Rational design, synthesis, and pharmacological properties of pyranochalcone derivatives as potent anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2012.05.005

24 derivatives (5a-x) derived from natural pyranochalcones (I and II) were designed and evaluated for their inhibitory potency on the production of nitric oxide (NO) in LPS-stimulated RAW264.7 cells. Among them, four compounds (5b, 5d, 5f, and 5h) exhibit

Full text of DOI:10.1016/j.ejmech.2012.05.005

European Journal of Medicinal Chemistry 54 (2012) 272e280
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Rational design, synthesis, and pharmacological properties of pyranochalcone
derivatives as potent anti-inflammatory agents
,
,
Fei Peng a 1, Guangcheng Wang a 1, Xiuxia Li a, Dong Cao a, Zhuang Yang b, Liang Ma a, Haoyu Ye a,
Xiaolin Liang a, Yan Ran c, Jinying Chen a, Jingxiang Qiu a, Caifeng Xie a, Chongyang Deng a, Mingli Xiang a,
Aihua Peng a, Yuquan Wei a, Lijuan Chen a
,
*
a State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Gaopeng Street, Chengdu 610041, China
b College of Chemistry, Sichuan University, Chengdu 610045, China
c Pharmacy College of West China of Sichuan University, Chengdu, Sichuan 610065, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< Two natural pyranochalcones and
their derivatives were synthesized
and evaluated for anti-inflammatory
activities.
< 5b showed remarkable in vitro and
in vivo anti-inflammatory activity
comparable to indomethacin.
< Molecular docking was employed to
investigate the binding mode of the
significant inhibitor 5b.
< Structureeactivity relationship is
discussed.
a r t i c l e i n f o
a b s t r a c t
Article history:
24 derivatives (5aex) derived from natural pyranochalcones (I and II) were designed and evaluated for
their inhibitory potency on the production of nitric oxide (NO) in LPS-stimulated RAW264.7 cells. Among
them, four compounds (5b, 5d, 5f, and 5h) exhibited more potent inhibitory effects on iNOS activity and
iNOS-mediated NO production than a positive control indomethacin. Furthermore, 5b could significantly
suppress the progression of carrageenan-induced hind paw edema compared to indomethacin at
a dosage of 10 mg/kg/day, and dose-dependently ameliorated the development of adjuvant-induced
arthritis (AIA) validated by arthritic scores and H&E staining of joints. In addition, docking study
confirmed that 5b was an iNOS inhibitor with binding to the active site of murine iNOS.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 3 March 2012
Received in revised form
28 April 2012
Accepted 3 May 2012
Available online 12 May 2012
Keywords:
Anti-inflammation
NO production
iNOS
Paw edema
Pyranochalcones
Adjuvant-induced arthritis
* Corresponding author. Tel.: þ86 28 85164063; fax: þ86 28 85164060.
E-mail address: lijuan170@hotmail.com (L. Chen).
1
Authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.005

F. Peng et al. / European Journal of Medicinal Chemistry 54 (2012) 272e280
273
activity [7], and the inhibition of matrix metalloproteinase-2
(MMP-2) secretion and nicotinamide adenine dinucleotide
(NADH), etc [8,9]. However, only few study focused on their
structural modifications associated with the potential anti-
inflammatory potency [10,11]. In our laboratory, two typical
representatives of pyranochalcones, 4-hydroxyloncarpin (I) and
300,300-dimethylpyrano[30,40]2,4,20-trihydroxychalcone (II), isolated
from Millettia ferruginea and Artocarpus communis, were found to
exhibit potent inhibitory activity against the production of NO in
LPS-stimulated RAW264.7 cells and potential anti-inflammatory
activity in vivo (Fig. 1).
As our continuing effort to explore for new anti-inflammatory
chemical entities [5,12e14], a series of pyranochalcone derivatives
were designed, synthesized and investigated for their inhibitory
effects on NO production in LPS-induced RAW 264.7 cells and iNOS
activity. Among these derivatives, four compounds (5b, 5d, 5f, and
5h) showed more potent inhibitory activity against NO production
Fig. 1. Structure of I, II, and pyranochalcone derivatives.
1. Introduction
Inflammation is a central feature of many pathological condi-
tions and always companies with physiological signs such as pain,
redness, swelling, and even loss of organ functions [1]. During the
progression of multiple inflammatory diseases, the activation of
macrophages is crucial through the excessive release of pro-
inflammatory cytokines and mediators such as nitric oxide (NO),
than a positive control indomethacin at a concentration of 10 mM
(78.4%, 81.3%, 80.4%, 79.9% vs. 59.2%, respectively). Furthermore, 5b
significantly suppressed the progression of inflammation in both
two experimental models of carrageenan-induced hind paw edema
and adjuvant-induced arthritis (AIA). Additionally, 5b was
confirmed as a potent iNOS inhibitor (IC50: 7.9 mM) with binding to
the active site of murine iNOS by docking study.
tumor necrosis factor (TNF)-
glandin E2 (PGE2) [2]. NO, being an endogenous free radical, is
produced by the oxidation of -arginine to -citrulline which
primarily catalyzed by inducible nitric oxide synthase (iNOS).
Normally, NO is at low level. While stimulated by pro-
a, interleukin (IL)-1b, and prosta-
L
L
a
2. Chemistry
inflammatory cytokines or lipopolysaccharide (LPS), the iNOS
could produce a high expression of NO and further induce joint
injury at the inflammatory site. Since the suppression of the over-
production of iNOS-mediated NO improves the incidence and
severity of chronic inflammatory diseases like rheumatoid arthritis,
inflammatory bowel disease, and psoriatic arthritis, iNOS could be
recognized as a drug target of anti-inflammatory agents [3e5].
Pyranochalcones are an abundant class of flavonoids and widely
distributed in nature. Comprehensive literatures have been repor-
ted that pyranochalcones possessed various pharmacological
activity including radical scavenging activity [6], antimicrobial
Two pyranochalcone occurrences I, II and 24 derivatives were
synthesized according to previous reported procedures with the
yield of 30e70% (Scheme 1) [15,16]. Briefly, the key intermediate 3
was obtained by the condensation of 2,4-dihydroxylacetophenone
(2) with 3-methylcrotonaldehyde (1) in anhydrous pyridine at
120 ꢀC in a good yield [17,18]. Then, two methods were developed
to synthesize these desired compounds. Method A: The mixture of
3, hydroxyl benzaldehydes protected by chloro(methoxy)methane
(MOMeCl), tetrabutyl ammonium bromide (TBAB), and sodium
hydroxide was carried out using the microwave apparatus for 5 min
Scheme 1. General synthesis for the twenty-six compounds. Reagents and conditions: (i) pyridine, reflux, 120 ꢀC, 12 h; (ii) a. various MOM-protected benzaldehydes, NaOH (50%),
TBAB, microwave, 5 min; b. MeOH, HCl (20%), reflux, 65 ꢀC, 1 h. (iii) DMF, NaH, MOMeCl, rt, 2 h; (iv) a. substituted aromatic aldehyde, MeOH, KOH (50%), rt, 24 h; b. MeOH, HCl
(20%), reflux, 65 ꢀC, 1 h.

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F. Peng et al. / European Journal of Medicinal Chemistry 54 (2012) 272e280
to give another intermediates, and then deprotected by a treatment
with 20% HCl in methanol at 60 ꢀC to give 5me5t [19]. Method B:
Compound 3 was initially protected by MOMeCl to give 4. The
ClaiseneSchmidt condensation between 4 and appropriate benz-
aldehydes without any hydroxyl group was performed in the
presence of 50% KOH solution to afford the relevant intermediates
and further deprotected in 20% HCl methanol solution at 60 ꢀC to
give I, II, 5ae5l and 5ue5x [20,21].
to slightly reduction in inhibitory activities due to the severe steric
effects. The NO inhibitory activity varied from 47 to 81% when
introducing halogen element on the B-ring such as 5fe5h and
5ue5w.
3.2. Four derivatives inhibited iNOS activity and their cytotoxicity
Four compounds 5b, 5d, 5f and 5h were chosen to investigate
both the inhibitory activity against iNOS and the cytotoxicity in
RAW 264.7 microphages. As exhibited in Table 2, compared to
indomethacin, they presented remarkable inhibitory activity
3. Biological results and discussion
against iNOS (IC50: 7.9, 6.2, 5.4, and 17.8 mM, respectively). To check
3.1. Inhibition of NO production in LPS-induced RAW 264.7 cells
whether the suppression of iNOS activity was related to cell
viability, a MTT assay was adopted. None of the four compounds
exerted any obvious cytotoxicity in RAW 264.7 microphages (with
After the stimulation of RAW 264.7 macrophages with LPS for
18 h, the inhibitory effects on NO production of twenty-six
compounds were indirectly measured by the analysis of nitrite
concentration using the Greiss reaction [22]. Indomethacin, a non-
steroidal anti-inflammatory drug, was used as a positive control
(Table 1).
or without LPS, IC50 > 80 mM).
3.3. Anti-inflammatory activity in carrageenan-induced hind paw
edema model
As shown in Table 1, four compounds (5b, 5d, 5f and 5h) showed
approximate inhibitory ability compared with I (76.9%) and II
(79.8%). Removal of hydroxyl groups on ring-B (5l 62.9%) led to
decrease in NO inhibitory activities. Compounds 5ne5q were more
potent than 5m (52.8%), 5r (61.2%), 5s (46.4%), 5t (51.2%), all of
which contained hydroxyl group. Introduction of two methoxyl
groups (5t) at position C-30 and C-50 led to 25.7% activity decrease
compared with compound I, whereas the replacement of 40-
hydroxyl (5t) with 40-methoxyl (5d) could improve the activity. The
above-mentioned results highlighted that the inhibitory activity of
these hydroxyl compounds were closely connected with the posi-
tion of hydroxyl groups, the electric density of substituents and the
steric hindrance.
Carrageenan-induced hind paw edema was used as an acute
inflammatory experimental model to test the in vivo anti-
inflammatory activities of 5b, 5d, 5f and 5h. Carrageenan injec-
tion caused a time-dependent increase of paw with a peak occur-
ring at 4 h (Fig. 2). Indomethacin (Indo), administered (i.p) at a dose
of 10 mg/kg prior to the carrageenan injection, significantly
reduced paw edema with inhibitory rate of 45.8% at 4 h. Treated
rats received the four compounds 30 min before carrageenan
injection at 10 mg/kg (i.p). At the time of maximum foot increase
(4 h), compound 5b significantly reduced carrageenan-induced
edema by the value of 52.4% compared with 34.4% of 5d, 9.0% of
5f, and 27.6% of 5h (P < 0.05; n ¼ 5). Moreover, 5b showed slightly
better inhibitory activity than that of indomethacin (45.8%).
Although the replacement of 40-hydroxyl with 40-methoxyl
group (5a) on B-ring resulted in a 3.0-fold decrease in the NO
inhibitory activity (26.6%) compared with I (76.9%), the introduc-
tion of two methoxyl groups at both C-30 and C-40 position on
B-ring (5b) could increase the activity to 78.4%, which confirmed by
5d with three methoxyl groups substituted at 30, 40 and 50 on B-ring
(81.3%). However, once C-20 position was substituted by methoxyl
group (5c and 5w), the activity slightly reduced in comparison with
I and II. Introduction of phenyl (5e), pyridyl (5j), diphenylamino
(5k) or N,N-diethylamino group (5w) at C-40 position of B-ring led
3.4. Anti-arthritic effects of 5b in AIA models
Since 5b showed effective inhibition of paw carrageenan-
induced hind edema, we chose it to assess the anti-arthritis
effects in AIA model. AIA was a form of (sub)chronic arthritis and
was widely used to test agents for anti-inflammatory activity.
Briefly, AIA Lewis rat were treated with 5b at a dose of 10, and
25 mg/kg/day following the appearance of first AIA signs. Results
indicated that the severity of AIA was dose-dependently improved
in 5b-treated animals (Fig. 3A). Arthritic score and body weight loss
were significantly reduced during the therapeutic process (Fig. 3B).
Histological evaluation by H&E staining from vehicle and 5b-
treated mice revealed the signs of severe arthritis in the control
animals. Treatment with 5b at a dosage of 25 mg/kg resulted in
a significant suppression of these histopathological changes (Fig. 4).
Table 1
Inhibitory effects of 5ae5x on the production of NO in LPS-induced RAW 264.7
macrophages. The cells were pretreated with 5ae5x at a concentration of 10
2 h, and then incubated with LPS (1 g/mL) for 18 h. The inhibition of LPS-treat group
was set as 100.0%, and the control as 0.0%. Inhibition (%) [LPS
mM for
m
¼
(OD540)] ꢁ [Compound (OD540)]/[LPS (OD540)] ꢁ Control (OD540)] ꢂ 100%. The
results are the means ꢃ SD (n ¼ 3, P < 0.05).
3.5. Docking study of 5b with murine iNOS
Compds.
NO inhibition
Compds.
NO inhibition
rate (%) ꢃ SD
rate (%) ꢃ SD
As the most potent molecule 5b, its interaction mode binding to
iNOS protein (PBD ID: 1r35) was demonstrated with the help of
Indomethacin
I
II
59.2 ꢃ 2.1
76.9 ꢃ 5.1
79.8 ꢃ 6.9
26.6 ꢃ 13.6
78.4 ꢃ 4.6
40.5 ꢃ 11.3
81.3 ꢃ 6.0
41.6 ꢃ 12.5
80.4 ꢃ 8.4
66.2 ꢃ 6.1
79.9 ꢃ 7.2
51.3 ꢃ 5.4
62.8 ꢃ 4.5
36.2 ꢃ 1.9
5l
62.9 ꢃ 5.1
52.8 ꢃ 5.8
74.8 ꢃ 6.7
73.5 ꢃ 7.4
76.9 ꢃ 6.6
74.2 ꢃ 6.7
61.2 ꢃ 5.4
46.4 ꢃ 5.9
51.2 ꢃ 7.1
61.5 ꢃ 2.4
73.9 ꢃ 6.2
56.7 ꢃ 7.7
69.9 ꢃ 6.2
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5a
5b
5c
5d
5e
5f
5g
5h
5i
Table 2
IC50 values on iNOS and cytotoxicity in RAW 264.7 cells. Cytotoxicity ¼ (1 ꢁ [Compounds
(OD570)] ꢁ Background (OD570)/[Control (OD570) ꢁ Background (OD570)]) ꢂ 100%.
Compds.
iNOS/IC50
M)
Cytoxic/IC50(þLPS)
M)
Cytoxic/IC50(ꢁLPS)
(mM)
(
m
(
m
Indomethacin
20.0
7.9
6.2
5.4
17.8
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
5b
5d
5f
5j
5k
5h

F. Peng et al. / European Journal of Medicinal Chemistry 54 (2012) 272e280
275
Fig. 2. Inhibitory effects of 5b, 5d, 5f and 5h on hind-paw edema induced by carrageenan in mice. The results are expressed as mean (SEM, where n ¼ 5 rats. P < 0.05). Inhibitory
rate ¼ 100 ꢂ (Increase Paw Thickness(control ꢁ Increase Paw Thickness(inhibited group)/Increase Paw Thickness(control group)).
group)
a proteineligand docking program FRED. Compound 5b included
three parts: The dimethyl-2H-chromenol moiety, propenone linker,
and dimethoxyphenyl group. The dimethyl-2H-chromenol moiety
and the linker of 5b occupied the polar domain. As demonstrated in
Fig. 5, the oxygen atom of the dimethyl-2H-pyran moiety formed
a hydrogen bond with the nitrogen atom of Arg375 and the
carbonyl oxygen atom worked as a hydrogen bond acceptor for the
polar hydrogen on Tyr367. Hydrogen atom of the hydroxyl group
provided a donor for the oxygen of Gln257. It was noted that there
were
p-cation interactions between the side chain N atoms on
Arg382 and the phenyl ring of 5b. The dimethoxyphenyl part
dropped into the non-polar pocket. And one oxygen atom of the
dimethoxyphenyl coordinates with Fe2þ of the cofactor HEM. In
summary, the computational result confirmed our hypothesis that
5b was a potent iNOS inhibitor with perfect binding to the active
site of murine iNOS.
4. Conclusion
In our study, twenty-six pyranochalcones have been synthe-
sized and evaluated for their inhibition of iNOS activity, and iNOS-
mediated NO production in LPS-stimulated RAW 264.7 cells. Four
derivatives (5b, 5d, 5f and 5h) exhibited potent inhibitory activity
and were chosen to investigate their anti-inflammation in two
inflammatory experimental models of carrageenan-induced hind
paw edema and adjuvant-induced arthritis. Among them, 5b dis-
played better inhibitory activity than indomethacin in carrageenan-
induced hind paw edema at a dose of 10 mg/kg. In AIA animal
model, oral administration of 5b inhibited the arthritic inflamma-
tion in a dose-dependent manner. All these data suggested that 5b
was a potential agent for the inflammatory disorders and further
the development of this compound may be of interest.
5. Experimental
5.1. Chemistry
Fig. 3. The evolution of arthritis scores (A) and variation of body weight (B) of AIA rats
following the treatment of vehicle, 5b (10 mg/kg, and 25 mg/kg) and indomethacin
(10 mg/kg). Results are means ꢃ S.E.M. from six rats.
All reagents were obtained from commercial sources and were used
without further purification unless indicated. N,N-dimethylformamide

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F. Peng et al. / European Journal of Medicinal Chemistry 54 (2012) 272e280
Fig. 4. H&E staining for the histological changes of joints from 5b-treated AIA rats. (A) Model group; (B) 5b-treated group at a dose of 10 mg/kg; (C) 5b-treated group at a dose of
25 mg/kg; (D) Indomethacin (Indo)-treated group at a dose of 10 mg/kg; (E) Normal group.
was dried by distillation from calcium hydride and stored on activated
molecular sieves (4 Å). Analytical thin-layer chromatography (TLC) was
run on silica gel GF245 plates (Qingdao Haiyang Chemical Plant, Qing-
dao, People’s Republic of China). Column chromatography was
accomplished on silica gel 60 (300e400 mesh). A microwave reactor
(MCL-III, 1300 W, alternating current) was used for reactions. NMR
spectra were obtained by a Varian DPX 400-MHz spectrometer with
TMS as the international standard. All chemical shifts are recorded in
parts per million (ppm). MS spectra (MS-ESI) were performed by a
Q-TOF Premier Mass Spectrometer (Micromass, Simonsway, Man-
chester, UK). The purities of all final synthetic compounds were carried
5.1.1. 1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)ethanone (3)
To a solution of 2,4-dihydroxyacetophenone (6.08 g, 40.0 mmol)
in dry pyridine (4 mL) was added 3-methylcrotonaldehyde (4.2 mL,
44.0 mmol). After being refluxed for 12 h, the pyridine was
removed by distillation in vacuo and the residue was separated by
silica gel column chromatography to get 3 as a yellow crystal. Yield:
50%. TLC (petroleum ether/ethyl acetate ¼ 15:1), Rf ¼ 0.25. 1H NMR
(400 MHz, CDCl3)
d: 1.45 (s, 6H), 2.54 (s, 3H), 5.57 (d, 1H,
J ¼ 10.0 Hz), 6.33 (d, 1H, J ¼ 8.8 Hz), 6.70 (d, 1H, J ¼ 10.0 Hz), 7.51 (d,
1H, J ¼ 8.8 Hz), 12.98 (s, 1H).
out by reverse-phase HPLC (X-terra, C18, 150 mm ꢂ 4.6 mm, 5
mm) using
5.1.2. 1-(5-(Methoxymethoxy)-2,2-dimethyl-2H-chromen-6-yl)
ethanone (4)
methanolewater (80:20) as mobile phase.
Sodium hydride was added to a yellow solution of 3 (2.18 g,
10 mmol) in N,N-dimethylformamide (100 mL). Then MOMeCl
(2.42 g, 30.0 mmol) was slowly dropped to the solution and stir-
red at room temperature for 2 h. The crude product was extracted
with ethyl acetate and purified by silica gel column chromatog-
raphy. Yellow liquid, yield: 60%. TLC (petroleum ether/ethyl
acetate ¼ 10:1), Rf ¼ 0.31. 1H NMR (400 MHz, CDCl3)
d: 1.44 (s, 6H),
2.57 (s, 3H), 3.52 (s, 3H), 5.01 (s, 2H), 5.67 (d, 1H, J ¼ 10.0 Hz), 6.61
(d, 1H, J ¼ 8.8 Hz), 6.66 (d, 1H, J ¼ 10.0 Hz), 7.51 (d, 1H, J ¼ 8.4 Hz).
5.1.3. Method A. Synthesis of I, II, 5ael and 5uex
All the reactions were performed under N2 atmosphere. The
mixture of 4 (1.0 equiv) and the corresponding benzaldehydes (1.0
equiv) in MeOH (15 mL for 1 mmol of 4) was stirred at 0 ꢀC and
warmed to room temperature until both were dissolved. Then the
KOH (50%) was added. The reaction was stirred at room tempera-
ture for 24 h until the benzaldehydes were consumed. The mixture
was poured to water (20 mL) and neutralized by the addition of 20%
HCl. A solid was separated out. Then the solid was filtered out and
dissolved in MeOH (3 mL) and HCl (20%, 2 mL) to reflux at 65 ꢀC for
1 h. Evaporation of solvent and purification by column chroma-
tography to get pure compounds.
Fig. 5. The interaction mode of 5b within the active site of murine iNOS (PDB ID: 1r35).
Residues in the active site are displayed in line style and colored differently. HEM and
5.1.3.1. 4-Hydroxyloncarpin (I). Yellow solid; Yield: 82%; HPLC:
100%. TLC (petroleum ether/ethyl acetate ¼ 10:1), Rf ¼ 0.33. 1H NMR
H4B are depicted by gray line. 5b is represented with stick. Hydrogen bonds and
p-
cation interaction formed between residues within the active site and 5b are high-
lighted. This figure was generated by DS Visualizer 2.5.
(400 MHz, CDCl3)
d
: 1.38 (s, 6H), 5.61 (d,1H, J ¼ 10.0 Hz), 6.39 (d,1H,

F. Peng et al. / European Journal of Medicinal Chemistry 54 (2012) 272e280
277
J ¼ 8.8 Hz), 6.77 (d, 1H, J ¼ 10.0 Hz), 6.90 (d, 2H, J ¼ 8.4 Hz), 7.46 (d,
1H, J ¼ 15.2 Hz), 7.58 (d, 2H, J ¼ 8.8 Hz), 7.73 (d, 1H, J ¼ 8.8 Hz), 7.86
(d, 1H, J ¼ 15.8 Hz), 13.78 (s, 1H). MS (ES), m/z: 321 (ESꢁ).
J ¼ 8.8 Hz), 6.76 (d, 1H, J ¼ 10.0 Hz), 7.10e7.14 (1H, m), 7.33e7.41
(3H, m), 7.53 (d, 1H, J ¼ 15.6 Hz), 7.70 (d, 1H, J ¼ 8.8 Hz), 7.80 (d,
1H, J ¼ 15.2 Hz), 13.58 (s, 1H). 13C NMR (100 MHz, CDCl3)
d: 28.40,
77.96, 108.48, 109.43, 113.97, 114.39, 115.78, 117.29, 121.62, 124.61,
128.19,130.48,130.71,137.06,142.66,160.09,161.02,191.55. MS (ES),
m/z: 323 (ESꢁ).
5.1.3.2. 300,300-Dimethylpyrano[30,40]2,4,20-trihydroxychalcone
(II). Orange solid; Yield: 61%; HPLC: 99%. TLC (petroleum ether/
ethyl acetate ¼ 10:1), Rf ¼ 0.28. 1H NMR (400 MHz, CDCl3)
d: 1.42 (s,
6H), 5.77 (d, 1H, J ¼ 10.0 Hz), 6.34 (dd, 1H, J ¼ 2.0 Hz, J ¼ 8.4 Hz),
6.39 (s, 1H), 6.42 (d, 2H, J ¼ 8.8 Hz), 7.22 (d, 1H, J ¼ 15.2 Hz), 7.79 (d,
1H, J ¼ 8.4 Hz), 8.07 (d, 1H, J ¼ 8.8 Hz), 8.14 (d, 1H, J ¼ 15.6 Hz), 10.08
(s, 1H), 10.31 (s, 1H), 13.78 (s, 1H). MS (ES), m/z: 337 (ESꢁ).
5.1.3.9. (E)-3-(4-Chlorophenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chro-
men-6-yl)prop-2-en-1-one (5g). Yellow solid; Yield: 82%; HPLC:
99.3%. TLC (petroleum ether/ethyl acetate ¼ 6:1), Rf ¼ 0.35. 1H NMR
(400 MHz, CDCl3)
d
: 1.47 (s, 6H), 5.59 (d,1H, J ¼ 10.0 Hz), 6.38 (d,1H,
J ¼ 8.8 Hz), 6.74 (d, 1H, J ¼ 10.0 Hz), 7.39 (d, 1H, J ¼ 8.4 Hz), 7.52 (d,
1H, J ¼ 15.2 Hz), 7.57 (d, 2H, J ¼ 8.8 Hz), 7.70 (d, 1H, J ¼ 8.8 Hz), 7.80
(d, 1H, J ¼ 15.6 Hz), 13.68 (s, 1H). MS (ES), m/z: 339 (ESꢁ).
5.1.3.3. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-
methoxyphenyl)prop-2-en-1-one (5a). Orange solid; Yield: 78%;
HPLC: 100%. TLC (petroleum ether/ethyl acetate ¼ 6:1), Rf ¼ 0.35. 1H
NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H), 3.86 (s, 3H), 5.58 (d, 1H,
5.1.3.10. (E)-3-(4-Bromophenyl)-1-(5-hydroxy-2,2-dimethyl-2H-
chromen-6-yl)prop-2-en-1-one (5h). Orange solid; Yield: 79%;
HPLC: 99.1%. TLC (petroleum ether/ethyl acetate ¼ 15:1), Rf ¼ 0.38.
J ¼ 10.0 Hz), 6.37 (d, 1H, J ¼ 8.8 Hz), 6.74 (d, 1H, J ¼ 10.0 Hz), 6.93 (d,
2H, J ¼ 8.4 Hz), 7.43 (d,1H, J ¼ 15.6 Hz), 7.60 (d, 2H, J ¼ 8.8 Hz), 7.71 (d,
1H, J ¼ 8.8 Hz), 7.84 (d, 1H, J ¼ 15.2 Hz), 13.80 (s, 1H). MS (ES), m/z:
335 (ESꢁ).
1H NMR (400 MHz, CDCl3)
d
: 1.47 (s, 6H), 5.59 (d, 1H, J ¼ 10.0 Hz),
6.38 (d, 1H, J ¼ 8.8 Hz), 6.74 (d, 1H, J ¼ 10.0 Hz), 7.49e7.57 (5H, m),
7.69 (d, 1H, J ¼ 8.8 Hz), 7.78 (d, 1H, J ¼ 15.2 Hz), 13.60 (s, 1H). 13C
5.1.3.4. (E)-3-(3,4-Dimethoxyphenyl)-1-(5-hydroxy-2,2-dimethyl-
2H-chromen-6-yl)prop-2-en-1-one (5b). Orange solid; Yield: 83%;
HPLC: 100%. TLC (petroleum ether/ethyl acetate ¼ 6:1), Rf ¼ 0.33. 1H
NMR (100 MHz, CDCl3) d: 28.41, 77.96, 108.45, 109.45, 113.99,115.81,
120.90,124.90,128.20,129.85,130.67,132.24,133.75,142.78,160.05,
161.02, 191.60. MS (ES), m/z: 383 (ESꢁ).
NMR (400 MHz, CDCl3) d: 1.47 (s, 6H), 3.94 (s, 3H), 3.97 (s, 3H), 5.57
(d, 1H, J ¼ 10.0 Hz), 6.38 (d, 1H, J ¼ 8.8 Hz), 6.75 (d, 1H, J ¼ 10.0 Hz),
6.90 (d,1H, J ¼ 8.4 Hz), 7.16 (d,1H, J ¼ 2.0 Hz), 7.24 (d,1H, J ¼ 2.0 Hz),
7.41 (d, 1H, J ¼ 15.2 Hz), 7.72 (d, 1H, J ¼ 8.8 Hz), 7.82 (d, 1H,
5.1.3.11. (E)-3-(2-Fluoro-4-methoxyphenyl)-1-(5-hydroxy-2,2-
dimethyl-2H-chromen-6-yl)prop-2-en-1-one (5i). Orange solid;
Yield: 80%; HPLC: 98.6%. TLC (petroleum ether/ethyl
J ¼ 15.2 Hz), 13.79 (s, 1H). 13C NMR (100 MHz, CDCl3)
d: 28.37, 56.00,
acetate ¼ 20:1), Rf ¼ 0.26. 1H NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H),
56.02, 77.81, 108.19, 109.45, 110.17, 111.14, 114.09, 115.92, 118.00,
123.34, 127.82, 128.11, 130.57, 144.45, 149.28, 151.55, 159.70, 160.94,
191.89. MS (ES), m/z: 365 (ESꢁ).
3.85 (s, 3H), 5.58 (d, 1H, J ¼ 10.0 Hz), 6.37 (d, 1H, J ¼ 8.8 Hz), 6.66
(dd, 1H, J ¼ 12.4 Hz, 2.4 Hz), 6.74 (d, 2H, J ¼ 9.6 Hz), 7.53 (d, 1H,
J ¼ 8.4 Hz), 7.58 (d, 1H, J ¼ 5.2 Hz), 7.69 (d, 1H, J ¼ 9.2 Hz), 7.89 (d,
1H, J ¼ 15.6 Hz), 13.75 (s, 1H). MS (ES), m/z: 353 (ESꢁ).
5.1.3.5. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (5c). Pale yellow solid; Yield:
81%; HPLC: 100%. TLC (petroleum ether/ethyl acetate ¼ 3:1),
5.1.3.12. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-
(pyrrolidin-1-yl)phenyl)prop-2-en-1-one (5j). Orange solid; Yield:
78%; HPLC: 99.0%. TLC (petroleum ether/ethyl acetate ¼ 6:1),
Rf ¼ 0.21.1H NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H), 3.92 (s, 3H), 3.93
(s, 3H), 3.96 (s, 3H), 5.58 (d, 1H, J ¼ 10.0 Hz), 6.36 (d, 1H, J ¼ 8.8 Hz),
6.53 (s, 1H), 6.75 (d, 1H, J ¼ 10.0 Hz), 7.11 (s, 1H), 7.50 (d, 1H,
J ¼ 15.2 Hz), 7.71 (d, 1H, J ¼ 8.8 Hz), 8.15 (d, 1H, J ¼ 15.2 Hz), 13.94 (s,
1H). MS (ES), m/z: 395 (ESꢁ).
Rf ¼ 0.37. 1H NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H), 2.04 (q, 4H,
J ¼ 6.0 Hz), 3.35 (d, 4H, J ¼ 6.0 Hz), 5.57 (d, 1H, J ¼ 10.0 Hz), 6.35 (d,
1H, J ¼ 8.8 Hz), 6.55 (d, 2H, J ¼ 8.0 Hz), 6.75 (d, 1H, J ¼ 10.0 Hz), 7.32
(d, 1H, J ¼ 15.2 Hz), 7.53 (d, 2H, J ¼ 8.4 Hz), 7.72 (d, 1H, J ¼ 8.8 Hz),
7.85 (d, 1H, J ¼ 15.2 Hz), 14.10 (s, 1H). MS (ES), m/z: 374 (ESꢁ).
5.1.3.6. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(3,4,5-
trimethoxy-phenyl)prop-2-en-1-one (5d). Orange solid; Yield: 78%;
HPLC: 100%. TLC (petroleum ether/ethyl acetate ¼ 6:1), Rf ¼ 0.33. 1H
5.1.3.13. (E)-3-(4-(Diphenylamino)phenyl)-1-(5-hydroxy-2,2-dimethyl-
2H-chromen-6-yl)prop-2-en-1-one (5k). Orange solid; Yield: 76%;
HPLC: 98.1%. TLC (petroleum ether/ethyl acetate ¼ 30:1), Rf ¼ 0.33. 1H
NMR (400 MHz, CDCl3) d: 1.47 (s, 6H), 3.90 (s, 3H), 3.93 (s, 6H), 5.59
(d, 1H, J ¼ 10.0 Hz), 6.38 (d, 1H, J ¼ 8.8 Hz), 6.75 (d, 1H, J ¼ 10.0 Hz),
NMR (400 MHz, CDCl3) d: 1.47 (s, 6H), 2.54 (s, 1H), 5.58 (d, 1H,
6.87 (s, 1H), 7.42 (d, 1H, J ¼ 15.2 Hz), 7.72 (d, 1H, J ¼ 8.8 Hz), 7.78 (d,
J ¼ 10.0 Hz), 6.36 (d, 1H, J ¼ 9.2 Hz), 6.74 (d, 1H, J ¼ 10.0 Hz), 7.01 (d,
2H, J ¼ 8.4 Hz), 7.09e7.16 (5H, m), 7.29e7.33 (4H, m), 7.40 (d, 1H,
J ¼ 15.2 Hz), 7.48 (d, 2H, J ¼ 8.4 Hz), 7.69 (d, 1H, J ¼ 8.8 Hz), 7.81 (d, 1H,
J ¼ 15.6 Hz), 13.76 (s, 1H). MS (ES), m/z: 474 (ESþ).
1H, J ¼ 15.6 Hz), 13.71 (s, 1H). 13C NMR (100 MHz, CDCl3)
d: 28.38,
56.25, 61.02, 77.89, 105.79, 108.28, 109.46, 114.04, 115.86, 119.47,
128.16, 130.31, 130.64, 140.57, 144.43, 153.50, 159.86, 160.97, 191.55.
MS (ES), m/z: 395 (ESꢁ).
5.1.3.14. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-
phenylprop-2-en-1-one (5l). Orange solid; Yield: 80%; HPLC:
100%. TLC (petroleum ether/ethyl acetate ¼ 30:1), Rf ¼ 0.31. 1H
5.1.3.7. (E)-3-([1,10-Biphenyl]-4-yl)-1-(5-hydroxy-2,2-dimethyl-2H-
chromen-6-yl)prop-2-en-1-one (5e). Orange solid; Yield: 81%;
HPLC: 100%. TLC (petroleum ether/ethyl acetate ¼ 15:1), Rf ¼ 0.38.
NMR (400 MHz, CDCl3)
d
: 1.47 (s, 6H), 5.59 (d, 1H, J ¼ 10.0 Hz),
1H NMR (400 MHz, CDCl3)
d
: 1.47 (s, 6H), 5.60 (d, 1H, J ¼ 10.0 Hz),
6.38 (d, 1H, J ¼ 9.2 Hz), 6.75 (d, 1H, J ¼ 10.0 Hz), 7.42e7.43 (3H, m),
7.55 (d, 1H, J ¼ 15.6 Hz), 7.64e7.66 (2H, m), 7.12 (d, 1H, J ¼ 8.8 Hz),
7.86 (d, 1H, J ¼ 15.6 Hz), 13.68 (s, 1H). MS (ES), m/z: 305 (ESꢁ).
6.39 (d, 1H, J ¼ 8.8 Hz), 6.76 (d, 1H, J ¼ 10.0 Hz), 7.39 (d, 1H,
J ¼ 7.6 Hz), 7.46e7.49 (2H, m), 7.59e7.68 (5H, m), 7.72e7.77 (3H, m),
7.90 (d, 1H, J ¼ 15.2 Hz), 13.72 (s, 1H). MS (ES), m/z: 381 (ESꢁ).
5.1.3.15. (E)-3-(4-Fluorophenyl)-1-(5-hydroxy-2,2-dimethyl-2H-
chromen-6-yl)prop-2-en-1-one (5u). Orange solid; Yield: 77%;
HPLC: 100%. TLC (petroleum ether/ethylacetate ¼ 20:1), Rf ¼ 0.30.1H
5.1.3.8. (E)-3-(3-Fluorophenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chro-
men-6-yl)prop-2-en-1-one (5f). Orange solid; Yield: 74%; HPLC:
100%. TLC (petroleum ether/ethyl acetate ¼ 15:1), Rf ¼ 0.34. 1H NMR
NMR (400 MHz, CDCl3)
1H, J ¼ 8.8 Hz), 6.74 (d,1H, J ¼ 10.0 Hz), 7.10 (t, 2H, J ¼ 8.8 Hz), 7.47 (d,
d
: 1.47 (s, 6H), 5.59 (d,1H, J ¼ 10.0 Hz), 6.38 (d,
(400 MHz, CDCl3)
d: 1.47 (s, 6H), 5.60 (d,1H, J ¼ 10.0 Hz), 6.39 (d,1H,

278
F. Peng et al. / European Journal of Medicinal Chemistry 54 (2012) 272e280
1H, J ¼ 15.6 Hz), 7.62e7.65 (2H, m), 7.70 (d,1H, J ¼ 8.8 Hz), 7.82 (d,1H,
J ¼ 15.6 Hz), 13.65 (s, 1H). MS (ES), m/z: 323 (ESꢁ).
6.36 (d, 1H, J ¼ 7.6 Hz), 6.74 (d, 1H, J ¼ 9.6 Hz), 6.89 (d, 1H,
J ¼ 7.2 Hz), 7.11 (d, 1H, J ¼ 6.4 Hz), 7.18 (s, 1H), 7.37 (d, 1H,
J ¼ 14.8 Hz), 7.69 (d, 1H, J ¼ 8.0 Hz), 7.73(d, 1H, J ¼ 7.2 Hz), 13.78 (s,
1H). MS (ES), m/z: 337 (ESꢁ).
5.1.3.16. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(3-
methoxyphenyl)prop-2-en-1-one (5v). Orange solid; Yield: 81%;
HPLC: 99.6%. TLC (petroleum ether/ethyl acetate
¼
6:1),
5.1.4.4. (E)-3-(3-Ethoxy-4-hydroxyphenyl)-1-(5-hydroxy-2,2-dimethyl-
2H-chromen-6-yl)prop-2-en-1-one (5p). Yellow solid; Yield: 34%;
HPLC: 100%. TLC (petroleum ether/ethyl acetate ¼ 10:1), Rf ¼ 0.31. 1H
Rf ¼ 0.35. 1H NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H), 3.86 (s, 3H),
5.59 (d, 1H, J ¼ 10.0 Hz), 6.38 (d, 1H, J ¼ 9.2 Hz), 6.74 (d, 1H,
J ¼ 10.0 Hz), 6.95 (dd, 1H, J ¼ 8.4, 2.4 Hz), 7.15 (s, 1H), 7.23 (d, 1H,
J ¼ 9.2 Hz), 7.32 (t, 1H, J ¼ 8.0 Hz), 7.52 (d, 1H, J ¼ 15.6 Hz),
7.71(d, 1H, J ¼ 8.8 Hz), 7.82 (d, 1H, J ¼ 15.6 Hz), 13.65 (s, 1H). MS
(ES), m/z: 335 (ESꢁ).
NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H), 4.17 (q, 2H, J ¼ 6.8 Hz), 5.59 (d,
1H, J ¼ 10.0 Hz), 6.00 (s, 1H), 6.37 (d, 2H, J ¼ 9.2 Hz), 6.74 (d, 1H,
J ¼ 10.0 Hz), 6.95 (d,1H, J ¼ 8.4 Hz), 7.11 (d,1H, J ¼ 1.6 Hz), 7.21 (dd,1H,
J ¼ 8.0, 2.0 Hz), 7.38 (d, 1H, J ¼ 15.6 Hz), 7.71 (d, 1H, J ¼ 8.8 Hz), 7.80 (d,
1H, J ¼ 15.2 Hz), 13.80 (s, 1H). MS (ES), m/z: 367 (ESþ).
5.1.3.17. (E)-3-(4-(Diethylamino)phenyl)-1-(5-hydroxy-2,2-dimethyl-
2H-chromen-6-yl)prop-2-en-1-one (5w). Orange solid; Yield: 76%;
HPLC: 100%. TLC (petroleum ether/ethyl acetate ¼ 20:1), Rf ¼ 0.33.1H
5.1.4.5. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(3-
hydroxy-4-methoxyphenyl)prop-2-en-1-one (5q). Pale yellow solid;
Yield: 37%; HPLC: 98.4%. TLC (petroleum ether/ethyl
NMR (400 MHz, CDCl3) d: 1.23 (6H, m) 1.47 (s, 6H), 3.40 (q, 4H,
J ¼ 7.2 Hz), 5.57 (d, 1H, J ¼ 10.0 Hz), 6.35 (d, 1H, J ¼ 9.2 Hz), 6.65 (d,
2H, J ¼ 8.8 Hz), 6.75 (d, 1H, J ¼ 10.0 Hz), 7.31 (d, 1H, J ¼ 15.6 Hz), 7.52
(d, 1H, J ¼ 8.8 Hz), 7.71 (d, 1H, J ¼ 9.2 Hz), 7.83 (d, 1H, J ¼ 15.2 Hz),
14.10 (s, 1H). MS (ES), m/z: 378 (ESþ).
acetate ¼ 10:1), Rf ¼ 0.32. 1H NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H),
3.95 (s, 3H), 5.58 (d, 1H, J ¼ 10.0 Hz), 5.66 (s, 1H), 6.37 (d, 1H,
J ¼ 8.8 Hz), 6.74 (d, 1H, J ¼ 10.0 Hz), 6.87 (d, 1H, J ¼ 8.4 Hz), 7.13 (dd,
1H, J ¼ 8.4, 1.6 Hz), 7.28 (d, 1H, J ¼ 1.2 Hz), 7.41 (d, 1H, J ¼ 15.2 Hz),
7.70 (d, 1H, J ¼ 8.8 Hz), 7.78 (d, 1H, J ¼ 15.6 Hz), 13.80 (s, 1H). MS
(ES), m/z: 351 (ESꢁ).
5.1.3.18. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(2,3,4-
trimethoxyphenyl)prop-2-en-1-one (5x). Yellow solid; Yield: 77%;
HPLC: 98.7%. TLC (petroleum ether/ethyl acetate ¼ 50:1), Rf ¼ 0.40.
5.1.4.6. (E)-3-(5-Chloro-2-hydroxyphenyl)-1-(5-hydroxy-2,2-dimethyl-
2H-chromen-6-yl)prop-2-en-1-one (5r). Yellow solid; Yield: 29%;
HPLC: 98.8%. TLC (petroleum ether/ethyl acetate ¼ 10:1), Rf ¼ 0.29. 1H
1H NMR (400 MHz, CDCl3)
d
: 1.44 (s, 6H), 3.90 (d, 6H, J ¼ 9.2 Hz),
3.97 (s, 3H), 5.58 (d, 1H, J ¼ 10.0 Hz), 6.37 (d, 1H, J ¼ 8.8 Hz), 6.72 (d,
1H, J ¼ 8.8 Hz), 6.75 (d, 1H, J ¼ 10.0 Hz), 7.37 (d, 1H, J ¼ 8.8 Hz), 7.60
(d, 1H, J ¼ 15.2 Hz), 7.70 (d, 1H, J ¼ 9.2 Hz), 8.03 (d, 1H, J ¼ 15.6 Hz),
13.84 (s, 1H). MS (ES), m/z: 397 (ESþ).
NMR (400 MHz, DMSO)
d
: 1.22 (s, 6H), 5.76 (d, 1H, J ¼ 10.0 Hz), 6.43
(d,1H, J ¼ 8.8 Hz), 6.63 (d,1H, J ¼ 10.0 Hz), 6.94 (d,1H, J ¼ 8.4 Hz), 7.30
(dd, 1H, J ¼ 8.4, 2.4 Hz), 7.98 (d, 1H, J ¼ 15.6 Hz), 8.08 (d, 1H,
J ¼ 10.0 Hz), 8.11 (s, 1H), 8.21 (d, 1H, J ¼ 9.2 Hz), 13.93 (s, 1H). MS (ES),
m/z: 355 (ESꢁ).
5.1.4. Method B. Synthesis of 5me5t
Compound 3 (1.0 equiv), the corresponding benzaldehydes (1.0
equiv) whose hydroxyl groups were protected by MOMeCl (1
equiv), TBAB (0.6 equiv) and NaOH (50%, 5 mL) were mixed in
a 25 mL flask. The flask with an air set pipe was put in the micro-
wave reactor. After 5 min, the mixture was neutralized by diluted
HCl, extracted with ethyl acetate (3 ꢂ 30 mL), washed with brine,
dried by Na2SO4, and concentrated in vacuo. Then MeOH (3 mL) and
HCl (20%, 2 mL) were added to the concentrated mixture. After
being refluxed at 65 ꢀC for 1 h, the solution was extracted with ethyl
acetate (3 ꢂ 30 mL). The solvent was evaporated and the residue
was purified by column chromatography to give pure compounds.
5.1.4.7. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(3-
hydroxyphenyl)prop-2-en-1-one (5s). Pale yellow solid; Yield:
33%; HPLC: 100%. TLC (petroleum ether/ethyl acetate ¼ 10:1),
Rf ¼ 0.32. 1H NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H), 5.76 (d, 1H,
J ¼ 10.0 Hz), 6.38 (d, 1H, J ¼ 8.8 Hz), 6.74 (d, 1H, J ¼ 10.0 Hz), 6.89
(d, 1H, J ¼ 8.0 Hz), 7.11 (s, 1H), 7.21 (d, 1H, J ¼ 7.6 Hz), 7.27 (t, 1H,
J ¼ 8.0 Hz), 7.51 (d, 1H, J ¼ 15.6 Hz), 7.70 (d, 1H, J ¼ 9.2 Hz), 7.78 (d,
1H, J ¼ 15.6 Hz), 13.65 (s, 1H). MS (ES), m/z: 321 (ESꢁ).
5.1.4.8. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-
hydroxy-3,5-dimethoxyphenyl)prop-2-en-1-one (5t). Pale yellow
solid; Yield: 36%; HPLC: 99.2%. TLC (petroleum ether/ethyl
5.1.4.1. (E)-1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-
hydroxy-3-methoxyphenyl)prop-2-en-1-one (5m). Pale yellow solid;
Yield: 36%; HPLC: 98.0%. TLC (petroleum ether/ethyl acetate ¼ 6:1),
acetate ¼ 10:1), Rf ¼ 0.35. 1H NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H),
3.97 (s, 6H), 5.59 (d, 1H, J ¼ 10.0 Hz), 5.86 (S, 1H), 6.38 (d, 1H,
J ¼ 9.2 Hz), 6.75 (d, 1H, J ¼ 10.0 Hz), 6.89 (s, 2H), 7.39 (d, 1H,
J ¼ 15.2 Hz), 7.72 (d, 1H, J ¼ 8.8 Hz), 7.79 (d, 1H, J ¼ 15.2 Hz), 13.79
(s, 1H). MS (ES), m/z: 383 (ESþ).
Rf ¼ 0.27. 1H NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H), 3.98 (s, 3H), 5.59
(d, 1H, J ¼ 10.0 Hz), 5.92 (s, 1H), 6.37 (d, 1H, J ¼ 8.8 Hz), 6.75 (d, 1H,
J ¼ 10.0 Hz), 6.95 (d, 1H, J ¼ 8.4 Hz), 7.12 (d, 1H, J ¼ 1.6 Hz), 7.22 (dd,
1H, J ¼ 8.0,1.6 Hz), 7.39 (d,1H, J ¼ 15.6 Hz), 7.71 (d,1H, J ¼ 8.8 Hz), 7.80
(d, 1H, J ¼ 15.2 Hz), 13.79 (s, 1H). MS (ES), m/z: 353 (ESþ).
5.2. Biological experiment
5.1.4.2. 3(E)-3-(2,5-Dihydroxyphenyl)-1-(5-hydroxy-2,2-dimethyl-
2H-chromen-6-yl)prop-2-en-1-one (5n). Pale yellow solid; Yield:
33%; HPLC: 100%. TLC (petroleum ether/ethyl acetate ¼ 10:1),
5.2.1. Cell culture
RAW264.7 cells (murine) were obtained from American Type
Culture Collection (ATCC, Rockville, MD, USA) and cultured in a 5%
CO2 humidified atmosphere at 37 ꢀC with RPMI 1640 containing
10% FBS.
Rf ¼ 0.28. 1H NMR (400 MHz, CDCl3)
d: 1.47 (s, 6H), 5.76 (d, 1H,
J ¼ 10.0 Hz), 6.42 (d, 1H, J ¼ 9.2 Hz), 6.63 (d,1H, J ¼ 10.0 Hz), 6.77 (s,
2H), 7.79 (d, 1H, J ¼ 15.6 Hz), 8.09e8.13 (2H, m), 8.93 (s, 1H), 9.67
(s, 1H), 14.02 (s, 1H). MS (ES), m/z: 337 (ESꢁ).
5.2.2. Measurement of NO production
NO production was indirectly assessed by measuring the nitrite
concentration in the culture medium by a colorimetric method
according to the Griess reaction. RAW264.7 cells (5 ꢂ 104 cells/well)
were cultured in 96-well plates and incubated for 24 h. Then the
cells were pretreated with different samples at the concentration of
5.1.4.3. (E)-3-(3,4-Dihydroxyphenyl)-1-(5-hydroxy-2,2-dimethyl-
2H-chromen-6-yl)prop-2-en-1-one (5o). Yellow solid; Yield: 38%;
HPLC, 100%. TLC (petroleum ether/ethyl acetate ¼ 10:1), Rf ¼ 0.30.
1H NMR (400 MHz, CDCl3)
d
: 1.47 (s, 6H), 5.58 (d, 1H, J ¼ 10.0 Hz),

F. Peng et al. / European Journal of Medicinal Chemistry 54 (2012) 272e280
279
10
mL final concentration of LPS and incubation for 22 h. After incu-
bation, 100 L of conditioned media was mixed with the same
m
M for 2 h. Cellular NO production was induced by adding a 1
m
g/
carrageenan treatment, and b is the volume of the right hind paw
before carrageenan treatment.
m
volume of Griess reagent and incubated for 10 min. The total
amount of nitrite present was calculated on the basis of the
absorbance of the sample at 540 nm (SpectraMax M5, Sunnyvale,
CA, USA). The cells were treated with PBS as the blank control and
5.2.8. Induction and assessment of AIA
This model was performed as previous reports [23]. Briefly, 12-
week-old, gonad-intact, male Lewis rats with six rats/group were
injected on the ventral side of the base of the tail with CFA. The
degree of arthritis severity was monitored daily and scored
according to the following disease indices: hind paw erythema,
hind paw swelling, tenderness of the joints, and movements, and
posture. An integer scale of 0 to 3 is used to quantify the level of
erythema (0, normal paw; 1, mild erythema; 2, moderate
erythema; 3, severe erythema) and swelling (0, normal paw; 1, mild
swelling; 2, moderate swelling; 3, severe swelling of the hind paw).
The maximal possible score per day is 12, and this score was seen in
all rats 8 d after CFA injection. A cohort of Lewis rats not injected
with CFA but receiving daily oral doses of vehicle (2% Tween 80/
0.5% methylcellulose) for 21 d were designated normal controls.
CFA-treated rats were dosed with vehicle for 7 d until full joint
inflammation developed and then were randomized into four
groups and treated for another 14 d. One group continued to be
dosed with vehicle, designated CFA hereafter. And one group was
treated with indomethacin (10 mg/kg). The other two groups were
treated with daily oral doses of 5b (10 and 25 mg/kg), designated
CFA-5b.
with LPS (1
mg/mL) as the positive control.
5.2.3. MTT assay for cell viability
The
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) staining method was used to test the cell viability.
Briefly, the cells with a density of 5 ꢂ 104 cells/well were added in
96-well plates and incubated with serum-free media in the pres-
ence of different concentrations of compounds 5b, 5d, 5f, 5h and
indomethacin for 24 h. 10 mL MTT (5 mg/mL in saline) was added
and incubation was continued for additional 4 h. The MTT in live
cells was converted into visible formazan crystals by mitochondrial
succinate dehydrogenase during incubation. The crystals were then
solubilized in DMSO and the absorbance of the mixture at 540 nm
was measured. Relative cell viability was calculated compared with
the absorbance of the blank control group.
5.2.4. Assay of iNOS activity
After treated with LPS (1
(1e100
M) for 2 h at 37 ꢀC, the culture supernatant was removed
and 100
L of NOS assay buffer (1ꢂ) were added to each well. Then
100 L of NOS assay reaction solution (50% NOS assay buffer, 39.8%
MilliQ water, 5%
mg/mL) and 5b, 5d, 5f, and 5h
m
m
5.2.9. Histologic assessment
m
Animals were sacrificed at the indicated time points (d) after the
induction of arthritis. Foot pads were dissected, fixed in neutral
buffered formal saline, and embedded in paraffin. Serial sections
(5
mm thickness) were cut and stained with H&E. The histological
changes were examined under microscope.
L-Arginine solution, 5% 0.1 mM NADPH, 0.2% DAF-
FMDA) was added and incubated for 2 h at 37 ꢀC. Fluorescence was
measured with SpectraMax M5 at excitation of 485 nm and emis-
sion of 528 nm.
5.2.5. Molecular docking assay
5.3. Statistical analysis
iNOS was chosen as the target receptor. The 3D structure of the
receptor was gained from Protein Data Bank (PDB ID: 1r35).
Conformers of molecules were created by the aid of Omega and the
up limit of conformer number for each molecule is set to 2000.
Compound 5b was docked to the active site of iNOS by employing
Experimental values are presented as the arithmetic means ꢃ SD.
An ANOVA test followed by Dunnett’s analysis was used to deter-
mined statistical differences. P < 0.05 was considered to be statis-
tically significant.
a
proteineligand docking program FRED. Scoring functions
Chemgauss version two, shapegauss and screenscore were used for
exhaustive searching, solid body optimizing and interaction
scoring.
Acknowledgments
The authors greatly appreciate the financial support from
National Key Technologies R&D Program of China (2012ZX09103101-
009).
5.2.6. Animals
The studies were performed on male ICR mice and Lewis which
were purchased from the animal breeding laboratories of Sichuan
University Animal Center (Sichuan, Chengdu, China). They were fed
with a standard laboratory diet and tap water and kept at
a constant temperature of 22 ꢀC, under 55.5% relative humidity and
a 12 h darkelight cycle for at least 2 weeks. All experimental
procedures were performed according to the NIH Guide for the Care
and Use of Laboratory Animals.
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