Synthesis and biological evaluations of novel apocynin analogues

Article abstract of DOI:10.1016/j.ejmech.2011.03.056

We have designed and synthesized a series of novel apocynin analogues, and evaluated their biological activity. Compound 10, an apocynin dimer analogue, compound 12, the lipoic acid (LA) and apocynin conjugate, were the most potent in protecting cells from lipopolysaccharide (LPS)-induced cytotoxicity, had significant activity scavenging ROS induced by LPS, and greatly decreased LPS-induced P67^phox protein expression. SAR analysis suggests that modification of apocynin can increase its activity. Our results demonstrate that arming apocynin with a powerful antioxidant such as lipoic acid is a valid strategy to design new apocynin analogues with enhanced biological activity.

Full text of DOI:10.1016/j.ejmech.2011.03.056

European Journal of Medicinal Chemistry 46 (2011) 2691e2698
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluations of novel apocynin analogues
,
,
,
b
,
a
a
a
,
,b
Xiaoyu Lu a 1, Sainan Wan a 1, Jie Jiang a , Xiaojian Jiang , Wenjing Yang , Pei Yu b, Lipeng Xu a
,
*
Zaijun Zhang a b, Gaoxiao Zhang a b, Luchen Shan a b, Yuqiang Wang a
,
,
,
,b,
*
a Institute of New Drug Research, College of Pharmacy, Jinan University, Guangzhou 510632, China
b Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drug Research, Guangzhou 510632, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed and synthesized a series of novel apocynin analogues, and evaluated their biological
activity. Compound 10, an apocynin dimer analogue, compound 12, the lipoic acid (LA) and apocynin
conjugate, were the most potent in protecting cells from lipopolysaccharide (LPS)-induced cytotoxicity,
had significant activity scavenging ROS induced by LPS, and greatly decreased LPS-induced P67phox
protein expression. SAR analysis suggests that modification of apocynin can increase its activity. Our
results demonstrate that arming apocynin with a powerful antioxidant such as lipoic acid is a valid
strategy to design new apocynin analogues with enhanced biological activity.
Received 16 November 2010
Received in revised form
21 March 2011
Accepted 25 March 2011
Available online 3 April 2011
Keywords:
Apocynin
NADPH oxidase
ROS
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antioxidant
1. Introduction
to its structureeactivity relationship (SAR). To uncover its SAR,
better understand mechanism(s) of action and find more potent
Apocynin (4-hydroxy-3-methoxyacetophenone, Fig. 1), a methox-
ycatechol first isolated from the roots of Apocynum cannabinum
(Canadian hemp) in 1883, was originally used as remedies for dropsy
and heart illness [1]. Recent investigations have found that apocynin is
an efficient inhibitor of NADPH oxidase [2e5]. Apocynin has been
investigated as a therapeutic candidate for inflammation-mediated
diseases, including jaundice, asthma [6,7] and cardiovascular
diseases [8].
NADPH oxidase inhibitors, we have designed and synthesized new
apocynin analogues (Fig. 2) and investigated their preliminary
biological activities in vitro and in cell cultures.
In this work, we first modified apocynin’s 5-position to
synthesize apocynin amides and amines, i.e., compounds 3, 6 and 8.
Since apocynin was converted to its dimer, we then synthesized an
apocynin dimer, i.e., compound 10 to help understand its mecha-
nism of action.
Although apocynin has been widely used as an inhibitor of
NADPH oxidase; the exact mechanism by which it inhibits NADPH
oxidase is still not well understood. Apocynin was found to be
metabolized into its corresponding dimer [9], trimer [10] or even
oligomer, which were considered to be the actual active component
inhibiting NADPH oxidase by disrupting the interaction of p47phox
with p22phox [7,9,10]. However, Heumueller et al. proposed
a different mechanism that apocynin is not an inhibitor of vascular
NADPH oxidase, but an antioxidant [11].
In order to increase antioxidative activity, we coupled two of the
most powerful antioxidants, i.e., a nitrone and a-lipoic acid (LA) to
apocynin to make compounds 11 and 12. Nitrones have been
widely used as spin traps in chemistry. In the last two decades, the
therapeutic effects of nitrones against atherosclerosis, septicemia,
stroke, and Alzheimer’s disease have been intensively investigated
[12,13]. Organic molecules incorporating a nitrone moiety act as
reactive oxygen species (ROS) scavenger against oxidative chal-
lenges. LA is biogenic and one of the most powerful antioxidants
known. LA directly terminates free radicals, chelates transition
metal ions (iron and copper), and increases cytosolic glutathione
and vitamin C levels [14,15]. These diverse actions suggest that LA
acts by multiple mechanisms both physiologically and pharmaco-
logically [16]. LA has been successfully employed in the treatment
of diabetic polyneuropathy for decades [14,17].
The biological effects and mechanism of action of apocynin are
extensively investigated; however, few reports are seen regarding
* Corresponding authors. Institute of New Drug Research, College of Pharmacy,
Jinan University, Guangzhou 510632, China. Tel.: þ86 20 3837 5006; fax: þ86 20
8522 4766.
E-mail addresses: jiejiang2008@gmail.com (J. Jiang), yuqiangwang2001@yahoo.
com (Y. Wang).
In order to evaluate the biological activity of the apocynin
analogues, we first investigated their protective activity against
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.056

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X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 2691e2698
O
CH3
O
CH3
H3CO
R2
H3CO
OR1
OH
R1
R2
Fig. 1. Structure of apocynin.
3a
3b
6a
6b
6c
8a
8b
8c
H
H
CH3(CH2)2CH2NHCH2
(CH3)3CNHCH2
LPS-induced cytotoxicity by the MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assay. We then investigated
the ROS-scavenging activity of compounds 10, 11 and 12 in RAW
264.7 cells by a fluorescence-based assay. Finally, we determined
their inhibitory activity against LPS-induced P67phox expression.
We herein report our experimental results.
CH3CH2CO
CH3CH2CONH
CH3CH2CH2CO
CH3CH2CH2CO NH
(CH3)2CHCONH
CH3CH2CONH
(CH3)2CHCO
H
H
H
2. Chemistry
CH3CH2CH2CO NH
(CH3)2CHCONH
We first synthesized apocynin amides and amines, 3a, 3b, 6ae6c
and 8aec (Scheme 1), to find their effects on biological activity.
Briefly, compound 2 was prepared by the ReimereTiemann reaction.
Butylamine and tert-butylamine were then conjugated with
compound 2, respectively, to afford the corresponding imines, which
were reducedimmediatelytoproducethemorestableamines 3aand
3b. Compound 1 was nitrated to give a nitro 4. Catalytic hydroge-
nation of the latter over 10% Pd/C in absolute ethanol afforded amine
5. Direct condensation of 5 with corresponding carboxylic acids
produced compounds 6aec. Protection of the hydroxyl group of 5 by
TBDMSeCl in dichloromethane gave compound 7. Amidation of 7
with various acid anhydrides in THF catalyzed by DMAP yielded the
corresponding amides, which were then treated with TBAF in THF
removing TBDMS to produce compounds 8aec.
O
CH3
10
11
12
H
H
H
HNH2C
OCH3
OH
O
HC
N
O
HN
S
S
Fig. 2. Structure of apocynin amides and amines.
Next, we synthesized an apocynin dimer 10. The latter was
structurally homologous to diapocynin, reported by Johnson et al.
to be the real active component in peroxidization process in vivo.
The synthesis of compound 10 is shown in Scheme 2. Treatment of
compound 2 with 5 produced Schiff base 9, which was reduced
using sodium borohydride afforded the target compound 10.
We then synthesized compounds 11 and 12. Both of them are
armed with a powerful antioxidative moiety, i.e., nitrone or LA. The
synthesis of 11 is shown in Scheme 3. Compound 2 was reacted
with N-tert-butylhydroxylamine, prepared on the day of use by
reduction of 2-methyl-2-nitropropane catalyzed by activated zinc,
to afford 11.
The synthesis of 12 is shown in Scheme 4. Amidation of
compound 7 with LA in THF catalyzed by EDCI and HOBT followed
by treatment with TBAF in THF afforded target 12.
O
CH3
O
CH3
O
CH3
O
CH3
H2, 10% Pd/C
EtOH
Anhydrides, DMAP
MeOH
65% HNO3, AcOH
75%
O
85%
85-87%
H3CO
H3CO
NO2
H3CO
N
R
H3CO
NH2
H
O
OH
4
O
R
OH
1
OH
5
6a: R = CH3CH2
i. 50% NaOH aqua, ethanol
ii. CHCl3, reflux
iii. HCl
6b: R = CH3CH2CH2
6c: R = (CH3)2CH
TBDMS-Cl, Imidazole
CH2Cl2
35%
O
93%
O
CH3
O
CH3
CH3
O
CH3
i. RNH2, MeOH
i. Anhydrides, DMAP, MeOH
ii. TBAF, THF
ii. NaBH4, MeOH
O
H
N
50-51%
CHO
42-45%
H3CO
H3CO
R
H3CO
NH2
OTBDMS
H3CO
N
R
H
OH
OH
OH
2
3a. R = CH3CH2CH2CH2
3b. R = (CH3)3C
8a: R = CH3CH2
7
8b: R = CH3CH2CH2
8c: R = (CH3)2CH
Scheme 1. Synthesis of apocynin amides and amines.

X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 2691e2698
O CH3
2693
O
CH3
O
CH3
O
CH3
O
CH3
O
CH3
EtOH, reflux
65%
NaB H4, MeOH
58%
H3CO
CHO H3CO
NH2
H3CO
N
OCH3
H3CO
N
OCH3
H
OH
2
OH
5
OH
OH
OH
OH
9
10
Scheme 2. Synthesis of compound 10.
O
CH3
O
CH3
3.2. Intracellular ROS-scavenging activity
C(CH3)3NHOH
EtOH, reflux
Next, we investigated the ROS-scavenging activity of
compounds 10e12 in RAW 264.7 cells by a fluorescence-based
assay (Fig. 4). RAW 264.7 cells were labeled with the fluorescence
probe 20,70-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA
enters cells and its DCFH moiety interacts with ROS to emit fluo-
rescence [24].
O
74%
H3CO
CHO
H3CO
C
N
H
OH
2
OH
11
Scheme 3. Synthesis of compound 11.
In the absence of any drug, cells treated with LPS showed strong
presence of ROS. At the tested concentrations (0.1, 1 and 10 mM),
apocynin (Apo) and compound 11 had minimum activity scav-
enging LPS-induced ROS. Compounds 10 and 12 dose-dependently
3. Biological evaluations and discussion
3.1. Protective effects against LPS-induced cytotoxicity
reduced ROS level. Recent studies have shown that both
a-lipoic
acid and apocynin can reduce intracellular reactive oxygen species
To determine the new compounds’ biological activities, we first
investigated their protective effect against LPS-induced cytotoxicity
by the MTT assay in RAW 264.7 macrophage cells by the procedure
described by Kim and Ha [18]. RAW 264.7 cells were cultured and
treated with various drugs for 1 h prior to addition of LPS. After 24 h
of incubation, cell viability was determined. The results are shown
in Fig. 3. Apocynin (Apo) had minimum protective effects against
LPS-induced cytotoxicity in this assay, and the result was consistent
with what had been reported in the literature [19e21]. The reason
may be that it needs to be converted into its active metabolites after
a peroxidation process in vivo [9].
The LA substituted analogue 12 is the most potent compound in
this assay. This finding confirmed that an antioxidant moiety could
ameliorate the LPS-induced cell damage (Fig. 3). The apocynin
dimer 10 also had strong protective effect against the LPS-induced
cell damage. Interestingly, the structure of compound 10 is analo-
gous to diapocynin (5,50-dehydrodiacetovanillone), which was
synthesized by oxidative coupling of apocynin [22]. Diapocynin is
a metabolite of apocynin, which has anti-inflammatory and anti-
oxidative properties. In LPS-stimulated PBMC cells diapocynin was
more effective than apocynin [23]. Further studies are needed to
confirm if apocynin dimer is the active form. These findings indi-
cate that compounds 10 and 12 may increase apocynin’s protective
effect in this assay.
[25,26]. Diapocynin can inhibit inflammatory factors such as TNF-a,
IL-10, and gp91phox mRNA expression in LPS-induced PBMC cells.
Compound 10 is analogous to diapocynin. It will be interesting to
find out if compound 10 and diapocynin work by the same
mechanisms.
3.3. Inhibition of LPS-induced P67phox protein expression
Macrophage RAW 264.7 cells were pre-treated with 0.1e10
apocynin (Apo), compounds 10e12 for 1 h, and were then stimu-
lated with LPS (1
g/ml) for 24 h. Expression of P67phox protein was
mM
m
detected by Western blot analysis (Fig. 5). As is shown in Fig. 5, LPS
induced considerable expression of P67phox proteins. Apo and its
nitrone analogue 11 did not reduce P67phox protein expression at
concentrations up to 10
mM. Others reported that Apo at 100
mM
O
CH3
O
CH3
i. LA, EDCI, HOBT
ii. TBAF, THF
O
51%
H3CO
N
H3CO
NH2
H
S
OTBDMS
7
OH
S
12
Scheme 4. Synthesis of compound 12.
120
100
80
60
40
20
0
Con
LPS
0.1 μΜ
1
10 μΜ
100 μΜ
*
*
μΜ
*
Con LPS Apo
3a
3b
6a
6b
6c
8a
8b
8c
10
11
12
Compouds
Fig. 3. Protective effects of Apo and its analogues against LPS-induced cytotoxicity in RAW 264.7 cells.

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X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 2691e2698
Fig. 4. ROS-scavenging effect of Apo and compounds 10e12 in RAW 264.7 cells.
effectively decreased P67phox expression in leptin-induced car-
diomyocytes and completely suppressed NADPH oxidase at 1 mM
[27e29]. Both the dimer 10 and the LA analogue 12 significantly
inhibited expression of P67phox protein dose-dependently from 0.1
oxidase subunits in diabetic rats can be reduced by a-lipoic acid
[30]. Kanegae et al. reported that diapocynin inhibited NADPH
oxidase more effectively than apocynin through gp91phox mRNA
expression [22]. The results demonstrated that compounds 10 and
12 are effective NADPH oxidase inhibitors.
to 10 mM. Bhatti et al. reported that kidney expression of NADPH
Fig. 5. Inhibition effect of Apo and compounds 10e12 against LPS-induced P67phox protein expression.

X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 2691e2698
2695
4. Conclusions
5.3.1. 1-(3-((Butylamino)methyl)-4-hydroxy-5-methoxyphenyl)
ethanone (3a)
In this study, we have synthesized a series of novel apocynin
analogues. Their biological activity was first evaluated by the MTT
assay against LPS-induced cytotoxicity. Among the new
compounds, the dimer 10 and the LA-substituent 12 were the most
potent protecting cells from LPS-induced cytotoxicity. Apocynin
was found to have minimum activity. Compounds 10 and 12
significantly reduced LPS-induced ROS production at a concentra-
Synthesized from
2
(200 mg, 1.03 mmol) and butylamine
(150 mg, 2.06 mmol). Brown solid (131 mg, 51% yield), mp:
161e163 ꢀC. 1H NMR (CDCl3, 400 MHz): 7.44 (d, 1H, J ¼ 1.6 Hz, ArH),
7.29 (d, 1H, J ¼ 1.6 Hz, ArH), 4.07 (s, 2H, ArCH2N), 3.93 (s, 3H, OCH3),
2.70e2.68 (t, 2H, J ¼ 7.1 Hz, NCH2), 2.53 (s, 3H, CH3CO), 1.56e1.51
(m, 2H, CH3CH2CH2CH2), 1.41e1.33 (m, 2H, CH3CH2CH2CH2),
0.93e0.90 (t, 3H, J ¼ 7.3 Hz, CH3CH2). MS (ESI) m/z 250 [M ꢂ H]þ,
252 [M þ H]þ. Analysis calculated for C14H21NO3$0.67H2O: C, 63.85;
H, 8.55; N, 5.32. Found: C, 63.68; H, 8.18; N, 5.11.
tion as low as 0.1 mM in RAW 264.7 cells. Compounds 10 and 12
greatly inhibited LPS-induced P67phox protein expression. These
results seem to support the notion that apocynin needs to be
converted to a dimer to be active [9,11]. Our results demonstrate
that coupling a powerful antioxidant moiety such as LA to apocynin
is a valid strategy for improving apocynin’s biological activity.
5.3.2. 1-(3-((tert-Butylamino)methyl)-4-hydroxy-5-methoxy
phenyl)ethanone (3b)
Synthesized from 2 (200 mg, 1.03 mmol) and tert-butylamine
(150 mg, 2.06 mmol). Brown solid (128 mg, 50% yield), mp:
176e178 ꢀC. 1H NMR (CDCl3, 400 MHz): 7.42 (d, 1H, J ¼ 1.6 Hz, ArH),
7.30 (d, 1H, J ¼ 1.6 Hz, ArH), 4.03 (s, 2H, ArCH2N), 3.92 (s, 3H, OCH3),
2.53 (s, 3H, CH3CO),1.23 (s, 9H, (CH3)3C). MS (ESI) m/z 250 [M ꢂ H]þ,
252 [M þ H]þ. Analysis calculated for C14H21NO3: C, 66.91; H, 8.42;
N, 5.57. Found: C, 66.83; H, 8.83; N, 5.67.
5. Experimental protocols
5.1. General methods
All chemicals (reagent grade) used were commercially available.
Melting points were measured using a Mel-Temp (X7L20, Beijing),
and are uncorrected. NMR spectra were recorded at ambient
temperature on a 400 MHz spectrometer (AV-400, Bruker) in
CDCl3, CD3OD or DMSO-d6. Electrospray ionization mass spectra
(ESI-MS) were obtained in the positive ion detection mode on
a Finnigan LCQ Advantage MAX mass spectrometer (Applied Bio-
systems, 4000 Q TRAP). Elemental analysis was performed at the
Jinan University Experimental Center.
5.4. 1-(4-Hydroxy-3-methoxy-5-nitrophenyl)ethanone (4)
Apocynin 1 (20 g, 120 mmol), dissolved in glacial acetic acid
(300 ml), was cooled to 0 ꢀC in an ice bath with stirring on. Nitric
acid (65%, 14 ml, 224 mmol) was then added slowly through
a dropping funnel. The reaction mixture was maintained at room
temperature for 2 h and was then poured into ice water (250 ml)
with stirring on for 10 min. After 30 min of standing, the reaction
mixture was filtered through a Buchner funnel and the residue was
washed with water, which was recrystallized in 95% ethanol to
afford 19.1 g (75% yield) of 4 as a yellow needle crystal, mp:
159e161 ꢀC. 1H NMR (CDCl3, 400 MHz): 11.13 (s, 1H, OH), 8.31 (d,
1H, J ¼ 1.6 Hz, ArH), 7.77e7.76 (d, 1H, J ¼ 1.6 Hz, ArH), 4.01 (s, 3H,
OCH3), 2.63 (s, 3H, CH3CO). MS (ESI) m/z 212 [M þ H]þ. Analysis
calculated for C9H9NO5$0.2H2O: C, 50.33; H, 4.41; N, 6.52. Found: C,
50.63; H, 4.23; N, 6.26.
5.2. 5-Acetyl-2-hydroxy-3-methoxybenzaldehyde (2)
To apocynin 1 (5 g, 30.1 mmol) and triethylamine (3 ml) in
ethanol (50 ml), was added a solution of sodium hydroxide (25 g) in
water rapidly. The resulting solution was heated to 70 ꢀC on a steam
bath. Chloroform (39 g, 300 mmol) was added dropwise. Stirring
was continued for 2 h after all the chloroform had been added. After
cooling to room temperature, the reaction mixture was acidified by
hydrochloric acid to pH ¼ 1. The chloroform phase was collected,
and the aqueous phase was extracted by dichloromethane
(20 ml ꢁ 3). The mixture of organic phase was washed with water
(10 ml ꢁ 3) and was then dried using anhydrous sodium sulfate.
Solvent was removed in vacuo and the product was purified by
column chromatography, eluting with ethyl acetate/petroleum
ether (2:3, v/v), to afford 2.2 g (35% yield) of 2 as a yellow powder,
mp: 156e157 ꢀC. 1H NMR (CDCl3, 400 MHz): 11.47 (s, 1H, OH), 10.00
(s, 1H, CHO), 7.83 (d, 1H, J ¼ 1.6 Hz, ArH), 7.74e7.73 (d, 1H, J ¼ 1.6 Hz,
ArH), 3.98 (s, 3H, OCH3), 2.62 (s, 3H, CH3CO). MS (ESI) m/z 193
[M ꢂ H]þ, 195 [M þ H]þ. Analysis calculated for C10H10O4$0.67H2O:
C, 58.25; H, 5.54. Found: C, 58.04; H, 5.78.
5.5. 1-(3-Amino-4-hydroxy-5-methoxyphenyl)ethanone (5)
To a solution of 4 (19.1 g, 90.0 mmol) in ethanol (300 ml) was
added 10% Pd/C (950 mg) and the reaction mixture was hydroge-
nated by hydrogen over night. The reaction mixture was filtered,
and the filtrate was washed with water (50 ml ꢁ 5). The organic
layer was dried by anhydrous sodium sulfate. Solvent was removed
in vacuo to afford 13.1 g (85% yield) of 5 as a brown powder, mp:
156e158 ꢀC. 1H NMR (CDCl3, 400 MHz): 7.06 (d, 1H, J ¼ 1.6 Hz, ArH),
7.03 (d, 1H, J ¼ 1.6 Hz, ArH), 5.90 (s, 1H, OH), 3.92 (s, 3H, OCH3), 2.52
(s, 3H, CH3CO). MS (ESI) m/z 180 [M ꢂ H]þ, 182 [M þ H]þ. Analysis
calculated for C9H11NO3$0.17H2O: C, 58.69; H, 6.20; N, 7.60. Found:
C, 58.98; H, 5.64; N, 7.57.
5.3. General procedure for synthesis of compounds 3a and 3b
Compound 2 (200 mg, 1.03 mmol) was first dissolved in meth-
anol (20 ml). A solution of alkylamine in methanol (5 ml) was
added dropwise and the reaction mixture was stirred at room
temperature for 1 h to afford a Schiff base intermediate. The reac-
tion mixture was then cooled to 0 ꢀC under an ice bath, and sodium
borohydride (13 mg, 0.34 mmol) was added in single portions with
vigorously stirred. Ice bath was removed and the reaction mixture
was stirred at room temperature for 2 h. Ice water (10 ml) was
added to dissolve the excess sodium borohydride. The reaction
mixture was extracted with dichloromethane (10 ml ꢁ 3) and the
organic layer was dried using anhydrous sodium sulfate. Solvent
was removed in vacuo to afford 3a and 3b.
5.6. General procedure for synthesis of compounds 6aec
To
a
solution of
5
(200 mg, 1.10 mmol), DMAP (134 mg,
l) in THF (20 ml) cooled to 0 ꢀC
1.10 mmol) and triethylamine (500
m
under nitrogen, was added a solution of excess acid anhydride in
THF (5 ml) dropwise. The reaction mixture was allowed to warm to
room temperature and stirred for 5 h. THF was removed in vacuo,
and ethyl acetate (20 ml) was added. The solution was washed with
water (5 ml ꢁ 3). The organic phase was dried using anhydrous
sodium sulfate, and the solvent was removed in vacuo. The residue
was purified by column chromatography, eluting with ethyl
acetate/petroleum ether (1:2, v/v), to afford 6aec.

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X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 2691e2698
5.6.1. 4-Acetyl-2-methoxy-6-propionamidophenyl propionate (6a)
Synthesized from 5 (200 mg, 1.10 mmol) and propionic anhy-
dride (715 mg, 5.50 mmol). White powder (274 mg, 85% yield), mp:
151e152 ꢀC. 1H NMR (DMSO-d6, 400 MHz): 9.42 (s, 1H, NH),
8.19e8.18 (d, 1H, J ¼ 1.6 Hz, ArH), 7.36 (d, 1H, J ¼ 1.6 Hz, ArH), 3.83
(s, 3H, OCH3), 2.67e2.62 (q, 2H, J ¼ 7.5 Hz, CH2CH3), 2.42e2.36 (q,
2H, J ¼ 7.5 Hz, CH2CH3), 1.18e1.14 (t, 3H, J ¼ 7.5 Hz, CH3CH2),
1.10e1.06 (t, 3H, J ¼ 7.5 Hz, CH3CH2). MS (ESI) m/z 292 [M ꢂ H]þ,
294 [M þ H]þ. Analysis calculated for C15H19NO5: C, 61.42; H, 6.53;
N, 4.78. Found: C, 61.31; H, 6.67; N, 4.55.
150e152 ꢀC. 1H NMR (DMSO-d6, 400 MHz): 10.03 (s, 1H, NH), 9.26
(s, 1H, OH), 8.10 (s, 1H, ArH), 7.30 (d, 1H, J ¼ 2.0 Hz, ArH), 3.87 (s, 3H,
OCH3), 2.49 (s, 3H, CH3CO), 2.45e2.40 (m, 2H, CH2CH3), 1.11e1.07 (t,
3H, J ¼ 7.5 Hz, CH2CH3). MS (ESI) m/z 236 [M ꢂ H]þ, 238 [M þ H]þ.
Analysis calculated for C12H15NO4: C, 60.75; H, 6.37; N, 5.90. Found:
C, 61.15; H, 6.80; N, 5.73.
5.8.2. N-(5-Acetyl-2-hydroxy-3-methoxyphenyl)butyramide (8b)
Synthesized from 7 (900 mg, 2.56 mmol) and butyric anhydride
(808 mg, 5.12 mmol). White powder (289 mg, 45% yield), mp:
200e202 ꢀC. 1H NMR (DMSO-d6, 400 MHz): 10.07 (s, 1H, NH), 9.31
(s, 1H, OH), 8.09 (s, 1H, ArH), 7.30 (d, 1H, J ¼ 2.0 Hz, ArH), 3.87 (s, 3H,
OCH3), 2.49 (s, 3H, CH3CO), 2.41e2.37 (t, 2H, J ¼ 7.3 Hz, CH2CH2CO),
1.66e1.57 (m, 2H, CH3CH2), 0.94e0.90 (t, 3H, J ¼ 7.4 Hz, CH3CH2).
MS (ESI) m/z 250 [M ꢂ H]þ, 252 [M þ H]þ. Analysis calculated for
C13H17NO4$0.25H2O: C, 61.04; H, 6.90; N, 5.48. Found: C, 61.07; H,
6.96; N, 5.88.
5.6.2. 4-Acetyl-2-butyramido-6-methoxyphenyl butyrate (6b)
Synthesized from 5 (200 mg, 1.10 mmol) and butyric anhydride
(870 mg, 5.50 mmol). White powder (307 mg, 87% yield), mp:
138e139 ꢀC. 1H NMR (DMSO-d6, 400 MHz): 9.43 (s, 1H, NH),
8.14e8.13 (d, 1H, J ¼ 1.6 Hz, ArH), 7.37 (d, 1H, J ¼ 1.6 Hz, ArH), 3.83
(s, 3H, OCH3), 2.62e2.58 (t, 2H, J ¼ 7.3 Hz, CH2CO), 2.37e2.33 (t, 2H,
J ¼ 7.3 Hz, CH2CO), 1.73e1.64 (m, 2H, CH3CH2), 1.64e1.55 (m, 2H,
CH3CH2), 1.01e0.97 (t, 3H, J ¼ 7.4 Hz, CH3CH2), 0.93e0.89 (t, 3H,
J ¼ 7.4 Hz, CH3CH2). MS (ESI) m/z 320 [M ꢂ H]þ, 322 [M þ H]þ.
Analysis calculated for C17H23NO5: C, 63.54; H, 7.21; N, 4.36. Found:
C, 63.36; H, 7.47; N, 4.23.
5.8.3. N-(5-Acetyl-2-hydroxy-3-methoxyphenyl)isobutyramide
(8c)
Synthesized from 7 (900 mg, 2.56 mmol) and isobutyric anhy-
dride (808 mg, 5.12 mmol). White powder (275 mg, 43% yield), mp:
135e136 ꢀC. 1H NMR (DMSO-d6, 400 MHz): 10.05 (s, 1H, NH), 9.23
(s, 1H, OH), 8.09 (d, 1H, J ¼ 1.6 Hz, ArH), 7.31e7.30 (d, 1H, J ¼ 1.6 Hz,
ArH), 3.87 (s, 3H, OCH3), 2.85e2.74 (m, 1H, (CH3)2CH), 2.49 (s, 3H,
CH3CO), 1.12e1.10 (d, 6H, J ¼ 6.8 Hz, (CH3)2CH). MS (ESI) m/z
250 [M ꢂ H]þ, 252 [M þ H]þ. Analysis calculated for C13H17
NO4$0.25H2O: C, 61.04; H, 6.90; N, 5.48. Found: C, 61.18; H, 6.98; N,
5.76.
5.6.3. 4-Acetyl-2-isobutyramido-6-methoxyphenyl isobutyrate (6c)
Synthesized from 5 (200 mg, 1.10 mmol) and isobutyric anhy-
dride (870 mg, 5.50 mmol). White powder (302 mg, 85% yield), mp:
160e162 ꢀC. 1H NMR (DMSO-d6, 400 MHz): 9.37 (s, 1H, NH),
8.02e8.01 (d, 1H, J ¼ 1.6 Hz, ArH), 7.39e7.38 (d, 1H, J ¼ 1.6 Hz, ArH),
3.83 (s, 3H, OCH3), 2.93e2.83 (m, 1H, (CH3)2CH), 2.76e2.66 (m, 1H,
(CH3)2CH), 2.58 (s, 3H, CH3CO), 1.26e1.24 (d, 6H, J ¼ 7.0 Hz,
(CH3)2CH), 1.10e1.08 (d, 6H, J ¼ 6.8 Hz, (CH3)2CH). MS (ESI) m/z 320
[M ꢂ H]þ, 322 [M þ H]þ. Analysis calculated for C17H23NO5: C,
63.54; H, 7.21; N, 4.36. Found: C, 63.26; H, 7.37; N, 4.21.
5.9. (Z)-1-(3-(5-Acetyl-2-hydroxy-3-methoxybenzylidene amino)-
4-hydroxy-5-methoxyphenyl)ethanone (9)
A solution of 2 (200 mg, 1.03 mmol) and 5 (190 mg, 1.03 mmol)
in ethanol was refluxed in an oil bath over night. Solvent was
removed in vacuo and the product was purified by column chro-
matography, eluting with ethyl acetate/methanol (10:1, v/v), to
afford 210 mg (60% yield) of a red powder 9, mp: 195e196 ꢀC. 1H
NMR (CDCl3, 400 MHz): 8.96 (s, 1H, ArCHN), 7.74 (s, 1H, ArH), 7.62
(s, 1H, ArH), 7.60 (s, 1H, ArH), 7.49 (s, 1H, ArH), 4.03 (s, 3H, OCH3),
4.01 (s, 3H, OCH3), 2.63 (s, 3H, CH3CO), 2.63 (s, 3H, CH3CO). MS(ESI)
m/z 356 [M ꢂ H]þ. Analysis calculated for C19H19NO6$1.75H2O: C,
58.68; H, 5.83; N, 3.60. Found: C, 58.85; H, 6.06; N, 3.57.
5.7. 1-(3-Amino-4-(tert-butyldimethylsilyloxy)-5-methoxy phenyl)
ethanone (7)
To a solution of 5 (500 mg, 2.76 mmol) in dichloromethane
(50 ml) cooled to 0e10 ꢀC in an ice bath, was added TBDMSeCl
(830 mg, 5.52 mmol) and imidazole (373 mg, 5.52 mmol). The
reaction mixture was stirred at room temperature for 3 h under
nitrogen. The reaction mixture was washed with water (10 ml ꢁ 3).
The organic layer was dried using anhydrous sodium sulfate.
Solvent was removed in vacuo to afford 900 mg (93% yield) of
a while power 7, mp: 112e114 ꢀC. 1H NMR (CDCl3, 400 MHz): 7.03
(d, 1H, J ¼ 1.6 Hz, ArH), 6.99e6.98 (d, 1H, J ¼ 1.6 Hz, ArH), 3.83 (s,
3H, OCH3), 2.52 (s, 3H, CH3CO), 1.01 (s, 9H, (CH3)3C), 0.21 (s, 6H,
(CH3)2C). MS (ESI) m/z 296 [M þ H]þ. Analysis calculated for
C15H25NO3Si: C, 60.98; H, 8.53; N, 4.74. Found: C, 61.14; H, 8.66; N,
4.77.
5.10. 1-(3-(5-Acetyl-2-hydroxy-3-methoxybenzylamino)-4-
hydroxy-5-methoxyphenyl)ethanone (10)
To a solution of 9 (200 mg, 0.56 mmol) in methanol (20 ml)
cooled to 0 ꢀC, was added sodium borohydride (10.6 mg,
0.28 mmol). The reaction mixture was stirred at room temperature
for 2 h. Ice water (10 ml) was added to dissolve the excess sodium
borohydride. The reaction mixture was extracted with dichloro-
methane (5 ml ꢁ 3) and the organic layer was dried using sodium
sulfate. Solvent was removed in vacuo to afford 110 mg (58% yield)
of a brown solid 10, mp: 200e203 ꢀC. 1H NMR (CDCl3, 400 MHz):
7.64e7.63 (d, 1H, J ¼ 1.6 Hz, ArH), 7.50 (d, 1H, J ¼ 1.6 Hz, ArH),
7.13e7.12 (d, 1H, J ¼ 1.6 Hz, ArH), 7.08e7.07 (d, 1H, J ¼ 1.6 Hz, ArH),
4.54 (s, 2H, ArCHN), 3.99 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 2.57 (s,
3H, CH3CO), 2.53 (s, 3H, CH3CO). 13C NMR (CDCl3 and CD3OD,
300 MHz): 199.0 (COMe), 198.5 (COMe), 149.8 (ArC), 147.3 (ArC),
146.4 (ArC), 138.8 (ArC), 136.6 (ArC), 128.4 (ArC), 128.3 (ArC), 125.2
(ArC), 123.6 (ArC), 108.9 (ArC), 106.6 (ArC), 102.2 (ArC), 55.6 (CH3O),
55.5 (CH3O), 42.5 (CH2), 25.5 (CH3), 25.4 (CH3). MS (ESI) m/z 360
[M þ H]þ. Analysis calculated for C19H21NO6$0.86H2O: C, 60.88; H,
6.11; N, 3.74. Found: C, 60.80; H, 6.11; N, 3.87.
5.8. General procedure for synthesis of compounds 8aec
To a solution of 7 (900 mg, 2.56 mmol) in THF was added acid
anhydride, DMAP (312 mg, 2.56 mmol) and triethylamine (750 ml).
The reaction mixture was stirred at room temperature for 3 h to
afford a hydroxyl protected amide intermediate. TBAF (900 mg,
2.56 mmol) was then added and the reaction was maintained at
room temperature for 1 h. After solvent was removed in vacuo, the
residue was purified by column chromatography, eluting with ethyl
acetate/petroleum ether (1:1, v/v), to afford 8aec.
5.8.1. N-(5-Acetyl-2-hydroxy-3-methoxyphenyl)propionamide (8a)
Synthesized from 7 (900 mg, 2.56 mmol) and propionic anhy-
dride (665 mg, 5.12 mmol). White powder (275 mg, 42% yield), mp:

X. Lu et al. / European Journal of Medicinal Chemistry 46 (2011) 2691e2698
2697
5.11. (Z)-N-(5-Acetyl-2-hydroxy-3-methoxybenzylidene)-2-
methylpropan-2-amine oxide (11)
100
with PBS for three times. Cells were treated with 40
24 h. MTT was then added (0.5 mg/ml). After incubation for 4 h, the
supernatant was removed and DMSO (150 l) was added. Absor-
m
M) for 1 h. Supernatant was removed, and cells were washed
mg/ml of LPS for
N-tert-Butylhydroxylamine was first prepared on the day of use
by using an activated zinc catalyst. 2-Methyl-2-nitropropane was
dissolved in ethanol, and the solution was cooled to 0 ꢀC in an ice
bath. Zinc dust (5.89 g, 90 mmol) was added. Glacial acetic acid
(10.8 g, 180 mmol) was then added dropwise at such a rate that the
temperature maintained below 10 ꢀC with vigorous stirring. Ice
bath was removed and the reaction mixture was stirred for 3 h at
room temperature. The zinc dust was filtered through a Buchner
funnel and the solvent was removed in vacuo. The residue was
dissolved in methanol for use in condensation with aldehyde 2. To
a solution of 2 (11.6 g, 60 mmol) in methanol (300 ml) was added
a solution of N-tert-butylhydroxylamine in methanol (50 ml). The
mixture was heated to reflux under nitrogen for 6 h. Solvent was
removed and the product was purified by column chromatography,
eluting with ethyl acetate/petroleum ether (2:1, v/v), to afford
11.8 g (74% yield) of a yellow powder 11, mp: 183e184 ꢀC. 1H NMR
(CDCl3, 400 MHz): 7.76 (s, 1H, CHNO), 7.53 (d, 1H, J ¼ 1.6 Hz, ArH),
7.43e7.42 (d, 1H, J ¼ 1.6 Hz, ArH), 3.93 (s, 3H, OCH3), 2.55 (s, 3H,
CH3CO), 1.65 (s, 9H, (CH3)3C). 13C NMR (DMSO-d6, 300 MHz): 196.1
(COMe), 155.2 (ArC), 150.2 (ArC), 138.8 (ArC), 128.0 (CHN), 127.8
(ArC), 117.1 (ArC), 112.7 (ArC), 70.5 (CMe3), 56.1 (CH3O), 28.0
(3 ꢁ CH3), 26.5 (CH3). MS (ESI) m/z 264 [M ꢂ H]þ, 266 [M þ H]þ.
Analysis calculated for C14H19NO4: C, 63.38; H, 7.22; N, 5.28. Found:
C, 63.37; H, 7.37; N, 5.25.
m
bance (OD) was recorded at 570 nm using a 96-well plate reader
(Bio-RAD, USA).
5.14. Fluorescence-based ROS-scavenging assay
RAW 264.7 cells were seeded in confocal plates. After 24 h,
which were treated with various concentrations of apocynin,
compounds 10e12 (0.1, 1 and 10
mM) in the absence or presence of
LPS (1 g/ml). Then the cells were incubated with 20
m
mM DCFH-DA
for 20 min at 37 ꢀC in CO2 incubator (5% CO2). After that, wash with
PBS three times, the fluorescence (ex488 nm, em525 nm) was
detected by using a laser scanning microscope Carl Zeiss Micro-
scope systems. Fluorescence quantitative determination was
assayed by a Berthold TriStar Multimode LB 941 microplate reader.
5.15. Western blot for P67phox expression
RAW 264.7 were pre-treated with various concentrations of
apocynin, compounds 10e12 (0.1, 1 and 10
mM) for 1 h and stimu-
lated with LPS (1 g/ml) for 24 h. Cytoplasmic extracts of the cells
m
were resolved on 12% SDS-acrylamide gel by electrophoresis, and
transferred to PVDF membranes (PALL). The membranes were
blocked in 5% (weight/vol) nonfat milk in Tris-buffered sali-
neeTbuffer, and then incubated with purified mouse anti-p67phox
5.12. (R)-N-(5-Acetyl-2-hydroxy-3-methoxyphenyl)-5-(1,2-
dithiolan-3-yl)pentanamide (12)
antibody (BD Transduction, 1:2000), b-actin antibody (Neomakers,
1:2000) at 4 ꢀC over night. After incubation with goat anti-mouse
IgG horseradish peroxidase linked antibodies (Santa Cruz, 1:2000)
at room temperature for 1 h, the blots were treated with ECL
To a solution of 5-a-lipoic acid (1.58 g, 7.68 mmol) in DMF
(20 ml), were added EDCI (1.46 g, 7.68 mmol) and HOBT (1.03 g,
7.68 mmol). The reaction mixture was stirred at room temperature
for 30 min. A solution of 7 (900 mg, 2.56 mmol) in DMF (10 ml) was
then added dropwise, and the reaction mixture was maintained
20 h at room temperature. Ethyl acetate (300 ml) was added, and
the reaction mixture was washed with ice water (50 ml ꢁ 3). The
organic layer was dried using anhydrous sodium sulfate and the
solvent was removed in vacuo to afford a hydroxyl protected amide
intermediate. TBAF (900 mg, 2.56 mmol) was then added, and the
reaction was maintained at room temperature for 1 h. After
removing the solvent, the product was purified by column chro-
matography, eluting with ethyl acetate/petroleum ether (3:2, v/v),
to afford 482 mg (51% yield) of a gray powder 12, mp: 115e116 ꢀC.
1H NMR (CDCl3, 400 MHz): 8.50 (s, 1H, NH), 7.63 (s, 1H, ArH), 7.37
(d, 1H, J ¼ 1.6 Hz, ArH), 3.96 (s, 3H, OCH3), 3.64e3.58 (m, 1H, SCH),
3.23e3.10 (m, 2H, SCH2), 2.59 (s, 3H, COCH3), 2.49e2.46 (t, 2H,
J ¼ 6.7 Hz, CH2CO), 1.98e1.89 (m, 2H, SCH2CH2), 1.86e1.78 (m, 2H,
CH2CH2O), 1.77e1.72 (m, 2H, CH2CH2CH), 1.61e1.53 (m, 2H,
CH2CH2CH2). 13C NMR (CDCl3, 300 MHz): 197.2 (COMe), 171.7
(CON), 146.8 (ArC), 139.3 (ArC), 129.4 (ArC), 125.2 (ArC), 115.6 (ArC),
105.6 (ArC), 56.3 (2C, CH3O, CH), 40.2 (CH2), 38.5 (CH2), 37.3 (CH2),
34.6 (CH2), 28.8 (CH2), 26.3 (CH3), 25.1 (CH2). MS(ESI) m/z 392
[M þ Na]þ. Analysis calculated for C17H23NO4S2: C, 55.26; H, 6.27; N,
3.79. Found: C, 55.23; H, 6.45; N, 3.61.
reagents (Pierce) and then exposed to X-ray films. b-Actin was used
as an internal loading control.
Acknowledgements
This work was supported in part by grants from the China
Natural Science Fund (30772642 and 30973618 to Y.W), Guangzhou
city (2008Z1-E691 to J.J) Science and Technology Funds as well as
the 211 Project of Jinan University.
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