Synthesis and anti-inflammatory evaluations of β-lapachone derivatives

Article abstract of DOI:10.1016/j.bmc.2012.10.047

β-Lapachone (β-LAPA), a natural product from the lapacho tree in South America, is a potential chemotherapeutic agent that exhibit a wide variety of pharmacological effects such as anti-virus, anti-parasitic, anti-cancer, and anti-inflammatory activities.

Full text of DOI:10.1016/j.bmc.2012.10.047

Bioorganic & Medicinal Chemistry 21 (2013) 523–531
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti-inflammatory evaluations of b-lapachone derivatives
Chih-Hua Tseng a, , Chih-Mei Cheng b, , Cherng-Chyi Tzeng a, Shin-I Peng a, Chiao-Li Yang a,
Yeh-Long Chen a,
⇑
a Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
b Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
b-Lapachone (b-LAPA), a natural product from the lapacho tree in South America, is a potential chemo-
therapeutic agent that exhibit a wide variety of pharmacological effects such as anti-virus, anti-parasitic,
anti-cancer, and anti-inflammatory activities. In order to discover novel anti-inflammatory agents, we
have synthesized a series of b-LAPA derivatives for evaluation. Among them, 4-(4-methoxyphenoxy)
Received 8 October 2012
Revised 29 October 2012
Accepted 31 October 2012
Available online 24 November 2012
naphthalene-1,2-dione (6b) was found to be able to inhibit NO and TNF-
a released in LPS-induced
Raw 264.7 cells. Inhibition of iNOS and COX-2 was also observed in compound 6b treated cells.
Mechanism studies indicated that 6b exhibited anti-inflammatory properties by suppressing the release
of pro-inflammatory factors through down-regulating NF-jB activation. In addition, it suppressed NF-jB
translocation by inhibiting the phosphorylation of p38 kinase. Our results also indicate that the inhibitory
effect of 6b on LPS-stimulated inflammatory mediator production in Raw 264.7 cell is associated with
the suppression of the NF-jB and MAPK signaling pathways. A low cytotoxicity (IC50 = 31.70 lM) and
Keywords:
b-Lapachone (b-LAPA)
Anti-inflammatory activity
iNOS
COX-2
NO
TNF-a
the potent anti-inflammatory activity exhibited by compound 6b make this compound a potential lead
for developing new anti-inflammatory agents. Further structural optimization of compound 6b is
on-going.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
quinones include lapachol, a-lapachone and b-lapachone (b-LAPA)
(Fig. 1) were isolated from the heartwood of the Bignoniaceae fam-
ily (Tabebuia sp) and evaluated for their biological activities.
Among them, b-LAPA was found to be able to inhibit the expres-
sion of nitric oxide (NO) and PGE2 in alveolar macrophages.14 In or-
der to discover novel drug candidates, we have synthesized certain
furo[30,20:3,4]naphtho[1,2-d]imidazole derivatives and evaluated
for their anti-inflammatory activities. Our results indicated that
(E)-2-(2-(5-nitrofuran-2-yl)vinyl)furo[30,20:3,4]naphtho[1,2-d]imi-
dazole (1) was capable of inhibiting iNOS expression, with an
Inflammation is a central feature of many pathophysiological
conditions in response to tissue injury and host defenses against
microbial challenge. Pro-inflammatory cells, mainly activated
macrophages, mediate most of the cellular and molecular patho-
physiology of inflammation by producing cytokines and other
pro-inflammatory molecules, including prostaglandins, enzymes,
and free radicals such as NO.1,2 Three isoforms of NOS exist, consti-
tutively expressed neuronal NOS (nNOS), endothelial NOS (eNOS),
and inducible isoform (iNOS).3 Activated macrophages transcrip-
tionally express inducible nitric oxide synthase (iNOS), which is
responsible for the prolonged and profound production of NO.4–6
The aberrant release of NO can lead to amplification of inflamma-
tion as well as tissue injury.7 Therefore, inhibition of neutrophils
and/or macrophages activation and the following release of inflam-
matory mediators provide a promising strategy for the develop-
ment of potential anti-inflammatory agents.
IC50 value of 0.52
naphtho[1,2-d]imidazole (2) exhibited
activity on LPS-induced PGE2 production, with an IC50 value of
0.047
M.15,16 We have also demonstrated that certain quinolin-2(1H)-
l
M while 2-(4-methoxyphenyl)-furo[30,20:3,4]
strongly inhibitory
a
l
one derivatives such as (E)-4-[2-cyclohex-2-enylidene]hydrazi-
nyl]quinolin-2(1H)-one (3) significantly suppressed LPS-induced
NO production and iNOS gene expression in macrophages.17 The
present study intends to explore novel b-LAPA derivatives which
possess significant anti-inflammatory potency and lower
cytotoxicity. In addition to the quinone (naphthalene-1,2-dione)
pharmacophores, their hydroxylamine-condensed derivatives have
also been synthesized and evaluated on the ground that many
hydroxylamine-condensed derivatives exhibited more potent
biological activities than that of their respective ketone precur-
sors.18–21
Many efforts had been devoted to the discovery of novel
anti-inflammatory agents for the past few years.8–13 The natural
⇑
Corresponding author. Tel.: +886 7 3121101x2684; fax: +886 7 3125339.
E-mail address: yeloch@kmu.edu.tw (Y.-L. Chen).
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.047

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C.-H. Tseng et al. / Bioorg. Med. Chem. 21 (2013) 523–531
O
O
O
O
O
OH
O
O
O
α-lapachone
lapachol
β-lapachone
H
N
H
O
H
N
NO2
N
N
O
OMe
N
O
O
HN
N
1
2
3
Figure 1. Structures of lapachol, b-lapachone, a-lapachone, and compounds 1–3.
(dC 178.04) and one oxime carbon (dC 143.66, it was shifted upfield
compared to 5a). The HMQC spectrum of compound 7a showed
that the resonance of 3-H (dC 97.41/dH 5.62) was shifted upfield
compared to that of 3-H of 5a (dC 100.41/dH 5.87). Further, the ex-
tremely deshielded NOH proton (dH 18.53) can be explained by it
being involved in a strong intramolecular hydrong bond to the
naphthalen-2-one C@O unit. This configurational fixation is also
confirmed by X-ray crystallographical analysis ( Fig. 3) of com-
pound 7a23, the yellow single crystal (0.12 ꢀ 0.08 ꢀ 0.04 mm3) of
7a was obtained by slow evaporation from MeOH/CH2Cl2 (10/90)
solution. In the crystal structure, 7a has a strong hydrogen bond
(O(2)-H. . .O(1) = 1.743 Å) between (Z)-configurational oxime
group and the C@O unit.
Accordingly, (Z)-1-hydroxyimino-4-(methoxyphenylamino) nap-
hthalene-1,2-dione (7b) was prepared by the condensation of
NH2OH with 4-(4-methoxyphenylamino)-naphthalene-1,2-dione
(5b) which in turn was obtained by the treatment of 4 and
4-methoxyaniline. Treatment of 4 with phenol under basic condi-
tion afforded 4-phenoxynaphthalene-1,2-dione (6a) in 48% yield.
4-(4-Methoxyphenoxy)-naphthalene-1,2-dione (6b) was obtained
in a moderate yield by the treatment of 4 with 4-methoxyphenol
2. Results and discussion
2.1. Chemistry
Treatment of sodium 3,4-dioxo-3,4-dihydronaphthalene-1-sul-
fonate (4) with aniline afforded 4-(phenylamino)naphthalene-
1,2-dione (5a) in 81% yield as described in Figure 2. The 1H NMR
spectrum of compound 5a showed a total of 11 proton signals,
which included ten aromatic methynes (dH 5.87 (3-H), 7.29–7.33
(3 Ar-H), 7.49–7.53 (2 Ar-HH), 7.75 (7-H), 7.87 (6-H), 8.05 (5-H),
8.33 (8-H)) and one broad amino-proton (dH 9.89), and the 13C
NMR spectrum of 5a showed two carbonyl carbons (dC 175.92,
181.08). A correlation between dC 100.41/dH 5.87 was also ob-
served in the HMQC spectrum of compound 5a. Condensation of
5a with NH2OH gave (Z)-1-hydroxyimino-4-(phenylamino)naph-
thalene-1,2-dione (7a)22 exclusively based on the spectra data.
The 1H NMR spectrum of compound 7a indicated ten aromatic
methynes (dH 5.62 (3-H), 7.40–7.44 (3 Ar-H), 7.54–7.58 (2 Ar-
HH), 7.68–7.74 (6- and 7-H), 8.24 (5-H), 8.38 (8-H)), one broad
amino-proton (dH 10.31), and one sharp oxime-proton (dH 18.53),
and the 13C NMR spectrum of 7a showed one carbonyl carbons
OH
N
O
O
O
NH2OH HCl,
K2CO3
H2N
R
HN
HN
2-EtOEtOH reflux
H2O
R
R
O
5a R = H (81%)
7a R = H (68%)
O
5b R = OMe (84%)
7b R = OMe (71%)
OH
N
O
O
SO3Na
O
NH2OH HCl,
K2CO3
4
HO
KOH, H2O
R
O
2-EtOEtOH reflux
O
R
R
6a
R = H (48%)
8b R = OMe (15%)
6b R = OMe (46%)
Figure 2. Synthesis of compounds 5a–8b.

C.-H. Tseng et al. / Bioorg. Med. Chem. 21 (2013) 523–531
525
2.3. Nitric oxide (NO)-scavenging effects
NO-scavenging effects of compounds 6a, 6b, and 8b were mea-
sured as previously described.24 Sodium nitroprusside (SNP)
(2.5 mM) was incubated alone or in combination with tested com-
pounds (10 lM) or 1400 W (10 lM) in light at room temperature
for 60 min. Results of Figure 4 indicated the co-incubation of SNP
with compound 6a, 6b, 8b, b-LAPA or 1400 W did not affect the
levels of nitrite.
2.4. Lipopolysaccharide (LPS)-induced iNOS and/or COX-2
protein
To address whether the inhibition of NO production was associ-
ated with decreased levels of iNOS and/or COX-2, we performed RT-
PCR and Western blot analysis for detection of protein levels. Raw
264.7 cells were treated with compound 6a, 6b, and 8b (0.5 lM),
respectively for 24 h. The protein levels of iNOS and COX-2 were de-
tected in whole cell lysates 24 h after LPS treatment. In iNOS protein
level, three compounds were approximately equally potent in the
inhibition of iNOS expression while b-LAPA and 1400 W were less
active (Fig. 5A). It was clear that the density ratio of LPS-induced
iNOS was significantly (⁄P <0.05 and ⁄⁄P <0.01) inhibited in a dose-
dependent manner by 6b and b-LAPA, respectively in the quantita-
tive determination. Both compounds were found to be more active
Figure 3. ORTEP view of (Z)-1-(hydroxyimino)-4-(phenylamino)naphthalen-2(1H)-
one (7a).
under the same reaction conditions. Hydroxylamine fails to give
definite products with 6a, but condenses with 6b gave (Z)-1-
hydroxyimino-4-(methoxyphenoxy)naphthalene-1,2-dione (8b)
as a sole product with low yield.
than 1400 W at a concentration of 2.0 lM (Fig. 5B). Therefore, our
results indicated the inhibition of NO production by 6b and b-LAPA
was associated at least partly with decreased levels of iNOS. Results
had also indicated the inhibition of NO production by 1400 W may
not be associated with decreased levels of iNOS.
2.2. Lipopolysaccharide (LPS)-induced nitric oxide production
As shown in Figure 5C, the LPS induced COX-2 level was signifi-
cantly inhibit by all the compoundstested and the inhibitoryactivity
Murine macrophage Raw 264.7 was treated with LPS (0.1 lg/
mL) for 24 h to induce NO production. The accumulation of nitrite,
a stable NO metabolite was assessed by Griess reaction. Results
from Table 1 indicated 4-(4-methoxyphenylamino)naphthalene-
1,2-dione (5b) was approximately twofold less active than b-LAPA
in the suppression of LPS-induced NO production. Replacement of
4-(4-methoxyphenylamino) moiety with its isosteric 4-(4-
methoxyphenoxy) counterpart did not affect the inhibitory activity
in which both 5b and 6b were able to suppress LPS-induced NO
decreased in an order of 6b > 6a > 8b at 0.5
approximately equal potent with 1400 W and was more active than
b-LAPA at a concentration of 0.5 M. Compound 6b and b-LAPAwere
lM. Compound 6b was
l
found to inhibited the LPS induced COX-2 level in a dose-dependent
manner as shown in Figure 5D. The inhibitory activity decreased in
an order of 6b > b-LAPA > 1400 W at 2.0 lM. Therefore, our results
indicated that the inhibition of NO production by 6b and b-LAPA
was associated at least partly with decreased levels of COX-2.
production with an IC50 value of 0.81 and 0.74 lM, respectively.
A comparable activity was observed for 5b and its 1-hydroxyimino
derivative 7b, while the 1-hydroxyimino derivative 8b was less ac-
tive than its parent dioxo precursor 6b.
To identify active potential drug candidates with low cytotoxic-
ity, Raw 264.7 cells were treated with these newly synthesized
compounds and b-LAPA, respectively for 24 h. Cytotoxicity was
determined by MTS assay and the results indicated that 4-phenyla-
mino derivatives 5b, 7a, and 7b were highly cytotoxic with an IC50
2.5. Lipopolysaccharide (LPS)-induced iNOS mRNA
Compound 6b was proved to be able to suppress LPS-induced
NO production through the inhibition of iNOS and COX-2 levels.
value of 1.84, 1.67, and 4.69
derivatives 6a, 6b, and 8b were less cytotoxic with an IC50 value
of 28.47, 31.70, and 20.63 M, respectively. Therefore, compounds
lM, respectively while 4-phenoxy
l
6a, 6b, and 8b were selected for further pharmacological
evaluations.
Table 1
Cytotoxicity and the inhibitory effect of b-lapachone (b-LAPA) derivatives on the
accumulation of nitric oxide induced by LPS in the culture media of RAW 264.7 cells
Compound
NO inhibition IC50
(l
M)
Cytotoxicity IC50 (lM)
5b
6a
6b
7a
7b
8b
b-LAPA
1400 W
0.81 0.11
0.85 0.55
0.74 0.16
0.63 0.20
0.87 0.05
1.29 0.05
0.47 0.04
0.20 0.02
1.84 0.08
28.47 3.61
31.70 2.65
1.67 0.13
4.69 0.30
20.63 2.31
4.06 0.03
47.47 1.89
Figure 4. NO-scavenging effects of 1400 W, b-LAPA, compounds 6a, 6b, and 8b. SNP
solution (2.5 mM) in PBS was incubated alone or with tested compound (10
1400 W (10 M) in the presence of light at room temperature for 60 min.
lM) or
l

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C.-H. Tseng et al. / Bioorg. Med. Chem. 21 (2013) 523–531
Figure 5. Inhibition of iNOS and COX-2 expression by b-LAPA analogues and 1400 W in LPS-stimulated RAW 264.7 cells. Cells (5 ꢀ 105 cells/well) were pretreated with the
indicated concentrations of tested compounds for 2 h before incubation with 0.1 lg/mL LPS for 24 h. Cell lysates were then prepared and Western blots were performed using
an antibody specific for murine iNOS (A, B) and COX-2 (C, D). Data are presented as means + S.D. mean of 3 separate experiments performed in triplicate. ⁄P <0.05 as compared
with control. ⁄⁄P <0.01 as compared with LPS alone.
We further investigated whether the inhibition of NO production
was associated with decreased levels of iNOS mRNA. As shown in
Figure 6, iNOS mRNA level was significantly decreased by 6b and
b-LAPA in a dose-dependent manner for 9 h after LPS treatment.
The inhibitory activity decreased in an order of 6b > b-LA-
same concentrations. These results indicated that reduced expres-
sion of iNOS and COX-2 by 6b and b-LAPA were liable for inhibition
of NO and TNF-a production.
2.7. Lipopolysaccharide (LPS)-induced MMP-9 expression
PA > 1400 W at 2.0
lM.
To determine whether compound 6b and b-LAPA affect the activ-
ity of MMP-9 in LPS-stimulated macrophages, Raw 264.7 cells were
incubated with different concentrations of 6b and b-LAPA, respec-
tively, followed by stimulation with LPS as described above and
the activity of MMP-9 was analyzed by gelatin zymography. We
found the activity of MMP-9 was significantly inhibited by the treat-
ment of 6b and b-LAPA in a dose dependent manner (Fig. 7B).
2.6. Lipopolysaccharide (LPS)-induced TNF-
a
production
To determine the effect of b-LAPA derivatives in the production
of pro-inflammatory cytokine TNF- production, we have analyzed
the effects of 6b and b-LAPA on pro-inflammatory cytokines, such
as TNF- . Raw 264.7 cells were incubated with 6b and b-LAPA,
respectively (0.5, 1.0, 2.0, or 5.0 M) in the presence or absence
of LPS (0.1 g/mL) for 24 h, and cytokine levels in the culture med-
ium were measured. As shown in Figure 7A, TNF- level was signif-
a
a
l
l
2.8. Effect of 6b on NF-jB activity
a
icantly decreased by 6b and b-LAPA in a concentration-dependent
NF-jB is a primary regulator of genes that are involved in the
manner, and compound 6b was more active than b-LAPA at the
production of pro-inflammatory cytokines and enzymes involved

C.-H. Tseng et al. / Bioorg. Med. Chem. 21 (2013) 523–531
527
Figure 6. Inhibitory effects of compound 6b on LPS-induced iNOS mRNA expres-
sion. Cells (5 ꢀ 105 cells/well) were pretreated with the indicated concentrations of
tested compounds for 2 h before incubation with 0.1 lg/mL LPS for 9 h. Total RNA
was prepared for RT-PCR analysis of iNOS mRNA expression in LPS-stimulated RAW
264.7 cells. GAPDH were used as internal controls for the RT-PCR assays. Data are
presented as means + S.D. mean of 3 separate experiments performed in triplicate.
⁄P <0.05 as compared with control. ⁄⁄P <0.01 as compared with LPS alone.
in the inflammatory process.25 To clarify the influence of 6b on NF-
j
B p65 nuclear translocation, the nuclear localization of NF-
Raw 264.7 cells was analyzed using laser confocal immunofluores-
cent microscopy (Fig. 8). The confocal images revealed that NF-
p65 was normally sequestered in the cytoplasm, and that nuclear
localization of NF- B p65was significantly induced after stimula-
tion of cells with LPS. The LPS-induced translocation of NF-
p65 was completely abolished after pre-treating the cells with
6b. Nuclear translocation of NF- B p65 was not observed in the
jB in
jB
Figure 7. Inhibitory effects of compound 6b on LPS-induced TNF-
a
, and MMP-9
j
expression. Cells (5 ꢀ 105 cells/well) were pretreated with the indicated concen-
jB
trations of tested compounds for 2 h before incubation with 0.1 lg/mL LPS for 24 h.
The amounts of TNF-a were determined using ELISA in culture medium. (A) The
j
medium was collected and used in zymography, activity was measured by scanning
the zymogram, quantified the intensity of the MMP-9 band and expressed the
intensity as % of control. (B) Data are presented as means + S.D. mean of 3 separate
experiments performed in triplicate. ⁄P <0.05 as compared with control. ⁄⁄P <0.01 as
compared with LPS alone.
cells after pre-treatment with cordycepin alone in the absence of
LPS stimulation. Taken together, these findings showed that the
NF-jB pathway was involved in the anti-inflammatory effect of
6b in LPS-stimulated Raw 264.7 cells.
and b-LAPA significantly attenuated the release of inflammatory
2.9. Effects of 6b and b-LAPA on the phosphorylation of p-38
kinase in LPS-stimulated Raw 264.7 cells
mediators (NO and TNF-
manner. These data indicated that 6b and b-LAPA target p38 kinase
and NF- B. These compounds inhibit iNOS, COX-2, and the forma-
tion of pro-inflammatory cytokine NO, TNF- , MMP-9. Although b-
LAPA was able to suppress LPS-induced NO production with an IC50
value of 0.47 M, its high cytotoxicity against the growth of RAW
264.7 cells with an IC50 value of 4.06 M limited its further devel-
a, MMP-9) in a concentration-dependent
j
The MAPK pathways are known to be important for the expres-
sion of iNOS and COX-2.26 Therefore, MAP kinases act as specific
targets for inflammatory responses. To test whether the inhibition
of inflammation by cordycepin is regulated through the MAP ki-
nase pathways, we examined the effect of 6b and b-LAPA on LPS-
induced phosphorylation of p38 kinase in Raw 264.7 cells using
Western blot analysis. As shown in Figure 9, 6b and b-LAPA atten-
uated the LPS-stimulated phosphorylation of p38 in a concentra-
tion-dependent manner. In contrast, the amounts of p38 kinase
were unaffected by either LPS or 6b treatment. These results sug-
gested that the MAPK pathways were involved during LPS-medi-
ated expression of inflammatory mediators.
a
l
l
opment as a potential drug candidate. We have identified a new
type of b-LAPA derivatives which are non cytotoxic and exhibited
the same anti-inflammatory mechanism with b-LAPA. Among
them, 6b may serve as a new lead for the discovery of potential
anti-inflammatory agents.
4. Experimental part
4.1. General
3. Conclusion
TLC: Precoated (0.2 mm) silica gel 60 F254 plates from EM
Laboratories, Inc.; detection by UV light (254 nm). Mp: electrother-
mal IA9100 digital melting-point apparatus; uncorrected. The
In summary, we have synthesized certain b-LAPA derivatives
and evaluated for their anti-inflammatory activities. Results indi-
cated that 4-(4-methoxyphenoxy)-naphthalene-1,2-dione (6b)

528
C.-H. Tseng et al. / Bioorg. Med. Chem. 21 (2013) 523–531
Figure 8. Effects of 6b on NF-jB activity in LPS-stimulated RAW 264.7 cell. Localization of NF-jB p65 was visualized with fluorescence microscopy after immunofluorescence
staining with NF-jB p65 antibody (red). Cells were stained with DAPI for visualization of nuclei (blue). White arrows indicate the nuclear localization of NF-kB.
ultraviolet–visible (UV–VIS) absorption spectra were recorded on a
Jasco V570 spectrometer. IR spectra were measured on PERKIN
ELMER System 2000 FT-IR spectrophotometer. 1H and 13C NMR
spectra: Varian-Unity-400 spectrometer at 400 and 100 MHz or
Varian-Gemini-200 spectrometer at 200 and 50 MHz, chemical
shifts d in ppm with SiMe4 as an internal standard (=0 ppm), cou-
pling constants J in Hz. Elemental analyses were carried out on a
Heraeus CHN–O-Rapid elemental analyzer, and results were within
0.4% of calculated values.
(400 MHz, CDCl3): 5.87 (br s, 1H, 3-H), 7.29–7.33 (m, 3H, Ar-H),
7.49–7.53 (m, 2H, Ar-H), 7.73–7.77 (m, 1H, 7-H), 7.86–7.89 (m,
1H, 6-H), 8.05 (d, 1H, J = 7.6 Hz 5-H), 8.33 (d, 1H, J = 8.0 Hz, 8-
H), 9.89 (br s, 1H, NH). 13C NMR (100 MHz, CDCl3): 100.41,
103.73, 120.00, 123.98, 126.01, 126.84, 128.19, 129.42 (2C),
131.52 (2C), 134.32, 137.87, 154.64, 175.92, 181.08. Anal. calcd
for C16H11NO2: C 77.10, H 4.45, N 5.62; found: C 77.01, H 4.70,
N 5.41.
4.1.2. 4-(4-Methoxyphenylamino)naphthalene-1,2-dione (5b)
4.1.1. 4-(Phenylamino)naphthalene-1,2-dione (5a)
From 4 as described for 5a, to give of 5b as a brown solid (1.13 g,
To a solution of sodium 3,4-dioxo-3,4-dihydronaphthalene-1-
sulfonate (4) (1.30 g, 5.0 mmol) in water (150 mL) was added
aniline (0.48 g, 5.5 mmol). The reaction mixture was stirred at
room temperature for 20 min (TLC monitoring). The solvent was
removed in vacuo and the resulting residue was crystallized from
EtOH to give 1.10 g (81%) of 5a as a red solid. Mp: 272–274 °C. UV
84% yield). Mp: 108–110 °C. UV kmax nm (log
e): 429 (3.51), 278
(4.17), 225 (4.34), 204(3.93) in MeOH. IR (KBr pellets) cmꢁ1
:
3328, 1582, 1522. 1H NMR (400 MHz, DMSO-d6): 3.83 (s, 3H,
OMe), 5.63 (s, 1H, 3-H), 7.04–7.07 (m, 2H, Ar-H), 7.26–7.29 (m,
2H, Ar-H), 7.68–7.72 (m, 1H, 7-H), 7.80–7.82 (m, 1H, 6-H), 8.05
(d, 1H, J = 7.6 Hz, 5-H), 8.30 (d, 1H, J = 8.0 Hz, 8-H), 9.80 (br s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6): 55.16, 99.96, 104.38, 114.40
(2C), 123.58, 125.58, 127.33 (2C), 127.93, 130.11, 131.12, 133.97,
kmax nm (log
e
): 460 (3.73), 278 (4.22), 243 (4.41), 206 (4.21) in
MeOH. IR (KBr pellets) cmꢁ1
:
3318, 1583, 1520. 1H NMR

C.-H. Tseng et al. / Bioorg. Med. Chem. 21 (2013) 523–531
529
ethanol (10 mL) was heated at reflux for 3 h (TLC monitoring). The
solvent was removed in vacuo and the residue was suspended in
H2O (20 mL). The resulting precipitate that separated was col-
lected, washed with H2O, and dried to give a crude solid. The crude
product was crystallized from EtOH to afford 7a as a red solid
(0.32 g, 68%). Mp: 226–228 °C. UV kmax nm (log
e): 411 (3.88),
340 (4.08), 269 (4.10), 214 (4.32) in MeOH. IR (KBr pellets) cmꢁ1
:
3443, 3303, 1592, 1517. 1H NMR (400 MHz, DMSO-d6): 5.62 (s,
1H, 3-H), 7.40–7.44 (m, 3H, Ar-H), 7.54–7.58 (m, 2H, Ar-H), 7.68–
7.74 (m, 2H, 6- and 7-H), 8.26–8.30 (m, 1H, 5-H), 8.36–8.40 (m,
1H, 8-H), 10.31 (br s, 1H, NH), 18.53 (s, 1H, NOH). 13C NMR
(100 MHz, DMSO-d6): 97.41, 122.47, 123.54, 124.02, 126.20 (2C),
127.33, 129.24, 129.67 (2C), 131.33, 131.54, 137.57, 143.66,
156.95, 178.04. Anal. calcd for C16H14N12O2: C 72.72, H 4.58, N
10.60; found: C 72.35, H 4.50, N 10.54.
4.1.6. (Z)-1-(Hydroxyimino)-4-(4-
methoxyphenylamino)naphthalen-2(1H)-one (7b)
From 4 as described for 7a, to give 0.38 g (71%) of 7b as a red
solid. Mp: 224–226oC. UV kmax nm (log
e): 417 (3.90), 347 (4.09),
Figure 9. Inhibitory effects of compounds 6b on LPS-induced p-p38 protein
expression. Cells (5 ꢀ 105 cells/well) were pretreated with the indicated concen-
270 (4.20), 224 (4.56) in MeOH. IR (KBr pellets) cmꢁ1: 3442,
3255, 1607, 1505. 1H NMR (400 MHz, DMSO-d6): d 3.83 (s, 3H,
OMe), 5.52 (s, 1H, 3-H), 7.08–7.12 (m, 2H, Ar-H), 7.32–7.36 (m,
2H, Ar-H), 7.66–7.73 (m, 2H, 6- and 7-H), 8.25–8.29 (m, 1H, 5-
H), 8.35–8.39 (m, 1H, 8-H), 10.24 (br s, 1H, NH), 18.61 (s, 1H,
NOH). 13C NMR (100 MHz, DMSO-d6): 55.40, 97.05, 114.81 (2C),
122.43, 123.43, 124.04, 127.64 (2C), 129.16, 130.13, 131.34,
131.44, 143.70, 157.41, 158.25, 177.65. Anal. calcd for
trations of tested compounds for 2 h before incubation with 0.1 lg/mL LPS for 24 h.
Proteins (15 lg) of total cell lysates were separated by 8% SDS–PAGE, p38 and p-p38
protein expression was detected by Western blot and analyzed by densitometry.
The relative density was calculated as the ratio of p-p38 expression to b-actin
expression. Data are presented as means + S.D.mean of 3 separate experiments
performed in triplicate. ⁄P<0.05 as compared with control. ⁄⁄P<0.01 as compared
with LPS alone.
C
17H14N2O3ꢂ0.2H2O: C 68.53, H 4.87, N 9.40; found: C 68.32, H
154.97, 157.90, 175.55, 181.31. Anal. calcd for C17H13NO3: C 73.11,
H 4.69, N 5.02; found: C 72.92, H 4.81, N 5.05.
4.82, N 9.16.
4.1.7. (Z)-1-(Hydroxyimino)-4-(4-
4.1.3. 4-Phenoxynaphthalene-1,2-dione (6a)
methoxyphenoxy)naphthalen-2(1H)-one (8b)
From 6b as described for 7b, to give of 8b a yellow solid (0.08 g,
To a solution of 4 (1.30 g, 5.0 mmol) in water (150 mL) was
added phenol (0.47 g, 5.5 mmol) and potassium hydroxide
(0.28 g, 5.0 mmol). The reaction mixture was stirred at room tem-
perature for 20 min (TLC monitoring). The solvent was removed in
vacuo and the resulting residue was crystallized from EtOH to give
0.62 g (48%) of 6a as a yellow solid. Mp: 163–165 °C. UV kmax nm
15% yield). Mp: 108–110 °C. UV kmax nm (log
e): 429 (3.51), 278
(4.17), 225 (4.34), 204 (3.93) in MeOH. IR (KBr pellets) cmꢁ1
:
3438, 1504, 1213. 1H NMR (400 MHz, CDCl3): 3.84 (s, 3H, OMe),
6.93–6.96 (m, 2H, Ar-H),7.00–7.06 (m, 2H, Ar-H), 7.26 (s, 1H, 3-
H), 7.68–7.82 (m, 2H, 6- and 7-H), 8.30 (d, 1H, J = 8.0 Hz, 5-H),
8.55 (d, 1H, J = 8.0 Hz, 8-H), 12.11 (s, 1H, NOH). 13C NMR
(100 MHz, CDCl3): 55.68, 102.61, 115.23 (2C), 120.84 (2C),
122.54, 125.25, 127.11, 127.91, 131.17, 131.47, 147.88, 149.44,
(loge): 401 (3.24), 251 (4.38), 218 (4.47) in MeOH. IR (KBr pellets)
cmꢁ1: 1646, 1564, 1363, 1231. 1H NMR (400 MHz, CDCl3): 5.67 (s,
1H, 3-H), 7.15–7.19 (m, 2H, Ar-H), 7.34–7.38 (m, 1H, Ar-H), 7.47–
7.52 (m, 2H, Ar-H), 7.67 (td, 1H, J = 7.6, 1.2 Hz, 7-H), 7.79 (td, 1H,
J = 7.6, 1.6 Hz, 6-H), 8.10 (dd, 1H, J = 7.6, 1.2 Hz, 5-H), 8.18 (dd,
1H, J = 7.6, 1.6 Hz, 8-H). 13C NMR (100 MHz, CDCl3): 106.40,
121.28 (2C), 124.90, 126.95, 129.41, 130.39 (2C), 130.49, 131.63,
131.91, 135.12, 152.33, 168.81, 179.29, 179.55. Anal. calcd for
151.90, 156.50, 178.35. Anal. calcd for
66.70, H 4.68, N 4.58; found: C 66.92, H 5.05, N 4.63.
C
17H13NO4ꢂ0.6H2O:
C
4.2. Biological testing
C16H10O3ꢂ0.3H2O: C 75.17, H 4.18; found: C 75.16, H 4.19.
4.2.1. Cell culture
Raw 264.7 cells, purchased from Food Industry Research and
Development Institute, Taiwan, were cultured in DMEM, supple-
mented with 5% fetal bovine serum, 100 units/mL of penicillin,
4.1.4. 4-(4-Methoxyphenoxy)naphthalene-1,2-dione (6b)
From 4 as described for 6a, to give 0.64 g (46%) of 6b as a yellow
solid. Mp: 174–175 °C. UV kmax nm (loge): 338 (3.46), 250 (4.39),
224 (4.55) in MeOH. IR (KBr pellets) cmꢁ1: 1649, 1563, 1363,
1193. 1H NMR (400 MHz, CDCl3): 3.84 (s, 3H, OMe), 5.67 (s, 1H,
3-H), 6.95–6.99 (m, 2H, Ar-H), 7.05–7.09 (m, 2H, Ar-H), 7.65 (td,
1H, J = 7.6, 1.2 Hz, 7-H), 7.77 (td, 1H, J = 7.6, 1.2 Hz, 6-H), 8.08
(dd, 1H, J = 8.0, 1.2 Hz, 8-H), 8.16 (dd, 1H, J = 7.6, 1.2 Hz, 5-H). 13C
NMR (100 MHz, CDCl3): 55.74, 106.24, 115.28(2C), 122.10 (2C),
124.89 (2C), 129.36, 130.47, 131.69, 131.85, 135.10, 145.67,
158.06, 169.28, 179.40, 179.56. Anal. calcd for C17H12O4: C 72.84,
H 4.32; found: C 72.84, H 4.34.
100 lg/mL of streptomycin, 2 mM L-glutamine, and 1 mM non-
essential amino acids in a 10-cm plate at a density of 1 ꢀ 106
cells/mL, at 37 °C in a humidified atmosphere containing 5% CO2.
4.2.2. Nitrite and TNF-
Nitrite was measured by adding 100
(1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihy-
drochloride in 5% phosphoric acid) to 100 L of medium for
a measurement
l
L of the Griess reagent27
l
5 min. The optical density at 550 nm (OD550) was measured with
a microplate reader. Concentrations were calculated by compari-
son with the OD550 of a standard solution of sodium nitrite pre-
pared in culture medium. The levels of TNF-a28 in the RAW264.7
cell culture medium were measured by Elisa assay kits according
to the manufacturer’s instructions (R&D).
4.1.5. (Z)-1-(Hydroxyimino)-4-(phenylamino)naphthalen-
2(1H)-one (7a)
A mixture of 5a (0.50 g, 2.0 mmol), hydroxylamineꢂHCl (0.16 g,
2.2 mmol), potassium carbonate (0.56 g, 4.0 mmol), and 2-ethoxy-

530
C.-H. Tseng et al. / Bioorg. Med. Chem. 21 (2013) 523–531
4.2.3. Cell viability
The gel was then examined and band intensities were measured
using a spectrophotometer (Lab work) and normalized against
GAPDH.
To evaluate whether the suppressive effect of b-lapachone
derivatives on nitrite production is related to cell viability. Raw
264.7 cells were treated with LPS for 24 h, and washed with
4.2.7. Gelatin zymography
Metalloproteinase activity was assayed by gelatin zymography.
Raw 264.7 cells were plated in 10 cm culture for 16–18 h before
500 lL of phosphate buffer solution (PBS) before resuspended in
1 mL of culture medium. Cell viability was assessed by the MTS29
staining method. Briefly, cells at 5 ꢀ 104 cells/well were seeded
into 96-well microtiter plates and treated with various reagents
for 24 h. Twenty microliters of MTS solution was added to the cells
which were then incubated at 37 °C for 1 h. Color was measured
spectrophotometrically in a microtiter plate reader at 490 nm
and used as a relative measure of viable cell number. The number
of viable cells following treatment was compared to solvent and
untreated control cells and used to determine the percent of con-
trol growth as (Abtreated/Abcontrol)ꢀ100, where Ab represents the
mean absorbance (n = 3). The concentration that killed 50% of cells
(IC50) was determined from the linear portion of the curve by cal-
culating the concentration of agent that reduced absorbance in
treated cells, compared to control cells, by 50%.
treatmentin. Cells were treated with or without LPS (0.1
lg/ml)
and b-LAPA, compounds 6b (0.5, 1.0, 2.0 M), and culture medium
l
was collected 24 h after initiation of the treatment. The medium
was concentrated using an Amicon Ultra 4 (Millipore). Equal
amount of concentrated medium was fractionated by SDS–PAGE
on gelatin gel (7.5% acrylamide,2 mg/ml gelatin, Sigma). Separated
protein was renatured by incubating the gel for 1 h with 2.5% Tri-
ton-X100, and then with renaturing buffer (40 mM Tris, pH 7.5,
200 mM NaCl, 10 mM CaCl2) overnight at 37 °C. Finally, the gel
was incubated for 16–18 h with gentle shaking at room tempera-
ture with a developing buffer containing 40 mM Tris pH 7.5,
200 mM NaCl, 10 mM CaCl2. Gels were stained in 0.1% coomassie-
blue (R250, Sigma) for 1 h and destained until the bands were
evident.32
4.2.4. NO-scavenging activity
To understand the inhibitory effects of compounds 6a, 6b and
8b on NO production, sodium nitroprusside (SNP)24 was freshly
prepared at 5 mM in PBS was incubated alone or in combination
4.2.8. Immunofluorescence staining and confocal laser
scanning microscopy study
The NF-jB p65 nuclear localization was detected by indirect
immunofluorescence assays using confocal microscopy. Raw
with 10 lM of the different compounds. SNP is an inorganic com-
plex where NO is found as NO+ and light irradiation is necessary for
the release of NO. Therefore, incubation mixtures were incubated
in light at room temperature, and nitrite levels were determined
after 60 min by Griess reaction.
264.7 cells were cultured directly on glass coverslips in 24-well
plates for 24 h. After stimulation with 0.1
0.5 M of b-LAPA or 6b, the cells were fixed with 4% paraformalde-
hyde in PBS, permeabilized with 0.2% triton X-100 in PBS. Poly-
clonal antibodies against anti-NF- B p65 (Santa Cruz) (1 g/well)
lg/mL of LPS and/or
l
j
l
4.2.5. Western blotting
were applied for 24 h, followed by an 1 h incubation with FITC-
conjugated anti-mouse Alexa fluor 568 (Invitrogen). The position
of the cell nucleus was determined with DAPI. After washing with
PBS, the coverslips were mounted with Mounting medium (DAKO),
and the fluorescence was visualized using an Olympus FluorViewÒ
FV100 laser scanning confocal microscope.33
Raw 264.7 cells were pretreated with tested compound for 2 h
before stimulation in the presence or absence of LPS (0.1 lg/mL).
Cell were harvest and lysed with RIPA lysis buffer(50 mM Tris–Cl,
pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% NP-40, 1% sodiumdeoxycho-
late, 1 mM phenylmethyl-sulfonylfluoride, 1 mM EDTA, no added
proteinase) 24 h after treatment. The total protein in the cell ly-
sates was assayed according to the method described by Brad-
ford.30 Equal amounts (20–50
lg) of protein were separated
4.3. Statistical analysis
using 8–15% SDS–PAGE. Proteins were transferred to Immobilon
PVDF membranes (Millipore) and subsequently blocked in 5% bo-
vine serum albumin (BSA)–Tris-Buffered Saline Tween (TBST,
100 mM Tris, pH 8.0, 150 mM NaCl and 0.1% Tween 20) for 1 h at
room temperature. Specific antibodies against iNOS, COX-2, p38
and p-p38 were diluted in 5% BSA–TBST. After incubation with
the appropriate primary antibody, membranes were incubated
for 1 h at room temperature with a secondary antibody conjugated
to horseradish peroxidase (1:10,000 dilution; Santa Cruz Biotech-
nology, Santa Cruz, CA). Following three washes in TBST, immuno-
reactive bands were visualized using the ECL detection system (ECL
Amersham).
The results were expressed as means S.D. Differences in mean
values between groups were analyzed by a one-way analysis of
variance (ANOVA) and Student’s t-test. For the septic shock assay,
we used the log-rank test. Statistical significance was assessed as p
<0.05 [⁄p <0.05; ⁄⁄p <0.01].
Acknowledgments
Financial support of this work by the National Science Council of
the Republic of China (NSC 99-2320-B-037-0-18-MY2) is gratefully
acknowledged. We also thank the National Center for High-Perfor-
mance Computing for providing computer resources and chemical
database services.
4.2.6. Isolation of total RNA and RT-PCR
Raw 264.7 cells treated with LPS (0.1 lg/mL) in the presence or
References and notes
absence of drug were homogenized with 1 mL of Trizol reagent
(InvitrogenTM), and total RNA was isolated according to the man-
ufacturer’s protocol. One microgram of RNA was used for the re-
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primers. Five microliters of cDNA were used for the amplification
of iNOS and GAPDH (glyceraldehyde 3-phosphate dehydrogenase)
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AGG CTG AGA-30 reverse:50-AGA CGC CAG TAG ACT CCA CGA C-
3030. The amplicons were electrophoresed on a 3% agarose gel.
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