Syntheses of NAMDA derivatives inhibiting NO production in BV-2 cells stimulated with lipopolysaccharide

Article abstract of DOI:10.1016/j.bmcl.2005.05.033

Sixteen derivatives of N-acetyl-3-O-methyldopamine (NAMDA), an inhibitor of BH₄ synthesis, were designed and synthesized. The ability of these derivatives to inhibit NO and BH₄ production by lipopolysaccharide- stimulated BV-2 microglial cells was determined. While NAMDA at 100 μM inhibited NO and BH₄ production by only about 20%, its catecholamide 8, indole 23 derivative, 13, and N-acetyl tetrahydroisoquinoline 25 inhibited the NO production by >50% at the same concentration. In particular, 13 and 25 inhibited both NO and BH₄ production to similar degrees, which suggested that these compounds might inhibit NO production by blocking BH ₄-dependent dimerization of the newly synthesized iNOS monomer.

Full text of DOI:10.1016/j.bmcl.2005.05.033

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3369–3373
Syntheses of NAMDA derivatives inhibiting NO production
in BV-2 cells stimulated with lipopolysaccharide
Jai Woong Seo,a Ekaruth Srisook,a Hyo Jin Son,b Onyou Hwang,b,*
Young-Nam Chac and Dae Yoon Chia,*
aDepartment of Chemistry, Inha University, 253 Yonghyundong Namgu, Inchon 402-751, Republic of Korea
bDepartment of Biochemistry and Molecular Biology, University of Ulsan College of Medicne, Seoul 138-736, Republic of Korea
cDepartment of Pharmacology and Toxicology, College of Medicine, Inha University, Inchon 402-751, Republic of Korea
Received 1 February 2005; revised 4 May 2005; accepted 10 May 2005
Available online 13 June 2005
Abstract—Sixteen derivatives of N-acetyl-3-O-methyldopamine (NAMDA), an inhibitor of BH4 synthesis, were designed and
synthesized. The ability of these derivatives to inhibit NO and BH4 production by lipopolysaccharide-stimulated BV-2
microglial cells was determined. While NAMDA at 100 lM inhibited NO and BH4 production by only about 20%, its
catecholamide 8, indole 23 derivative, 13, and N-acetyl tetrahydroisoquinoline 25 inhibited the NO production by >50%
at the same concentration. In particular, 13 and 25 inhibited both NO and BH4 production to similar degrees, which sug-
gested that these compounds might inhibit NO production by blocking BH4-dependent dimerization of the newly synthesized
iNOS monomer.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
pend on Ca2+ concentration and constantly produces a
large amount of NO.4 The iNOS is not expressed under
normal conditions but is induced in response to invading
pathogens (e.g., bacteria, fungus, and virus) and various
cytokines. The NO overproduced by iNOS is implicated
in various diseases, such as stroke,5 AlzheimerÕs disease,6
ParkinsonÕs disease,7 artherosclerosis,8 and septic
shock.9 Intensive efforts have been made to identify or
synthesize selective inhibitors that control enzymatic
activity of iNOS for effective attenuation of NO
overproduction.10
Nitric oxide radical (NO) is a small, membrane-perme-
able, and yet reactive gaseous metabolite. It is produced
from L-arginine by nitric oxide synthase (NOS), widely
distributed in biosystems from plants to mammals.
NO was originally recognized as the endothelium-de-
rived relaxing factor in vasculature,1 but was later found
to serve as an important physiological mediator in car-
diovascular, immune, and nervous systems.2 While the
small amount of NO produced intermittently by the
endothelial NOS (eNOS, also referred to as type-3)
and neuronal NOS (nNOS, type-1) serves as a key medi-
ator in many physiological functions, the large amount
of NO produced constantly by inducible NOS (iNOS,
type-2) is associated with various diseases.3
The NO producing, functional NOS enzyme is a
homodimer. Each monomer is composed of two do-
mains: a reductase domain containing the binding sites
for FMN, FAD, and NADPH and an oxygenase do-
main containing the binding sites for heme (iron proto-
porphyrin) and tetrahydrobiopterin (BH4).3,11 Binding
of all these cofactors to their respective sites is essential
for effective generation of NO. Among the cofactors,
BH4 plays an essential role in dimerization of the mono-
mers and therefore in catalytic activity.12 Thus, inhibi-
tion of iNOS dimerization even after an increased
number of the monomers during iNOS induction would
prevent the NO overproduction and reduce NO-related
damages.
Among the three isozymes, eNOS and nNOS are ex-
pressed constitutively and are activated by increased
cytosolic Ca2+. On the other hand, iNOS does not de-
Keywords: NAMDA; Nitric oxide; Inhibition of NO; Inhibition of
tetrahydrobiopterin; Nitric oxide synthase.
*
Corresponding authors. Tel.: +82 2 3010 4279; fax: +82 2 3010 4248
(O.H.); tel.: +82 32 860 7686; fax: +82 32 867 5604 (D.Y.C.); e-mail
addresses: oyhwang@amc.seoul.kr; dychi@inha.ac.kr
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.033

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J. W. Seo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3369–3373
In this connection, Cho et al. reported that N-acetyl-
3-O-methyldopamine (NAMDA, 1) attenuated NO gen-
eration via inhibition of BH4 production in BV-2
microglial cells that had been stimulated with lipopoly-
saccharide (LPS).13 However, large doses (1–3 mM) of
NAMDA were required to observe this effect, obviating
its application in clinical situations. Thus, toward effec-
tive inhibition of BH4 and NO production, we designed
and synthesized 16 derivatives using NAMDA as a lead
compound. Based on the activities of these derivatives,
we provide information on the structure–activity rela-
tionship (SAR) of NAMDA.
2. Chemistry
Chemical structure of NAMDA was modified in three
directions: variation of acetamide to various alkyl-ami-
no moieties (Method A), homologation by inserting
one more carbon (Method B), and cyclization of the
flexible acetamide group to construct a rigid NAMDA
structure (Method C). This methodological scheme is
shown in Figure 1.
Scheme 1. Reagents and conditions: (a) NaCN, DMF, 130 ꢁC, 20 h;
(b) H2, Pd/C, HCl, EtOH, rt, 12 h; (c) Ac2O, Et3N, CH2Cl2, rt, 30 min;
(d) (S)-2-naphthylmethyl thioacetimidate hydrobromide, Et3N, EtOH,
rt, 3 h; (e) trimethylorthoformate, reflux, 2 h; (f) Ac2O, Et3N, CH3CN,
rt, 1 h.
First, to generate various alkyl-amino derivatives of
NAMDA (Method A), a large amount of 3-O-methyl-
dopamine hydrochloride (4) was required as a key inter-
mediate of NAMDA derivatives. Compound 4 was
obtained from 4-hydroxy-3-methoxybenzyl alcohol (2)
by cyanation and hydrogenation using palladium as a
catalyst, according to previously reported method.14
As shown in Scheme 1, the reaction of 4 with naph-
thylmethyl thioacetamide gave the acetamidine 5 with
70% yield and trimethylorthoformate yielded formam-
ide 6 with 71%, respectively. Alternatively, acetylation
of 3-hydroxytryptamine produced the catecholamide 8
with 90% yield.
Next, to introduce the cyclic alkyl-amino derivatives to
NAMDA, 4 was reacted with diphosgene to produce
isocyanate and then addition of corresponding piperi-
dine and morphorine produced 9 and 10 with 44% and
75% yield, respectively. Other compounds (12–15) were
synthesized from 4-hydroxy-3-methoxybenzyl cyanide
(3). Conversion of nitrile to carboxylic acid produced
11. Further reactions for the amide formation were con-
ducted by adding piperidine and pyrrolidine to obtain
12 and 13, and following reduction of each carbonyl
group yielded 14 and 15, respectively, as shown in
Scheme 2. Another homologation derivative, phenylpro-
pylacetamide 19, was prepared by condensation of 16
with acetonitrile, hydrogenation using palladium under
acidic condition, and then acetylation (Scheme 3).
Scheme 2. Reagents and conditions: (a) diphosgene, CHCl3, reflux,
8 h; (b) piperidine or morpholine, CHCl3, rt, 2 h; (c) KOH, EtOH,
reflux, overnight; (d) piperidine or pyrrolidine, 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDCI), p-dimethylamino-
pyridine (DMAP), DMF, rt, 3 h; (e) LAH, THF, reflux, 2 h.
To obtain the cyclized derivatives of NAMDA (Method
C), the modified Leimgruber–Batcho indole synthesis15
(Scheme 4) and Pictet–Spengler tetrahydroisoquinoline
synthesis16 methods (Scheme 5) were used. Protection
of hydroxyl group on 16 with benzyl bromide gave 20
and nitration produced regioselective 2-nitrobenzalde-
hyde 21. Following Henry reaction, nitro-condensation,
and palladium catalyzed reduction with ammonium for-
Figure 1. Structure and three methods for the modification of
NAMDA.

J. W. Seo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3369–3373
3371
tal calf serum and penicillin–streptomycin at 37 ꢁC in a
humidified incubator under 5% CO2. For experiments,
the cells were plated on polystyrene tissue culture dish-
es at a density of 2 · 105 cells/well in 24-well culture
plates. After 24 h, the cells were changed into fresh
medium and treated with bacterial LPS (0.2 lg/mL).
The NAMDA derivatives (various concentrations dis-
solved in DMSO) were added at 15 min prior to the
LPS treatment. After 24 h, the medium was taken to
measure NO and BH4 and lactate dehydrogenase
(LDH) activity.
Scheme 3. Reagents and conditions: (a) KOH, CH3CN, reflux,
overnight; (b) H2, Pd/C, HCl, EtOH, rt, 12 h; (c) Ac2O, Et3N, CH2Cl2,
rt, 30 min.
3.2. NO production
Accumulated nitrite, a stable oxidation metabolite of
NO, was measured by the Griess reaction.17 Briefly,
200 lL aliquots of the culture medium were mixed with
100 lL of Griess reagent (1% sulfanilamide, 0.1% naph-
thylethylenediamine dihydrochloride, and 2.5% H3PO4)
in a 96-well microtiter plate, and the absorbance was
read at 540 nm. The effect of each compound on NO
production was expressed as the percent of NO pro-
duced by the LPS-treated control cells.
3.3. BH4 Production
Scheme 4. Reagents and conditions: (a) benzyl bromide, K2CO3,
acetone, reflux, 4 h; (b) HNO3, H2SO4, AcOH, rt, 1 h; (c) NH4OAc,
CH3NO2, reflux, 2 h; (c) Pd/C, HCOONH4, formic acid, methanol, rt,
9 h.
BH4 produced was determined according to the method
reported previously.18 To the 900 lL aliquot of culture
medium, 100 lL of 1 M phosphoric acid and 200 lL
of acidic iodine solution (0.5% I2 and 1.0% KI in
0.2 M trichloroacetic acid) were added and incubated
for 1 h in the dark. The oxidation reaction was terminat-
ed by addition of 0.1 mL of 0.1% ascorbic acid. The
reaction mixture was then centrifuged for 15 min at
8000g and the supernatant was diluted with distilled
water. BH4 was separated isocratically with 5% metha-
nol as mobile phase using HPLC and detected by a fluo-
rescence detector (Waters, Boston, MA, USA). BH4
standard curve was prepared every time. The BH4 con-
tent was calculated using Waters 991 computerized inte-
grator system as nanogram of BH4 per milligram of
cellular protein and expressed as percent of LPS-treated
control.
Scheme 5. Reagetns and conditions: (a) HCHO, H2O, HCl, 70 ꢁC, 1 h;
(b) Ac2O, Et3N, CH2Cl2, rt, 30 min; (c) trimethylorthoformate,
concentrated HCl (cat), reflux, overnight; (d) Bz2O, Et3N, CH2Cl2,
rt, 30 min.
3.4. Determination of cytotoxicity by LDH activity
Cytotoxicity of the newly synthesized compounds
was assessed by determining the activity of LDH re-
leased into the culture medium. LDH activity was
determined using the CytoTox 96R Non-Radioactive
Cytotoxicity Assay kit (source) and the absorbance
mate in formic acid gave an indole 23. Tetrahydroiso-
quinoline 24 was prepared by reaction of 4 with formal-
dehyde in acidic solution and 24 was derivatized further
to yield 25 by reacting 24 with acetic anhydride, to yield
26 by reacting with trimethylorthoformate, and to yield
27 by reacting with benzoic anhydride.
was read at 490 nm using
a spectrophotometer.
Cytotoxicity values were expressed as percent of
LDH released by the NAMDA derivatives compared
with that released from LPS-treated control cells
and thus, numbers approaching 100 indicate no
toxicity.
3. Biological studies
3.1. Cell culture
4. Results and discussion
BV-2 cells, a murine microglial cell line, were grown
and maintained in DMEM supplemented with 10% fe-
Sixteen new derivatives of NAMDA were synthesized.
As compounds with cytotoxicity can give misleading

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J. W. Seo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3369–3373
Table 1. Effects on the production of NO and BH4 and cell survivala
results on NO production and will not be a useful inhib-
itor, we examined their cytotoxicity by determining
LDH activity released by the BV-2 cells that had been
treated with each compound at 100 lM. As shown in
Table 1, in general, none of the derivatives were signifi-
cantly more toxic than NAMDA.
b
No
Compound
Nitriteb LDHc BH4
(%)
(%)
(%)
LPS
DAHPd
1
100
100
100
109
84.0
73.4
81.48
We also tested the newly synthesized compounds for
their inhibitory effects on LPS-induced NO overproduc-
tion in BV-2 microglial cells. As shown in Table 1, the
inhibitory effect of NAMDA (100 lM) was 84.0% of
that produced by the LPS-treated control cells. This
was similar to that reported previously.13 Compounds
numbered 8, 13, 17, 19, 23, and 25 had a stronger inhib-
itory effect than NAMDA.
5
96.9
74.8
23.6
95.3
99.1
90.3
56.1
85.9
84.4
54.0
77.5
54.9
106.5
45.6
128.8
84.9
96.4
90.6
6
8
87.9
89.64
Among the compounds (5, 6, 8, 9, 10, 12, 13, 14, and 15)
synthesized by Method A, 5 and 6 have structural simi-
larities with NAMDA and produced similar inhibitory
effects on NO production as NAMDA. Compound 8,
a demethylated catechol amide, had a strong inhibitory
effect and decreased the NO production by up to 23.6%
of untreated LPS-activated control. The azacycloalkyl
substituted NAMDA derivatives (9, 10, 12, 14, and 15)
synthesized by Method B had similar inhibitory effects
on NO production as NAMDA.
9
97.6
10
12
13
14
15
17
19
23
24
25
26
27
88.5
81.1
68.8
38.46
However, for an unknown reason, compound 13 inhib-
ited the NO production to a greater degree (39.5% of
that produced by LPS-treated control cells). Both the
cinnamonitrile 17 and one carbon inserted NAMDA
homologue 19 showed some improvement of the inhibi-
tory effect. The cyclized compounds synthesized by
Method C had interesting effects on NO production:
while the compounds with less polar group (23, 25,
and 27) exerted greater inhibition, those with polar
group (24, 26) had no inhibitory effect. Both the lipo-
philic indole 23 and N-acetyltetrahydroisoquinoline 25
derivatives are expected to be permeable and to strongly
inhibit the NO production. However, the indole deriva-
tive 23 was not stable under the atmospheric conditions
and needs to be further modified for stabilization.
78.2
83.7
100.4
98.6
115.9
75.4
As we sought for inhibitors of NO production that were
significantly more effective than NAMDA, and the BH4
assay method was rather time- and labor-consuming, we
selected only those with >50% inhibitory effects on NO
production (e.g., 8, 13, and 25) and determined their ef-
fects on BH4 production. Among these three com-
pounds, 8 had a <20% inhibitory effect, similar to
NAMDA. Compounds 13 and 25 on the other hand
showed >50% inhibitory effects. The finding that the
inhibitory effect of 8 on NO production (>75%) was
much greater than that on the BH4 production suggest-
ed that 8 might inhibit NO production at other steps.
This may be explained by the fact that catecholamines
bind at both the L-arginine and BH4 binding sites and
therefore both compete with the substrates and antago-
nize NO production.19 Compounds 13 and 25 exhibited
similar degrees of inhibition on both NO and BH4 pro-
duction. Thus, these compounds may suppress NO pro-
duction by inhibiting BH4 production, and thus, iNOS
dimerization. As NAMDA is known to be travel across
106.8
108.1
104.7
46.23
a All values were obtained from triplicate experiments.
b Amount of nitrite and BH4 in the presence of each drug in LPS-
induced BV-2 cells, presented as percent of LPS-activated BV-2.
c Cytotoxicity of each drug assessed by lactate dehydrogenase (LDH)
activity released into the medium, presented as percent of LPS-acti-
vated BV-2.
d DAHP (2,4-diamino-6-hydroxypyrimidine)—an inhibitor of GTP
cyclohydrolase I.20

J. W. Seo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3369–3373
3373
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have similar lipophilicity to NAMDA, it is noteworthy
that compounds 13 and 25 are expected to travel across
BBB.
In conclusion, we designed and synthesized 16 NAMDA
derivatives by three different methods and identified lead
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Acknowledgments
This work was supported by an SRC grant from KOSEF
provided at the Nitric Oxide Radical Toxicology Re-
search Center (NORTEREC) and the Brain Research
Center of the 21st Century Frontier Research Program
(M103KV010006 03K2201 00630) funded by the Korea
Ministry of Science and Technology. We thank the Korea
Basic Science Institute (Daegu, Korea) for the analyses
using high-resolution mass spectra to identify chemical
structures of our newly synthesized compounds.
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