Structure-activity relationships of 2-aminothiazole derivatives as inducible nitric oxide synthase inhibitor

Article abstract of DOI:10.1248/cpb.52.634

Nitric oxide synthase (NOS) has been divided into two major sub-enzymes, i.e. inducible NOS (iNOS) and constitutive NOS (cNOS). Although nitric oxide (NO) plays an important role as host defense mediator, excessive production of NO by iNOS has been involved in the pathology of many inflammatory diseases. Recently, we reported that the 2-imino-1,3-oxazolidine (1a) weakly inhibits iNOS and that introduction of an alkyl moiety on the oxazolidine ring of 1a enhances the inhibitory activity and selectivity for iNOS. In our search for better iNOS inhibitors, we focused our efforts on the 2-aminothiazole scaffold 3 as it possesses a ring similar to that of 1a. In this study, we evaluated the inhibitory activity of a series of 2-aminothiazole derivatives against both iNOS and neuronal NOS (nNOS). Our results show that introduction of appropriately-sized substituents at the 4- and 5-position of the 2-aminothiazole ring improves the inhibitory activity and selectivity for iNOS. We also found that the selectivity of 5a [5-(1-methyl)ethyl-4-methylthiazol-2-ylamine] and 5b [5-(1,1-dimethyl)ethyl-4-methylthiazol-2-ylamine] for iNOS was similar to that of oxazolidine derivative 1b (4-methyl-5-propyl-2-imino-1,3-oxazolidine) and much higher than that of L-NAME. However, we could not enhance the inhibitory activity against iNOS by introducing an alkyl substituent into the 2-aminothiazole ring as we could in the case of oxazolidine one. On the other hand, introduction of bulky or hydrophilic substituent at any position of the 2-aminothiazole ring remarkably decreased or even abolished the inhibitory activity against NOS.

Full text of DOI:10.1248/cpb.52.634

634
Notes
Chem. Pharm. Bull. 52(5) 634—637 (2004)
Vol. 52, No. 5
Structure–Activity Relationships of 2-Aminothiazole Derivatives as
Inducible Nitric Oxide Synthase Inhibitor
c
Shigeo UEDA, ,a Hideo TERAUCHI,a,b Motoji KAWASAKI,a Akihiro YANO,c and Motoharu IDO
*
a Chemistry Research Laboratories, Dainippon Pharmaceutical Co., Ltd.; 33–94 Enoki-cho, Suita, Osaka 564–0053,
b
Japan: Pharmaceutical Research & Technology Center, Dainippon Pharmaceutical Co., Ltd.; 1–5–51 Ebie, Fukushima-
ku, Osaka 553–0001, Japan: and c Pharmacology & Microbiology Research Laboratories, Dainippon Pharmaceutical Co.,
Ltd.; 33–94 Enoki-cho, Suita, Osaka 564–0053, Japan.
Received January 7, 2004; accepted February 10, 2004; published online February 13, 2004
Nitric oxide synthase (NOS) has been divided into two major sub-enzymes, i.e. inducible NOS (iNOS) and
constitutive NOS (cNOS). Although nitric oxide (NO) plays an important role as host defense mediator, excessive
production of NO by iNOS has been involved in the pathology of many inflammatory diseases. Recently, we re-
ported that the 2-imino-1,3-oxazolidine (1a) weakly inhibits iNOS and that introduction of an alkyl moiety on the
oxazolidine ring of 1a enhances the inhibitory activity and selectivity for iNOS. In our search for better iNOS in-
hibitors, we focused our efforts on the 2-aminothiazole scaffold 3 as it possesses a ring similar to that of 1a. In
this study, we evaluated the inhibitory activity of a series of 2-aminothiazole derivatives against both iNOS and
neuronal NOS (nNOS). Our results show that introduction of appropriately-sized substituents at the 4- and 5-po-
sition of the 2-aminothiazole ring improves the inhibitory activity and selectivity for iNOS. We also found that
the selectivity of 5a [5-(1-methyl)ethyl-4-methylthiazol-2-ylamine] and 5b [5-(1,1-dimethyl)ethyl-4-methylthiazol-
2-ylamine] for iNOS was similar to that of oxazolidine derivative 1b (4-methyl-5-propyl-2-imino-1,3-oxazolidine)
and much higher than that of L-NAME. However, we could not enhance the inhibitory activity against iNOS by
introducing an alkyl substituent into the 2-aminothiazole ring as we could in the case of oxazolidine one. On the
other hand, introduction of bulky or hydrophilic substituent at any position of the 2-aminothiazole ring remark-
ably decreased or even abolished the inhibitory activity against NOS.
Key words nitric oxide; inducible nitric oxide synthase inhibitor; thiazole; neuronal nitric oxide synthase inhibitor; neuronal
nitric oxide synthase (nNOS); 2-aminothiazole
Nitric oxide synthase (NOS) oxidizes one of the two synthesized and investigated in connection with a number of
equivalent terminal guanidino nitrogens of L-arginine using inflammatory diseases. However, these analogues have not
NADPH and oxygen as substrates, as well as flavin adenine been approved for the treatment of inflammatory diseases
dinucleotide, flavin mononucleotide, tetrahydrobiopterin and due to their undesired inhibition of cNOS.
haem as cofactors to produce L-citrulline and the biologically
In addition to the L-amino acid analogues described above,
active free radical nitric oxide (NO).1—3) In general, NOS has a number of non-amino acid NOS inhibitors have been inves-
been divided into two major sub-enzymes, i.e. a constitutive tigated.20—28) Recently, we reported that the 2-imino-1,3-oxa-
NOS (cNOS) which requires Ca2ꢀ/Calmodulin for its activa- zolidine (1a) (Chart 1) weakly inhibits iNOS and that intro-
tion, and an inducible NOS (iNOS) which is independent of duction of an alkyl moiety on its oxazolidine ring enhances
Ca2ꢀ/Calmodulin for its activation. In addition, cNOS has the inhibitory activity and selectivity for iNOS (1b, Chart 1,
further been divided into neuronal NOS (nNOS) found Table 2).29) In our search for better iNOS inhibitors, we fo-
mainly in the brain, and endothelial NOS (eNOS) predomi- cused our efforts on the 2-aminothiazole scaffold 3 as it pos-
nantly present in the vascular endothelium. It has been re- sesses a ring similar to that of 1a. Although the 4,5-di-
ported that nNOS generates NO that functions as a neuro-
transmitter regulating neuronal transmission,4) and that
eNOS generates low concentration of NO which lowers
blood pressure.5) On the other hand, iNOS, which is
expressed in activated macrophage in response to inflamma-
tory cytokines and lipopolysaccharides (LPS), generates NO
that has cytotoxic/cytostatic activity against intracellular
pathogens including viruses and therefore plays an important
role as host defense mediator.6) However, excessive produc-
tion of NO by iNOS has been involved in numerous disease
states such as septic shock,7) rheumatoid arthritis,8) hypoten-
sion,9) chronic ileitis10) and autoimmune diabetes.11)
There was a main approach that could potentially be ex-
ploited to inhibit excessive production of NO, and analogues
of L-arginine, L-lysine, L-ornithine, L-citrulline and guanidine
such as NG-methyl-L-arginine,12) NG-nitro-L-arginine,13) NG-
nitro-L-arginine methylester (L-NAME),14) N6-(1-iminoethyl)-
L-lysine,15) N5-(1-iminoethyl)-L-ornithine,16) L-thiocitrulline,17)
S-alkyl-L-thiocitrulline18) and aminoguanidine19) have been Chart 1. Design of 2-Aminothiazole Derivatives
∗ To whom correspondence should be addressed. e-mail: shigeo-ueda@dainippon-pharm.co.jp
© 2004 Pharmaceutical Society of Japan

May 2004
635
methylthiazol-2-ylamine (2) has previously been reported to groups were inserted into the 4-position and 5-position of the
inhibit iNOS20) to our knowledge, derivatives of 2-aminothia- 2-aminothiazole ring, respectively, the inhibitory activity
zole scaffold have hardly been studied in connection with against iNOS was improved and the selectivity for iNOS over
iNOS inhibition. Herein, we report the synthesis and struc- nNOS was greatly increased (5a: IC50ꢁ0.6 mM, 4.7-fold
ture–activity relationships (SARs) for NOS inhibition of a stronger than that of 2; nNOS/iNOSꢁ23, selectivity of 5a for
series of 2-aminothiazole derivatives.
iNOS 26-fold higher than that of 2). An additional insertion
of a methyl group into the isopropyl group of 5a significantly
decreased nNOS inhibition, thus the selectivity of 5b for
Chemistry
As illustrated in Chart 2, annulation of ketone 4a—g with iNOS was the highest among the synthesized compounds
thiourea using I2 led to a mixture of the 2-aminothiazole (5b: nNOS IC50 value of 100 mM, nNOS/iNOSꢁ33, selectiv-
regio isomers 5 and 6, which were separated by column chro- ity of 5b for iNOS 37-fold higher than that of 2). When the
matography with except for 6d (Table 1).
length of side chain at the 5-position of the thiazole ring in-
Next, as shown in Chart 3, the 2,4,5-trisubstituted-2- creased, the inhibitory activity against iNOS decreased and
aminothiazole derivatives were synthesized. Reaction of a- that against nNOS was completely abolished (5c: iNOS IC50
chlorocyclopentanone with phenylthiourea and that of 5a
with isopropylisothiocyanate easily gave the 2-phenylamino
Table 2. Inhibitory Activity of Mono, Di and Trisubstituted 2-Aminothia-
zole Derivatives
derivative 7 and the thiourea derivative 8, respectively.30)
Inhibitory activitya)
Selectivityc)
Results and Discussion
The inhibitory activity of the synthesized compounds
(5a—g, 6a—c, g, 7, 8) against iNOS and nNOS was evalu-
ated according to previously reported procedures31,32) with
some modifications and their selectivity was determined
from nNOS IC50 value/iNOS IC50 value ratio.
Compound
iNOS IC50
nNOS IC50 nNOS/iNOS
(mM)b)
(mM)b)
0.6
14
23
33
As shown in Table 2, when a methyl and an isopropyl
3.0
100
e)
54
n.s. @100d)
—
6.2
28
4.5
2.1
0.7
1.3
3.1
3
12
21
25
Chart 2. Synthesis of 2-Aminothiazole Derivatives (5, 6) from Ketone 4
and Thiourea
14
4.1
5.5
17
Table 1. Synthesis of Mono and Disubstituted 2-Aminothiazole Deriva-
tives (5, 6) from Ketone 4 and Thiourea
5.5
Compound
Starting
material
Yield (%)
Salt
9.3
28
4
(R1, R2)
5
6
5
6
e)
n.s. @100d)
n.s. @100d)
—
b)
b)
b)
a
b
c
d
e
f
H, iPr
H, tBu
19
10
38
13
28
39
HCl
b)
e)
e)
n.s. @100d)
n.s. @100d)
n.s. @100d)
n.t.f)
—
—
H, nBu
11
b)
a)
Et, Me
cPnc)
32
76
b)
cHexd)
HCl
b)
g
H, CH2CO2Et
10
3
HCl
e)
n.s. @100d)
n.s. @100d)
—
a) Not isolated purely. b) Free amine. c) Cyclopentyl. d) Cyclohexyl.
1a29)
1b29)
2
7.9
0.041
2.8
34
4.3
22
0.86
0.0027
0.92
2.4
0.8
L-NAME
300
a) IC50 values for iNOS and nNOS were determined by testing each compound at
eight concentrations. b) iNOS and nNOS activity was evaluated according to previ-
ously reported methods with some modifications.31,32) c) Selectivity was defined as
the ratio of IC50 value of nNOS to iNOS. d) n.s.ꢁno significant effect (ꢂ5% inhibi-
tion). e) Not determined. f) Not tested.
Chart 3. Synthesis of Trisubstituted 2-Aminothiazole Derivatives (7, 8)

636
Vol. 52, No. 5
general procedure.
4-Methyl-5-(1-methyl)ethylthiazol-2-ylamine (5a) and 4-(2-Methyl)-
value of 54 mM; nNOS IC50 value of n.s. @100 mM). There-
fore, it is suggested that an increase of the length of side
chain at the 5-position can be tolerated in iNOS inhibition
but not in nNOS. Monosubstituted compounds, on the other
hand, generally displayed moderate inhibitory activity against
both iNOS and nNOS (6a—c). Unexpectedly, the inhibitory
activity against iNOS of 5d, a thiazole analogue of 1b, was
much weaker than that of 1b. Therefore, it is suggested that a
structurally planar ring is not favorable for strong iNOS inhi-
bition.21)
propylthiazol-2-ylamine (6a) from 4-Methyl-2-pentanone (4a) 5a:
Yield, 19%. mp 65—67 °C (hexane) (lit.34) 69—70 °C). 6a: yield, 28%. H-
1
NMR (300 MHz, CDCl3) d: 0.91 (6H, d, Jꢁ6.6 Hz), 1.95 (1H, m), 2.39 (2H,
d, Jꢁ6.6 Hz), 5.11 (2H, br), 6.07 (1H, s). 6a·HCl, mp 160—162 °C
(EtOH/Et2O). Anal. Calcd for C7H12N2S·0.95HCl·0.15H2O: C, 43.43; H,
6.90; N, 14.47; S, 16.56; Cl, 17.40. Found: C, 43.68; H, 6.81; N, 14.52; S,
16.29; Cl, 17.54. EI-MS m/z: 157 (Mꢀ1).
5-(1,1-Dimethyl)ethyl-4-methylthiazol-2-ylamine (5b) and 4-(2,2-Di-
methyl)propylthiazol-2-ylamine (6b) from 4,4-Dimethyl-2-pentanone
(4b) 5b: Yield, 10%. mp 70—71 °C (hexane). 1H-NMR (300 MHz, CDCl3)
d: 1.35 (9H, s), 2.23 (3H, s), 4.60 (2H, br). Anal. Calcd for C8H14N2S: C,
56.43; H, 8.29; N, 16.45; S, 18.83. Found: C, 56.56; H, 8.55; N, 16.53; S,
19.09. EI-MS m/z: 171 [Mꢀ1]ꢀ. 6b: Yield, 39%. mp 69—70 °C (hexane).
1H-NMR (300 MHz, CDCl3) d: 0.95 (9H, s), 2.42 (2H, s), 4.89 (2H, br),
Next, we evaluated the inhibitory activity of other 2-
aminothiazole derivatives against NOS.
The inhibitory activity of the bicyclic compounds (5e, f)
against iNOS were similar to that of 2. On the other hand, in- 6.08 (1H, s). Anal. Calcd for C8H14N2S·0.1H2O: C, 55.84; H, 8.32; N,
16.28; S, 18.63. Found: C, 55.81; H, 8.24; N, 16.27; S, 18.94. EI-MS m/z:
troduction of substituent at the 2-amino group (7, 8) abol-
ished the inhibitory activity against both iNOS and nNOS.
These findings suggest that strong iNOS inhibition requires a
non-substituted 2-amino group on the thiazole ring. Hy-
drophilically substituted 2-aminothiazole derivatives (5g, 6g)
also displayed no significant inhibition against both iNOS
and nNOS. As iNOS oxygenase haem domain has been re-
ported to form a hydrophobic pocket,33) the contrastive hy-
drophilicity of 5g and 6g is assumed to be responsible for the
abolishment of inhibitory activity against iNOS.
171 [Mꢀ1]ꢀ.
5-Butyl-4-methylthiazol-2-ylamine (5c) and 4-Pentylthiazol-2-ylamine
(6c) from 2-Heptanone (4c) 5c: Yield, 38%. mp 38—40 °C (hexane)
(lit.35) 41—42 °C). 6c: Yield, 11%. mp 36—37 °C (hexane) (lit.36) 45—
46 °C).
5-Methyl-4-propylthiazol-2-ylamine (5d) from 2-Hexanone (4d)
Yield, 13%. mp 58—60 °C (hexane, 4 times). H-NMR (300 MHz, CDCl3)
1
d: 0.92 (3H, t, Jꢁ7.2 Hz), 1.55—1.67 (2H, m), 2.19 (3H, s), 2.41 (2H, t,
Jꢁ7.4 Hz), 4.75 (2H, br). Anal. Calcd for C7H12N2S: C, 53.81; H, 7.74; N,
17.93; S, 20.52. Found: C, 53.82; H, 7.80; N, 17.81; S, 20.31. EI-MS m/z:
157 [Mꢀ1]ꢀ.
4,5,6-Trihydrocyclopenta[d]thiazol-2-ylamine (5e) from Cyclopen-
tanone (4e) Yield, 32%. mp 90—92 °C (hexane) (lit.37) 92.5—94 °C).
4,5,6,7-Tetrahydrobenzo[d]thiazol-2-ylamine (5f) from Cyclohexa-
none (4f) Yield, 76%. 5f·HCl, mp 240—243 °C (EtOH/Et2O) (lit.37)
248—251 °C).
Conclusion
In this study, we evaluated the inhibitory activity of a se-
ries of 2-aminothiazole derivatives against both iNOS and
nNOS. Our results show that introduction of appropriately-
sized substituent at the 4- and 5-position of the 2-aminothia-
zole ring improves the inhibitory activity and selectivity for
iNOS. We also found that the selectivity of 5a and 5b for
iNOS was similar to that of oxazolidine 1b and much higher
than that of L-NAME. However, we could not enhance the in-
hibitory activity against iNOS by introducing an alkyl sub-
stituent into the 2-aminothiazole ring as we could in the case
of oxazolidine one. On the other hand, introduction of bulky
or hydrophilic substituent at any position of the 2-aminothia-
zole ring remarkably decreased or even abolished the in-
hibitory activity against NOS.
(2-Amino-4-methylthiazol-5-yl)acetic Acid Ethyl Ester (5g) and 3-(2-
Aminothiazol-4-yl)propionic Acid Ethyl Ester (6g) from 4-Oxopentanoic
Acid Ethyl Ester (4g) 5g: Yield, 10%. mp 114—115 °C (CH3CN). 1H-
NMR (200 MHz, DMSO-d6) d: 1.19 (3H, t, Jꢁ7.3 Hz), 1.96 (3H, s), 3.56
(3H, s), 4.07 (2H, q, Jꢁ7.0 Hz), 6.68 (2H, br). Anal. Calcd for C8H12N2O2S:
C, 47.98; H, 6.04; N, 13.99; S, 16.01. Found: C, 47.63; H, 6.18; N, 13.98; S,
16.10. EI-MS m/z: 201 [Mꢀ1]ꢀ. 6g: Yield, 3%. 6g·HCl, mp 103—104 °C
(EtOH/Et2O). 1H-NMR (200 MHz, DMSO-d6) d: 1.18 (3H, t, Jꢁ7.3 Hz),
2.63—2.82 (4H, m), 4.07 (2H, q, Jꢁ7.0 Hz), 6.53 (1H, s), 9.22 (3H, br).
Anal. Calcd for C8H12N2O2S·HCl: C, 40.59; H, 5.54; N, 11.83; S, 13.55; Cl,
14.98. Found: C, 40.95; H, 5.55; N, 11.65; S, 13.85; Cl, 14.88. EI-MS m/z:
201 [Mꢀ1]ꢀ.
N-Phenyl-N-4,5,6-trihydrocyclopenta[d]thiazol-2-ylamine (7) A mix-
ture of N-phenylthiourea (6.4 g, 42 mmol) and 2-chlorocyclopentanone
(5.0 g, 42 mmol) in 200 ml of MeOH was heated at 60 °C for 18 h. The sol-
vent was removed in vacuo and the residue was partitioned into saturated
aqueous sodium hydrogen bicarbonate solution and AcOEt. The organic
phase was dried over MgSO4 and the solvent was evaporated under reduced
pressure, and then the residue was purified by column chromatography on
silica gel using MeOH/CHCl3 (1/100) as eluent to give 7 (0.32 g, 3%), mp
121—123 °C (CH3CN). 1H-NMR (200 MHz, CDCl3) d: 2.37—2.46 (2H, m),
2.72—2.85 (4H, m), 7.03 (1H, m), 7.29—7.38 (4H, m). Anal. Calcd for
C12H12N2S·0.1H2O: C, 66.08; H, 5.64; N, 12.84; S, 14.70. Found: C, 66.16;
H, 5.59; N, 12.81; S, 14.79. EI-MS m/z: 217 [Mꢀ1]ꢀ.
N-(1-Methyl)ethyl-Nꢀ-[5-(1-methyl)ethyl-4-methylthiazol-2-
yl]thiourea (8) A mixture of 5a (2.0 g, 13 mmol) and isopropylisothio-
cyanate (1.3 g, 13 mmol) in 30 ml of toluene was refluxed for 16 h. The sol-
vent was removed under reduced pressure and the residue was purified by
column chromatography on silica gel using MeOH/CHCl3 (1/10) as eluent to
give 8 (1.1 g, 35%), mp 186—187 °C (CH3CN). 1H-NMR (200 MHz,
DMSO-d6) d: 1.18 (6H, d, Jꢁ6.9 Hz), 1.21 (6H, d, Jꢁ7.2 Hz), 2.16 (3H, s),
3.13 (1H, m), 4.31 (1H, m), 9.78 (1H, br), 11.32 (1H, br). Anal. Calcd for
C11H19N3S2: C, 51.32; H, 7.44; N, 16.32; S, 24.91. Found: C, 51.40; H, 7.57;
N, 16.30; S, 24.62. EI-MS m/z: 258 [Mꢀ1]ꢀ.
In Vitro Biological Assay. Preparation of Partially Purified iNOS
Enzyme and Determination of Inhibitory Activity against iNOS31)
RAW 264.7 macrophage cells were grown in Dulbecco’s modified eagles
medium supplemented by 10% fetal bovine serum under 5% CO2 atmo-
sphere at 37 °C. To this medium were added lipopolysaccharide and inter-
feron-g to make a final concentration of 0.2 mg/ml and 100 unit/ml respec-
Experimental
General Melting points were recorded on a Yanagimoto micro-melting
point apparatus MP-J3 without correction. 1H-NMR spectra were deter-
mined in the indicated solvent on a Varian Jemini (200 MHz) or a JEOL
JNM-LA300 (300 MHz) spectrometer. Chemical shifts are expressed as d
(ppm) values downfield from tetramethylsilane as an internal standard. Split-
ting patterns are designated as s (singlet), d (doublet), t (triplet), q (quartet),
m (mutiplet), and br (broad). Coupling constants are given in hertz. EI-MS
were recorded on a JEOL JMS D-300 or a Hitachi M-80-B mass spectrome-
ter. Column chromatography was carried out on Kieselgel 60 (Merck; 70—
230 mesh).
General Procedure for Synthesis of 2-Aminothiazole Derivatives
A
mixture of ketone 4 (46 mmol), thiourea (93 mmol), and I2 (46 mmol) was
heated at 100 °C with vigorous stirring for 8 h. To this reaction mixture was
added hot water (100 ml) and charcoal (5 g). After stirring for 15 min, the
charcoal was filtered off and to the filtrate was added 28% aqueous ammonia
solution (50 ml). Extraction with two 50-ml portions of ether followed by
drying of the combined organic extracts with MgSO4, and evaporation under
reduced pressure gave a mixture of 5 and 6. The mixture was purified by col-
umn chromatography on silica gel using MeOH/CHCl3 (1/100) as eluent to
give a free amine, or as hydrochloride salt by treatment with ethanolic 4 N
hydrochloric acid solution.
The following 2-aminothiazole derivatives (5a—g, 6a—c, g) were synthe-
sized from the corresponding starting materials (4a—g) according to the

May 2004
637
tively. The grown cells were then collected and mixed with Tris and dithio- 12) Wright C. W., Rees D. D., Moncada S., Cardiovasc. Res., 26, 48—57
threitol to make a final concentration of 50 mM and 100 mM respectively at
pH 7.5. The mixture was centrifuged at 10000ꢃg for 30 min, and the super-
natant was added to Dowex HCR-W2 at 4 °C and stirred for 30 min at the
same temperature. This solution was used as crude enzyme. NOS activity
was measured by monitoring the conversion level of L-citrulline from L-argi-
(1992).
13) Wang Y. X., Poon C. I., Pang C. C. Y., J. Pharmacol. Exp. Ther., 265,
112—119 (1993).
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H. A., Circulation, 99, 271—275 (1999).
nine. To 70 ml of the crude enzyme solution was added, 20 ml of Tris (pH 15) Feder L. S., Stelts D., Chapman R. W., Manfra D., Crawley Y., Jones
7.5) including 1 mM of NADPH, 10 ml of test-compound, 20 ml of 10 mCi/ml
L-[H3]-arginine and 80 ml of Tris (pH 7.5). The resulting mixture was incu-
bated at 37 °C for 30 min, after which 20 ml of 0.1 M (pH 5.0) including 2 mM
ethylenediamine-N,N,Nꢄ,Nꢄ-tetraacetic acid (EDTA) and 2 mM O,Oꢄ-bis(2-
H., Minnicozzi M., Fernandez X., Paster P., Egan R. W., Kreutner W.,
Kung T. T., Am. J. Respir. Cell. Mol. Biol., 17, 436—442 (1997).
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J. Pharmacol., 101, 746—752 (1990).
aminoethyl)ethyleneglycol-N,N,Nꢄ,Nꢄ-tetraacetic acid (EGTA) were added. 17) Frey C., Narayanan K., McMillan K., Spack L., Gross S. S., Masters
To the resulting solution was added Dowex 50W-8X, and the whole was
stirred for 30 min. L-[H3]-Citrulline in the supernatant was evaluated by scin-
tillation counting.
B. S., Griffith O. W., J. Biol. Chem., 269, 26083—26091 (1994).
18) Narayanan K., Spack L., McMillan K., Kilbourn R. G., Hayward M.
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21) Wolff D. J., Gribin B. J., Arch. Biochem. Biophys., 311, 300—306
(1994), according to this reference, it is indicated that structurally pla-
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mol order.
22) Shearer B. G., Lee S., Oplinger J. A., Frick L. W., Garvey E. P., Furine
E. S., J. Med. Chem., 40, 1901—1905 (1997).
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Preparation of Partially Purified nNOS Enzyme and Determination
of Inhibitory Activity against nNOS32) Male wistar rat (150—170 g)
cerebella were homogenized in 50 mM of N-2-hydroxyethylpiperazine-Nꢄ-2-
ethanesulufonic acid (pH 7.1) including 0.1 mM phenylmethylsulfonyl fluo-
ride, 12.5 mM 2-mercaptoethanol and 0.5 mM EDTA, and centrifuged at
10000ꢃg for 1 h. To the supernatant was added Dowex HCR-W2 and the re-
sulting mixture was stirred for 30 min at 4 °C. The final supernatant was
used as crude enzyme. NOS activity was measured by monitoring the con-
version level of L-citrulline from L-arginine. To 100 ml of the crude enzyme
solution was added 60 ml of 50 mM Tris (pH 7.5) including 1 mM NADPH,
2 mM CaCl2 and 300 nM calmodulin, 10 ml of test-compound, 20 ml of
10 mCi/ml L-[H3]-arginine and 10 ml of Tris (pH 7.5). The resulting mixture
was incubated at 37 °C for 30 min, after which 200 ml of 0.1 M sodium ac-
etate buffer (pH 5.0) including 2 mM EDTA and 2 mM EGTA were added. To
the resulting solution was added Dowex 50W-8X, and the whole was stirred
for 30 min. L-[H3]-Citrulline in the supernatant was evaluated by scintillation
counting.
24) Garvey E. P., Oplinger J. A., Furfin E. S., Kiff R. J., Laszlo F., Whittle
B. J. R., Knowles R. G., J. Biol. Chem., 272, 4959—4963 (1997).
Acknowledgement The thorough and helpful comments on preliminary
versions of the article are gratefully acknowledged to Dr. Nobuhide Wata- 25) Nakane M., Klinghofer V., Kuk J., Donnelly J., Budzik G., Pollock J.,
nabe.
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