Synthesis and biological evaluation of novel semi-conservative monocarbonyl analogs of curcumin as anti-inflammatory agents

Article abstract of DOI:10.1039/c5md00114e

Curcumin, a natural product, has been shown to exhibit notable anti-inflammatory activities. However, the clinical application of curcumin was restricted by its poor stability and bioavailability. We have reported a series of monocarbonyl analogs of curcumin (MACs) previously. In the present study, we synthesized 32 semi-conservative MACs and evaluated their anti-inflammatory effects in RAW 264.7 macrophages. Most of them showed enhanced abilities to inhibit the lipopolysaccharide (LPS)-induced expression of tumor necrosis factor alpha (TNF-α). The preliminary structure-activity relationship was discussed and the preliminary anti-inflammatory mechanism was also explored. The most potent analog compound, WZ35, exhibits significant protection against LPS-induced death in septic mice. These findings suggest that this kind of curcumin derivative may be used to develop promising anti-inflammatory agents to treat inflammatory diseases.

Full text of DOI:10.1039/c5md00114e

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ARTICLE
Synthesis and biological evaluation of novel semi-conservative
mono-carbonyl analogs of curcumin as anti-inflammatory agents
Received 00th January 20xx,
Accepted 00th January 20xx
Zhe Wang,#a Peng Zou,#a Chenglong Li,b,a Wenfei He,a Bing Xiao,a Qilu Fang,a Wenbo Chen,a Suqing
Zheng,a Yunjie Zhao,*a Yuepiao Cai,*a and Guang Lianga
DOI: 10.1039/x0xx00000x
Curcumin, a natural product, has been shown to possess notable anti-inflammatory activities. However, the clinical
application of curcumin was restricted by its poor stability and bioavailability. We have reported a series of mono-carbonyl
analogs of curcumin (MACs) previously. In the present study, we synthesized 32 semi-conservative MACs and evaluated
their anti-inflammatory effects in RAW 264.7 macrophages. Most of them showed enhanced abilities to inhibit the
lipopolysaccharide (LPS)-induced expression of tumor necrosis factor alpha (TNF-α). The preliminary structure-activity
relationship was discussed and the preliminary anti-inflammatory mechanism was also explored. The most potent analog
compound WZ35 exhibits significant protection against LPS-induced death in septic mice. These findings suggest that this
kind of curcumin derivative may be used to develop promising anti-inflammatory agents to treat inflammatory diseases.
www.rsc.org/
inflammatory
agents.
Curcumin
(1,7-bis(p-hydroxy-m-
1. Introduction
methoxyphenyl)-1,6-heptdiene-3,5-dione), a yellow powder, is
the major component in Curcuma longa. It has been
demonstrated to possess multiple pharmacological activities
and medicinal applications, such as anti-inflammatory,16-18 anti-
cancer,19, 20 and anti-oxidation 21, 22. In addition, clinical trials
Numerous studies have proved that the inflammatory response
plays an important role in the pathological processes of various
diseases, including sepsis,1 cancer,2 diabetes,3 and
inflammatory bowel4. In particular, sepsis, an excessive and
irregular systemic inflammatory response syndrome against
serious infection, is believed to be related to an exacerbated
release of pro-inflammatory cytokines, such as TNF-α.5, 6 TNF-
α, a representative and multifunctional cytokine, is produced
primarily by activated monocytes/macrophages and is crucial in
the initiation and continuation process of inflammation and
immunity.7, 8 The expression of TNF-α is regulated by nuclear
factor (NF-κB), which is a key transcription factor of
lymphocytes and macrophages.9 Thus, an effective NF-κB
inhibitor is considered to be a potential anti-inflammatory drug
manifest that curcumin does not lead to discernible toxicities
24
even under a high-dose.23,
However, its poor metabolic
stability and low bioavailability have dramatically restricted its
therapeutic application. Several studies have found that the β-
diketone moiety in the structure of curcumin contributes to its
26
instability in vivo and leads its rapid degradation.25,
Previously, our research group has synthesized several different
Mono-carbonyl Analogs of Curcumin (MACs), most of which
possess enhanced anti-inflammatory properties and stability
compared to curcumin.27-30
More recently, some studies suggested that asymmetric
MACs are able to improve survival from lethal sepsis in septic
mouse model.31, 32 However, there are big structural differences
between the reported asymmetric MACs and curcumin. For the
purpose of studying more structurally conserved curcumin
analogues, in our ongoing research for anti-inflammatory
MACs, we focused on a new mono-carbonyl analog of
curcumin containing the same 4-hydroxy-3-methoxyphenyl on
curcumin, named as semi-conservative MAC. This moiety, 4-
hydroxy-3-methoxyphenyl, is common structural unit of many
biologically active nature products, such as curcumin, vanillin,
ferulic acid, guaiacol, eugenol and so on. Our pervious results
and some other papers showed the presence of 4-hydroxy-3-
candidate for the treatment of inflammation-related diseases.10,
11
Traditional
non-steroidal
anti-inflammatory
drugs
(NSAIDs), acting as inhibitors of the enzyme cyclooxygenase,
are widely used for the treatment of acute or chronic conditions
where pain and inflammation are present.12, 13 However, long
term use of NSAIDs is limited by their serious gastrointestinal
15
side effects.14,
The risk of gastric ulceration increases
dramatically with long therapy duration and high doses. As a
result, it’s urgent to develop novel and potential anti-
a.Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou
Medical University, Wenzhou, Zhejiang 325035, China
methoxyphenyl is essential to obtain high biological activity.29,
E-Mail: ypcai@wmu.edu.cn, aabye1100@aliyun.com
30, 33
b.Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio
State University, Columbus, Ohio 43210, United States
Here,
a series of semi-conservative MACs were
synthesized and their corresponding anti-inflammatory
activities were tested in mouse RAW 264.7 macrophages. Most
#.These authors contribute equally to this work
†
Electronic supplementary information (ESI) available
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of them showed enhanced abilities to inhibit the LPS-induced hours, and then treated with LPS (0.5 μg/mL) fVoierw2A2rticlheoOunlrinse.
DOI: 10.1039/C5MD00114E
expression of TNF-α. Especially, the compound WZ35, a TNF-α levels in the culture media were measured by ELISA and
potent NF-κB inhibitor, exhibited a significant therapeutic were normalized by the total protein. The results were
effect in the LPS-induced septic mouse model.
expressed as the percent of LPS control. Each bar represents
mean ± standard error of the mean (SEM) of 3-5 independent
experiments. Statistical significance relative to LPS group was
indicated, *p< 0.05, **p< 0.01.
2. Results and Discussion
2.1.Chemistry
Methyl thiazolyl tetrazolium (MTT) was applied to measure
the cytotoxicity and safety of analogues in the human normal
hepatic cell line, HL-7702, after 24 hours treatment of the cells
with compounds at a concentration of 10 μM. As shown in Fig.
2B, no compounds displayed apparent toxicity in hepatic cells,
suggesting that they are relatively safe.
The synthetic routes and structure of semi-conservative
curcumin derivatives are shown in Fig. 1. Intermediate
3 was
prepared from vanillin by protection of the alcohol group using
3,4-dihydro-2H-pyran in the presence of pyridinium p-
toluenesulfonate (PPTS) in dichloromethane and subsequent
aldol condensation with acetone in basic condition. Conversion
of intermediate
3 to semi-conservative MACs except WZ21
2.3.Preliminary structure activity relationship
(SAR)
and WZ26 were achieved by aldol condensation with different
substituted aromatic aldehydes in basic condition, followed by
hydrolysis reaction. WZ21 and WZ26 were prepared in acidic
condition directly.
As shown in Fig. 1, all synthetic semi-conservative MACs are
composed of two parts including ring A and ring B. Obviously,
all the structural differences of synthetic compounds are
presented on ring B while the ring A remains all the same. They
suppressed TNF-α with inhibition ratios ranging from 6% to
96%, and most of them showed better inhibitory activity than
curcumin. To investigate the SAR on ring B, the influence of
group size, electronic properties, and position were tested by
modifying the substituents of analogs. Obviously, the
heterocycle and long chain containing analogs, such as
Insert Fig. 1
Fig. 1 Structures and synthesis of MACs compounds. Reagents
and conditions: (i) 3,4-dihydro-2H-pyran, PPTS, CH2Cl2, rt; (ii)
acetone, NaOH, EtOH, rt; (iii) NaOEt, EtOH, rt; (iii’) HCl(g), EtOH,
rt; (iv) HCl, THF, rt.
compounds WZ28
, WZ33, WZ29, WZ31 and WZ36,
2.2.Anti-inflammatory and cytotoxic evaluation of
synthetic semi-conservative MACs
exhibited relatively weaker activities which indicate that too
large substituents at ring B are unfavorable for the anti-
inflammatory activities. On the other hand, most of mono-
The stimulation of macrophages with the bacterial endotoxin
LPS induces a pro-inflammatory process through the activation
of cytokine expression and release.34 Curcumin and 32 semi-
conservative MACs were evaluated for their ability to inhibit
TNF-α release in mouse RAW 264.7 macrophages.
Macrophages were incubated with the compounds at a
concentration of 10 μM for 2 hours and then stimulated with
LPS for 22 hours. The quantities of TNF-α in the media were
detected by Enzyme-Linked Immunosorbant Assay (ELISA)
and the protein concentrations of cells harvested from the
corresponding cultural plates were used to normalize the
cytokines level.
substituted analogs (compounds WZ01, WZ03, WZ17 and
WZ21) also displayed less activity than di- or tri-substituted
analogs, which suggested that groups with little steric hindrance
at ring B are also unfavorable for the activities. In summary, the
size of substituents may play a crucial role in the anti-
inflammatory activity of the asymmetrical monocarbonyl
curcumin analogs.
2.4.Active compounds inhibit the LPS-induced
cytokine release in a dose-dependent manner
To further confirm the anti-inflammatory properties of the
The results of the anti-inflammation assay are illustrated in
Fig. 2A. The initial screening indicates that the majority of
semi-conservative MACs showed more intense anti-
inflammatory activities than curcumin at the concentration of
10 μM. Among the 32 compounds, 31 showed better inhibitory
effects than curcumin on LPS-induced TNF-α expression.
active compounds, compounds WZ08, WZ12, WZ19, WZ32,
WZ35, and WZ37 were selected to investigate their inhibitory
activity against LPS-stimulated TNF-α and interleukin-6 (IL-6)
release in a dose-dependent manner. Both RAW 264.7 and
primary mouse peritoneal macrophages were pretreated at
different concentrations (1.1, 3.3 and 10.0 μM) of the six active
analogs for 2 hours and then were incubated with LPS (0.5
μg/mL) for 22 hours. As illustrated in Fig. 3, all of six
compounds exhibited a dose-dependent inhibition activity
against LPS-induced TNF-α and IL-6 release in the two
different types of macrophages. The result further suggested the
potential of these semi-conservative MACs as anti-
inflammatory agents.
Particularly, compounds WZ4, WZ7, WZ12, and WZ35
almost completely inhibited the release of TNF-α.
Insert Fig. 2
Fig 2Curcumin and its analogs inhibited LPS-induced TNF-α (A)
secretion in RAW 264.7 macrophages and the cytotoxic
evaluation (B) in HL-7702 cells. Macrophages were plated at a
density of 4.0 x 105/plate at 37 °C and 5% CO2 overnight. Cells
Insert Fig. 3
were pre-treated with curcumin or its analogs (10 μM) for 2
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In addition, to clarify the mechanism of anti-inflammatory
View Article Online
DOI: 10.1039/C5MD00114E
Fig. 3 Six active MACs inhibited LPS-induced TNF-α and IL-6 effects of the most active compound WZ35, we try to identify
release in a dose-dependent manner. Macrophages were the possible signaling pathways responsible for the production
plated at a density of 4× 105/plate overnight in 37 °C and 5% of pro-inflammatory cytokines that are inhibited by our
CO2. Cells were pretreated with active compounds in a series compounds. We tested the effect of WZ35 on NF-κB known to
concentration of 1.1 μM, 3.3 μM, and 10 μM for 2 hours and be activated by LPS. NF-κB is a nuclear transcriptional factor
subsequently incubated with or without LPS (0.5 μg/mL) for 22 and plays a crucial role in regulating immune response and
hours. TNF-α and IL-6 levels in the culture medium of RAW inflammation.37 While in an inactivated state, NF-κB is located
264.7 macrophages (A and B) and primary macrophages (C and in the cytosol and complex with the inhibitory protein IκB-α. In
D
) were measured by ELISA and were normalized by the total response to LPS, IκB kinase β phosphorylates cytoplasmic IκB-
protein. The results were presented as the percent of LPS α, which results in ubiquitination, dissociation of IκB-α from
control. Each bar represents the mean ±SEM of three NF-κB. The activated NF-κB is then translocated into the
independent experiments. Statistical significance relative to nucleus to promote the transcription of inflammatory genes.38, 39
the LPS group was indicated, *p< 0.05, **p< 0.01.
Since the activation of NF-κB is regulated by the degradation of
IκB-α, we evaluated the effects of the analog on IκB-α
2.5. Active compounds inhibited LPS-induced degradation in LPS-stimulated RAW 264.7 cells. As shown in
inflammatory gene expression
Fig. 5A-B, WZ35 dose-dependently reduced the LPS-induced
Apart from TNF-α and IL-6, interleukin-1 beta (IL-1β) and IκB-α degradation. The amount of P65 decreased in nucleus
cyclooxygenase-2 (COX-2) are two other important (P65N) but increased in cytoplasm (P65C) by increasing
inflammatory cytokines.35 The mRNAs levels of the pro- concentration of the compound. Furthermore, we also detected
inflammatory cytokines, such as TNF-α, IL-6, IL-1β, and COX- the binding ability of NF-κB with DNA in nucleus of LPS-
2, are important and sensitive indicators of inflammation. stimulated RAW264.7 cells, which pretreated with a different
During the inflammatory response, the expression of mRNAs concentration of WZ35 by EMSA assay. As shown in Fig. 5C
,
for these inflammatory cytokines will be markedly up- WZ35 significantly inhibited the NF-κB binding to DNA in a
regulated.36 We tested the inhibitory effects of these six dose-dependent manner. These results demonstrate that
compounds on inflammatory gene transcription in LPS- compound WZ35 inhibit the LPS-induced activation of NF-κB,
stimulated macrophages. As shown in Fig. 4A, the LPS- which may be involved in the anti-inflammatory actions.
induced up-regulated mRNA level of TNF-α, IL-6, IL-1β, and
COX-2 could be significantly reduced through pretreatment
with these six analogs, respectively. Furthermore, the results in
Insert Fig. 5
the Fig. 4B indicated that the best compound WZ35 possessed Fig. 5 Compound WZ35 inhibited LPS-induced NF-κB activation
the inhibitory activities against the up-regulated mRNA level in in RAW 264.7 macrophages. (A) Macrophages were pretreated
a
dose-dependent manner. The chemical stability of with WZ35 at 2.5 μM, 5 μM, and 10 μM or vehicle control for 2
representative compounds, WZ19 and WZ35 were detected hours and then treated with LPS (0.5 μg/mL) for 1 hour. The
and measured by UV-visible absorption in phosphate buffer. level of IκB-α and P65 were examined using a specific antibody
The results indicate that MACs are more stable than curcumin with GAPDH or LaminB as the loading control. (B) The column
in the buffer at physiological pH 7.4 (Fig. S1)
.
figures show the normalized optical density as times of the LPS
group. Bars represent the mean ± SE M of the three
independent experiments (*p< 0.05, **p< 0.01 versus the LPS
group). (C) Macrophages were pretreated with WZ35 (1 μM,
Insert Fig. 4
Fig. 4 Six active MACs inhibited LPS-induced mRNA of 2.5 μM, 5 μM, and 10 μM) or vehicle (DMSO) for 2 hours and
inflammatory mediators release in RAW 264.7 macrophages. then stimulated with LPS (0.5 μg/mL) for 1 hours, and then
Macrophages were pre-treated with (A) compounds WZ08
WZ12 WZ32 WZ35, and WZ37 at 10 μM, (B) WZ35 at 2.5 μM, by EMSA kit.
5 μM, and 10 μM or vehicle control for 2 hours and then
,
detected the bind ability between NF-κB and DNA in nucleus
,
,
stimulated with LPS (0.5 μg/mL) for 6 hours. Macrophages 2.7.Active compound WZ35 inhibited LPS-induced
were plated at a density of 4.0×105/plate for overnight in 37 °C septic death in mice
and 5% CO2. The mRNA levels of inflammatory mediators IL-6, It is reported that LPS, a representative endotoxin, has been
TNF-α, IL-1β and COX-2 were quantified by RT-qPCR. The implicated as a main cause of sepsis.40, 41 Lastly, we further
mRNA values for each gene was normalized to internal control determined whether compound WZ35 is able to attenuate LPS-
β-actin mRNA and were expressed as a ratio to dimethyl induced septic death in vivo. C57BL/6 mice were injected
sulfoxide (DMSO). Each bar represents mean ± SEM of 3-5 intravenously with the compounds WZ35 at a dosage of 10
independent experiments. Statistical significance relative to mg/kg, 15 minutes later 20 mg/kg of LPS was injected
LPS group was indicated, *p< 0.05, **p< 0.01.
intraperitoneally and then the survival rates were recorded for 7
days. As shown in Fig. 6, 100% of the animals treated with
2.6.Active compounds inhibited the LPS-induced LPS alone died within 50 hours because of septic shock. The
activation of NF-κB
survival rates were increased from 0% to 55% by pre-treatment
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with WZ35. These data suggest that WZ35 exhibits anti- J = 10.2 Hz, 1H, Ar2-H3), 7.64(d, J = 8.4 Hz, 1H, Ar2-H6), 7.37-
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inflammatory activity in vivo.
Insert Fig. 6
7.34(m, 2H, Ar2-H4,5), 7.24(d, J = 7.8DHOzI:,101.1H03, 9A/Cr51M-CDH00=1C14)E,
7.20(d, J = 7.8 Hz, 1H, Ar1-C=CH), 7.12(s, 1H, Ar1-H2), 7.00(d,
J = 16.2 Hz, 1H, Ar2-C=CH), 6.97(d, J = 3.6 Hz, 1H, Ar1-H5),
6.95(d, J = 3.6 Hz, 1H, Ar1-H6), 5.92(s, 1H, Ar1-OH), 3.96(s,
Fig.6 Compound WZ35 improve survival of mice subjected to a 3H, Ar1-OCH3). ESI-MS m/z: 359.3(M+1)+, calcd for
lethal dose of LPS. Mice were intravenously injected with 10 C18H15BrO3: 358.02.
mg/kg WZ35 15 minutes before intraperitoneal injection of
LPS 20 mg/kg. Survival rates were recorded for 7 days after the (1E,4E)-1-(3-bromophenyl)-5-(4-hydroxy-3-
LPS injection at the interval of 1 day. n=9 animals in each methoxyphenyl)penta-1,4-dien-3-one (WZ02
group. Statistical significance relative to LPS group was Yellow powder, 65% yield, mp 97.2-99.8 °C. H NMR (600
)
1
indicated, ** p<0.01.
MHz, CDCl3): δ = 7.70(s, 1H, Ar2-H2), 7.69(d, J = 15.6 Hz, 1H,
Ar2-CH=C), 7.64(d, J = 16.2 Hz, 1H, Ar1-CH=C), 7.30-7.27(m,
3H, Ar2-H4,5,6), 7.19(d, J = 7.8 Hz, 1H, Ar1-H5), 7.12(s, 1H,
Ar1-H2), 7.09(d, J = 16.2 Hz, 1H, Ar1-C=CH), 6.96(d, J = 7.8
Hz, 1H, Ar1-H6), 6.90(d, J = 15.6 Hz, 1H, Ar2-C=CH), 5.92(s,
1H, Ar1-OH), 3.97(s, 3H, Ar1-OCH3). ESI-MS m/z: 359.4
(M+1)+, calcd for C18H15BrO3: 358.02.
3. Experimental Section
3.1.Chemistry
In general, reagents, solvents, and other chemicals were used as
purchased without further purification. All reagents for
synthesis were obtained from Sigma Aldrich and Fluka. Thin-
layer chromatography (TLC) was performed on Kieselgel 60
(1E,4E)-1-(4-bromophenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ03
)
1
Yellow powder, 56% yield, mp 101.2-104.8 °C. H NMR (600
MHz, CDCl3): δ = 7.68(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.66(d,
J = 16.2 Hz, 1H, Ar1-CH=C), 7.55(d, J = 8.4 Hz, 2H, Ar2-H2,6 ),
7.48(d, J = 8.4 Hz, 2H, Ar2-H3,5), 7.19(d, J = 8.4 Hz, 1H, Ar1-
H5), 7.12(s, 1H, Ar1-H2), 7.08(d, J = 16.2 Hz, 1H, Ar1-C=CH),
6.96(d, J = 8.4 Hz, 1H, Ar1-H6), 6.90(d, J = 16.2 Hz, 1H, Ar2-
C=CH), 5.91(s, 1H, Ar1-OH), 3.96(s, 3H, Ar1-OCH3). ESI-MS
m/z: 359.3 (M+1)+, calcd for C18H15BrO3: 358.02.
F
254 plates and flash column chromatography (medium pressure
liquid chromatography) purifications were carried out using
Merck silica gel 60 (230-400 mesh ASTM) (Merck KGaA,
Darmstadt, Germany). Melting points were determined on a
1
Fisher-Johns melting apparatus and were uncorrected. H NMR
spectra were recorded on a Bruker 600 MHz instruments. The
chemical shifts were presented in terms of parts per million
with TMS as the internal reference. Electron-spray ionization
mass spectra in positive mode (ESI-MS) data were recorded on
a Bruker Esquire 3000t spectrometer. All general chemicals
were the highest available grade.
(1E,4E)-1-(2-bromo-5-fluorophenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ04
)
1
Yellow powder, 61% yield, mp 175.1-177.4 °C. H NMR (600
MHz, CDCl3): δ = 7.98(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.70(d,
J = 15.6 Hz, 1H, Ar1-CH=C), 7.60(d, J = 9.0 Hz, 1H, Ar2-H3),
7.40(d, J = 9.0 Hz, 1H, Ar2-H4), 7.19(d, J = 8.4 Hz, 1H, Ar1-H5),
7.12(s, 1H, Ar1-H2), 7.00(s, 1H, Ar2-H6), 6.94(d, J = 15.6 Hz,
1H, Ar1-C=CH), 6.93(d, J = 16.2 Hz, 1H, Ar2-C=CH), 6.89(d, J
= 8.4 Hz, 1H, Ar1-H6), 5.91(s, 1H, Ar1-OH), 3.96(s, 3H, Ar1-
OCH3). ESI-MS m/z: 377.0 (M+1)+, calcd for C18H14BrFO3:
376.01.
3.1.1.Synthesis of WZ01-WZ20, WZ25, and WZ28-WZ36
A solution of intermediate compound
3 (0.002 mol) and various
aromatic aldehydes (0.002 mol) in EtOH (30 mL) was added
NaOEt (1 mL) and then stirred at room temperature for 3 – 4
hours. The resulting mixture was diluted with H2O (15 mL) and
extracted with EtOAc. The combined organic layers were
washed with brine (15 mL) and dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The residue
was further purified by chromatography on silica gel to afford
target semi-conservative MACs.
(1E,4E)-1-(2-bromo-6-fluorophenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ05
)
1
Yellow powder, 66% yield, mp 151.5-157.9 °C. H NMR (600
MHz, CDCl3): δ = 7.86(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.68(d,
J = 16.2 Hz, 1H, Ar1-CH=C), 7.47(d, J = 7.8 Hz, 1H, Ar2-H3),
7.32(d, J = 16.2 Hz, 1H, Ar2-H5), 7.20-.7.17(m, 2H, Ar2-H4,
Ar1-H5), 7.13-7.10(m, 2H, Ar1-H2, Ar1-C=CH), 6.95(d, J = 8.4
Hz, 1H, Ar1-H6), 6.90(d, J = 16.2 Hz, 1H, Ar2-C=CH), 5.91(s,
1H, Ar1-OH), 3.97(s, 3H, Ar1-OCH3). ESI-MS m/z: 377.0
(M+1)+, calcd for C18H14BrFO3: 376.01.
3.1.2.Synthesis of WZ01-WZ20, WZ25, and WZ28-WZ36
A solution of intermediate compound
3 (0.002 mol) and various
aromatic aldehydes (0.002 mol) in EtOH (30 mL) was stirred at
room temperature. Subsequently, HCl (gas) was bubbled into
the mixture for 30 minutes, and the resulting reaction solution
was stirred for 4 hours at room temperature. The mixture was
post-processed the same as 3.1.1.
(1E,4E)-1-(2-bromophenyl)-5-(4-hydroxy-3-
(1E,4E)-1-(4-bromo-3-fluorophenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ01
Yellow powder, 53% yield, mp 95.5-98.4 °C. H NMR (600
MHz, CDCl3): δ = 8.06(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.68(d,
)
methoxyphenyl)penta-1,4-dien-3-one (WZ06
Yellow powder, 48% yield, mp 93.2-95.7 °C. H NMR (600
MHz, CDCl3): δ = 7.69(d, J = 15.6, 1H, Ar2-CH=C), 7.61(d, J
)
1
1
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=15.6, 1H, Ar1-CH=C), 7.60(d, J = 7.2, 1H, Ar2-H5), 7.36(d, J J = 16.4 Hz, 1H, Ar1-CH=C), 7.70-7.66(m, 4H,VieAwrA2r-tHicle2,O3,n5,l6in)e,
= 7.2, 1H, Ar2-H6), 7.18(d, J = 8.4, 1H, Ar1-H5), 7.12(s, 1H, 7.20(d, J =16.4 Hz, 1H, Ar1-C=CH), 7.16(d, J = 8.4 Hz, 1H,
DOI: 10.1039/C5MD00114E
Ar1-H2), 7.08(d, J =15.6, 1H, Ar1-C=CH), 6.96(d, J = 8.4, 1H, Ar1-H5), 7.12(s, 1H, Ar1-H2), 6.96(d, J = 16.4 Hz, 1H, Ar2-
Ar1-H6), 6.89(d, J =15.6, 1H, Ar2-C=CH), 6.86(s, 1H, Ar2-H2), C=CH), 6.92(d, J = 8.4 Hz, 1H, Ar1-H6), 5.93(s, 1H, Ar1-OH),
5.91(s, 1H, Ar1-OH), 3.97(s, 3H, Ar1-OCH3). ESI-MS m/z: 3.97(s, 3H, Ar1-OCH3). ESI-MS m/z: 349.5 (M+1)+, calcd for
376.8 (M+1)+, calcd for C18H14BrFO3: 376.01.
C19H15F3O3: 348.10.
(1E,4E)-1-(2,5-dibromophenyl)-5-(4-hydroxy-3-
(1E,4E)-1-(2-fluoro-4-methoxyphenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ07
)
methoxyphenyl)penta-1,4-dien-3-one (WZ12)
1
1
Yellow powder, 71% yield, mp 95.2-98.0 °C. H NMR (600 Yellow powder, 72% yield, mp 92.8-95.0 °C. H NMR (600
MHz, CDCl3): δ = 7.96(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.82(s, MHz, CDCl3): δ = 8.01(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.69(d,
1H, Ar2-H6), 7.70(d, J = 15.6 Hz, 1H, Ar1-CH=C), 7.50(d, J = J =16.2 Hz, 1H, Ar1-CH=C), 7.52(d, J =8.4 Hz, 1H, Ar2-H6),
8.4 Hz, 1H, Ar1-H5), 7.36(d, J = 8.4 Hz, 1H, Ar2-H4), 7.20(d, J 7.21(d, J = 8.4 Hz, 1H, Ar1-H5), 7.20(s, 1H, Ar2-H3), 7.12(s, 1H,
= 8.4 Hz, 1H, Ar2-H3), 7.12(s, 1H, Ar1-H2), 7.01(d, J = 15.6 Hz, Ar1-H2), 6.96(d, J =8.4 Hz, 1H, Ar1-H6), 6.95(d, J = 16.2 Hz,
1H, Ar1-C=CH), 6.96(d, J = 8.4 Hz, 1H, Ar1-H6), 6.92(d, J = 2H, Ar1-C=CH, Ar2-C=CH), 6.82(d, J = 8.4 Hz, 1H, Ar2-H5),
16.2 Hz, 1H, Ar2-C=CH), 5.92(s, 1H, Ar1-OH), 3.97(s, 3H, Ar1- 5.90(s, 1H, Ar1-OH), 3.97(s, 3H, Ar1-OCH3), 3.85(s, 3H, Ar2-
OCH3). ESI-MS m/z: 435.7, calcd for C18H14Br2O3: 435.93.
OCH3). ESI-MS m/z: 329.4 (M+1)+, calcd for C19H17FO4:
328.11.
(1E,4E)-1-(3-bromo-4-methoxyphenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ08
Yellow powder, 76% yield, mp 133.2-135.4 °C. H NMR (600 methoxyphenyl)penta-1,4-dien-3-one (WZ13
)
(1E,4E)-1-(2-fluoro-5-methoxyphenyl)-5-(4-hydroxy-3-
1
)
MHz, CDCl3): δ = 7.86(s, 1H, Ar2-H2), 7.68(d, J = 15.6 Hz, 1H, Yellow powder, 70% yield, mp 106.2-108.5 °C. H NMR (600
Ar2-CH=C), 7.62(d, J = 15.6 Hz, 1H, Ar1-CH=C), 7.51(d, J = MHz, CDCl3): δ = 7.79(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.69(d,
8.4 Hz, 1H, Ar1-H5), 7.18(d, J = 7.8 Hz, 1H, Ar2-H6), 7.12(s, J = 15.6 Hz, 1H, Ar1-CH=C), 7.19(d, J = 8.4 Hz, 1H, Ar1-H5),
1H, Ar1-H2), 6.98(d, J = 15.6 Hz, 1H, Ar1-C=CH), 6.95(d, J = 7.15(d, J = 15.6 Hz, 1H, Ar1-C=CH), 7.13(s, 1H, Ar1-H2),
7.8 Hz, 1H, Ar2-H5), 6.92(d, J = 8.4 Hz, 1H, Ar1-H6), 6.90(d, J 7.09(d, J = 5.4 Hz, 1H, Ar2-H3), 7.05(d, J = 8.4 Hz, 1H, Ar1-H6),
= 15.6 Hz, 1H, Ar2-C=CH), 5.90(s, 1H, Ar1-OH), 3.96(s, 3H, 6.94(d, J = 16.2 Hz, 1H, Ar2-C=CH), 6.93(s, 1H, Ar2-H6),
Ar2-OCH3), 3.95(s, 3H, Ar1-OCH3). ESI-MS m/z: 388.1, calcd 6.90(d, J =5.4 Hz, 1H, Ar2-H4), 5.91(s, 1H, Ar1-OH), 3.97(s,
1
for C19H17BrO4: 388.03.
3H, Ar1-OCH3), 3.83(s, 3H, Ar2-OCH3). ESI-MS m/z: 329.3
(M+1)+, calcd for C19H17FO4: 328.11.
(1E,4E)-1-(2-bromo-5-methoxyphenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ09
)
(1E,4E)-1-(2-fluoro-3-(trifluoromethyl)phenyl)-5-(4-hydroxy-3-
Yellow oil, 78% yield. 1H NMR (600 MHz, CDCl3): δ = 8.00(d, methoxyphenyl)penta-1,4-dien-3-one (WZ14
)
1
J = 15.6 Hz, 1H, Ar2-CH=C), 7.69(d, J = 15.6 Hz, 1H, Ar1- Brown powder, 68% yield, mp 74.6-76.1 °C. H NMR (600
CH=C), 7.52(d, J = 9.0 Hz, 1H, Ar2-H3), 7.12(s, 1H, Ar1-H2), MHz, CDCl3): δ = 7.81(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.69(d,
7.07(d, J =15.6 Hz, 1H, Ar1-C=CH), 6.97(d, J = 8.6 Hz, 1H, J = 16.2 Hz, 1H, Ar1-CH=C), 7.64(t, J = 7.8 HZ, 1H, Ar2-H4),
Ar1-H5), 6.95(s, 1H, Ar2-H6), 6.93(d, J = 8.6 Hz, 1H, Ar1-H6), 7.30(t, J = 7.8 HZ, 1H, Ar2-H6), 7.24(d, J =16.2 Hz, 1H, Ar1-
6.90(d, J = 15.6 Hz, 1H, Ar2-C=CH), 6.83(d, J = 9.0 Hz, 1H, C=CH), 7.19(d, J = 8.4 Hz, 1H, Ar1-H5), 7.12(s, 1H, Ar1-H2),
Ar2-H4), 5.91(s, 1H, Ar1-OH), 3.96(s, 3H, Ar2-OCH3), 3.85(s, 7.06-7.03(m, 1H, Ar2-H5), 6.96(d, J = 8.4 Hz, 1H, Ar1-H6),
3H, Ar1-OCH3). ESI-MS m/z: 389.3 (M+1)+, calcd for 6.92(d, J = 16.2 Hz, 1H, Ar2-C=CH), 5.94(s, 1H, Ar1-OH),
C19H17BrO4: 388.03.
3.97(s, 3H, Ar1-OCH3). ESI-MS m/z: 367.0 (M+1)+, calcd for
C19H14F4O3: 366.09.
(1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(2-
(trifluoromethyl)phenyl)penta-1,4-dien-3-one (WZ10
Yellow powder, 81% yield, 90.4-93.2 °C. H NMR (600 MHz, methoxyphenyl)penta-1,4-dien-3-one (WZ15
)
(1E,4E)-1-(2,5-difluorophenyl)-5-(4-hydroxy-3-
1
)
1
CDCl3): δ = 8.04(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.70(d, J = Brown powder, 72% yield, mp 104.3-107.4 °C. H NMR (600
16.2 Hz, 1H, Ar1-CH=C), 7.64(d, J = 8.4 Hz, 1H, Ar2-H6), MHz, CDCl3): δ = 7.77(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.69(d,
7.74-7.36(m, 3H, Ar2-H3,4,5), 7.24(d, J = 7.8 Hz, 1H, Ar1-H5), J = 16.2 Hz, 1H, Ar1-CH=C), 7.19(d, J =8.4 Hz, 1H, Ar1-H5),
7.20(d, J = 7.8 Hz, 1H, Ar1-H6), 7.12(s, 1H, Ar1-H2), 7.00(d, J 7.15(d, J = 16.2 Hz, 1H, Ar1-C=CH), 7.12(s, 1H, Ar1-H2), 7.10-
= 16.2 Hz, 1H, Ar1-C=CH), 6.96(d, J = 16.2 Hz, 1H, Ar2- 7.06(m, 3H, Ar2-H3,4,6), 6.96(d, J = 8.4 Hz, 1H, Ar1-H6), 6.92(d,
C=CH), 5.92(s, 1H, Ar1-OH), 3.97(s, 3H, Ar1-OCH3). ESI-MS J = 16.2 Hz, 1H, Ar2-C=CH), 5.92(s, 1H, Ar1-OH), 3.97(s, 3H,
m/z: 349.8 (M+1)+, calcd for C19H15F3O3: 348.10.
Ar1-OCH3). ESI-MS m/z: 317.1 (M+1)+, calcd for C18H14F2O3:
316.09.
(1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(4-
(trifluoromethyl)phenyl)penta-1,4-dien-3-one (WZ11
Yellow powder, 84% yield, mp 116.1-119.5 °C. H NMR (600 methoxyphenyl)penta-1,4-dien-3-one (WZ16
MHz, CDCl3): δ = 7.73(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.71(d,
)
(1E,4E)-1-(2-fluoro-5-nitrophenyl)-5-(4-hydroxy-3-
1
)
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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COMMUNICATION
Journal Name
1
1
Yellow powder, 42% yield, mp 113.1-116.8 °C. H NMR (600 Yellow powder, 43% yield, mp 78.4-80.1 °C. H NMR (600
View Article Online
MHz, CDCl3): δ = 8.27(d, J = 9.6 Hz, 1H, Ar2-H4), 7.83(d, J = MHz, CDCl3): δ = 7.68(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.66(d,
DOI: 10.1039/C5MD00114E
16.2 Hz, 1H, Ar2-CH=C), 7.73(d, J = 16.2 Hz, 1H, Ar1-CH=C), J = 16.2 Hz, 1H, Ar1-CH=C), 7.46-7.41(m, 1H, Ar2-H5), 7.19-
7.69(s, 1H, Ar2-H6), 7.45(d, J = 9.6, 1H, Ar2-H3), 7.21(d, J =8.4 7.16(m, 2H, Ar1-H5, Ar1-H6), 7.12(s, 1H, Ar1-H2), 7.06(d, J
Hz, 1H, Ar1-H5), 7.13(s, 1H, Ar1-H2), 7.07(d, J = 16.2 Hz, 1H, =16.2 Hz, 1H, Ar1-C=CH), 6.95(d, J =7.8 Hz, 1H, Ar2-H6),
Ar1-C=CH), 6.93(d, J = 8.4 Hz, 1H, Ar1-H6), 6.59(d, J = 16.2 6.92(d, J =16.2 Hz, 1H, Ar2-C=CH), 6.90-6.87(m, 1H, Ar2-H4),
Hz, 1H, Ar2-C=CH), 5.89(s, 1H, Ar1-OH), 3.94(s, 3H, Ar1- 5.84(s, 2H, Ar1-OH, Ar2-OH), 3.96(s, 3H, Ar1-OCH3). ESI-MS
OCH3). ESI-MS m/z: 344.3 (M+1)+, calcd for C18H14FNO5: m/z: 297.3 (M+1)+, calcd for C18H16O4: 296.10.
343.09.
(1E,4E)-1-(2,4-bis(trifluoromethyl)phenyl)-5-(4-hydroxy-3-
(1E,4E)-1-(2-chlorophenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ17
methoxyphenyl)penta-1,4-dien-3-one (WZ25
Yellow powder, 78% yield, mp 147.9-150.3 °C. H NMR (600
)
1
)
Yellow oil, 67% yield. 1H NMR (600 MHz, CDCl3): δ = 8.11(d, MHz, CDCl3): δ = 7.66(d, J = 15.6 Hz, 1H, Ar2-CH=C), 7.65(d,
J = 16.2 Hz, 1H, Ar2-CH=C), 7.69(d, J = 15.6 Hz, 1H, Ar1- J = 15.6 Hz, 1H, Ar1-CH=C), 7.25(s, 1H, Ar2-H3)
，7.18(d, J =
CH=C), 7.45(d, J = 9.0 Hz, 1H, Ar2-H3), 7.35-7.30(m, 3H, Ar2- 7.8 Hz, 1H, Ar1-H5), 7.12(s, 1H, Ar1-H2), 7.11(d, J = 8.4 Hz,
H4,5,6), 7.19(d, J =7.8 Hz, 1H, Ar1-H5), 7.12(s, 1H, Ar1-H2), 1H, Ar2-H5), 6.96(d, J = 15.6 Hz, 1H, Ar1-C=CH), 6.95(d, J =
7.05(d, J =7.8 Hz, 1H, Ar1-H6), 6.97-6.94(m, 2H, Ar1-C=CH, 7.8 Hz, 1H, Ar1-H6), 6.90(d, J = 15.6 Hz, 1H, Ar2-C=CH),
Ar2-C=CH), 5.90(s, 1H, Ar1-OH), 3.97(s, 3H, Ar1-OCH3). ESI- 6.87(d, J = 8.4 Hz, 1H, Ar2-H6), 5.94(s, 1H, Ar1-OH), 3.96(s,
MS m/z: 315.1 (M+1)+, calcd for C18H15ClO3: 31407.
3H, Ar1-OCH3). ESI-MS m/z: 417.3 (M+1)+, calcd for
C20H14F6O3: 416.08.
(1E,4E)-1-(2,3-dichlorophenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ18
Yellow powder, 66% yield, mp 107.2-109.5 °C. H NMR (600 methoxyphenyl)penta-1,4-dien-3-one (WZ26
)
(1E,4E)-1-(2-bromo-4-hydroxyphenyl)-5-(4-hydroxy-3-
1
)
1
MHz, CDCl3): δ = 8.09(d, J = 15.6 Hz, 1H, Ar2-CH=C), 7.70(d, Yellow powder, 47% yield, mp 137.9-142.5 °C. H NMR (600
J = 15.6 Hz, 1H, Ar1-CH=C), 7.61(d, J = 7.8 Hz, 1H, Ar2-H4), MHz, CDCl3): δ = 8.02(d, J = 15.6 Hz, 1H, Ar2-CH=C), 7.98(s,
7.51-7.43(m, 2H, Ar2-H5,6), 7.19(d, J = 7.8 Hz, 1H, Ar1-H5), 1H, Ar2-H3), 7.91(d, J = 8.4 Hz, 1H, Ar2-H6), 7.86(d, J = 8.4 Hz,
7.12(s, 1H, Ar1-H2), 7.03(d, J = 15.6 Hz, 1H, Ar1-C=CH), 1H, Ar2-H5), 7.70(d, J = 15.6 Hz, 1H, Ar1-CH=C), 7.19(d, J =
6.96(d, J = 7.8 Hz, 1H, Ar1-H6), 6.94(d, J = 15.6 Hz, 1H, Ar2- 8.4 Hz, 1H, Ar1-H5), 7.12(s, 1H, Ar1-H2), 7.06(d, J = 15.6 Hz,
C=CH), 5.91(s, 1H, Ar1-OH), 3.97(s, 3H, Ar1-OCH3). ESI-MS 1H, Ar1-C=CH), 6.96(d, J = 8.4 Hz, 1H, Ar1-H6), 6.91(d, J =
m/z: 349.0 (M+1)+, calcd for C18H14Cl2O3: 348.03.
15.6 Hz, 1H, Ar2-C=CH), 5.96(s, 1H, Ar1-OH), 3.96(s, 3H, Ar1-
OCH3). ESI-MS m/z: 375.4 (M+1)+, calcd for C18H15BrO4:
374.02.
(1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(2,4,5-
trimethoxyphenyl)penta-1,4-dien-3-one (WZ19
Yellow powder, 87% yield, mp 165.7-167.4 °C. H NMR (600 (1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(indolin-5-yl)penta-
MHz, CDCl3): δ = 8.04(d, J = 15.6 Hz, 1H, Ar2-CH=C), 7.66(d, 1,4-dien-3-one (WZ28
)
1
)
1
J = 15.6 Hz, 1H, Ar1-CH=C), 7.19(d, J =8.4 Hz, 1H, Ar1-H5), Yellow powder, 68% yield, mp 110.5-113.9 °C. H NMR (600
7.11(s, 2H, Ar1-H2, Ar2-H6), 6.98(d, J =15.6 Hz, 2H, Ar1-C=CH, MHz, CDCl3): δ = 7.71(d, J = 15.6 Hz, 1H, Ar2-CH=C), 7.66(d,
Ar2-C=CH), 6.94(d, J = 8.4 Hz, 1H, Ar1-H6), 6.52(s, 1H, Ar2- J = 15.6 Hz, 1H, Ar1-CH=C), 7.51(d, J = 8.4 Hz, 1H, Ar2-H6),
H3), 5.91(s, 1H, Ar1-OH), 3.96(s, 3H, Ar1-OCH3), 3.94(s, 3H, 7.17(d, J = 8.4 Hz, 1H, Ar1-H5), 7.12(s, 1H, Ar1-H2), 6.94(d, J
Ar2-OCH3), 3.91(s, 3H, Ar2-OCH3), 3.89(s, 3H, Ar2-OCH3). = 15.6 Hz, 1H, Ar1-C=CH), 6.93(s, 1H, Ar2-H1), 6.87(d, J =
ESI-MS m/z: 371.1 (M+1)+, calcd for C21H22O6: 370.14.
15.6 Hz, 1H, Ar2-C=CH), 6.55(d, J = 8.4 Hz, 1H, Ar2-H5),
5.88(s, 1H, Ar1-OH), 3.97(s, 1H, Ar2-NH), 3.96(s, 3H, Ar1-
OCH3), 3.36(m, 2H, Ar2-N-CH2), 2.04(m, 2H, Ar2-CH2). ESI-
MS m/z: 322.4 (M+1)+, calcd for C20H19NO3: 321.14.
(1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(2,4,6-
trimethoxyphenyl)penta-1,4-dien-3-one (WZ20
Yellow powder, 90% yield, mp 103.5-105.7 °C. H NMR (600
MHz, CDCl3): δ = 8.16(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.63(d, 4-((1E,4E)-5-(4-hydroxy-3-methoxyphenyl)-3-oxopenta-1,4-
J = 15.6 Hz, 1H, Ar1-CH=C), 7.17(d, J = 8.4 Hz, 1H, Ar1-H5), dien-1-yl)phenyl acetate (WZ29
)
1
)
7.12(s, 1H, Ar1-H2), 6.95(d, J =15.6 Hz, 1H, Ar1-C=CH), Yellow oil, 52% yield. 1H NMR (600 MHz, CDCl3): δ = 7.90(d,
6.90(d, J =16.2 Hz, 1H, Ar2-C=CH), 6.84(d, J = 8.4 Hz, 1H, J = 15.6 Hz, 1H, Ar2-CH=C), 7.69(d, J = 15.6 Hz, 1H, Ar1-
Ar1-H6), 6.14(s, 2H, Ar2-H3,5), 5.86(s, 1H, Ar1-OH), 3.91(s, 3H, CH=C), 7.45(d, J = 7.8 Hz, 2H, Ar2-H2, Ar2-H6), 7.19(d, J = 8.4
Ar1-OCH3), 3.86(s, 3H, Ar2-OCH3), 3.83(s, 3H, Ar2-OCH3), Hz, 1H, Ar1-H5), 7.14(s, 1H, Ar1-H2), 7.09(d, J = 7.8 Hz, 2H,
3.79(s, 3H, Ar2-OCH3). ESI-MS m/z: 371.1 (M+1)+, calcd for Ar2-H3, Ar2-H5), 7.07(d, J = 15.6 Hz, 1H, Ar1-C=CH), 6.94(d, J
C21H22O6: 370.14.
= 8.4 Hz, 1H, Ar1-H6), 6.59(d, J = 15.6 Hz, 1H, Ar2-C=CH),
5.90(s, 1H, Ar1-OH), 3.94(s, 3H, Ar1-OCH3), 2.37(s, 3H, Ar2-
OOC-CH3). ESI-MS m/z: 339.5 (M+1)+, calcd for C20H18O5:
338.12.
(1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(3-
hydroxyphenyl)penta-1,4-dien-3-one (WZ21
)
6 | J. Name., 2012, 00, 1-3
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MedChemComm
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COMMUNICATION
(1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(4-
isopropylphenyl)penta-1,4-dien-3-one (WZ31
Yellow powder, 76% yield, mp 133.8-136.1 °C. H NMR (600 methoxyphenyl)penta-1,4-dien-3-one (WZ36
View Article Online
DOI: 10.1039/C5MD00114E
)
(1E,4E)-1-(4-(allyloxy)phenyl)-5-(4-hydroxy-3-
1
)
1
MHz, CDCl3): δ = 7.70(d, J = 15.6 Hz, 1H, Ar2-CH=C), 7.66(d, Yellow powder, 83% yield, mp 99.8-102.5 °C. H NMR (600
J = 15.6 Hz, 1H, Ar1-CH=C), 7.56(d, J = 9.0 Hz, 2H, Ar2-H2, MHz, CDCl3): δ = 7.57(d, J = 9.0 Hz, 1H, Ar2-CH=C), 7.45(d,
Ar2-H6), 7.18(d, J = 7.8 Hz, 1H, Ar1-H5), 7.12(s, 1H, Ar1-H2), J = 16.2 Hz, 1H, Ar1-CH=C), 7.06(s, 1H, Ar1-H2), 6.97(d, J =
7.09(d, J = 9.0 Hz, 2H, Ar2-H3, Ar2-H5), 6.97(d, J = 15.6 Hz, 16.2 Hz, 1H, Ar1-H5), 6.95(d, J = 9.0 Hz, 1H, Ar1-C=CH),
1H, Ar1-C=CH), 6.95(d, J = 7.8 Hz, 1H, Ar1-H6), 6.59(d, J = 6.91(d, J = 16.2 Hz, 1H, Ar2-C=CH), 6.98-6.90(m, 4H, Ar2-
15.6 Hz, 1H, Ar2-C=CH), 5.91(s, 1H, Ar1-OH), 3.96(s, 3H, Ar1- H2,3,5,6), 6.59(d, J = 16.2 Hz, Ar1-H6 ), 6.08-6.02(m, 1H, Ar2-O-
OCH3), 3.74-3.70(m, 1H, Ar2-CH), 1.24(d, J = 7.2 Hz, 6H, Ar2- C-CH=C), 5.93(s, 1H, Ar1-OH), 5.45-5.31(m, 2H, Ar2-O-C-
C(CH3)2). ESI-MS m/z: 323.5 (M+1)+, calcd for C21H22O3: C=CH), 4.95(d, J = 5.4 Hz, 2H, Ar2-O-CH2-C=C), 3.93(s, 3H,
322.16.
Ar1-OCH3). ESI-MS m/z: 337.4 (M+1)+, calcd for C21H20O4:
336.14.
(1E,4E)-1-(6-bromobenzo[d][1,3]dioxol-5-yl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ32
)
(1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-mesitylpenta-1,4-
Yellow oil, 69% yield. 1H NMR (600 MHz, CDCl3): δ = 7.72(d, dien-3-one (WZ37
)
1
J = 16.2 Hz, 1H, Ar2-CH=C), 7.68(d, J = 15.6 Hz, 1H, Ar1- Yellow powder, 78% yield, mp 123.2-125.5 °C. H NMR (600
CH=C), 7.56(s, 1H, Ar2-H3), 7.54(s, 1H, Ar2-H6), 7.28(d, J = MHz, CDCl3): δ = 8.00(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.65(d,
8.4 Hz, 1H, Ar1-H5), 7.18(d, J = 8.4 Hz, 1H, Ar1-H6), 7.12(s, J = 15.6 Hz, 1H, Ar1-CH=C), 7.44(d, J = 15.6 Hz, 1H, Ar1-
1H, Ar1-H2), 7.05(d, J = 15.6 Hz, 1H, Ar1-CH=C), 6.93(d, J = C=CH), 7.17(d, J = 8.4 Hz, 1H, Ar1-H5), 7.10(s, 1H, Ar1-H2),
16.2 Hz, 1H, Ar2-CH=C), 6.91(s, 2H, Ar2-O-CH2-), 5.93(s, 1H, 6.92(s, 2H, Ar2-H3,5), 6.88(d, J = 8.4 Hz, 1H, Ar1-H6), 6.71(d, J
Ar1-OH), 3.96(s, 3H, Ar1-OCH3). ESI-MS m/z: 403.5 (M+1)+, = 16.2 Hz, 1H, Ar2-C=CH), 5.892(s, 1H, Ar1-OH), 3.96(s, 3H,
4
calcd for C19H15BrO5: 402.01.
Ar1-OCH3), 2.29(s, 6H, Ar2-CH32,6), 2.21(s, 3H, Ar2-CH3 ).
ESI-MS m/z: 323.6 (M+1)+, calcd for C21H22O3: 322.16.
(1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(naphthalen-2-
yl)penta-1,4-dien-3-one (WZ33
)
3.2.Animals
1
Yellow powder, 73% yield, mp 195.7-198.0 °C. H NMR (600 Male C57BL/6 mice weighing 18–22 g were obtained from the
MHz, CDCl3): δ = 7.91-7.84(m, 7H, Ar2-H2-8), 7.77(d, J = 7.2 Animal Center of Wenzhou Medical University (Wenzhou,
Hz, 1H, Ar2-CH=C), 7.72(d, J = 16.2 Hz, 1H, Ar1-CH=C), China). The animals were housed under a constant room
7.22(d, J = 7.2 Hz, 1H, Ar2-C=CH), 7.20(d, J = 8.4 Hz, 1H, temperature with a 12:12 hours light-dark cycle and fed with a
Ar1-H5), 7.14(s, 1H, Ar1-H2), 6.98(d, J = 8.4 Hz, 1H, Ar1-H6), standard rodent diet and water. The animals were acclimatized
6.97(d, J = 16.2 Hz, 1H, Ar1-C=CH), 5.91(s, 1H, Ar1-OH), to the laboratory for at least 7 days before being used in the
3.97(s, 3H, Ar1-OCH3). ESI-MS m/z: 331.2 (M+1)+, calcd for experiments. Protocols involving the use of animals were
C22H18O3: 330.13.
approved by the Wenzhou Medical University Animal Policy
and Welfare Committee (Approval documents:
2012/APWC/0102).
(1E,4E)-1-(2,4-dinitrophenyl)-5-(4-hydroxy-3-
methoxyphenyl)penta-1,4-dien-3-one (WZ34
)
1
Brown powder, 49% yield, mp 122.7-126.5 °C. H NMR (600 3.3.Cells and reagents
MHz, CDCl3): δ = 8.11(s, 1H, Ar2-H3), 8.08(d, J = 9.6 Hz, 1H, RAW 264.7 macrophages were incubated in DMEM medium
Ar2-H5), 7.72(d, J = 16.2 Hz, 1H, Ar2-CH=C), 7.70(d, J = 9.6 (Gibco, Eggenstein, Germany) supplemented with 10% FBS
Hz, 1H, Ar2-H6), 7.56(d, J = 14.4 Hz, 1H, Ar1-CH=C), 7.20(d, J (Hyclone, Logan, UT), 100 U/mL penicillin, and 100 mg/mL
= 8.4 Hz, 1H, Ar1-H5), 7.12(s, 1H, Ar1-H2), 6.96(d, J = 14.4 Hz, streptomycin at 37 °C with 5% CO2. LPS purchased from
1H, Ar1-C=CH), 6.95(d, J = 8.4 Hz, 1H, Ar1-H6), 6.91(d, J = Sigma was dissolved in PBS. Curcumin and its analogues were
16.2 Hz, 1H, Ar2-C=CH), 5.93(s, 1H, Ar1-OH), 3.96(s, 3H, Ar1- dissolved in DMSO before use. For peritoneal macrophage
OCH3). ESI-MS m/z: 371.1 (M+1)+, calcd for C18H14N2O7: preparation, ICR mice were stimulated by intraperitoneal (i.p.)
370.08.
injection of 3 ml thioglycollate solution (0.3 g beef extract, 1 g
tryptone, 0.5g sodium chloride, and 6 g soluble starch were
dissolved and boiled in 100mL water. Before used, the solution
was filtrated with 0.22 μm filter.) per mouse and kept in
(1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(2-
nitrophenyl)penta-1,4-dien-3-one (WZ35
Yellow powder, 61% yield, mp 99.0-111.4 °C. H NMR (600 pathogen-free conditions for
)
1
3
days before peritoneal
MHz, CDCl3): δ = 8.10(d, J = 16.2 Hz, 1H, Ar2-CH=C), 8.00(d, macrophage isolation. Total peritoneal macrophages were
J = 9.6 Hz, 1H, Ar2-H3), 7.72(d, J = 15.6 Hz, 1H, Ar1-CH=C), harvested by washing the peritoneal cavity with PBS 8 mL per
7.71-7.67(m, 3H, Ar2-H4,5,6), 7.56(d, J = 15.6 Hz, 1H, Ar1- mouse), centrifuged, then the pellet was re-suspended in RPMI-
C=CH), 7.20(d, J = 8.4 Hz, 1H, Ar1-H5), 7.12(s, 1H, Ar1-H2), 1640 medium (Gibco, Eggenstein, Germany) with 10% FBS
6.96(d, J = 8.4 Hz, 1H, Ar1-H6), 6.91(d, J = 16.2 Hz, 1H, Ar2- (Hyclone, Logan, UT), 100 U/mL penicillin, and 100 mg/mL
C=CH), 5.93(s, 1H, Ar1-OH), 3.96(s, 3H, Ar1-OCH3). ESI-MS streptomycin. Nonadherent cells were removed by washing
m/z: 326.7 (M+1)+, calcd for C18H15NO5: 325.10.
with medium at 3 hours after seeding. Experiments were
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undertaken after the cells adhered firmly to the culture plates. After treated, protein samples at 50 μg/lane were resolved by
View Article Online
Before used, peritoneal macrophages were cultured in RPMI- SDS-PAGE and the separated proteins DwOeI:re10.t1r0a3n9s/fCe5rMreDd00o1n14toE
1640 medium on 35 mm plates at a density of 4.0×105/plate and nitrocellulose membranes by the wet transfer method using a
maintained at 37 °C in a 5% CO2-humidified air.
Bio-Rad electrotransfer apparatus. Following transfer the blots
were blocked with 5% nonfat milk in Tris buffer saline
containing 0.1% Tween 20 and then incubated with primary
antibodies followed by secondary antibodies. Proteins were
3.4.Determination of TNF-αand IL-6
RAW 264.7 macrophages were treated with LPS (0.5 μg/mL) in visualized by using the enhanced chemiluminescence system.
the presence or absence of compounds for 22 hours. The culture Western blot data presented are representative of those obtained
media were collected and centrifuged (1,000 rpm) at 4 °C for 5 in at least three separate experiments.
minutes, and the supernatant was collected. TNF-α and IL-6
levels in the medium were determined with an ELISA kit 3.8.Preparation of Nuclear Extracts and
(eBioScience, Inc.) according to the manufacturer’s instructions. Electrophoretic mobility shift assay (EMSA)
The total amount of the inflammatory factor in the medium was Nuclear protein was extracted by using nuclear and cytoplasmic
normalized to the total protein quantity of the viable cell pellets. extraction reagents (10 μg) and then was incubated with 1 μg of
poly (deoxyinosinic-deoxycytidylic acid) in binding buffer for
3.5.MTT assay
30 minutes at 4 °C. DNA binding activity was confirmed with a
HL-7702 cells were seeded into 96-well plates at a density of biotin-labeled oligonucleotide bio-NF-κB probe using an
5000 cells per well in 1640 medium, supplemented with 5% EMSA kit.
heat-inactivated serum, 100 U/mL penicillin, and 100 μg/mL
streptomycin. The cells were maintained at 37 °C in a 3.9.LPS-induced inflammatory mortality in
humidified atmosphere containing 5% CO2. All experiments C57BL/6 mice
were carried out 24 hours after cells were seeded. Tested The compounds WZ35 were first dissolved with macrogol-15-
compounds were dissolved in DMSO and diluted with 1640 hydroxystearate (a nonionic solubilizer for injection from
medium to the final concentrations of 10 μM. The cells were BASF [Ludwigshafen, Germany]) with or without medium-
incubated with test compounds for 24 hours before the MTT chain triglycerides (MCT) from BASF in a water bath at 37 °C.
assay. A fresh solution of MTT (5 mg/mL) prepared in NaCl The concentration of the compounds was 2 mg/mL. The
solution (0.9%) was added to each single well of the 96-well concentration of the solubilizer ranged from 5%–10% and that
plate. The plates were then incubated in a CO2 incubator for 4 of MCT ranged from 0.5%–2% in the final solution. For the
hours, cells dissolved with 150 μL DMSO, and then analyzed in vehicle, the mixture of solubilizer and MCT was prepared at 10%
a multi-well-plate reader at 490 nM.
and 2%, respectively. Male C57BL/6 mice weighing 18–22 g
were pretreated with compound WZ35 (10 mg/kg) in a water
solution by intravenous injection 15 minutes before the
3.6.Real-time quantitative PCR
Cells were homogenized in TRIZOL kit (Invitrogen, Carlsbad, intraperitoneal injection of LPS (20 mg/kg). The control
CA) for extraction of RNA according to manufacturer’s animals received a similar volume (200 μL) of the vehicle. The
protocol. Both reverse transcription and quantitative PCR were mortalities were recorded for 7 days.
carried out using a two-step M-MLV Platinum SYBR Green
qPCR SuperMix-UDG kit (Invitrogen, Carlsbad, CA).
Eppendorf Mastercycler ep realplex detection system
(Eppendorf, Hamburg, Germany) was used for q-PCR analysis.
The primers of genes including TNF-α, IL-6, IL-1β, and β-actin
were synthesized by Invitrogen. The primer sequences of
mouse genes used are shown as follows:
4. Conclusions
In summary, we synthesized 32 semi-conservative MACs and
evaluated their anti-inflammatory activities in LPS-stimulated
RAW 264.7 macrophages. The majority of the compounds
presented enhanced anti-inflammatory activities compared to
curcumin. The SAR analysis suggests that the large substituents
are unfavorable for anti-inflammatory activity. The six active
compounds WZ08, WZ12, WZ19, WZ32, WZ35, and WZ37
Mouse TNF-α sense: 5’-TGATCCGCGACGTGGAA-3’
Mouse TNF-α antisense: 5’-ACCGCCTGGAGTTCTGGAA-3’
Mouse IL-6 sense: 5’-CCAAGAGGTGAGTGCTTCCC-3’
Mouse IL-6 antisense: 5’-CTGTTGTTCAGACTCTCTCCCT-3’
Mouse IL-1β sense: 5’-ACTCCTTAGTCCTCGGCCA-3’
Mouse IL-1β antisense: 5’-CCATCAGAGGCAAGGAGGAA-3’
Mouse COX-2 sense 5’-TGGTGCCTGGTCTGATGATG-3’
Mouse COX-2 antisense: 5’-GTGGTAACCGCTCAGGTGTTG-3’
Mouse β-actin sense: 5’-CCGTGAAAAGATGACCCAGA-3’
Mouse β-actin antisense: 5’-TACGACCAGAGGCATACAG-3’
The amount of each gene was determined and normalized by
the amount of β-actin.
not only exhibited dose-dependent inhibition on TNF-α and IL-
6 production, but also showed significant inhibition on
inflammatory gene expression. Furthermore, we have
confirmed that the NF-κB pathway was to be involved in the
anti-inflammatory activity of the most active compounds
WZ35 and pretreatment of WZ35 could improve the survival
rate in LPS-induced acute inflammatory mouse model. Overall,
these results indicate that semi-conservative MACs may be
treated as potential agents for the treatment of acute
inflammatory diseases.
3.7.Western blot
8 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5MD00114E
Fig. 1
Fig. 1 Structures and synthesis of MACs compounds. Reagents and conditions: (i)
3,4-dihydro-2H-pyran, PPTS, CH2Cl2, rt; (ii) acetone, NaOH, EtOH, rt; (iii) NaOEt, EtOH, rt; (iii’)
HCl(g), EtOH, rt; (iv) HCl, THF, rt.

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DOI: 10.1039/C5MD00114E
Fig. 2
Fig. 2 Curcumin and its analogs inhibited LPS-induced TNF-α (A) secretion in RAW 264.7
macrophages and the cytotoxic evaluation (B) in HL-7702 cells. Macrophages were plated at a
density of 4.0 x 105/plate at 37 °C and 5% CO2 overnight. Cells were pre-treated with curcumin or
its analogs (10 μM) for 2 hours, and then treated with LPS (0.5 μg/mL) for 22 hours. TNF-α levels
in the culture media were measured by ELISA and were normalized by the total protein. The
results were expressed as the percent of LPS control. Each bar represents mean ± standard error of
the mean (SEM) of 3-5 independent experiments. Statistical significance relative to LPS group
was indicated, *p < 0.05, **p < 0.01.

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DOI: 10.1039/C5MD00114E
Fig. 3
Fig. 3 Six active MACs inhibited LPS-induced TNF-α and IL-6 release in a dose-dependent
manner. Macrophages were plated at a density of 4× 105/plate overnight in 37 °C and 5% CO2.
Cells were pretreated with active compounds in a series concentration of 1.1 μM, 3.3 μM, and 10
μM for 2 hours and subsequently incubated with or without LPS (0.5 μg/mL) for 22 hours. TNF-α
and IL-6 levels in the culture medium of RAW 264.7 macrophages (A and B) and primary
macrophages (C and D) were measured by ELISA and were normalized by the total protein. The
results were presented as the percent of LPS control. Each bar represents the mean ± SEM of three
independent experiments. Statistical significance relative to the LPS group was indicated, *p <
0.05, **p < 0.01.

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DOI: 10.1039/C5MD00114E
Fig. 4
Figure 4. Six active MACs inhibited LPS-induced mRNA of inflammatory mediators release in
RAW 264.7 macrophages. Macrophages were pre-treated with (A) compounds WZ08, WZ12,
WZ32, WZ35, and WZ37 at 10 μM, (B) WZ35 at 2.5 μM, 5 μM, and 10 μM or vehicle control
for 2 hours and then stimulated with LPS (0.5 μg/mL) for 6 hours. Macrophages were plated at a
density of 4.0×105/plate for overnight in 37 °C and 5% CO2. The mRNA levels of inflammatory
mediators IL-6, TNF-α, IL-1β and COX-2 were quantified by RT-qPCR. The mRNA values for
each gene was normalized to internal control β-actin mRNA and were expressed as a ratio to
dimethyl sulfoxide (DMSO). Each bar represents mean ± SEM of 3-5 independent experiments.
Statistical significance relative to LPS group was indicated, *p < 0.05, **p < 0.01.

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DOI: 10.1039/C5MD00114E
Fig. 5
Fig. 5 Compound WZ35 inhibited LPS-induced NF-κB activation in RAW 264.7 macrophages.
(A) Macrophages were pretreated with WZ35 at 2.5 μM, 5 μM, and 10 μM or vehicle control for
2 hours and then treated with LPS (0.5 μg/mL) for 1 hour. The level of IκB-α and P65 were
examined using a specific antibody with GAPDH or LaminB as the loading control. (B) The
column figures show the normalized optical density as times of the LPS group. Bars represent the
mean ± SE M of the three independent experiments (*p < 0.05, **p < 0.01 versus the LPS group).
(C) Macrophages were pretreated with WZ35 (1 μM, 2.5 μM, 5 μM, and 10 μM) or vehicle
(DMSO) for 2 hours and then stimulated with LPS (0.5 μg/mL) for 1 hours, and then detected the
bind ability between NF-κB and DNA in nucleus by EMSA kit.

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Fig. 6
Fig. 6 Compound WZ35 improve survival of mice subjected to a lethal dose of LPS. Mice were
intravenously injected with 10 mg/kg WZ35 15 minutes before intraperitoneal injection of LPS 20
mg/kg. Survival rates were recorded for 7 days after the LPS injection at the interval of 1 day. n=9
animals in each group. Statistical significance relative to LPS group was indicated, ** p<0.01.