Synthesis and biological activity of new phthalimides as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2017.04.027

The overproduction of nitric oxide (NO) plays an important role in a variety of pathophysiological processes, including inflammation. Therefore, the suppression of NO production is a promising target in the design of anti-inflammatory agents. In the prese

Full text of DOI:10.1016/j.bmc.2017.04.027

Accepted Manuscript
Synthesis and biological activity of new phthalimides as potential anti-inflam-
matory agents
Duc-Hiep Bach, Jian-Yu Liu, Won Kyung Kim, Ji-Young Hong, So Hyun Park,
Donghwa Kim, Si-Ning Qin, Thi-Thu-Trang Luu, Hyen Joo Park, Yong-Nan
Xu, Sang Kook Lee
PII:
S0968-0896(17)30368-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.027
BMC 13697
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
23 February 2017
18 April 2017
21 April 2017
Please cite this article as: Bach, D-H., Liu, J-Y., Kim, W.K., Hong, J-Y., Park, S.H., Kim, D., Qin, S-N., Luu, T-
T., Park, H.J., Xu, Y-N., Lee, S.K., Synthesis and biological activity of new phthalimides as potential anti-
inflammatory agents, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.04.027
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Synthesis and biological activity of new phthalimides as potential anti-
inflammatory agents
a,1
Duc-Hiep Bach , Jian-Yu Liu , Won Kyung Kim , Ji-Young Hong , So Hyun Park ,
b,1
a
a
a
a
b
a
Donghwa Kim , Si-Ning Qin , Thi-Thu-Trang Luu, Hyen Joo Park , Yong-Nan Xu *, and
b,
a,
Sang Kook Lee *
a
College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul,
1
51-741, Korea
b
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang
10016, People’s Republic of China
1

ABSTRACT
The overproduction of nitric oxide (NO) plays an important role in a variety of
pathophysiological processes, including inflammation. Therefore, the suppression of NO
production is a promising target in the design of anti-inflammatory agents. In the present
study, a series of phthalimide analogs was synthesized, and their anti-inflammatory
activities were evaluated using lipopolysaccharide (LPS)-stimulated NO production in
cultured murine macrophage RAW264.7 cells. A structure-activity relationship study showed
that the free hydroxyl group at C-4 and C-6 and the bulkiness of the N-substituted alkyl chain
are associated with biological activity. Among the series of phthalimide derivatives,
compound IIh exhibited potent inhibitory activity, with an IC50 value of 8.7 µg/mL. Further
study revealed that the inhibitory activity of compound IIh was correlated with the down-
regulation of the mRNA and protein expression of LPS-stimulated inducible nitric oxide
synthase (iNOS). Compound IIh also suppressed the induction of the pro-inflammatory
cytokines tumor necrosis factor-α and interleukin-1β in LPS-stimulated RAW 264.7 cells.
The anti-inflammatory activity of compound IIh was also found to be associated with the
suppression of the Toll-like receptor (TLR)4 signaling pathway by down-regulating the
activation of interferon regulatory factor 3 (IRF-3) and interferon-β and signal transducer
expression. These findings demonstrate that novel phthalimides might be potential candidates
for the development of anti-inflammatory agents.
Keywords: phthalimides, anti-inflammation, iNOS, Toll-like receptor (TLR) 4

1
. INTRODUCTION
Inflammation is a complex biological response of the vascular tissue to harmful stimuli,
such as pathogens, damaged cells, or irritants [1]. Consequently, the initiation and
progression of many diseases, such as cardiovascular disease, cancer, obesity and insulin
resistance, are highly associated with inflammatory responses [2,3]. Macrophages are one of
the most dominant and widely distributed inflammatory cells and are involved in the
initiation and maintenance of the acute inflammatory response [4]. Lipopolysaccharide (LPS),
an endotoxin localized in the outer membrane of Gram-negative bacteria, can elicit
significant immune and inflammatory responses in macrophages [5]. In the process of LPS-
mediated inflammation, Toll-like receptors (TLRs) are key components. In particular, TLR4
is activated by the interaction with LPS, and activated TLR4 leads to the expression of
several pro-inflammatory cytokines and mediators of inflammation, such as nitric oxide (NO)
[
6,7]. LPS/TLR4 signaling has been divided into two major downstream pathways: the
myeloid differentiation factor 88 (MyD88)-dependent pathway and the MyD88-independent
pathway. The activation of the MyD88-dependent pathway contributes to the early phase of
NF-κB activation and pro-inflammatory cytokine expression, while the MyD88-independent
pathway mediates the late phase of NF-κB activation and the induction of interferon (IFN)-
inducible genes [8].
A variety of classes of compounds from natural or synthetic sources have been shown to be
potential inhibitors of LPS-stimulated NO production in macrophages [1-3]. Phthalimides
have been reported to have antifungal, anticonvulsant, analgesic, hypolipidemic, antitumor
and anti-inflammatory activities [4, 5]. In the present study, we synthesized a series of novel
phthalimide derivatives and evaluated the biological activity of LPS-induced NO production.
We also further explored the underlying molecular mechanism responsible for the anti-

inflammatory activity using an active phthalimide analog.
2
. RESULTS AND DISCUSSION
2.1. Chemistry
The synthesis of a series of novel phthalimide derivatives is outlined in Scheme 1.
Scheme 1. The synthesis of N-substituted phthalimide analogs. (A) Procedure for the synthesis of
type I and II N-substituted phthalimide analogs. (B) Procedure for the synthesis of type III N-substituted
phthalimide analogs.
Intermediate 2 was prepared using 3,5-dimethoxybenzoic acid and glyoxylic acid. In this

procedure, sulfuric acid was diluted with ten times amount of acetic acid to avoid the
carbonization of product induced by concentrated sulfuric acid, and 2 was yielded as 64.2%.
Decarboxylation was then performed in dimethyl phthalate (DMP) at 180~185 °C to afford 3
with the yield of 95.7%. 3 was oxidated to be substituted phthalic acid (4, 93.6%) and then
dehydrated to afford anhydride 5 (90.2%). The intermediate 6 was prepared by ammonolysis
and condensation of 5 and urea.
Target compounds Ia-Im were synthesized using compound 5 and different
halohydrocarbons with yields ranging from 62% to 80%. IIa-IIm were synthesized by
demethylation with Ia-Im, respectively, with yields from 60% - 70%.
Compound 5 was reacted with ethanolamine at 185°C to afford the intermediate 7
(
52.2%) and target compounds IIIa-IIIc were synthesized using different acyl chloride as
acylation reagent to yield from 60% to 73%.
The data of NMR spectroscopy and ESI-MS spectra analysis of the target compounds
(
Supplementary data) reflected the assigned structures of the compounds.
.2. Inhibitory activity of NO production by phthalimide derivatives and structure-
2
activity relationship
The overproduction of NO by inducible nitric oxide synthase (iNOS) is significant in
inflammatory processes. Therefore, high levels of NO are considered to be a biomarker for
inflammatory disorders and a useful target for the procurement of anti-inflammatory agents.
The synthetic phthalimide analogs were initially evaluated for inhibitory effects on the
production of NO in LPS-stimulated RAW264.7 macrophage cells. When the cells were
treated with LPS (1 µg/mL) for 22 h, the NO production was markedly stimulated from the
basal level of 0.46 ± 0.2 to 25.3 ± 0.3 µM. The positive control, AMT, showed 98.0 %
inhibition at the test concentration of 50 µM under the same experimental conditions. The
inhibitory activity of the synthetic phthalimide analogs (Ia-Im, IIa-IIm, IIIa-IIIc) is

summarized in Table 1.
Table 1. Structures and inhibitory effects on nitric oxide production of compounds Ia-Im, IIa-IIm a
nd IIIa-IIIc in LPS-induced RAW 264.7 macrophages.
a
Nitrate
µM)
% control of
NO production
Comp
R
R′
R′′
(
LPS -
LPS +
-
-
-
-
-
-
-
0.46
25.31
0.5
-
-
b
-
-
c
(
+)
Ia
Ib
Ic
Id
Ie
If
2.0
-C
-C
2
H
5
7
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
3
-OCH
3
3
3
3
3
3
3
3
3
3
3
3
3
10.81
23.24
24.34
19.70
21.45
18.60
23.01
20.99
18.87
18.65
20.79
20.16
18.43
21.12
20.71
20.47
18.24
21.07
11.30
1.98
43.5
93.5
97.9
79.3
86.3
74.8
92.6
84.5
75.9
75.1
83.7
81.1
74.2
85.0
83.3
82.4
73.4
84.8
45.5
8.0
3
H
3
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OCH
-OH
-CH(CH
-C
CH(CH
3
)
2
3
4
H
9
3
-CH
2
3
)
2
3
-C
5
H
11
13
15
3
Ig
Ih
Ii
-C
-C
6
7
H
H
3
3
-CH
-CH
-CH
-CH
-CH
2
-C
-C
-C
-C
6
H
5
-F(o)
3
Ij
2
6
H
5
-F(m)
-Br(o)
-Br(p)
3
Ik
Il
2
6
H
H
5
3
2
6
5
3
Im
IIa
IIb
IIc
IId
IIe
IIf
2
-C
6
H
5
-CH
3
(m)
-OCH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
3
-C
2
H
5
7
-C
3
H
-OH
-CH(CH
-C
CH(CH
3
)
2
-OH
4
H
9
-OH
-CH
2
3
)
2
-OH
-C
5
H
11
13
15
-OH
IIg
IIh
IIi
-C
-C
6
7
H
H
-OH
-OH
0.72
2.9
-CH
-CH
-CH
-CH
2
-C
-C
-C
-C
-C
6
H
5
-F(o)
-OH
11.76
10.46
9.65
47.3
42.1
38.8
32.4
41.7
97.1
101.9
48.9
99.8
IIj
2
6
H
5
-F(m)
-Br(o)
-Br(p)
-OH
IIk
IIl
2
6
H
H
5
-OH
2
6
5
-OH
8.04
IIm
IIIa
IIIb
IIIc
7
-CH
2
6
H
5
-CH
-O-CO-CH
-O-CO-C
-O-CO-CH
-CH -CH -OH
3
(m)
-OH
10.36
24.13
25.31
12.14
24.80
-C
-C
-C
2
H
4
3
-OCH
-OCH
-OCH
-OCH
3
-OCH
-OCH
-OCH
-OCH
3
3
3
3
2
H
4
2
H
5
3
2
H
4
2
Cl
3
2
2
3
a
b
c
Comp: compound 20 µg/mL,
LPS +: 1 µg/mL, (+): 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine
hydrochloride (AMT: positive control, 50 µM)

Among the tested compounds, 12 phthalimides inhibited the LPS-stimulated NO production
by at least 50 % at the test concentration of 20 µg/mL. Regarding the N-alkyl chain, the
biological activity varied depending on the length of the carbon chain and the substitution
patterns. Activity decreased when the number of carbon atoms exceeded 8 in the N-alkyl
chain. The introduction of halides or ester groups into the alkyl chain also decreased the
activity. The substitution of a methoxy group in the R′and R″positions decreased the
activity compared to the free hydroxyl group in the position. The C6- to C7-alkyl chains
maintained the biological activity without any substitutions in the alkyl chain and the R′and
R″groups. Among the tested compounds, the C7-alkyl chain with a free hydroxyl group in
the R′and R″, compound IIh, was the most active in the inhibition of NO production.
Therefore, the inhibitory activity of NO production by phthalimides is dependent on the size
of the carbon chain, the spatial geometry of the N-alkyl chain and the substitutions at the
aromatic R′and R″positions.
2.3. Mechanism of action studies with compound IIh
Based on the SAR study of phthalimide analogs, compound IIh (Figure 1), which showed
the highest activity (Table 1), was selected for further detailed mechanism of action studies in
RAW264.7 cells.
Figure 1. The chemical structure of compound IIh.

Compound IIh exhibited concentration-dependent inhibition of LPS-induced NO production
in RAW264.7 cells, with an IC50 value of 8.7 µg/mL (Figure 2A).
Figure 2. The effects of compound IIh on NO production and iNOS expression in RAW 264.7 cells.
(A) Cells were treated with LPS (1 µg/mL) in the presence or absence of various concentrations of compound
IIh for 22 h. Nitrite was measured using the Griess reaction. Data are presented as the means ± SD of three
independent experiments. (B) RAW 264.7 cells were pretreated with various concentrations of compound IIh
for 30 min and then stimulated with 1 µg/mL LPS for 16 h. The iNOS protein level was analyzed using western
blotting. (C) RAW 264.7 cells were pretreated with different concentrations of compound IIh for 30 min and

then stimulated with 1 µg/mL LPS for 4 h. The iNOS mRNA expression was analyzed using real-time PCR. The
values are expressed as the means ± SD of triplicate tests.
Employing MTT assay the cytotoxic effect of compound IIh was also analyzed at the test
concentrations of up to 40 µg/mL. The compound did not show any significant cell viability
at the concentrations of up to 20 µg/mL (> 95% survival), but the compound IIh at the test
concentration of 40 µg/mL affected to the cell viability (approximately 17% survival).
Therefore, further study for the mechanism of action in the anti-inflammatory activity with
the compound IIh was observed at the test concentrations of up to 20 µg/mL. Thereby, the
inhibition of NO production by compound IIh was not attributable to cytotoxic effects at the
test concentrations. To further elucidate the possible mechanisms of active phthalimides, the
suppression of NO production by compound IIh was investigated in relation to the levels of
iNOS protein and mRNA expression in the LPS-stimulated RAW 264.7 cells. The RAW264.7
cells were incubated with LPS (1 µg/mL) in the presence or absence of various
concentrations of compound IIh. After 4 h and 12 h of treatment, total RNA and protein were
isolated for real-time PCR and western blot analysis, respectively. The iNOS protein level
was markedly increased in LPS-induced RAW264.7 cells, but co-treatment with compound II
h significantly suppressed the iNOS protein level in a concentration-dependent manner
(
Figure 2B). In addition, treatment with compound IIh significantly suppressed iNOS
mRNA expression in LPS-stimulated RAW264.7 cells in a concentration-dependent manner
Figure 2C). These findings suggest that the inhibition of NO production by compound IIh
(
might be correlated with its suppressive effect on LPS-induced iNOS protein and mRNA
expression.
Because inflammation involves the activation of pro-inflammatory cytokines, such as IL-
1
β and TNF-α [6], the effects of compound IIh on cytokine expression were also investigated

in LPS-stimulated RAW264.7 cells. As shown in Figure 3A, treatment with compound IIh
for 4 h significantly suppressed the LPS-stimulated expression of TNF-α and IL-1β.
Figure 3. The effect of compound IIh on LPS-stimulated pro-inflammatory cytokine protein and
mRNA expression in RAW 264.7 cells. (A) & (B) RAW 264.7 cells were pretreated with various concentrations
of compound IIh for 30 min and then stimulated with LPS (1 µg/mL) for 4 h. After incubation, total protein (A)
and mRNA (B) were extracted and then further analyzed using western blotting and real-time PCR, respectively.
The values are expressed as the means ± SD of triplicate tests.
In addition, the overexpression of IL-1β mRNA by LPS (1 µg/mL) was significantly down-
regulated by treatment with compound IIh in a concentration-dependent manner (Figure 3B).
These findings suggest that the anti-inflammatory activity of compound IIh is associated in
part with the suppression of pro-inflammatory cytokine expression.
NF-κB is a major transcription factor that modulates the expression of pro-inflammatory
proteins (e.g., iNOS) and pro-inflammatory cytokines (e.g., TNF-α and IL-1β) induced by
LPS [7]. To further investigate whether compound IIh is able to affect the transcriptional
activity of NF-κB, a reporter gene assay (SEAP) was performed in LPS-stimulated RAW
2
64.7 cells. As shown in Figure 4A, in response to the stimulation with LPS for 16 h, the NF-
κB transcriptional activity increased by approximately 5.5-fold, but co-treatment with

compound IIh effectively suppressed NF-κB transcriptional activity.
The translocation of NF-κB to the nucleus is preceded by the phosphorylation and
proteolytic degradation of IκBα by IκB kinases [8]. In general, in unstimulated cells, NF-κB
subunits (p50 and/or p65) form a complex with an inhibitory factor, IκBα, in the cytosol and
thus are inactivated [5]. However, when stimulated by pro-inflammatory signaling, IκBα is
phosphorylated by IκB kinases (IKKs) and then subject to ubiquitin-mediated degradation [6].
These events lead to the translocation of free NF-κB into the nucleus, where it serves as a
transcription factor. Accordingly, the higher levels of phosphorylated IκBα induced by LPS
were reduced by treatment with compound IIh, but the lower level of total IκBα recovered
with compound IIh treatment for 30 min (Figure 4B).
Figure 4. The effect of compound IIh on the MyD88-dependent signaling pathway in RAW 264.7
cells. (A) The effect of compound IIh on LPS-stimulated NF-ĸB transcriptional activity. RAW-SEAP cells
were incubated in a 12-well plate for 24 h and pretreated with compound IIh for 2 h. Subsequently, the cells
were incubated with LPS (1 µg/mL) for 16 h. For secreted alkaline phosphatase activity (SEAP), relative
fluorescence units (RFU) were measured using a 96-well fluorometer. (B) The effect of compound IIh on NF-

κB and its related proteins. RAW 264.7 cells were pretreated with various concentrations of compound IIh and
then stimulated with LPS (1 µg/mL) for different lengths of time NF-κB (p50 and p65) and IKK (α and β) for 16
h, and IκBα and p-IκBα for 30 min. Proteins were analyzed using western blotting.
In addition, the increased level of IKKβ protein caused by LPS was reduced by treatment
with compound IIh for 16 h. These data suggest that compound IIh effectively suppresses
LPS-induced NF-κB activation by blocking the transcriptional activity of NF-κB, up-
regulating IκBα, and inhibiting IKKβ.
In an additional regulation of iNOS expression, the TRIF-mediated signal transduction
pathway is highly involved in inflammatory responses. The adaptor protein, TRIF-related
adaptor molecule (TRAM), together with TIR domain-containing adaptor-inducing IFN-β
(
TRIF), contributes to the activation of interferon regulatory factor 3 (IRF3) and subsequently
induces the IRF3-dependent expression of genes such as interferon-β (IFN-β) [9]. Therefore,
the effects of compound IIh on TRIF-mediated signaling pathways were also examined in
LPS-stimulated RAW264.7 cells. Compound IIh significantly suppressed the LPS-stimulated
396
activation of IRF3 (p-Ser ) (Figure 5A) and suppressed the mRNA expression of IFN-β
(
Figure 5B), suggesting the involvement of the inhibition of the TRIF-mediated pathway in
the regulation of iNOS expression by compound IIh.
Recent studies have demonstrated that heme-oxygenase 1 (HO-1), an anti-oxidative protein,
acts as a major protective factor through its antioxidant, anti-apoptotic, and anti-inflammatory
properties [10-12]. The induction of HO-1 also enables its anti-inflammatory function via the
down-regulation of iNOS and NO production in macrophages [13-17]. Therefore, we also
investigated whether the anti-inflammatory activity of compound IIh is in part associated
with the up-regulation of HO-1 expression. At the test concentration of 20 µg/mL, compound
IIh significantly up-regulated HO-1 protein expression (Figure 5C).

Figure 5. The effect of compound IIh on MyD88-independent signaling pathway in RAW 264.7
cells. (A) and (B) RAW 264.7 cells were pretreated with various concentrations of compound IIh for 30 min
and then stimulated with LPS (1 µg/mL) for 30 min (A) and 4 h (B). Subsequently, the expression of proteins
and mRNA were further analyzed by western blot and real-time PCR, respectively. (C) The effect of compound
IIh on heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. RAW 264.7 cells were pretreated with
compound IIh for 30 min and subsequently stimulated with LPS (1 µg/mL) for 16 h. The level of HO-1 protein
was further analyzed using western blotting.
These findings suggest the involvement of HO-1 in the regulation of iNOS expression and
NO production by compound IIh in LPS-stimulated macrophage cells.
3
. EXPERIMENTAL SECTION

Bruker ARX-300 and AV-600 spectrophotometers (Bruker Corporation, Germany) at 300
1 13
MHz were used to obtain H- and C-NMR spectra. Mass spectra were obtained using a
6
890 GC-5975 spectrometer (Agilent Technologies, US) using positive electrospray
ionization. High Performance Liquid Chromatography (HPLC) was performed on a JAI
LC9103 Recycling preparative HPLC (Japan Analytical Industries, Japan) equipped with a
JAIGEL-ODS-AP-P column and a JAIGEL-GS310 column using a JAI refractive index
detector and a JAI UV-3702 detector with a Multichro 2000 workstation. TLC was performed
2 4
on pre-coated GF254 plates (Merk, Germany) and detected by spraying with 10 % H SO
followed by heating. The purity of all compounds screened in the biological assays was
examined using HPLC analysis and was found to be not less than 95 %.
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), sodium
pyruvate, L-glutamine, antibiotic-antimycotic solution, and trypsin-EDTA were purchased
from Invitrogen Co. (Grand Island, NY, USA). Lipopolysaccharide (E. coli 0111: B4), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and other chemicals were
purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated. iNOS,
3
2/36
COX-2, IL-1β, IKKα/β, p50, p65, IRF3, IκB-α, and p-IκB-α (Ser
) antibodies were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against
3
96
TNF-α, and p-IRF3 (Ser ) were purchased from Cell Signaling Technology (Beverly, MA,
USA). Anti-(HO)-1(ADI-SPA-895) antibody was obtained from Enzo Life Sciences. Gene-
specific primers were synthesized by Bioneer (Daejeon, Korea). AMV reverse transcriptase,
the dNTP mixture, random primers, RNasin, and Taq polymerase were purchased from
Promega (Madison, WI, USA).
3
.1. General procedure for the synthesis of N-substituted phthalimideanalogs (Ia-Im, II
a-IIm, and IIIa-IIIc)

3
.1.1. Procedure for the synthesis of type I N-substituted phthalimide analogs (Ia-Im)
Procedures for the synthesis of intermediates 2-6 were using previously published
protocol with slight modifications [18]. Briefly, a total of 1.82 g (0.01 mol) of 3,5-
dimethoxybenzoic acid and 1.48 g (0.02 mol) of glyoxylic acid were dissolved in acetic acid
(
2 2 4
10 mL) under N , and 1.0 mL (0.01 mol) H SO (diluted with acetic acid beforehand) was
added carefully. The solution was then stirred for 5 h at 50°C. Then intermediate 2 (1.38 g,
6
4.2%) was afforded after recrystallization with EtOAc.
Then, 2.38 g (0.01 mol) of compound 2 was suspended in 7 mL dimethyl phthalate
(
DMP) and was stirred at 180~185°C until the solid was dissolved successfully. The solution
was then cooled, and 5 mL MeOH was added. After stirring for 20 min, the residue was
washed with Et O three times. The intermediate 3 (1.84 g, 95.7%) was afforded as yellow
needles after recrystallization with CH Cl
2
2
2
.
A total of 1.94 g (0.01 mol) of compound 3 was dissolved in 50 mL NaOH (8%, m/m).
Then, 1.59 g KMnO was added, and the mixture was stirred at ambient temperature for 24 h.
Next, 2 mL EtOH was added to remove surplus KMnO , and white crystals of 4 (2.10 g,
3.6%) were afforded after acidification with HCl and washing with water.
4
4
9
A total of 2.26 g (0.01 mol) of compound 4 and 7.14 g (0.07 mol) of acetic anhydride
were mixed and reacted for 2 h at 140°C. The intermediate 5 (1.91 g, 90.2%) was afforded as
white needles after recrystallization with EtOAc.
Then, 2.08 g (0.01 mol) of compound 5, 0.30 g (0.005 mol) of urea and 2.0 mL of
dimethylbenzene were mixed and reacted for 2 h at 140 °C. The mixture was dissolved in
EtOH and refluxed for 1.5 h at 78 °C. The intermediate 6 (2.04 g, 91.5%) was afforded as
white needles after recrystallization with EtOAc.

Finally, 2.07 g (0.01 mol) of compound 6 was heated and dissolved in 15 mL anhydrous
N,N-dimethylformamide, and then 6.21 g of anhydrous K CO was added. After stirring for
0 min, halohydrocarbon (0.01 mol) was added and stirred for 0.5 h at 115°C. The products
2
3
2
Ia-Im were afforded after recrystallization with EtOAc.
Ia N-ethyl-3,5-dimethoxyphthalimide
+
1
m.p.: 147~148 °C; MS m/z: 235.1 [M] ; H-NMR (300 MHz, CDCl
H-6), 6.64(1H, d, J=1.8 Hz, H-4), 3.99(3H, s, OCH ), 3.93(3H,s, OCH
N-CH ), 1.26(3H, t, J=7.2 Hz, CH ).
3
) δ: 6.98(1H, J=2.1 Hz,
3
3
), 3.70(2H,q, J=7.2 Hz,
2
3
Ib N-propyl-3,5-dimethoxyphthalimide
+
1
m.p.: 119~120 °C;MS m/z: 249.1 [M] ; H-NMR (600 MHz, CDCl
3
) δ: 6.97(1H, d, J=0.9 Hz,
), 3.92(3H, s, OCH ), 3.60(2H, t, J=3.6
), 0.93(3H, t, J=3.6 Hz, CH ). 13C-NMR (600 MHz, CDCl ) δ:
68.11, 166.95, 166.45, 157.94, 136.41, 110.49, 103.55, 100.26, 56.28, 56.18, 39.36, 21.88,
1.30.
Ic N-isopropyl-3,5-dimethoxyphthalimide
H-6), 6.63(1H, d, J=0.9 Hz, H-4), 3.97(3H, s, OCH
3
3
Hz, N-CH ), 1.68(2H, m, CH
2
2
3
3
1
1
+
1
m.p.: 197~198 °C; MS m/z: 249.2 [M] ; H-NMR (300 MHz, CDCl
H-6), 6.59(1H, d, J=1.8 Hz, H-4), 3.94(3H, s, OCH ), 3.88(3H, s, OCH
). C-NMR (600 MHz, CDCl ) δ: 167.92, 166.91, 166.37,
57.89, 136.41, 110.50, 103.49, 99.98, 56.24, 56.16, 42.73, 20.10.
3
) δ: 6.90(1H, d, J=1.8 Hz,
3
3
), 4.45(1H, m, N-CH),
1
3
1
1
.43(6H, d, J=6.9 Hz, 2×CH
3
3
Id N-butyl-3,5-dimethoxyphthalimide
+
1
m.p.: 117~118 °C; MS m/z: 263.1 [M] ; H-NMR (300 MHz, CDCl
H-6), 6.60(1H, d, J=2.1 Hz, H-4), 3.95(3H, s, OCH ), 3.90(3H, s, OCH
Hz, N-CH ), 1.61(2H, m), 1.32(2H, m), 0.91(3H, t, J=7.2 Hz, CH
CDCl ) δ: 168.07, 166.93, 166.43, 157.92, 136.41, 110.50, 103.52, 100.24, 56.27, 56.17,
7.52, 30.63, 20.01, 13.65.
3
) δ: 6.93(1H, d, J=2.1 Hz,
3
3
), 3.60(2H, q, J=7.2
1
3
2
3
). C-NMR (600 MHz,
3
3

Ie N-isobutyl-3,5-dimethoxyphthalimide
+
1
m.p.: 140~141 °C; MS m/z: 263.2 [M] ; H-NMR (300 MHz, CDCl
3
) δ: 6.96(1H, d, J=1.5 Hz,
), 3.92(3H, s, OCH ), 3.45(2H, q, J=7.2
). C-NMR (600 MHz, CDCl
δ: 168.27, 167.09, 166.46, 157.95, 136.29, 110.40, 103.58, 100.25, 56.28, 56.18, 45.06, 27.78,
0.09.
If N-amyl-3,5-dimethoxyphthalimide
H-6), 6.63(1H, d, J=1.5 Hz, H-4), 3.98(3H, s, OCH
3
3
1
3
Hz, N-CH ), 2.10(1H, m, CH), 0.92(6H, d, J=6.9 Hz, 2×CH
2
3
3
)
2
+
1
m.p.: 110~112 °C; MS m/z: 277.2 [M] ; H-NMR (300 MHz, CDCl
H-6), 6.60(1H, d, J=1.8 Hz, H-4), 3.95(3H, s, OCH ), 3.89(3H, s, OCH
Hz, N-CH ), 1.62(2H, m), 1.24~1.35(4H, m), 0.85(3H, t, J=6.9 Hz, CH
MHz, CDCl ) δ: 168.07, 166.92, 166.43, 157.92, 136.42, 110.50, 103.52, 100.24, 56.27,
6.17, 37.78, 28.92, 28.25, 22.28, 13.93.
Ig N-hexyl -3,5-dimethoxyphthalimide
3
) δ: 6.94(1H, d, J=1.8 Hz,
3
3
), 3.59(2H, q, J=7.2
1
3
2
3
). C-NMR (600
3
5
+
1
m.p.: 104~105 °C; MS m/z: 291.2 [M] ; H-NMR (300 MHz, CDCl
H-6), 6.60(1H, d, J=1.8 Hz, H-4), 3.99(3H, s, OCH ), 3.93(3H, s, OCH
Hz, N-CH ), 1.65(2H, m), 1.31(6H, s), 0.88(3H, t, J=6.6 Hz, CH
CDCl3) δ: 168.08, 166.93, 166.44, 157.93, 136.44, 110.53, 103.54, 100.25, 56.29, 56.18,
7.83, 31.40, 28.54, 26.48, 22.50, 14.02.
Ih N-heptyl -3,5-dimethoxyphthalimide
3
) δ: 6.94(1H, d, J=1.8 Hz,
3
3
), 3.63(2H, q, J=7.2
1
3
2
3
). C-NMR (600 MHz,
3
+
1
m.p.: 89~91 °C; MS m/z: 305.2 [M] ; H-NMR (300 MHz, CDCl
H-6), 6.60(1H, d, J=1.8 Hz, H-4), 3.94(3H, s, OCH ), 3.89(3H, s, OCH
Hz, N-CH ), 1.61(2H, m), 1.21~1.31(6H, m), 0.84(3H, t, J=6.6 Hz, CH
MHz, CDCl ) δ: 168.06, 166.92, 166.43, 157.92, 136.43, 110.51, 103.53, 100.24, 56.27,
6.17, 37.83, 31.68, 28.88, 28.38, 26.77, 22.57, 14.04.
Ii N-(2-fluorobenzyl)-3,5-dimethoxyphthalimide
3
) δ: 6.93(1H, d, J=1.8 Hz,
3
3
), 3.59(2H, q, J=7.2
1
3
2
3
). C-NMR (600
3
5

+
+
+ 1
m.p.: 181~183 °C; MS m/z: 316.1 [M+H] , 338.0 [M+Na] , 653.1 [2M+Na] ; H-NMR (300
MHz, CDCl ) δ: 7.16~7.33(2H, m, Ar-H), 6.96~7.05 (2H, m, Ar-H), 6.95(1H, d, J=1.8 Hz, H-
), 6.60(1H, d, J=1.8 Hz, H-6), 4.85(2H, s, CH
NMR (600 MHz, CDCl ) δ: 167.44, 166.65, 166.32, 160.58, 158.17, 136.27, 130.09, 129.33,
24.10, 123.43, 115.44, 110.41, 103.73, 100.53, 56.32, 56.22, 35.03.
3
1
3
6
2 3 3
), 3.94(3H, s, OCH ), 3.89(3H, s, OCH ). C-
3
1
Ij N-(3-fluorobenzyl)-3,5-dimethoxyphthalimide
+
+
+ 1
m.p.: 144~145 °C; MS m/z: 316.0 [M+H] , 338.0 [M+Na] , 653.1 [2M+Na] ; H-NMR (300
MHz, CDCl
.60(1H, d, J=1.8 Hz, H-4), 4.74(2H, s, CH
NMR (600 MHz, CDCl ) δ: 167.51, 166.68, 166.35, 162.28, 158.18, 138.97, 136.23, 130.10,
24.18, 115.53, 114.64, 110.33, 103.69, 100.58, 56.32, 56.23, 40.83.
Ik N-(2-bromobenzyl)-3,5-dimethoxyphthalimide
3
) δ: 7.07~7.24(3H, m, Ar-H), 6.90(1H, m, Ar-H), 6.94(1H, d, J=1.8 Hz, H-6),
1
3
6
2 3 3
), 3.94(3H, s, OCH ), 3.89(3H, s, OCH ). C-
3
1
+
1
m.p.: 205~206 °C; MS m/z: 376.0 [M+H] ; H-NMR (300 MHz, CDCl
3
) δ: 7.52(1H, dd,
J=0.9, 6.9 Hz, Ar-H), 7.05~7.22(3H, m, Ar-H), 6.97(1H, d, J=1.8 Hz, H-6), 6.63(1H, d, J=1.8
13
Hz, H-4), 4.89(2H, s, CH
CDCl ) δ: 167.55, 166.74, 166.33, 158.25, 136.22, 135.29, 132.86, 128.89, 128.35, 127.47,
22.77, 110.37, 103.83, 100.62, 56.35, 56.26, 41.60.
2 3 3
), 3.95(3H, s, OCH ), 3.90(3H, s, OCH ). C-NMR (600 MHz,
3
1
Il N-(4-bromobenzyl)-3,5-dimethoxyphthalimide
+
m.p.: 186~187 °C; MS m/z: 376.0 [M+H] ; 1H-NMR (300 MHz, CDCl
3
) δ: 7.40(2H, d,
J=1.8 Hz, Ar-H), 7.38(2H, d, J=2.1 Hz, Ar-H), 6.93(1H, d, J=1.8 Hz, H-6), 6.59(1H, d, J=2.1
13
Hz, H-4), 4.70(2H, s, CH
CDCl ) δ: 167.52, 166.68, 166.39, 158.17, 136.24, 135.63, 131.70, 130.50, 121.75, 110.33,
03.67, 100.58, 56.32, 56.23, 40.75.
Im N-(4-methybenzyl)-3,5-dimethoxyphthalimide
2 3 3
), 3.93(3H, s, OCH ), 3.88(3H, s, OCH ). C-NMR (600 MHz,
3
1
+
1
m.p.: 148~149 °C; MS m/z: 311.1 [M] ; H-NMR (300 MHz, CDCl ) δ: 7.12~7.21(3H, m,
3

Ar-H), 7.03(1H, d, J=7.2 Hz, Ar-H), 6.93(1H, d, J=1.8 Hz, H-6), 6.59(1H, d, J=2.1 Hz, H-4),
1
3
4
.72(2H, s, CH
MHz, CDCl ) δ: 167.66, 166.54, 166.54, 158.08, 138.25, 136.57, 136.40, 129.33, 128.46,
28.41, 125.74, 110.50, 103.61, 100.45, 56.31, 56.20, 41.35, 21.35.
.1.2. Procedure for the synthesis of type II N-substituted phthalimide analogs (IIa-IIm)
2 3 3 3
), 3.94(3H, s, OCH ), 3.88(3H, s, OCH ), 2.28(3H, s, CH ). C-NMR (600
3
1
3
2 2
A total of 0.01 mol of Ia-Im was dissolved in CH Cl . Then, 50 mL (0.05 mol) of
aqueous borontribromide (1.0 mol/L) was added dropwise at -78°C. The reaction was kept at
ambient temperature for 2 h. The reaction was carefully quenched with water at low
2 2
temperature, and the mixture was then extracted with CH Cl . After the solvent was removed,
the products IIa-IIm were afforded after recrystallization with MeOH.
IIa N-ethyl-3,5-dihydroxyphthalimide
+ + 1
m.p.: 250~252 °C; MS m/z: 208.1 [M+H] , 230.1 [M+Na] ; H-NMR (300 MHz, C
5 5
D N) δ:
1
2.66(2H, brs, 2×OH), 7.19(1H, d, J=1.8 Hz, H-6), 6.93 (1H, d, J=2.1 Hz, H-4), 3.68(2H, q,
1
3
J=7.2 Hz, N-CH
67.43, 165.93, 158.06, 136.76, 108.59, 107.97, 103.89, 32.28, 13.97.
IIb N-propyl-3,5-dihydroxyphthalimide
m.p.: 194~197 °C; MS m/z: 222.1 [M+H] , 244.0 [M+Na] ; H-NMR (600 MHz, C
3.04(2H, brs, 2×OH), 7.26(1H, d, J=1.8 Hz, H-6), 6.98(1H, d, J=1.2 Hz, H-4), 3.67(2H, t,
2 3 5 5
), 1.16(3H, t, J=7.2 Hz, CH ). C-NMR (600 MHz, C D N) δ:168.14,
1
+
+ 1
5
5
D N) δ:
1
1
3
J=7.2 Hz, N-CH
N) δ: 168.42, 167.69, 165.96, 158.09, 136.67, 108.64, 107.89, 103.91, 39.00, 22.16,
1.21.
IIc N-isopropyl-3,5-dihydroxyphthalimide
2 2 3
), 1.70(2H, m, CH ), 0.84(3H, t, J=7.2 Hz, CH ). C-NMR (600 MHz,
5 5
C D
1
+
+ 1
m.p.: 190~193 °C; MS m/z: 222.1 [M+H] , 244.1 [M+Na] ; H-NMR (300 MHz, DMSO) δ:
1
0.68(2H, brs,2×OH), 6.56(1H, d, J=1.8 Hz, H-6), 6.47(1H, d, J=1.8 Hz, H-4), 4.24(1H, m,

1
3
N-CH), 1.30(6H, d, J=6.9 Hz, 2×CH
64.68, 157.24, 136.06, 107.78, 106.83, 103.08, 42.01, 20.36.
IId N-butyl-3,5-dihydroxyphthalimide
m.p.: 175~176 °C; MS m/z: 236.1 [M+H] ，258.0 [M+Na] ; 1H-NMR (300 MHz, C
2.80(2H, brs, 2×OH), 7.26(1H, d, J=2.1 Hz, H-6), 6.98(1H, d, J=2.1 Hz, H-4), 3.71(2H, t,
3
). C-NMR (600 MHz, DMSO) δ: 167.93, 166.73,
1
+
+
5
5
D N) δ:
1
1
3
J=7.2 Hz, N-CH
NMR (600 MHz, C
7.14, 30.87, 20.06, 13.53.
IIe N-isobutyl-3,5-dihydroxyphthalimide
2
), 1.65(2H, m, CH
2 2 3
), 1.26(2H, m, CH ), 0.81(3H, t, J=7.5 Hz, CH ). C-
5
D
5
N) δ: 168.40, 167.68, 165.96, 158.09, 136.70, 108.63, 107.91, 103.91,
3
+
+ 1
m.p.: 222~224 °C; MS m/z: 236.1 [M+H] , 258.0 [M+Na] ; H-NMR (300 MHz, DMSO) δ:
1
0.75(1H, s, OH-3), 10.66(1H, s, OH-5), 6.60(1H, d, J=1.8 Hz, H-6), 6.49(1H, d, J=1.8 Hz,
13
H-4), 3.22(2H, d, J=7.2 Hz, N-CH
NMR (600 MHz, DMSO) δ: 168.25, 166.98, 164.75, 157.28, 135.95, 107.82, 106.77, 103.33,
4.67, 27.82, 20.37.
IIf N-amyl-3,5-dihydroxyphthalimide
m.p.: 194~196 °C; MS m/z: 250.1 [M+H] , 272.1 [M+Na] , 521.2 [2M+H] ; H-NMR (300
MHz, C N) δ: 13.04(2H, brs, 2×OH), 7.27(1H, d, J=1.8 Hz, H-6), 6.99(1H, d, J=1.8 Hz, H-
), 3.71(2H, t, J=7.2 Hz, N-CH ), 1.68(2H, m, CH ), 1.20~1.25(4H, m, 2×CH ), 0.77(3H, t,
N) δ: 169.91, 169.18, 167.46, 159.59, 138.20,
2 3
), 1.89(1H, m, CH), 0.78(6H, d, J=6.6 Hz, 2×CH ). C-
4
+
+
+ 1
5
D
5
4
2
2
2
1
3
J=7.2 Hz, CH
3
). C-NMR (600 MHz, C
5
D
5
110.13, 109.41, 105.41, 38.91, 30.48, 30.05, 23.76, 15.31.
IIg N-hexyl-3,5-dihydroxyphthalimide
+
+
+ 1
m.p.: 185~188 °C; MS m/z: 264.1 [M+H] , 286.0 [M+Na] , 549.2 [2M+Na] ; H-NMR (600
MHz, C
H-4), 3.68(2H, t, J=6.6 Hz, N-CH
×CH ), 0.73(3H, t, J=6.6 Hz, CH
5
D
5
N) δ: 12.99(2H, brs, 2×OH), 7.22(1H, d, J=1.8 Hz, H-6), 6.94 (1H, d, J=1.8 Hz,
), 1.65(2H, m, CH ), 1.21(2H, m, CH ), 1.11~1.13(4H, m,
). C-NMR (600 MHz, C N) δ: 168.42, 167.70, 165.97,
2
2
2
1
3
2
2
3
5 5
D

1
58.10, 136.72, 108.64, 107.92, 103.92, 37.46, 31.34, 28.84, 26.57, 22.51, 13.88.
IIh N-heptyl-3,5-dihydroxyphthalimide
m.p.: 175~176 °C; MS m/z: 278.1 [M+H] , 300.1 [M+Na] , 577.2 [2M+Na] ; H-NMR (600
MHz, C N) δ: 12.95(2H, brs, 2×OH), 7.20(1H, s, H-6), 6.92(1H, s, H-4), 3.68(2H, t, J=7.2
Hz, N-CH
NMR (600 MHz, C
7.48, 31.67, 28.89, 28.88, 26.89, 22.55, 13.96.
+
+
+ 1
5
D
5
13
2
), 1.65(2H, m, CH
2 2 3
), 1.08~1.22(8H, m, 4×CH ), 0.73(3H, t, J=6.9 Hz, CH ). C-
5
D
5
N) δ: 168.43, 167.70, 165.97, 158.11, 136.72, 108.64, 107.92, 103.92,
3
IIi N-(2-fluorobenzyl)-3,5-dihydroxyphthalimide
+
+ 1
m.p.: 217~219 °C; MS m/z: 288.0 [M+H] , 310.0[M+Na] ; H-NMR (600 MHz, DMSO) δ:
1
6
1
1
0.87(1H, s, OH-3), 10.75(1H, s, OH-5), 7.32~7.07 (4H, m, Ar-H), 6.63(1H, d, J=1.8 Hz, H-
1
3
), 6.51(1H, d, J=1.8 Hz, H-4), 4.66(2H, s, CH ). C-NMR (600 MHz, DMSO) δ: 167.67,
2
66.28, 164.96, 160.25, 157.54, 135.95, 129.87, 129.83, 124.97, 124.28, 115.78, 107.98,
06.84, 103.60, 39.99.
IIj N-(3-fluorobenzyl)-3,5-dihydroxyphthalimide
+
+ 1
m.p.: 196~197 °C; MS m/z: 288.0 [M+H] , 310.0 [M+Na] ; H-NMR (600 MHz, DMSO) δ:
1
0.86(1H, s, OH-3), 10.74(1H, s, OH-5), 7.33(1H, q, J=7.2 Hz, Ar-H), 7.08~7.02(3H, m, Ar-
1
3
H), 6.64(1H, d, J=1.5 Hz, H-6), 6.51(1H, d, J=1.5 Hz, H-4), 4.63(2H, s, CH
MHz, DMSO) δ: 167.81, 166.44, 164.97, 162.60, 157.57, 140.4, 135.97, 131.05, 123.64,
14.61, 114.47, 107.98, 106.81, 103.66, 40.31.
2
). C-NMR (600
1
IIk N-(2-bromobenzyl)-3,5-dihydroxyphthalimide
+
+ 1
m.p.: 231~233 °C; MS m/z: 348.0 [M+H] , 369.9 [M+Na] ; H-NMR (600 MHz, DMSO) δ:
1
0.89(1H, s, OH-3), 10.76(1H, s, OH-5), 7.61(1H, dd, J=0.9, 6.9 Hz, Ar-H), 7.16~7.31(2H, m,
Ar-H), 7.03(1H, dd, J=1.2, 6.3 Hz, Ar-H), 6.66(1H, d, J=1.8 Hz, H-6), 6.54(1H, d, J=2.1 Hz,
1
3
H-4), 4.64(2H, s, CH
2
). C-NMR (600 MHz, DMSO) δ: 167.79, 166.35, 165.02, 157.63,
1
36.00, 135.83, 133.07, 129.70, 128.45, 128.19, 122.05, 108.03, 106.90, 103.71, 41.26.

IIl N-(4-bromobenzyl)-3,5-dihydroxyphthalimide
+
+ 1
m.p.: 233~235 °C; MS m/z: 348.0 [M+H] , 369.9 [M+Na] ; H-NMR (600 MHz, DMSO) δ:
1
0.86(1H, s, OH-3), 10.74(1H, s, OH-5), 7.61(2H, d, J=8.4 Hz, Ar-H), 7.03(2H, d, J=8.7 Hz,
1
3
Ar-H), 6.63(1H, d, J=1.8 Hz, H-6), 6.51(1H, d, J=2.1 Hz, H-4), 4.59(2H, s, CH
600 MHz, DMSO) δ: 167.78, 166.41, 164.96, 157.56, 137.01, 135.96, 131.89, 130.01,
20.86, 107.97, 106.79, 103.63, 40.22.
2
). C-NMR
(
1
IIm N-(3-methybenzyl)-3,5-dihydroxyphthalimide
+
+ 1
m.p.: 226~289 °C; MS m/z: 284.0 [M+H] , 306.0 [M+Na] ; H-NMR (600 MHz, DMSO) δ:
1
0.85(1H, s, OH-3), 10.73(1H, s, OH-5), 7.15(1H, t, J=7.8 Hz, Ar-H), 7.03~6.97(3H, m, Ar-
H), 6.62(1H, d, J=1.8 Hz, H-6), 6.50(1H, d, J=1.8 Hz, H-4), 4.56(2H, s, CH ), 2.22(3H, s,
). C-NMR (600 MHz, DMSO) δ: 167.83, 166.52, 164.92, 157.49, 138.17, 137.54,
36.01, 128.90, 128.38, 128.25, 124.83, 107.95, 106.84, 103.58, 40.75, 21.40.
.1.3. Procedure for the synthesis of type III N-substituted phthalimide analogs (IIIa-
2
1
3
CH
3
1
3
IIIc)
A total of 2.08 g (0.01 mol) of compound 5 and 0.61 g (0.01 mol) of ethanolamine were
mixed and reacted for 2 h at 185°C. The yellow solid was then chromatographed (silica gel;
CH Cl : MeOH, 9:1) to afford the intermediate 7 (1.31 g, 52.2%) as a white solid.
2
2
Then, 2.51 g (0.01 mol) of compound 7 and 4-dimethylaminopyridine (DMAP) were
dissolved in 20 mL of CH Cl , and 0.01 mol of acyl chloride was added dropwise in an ice
2
2
3
bath and stirred for 6 h at ambient temperature. After the reaction, saturated NaHCO was
added, and the organic layer was evaporated to afford the compounds IIIa-IIIc.
IIIa N-(2-acetyloxyethyl)-3,5-dimethyoxyphthalimide
+
m.p.: 184~185 °C; MS m/z: 293.1 [M] ; H-NMR (300 MHz, CDCl
1
3
) δ: 6.93(1H, d, J=1.8 Hz,
), 3.89(3H,
H-6), 6.60(1H, d, J=1.8 Hz, H-4), 4.25(2H, t, J=5.4 Hz, OCH ), 3.94(3H, s, OCH
2
3

1
3
s, OCH
3
), 3.86(2H, t, J=5.4 Hz, N-CH
70.88, 167.80, 166.64, 166.44, 158.13, 136.22, 110.37, 103.75, 100.46, 61.62, 56.33, 56.23,
6.80, 20.83.
2 3 3
), 1.99(3H, s, CH ). C-NMR (600 MHz, CDCl ) δ:
1
3
IIIb N-(2-propionyloxyethyl)-3,5-dimethyoxyphthalimide
+
m.p.: 124~125 °C; MS m/z: 307.1 [M] ; H-NMR (300 MHz, CDCl
1
3
) δ: 6.93(1H, s, H-6),
), 3.89(3H, s, OCH ),
). C-NMR (600
6
3
.60 (1H, s, H-4), 4.26(2H, t, J=4.8 Hz, OCH
2
), 3.94(3H, s, OCH
3
3
1
3
.85(2H, m, N-CH ), 2.26(2H, q, J=7.5 Hz, CH ), 1.06(3H, t, J=7.5 Hz, CH
2
2
3
MHz, CDCl3) δ: 174.24, 167.78, 166.62, 166.43, 158.12, 136.23, 110.38, 103.74, 100.43,
1.46, 56.33, 56.22, 36.85, 27.38, 8.93.
IIIc N-(2-chloroacetyloyethyl)-3,5-dimethyoxyphthalimide
6
+
+
+ 1
m.p.: 143~144 °C; MS m/z: 328.0 [M+H] , 350.0 [M+Na] , 677.0 [2M+Na] ; H-NMR (300
MHz, CDCl ) δ: 6.94(1H, d, J=1.8 Hz, H-6), 6.61(1H, d, J=2.1 Hz, H-4), 4.38(2H, t, J=5.1
Hz, OCH ), 4.02(2H, s, CH -Cl), 3.95(3H, s, OCH ), 3.92(3H, s, OCH ), 3.89(2H, m, N-
). C-NMR (600 MHz, CDCl3) δ: 167.76, 167.25, 166.73, 166.37, 158.19, 136.11,
10.24, 103.80, 100.57, 63.38, 56.35, 56.25, 40.78, 36.52.
.2 Methods
3
2
2
3
3
1
3
CH
2
1
3
3
.2.1. Cell culture
Mouse macrophage RAW 264.7 cells, obtained from the American Type Culture Collection
(
(
ATCC, Rockville, MD, USA), were cultured in Dulbecco's Modified Eagle's Medium
DMEM) supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 100 units/mL
penicillin, 100 µg/mL streptomycin, and antibiotics-antimycotics (0.25 µg/mL amphotericin
B). The cells were incubated at 37 °C with 5 % CO in humidified air.
2
3
.2.2. Measurement of nitrite production

Nitric oxide (NO) has a short half-life and is oxidized to stable nitrite. Consequently, to
evaluate the inhibitory activity of the test compounds on LPS-induced NO production, RAW
5
64.7 cells were plated at a density of 2 × 10 per well in a 24-well culture plate and
2
2
incubated in a 37°C humidified incubator with 5% CO in air for 24 h. The cells were washed
twice with PBS, fresh medium was added, and the cells were then incubated with 1 µg/mL
LPS in the presence or absence of the test compounds for 22 h. The amount of nitrite released
in the culture media was measured using the Griess reaction (180 µL of Griess reagent [1:1
mixture (v/v) of 1% sulfanilamide in 5%
H
3
PO
4
and 0.1% N-(1-naphthyl)
ethylenediaminedihydrochloride solution]). Serially diluted sodium nitrite was used as a
standard, and the absorbance was measured at 540 nm. The IC50 value, which is the sample
concentration resulting in 50% inhibition of NO production, was determined using nonlinear
regression analysis (% inhibition vs concentration).
3
.2.3. Cell viability assay (MTT)
After the Griess reaction, MTT solution (final concentration of 500 µg/mL) was added to
each well and further incubated for 3 h at 37°C. The medium was discarded, and dimethyl
sulfoxide (DMSO) was added to each well to dissolve the generated formazan. The
absorbance was measured at 570 nm, and the percent survival was determined by comparison
to a control (CTL) group.
3
.2.4. Reporter gene (SEAP; Secreted Embryonic Alkaline Phosphatase) assay
To determine the effect of the test compounds on the activation of NF-κB, a reporter gene
assay was performed. Briefly, the cells were pre-treated with the test compounds for 2 h and
then further induced with LPS for an additional 16 h. Cell culture supernatants were heated at

6
2
5°C for 5 min and reacted with SEAP assay buffer [2 M diethanolamine, 1 mM MgCl , 500
µM 4-methylumbelliferyl phosphate (MUP)] in the dark at 37°C for 1 h. In addition,
fluorescence from the product of SEAP/MUP reaction was measured in relative fluorescence
units using a 96-well plate fluorometer with excitation at 360 nm and emission at 449 nm and
normalized to protein concentration. Data are presented as the proportion relative to vehicle-
treated control cells without LPS.
3
.2.5. Western blot analysis
5
RAW 264.7 cells (3 × 10 cells/mL in a 60 mm dish) were treated with various
concentrations of QSN-19 for the indicated times. Western blot analysis was carried out as
described previously [19].
3
.2.6. Real time-polymerase chain reaction (RT-PCR)
RAW 264.7 cells were stimulated with 1 µg/mL LPS in the presence or absence of QSN-19
for 4 h. Then, RT-PCR analysis was carried out as described previously[19]. The following
sequences were used: iNOS F5' -ATGTCCGAAGCAAACATCAC-3'; iNOS R5' -
TAATGTCCAGGAAGTAGGTG-3'; IL-1β F5′- TGCAGAGTTCCCCAACTGG TACATC-
3
′; IL-1β R5′- GTGCTGCCTAATGTCCCCTTGAATC-3′; IFN-β F5' -CATTT
CCGAATGTTCGTCCT-3'; IFN-β R5' -CAC AGCCCTCTCCATCAACTA-3'; β-actin F5 ′-
TGTGATGGTGGGAATGGGTCAG-3′; β-actin R5 ′-TTTGATGTCACGCACGATT TCC-
3
′. β-actin was used as an internal standard.
3
.2.7. Statistical Analysis

All experiments were repeated at least three times. Data are presented as the means ±
standard deviation(SD) for the indicated number of independently performed experiments.
Student's t-test (SigmaStat 3.1, Systat Software Inc.) was used for the determination of
statistical significance. The difference was considered statistically significant when p (*)
<0.05, (**) < 0.005, (***) < 0.0001.
4
. CONCLUSIONS
Figure 6. A possible mechanism by which compound IIh suppresses nitric oxide (NO) production in
the lipopolysaccharide (LPS)/TLR4 signaling pathway. (Red arrow: the effect of compound IIh)

For treating inflammation, a variety of phytochemicals have been evaluated for their
potential efficacy as anti-inflammatory agents, and several compounds have been identified
as selective inhibitors of iNOS. In the present study, we designed and evaluated a series of
phthalimides as inhibitors of NO production by iNOS. Several compounds showed potent
inhibitory activity towards LPS-induced NO production. The present study suggests that the
suppression of iNOS mRNA transcription is, in part, related to the inhibitory activity of
phthalimides. Additionally, the details mechanism of IIh also is investigated (Figure 6). The
inhibitors in this new series of are suggested as lead compounds for the development of
potent and selective inhibitors for potential therapeutic use. Based on these findings, future
studies aimed at producing more potent inhibitors are currently in progress in our laboratory.

AUTHOR INFORMATION
*Corresponding authors.
E-mail addresses: sklee61@snu.ac.kr (S.K.Lee), ynxu@syphu.edu.cn (Y.-N. Xu).
S.K.Lee and Y.-N.Xu contributed equally to this work as corresponding authors.
1
B.D. Hiep and J.-Y.Liu contributed equally to this work.
ACKNOWLEDGMENT
This work was supported by the National Research Foundation of Korea (NRF) grant
founded by the Korean Government (MEST) (MRC No. 2009-0083533).
CONFLICT OF INTEREST
The authors declare no conflict of interest

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