom of Arg 388 and a hydrogen atom of pyr-
˚
idinium ion was located at 1.44 A from an oxygen atom of
˚
a-NH3 was located at 1.55 A from heme propionate, an
Glu 377. On the other hand, in the case of 10, hydrogen of
˚
˚
oxygen of carboxylate was located at 2.24 A from hydro-
þ
2.3. Docking study
a-NH3 was located at 1.61 A from heme propionate, and
To study the binding modes of 8 and 10 to the active site,
we calculated the minimum energy conformations of 8
and 10 docked into a model based on the crystal structure
of human iNOS (PDB code 1NSI)23 using the software
package MacroModel 8.1, and we compared the results
with those obtained for L-NIL (3) (Fig. 4). The obtained
structures were similar in their amino acid backbone
orientation and showed good overlap with 3 in the iNOS
active site (Fig. 4a). There is a high degree of similarity
among the binding modes to iNOS of the amino acid
backbone in 8 and 10. However, in the case of 10, the ami-
no group on the pyridine ring cannot be overlapped with
that of 3 or 8 (Fig. 4b). This result is consistent with the
difference of NOS-inhibitory activity between 8 and 10.
gen of Arg 388. The pyridinium ion of 10 was located too
far from Glu 377 for hydrogen-bonding to occur. It is sug-
gested that 10 has weak affinity for the binding site be-
cause it has less hydrogen-bonding and ionic interaction
with iNOS residues than does 8. These results are consis-
tent with the difference of inhibitory activity between 8
and 10. We also calculated the minimum energy confor-
mations of 21 (Fig. 7), which are known as iNOS selective
inhibitor (IC50 values for human NOS; nNOS 177 lM,
iNOS 2.19 lM, and eNOS 544 lM),24 docked into a mod-
el. We calculated hydrogen-bonding interactions between
iNOS residues and the inhibitor 21 to compare with 8 and
10. In the case of 21, hydrogen atom of a-NHþ3 was locat-
˚
ed at 1.64 A from heme propionate, an oxygen atom of
Figure 4. Superposition of the minimum energy conformations of 3, 8, and 10 in the iNOS active site. iNOS protein is not shown for the sake of
clarity. Heme is displayed in ball and stick style. View of the conformations of (a) 3 and 8; (b) 3 and 10 docked into the iNOS catalytic center.
Figure 5. Superposition of the minimum energy conformations of 8 and 10. View of the conformation of (a) 8 and (b) 10 docked into the iNOS
˚
catalytic center. Residues within 5 A from the ligand are displayed using wire graphics and heme is displayed in ball and stick style.

R. Ijuin et al. / Bioorg. Med. Chem. 14 (2006) 3563–3570
3567
Figure 6. Hydrogen-bonding interactions of (a) 8 and (b) 10 with the iNOS active site.
inhibitory activity toward all of the human NOS iso-
zymes and could be a useful scaffold for developing nov-
el human NOS inhibitors. Further investigation of the
structure–activity relationship is in progress in our labo-
ratory and may lead to subtype-selective iNOS
inhibitors.
Figure 7. Structure of GW274150.
˚
carboxylate was located at 5.11 A from a hydrogen atom
of Arg 388, and a hydrogen atom of ammonium ions were
4. Experimental
4.1. Chemistry
˚
located at 1.42 and 1.48 A from oxygen atoms of Glu 377.
This result suggested that the tight binding to Glu 377 is
important for the inhibition of NOS isozymes and the
side-chain length of 8 is slightly short to bind two hydro-
gen atoms of pyridinium ion. However, two Arg residues
(Arg 266 and Arg 388) are located near the carboxylate,
and they might interact with carboxylate anion by ionic
interaction. We further calculated the length between
nitrogen atom of pyridine or iminoethyl moiety and car-
bon atom of carboxylate. The distance between nitrogen
4.1.1. General. All manipulations were carried out under
an argon atmosphere unless otherwise noted. Argon gas
was dried by passage through P2O5 (Merck, SICA-
PENT). NMR spectra were recorded on a JEOL
1
GSX-400 spectrometer (500 MHz for H, 100 MHz for
13C), JEOL JMN-LA500 spectrometer (400 MHz for
1H, 125 MHz for 13C), and JEOL JMN-AL500 spec-
trometer (500 MHz for 1H, 125 MHz for 13C). Chemical
shifts are reported in d parts per million referenced to an
˚
˚
atom and carbon atom is 7.06 A (8) and 8.13 A (21),
respectively. The distance between guanidium moiety
1
and nitrogen atom of a-NHþ3 is 5.66 A (8) and 7.38 A
(21), respectively. These results suggest that the longer
side chain might be partially responsible for the better
selectivity.
˚
˚
internal tetramethylsilane standard for H NMR. Chlo-
roform-d1 (d 77.0 for 13C) was used as an internal refer-
ence for 13C NMR. 1H and 13C NMR spectra were
recorded in CDCl3 at 25 °C unless otherwise noted. IR
spectra were obtained with a Jasco FT/IR-680 spectrom-
eter, and values are expressed in cmꢀ1. Mass spectra
were recorded on a JEOL JMS-LCmate and Bruker
Daltonics APEX II mass spectrometer.
3. Conclusion
In summary, we have designed and synthesized various
S-picolylcysteine-based compounds and examined their
structure–activity relationship as human NOS inhibi-
tors. Compound 8 had potent inhibitory activity toward
all the human NOS isozymes, while 10 had low inhibito-
ry activity. The results suggest that the amidino moiety
of 8 is tightly bound to Glu 377 (iNOS) and the amino
acid moiety interacts with Arg 388 (iNOS) and heme
propionate, whereas the structure of 10 is less favorable
for such interactions. Docking calculations were consis-
tent with this view. In addition, the 2-methylpyridine
derivative 11 had low activity, probably because of unfa-
vorable interaction of the methyl group on the pyridine
moiety with the human NOS binding site. This result
suggests that an aminopyridine moiety at an appropriate
position is favorable for recognition by human NOS.
Compound 8 is a stable compound with potent
4.1.2. Materials. DMF (N,N-dimethylformamide) and
dichloromethane were dried over calcium hydride and
distilled prior to use. THF (tetrahydrofuran) and diethyl
ether were dried over sodium benzophenone ketyl and
distilled prior to use. Triethylamine and diisopropyleth-
ylamine were dried over KOH and distilled prior to use.
Isobutene, di-tert-butyl dicarbonate (Boc2O), picoline,
2-amino-4-picoline, 2-amino-6-picoline, 6-amino-3-pico-
line, and 2-6-lutidine were purchased from Tokyo Kasei
Kogyo Co., Ltd. L-Cystine was purchased from Nacalai
Tesque, Inc.
4.1.3. General method for preparation of N,N-bis-
tert-butyloxycarbonylamino-methylpyridines (14a–c). A
typical procedure, for the preparation of N,N-bis-tert-
butyloxycarbonyl-2-amino-6-methylpyridine (14a) as an

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R. Ijuin et al. / Bioorg. Med. Chem. 14 (2006) 3563–3570
example, is as follows. To a solution of 540 mg (5 mmol)
of 2-amino-6-picoline and 12.2 mg (0.1 mmol) of DMAP
in 10 mL of CH2Cl2 was added 1.05 mL of N,N-diisopro-
pylethylamine at room temperature and the mixture was
cooled to 0 °C. Boc2O 2.62 g (12 mmol) in 5 mL of
CH2Cl2 was added and the reaction mixture was stirred
for 6 h at room temperature. The reaction mixture was
separated with 5% citric acid three times and then washed
with sat. NaHCO3 and brine. The organic phase was dried
over Na2SO4 and the solvent was removed under reduced
pressure. The residue was chromatographed on silica gel
(eluent: hexane/EtOAc = 6:1) to give 14a as a white solid,
338 mg (22%). The mono-Boc-protected compound was
also obtained as a by-product, 415 mg (40%).
and the mixture was stirred for 18 h at room tempera-
ture. The solvent was removed under reduced pressure.
The residue was chromatographed on silica gel (eluent:
CH2Cl2/ethyl acetate = 9:1) to give 19a as a colorless
oil, 101 mg (35% in two steps).
4.1.5.1.
(R)-N-tert-Butoxycarbonyl-2-amino-3-{[20-
(N,N-bis-tert-butoxycarbonyl)aminopyrid-60-yl]methyl-
sulfanyl}propionic acid O-tert-butyl ester (19a). Yield
35% as a colorless oil. 1H NMR (CDCl3) d 1.45 (s,
36H), 2.90 (m, 2H), 3.83 (s, 2H), 4.41 (m, 1H), 5.41
(br, 1H), 7.13 (d, J = 7.8 Hz, 1H), 7.25 (d, J = 7.8 Hz,
1H), 7.70 (t, J = 7.8 Hz, 1H); MS (FAB) 606 (M+Na),
584 (M+H).
4.1.3.1.
N,N-Bis-tert-butyloxycarbonyl-2-amino-6-
4.1.5.2.
(R)-N-tert-Butoxycarbonyl-2-amino-3-{[20-
methylpyridine (14a). Yield 22% as a white solid. 1H
NMR (CDCl3) d 1.44 (s, 18H), 7.03 (d, J = 7.8 Hz,
1H), 7.60 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H);
MS (FAB) 331 (M+Na), 309 (M+H).
(N,N-bis-tert-butoxycarbonyl)aminopyrid-40-yl]methyl-
sulfanyl}propionic acid O-tert-butyl ester (19b). Yield
58% as a colorless oil. 1H NMR (CDCl3) d 1.45 (s,
18H), 1.46 (s, 9H), 1.47 (s, 9H), 2.79 (dd, J = 5.6 Hz,
31.2 Hz, 2H), 3.73 (s, 3H), 4.41 (m, 1H), 5.33 (br, 1H),
7.20 (d, J = 5.1 Hz, 1H), 7.22 (s, 1H), 8.41 (d, J = 5.1
Hz, 1H); MS (FAB) 606 (M+Na), 584 (M+H).
4.1.3.2.
N,N-Bis-tert-butyloxycarbonyl-2-amino-4-
1
methylpyridine (14b). Yield 33% as a colorless oil. H
NMR (CDCl3) d 1.45 (s, 18H), 7.04 (m, 2H), 8.34 (d,
J = 5.4 Hz, 1H); MS (FAB) 331 (M+Na), 309 (M+H).
4.1.5.3.
(R)-N-tert-Butoxycarbonyl-2-amino-3-{[20-
(N,N-bis-tert-butoxycarbonyl)aminopyrid-50-yl]methyl-
sulfanyl}propionic acid O-tert-butyl ester (19c). Yield
53% as a colorless oil. 1H NMR (CDCl3) d 1.44 (s,
18H), 1.46 (s, 9H), 1.47 (s, 9H), 2.81 (dd, J = 5.6 Hz,
31.2 Hz, 2H), 3.77 (s, 3H), 4.43 (m, 1H), 5.30 (br, 1H),
7.20 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 8.40
(s, 1H); MS (FAB) 584 (M+H).
4.1.3.3.
N,N-Bis-tert-butyloxycarbonyl-6-amino-3-
methylpyridine (14c). Yield 24% as a white solid. 1H
NMR (CDCl3) d 1.44 (s, 18H), 2.35 (s, 3H), 7.09 (d,
J = 8.2 Hz, 1H), 7.53 (dd, J = 2.1 Hz, 8.2 Hz, 1H), 8.31
(d, J = 2.1 Hz, 1H); MS (FAB) 331 (M+Na), 309 (M+H).
4.1.4. General method for preparation of bromomethyl-
pyridines (15a–c and 17d,e). A typical procedure, for the
preparation of 2-bromomethylpyridine (17d) as an exam-
ple, is as follows. A solution of 0.5 mL (5 mmol) of pico-
line, 1.43 g (8 mmol) of N-bromosuccinimide, and
19.4 mg (0.08 mmol) of benzoyl peroxide in 40 mL CCl4
was stirred at room temperature and then irradiated with
a halogen lamp under reflux for 6 h. The reaction mixture
was cooled and the precipitate was filtered off. The filtrate
was concentrated under reduced pressure, and the residue
was chromatographed on silica gel (eluent: CH2Cl2) to
give 240 mg (28%) of 17d.
4.1.5.4. (R)-N-tert-Butoxycarbonyl-2-amino-3-{(pyrid-
20-yl)methylsulfanyl}propionic acid O-tert-butyl ester
(19d). Yield 35% as a colorless oil. H NMR (CDCl3)
d 1.45 (s, 18H), 2.93 (d, J = 5.4 Hz, 2H), 3.88 (s, 2H),
4.42 (m, 1H), 5.68 (br, 1H), 7.17 (m, 1H), 7.32 (m,
1H), 7.66 (m, 1H), 8.55 (m, 1H); MS (FAB) 369 (M+H).
1
4.1.5.5.
(R)-N-tert-Butoxycarbonyl-2-amino-3-
{(20methyl-pyrid-60-yl)methylsulfanyl}propionic acid O-
tert-butyl ester (19e). Yield 36% as a colorless oil. H
1
NMR (CDCl3) d 1.45 (s, 18H), 2.56 (s, 3H), 2.91 (m,
2H), 3.84 (s, 2H), 4.43 (m, 1H), 5.98 (br, 1H), 7.03 (d,
J = 7.8 Hz, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.54 (t,
J = 7.8 Hz, 1H); MS (FAB) 383 (M+H).
4.1.4.1. 2-Bromomethylpyridine (17d). Yield 28% as a
1
brown oil. H NMR (CDCl3) d 4.57 (s, 2H), 7.23 (m,
1H), 7.45 (m, 1H), 7.72 (m, 1H), 8.61 (m, 1H); FAB-
MS was not detectable.
4.1.6. Preparation of (R)-N-tert-butoxycarbonyl-2-ami-
no-3-(pyrid-20-ylmethylsulfonyl)propionic acid O-tert-bu-
tyl ester (20). To a solution of 66 mg (0.18 mmol) of 19a
in 5 mL of CH2Cl2 was added 78 mg (0.45 mmol) of m-
CPBA and the mixture was stirred at room temperature
for 3 h. It was then diluted with CH2Cl2 and washed
with saturated NaHCO3 three times and brine once.
The organic layer was dried over Na2SO4 and concen-
trated under reduced pressure. The residue was
chromatographed on silica gel (eluent: CH2Cl2/
EtOAc = 1:1) to give 20 as a colorless oil, 23.4 mg
(32%).
4.1.4.2. 2-Bromomethyl-6-methylpyridine (17e). Yield
1
36% as a brown powder. H NMR (CDCl3) d 2.56 (s,
3H), 4.52 (s, 2H), 7.07 (d, J = 7.6 Hz, 1H), 7.24 (d,
J = 7.6 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H); FAB-MS was
not detectable.
4.1.5. Preparation of (R)-N-tert-butoxycarbonyl-2-ami-
no-3-[(20-substituted-pyridyl)methylsulfanyl]propionic
acid O-tert-butyl esters (19a–e). A typical procedure, for
the preparation of 19a as an example, is as follows. To a
solution of 193 mg (0.5 mmol) of 15a and 348 lL
(2.0 mmol) of N,N-diisopropylethylamine in 0.5 mL of
CH2Cl2 was added 139 mg (0.5 mmol) of 18 at 0 °C,
1H NMR (CDCl3) d 1.45 (s, 9H), 1.46 (s, 9H), 3.64 (m,
2H), 4.50 (m, 2H), 4.66 (m, 1H), 5.99 (br, 1H), 7.33 (m,

R. Ijuin et al. / Bioorg. Med. Chem. 14 (2006) 3563–3570
3569
1H), 7.46 (d, J = 7.6 Hz, 1H), 7.77 (m, 1H), 8.64 (m,
1H); MS (FAB) 423 (M+Na), 401 (M+H).
4.1.7.5. (R)-2-Amino-3-[(20-methyl-pyrid-60-yl)meth-
ylsulfanyl]propionic acid dihydrochloride (11). Yield
quant. as a brown powder. H NMR (CD3OD) d 2.83
1
4.1.7. Preparation of (R)-2-amino-3-[(20-substituted-pyr-
idyl)methylsulfanyl]propionic acid dihydrochlorides (7–
12). A typical procedure, for the preparation of 7 as an
example, is as follows. HCl-saturated 1,4-dioxane solu-
tion (5 mL) was added to 51 mg (0.14 mmol) of 18d at
0 °C and the mixture was stirred for 12 h at room tem-
perature. The solvent was removed under reduced pres-
sure and 40 mg (quantitative) of the desired compound 7
was obtained. Upon recrystallization from MeOH–
ether, 12 mg of 7 was obtained as a light yellow solid.
Purity was confirmed by HPLC.
(s, 3H), 3.17 (m, 2H), 4.26 (m, 2H), 4.37 (m, 1H), 7.84
(m, 1H), 7.94 (m, 1H), 8.44 (m, 1H); 13C NMR (CD3OD)
d 26.4, 32.6, 33.3, 53.2, 126.0, 127.9, 147.9, 156.4; IR
(KBr) 1636, 2361, 3420 cmꢀ1; MS (FAB) 227 (M+H);
HR-MS (ESI) Calcd for C10H15N2O2S: 227.0849
(M+H). Found: 227.0876.
4.1.7.6. (R) 2-Amino-3-(pyrid-20-ylmethylsulfonyl)pro-
pionic acid dihydrochloride (12). Yield quant. as a brown
1
powder. H NMR (CD3OD) d 3.84 (m, 1H), 4.18 (m,
1H), 4.74 (m, 1H), 7.94 (m, 1H), 8.03 (d, J = 7.8 Hz,
1H), 8.45 (m, 1H), 8.87 (m, 1H); 13C NMR (CD3OD)
d 53.4, 127.6, 130.4, 145.4, 146.7, 168.8; IR (KBr)
1636, 2360, 3445 cmꢀ1; MS (FAB) 245 (M+H); HR-
MS (ESI) Calcd for C9H13N2O4S: 245.05941 (M+H).
Found: 245.0625.
4.1.7.1. (R)-2-Amino-3-[(pyrid-20-yl)methylsulfanyl]-
propionic acid dihydrochloride (7). Yield quant. as a
brown powder. 1H NMR (CD3OD) d 3.08 (dd,
J = 7.6 Hz, 14.9 Hz, 1H), 3.21 (dd, J = 4.4 Hz, 14.9 Hz,
1H), 4.27 (s, 2H), 4.35 (t, J = 4.4 Hz, 1H), 7.97 (m,
J = 7.3 Hz, 7.8 Hz, 1H), 8.08 (d, J = 7.8 Hz, 1H), 8.56
(d, J = 7.8 Hz, 1H), 8.82 (d, J = 7.3 Hz, 1H); 13C
NMR (CD3OD) d 32.7, 33.7, 53.2, 127.2, 128.9, 143.6,
4.2. Measurement of NOS-inhibitory activity
4.2.1. Materials. Ex-cell 420 was purchased from JRH
Biosciences. Hemin was purchased from Tokyo Kasei
Kogyo Co., Ltd. Riboflavin, nicotinic acid, BH4, DTT,
and PMSF were purchased from Wako Pure Chemical
Industries, Ltd, and Tris–HCl, EGTA, FAD, and
FMN from Nacalai Tesque, Inc. CHAPS was purchased
from Dojindo Laboratories. Pepstatin, leupeptin, and
chymostatin were purchased from Peptide Institute,
Inc. 20,50-ADP Sepharose was purchased from Pharma-
cia Biotech and NADPH from Oriental Yeast Co., Ltd.
148.3, 154.7, 170.0; IR (KBr) 1734, 2360, 3425 cmꢀ1
;
MS (FAB) 235 (M+Na), 213 (M+H); Anal. Calcd for
C9H12N2O2SÆ2HCl: C, 37.90; H, 4.95; N, 9.82. Found:
C, 37.33; H, 5.03; N, 9.63.
4.1.7.2.
(R)-2-Amino-3-[(20-aminopyrid-60-yl)meth-
ylsulfanyl]propionic acid dihydrochloride (8). Yield
1
quant. as a light brown powder. H NMR (CD3OD) d
3.03 (m, 1H), 3.14 (m, 1H), 3.94 (s, 2H), 4.31 (m, 1H),
6.86 (d, J = 7.3 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 7.86
(dd, J = 7.3 Hz, 8.3 Hz, 1H); 13C NMR (CD3OD) d
32.2, 33.2, 54.2, 113.6, 113.7, 117.6, 145.4, 146.7; IR
(KBr) 1662, 2361, 3397 cmꢀ1; MS (FAB) 228 (M+H);
Anal. Calcd for C9H13N3O2SÆ2HClÆ0.6H2O: C, 34.76;
H, 5.25; N, 13.51. Found: C, 34.99; H, 5.91; N, 12.91.
4.2.2. Expression and purification of human NOS iso-
zymes. Human NOS isozymes were expressed in Sf-9 (a
Spodoptera frugiperda insect cell line) as previously
reported.15 Monolayer cultures of Sf-9 cells were infected
with human NOS recombinant baculovirus and incubat-
ed for 72 h at 27 °C in Ex-cell 420 insect serum-free medi-
um supplemented with 1.5 lg/mL hemin, 1 lM
riboflavin, 5 lM nicotinic acid, and 10 lM BH4. For
iNOS, Sf-9 cells were co-infected with the CaM baculovi-
rus. The cells were harvested by centrifugation and
homogenized in five volumes of ice-cold buffer (Tris–
HCl 50 mM, pH 7.5, containing 1 mM EGTA, 1 mM
DTT, 1 mM PMSF, 1 lg/mL pepstatin, 1 lg/mL leupep-
tin, 20 lM chymostatin, 5 lM FAD, 5 lM FMN, and
10 lM BH4; for eNOS, 10 mM CHAPS was included).
The homogenate was centrifuged at 40,000g for 30 min.
The supernatant fractions were applied to a 2 mL 20,50-
ADP Sepharose affinity column. The column was washed
with 0.5 M NaCl and eluted with 10 mM NADPH. The
eluted fraction was concentrated to 0.2 mg/mL with a
Centricon 30. For eNOS, 10 mM CHAPS was included
in the buffer to obtain sufficient solubility of the enzyme.
4.1.7.3.
(R)-2-Amino-3-[(20-aminopyrid-40-yl)meth-
ylsulfanyl]propionic acid dihydrochloride (9). Yield
1
quant. as an off-white powder. H NMR (CD3OD) d
3.00 (dd, J = 7.6 Hz, 14.9 Hz, 1H), 3.11 (dd,
J = 4.3 Hz, 14.9 Hz, 1H), 3.86 (s, 2H), 4.26 (dd,
J = 4.3 Hz, 7.6 Hz, 1H), 6.94 (d, J = 6.7 Hz, 1H), 7.23
(s, 1H), 7.80 (d, J = 6.7 Hz, 1H); 13C NMR (CD3OD)
d 32.4, 35.8, 53.2, 113.9, 114.7, 136.6, 155.7, 157.7,
170.2; IR (KBr) 1669, 2360, 3427 cmꢀ1; MS (FAB)
228 (M+H); Anal. Calcd for C9H13N3O2SÆ2HClÆ0.75-
H2O: C, 34.46; H, 5.30; N, 13.39. Found: C, 34.48; H,
5.67; N, 12.91.
4.1.7.4.
(R)-2-Amino-3-[(20-aminopyrid-50-yl)meth-
ylsulfanyl]propionic acid dihydrochloride (10). Yield
1
quant. as a brown powder. H NMR (CD3OD) d 2.98
(dd, J = 7.6 Hz, 14.9 Hz, 1H), 3.09 (dd, J = 4.2 Hz,
14.9 Hz, 1H), 3.79 (s, 2H), 4.27 (dd, J = 4.2 Hz,
7.6 Hz, 1H), 7.04 (d, J = 9.4 Hz, 1H), 7.83 (s, 1H),
8.00 (d, J = 9.4 Hz, 1H); 13C NMR (CD3OD) d 32.4,
33.4, 53.2, 115.5, 124.1, 135.0, 144.9, 146.7, 155.2; IR
(KBr) 1671, 2360, 3853 cmꢀ1; MS (FAB) 228 (M+H);
Anal. Calcd for C9H13N3O2SÆ2HClÆH2O: C, 33.97; H,
5.39; N, 13.21. Found: C, 34.38; H, 5.57; N, 12.85.
4.2.3. Enzyme assay and Ki values. Recombinant human
NOS activity was measured by monitoring the conversion
of [14C]-L-citrulline as previously reported.15 Enzyme
solution (5 lL) was added to 25 lL of buffer containing
50 mM HEPES (pH 7.4), 4.2–16.7 lM [14C]-L-arginine,
18 mM CaCl2, 150 lM DTT, 10 lg/mL CaM, 15 lM
BH4, 1 mM EGTA, 15 lM FAD, 15 lM FMN, 0.9 mg/

3570
R. Ijuin et al. / Bioorg. Med. Chem. 14 (2006) 3563–3570
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mL BSA, 1.5 mM NADPH, and the test compound in the
concentration range from 1 pM to 1 mM. After incuba-
tion for 10 min at 37 °C, the reaction was terminated by
adding 20 lL of 5 mM cold L-arginine and 5 mM L-citrul-
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Docking was performed using MacroModel 8.1 soft-
ware. Coordinates of rat nNOS, human iNOS, and
human eNOS complexed with arginine or hydroxyargi-
nine were taken from the Protein Data Bank (PDB code
1NSI) and hydrogen atoms were added computationally
at appropriate positions. The structures of L-NIL (3)
and compounds 8, 10, and 21 docked to NOS were con-
structed by molecular mechanics (MM) energy minimi-
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determined manually. The conformations of 3, 8, 10,
and 21 in the active site were minimized by MM calcu-
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Acknowledgments
We thank Dr. Takayoshi Suzuki and Professor Naoki
Miyata of this university for the docking study, and
Mr. Takehiro Yamane for assistance in synthesizing
protected picolines. We are grateful to Uehara Memori-
al Foundation for financial support.
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