Development of resveratrol-curcumin hybrids as potential therapeutic agents for inflammatory lung diseases

Article abstract of DOI:10.1016/j.ejmech.2016.09.033

Acute lung injury (ALI) is a major cause of acute respiratory failure in critically-ill patients. Resveratrol and curcumin are proven to have potent anti-inflammatory efficacy, but their clinical application is limited by their metabolic instability. Here, a series of resveratrol and the Mono-carbonyl analogs of curcumin (MCAs) hybrids were designed and synthesized by efficient aldol construction strategy, and then screened for anti-inflammatory activities in vitro and in vivo. The results showed that the majority of analogs effectively inhibited the LPS-induced production of IL-6 and TNF-α. Five analogs, a9, a18, a19, a20 and a24 exhibited excellent anti-inflammatory activity in a dose-dependent manner along with low toxicity in vitro. Structure activity relationship study revealed that the electron-withdrawing groups at meta-position and methoxyl group ([sbnd]OCH₃) at the para position of the phenyl ring were important for anti-inflammatory activities. The most promising compound a18 decreased LPS induced TNF-α, IL-6, IL-12, and IL-33 mRNA expression. Additionally, a18 significantly protected against LPS-induced acute lung injury in the in vivo mouse model. The research of resveratrol and MCAs hybrids could bring insight into the treatment of inflammatory diseases and compound a18 may serve as a lead compound for the development of anti-ALI agents.

Full text of DOI:10.1016/j.ejmech.2016.09.033

Accepted Manuscript
Development of resveratrol-curcumin hybrids as potential therapeutic agents for
inflammatory lung diseases
Jialing Pan, Tingting Xu, Fengli Xu, Yali Zhang, Zhiguo Liu, Wenbo Chen, Weitao Fu,
Yuanrong Dai, Yunjie Zhao, Jianpeng Feng, Guang Liang
PII:
S0223-5234(16)30763-2
10.1016/j.ejmech.2016.09.033
EJMECH 8899
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 June 2016
Revised Date: 8 September 2016
Accepted Date: 9 September 2016
Please cite this article as: J. Pan, T. Xu, F. Xu, Y. Zhang, Z. Liu, W. Chen, W. Fu, Y. Dai, Y. Zhao,
J. Feng, G. Liang, Development of resveratrol-curcumin hybrids as potential therapeutic agents
for inflammatory lung diseases, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.09.033.
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ACCEPTED MANUSCRIPT
Development of resveratrol-curcumin hybrids as potential
therapeutic agents for inflammatory lung diseases
a
Jialing Pan a, #, Tingting Xu b, #, Fengli Xu b, Yali Zhang a, Zhiguo Liu a, Wenbo Chen , Weitao Fu a, Yuanrong Dai
b, Yunjie Zhao a, *, Jianpeng Feng a, *, Guang Liang a
a Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University, 1210
University Town, Wenzhou, Zhejiang 325035, China
b Department of Respiration, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang
325027, China
Graphical abstract:
Keywords: Resveratrol, MCAs, parallel, anti-inflammatory, acute lung injury.

ACCEPTED MANUSCRIPT
Development of resveratrol-curcumin hybrids as potential
therapeutic agents for inflammatory lung diseases
a
Jialing Pan a, #, Tingting Xu b, #, Fengli Xu b, Yali Zhang a, Zhiguo Liu a, Wenbo Chen , Weitao Fu a, Yuanrong Dai
b, Yunjie Zhao a, *, Jianpeng Feng a, *, Guang Liang a
a Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University, 1210
University Town, Wenzhou, Zhejiang 325035, China
b Department of Respiration, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang
325027, China
# These authors contribute equally to this work
*Corresponding author:
Yunjie Zhao, Ph.D, Associate Professor
School of Pharmaceutical Sciences, Wenzhou Medical University. 1210 University Town, Wenzhou, Zhejiang
325035, China
Tel: (+86)-577-86699892; Fax: (+86)-577-86699892
E-mail: aabye1100@aliyun.com
Jianpeng Feng, Ph.D, Associate Professor
Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University. 1210
University Town, Wenzhou, Zhejiang 325035, China
Tel: (+86)-577-86699892; Fax: (+86)-577-86699892
E-mail: chemime@163.com

ACCEPTED MANUSCRIPT
Abstract: Acute lung injury (ALI) is a major cause of acute respiratory failure in critically-ill
patients. Resveratrol and curcumin are proven to have potent anti-inflammatory efficacy, but their
clinical application is limited by their metabolic instability. Here, a series of resveratrol and the
Mono-carbonyl analogues of curcumin (MCAs) hybrids were designed and synthesized by
efficient aldol construction strategy, and then screened for anti-inflammatory activities in vitro and
in vivo. The results showed that the majority of analogs effectively inhibited the LPS-induced
production of IL-6 and TNF-α. Five analogs, a9, a18, a19, a20 and a24 exhibited excellent
anti-inflammatory activity in a dose-dependent manner along with low toxicity in vitro. Structure
activity relationship study revealed that the electron-withdrawing groups at meta-position and
methoxyl group (-OCH3) at the para position of the phenyl ring were important for
anti-inflammatory activities. The most promising compound a18 decreased LPS induced TNF-α,
IL-6, IL-12, and IL-33 mRNA expression. Additionally, a18 significantly protected against
LPS-induced acute lung injury in the in vivo mouse model. The research of resveratrol and MCAs
hybrids could bring insight into the treatment of inflammatory diseases and compound a18 may
serve as a lead compound for the development of anti-ALI agents.
Keywords: Resveratrol, MCAs, parallel, anti-inflammatory, acute lung injury.

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1. Introduction
Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is a critical disease
complicated with systemic inflammatory responses in the airspaces and lung parenchyma, which
involves the dysfunction of endothelial and epithelial barriers of the lung, the damage of alveolar–
capillary membrane, excessive transepithelial leukocyte migration, pulmonary edema, and
overproduction of pro-inflammatory mediators [1 5]. Clinically, ALI is a life-threatening problem
−
and leads to multiple organ dysfunctions syndrome (MODS), with significant incidences and
mortalities in critically ill patients [2, 6]. Despite recent advances in which several candidate therapy
strategies have been applied for ALI [1, 7, 8], the treatments remain ineffective with significant and
unacceptable mortality rates [9, 10]. Therefore, highly effective drugs and therapies are required
urgently for the treatment of ALI.
The pathologies of ALI have been generally defined as uncontrolled and excess release of
pro-inflammatory mediators such as cytokines, chemokines, adhesion molecules and bioactive
lipid products [1]. Lipopolysaccharide (LPS), a major constituent of outer membranes of
Gram-negative bacteria, is a pivotal risk factor and prominent stimulus for inflammatory mediator
release by activated macrophages [11]. It is generally used as a well-suited and reproducible model
for preliminary pharmacological evaluation of novel drugs or other therapeutic agents [12]. LPS
works by activating its major receptor TLR4, triggering the innate immune systems [13], with
subsequent activation of nuclear factor (NF)-ꢀB and other signaling molecules. The activated
NF-ꢀB results in a robust production of proinflammatory cytokines such as tumor necrosis
factor-ꢁ (TNF-ꢁ), interleukin-1ꢂ (IL-1ꢂ) and interleukin-6 (IL-6) [14]. Accumulating evidence
indicates that pro-inflammatory cytokines play a critical role in inflammation-induced lung injury
by promoting a cascade of events leading to the occurrencef and progression of ALI. Significantly,
the pharmacological inhibition of inflammatory cytokines reduces the severity of lung injury in
several experimental models [15 16]
.
−

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Resveratrol (3,5,4'-trihydroxy-trans-stilbene), a natural product with low toxicity found in
grapes and various medical plants, has been extensively investigated as anti-inflammatory,
anti-oxidant, anti-cancer, immune regulation, cardioprotective, antidiabetic, and neuroprotective
effects [17 21]. Furthermore, resveratrol interacts with numerous targets within the immune system,
−
such as the NF-ꢀB pathway that results in inhibiting inflammation [22]. However, the biological
activity of resveratrol is limited by its photosensitivity and metabolic instability [23]. Structure
optimization research that is focused on improving the anti-inflammatory and pharmacokinetic
properties has been attempted by many groups [24 29]
.
−
Curcumin is a natural yellow product derived from the turmeric rhizome of the herb Curcuma
Longa, which is shown to also have protective biological activities, such as anti-inflammation,
anti-oxidation, and cardiovascular protection [30-32]. Curcumin can reduce inflammation by
inhibiting release of cytokines such as TNF-ꢁ and IL-6 as reported in both in vitro and in vivo
experimental models [33, 34]. The natural safety and pharmacological efficacy of curcumin make it a
potential agent for the treatment and prevention of a wide variety of human diseases, however, the
clinical application of curcumin is dramatically limited due to its low stability and poor
bioavailability [35]. Evidence suggests that the 1,3-diketone moiety in the structure of curcumin
mainly contributes to its instability under physiological conditions, inducing its rapid degradation
and metabolism [36]. Therefore, several mono-carbonyl analogs of curcumin (MACs), which have
only single carbonyl curcumin analogs, showed greater stability and bioactivity than those of
curcumin [37], some MACs with potential anti-inflammatory activity are shown in Figure 1.
Molecular hybridization [38] is an important molecular modification strategy in the search for new
biologically active compounds, and has been widely used in the design of many therapeutic
classes. The synthesis of hybrids of bioactive compounds with the combined properties of their
individual components has emerged as a fast growing methodology in medicinal chemistry [39-41]
.
The hybridation design strategy is outlined in Figure 2, with the anti-inflammatory efficacy of
resveratrol and stability and bioactivity of MACs, we hypothesize that a hybrid of resveratrol and
MACs analogs would have improved pharmacodynamics and pharmacokinetics properties, and
thereby improved therapeutic value. As shown in Figure 3, the synthesis and screening of 6
hybrids with different linker groups for inhibiting the release of TNF-ꢁ and IL-6 were explored.
One
of
the
compounds,
(1E,4E)-1-{2,4-dimethoxy-6-[(E)

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-4-methoxystyryl]phenyl}-5-(2-trifluoromethylphenyl) penta-1,4-dien-3-one, a1, with penta-1, 4-
dien-3-one linker, was chosen for the structure modification and SAR (structure activity
relationship) studies. It was found that the majority of a1 derivatives had greater
anti-inflammatory activities than that of resveratrol in a dose-dependenet manner. Specially, a18,
(1E,4E)-1-{2,4-dimethoxy-
6-[(E)-4-methoxystyryl]phenyl}-5-(2,3-dimethoxyphenyl)
penta-1,4-dien-3-one, with the best in vitro activities among the a1 derivatives, was tested as a
novel anti-inflammatory agent for treatment of ALI.
2. Chemistry
The synthesis of general resveratrol aldehyde fragment, (E)-2,4-dimethoxy-6-(4-methoxy
[42]
styryl)benzaldehyde, 8 is shown in Scheme 1. According to the method described by Snyder
,
permethylated resveratrol 7 was obtained by treatment of resveratrol with iodomethane (MeI) in
the presence of K2CO3 with 68% yield. With the Vilsmeier reaction of 7 by using POCl3 and DMF
afforded the key aldehyde intermediate 8 with 60% yield.
The synthetic procedures of six different hybrid skeletons, a1-f1, are outlined in Scheme 2.
The aldol condensation of acetone and 2-trifluoromethylbenzaldehyde 9 generated α,β-unsaturated
ketone 10 in a sufficient amount of K2CO3 with 65% yield. Then, the first skeleton a1 was
generated by concentrating aldehyde 8 and ketone 10 in the presence of 20% NaOH with 52.3%
yield.
The skeleton analogs b1 and c1 with aliphatic ring fragment were also synthesized through
the aldol condensation strategy as with a1. In order to avoid the occurrence of symmetrical double
side condensation products, commercially available cyclopentanone 11 and cyclohexanone 12
were activated with morpholine, and the resulting enamines 13 and 14 were condensed with
aldehyde 8 to obtain the desired cyclopentanone skeleton derivative b1 or cyclohexanone skeleton
analogs c1 with 33% and 28% yield, respectively.
The heterocylco derivatives d1-f1 were obtained according to the method of a1. Without the
procedure of activation, the unilateral condensation products 18-20 were obtained by the aldol
reaction of heterocyclic ketones 15-17 with aldehyde 9 in the presence of pyrrolidine. The
following aldol reaction of the resulting α,β-unsaturated ketone 18-20 with the common aldehyde
8 completed the synthesis of other three scaffold analogs d1-f1.

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The general synthesis method of compounds a2-a25 is described in Scheme 3. The reaction
of aldehyde 8 with acetone generated the unsaturated ketone 21, which was reacted with a variety
of substituted aldehydes to give the expected target products with yield of 25%-81%. All
compounds were purified by recrystallization or column chromatography. Their structures were
characterized by 1H NMR and 13C NMR.
3. Results and discussion
3.1. Screening for inhibitory activity against LPS-induced TNF-ꢁ and IL-6 release
TNF-α and IL-6 are two well-known pro-inflammatory cytokines. They play important roles in
the pathological development of many inflammatory diseases. RAW 264.7 macrophages provide
useful cell models in the investigation of anti-inflammatory activities, since their stimulation by
lipopolysaccharides (LPS) induces the upregulation and secretion of pro-inflammatory cytokines
such asTNF-α and IL-6. In order to evaluate the anti-inflammatory activities of the synthetic
compounds, RAW 264.7 macrophages were pre-incubated with the compounds (10 µM) for 30
min and subsequently treated with LPS (0.5 µg mL−1) for 24 h. The conditioned medium was
collected, the IL-6 and TNF-α in the media was quantified by enzyme-linked immunosorbent
assay (ELISA) and normalized by protein concentration of cells harvested from the respective
culture plates.
The anti-inflammatory activities of six of the skeleton analogs are shown in Figure 4. The
results indicated that compound a1 with penta-1,4-dien-3-one linker was more effective in
preventing expression of IL-6 or TNF-α compared with the control compound resveratrol or other
5 skeleton analogs. Therefore, a1 was chosen as the potential leading compound for further
modification and optimization. Figure 5 shows the potential anti-inflammatory activity assessment
of 25 structure derivatives. The screening results indicated that most of the analogs attenuated the
LPS-induced TNF-α and IL-6 secretion at a dosage of 10 µM, with greater potency in inhibiting
IL-6 than TNF-α. Notably, 20 of the 25 synthetic compounds show inhibition of IL-6 secretion at
or greater than 50%. It is noteworthy that compound a9, a18, a19, a20, a24 exhibited a stronger
inhibition of both LPS-induced IL-6 and TNF-α production compared with the referecce
compound resveratrol and C66.
The most promising compounds from those tested, a9, a18, a19, a20, a24, were selected for

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further assessment of their dose-dependent inhibitory effects against LPS-induced IL-6 release.
Macrophages were pretreated with a9, a18, a19, a20, or a24, in a series of concentrations (1, 2.5,
5 and 10 µM) for 30 min and subsequently incubated with LPS (0.5 µg mL−1) for 24 h. As shown
in Figure 6, these compounds significantly decreased IL-6 secretion in a concentration-dependent
manner, indicating potent anti-inflammatory activities.
3.2. Assessment of toxicity and chemical stability of active compounds
As the primary component of Traditional Chinese Medicine, resveratrol and curcumin show
good safety profiles and have been extensively used in animal studies. We evaluated the
cytotoxicity of compounds a1–a25 using the MTT assay on the normal human hepatic cell line
HL-7702. As shown in Figure 7, all of the analogs shown low toxicity at a concentration of 10 µM,
indicating that they are relatively safe. However, because of poor stability, the clinical applications
of curcumin and resveratrol have been limited significantly. Therefore, the chemical stability of
the potential candidate compounds a9, a18, a19, a20, a24 and reference compound resveratrol
was also tested in phosphate buffer (pH = 7.4) containing 2% DMSO using the ultraviolet-visible
(UV-visible) absorption spectra at different times (0, 5, 10, 15, 20 and 25 min). The results
indicated that the optical density (OD) values of the maximal absorption peak of a9, a24 and
resveratrol declined rapidly, but not analogs a18, a19, and a20 (Figure 8), indicating the latter
analogs with greater chemical stability than control compound resveratrol.
3.3. The active compounds suppress LPS-induced pro-inflammatory gene expression in
macrophages
We evaluated the potency of the active synthetic compounds on the inhibition of inflammatory
gene expression to further confirm their anti-inflammatory effects. Mouse peritoneal macrophages
were pretreated with vehicle, resveratrol or active compounds at a concentration of 10 µM,
incubated with LPS (0.5µg mL−1) for 6 h, and mRNA level of pro-inflammatory genes was
determined by RT-qPCR. As shown in Figure 9A-D, LPS up-regulated the mRNA expression of
pro-inflammatory cytokines, including IL-6 (A), IL-12 (B), IL-33 (C) and TNF-α (D), while a18
markedly suppressed their mRNA expression (p<0.01). The data indicate that a18 was a potent
inhibitor of LPS-induced mRNA overexpression of inflammatory genes in macrophages.
3.4. Quantitative evaluation of structure activity relationship (SAR)

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The SAR studies evaluated the structure-functure relationship of the linkers and substituents
with anti-inflammatory activities. Among the six skeleton analogs, a1-f1, compound a1 with the
noncyclic acetone linker displayed the most potent activities, likely attributed to the cyclic linker’s
relative strong rigidity in limiting the sensitivity of bond rotation and decreasing the binding
capacity with target proteins. Therefore, a1 was chosen as the leading skeleton compound, and
other 24 analogs were synthesized by replacement of the 2-trifluoromethyl- benzaldehyde with a
variety of aldehydes.
The anti-inflammatory activities of the compounds are summarized in Figures 4 and 5, showing
that most of the analogs exhibited remarkable inhibition of IL-6 with inhibition ratios ranging
from 27% to 98%. The results clearly indicate the relation of the position and number of subtituent
groups with anti-inflammatory activities. As for the mono substituent analogs, compounds with
the electric neutrality group such as a12 had less activity. However, with an electron-withdrawing
group at the 2-position of phenyl ring resulted to enhancement of the biological activity as
compared with electron-donating group (a14). Furthermore, when the group was transferred to the
3-positon of the phenyl ring, such as compound a3, the activities were increased. The 4-position
substituent group of the phenyl ring was significantly linked to imparting the inhibitory activity
ranging from 36% to 92.5%. The presence of the hydrophilic group such as in a13 and a20, was
associated with greater activity compared to the hydrophobic group (OEt, OBut and OAll). With
analogs with two or more substituent groups in the phenyl group, except a22, the introduction of
electron-withdrawing group at the ortho-position and electron-donating group at meta-position
group resulted in enhanced anti-inflammatory activities (a19, a21 and a23). Based on these SAR
analyses, we conclude that electron-withdrawing substituted compounds are more active than the
electron-donating ones. Additionally, the introduction of 2-methylthiophene (a24) on a right-hand
phenyl unit yielded good consistent activity (IC50 (IL-6) < 4.328 µM), suggesting that the
heterocyclic ring contributed to the anti-inflammatory activity.
To further demonstrate the SAR of these compounds in the inhibition of TNF-α and IL-6
expression, a quantitative SAR (QSAR) was calculated for 25 synthetic resveratrol-curcumin
hybrids. Due to their relatively low activity, SAR of these compounds in the inhibition of TNF-α
and IL-6 eliminated a5, a11 and a24.The results represented by the scatterplot of predicted versus
experimental values are shown in Figure 10. Using three different variables, the statistically

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significant models were obtained for the anti-TNF-α and IL-6 activities of compounds, with
relatively high regression coefficients (R2) of 0.8402 and 0.7595, respectively. The QSAR results
indicate that the electro-negativity and molecular polarizability may play an important role in the
anti-TNF-α activity, while polarizability plays an important role in the IL-6 inhibitory activity.
Taken together, the SAR and QSAR results on the TNF-α and IL-6-inhibitory activities of the
resveratrol and MCAs hybrids may provide valuable information for the further design of novel
anti-inflammatory agents.
3.4. Active compound a18 reduced rat lung pathological changes in response to LPS
challenge
In order to more fully understand the mechanisms underlying the protective effects of a18, we
investigated further a18 in its ability to protect against LPS-induced ALI in mice. The lung
Wet/Dry ratio (W/D), inflammatory cell count and total protein in BALF were determined to
evaluate whether a18 was able to reduce or prevent lung injury in ALI mice. As shown in Figure
11A-D, the concentration of total protein, the number of total cells and of neutrophils in BALF,
and the lung W/D were significantly increased after LPS challenge (5 mg/kg) compared with the
control group. The result indicated that pretreatment with a18 (5 mg/kg) for a week effectively
reduced the concentration of the total protein, the number of total cells and neutrophils in BALF
and the lung W/D. For evaluation of lung histopathology changes in LPS-induced ALI mice, lung
tissues were sampled after tracheal instillation with LPS (5 mg/kg) and treatment with or without
a18 for 6 h, and prepared for histological assessment. LPS challenge induced notable pathologic
changes, such as inflammatory cell infiltration, interstitial edema and interalveolar septal
thickening compared with the control, which presented a normal structure with no histopathologic
changes under light microscope (Figure 11E). Significantly, the administration of a18 (10 µM)
effectively reduced the airspace inflammation Taken together, these results indicated that a18
protected against lung injury in the ALI mice, and further corroborated our structure-function
findings of the anti-inflammatory activitites of the synthetic compounds.
3.5. A18 reduced lung pro-inflammatory cytokine content in ALI
Increases of pro-inflammatory cytokines, occurring in the early phase of an inflammatory
response, play a critical role in ALI, meanwhile, both TNF-α and IL-6 are crucial cytokines in ALI.
In this study, the efficacy of a18 preventing increases of TNF-α and IL-6 levels from ALI mice

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were determined. As shown in Figure 12A-D, the inflammatory cytokine levels were increased
significantly in BALF and serum of mice after LPS challenge compared with the control group.
However, the administration of a18 prevented the LPS-induced increases of TNF-α and IL-6 in
BALF and serum. These results suggest that the a18-mediated inhibition of inflammatory
cytokines might attribut to inhibition of leukocytic extravasation/activation, a major source of
cytokines in BALF. In order to confirm these findings, The immunohistochemical analysis of
CD68, a macrophage marker, indicated that increased CD68-positive macrophages were detected
in lung sections from LPS-challenged mice, whereas the number of CD68-positive macrophages
from a18 pretreated LPS-challenged mice was similar to that of controlmice (Figure 12E). The
results indicated that a18 has valuable therapeutic potential for pulmonary inflammatory diseases.
4. Conclusion
In summary, our findings indicated that a18 was highly effective in reducing the LPS-induced
increases in cytokine mRNA expression and protein secretion in both in vitro and in vivo
experimental models. We conclude that a18 not only has the biological activity, but also the
physical properties of efficacy and stability, as a viable e potential candidate for the treatment of
ALI. Further research of mechanisms and structure optimization based on the lead compound of
a18 are underway by our team.
5. Experimental section
5.1. Chemistry
5.1.1. General
In general, reagents, solvents, and other chemicals were used as purchased without further
purification. All reagents for synthesis were obtained from Aladdin, J&K, Energe Chemical.
Thin-layer chromatography (TLC) was performed on Kieselgel 60 F254 plates and column
chromatography purifications were carried on using Merck silica gel 60 (200-300 mesh). Melting
1
points were determined on a Fish-Johns melting apparatus. HNMR and 13CNMR spectra data
were recorded on a Bruker 600 MHz instruments. The chemical shift was presented in terms of
parts per million with TMS as the internal reference. Electron-spray ionization mass spectra
(ESI-MS) data were recorded on a Bruker Esquire 3000t spectrometer. All reactions were
monitored by the silica gel TLC.
5.1.2. Synthesis of permethylated Resveratrol 7 [42]

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To a solution of resveratrol (1.00 g, 4.38 mmol, 1.0 equiv) in dry acetone (25 mL) was added
K2CO3 (5.45 g, 39.4 mmol, 9.0 equiv). After stirring the reaction mixture for 30 min, CH3I (2.45
mL, 39.4 mmol, 9.0 equiv) was added dropwise by syringe. Then the reaction mixture was stirred
at 40 oC in the dark for 12h. The reaction mixtures were monitored by TLC until completion. After
being quenched with saturated aqueous NH4C1 (25 mL), the reaction contents were poured into
water (10 mL), extracted with EtOAc (3 × 30 mL). The combined organic extracts were then
washed with water (30 mL) and brine (30 mL), dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was further purified by column chromatography
on silica gel (hexanes/EtOAc = 8:1) to afford the permethylated Resveratrol 7 (white solid, 0.81g,
68.6%).
5.1.3. Synthesis of (E)-2, 4-dimethoxy-6-(4-methoxystyryl)benzaldehyde 8[43]
To a solution of 0.54 g compound 7 (2 mmol, 1.0 equiv) in dry DMF (10 mL) was added
dropwise POCl3 (0.4 mL, 4.2 mmol, 2.1 equiv) slowly while cooling in an ice-water bath. After
stirring at room temperature for 1 h, the solution was added to ice-water and kept stirring 30 min,
the solid precipitated, filtered. The residue was purified by column chromatography on silica gel
(hexanes/EtOAc/CH2Cl2 = 8:1:1) to generate 8 (yellow solid, 0.36 g, 60.4%).
5.1.4. Synthesis of (E)-4-(2-trifluoromethylphenyl)but-3-en-2-one 10[44]
To a solution of 2-trifluoromethylbenzaldehyde 9 (0.52 g, 3 mmol, 1.0 equiv) in acetonitrile (10
mL) was added K2CO3 (1.24 g, 9 mmol, 3.0 equiv) and acetone (2.2 mL, 30 mmol, 10.0 equiv)
sequentially. Then the reaction mixture was stirred at room temperature for 2 h. The resulting
solution was quenched with water (10 mL), extracted with EtOAc (3 × 30 mL). The combined
organic layers were washed with brine (10 mL), dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by flash column chromatography
on silica gel (hexanes/EtOAc = 5: 1) to afford the single side concentration product 10 (yellow oil,
0.48g, 65.6%).
5.1.5. Synthesis of morpholine enamines 11 and 12[45]
To a solution of morpholine (10.5 mL, 0.12 mol, 1.2 equiv) in dry toluene (70 mL) was added
cyclopentanone (8.9 mL, 0.10 mol, 1.0 equiv) or cyclohexanone (10.4 mL, 0.10 mol, 1.0 equiv),
followed by p-toluenesulfonic acid (0.20 g, 1.16 mmol, 0.016 equiv) at room temperature. After

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refluxing for 5 h, the resulting solution was cooled and then concentrated under reduced pressure
to give corresponding morpholine enamines 11 and 12 with yield of 60% and 85% respectively.
5.1.6. Synthesis of singe side α, β-unsaturated ketones 13 and 14[45]
To a stirring solution of morpholine enamine 11 (2.30 g, 15 mmol, 3.0 equiv) or 12 (2.51 g, 15
mmol, 3.0 equiv) in Ethanol (15 mL) was added 2-trifluoromethylbenzaldehyde 9 (0.87g, 5 mmol,
1.0 equiv). After refluxing for 5 h, the resulting mixture was acidified by a solution of 5% HCl,
and then extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine
and dried over anhydrous MgSO4, filtered, and concentrated under vacuum; the residue was
further purified by column chromatography on silica gel (hexanes/EtOAc = 15:1) to produce
corresponding unsaturated ketones 13 and 14 with yield of 50.2% and 40.6% respectively.
5.1.7. General synthesis procedure for unsaturated ketones 20-22
To a solution of different heterocyclic ketones 15-17 (2.4 mmol, 1.2 equiv) in CH2Cl2 (5 mL)
was added pyrrolidine (0.34 g, 4.8 mmol, 2.4 equiv), and the reaction mixture were stirred for 5
min. Then 2-trifluoromethylbenzaldehyde (0.35 g, 2.4 mmol, 1.2 equiv) was added and refluxed
for 4 h. The resulting solution was concentrated under reduced pressure, the residue was purified
by column chromatography on silica gel (hexanes/EtOAc = 8:1) to afford corresponding products
18-20 with yield 48.9%, 45.6% and 32.5% respectively.
5.1.7.1. (Z)-3-(2-trifluoromethylbenzylidene)tetrahydro-4H-thiopyran-4-one (18).Yellow oil, 48.9%
yield. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 7.749 (1H, d, J = 7.8 Hz, H-3'), 7.688 (1H, t, J =
7.8 Hz, H-5'), 7.543 (1H, t, J = 7.8 Hz, H-4'), 7.427 (1H, s, H-7'), 7.419 (1H, d, J = 7.8 Hz, H-6'),
3.598 (2H, s, H-2), 3.014 (2H, t, J = 6.0 Hz, H-5), 2.851 (2H, t, J = 6.0 Hz, H-6). 13C NMR (150
MHz, DMSO-d6) δ (ppm): 197.8, 137.5, 132.9, 132.4, 130.6, 129.1, 128.7, 126.0, 124.8, 122.9,
41.0, 27.5, 24.9. ESI-MS m/z: 273.1 (M+H)+, calcd for C13H11F3OS: 272.0.
5.1.7.2. (E)-3-(2-trifluoromethylbenzylidene)tetrahydro-4H-pyran-4-one (19).Yellow oil, 45.6%
yield. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 7.777 (1H, s, H-7'), 7.728 (1H, d, J = 7.8 Hz,
H-3'), 7.552 (1H, t, J = 7.8 Hz, H-5'), 7.468 (1H, t, J = 7.8 Hz, H-4'), 7.130 (1H, d, J = 7.2 Hz,
H-6'), 4.571 (2H, s, H-2), 4.072 (2H, t, J = 6.0Hz, H-6), 2.715 (2H, t, J = 6.0 Hz, H-5). 13C NMR
(150 MHz, DMSO-d6) δ (ppm): 195.5, 136.4, 132.6, 130.8, 129.4, 129.3, 126.2, 126.1, 125.3,
123.0, 67.1, 64.8, 41.0. ESI-MS m/z: 257.1 (M+H)+, calcd for C13H11F3O2: 256.1.

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5.1.7.3. (E)-1-propyl-3-(2-trifluoromethylbenzylidene)piperidin-4-one (20).Yellow oil, 32.5%
yield. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 7.820 (1H, d, J = 7.8 Hz, H-3'), 7.552 (1H, t, J =
7.8 Hz, H-5'), 7.550 (1H, s, H-7'), 7.537 (1H, t, J = 7.8 Hz, H-4'), 7.401 (1H, d, J = 7.8 Hz, H-6'),
3.411 (2H, s, H-2), 2.754 (2H, t, J = 6.0 Hz, H-5), 2.546 (2H, t, J = 6.0 Hz, H-6), 2.299 (2H, t, J =
7.2 Hz, N-CH2CH2CH3 ), 1.280-1.341 (2H, m, N-CH2CH2CH3), 0.734 (3H, t, J = 7.2 Hz,
N-CH2CH2CH3). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 197.0, 136.1, 132.9, 132.2, 130.4,
129.6, 128.8, 125.9, 124.8, 122.9, 58.5, 54.1, 49.6, 38.5, 19.7, 11.2. ESI-MS m/z: 298.1 (M+H) +,
calcd for C16H18F3NO: 297.1.
5.1.8. General procedure for synthesis of different scaffold analogs a1-f1
To a stirring solution of different unsaturated ketones 10, 15-16 and 20-22 (1.2 mmol, 1.2 equiv)
in EtOH were added 10 drops a solution of 20% NaOH, then aldehyde 8 (0.30 g, 1 mmol, 1 equiv)
in CH2Cl2 (5 mL) was added dropwise slowly. The resulting solution was extracted with EtOAc (3
× 10 mL), the combined organic layers were washed with brine and dried over anhydrous MgSO4,
filtered, and concentrated under vacuum, the residue was further purified by column
chromatography on silica gel, hexanes/EtOAc) to afford corresponding scaffold analogs a1-f1
with yield of 30.8%-52.3%.
5.1.8.1. (1E,4E)-1-{2,4-dimethoxy-6-[(E)-4-methoxystyryl]phenyl}-5-(2-trifluoromethylphenyl)
penta-1,4-dien-3-one (a1). Yellow powder, 52.3% yield, mp: 105.7~108.1 oC. 1H-NMR (600 MHz,
DMSO-d6) δ (ppm): 8.140 (1H, d, J = 7.8 Hz, H-3''), 8.037 (1H, d, J = 15.6 Hz, H-5), 7.828 (2H,
m, H-1, H-6''), 7.748 (1H, t, J = 7.8 Hz, H-5''), 7.641 (1H, t, J = 7.8 Hz, H-4''), 7.575 (2H, d, J =
9.0 Hz, H-2''', H-6'''), 7.447 (1H, d, J = 15.6 Hz, H-8'''), 7.381 (1H, d, J = 15.6 Hz, H-4), 7.118 (1H,
d, J = 16.2 Hz, H-2), 7.096 (1H, d, J = 15.6 Hz, H-7'''), 6.959 (2H, d, J = 8.4 Hz, H-3''', H-5'''),
6.847 (1H, s, H-5'), 6.617 (1H, s, H-3'), 3.918 (3H, s, OCH3), 3.891 (3H, s, OCH3), 3.780 (3H, s,
OCH3). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 188.1, 160.6, 159.2, 158.2, 139.2, 139.1, 138.1,
137.7, 134.1, 132.7, 132.4, 131.3, 130.7, 130.2, 129.5, 128.7, 127.9×2, 127.4, 126.0, 123.8, 115.9,
114.2×2, 101.3, 97.5, 55.6, 55.3, 55.1. ESI-MS m/z: 494.4 M+, calcd for C29H25F3O4: 494.2.
5.1.8.2.
(2E,5E)-2-{2,4-dimethoxy-6-[(E)-4-methoxystyryl]benzylidene}-5-(2-trifluoromethyl
1
benzylidene)cyclopentanone (b1). Yellow oil, 32.7% yield. H NMR (600 MHz, DMSO-d6) δ
(ppm): 7.841 (1H, d, J = 7.8 Hz, H-3''), 7.779 (1H, t, J = 7.8 Hz, H-5''), 7.606 (1H, t, J = 8.4 Hz,
H-4''), 7.599 (2H, s, H-7', H-7''), 7.507 (2H, d, J = 8.4 Hz, H-2''', H-6'''), 7.410 (1H, d, J = 8.4 Hz,

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H-6''), 7.231 (1H, d, J = 16.2 Hz, H-8'''), 6.934 (2H, d, J = 7.8 Hz, H-3''', H-5'''), 6.914 (1H, s,
H-5'), 6.892 (1H, d, J = 16.2 Hz, H-7'''), 6.560 (1H, s, H-3'), 3.875 (3H, s, OCH3), 3750 (3H, s,
OCH3),3.723 (3H, s, OCH3), 2.853 (2H, t, J = 6.6 Hz, H-3), 2.438 (2H, t, J = 6.0 Hz, H-4). 13C
NMR (150 MHz, DMSO-d6) δ (ppm): 188.1, 160.6, 159.2, 158.5, 139.3, 139.2, 138.1, 137.7,
134.1, 132.7, 132.4, 131.3, 130.7, 130.2, 129.5, 128.7, 127.9×2, 126.0, 126.0, 123.8, 115.9,
114.2×2, 101.3, 97.5, 55.6, 55.3, 55.1, 28.2, 27.8. ESI-MS m/z: 520.1 M+, calcd for C31H27F3O4:
520.2.
5.1.8.3. (2E,6E)-2-{2,4-dimethoxy-6-[(E)-4-methoxystyryl]benzylidene}-6-(2-trifluoromethylben
zylidene)cyclohexanone (c1). Yellow power, 28.3% yield, mp: 141.2~143.8 oC. 1H NMR (600
MHz, DMSO-d6) δ (ppm): 7.822 (1H, d, J = 8.4 Hz, H-3''), 7.768 (1H, s, H-7'), 7.7010-7.736 (2H,
m, H-4'', H-5''), 7.631 (1H, s, H-7''), 7.542 (1H, d, J = 7.8 Hz, H-6''), 7.488 (2H, d, J = 8.4 Hz,
H-2''', H-6'''), 7.235 (1H, d, J = 16.2Hz, H-8'''), 6.935 (2H, d, J = 8.4 Hz, H-3''', H-5'''), 6.887 (1H,
s, H-5'), 6.812 (1H, d, J = 16.2 Hz, H-7'''), 6.548 (1H, s, H-3'), 3.863 (3H, s, OCH3), 3.798 (3H, s,
OCH3), 3.765 (3H, s, OCH3), 2.701 (2H, t, J = 5.4 Hz, H-3), 2.600(2H, t, J = 5.4 Hz, H-5),
13
1.507-1.548 (2H, m, H-4). C NMR (150 MHz, DMSO-d6) δ (ppm): 188.1, 160.6, 159.2, 158.5,
139.2, 138.1, 137.7, 132.7, 132.4, 131.7, 131.3, 130.7, 130.2, 129.5, 128.7×2, 127.9, 127.7, 127.2,
123.8, 115.9, 114.2×2, 113.6, 101.3, 97.5, 55.6, 55.3, 55.1, 28.1, 27.7, 22.4.ESI-MS m/z: 534.9
(M+H) +, calcd for C32H29F3O4: 534.2.
5.1.8.4. (3Z,5Z)-3-{2,4-dimethoxy-6-[(E)-4-methoxystyryl]benzylidene}-5-(2-trifluoromethylbenz
1
ylidene)dihydro-2H-thiopyran-4(3H)-one (d1). Yellow oil, 40.5% yield. H NMR (600 MHz,
DMSO-d6) δ (ppm): 7.867 (1H, s, H-7'), 7.854 (1H, d, J = 8.4 Hz, H-3''), 7.754 (1H, s, H-7''),
7.740 (1H, t, J = 7.8 Hz, H-5''), 7.628 (1H, t, J = 7.8 Hz, H-4''), 7.546 (2H, d, J = 8.4 Hz, H-2''',
H-6'''), 7.415 (1H, d, J = 7.8 Hz, H-6''), 7.223 (1H, d, J = 16.2Hz, H-8'''), 6.954 (2H, d, J = 8.4 Hz,
H-3''', H-5'''), 6.942 (1H, s, H-5'), 6.848 (1H, d, J = 16.2 Hz, H-7'''), 6.574 (1H, s, H-3'), 4.662 (2H,
s, H-2), 4.250 (2H, s, H-6), 3.876 (3H, s, OCH3), 3.825 (3H, s, OCH3), 3.778 (3H, s, OCH3). 13C
NMR (150 MHz, DMSO-d6) δ (ppm): 184.1, 161.3, 159.4, 158.7, 139.0, 136.1, 133.9, 132.7,
132.6, 132.4, 131.7, 131.4, 131.0, 130.4, 129.5, 129.4, 128.2×2, 126.3, 123.3, 114.3×2, 102.0,
97.5, 68.2, 67.5, 55.7, 55.4, 55.1, 27.5, 24.9. ESI-MS m/z: 553.2 (M+H) +, calcd for C31H27F3O4S:
552.2.

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5.1.8.5. (3E,5E)-3-{2,4-dimethoxy-6-[(E)-4-methoxystyryl]benzylidene}-1-propyl-5-(2-trifluoro
methylbenzylidene)piperidin-4-one (f1).Yellow oil, 30.8% yield. 1H NMR (600 MHz, DMSO-d6)
δ (ppm): 7.815 (2H, d, J = 8.4 Hz, H-2''', H-6'''), 7.716 (1H, t, J = 7.2 Hz, H-4''), 7.677 (1H, s,
H-7'), 7.584 (1H, t, J = 7.8 Hz, H-5''), 7.452-7.484 (3H, m, H-3'', H-6'', H-7''), 7.222 (1H, d, J =
15.6 Hz, H-8'''), 6.936 (1H, s, H-5'), 6.917 (2H, d, J = 8.4 Hz, H-3''', H-5'''), 6.822 (1H, d, J = 15.6
Hz, H-7'''), 6.545 (1H, s, H-3'), 3.854 (3H, s, OCH3), 3.787 (3H, s, OCH3), 3.779 (3H, s, OCH3),
3.504 (2H, s, H-6), 3.207 (2H, s, H-2), 2.135 (t, 2H, J = 7.2 Hz, N-CH2CH2CH3 ), 1.015-1.077 (m,
2H, N-CH2CH2CH3), 0.555 (t, 3H, J = 7.2 Hz, N-CH2 CH2CH3). 13C NMR (150 MHz, DMSO-d6)
δ (ppm): 185.9, 160.8, 159.2, 158.5, 137.9, 136.1, 135.6, 134.7, 133.1, 132.6, 132.5, 131.8, 131.4,
131.1, 130.8×2, 129.4, 129.1, 127.9×2, 123.6, 115.0, 114.2×2, 101.5, 97.5, 57.2, 55.6, 55.3, 55.1,
53.9, 53.3, 19.6, 11.3. ESI-MS m/z: 578.3 (M+H) +, calcd for C34H34F3NO4: 577.2
5.1.9. Synthesis of 3-buten-2-one derivative 21
To a stirring solution of Compound 8 (0.30 g, 1 mmol, 1 equiv) in acetone (10 mL) was added
dropwise 1 mL of 20% (w/v) NaOH slowly. After stirring 5 h at room temperature, the resulting
solution was poured into water, the solid precipitated, filtered, the cake was washed with water
and ethanol, and recrystallized from ethanol to give key intermediate compound 21 (light yellow
solid, 0.29 g, 85.4%).
(E)-4-{2,4-dimethoxy-6-[(E)-4-methoxystyryl]phenyl}but-3-en-2-one (21).Yellow powder, 70.9%
yield, mp: 108.0-110.3 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 7.774 (1H, d, J = 16.2 Hz,
H-4), 7.560 (2H, d, J = 8.4 Hz, H-2'', H-6''), 7.304 (1H, d, J = 16.2 Hz, H-8''), 7.072 (1H, d, J =
16.2 Hz, H-3), 6.968 (2H, d, J = 9.0 Hz, H-3'', H-5''), 6.822 (1H, s, H-5'), 6.598 (1H, d, J = 16.2
Hz, H-7''), 6.591 (1H, s, H-3'), 3.871 (3H, s, OCH3), 3.783 (3H, s, OCH3), 3.324 (3H, s, OCH3),
2.29(3H, s, H-1). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 198.1, 161.3, 160.2, 159.2, 140.7,
136.8, 131.8, 129.8, 129.4, 128.1, 127.9×2, 124.3, 114.2×2, 114.1, 103.9, 97.7, 55.8, 55.4, 55.1.
ESI-MS m/z: 339.1 (M+H) +, calcd for C21H22O4: 338.2.
5.1.10. General procedure for synthesis of compounds a2-a25
To a stirring solution of various substituent aldehydes (0.6 mmol, 1.2 equiv) in EtOH (10 mL)
20% NaOH (10 drops) was added, then the intermediate 23 (169.2 mg, 0.5 mmol, 1.0 equiv) was
added dropwise. The reaction mixture was stirred at room temperature overnight, with some of
products precipitated. The precipitate was washed with water and subsequently with cold ethanol,

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and dried in vacuum. The other products which cannot be precipitated were quenched with
saturated aqueous NH4Cl solution (10 mL) and extracted with EtOAc (3 × 10 mL). 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 furnish target a2-a25 with yield of 25.2%-80.8%.
5.2. Animals
Male C57BL/6 mice (n=8 per group) weighing 18-22 g were obtained from the Animal Center
of Wenzhou Medical University (Wenzhou, China). All animals were housed at a constant room
temperature with a 12:12 h light-dark cycle and fed with a standard rodent diet and water. After
acclimatization to the laboratory for 7 days, mice were randomly assigned to the vehicle control or
LPS-challenge group, the latter as a model for ALI/ARRDS. All animal care and experimental
procedures were approved by theWenzhou Medical University Animal Policy and Welfare
Committee (Approval Document No.wydw2014-0062).
5.3. Cells and reagents
RAW 264.7 macrophages were incubated in DMEM medium (Gibco, Eggenstein, Germany)
supplemented with 10% FBS (Hyclone, Logan, UT), 100 U/ml penicillin, and 100 mg/ml
streptomycin at 37 oC with 5% CO2. LPS purchased from Sigma was dissolved in PBS. Curcumin
and its analogs were dissolved in DMSO before use. For peritoneal macrophage preparation, ICR
mice were stimulated by intraperitoneal injection of 1.5 mL 6% thioglycolate broth (0.3 g beef
extract, 1.0 g tryptone, 0.5 g sodium chloride, and 6.0 g soluble starch dissolved and boiled in 100
ml water.) and kept in pathogen-free conditions for 3 days before mouse peritoneal macrophage
isolation. Total peritoneal macrophages were harvested by washing the peritoneal cavity with
RPMI-1640 medium (Gibco, Eggenstein, Germany, 13 mL per mouse), centrifuged, the pellet was
resuspended in RPMI-1640 medium (Gibco, Eggenstein, Germany) with 10% FBS (Hyclone,
Logan, UT), and 1% penicillin/streptomycin. Nonadherent cells were removed by washing with
medium at 4 h after seeding. Experiments were undertaken after the cells adhered firmly to the
culture plates. Before used, peritoneal macrophages were cultured in RPMI-1640 medium on 35
mm plates at a density of 5.0 ×105/plate and maintained at 37oC in a 5% CO2-humidified air.
5.4. Determination of TNF-α and IL-6

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After treatment of cells with indicated compounds and LPS, the TNF-α and IL-6 levels in
medium were determined with an ELISA kit (eBioScience, San Diego, CA) according to the
manufacturer’s instructions. Briefly, cells were pretreated with 10 mM of vehicle, resveratrol or
hybrid compounds or analogs for 30 min, and then treated with LPS (0.5 mg/ml) for 24 h. After
treatment, the conditioned media and cells were collected separately. The levels of TNF-α and
IL-6 in the media were determined by ELISA. The total protein concentrations of the viable cell
pellets were determinea using Bio-Rad protein assay reagents. The amounts of TNF-α and IL-6
were normalized to the total proteins in cells.
5.5. MTT (methyl thiazolyl tetrazolium) assay
HL-7702 cells were seeded into 96-well plates at a density of 5000 cells per well in 1640
medium, supplemented with 5% heat-inactivated serum, 100 U/ml penicillin, and 100 mg/ml
o
streptomycin. The cells were maintained at 37 C in 5% CO2. Test compounds were dissolved in
DMSO and diluted with 1640 medium to the final concentrations of 10 µM. Cells were incubated
with test compounds for 24 h before the MTT assay. A fresh solution of MTT (5 mg/ml) prepared
in NaCl solution (0.9%) was added to each well of the 96-well plate. The plate was then incubated
in a CO2 incubator for 4 h, cells dissolved with 150 mL DMSO, and analyzed in a multi-well-plate
reader at 490 nm.
5.6. UV-visible absorption spectra of Curcumin and its analogs
Absorbance readings were taken from 250 to 600 nm using a spectrum Max M5 (Molecular
Devices, USA). A stock solution of 1 mM curcumin or its analogs was prepared and diluted by
phosphate buffer (pH 7.4) to a final concentration of 20 µM. In the experiments where degradation
of curcumin was recorded, the absorption spectra were collected for over 25 min at 5 min intervals.
The UV-visible absorbance spectrum was measured at 25 oC at varying time interval in a 1 cm
path-length quartz cuvette.
5.7. Real-time quantitative PCR
Tissues and cells were homogenized using a TRIZOL (Invitrogen, Carlsbad, CA) for extraction
of RNA according to each manufacturer's protocol. Both reverse transcription and quantitative
PCR were 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,

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Hamburg, Germany) was used for q-PCR analysis. The primers of genes were synthesized by
Invitrogen. The primer sequences of mouse genes used are shown as follows:
IL-6 sense: 5′-CCAAGAGGTGAGTGCTTCCC-3′;
IL-6 antisense: 5′- CTGTTGTTCAGACTCTCTCCCT-3′;
IL-12 sense: 5′-GGAAGCACGGCAGCAGAATA-3′;
IL-12 antisense: 5′-AACTTGAGGGAGAAGTAGGAATGG-3′;
IL-33sense: 5′-GATGGGAAGAAGGTGATGGTG-3′;
IL-33 antisense: 5′-TTGTGAAGGACGAAGAAGGC-3′;
TNF-α sense: 5′-TGATCCGCGACGTGGAA-3′;
TNF-α antisense: 5′-ACCGCCTGGAGTTCTGGAA-3′;
GAPDH sense: 5′-AGGTCGGTGTGAACGGATTTG-3′;
GAPDH antisense: 5′-TGTAGACCATGTAGTTGAGGTCA-3′;
The amount of each gene was determined and normalized by the amount of GAPDH.
5.8. Quantitative SAR (QSAR) model
All these products were drawn with the Maestro (Schrödinger, LLC), and were saved in the
SDF format. These compounds were calculated with the ChemoPy descriptor calculation program
[46]. Using the program, about 1000 molecular descriptors based on molecular structure were
obtained. Evaluating the calculated molecular descriptors, those that stayed constant for all
molecules were eliminated and pairs of variables with a correlation coefficient greater than 0.75
were classified as inter-correlated and one in each correlated pair was deleted.
5.8.1. Multiple linear regression (MLR) analysis
MLR analysis was a statistical technique that uses several explanatory variables to predict the
outcome of a response variable [5]. The goal of multiple linear regression (MLR) is to model the
relationship between the explanatory and response variables. In our present study, MLR was
performed using R program, a powerful tool for statistical computing and graphics, to derive
QSAR models. The biological data used in this study were their TNF-α- or IL-6-inhibitory rates
when compared to LPS alone group. Compounds with negative values were abandoned because of
their pro-inflammatory activities. The inhibition rates against TNF-α and IL-6 release, named as
IRTNF-α and IRIL-6, respectively, were used as dependent variables in the linearization procedure.
Subsequently, Stepwise Multiple Linear Regression (Stepwise-MLR) was used to select the
significant descriptors. The most relevant descriptors were used as independent variables.
5.8.2. Validation of the models

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Validation of the lineal models is required for testing the predictive ability and generalizing the
methods by cross-validation. The leave-one-out (LOO) procedure was employed. When a data
point was removed from the analyzed set, the regression was recalculated, and then the predicted
value for that point was compared to its actual value. This process was repeated until each datum
had been omitted once and then the sum of squares of these deletion residuals could be used to
calculate q2, an equivalent statistic to R2.
5.9. LPS-induced inflammatory mortality in C57BL/6 mice
The 24 Male C57BL/6 mice were randomly divided into three groups on average, including the
control group, LPS group, and a18+LPS group. The mice in the a18+LPS group were pretreated
with a18 (5 mg/kg 0.5% CMC-Na), for consecutive 7 days, and the Sham-Vehicle group and
LPS-Vehicle group were given the same volume of 0.5% CMC-Na (1mL/kg). After being
anesthetized by diethyl ether, the mice from the LPS group and a18+LPS group were
intratracheally instilled with LPS (5 mg/kg, 1 mL/kg PBS) to induce lung injury. The control mice
were given PBS (1mL/kg). Mice were killed under ether anesthesia at 6 h after LPS instillation.
Bronchoalveolar lavage fluid (BALF), serum and lung tissues were collected for further tests.
5.9.1. Inflammation cytokine in BALF
Thoracotomy and ligation of the left lung were performed. The left lung was infused four times
with 0.8 mL phosphate buffered saline (PBS) in order to obtain BALF. The collected BALF was
centrifuged for 10 min at 1,000 rpm. Cell-free supernatant was used for measurement of target
protein and cytokines. The cell pellets obtained from BALF were washed and re-suspended in 50
µL PBS for the determination of the total number of cells and cell differentiation. The total
number of cells in BALF was counted by a standard hemocytometer, and neutrophils were
confirmed by counting 200 cells on a smear prepared by Wright-Giemsa staining. The protein
concentration in the supernatant was determined using a BCA protein assay (Pierce, Rockford, IL,
USA).The relative amount of IL-6 and TNF-α in the supernatant of BALF was determined with an
ELISA kit as above.
5.9.2. Inflammation cytokine in Serum
Blood samples were collected from aorta ventralis using a heparinized syringe with a needle and
centrifuged at 3000 rpm at 4°C for 10 min. The serum was collected for subsequent analysis. The
relative amount of IL-6 and TNF-α in the serum was determined with an ELISA kit as above.

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5.9.3. Lung wet/dry weight ratio
The superior lobes of the right lungs were excised, blotted dry, and weighed to obtain the “wet”
o
weight. Then the lungs were placed in an oven at 60 C for more than 48 h until getting constant
weight as dry weight. The ratio of the wet lung to the dry lung was calculated to assess tissue
edema.
5.9.4. Histopathologic evaluation
Lung tissues were fixed in 4% paraformaldehyde solution, embedded in paraffin, and sectioned
at 5 µm. After dehydration, sections were stained with H&E and CD68 staining. To evaluate the
histopathological damage, each image of the sections was examined under a light microscope (400
amplification; Nikon).
5.10. Statistical analysis
The results are presented as means SD. Student’s t-test was employed to analyze the differences
between sets of data. Statistics were performed using GraphPad Pro (GraphPad, San Diego, CA).
P values less than 0.05 (p < 0.05) were considered indicative of significance. All experiments were
repeated at least three times.
Acknowledgement
Financial support was provided by the National Natural Science Foundation of China–China
(81402783, 21272179 and 21472142), Wenzhou Public Welfare Science and Technology Project
– China (2014Y0398), Zhejiang Provincial Natural Science Foundation – China (LQ14H310003),
National ‘863’Project – China (2011AA02A113).
Supplementary data
Supplementary data associated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2015.xx.xxx.
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(2000) 1334-1349.

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Figure Legends
Figure 1. Mono-carbonyl curcumin analogues with potential anti-inflammatory activity
Figure 2. Design strategy based on hybrid resveratrol and Mono-Carbonyl Analogs of Curcumin
(MCAs)
Figure 3. Optimization and confirmation of the structure scaffold
Scheme 1. Synthetic route for resveratrol aldehyde fragment 8
Scheme 2. Synthesis of different scaffold analogs a1-f1.
Scheme 3. General synthetic routes for compounds a2-a25
Figure 4. Six different scaffold derivatives inhibited LPS-induced IL-6 (A) and TNF-α (B)
o
secretion in MPM. Macrophages were plated at a density of 5.0 ×105/plate at 37 C and 5% CO2
overnight. Cells were pre-treated with vehicle, resveratrol and hybrids compounds (10 mM) for 30
min, and subsequently treated with LPS (0.5 mg/mL) for 24 h. The culture media was collected
and the inflammatory cytokines, TNF-α and IL-6 in media were detected by ELISA and
normalized by total protein concentration.The results were expressed as the percent of LPS group.
Each bar represents mean ± SEM of 3 independent experiments. Statistical significance relative to
LPS group was indicated,*p<0.05, **p<0.01.
Table 1. Chemical structures and yields of compounds a1-a25
Figure 5. Resveratrol, C66 and MCAs hybrids a1-a25 inhibited LPS-induced TNF-α (A) and IL-6
o
(B) secretion in MPM. Macrophages were plated at a density of 5.0 ×105/plate at 37 C and 5%
CO2 overnight. Cells were pre-treated with vehicle, resveratrol and hybrids compounds (10 mM)
for 30 min, and subsequently treated with LPS (0.5 mg/mL) for 24 h. The culture media was
collected and the inflammatory cytokines, TNF-α and IL-6 in media were detected by ELISA and

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normalized by total protein concentration. The results were expressed as the percent of LPS group.
Each bar represents mean±SEM of 3 independent experiments. Statistical significance relative to
LPS group was indicated, *p<0.05, **p<0.01.
Figure 6. Active compounds inhibited LPS-induced inflammatory cytokines in a dose-dependent
manner. MPM (5 × 105/plate) were pretreated with vehicle, resveratrol (10 µM) or compounds at
indicated concentrations (1, 2.5, 5 and 10 µM) for 30 min. Then, cells were incubated with LPS
(0.5 µg/mL) for 24 h. IL-6 production in the supernatants was measured by ELISA. Each bar
represents mean ± SEM of 3 independent. Statistical significance relative to the LPS group was
indicated, *p < 0.05, **p < 0.01.
Figure 7. The cytotoxic evaluation in HL-7702 of a1-a25. MPM were plated at a density of 5000
o
per plate in 96-well plates for 24 h in 37 C and 5% CO2. Then, cells were treated with the
compounds (10 µM) for 24 h. After that, the viability of cells was measured by the MTT assay.
Figure 8. UV-visible absorption spectra of resveratrol, a3 (A), a18 (B), a19 (C), a20 (D) and a24
(E) in phosphate buffer (pH 7.4) containing 2% dimethyl sulfoxide.
Figure 9. Resveratrol and active compounds inhibited the inflammatory genes expression induced
by LPS in MPM. Cells were plated at a density of 1.0 × 106/plate overnight in 37 oC and 5% CO2.
MPM were pretreated with 10 µM vehicle, resveratrol or five active compounds for 30 min and
subsequently incubated with LPS (0.5 µg/mL) for 6 h. Cells were collected, and the total RNA
was extracted. The mRNA levels of IL-6, IL-12, IL-33 and TNF-α were detected by QPCR (A–D).
The results were presented as the percent of LPS control. Each bar represents the mean ±SEM of
the three independent experiments. Statistical significance relative to the LPS group was indicated,
*p < 0.05; **p < 0.01.
Figure 10. Plots of predicted activity against the corresponding experimental activity on TNF-α
(A) and IL-6 (B) inhibition. N, the number of compounds taken into account in the regression; R2,
the multiple correlation coefficient; Radj2, adjusted multiple correlation coefficient; s, residual
standard error; and the F value is related to the F-statistic analysis (Fischer test). The numbers in

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parentheses
mean
the
standard
deviation
of
the
coefficients.
(A)
IRTNF-α=277.4931(±46.0802)+72.5837(±11.0545)MoRSEE17+54.6398(±8.0133) MoRSEP4
+
8.0101(±0.8773) RDFE9, N=22, R2=0.8402, Radj2=0.8135, s=13.93, F3,18=31.54, p=2.218e-7. (B)
IRIL-6=199.545(±30.354)-180.459(±26.795) GATSp6+ 11.353(±1.881) RDFP13 -12.921(±2.391)
RDFP7, N=22, R2=0.7595, Radj2=0.7194, s=13.93, F3,18=18.95, p=8.389e-6.
Figure 11. A18 attenuate the LPS-induced lung inflammation in mice. Mice were given an
intragastric administration of a18 (5 mg/kg/day), or 0.5% CMC-Na 7 days prior to an intranasal
administration of LPS (5 mg/kg), then mice were anaesthetized and killed at 6 h after LPS
challenged. Bronchoalveolar lavage fluid and lung tissues were collected for further tests. Effects of
a18 on the total protein concentration (A)the relative amount of cells in BALF (B) the number of
neutrophils in BALF (C)and the lung W/D ratio (D) of LPS-induced ALI mice. (E) Effects of a18
on histopathological changes in lung tissues in ALI model induced by LPS (H&E staining 200 ×).
Figure 12. A18 attenuate the LPS-induced lung inflammation in mice. Mice were given an
intragastric administration of a18 (5 mg/kg/day), or 0.5% CMC-Na 7 days prior to an intranasal
administration of LPS(5 mg/kg),then mice were anaesthetized and killed at 6 h after LPS
challenged, Bronchoalveolar lavage fluid and lung tissues were collected for further tests. (A) The
amount of TNF-α in BALF. (B) The amount of IL-6 in BALF. (C) The amount of TNF-α in serum.
(D) The amount of IL-6 in BALF. (E) The changes of macrophage infiltration inlung tissues after
LPS treatment and a18 pretreatment. (Immunohistochemical of CD68 staining 200X) Statistical
significance relativeto the LPS group was indicated, *p < 0.05; **p < 0.01.

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Table 1