New MD2 inhibitors derived from curcumin with improved anti-inflammatory activity

Article abstract of DOI:10.1016/j.ejmech.2018.02.008

An overactive Toll-like receptor (TLR) signaling complex is a significant pathogenic factor of acute and chronic inflammatory diseases. The natural product curcumin is reported to inhibit the TLR4 co-receptor, MD2 (myeloid differentiation protein 2), but

Full text of DOI:10.1016/j.ejmech.2018.02.008

Accepted Manuscript
New MD2 inhibitors derived from curcumin with improved anti-inflammatory activity
Yali Zhang, Zhiguo Liu, Jianzhang Wu, Bin Bai, Hongjin Chen, Zhongxiang Xiao,
Lingfeng Chen, Yunjie Zhao, Hazel Lum, Yi Wang, Hong Zhang, Guang Liang
PII:
S0223-5234(18)30130-2
10.1016/j.ejmech.2018.02.008
EJMECH 10185
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 December 2017
Revised Date: 24 January 2018
Accepted Date: 3 February 2018
Please cite this article as: Y. Zhang, Z. Liu, J. Wu, B. Bai, H. Chen, Z. Xiao, L. Chen, Y. Zhao,
H. Lum, Y. Wang, H. Zhang, G. Liang, New MD2 inhibitors derived from curcumin with improved
anti-inflammatory activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.02.008.
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ACCEPTED MANUSCRIPT

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New MD2 inhibitors derived from curcumin with improved anti-inflammatory activity
Yali Zhanga,b,1, Zhiguo Liua,1, Jianzhang Wua, Bin Baia, Hongjin Chena, Zhongxiang Xiaob,
Lingfeng Chena, Yunjie Zhaoa, Hazel Luma, Yi Wanga, Hong Zhangc, *, Guang Lianga,b,*
aChemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China
bDepartment of Pharmacy, Affiliated Yueqing Hospital, Wenzhou Medical University, Wenzhou,
Zhejiang 325000, China
cThe Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, China
1 These authors contributed equally to this work.
*Corresponding author:
Guang Liang, Ph.D, Professor
Address: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou
Medical University, Wenzhou 325035, China
Tel: +86-577-86699892; Fax: +86-577-86689982;
E-mail: wzmcliangguang@163.com
And
Hong Zhang, Professor
Address: The Second Affiliated Hospital of Soochow University. Suzhou, 215004, China.
Tel: +86-13814882907
E-mail: szzhanghong126@126.com

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Abstract
An overactive Toll-like receptor (TLR) signaling complex is a significant pathogenic factor of
acute and chronic inflammatory diseases. The natural product curcumin is reported to inhibit the
TLR4 co-receptor, MD2 (myeloid differentiation protein 2), but its low in vivo bioavailability limits
its therapeutic potential. We developed new curcumin analogs (MACs) with removal of the
β-diketone moiety and substituted residues in benzene rings, and identify these as potential MD2
inhibitors with improved inhibition potency and stability over that of curcumin. Specifically, MAC
17 and 28 showed the highest anti-inflammatory activity, with >90% inhibition of LPS-stimulated
cytokine secretion from macrophages, and protected against LPS-induced acute lung injury and
sepsis. The MACs inhibited the TLR4-MD2 signaling complex through competition with LPS for
binding on MD2, likely at Arg90. Our findings indicated that MAC 17 and 28 are promising
candidates for future development as therapeutic drugs for inflammatory diseases with an
endotoxin etiology.
Key words: Curcumin; Acute lung injury; MD2; Inflammation; Sepsis

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1. Introduction
Cumulative evidence from the past two decades indicates that an overactive Toll-like receptor
(TLR) signaling complex can lead to uncontrolled inflammation and excessive
accumulation/activation of leukocytes, a key factor in the pathogenesis of a wide range of acute and
chronic inflammatory diseases. TLRs are pattern recognition receptors known to bind a spectrum of
ligands related to infectious agents [1]. Among these, TLR4 is the major receptor for the
Gram-negative bacterial product, lipopolysaccharide (LPS), a potent activator of immune cells, and
its activation by LPS leads to secretion of pro-inflammatory cytokines and activation of
transcription factors such as NF-κB. Moreover, the persistent or uncontrolled activation by LPS is
responsible for clinical consequences in diseases such as sepsis, endotoxemia, and acute lung injury
(ALI). As such, much research efforts, including our own laboratory, have focused in developing
therapeutic strategies to inhibit activation of the TLR4 signaling complex.
Structural studies indicate that TLR4 forms a heterodimer with its co-receptor, myeloid
differentiation protein 2 (MD2) on the cell surface, and its activation by LPS requires LPS binding
to MD2, leading to dimerization of the TLR4-MD2 complex, which allows for recruitment of
adaptor proteins to the TLR4 intracellular domains [2]. The subsequent recruitment of the adaptor
protein myeloid differentiation factor 88 (MyD88) signals activation of transcription factors,
NF-κB and AP-1, or the recruitment of MyD88-independent adaptors which signals interferon
response factors [2]. The importance of MD2 in TLR4 activation is clearly supported by
experimental models. MD2 knock-out mice survive endotoxic shock, and the depletion of MD2
impairs localization of TLR4 to the cell surface [3]. Furthermore, the interference of the MD2
binding/active sites by covalent bond formation abrogates LPS signaling at the cell level and in
mice [4].
Limited evidence suggests that curcumin, a natural product derived from the spice turmeric,
has anti-inflammatory activity that is linked to MD2 inhibition [5]. Curcumin has well-known
anti-inflammatory, anti-proliferative, anti-angiogenic, proapoptotic, immune-modulatory, as well as
oxygen radical scavenging properties [6-8]. Although a host of experimental animal models and
clinical trials have demonstrated curcumin’s safety and efficacy, its low solubility, chemical
instability, and rapid metabolism have limited its in vivo bioavailability, and therefore, making its
potential clinical value uncertain [9]. Thus, research priority has been focused in development of

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curcumin analogs with improved stability, efficacy, and potency while retaining its safety
characteristics [10]. We have developed series of curcumin analogs by removal of the β-diketone
moiety, believed to be a major contributor to curcumin’s instability and rapid degradation, yielding
the more chemically stable mono-carbonyl analogs of curcumin (MACs) [11-13]. Moreover, these
MACs protected against LPS-induced sepsis in mice [11, 12] and ALI in rats [13], indicative of
effective in vivo bioavailability. Recent evidence indicates that curcumin analogs which are
effective anti-inflammatory agents show direct interactions at sites within the MD2 pocket,
suggesting that these interactions are the basis of inhibition of the TLR4-MD2 signaling complex
[14, 15]. The goals of this study were to develop a series of new MACs with substituted residues
in the benzene rings that may alter interactions within the MD2 pocket, and identify the MACs as
potential MD2 inhibitors with improved inhibition potency and stability over that of curcumin.
2. Results
2.1. Chemical profile of curcumin analogs
As reported previously [11-13], the deletion of the central beta-diketone may improve
pharmacokinetic profiles of curcumin. Therefore, a series of 5-carbon linker-containing
mono-carbonyl analogues of curcumin were designed and synthesized. Different substituents in the
benzene rings and central linkers were designed to investigate and discuss the structure-activity
relationship. The synthesis essentially involved an aldol condensation of the appropriate aromatic
aldehyde with central linker in an alkaline medium at room temperature, respectively. We generated
35 heterocyclic MACs (Scheme 1). Generally, the coupling reaction yielded the respective products
in high yields. The chemical properties (yield and spectral data) of the MACs are described in
Materials and Methods. The chemical stability of MACs 9, 17, 26, 28, and 30 was evaluated by UV
absorbance spectroscopy. The UV absorption spectra of curcumin progressively decayed over 25
min, but the analogs, except for MAC 26, were stable and did not decayed over time (Fig. S1),
indicating high stability of MACs.
2.2. Screening of curcumin derivatives for anti-inflammatory activity
The generated 35 curcumin derivatives were initially screened for their inhibition of
LPS-stimulated secretion of IL-6 and TNF-α in freshly isolated MPMs (Materials and Methods).

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Results indicated that pretreatment with MAC (10 µM) for 30 min inhibited secretion of the
cytokines from LPS-activated MPMs, with the degree of inhibition ranging from no inhibition
to >90% (Fig. 1). Of the 35 curcumin analogs, the majority showed greater inhibition than
curcumin for the LPS-induced secretion of IL-6 or TNA-α; whereas 2 (MAC 7 and MAC 16)
lacked ability to inhibit the respective cytokine secretion (Fig. 1). Based on results of this initial
screening of the curcumin derivatives, six of the most active analogs (showing >80% inhibition)
(MACs 9, 17, 26, 28, 29, 30) were chosen for further assessment of their inhibition of
pro-inflammatory activities of LPS-activated macrophages. The selected MACs were evaluated for
their inhibition of LPS-stimulated cytokines in MPMs using a range of 1-10 µM. The general
inhibitory potency on cytokine secretion of these six curcumin analogs was consistently greater
than curcumin, having significantly greater inhibitory effects at 5 µM than curcumin at 10 µM.
Specifically, the inhibition of IL-6 secretion was >50% at 2.5 µM for the six curcumin analogs (Fig.
2A), whereas inhibition of TNF-α secretion was at 5.0 µM (Fig. 2B). Both MACs 17 and 28
showed the greatest inhibition potency, which inhibited >90% of the LPS-induced secretion of IL-6
at 1 µM (Fig. 2A). MACs 17 and 28 (10 µM) also significantly inhibited the LPS-induced mRNA
expression of TNF-α, IL-6, IL-1β and COX-2, which was substantially more effective than
curcumin at the same concentration (Fig. 2C).
2.3. Protective effects of MAC 17 or 28 in animal models of LPS-induced acute lung injury and
sepsis
We selected MACs 17 and 28 based on their overall high inhibitory potency and stability for
testing their anti-inflammatory activity in vivo in a rat model of LPS-induced ALI. The LPS
challenge induced ~20% increase of the lung wet/dry ratio, indicating presence of pulmonary
edema (Fig. 3A). However, rats treated with MAC 17 or 28 effectively prevented development of
pulmonary edema, with wet/dry ratios at control (absence of LPS) values (Fig. 3A). Evaluation of
histological lung sections was consistent with the protective effects of MACs 17 and 28, which was
associated with relatively normal tissue morphology (Fig. 3B). LPS induced a 6-fold increase in
cell number from BALF, whereas rats treated with MACs 17 or 28 showed no increase in cell
number in the BALF (Fig. 3C). The results indicated that both curcumin analogs prevented
leukocytic extravasation in lungs. Extravascular neutrophils are important factors contributing to

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ALI. LPS induced >2-fold increase over control in lung tissue MPO activity, indicating significant
increase in neutrophil extravasation, however, MAC 17 or 28 reduced ~40% of the extravasation
(Fig. 3D). ALI is also associated to with mobilization of macrophages to lungs. We detected
increased immunohistochemical localization of CD68, marker protein of monocytes/macrophages,
in lung tissues from the LPS-challenged rats (Fig. 3E). As expected, treatment with either MAC 17
or 28 reduced tissue content of CD68 (Fig. 3E), indicating protective effects of the curcumin
analogs. We found that the increased total cells in the BALF (Fig. 3C) from LPS challenge was
associated with increased TNF-α in the BALF (Fig. 3F) as well as increases in circulating TNF-α
(Fig. 3G), but only compound 28 reduced the TNF-α increases (Fig. 3F-G). The LPS challenge also
increased lung tissue mRNA content of cytokines (TNF-α, IL-6, IL-1β) (Fig. 3H-J, respectively),
adhesion molecules (ICAM-1, VCAM-1) (Fig. 3K-L, respectively), and chemokine, MCP-1 (Fig.
3M). Treatment with compound 28 significantly inhibited the LPS-induced increases in tissue
mRNA content of the cytokines, adhesion molecules and chemokine (Fig. 3H-M); whereas
inhibition by MAC 17 was more selective and less potent for TNF-α, IL-6, and MCP-1 (Fig. 3H-M).
The findings indicated that the protective effects of MACs 17 and 28 were associated with reduced
inflammatory cell infiltration and reduced tissue content of pro-inflammatory molecules.
A mouse model of sepsis was used to evaluate protective effects of MACs 17 and 28 on
LPS-induced septic shock. Within 2 days of LPS injection, 30% of the mice survived, which further
decreased to 10% from days 4-7 (Fig. 4). Treatment with MAC 17 improved survival to 70%, and
with 28 to 90% within the same period (Fig. 4).
2.4. Curcumin analog MAC 17 or 28 directly binds on MD2
Binding with purified MD2: We and others have reported that curcumin [5, 16] and curcumin
analog L48H37 [14] directly interact with MD2, the cofactor of TLR4, implicating a potential
inhibition mechanism. Therefore, these newly identified curcumin analogs with similar structure
likely have similar binding interactions with MD2. We first determined the binding affinity of the
curcumin analogs with recombinant purified human MD2 (rhMD2) by SPR analysis. Results
confirmed that MACs 17 and 18 bound rhMD2 directly with KD values of 71.1 and 59.5 µM,
respectively (Fig. 5A-B, respectively).
Competition with LPS binding to MD2: We next tested whether binding of the curcumin

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analogs with MD2 interfere with LPS interactions with the TLR4 receptor complex at the cell
surface. HUVECs were incubated with FITC-LPS in the presence or absence of pre-incubation with
either MAC 17 or 28. Flow cytometric analysis indicated that FITC-LPS significantly bound on
HUVECs, increasing the MFI from 2 in control to 17 (Fig. 5C-D). The pretreatment with either
MAC 17 or 28 (0.1-10 µM) reduced MFI by 3-fold (Fig. 5C-D), indicating decreased LPS binding
and, thereby prevented TLR4 activation. Notably, inhibition at the lowest curcumin analog
concentration of 0.1 µM was to a similar extent as 10 µM. We suspected that a reduced TLR4
activation would correspond to decreased complex formation with MD2. This was confirmed in
co-immunoprecipitation (co-IP) assays in which MAC 17 or 28 prevented the LPS-induced
TLR4-MD2 complex dimerization in macrophages (Fig. 5E-F) as well as from lung tissues (Fig.
5G-H). The findings suggested that the curcumin analogs competed with LPS for binding to MD2,
thereby impairing the LPS-activated TLR4-MD2 complex formation. We explored the relationship
of the curcumin analogs with the LPS interaction on MD2 using in vitro biotin-streptavidin-based
ELISA. Biotin-LPS was observed to directly bind rhMD2, and in the presence of MAC 17 or 28,
the bound biotin-LPS was reduced by 50% (Fig. 5I). We further tested this binding competition by
MAC 17 or 28 on MD2 using the fluorescent probe bis-ANS, which has the structural motif of lipid
A of LPS and binds to the hydrophobic binding site of MD2 [17]. The binding of bis-ANS to
rhMD2 showed maximum fluorescence at 480 nm, and was reduced when incubated with either
MAC 17 (Fig. 5J) or 28 (Fig. 5K) in a dose-dependent manner. These findings support a direct
binding of MAC 17 or 18 on MD2 at overlapping sites with LPS.
MAC 17 or 28 interacts with Arg90 of MD2: The specific binding of MAC 17 or 28 on MD2
was analyzed using Molecular Docking software. Results indicated that MACs 17 and 28 were
predicted to dock within the hydrophobic pocket of the MD2 crystal structure, forming a hydrogen
bond with Arg90 residue of MD2 (Fig. 6A and B, respectively). To test the significance of Arg90 of
MD2 in binding with MAC 17 or 28, we constructed a mutant protein, replacing Arg with Ala,
rhMD2R90A. Biotin-LPS bound the rhMD2R90A, as indicated by the increased fluorescence detected,
however, incubation with either MAC 17 or 28 did not reduced this fluorescence (Fig. 6C),
indicating inability of the curcumin analogs to bind rhMD2 R90A. Moreover, SPR analysis showed
that MACs 17 and 28 did not bind rhMD2R90A (Fig. 6D and E, respectively). The results identified
Arg90 on MD2 as the crucial residue for binding MAC 17 or 28.

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2.5. MAC 17 or 28 inhibits MAPK and NF-κB signaling
Our finding that MAC 17 or 28 inhibited binding of LPS on MD2 was consistent with loss of
complex formation of MD2 with TLR4, thereby inhibition of TLR4 activation. The key
downstream signaling molecules of TLR4 are ERK and NF-κB, and we suspect that MAC 17 or 28
inhibited the LPS-activation of these signals. Results indicated that MAC 17 or 28 were potent
inhibitors of LPS-induced activation of ERK, with significant inhibition of ERK activation at the
low dose of 2.5 µM (Fig. 7A). We used multiple approaches to assess the inhibition of LPS-induced
NF-κB activation. MAC 17 or 28 inhibited LPS-induced IκB degradation (Fig. 7B) and nuclear
translocation of NF-κB (Fig. 7C) by 5 µM of the analogs, and were effectively abrogated at 10 µM.
The curcumin analogs were also effective in blocking LPS-induced NF-κB activity as evaluated by
EMSA (Fig. 7D). Moreover, in RAW264.7 macrophages stably expressed with the reporter NF-κB
luciferase construct, MAC 17 or 28, prevented LPS-activation of NF-κB promoter activity at the
low dose of 2.5 µM (Fig. 7E). These findings indicated that MAC 17 or 28 was an effective
inhibitor of key signaling cascades downstream of TLR4, and is consistent with an inhibition
mechanism at the MD2-TLR4 complex. This thesis was further supported by our finding that direct
treatment of purified signaling kinases (i.e., ERK2, IRAK1, or IKKβ) with curcumin analog MAC
17 or 28 had minimal effect on their activity (Fig. S2).
3. Discussion
We generated new curcumin analogs, MACs, which are selective MD2 inhibitors, chemically
stable, with improved anti-inflammatory activity. MACs 17 and 28 were identified as having the
highest degree of anti-inflammatory activity among the MACs, showing >90% inhibition of
LPS-stimulated cytokine secretion from MPMs, and had significantly greater inhibition than
curcumin at same doses. Both effectively protected against LPS-induced ALI and sepsis in animal
models, which were likely attributed to inhibition of the TLR4-MD2 signaling complex through
direct competition with LPS for binding on MD2. MACs 17 and 28 are promising candidates for
future development as therapeutic drugs for acute and chronic inflammatory diseases with an
endotoxin etiology.
MAC 17 or 28 were highly effective in preventing LPS-induced ALI in mice, indicating that
there was sufficient in vivo bioavailability of the analogs. Curcumin is known for its low in vivo

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bioavailability due to its poor absorption, rapid metabolism, and rapid systemic elimination [9],
thereby necessitating administration of high doses, i.e., up to 400 mg/kg [9, 18]. In our study, mice
were administered orally with 20 mg/kg of MAC 17 or 28, which was sufficient to prevent
development of ALI. The finding was consistent with the chemical stability observed from our in
vitro studies, suggesting that MACs 17 and 28 were minimally degraded during transit from the
digestive tract to lung tissues. However, likely additional factors could contribute to the in vivo
bioavailability. Both MACs have significantly greater inhibition potency than curcumin, thereby
reducing the need for high doses of the curcumin analogs to achieve inhibition. Moreover, the
MACs, being structurally modified, were likely not as readily metabolized as curcumin. These
properties of MACs will be important therapeutic considerations, particularly since oral
administration of curcumin in humans have a significantly lower (2-3 orders in magnitude) in vivo
bioavailability than experimental animal models [9].
Our results point to an inhibition mechanism by MACs 17 and 28 which was through direct
binding on MD2 which competed with LPS binding. This conclusion was based on several pieces
of data obtained from cell-free studies using purified proteins as well as cell-based assays: i) MACs
bound to purified rhMD2 with micromolar KD values, ii) biotin-LPS bound to rhMD2 was
competed off with MACs, iii) the fluorescent bis-ANS (structurally similar to lipid A of LPS)
bound to rhMD2 was competed off with MACs, and iv) MAC 17 or 28 decreased the amount of
FITC-LPS bound to cell surface. These independent observations clearly indicated that MACs 17
and 28 were able to not only bind directly on MD2, but bind to overlapping sites of LPS. The
functional significance of the ability of MACs 17 and 28 to compete off LPS bound to MD2 was
preventing the formation of the dimerization of the TLR4-MD2 complex, and thereby preventing
activation of signaling pathways. Direct evidence in support of this regulation was our finding that
MAC 17 or 28 reduced the MD2 co-immunoprecipitated TLR4 proteins in LPS-stimulated
macrophages as well as lung tissues from ALI mice. The findings support a previous report by us
[14] and others [15] that curcumin analogs, as with curcumin [5], exhibit anti-inflammatory
properties through direct competitive binding on MD2 with LPS.
Not surprisingly, MACs 17 and 28 inhibited ERK and NF-κB in MPMs stimulated by LPS,
which are major regulatory pathways downstream of the TLR4-MD2 complex. ERK is
serine/threonine kinase that regulate a wide range of fundamental cell activities, such as

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proliferation, gene expression, differentiation, and an important target is the transcription factor,
AP-1 [19]. NF-κB is a pleiotropic transcription factor not only of inflammatory responses, but also
broader cellular activities, including cell proliferation, and cell death [20]. The MACs also
effectively inhibited LPS-activated NF-κB as assessed by IκB degradation, nuclear translocation of
the p65 subunit, EMSA, and NF-κB luciferase reporter assay. Thus, the effective and significant
inhibition of the inflammatory injury observed in the rat model of ALI as well as in the septic mice
can be accounted for by inhibition at the level of the TLR4-MD2 complex by MACs 17 and 28,
underscoring the importance of MD2 in controlling TLR4 activity.
Our molecular simulation analysis of the binding mode of MAC 17 or 28 with MD2 predicted
that the analogs fit into the hydrophobic pocket of MD2, forming hydrogen bonds with Arg90 of
MD2. The Arg90 appears essential for MACs 17 and 28 binding since mutation to alanine
(rhMD2R90A) abrogated MACs 17 and 28 binding on rhMD2R90A as well as competing off
biotin-LPS-bound on rhMD2R90A. Both MACs 17 and 28 formed hydrogen bond with Arg90 of
MD2 despite structural difference between the two analogs, in which O or NCH3, respectively, was
inserted into the “X” position of the center benzene ring (Scheme 1). Additionally, both MACs
showed similar inhibition potency of cytokine secretion (~90% inhibition), indicating that the
substitution of O (MAC 17) or NCH3 (MAC 28) did not impact hydrogen bond formation nor
bioactivity. However, substitution of S at the “X” position (MAC 4) (Scheme 1) significantly
lowered its inhibition potential for IL-6 and TNF-α secretion (60% and 25% inhibition,
respectively), and as did an “N” substitution forming the curcumin analog L48H37 reported in our
previous publication [14]. Interestingly, the 5-OCH3 residue of the benzene rings (MAC 17) appear
essential for the high anti-inflammatory activity since its removal (in MAC 20) resulted in
significant loss of activity, but insertion of 2-OCH3 (MAC 12) or 2,3-OCH3 (MAC 13) restored
activity (Fig. 1, Scheme 1). These observations suggest that 3,4,5-OCH3, 2,3-OCH3, and 2-OCH3
were structural significant configurations imparting high anti-inflammatory activity. Although we
do not fully understand the reasons why the O or NCH3 substitutions in the center ring conferred
greater bioactivity than either S or N substitutions, and that the 3,4,5-OCH3, 2,3-OCH3, and
2-OCH3 substitutions were favorable for high bioactivity, one possibility is that these
configurations provided lower binding energy, and, therefore, allowed for more efficient
displacement of LPS from MD2.

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In summary, we generated new curcumin analogs, MACs, which are selective MD2 inhibitors,
chemically stable, with improved anti-inflammatory activity. MACs 17 and 28 were identified as
having the highest degree of anti-inflammatory activity among the MACs, showing >90%
inhibition of LPS-stimulated cytokine secretion from MPMs, and had significantly greater
inhibition than curcumin at same doses. Both effectively protected against LPS-induced ALI and
sepsis in animal models, which were likely attributed to inhibition of the TLR4-MD2 signaling
complex through direct competition with LPS for binding on MD2. Our findings indicate that
MACs 17 and 28 are promising candidates for future development as therapeutic drugs for acute
and chronic inflammatory diseases with an endotoxin etiology.
4. Materials and Methods
4.1. Reagents
Chemical reagents and LPS were purchased from Sigma (St. Louis, MO). RPMI-1640 medium,
DMEM medium and FBS were obtained from Gibco (Gibco, Eggenstein, Germany). Mouse IL-6
ELISA kit, mouse TNF-α ELISA kit and anti-rhMD2 antibody were purchased from EBioscience
(eBioScience, San Diego, CA). Trizol-reagent was purchased from invitrogen (Invitrogen, Carlsbad,
CA). Chemiluminescent EMSA kit, biotin-labeled NF-κB probe and NF-κB p65 translocation kit
were obtained from Beyotime Biotechnology (Beyotime Biotech, Nantong, China). Luciferase
detection kit was purchased from Promega (Promega, Beijing, China). Anti-GAPDH, anti-IκBα,
anti-p-ERK, anti-ERK, anti-TLR4 and anti-MD2 antibodies were purchased from Santa Cruz
(Santa Cruz, CA, USA), and anti-p-P38, anti-P38, anti-p-JNK and anti-JNK antibodies from Cell
Signalling (Danvers, MA, USA).
4.2. Synthesis of curcumin analogs
Curcumin analogs were synthesized based on removal of the β-diketone moiety and with
substituted residues in the benzene rings (Scheme 1). To a stirred solution of 2.0 equivalent
different aldehydes and 1.0 equivalent various ketones in anhydrous ethanol added 40% NaOH or
o
HCl gas as catalyst at room temperature or 8 C. The reaction was monitored by TLC and after
completion was poured into water, then the precipitated product was filtered and washed with 5%
HAc-H2O, dried in vacuum, and the crude solids were recrystallized or chromatographed by on

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silica gel (hexanes/EtOAc) or (CH2Cl2/ methanol) to give target compounds. Compounds were
1
13
recrystallized from EtOH or EtOH/CH2Cl2 (2:1) solvent. The H-NMR and C-NMR spectrum of
MACs were shown in Supporting Information.
4.2.1. (3E,5E)-3,5-Bis(4-methoxybenzylidene)-1-propylpiperidin-4-one (1): Yellow solid, 75.4%
o
o
1
yield, mp 130.9-133.6 C [191.0-193.0 C, Lit [21]]. H-NMR (600MHz, CDCl3), δ: 7.79 (s, 2H,
Ar-CH=C×2), 7.37 (d, J = 8.4 Hz, 4H, Ar-H2×2, Ar-H6×2), 6.95 (d, J = 8.4 Hz, 4H, Ar-H3×2,
Ar-H5×2), 3.85 (s, 6H, 4-OCH3×2), 3.83 (s, 4H, CH2-N-CH2), 2.54 (t, J = 9.0 Hz, 2H, N-CH2), 1.49
(dd, J = 9.0 Hz, 2H, N-C-CH2), 0.86 (t, J = 7.2 Hz, 3H, CH3). ESI-MS m/z: 378.2 (M+H)+, calcd
for C24H27NO3: 377.2.
4.2.2. (3E,5E)-3,5-Bis(2-fluorobenzylidene)-1-propylpiperidin-4-one (2): Light yellow solid, 96.3%
o
1
yield, mp 87.0-89.9 C. H-NMR (600MHz, CDCl3), δ: 7.90 (s, 2H, Ar-CH=C×2), 7.37~7.38 ( m,
2H, Ar-H6×2), 7.28~7.31 (m, 2H, Ar-H4×2), 7.18~7.20 (m, 2H, Ar-H5×2), 7.12~7.15 ( m, 2H,
Ar-H3×2 ), 3.72 (s, 4H, CH2-N-CH2), 2.45 (t, J = 7.8 Hz, 2H, N-CH2), 1.39~1.42 (m, 2H,
13
N-C-CH2), 0.83 (t, J = 7.2 Hz, 3H, CH3). C-NMR (CDCl3): 186.2, 161.2, 159.6, 134.5, 130.4,
130.3, 130.2, 129.0, 123.5, 122.8, 122.7, 115.5, 115.4, 58.4, 54.2, 19.9, 11.2. ESI-MS m/z: 354.2
(M+H)+, calcd for C22H21F2NO: 353.16.
4.2.3. (3Z,5Z)-3,5-Bis(3,4-dihydroxybenzylidene)-tetrahydrothiopyran-4-one (3): Kelly solid,
20.4% yield，mp 223.3-224.6 oC [223.0-225.0 oC, Lit [22]]. 1H-NMR (600MHz, DMSO) δ: 7.44 (s,
2H, Ar-CH=C×2), 6.94 (d, J = 1.8 Hz, 2H, Ar-H2×2), 6.86 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 2H,
Ar-H6×2), 6.81 (d, J = 7.8 Hz, 2H, Ar-H5×2), 3.95 (s, 4H, CH2-S-CH2). ESI-MS m/z: 355.0 (M+H)
+, calcd for C19H16O5S: 356.07.
4.2.4. (3Z,5Z)-3,5-Bis(3,4,5-trimethoxybenzylidene)-tetrahydrothiopyran-4-one (4): Yellow solid,
41.0% yield, mp 208.5~210.6 oC. 1H-NMR (600MHz, CDCl3), δ: 7.71 (s, 2H, Ar-CH=C×2), 6.63 (s,
4H, Ar-H2×2, Ar-H6×2 ), 3.95 (s, 4H, CH2-S-CH2), 3.94 (s, 6H, 4-OCH3×2), 3.89 (s, 12H,
13
3-OCH3×2, 5-OCH3×2). C-NMR (CDCl3): 188.3, 152.7, 138.6, 136.6, 132.8, 130.1, 107.0, 60.5,
55.8, 29.68. ESI-MS m/z: 473.2 (M+H)+, calcd for C25H28O7S: 472.16.
4.2.5. (3Z,5Z)-3,5-Bis(3,4-dimethoxybenzylidene)-tetrahydrothiopyran-4-one (5): Yellow solid,
o
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1
52.2% yield, mp 158.2~161.1 C [165.0-166.0 C, Lit [23]]. H-NMR (600MHz, DMSO-d6), δ:
7.58 (s, Ar-CH=C×2), 7.12~7.14 (m, 4H, Ar-H2×2, Ar-H6×2), 7.04 (d, J = 9.0 Hz, 2H, Ar-H5×2),
4.02 (s, 4H, CH2-S-CH2), 3.81 (s, 6H, 3-OCH3×2, 4-OCH3×2). ESI-MS m/z: 413.3 (M+H)+, calcd

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for C23H24O5S: 412.13.
4.2.6. (3E,5E)-3,5-Bis(2-chlorobenzylidene)-tetrahydropyran-4-one (6): Light yellow solid, 58.7%
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1
yield, mp 207.4~209.9 C. H-NMR (600MHz, CDCl3), δ: 8.05 (s, 2H, Ar-CH=C×2), 7.47 (dd, J1
=2.0 Hz, J2 = 7.5 Hz, 2H, Ar-H3×2), 7.28~7.34 (m, 4H, Ar-H6×2, Ar-H4×2), 7.12 (dd, J1 =2.0 Hz, J2
= 7.5 Hz, 2H, Ar-H5×2), 4.78 (d, J = 2.0 Hz, 4H, CH2-O-CH2). ESI-MS m/z: 345.3 (M+H)+, calcd
for C19H14Cl2O22: 344.04.
4.2.7. (3E,5E)-3,5-Bis(4-methoxybenzylidene)-tetrahydropyran-4-one (7): Yellow solid, 46.3%
o
o
1
yield, mp 174.6~177.3 C [170.0-172.0 C, Lit [24]]. H-NMR (600MHz, CDCl3), δ: 7.79 (s, 2H,
Ar-CH=C×2), 7.29 (d, 4H, Ar-H2×2, Ar-H6×2), 6.95 (d, 4H, Ar-H3×2, Ar-H5×2), 4.93 (d, J = 1.2 Hz,
4H, CH2-O-CH2), 3.852 (s, 6H, 4-OCH3×2). ESI-MS m/z: 337.3 (M+H)+, calcd for C21H20O4:
336.14.
4.2.8. (3E,5E)-3,5-Bis(4-hydroxybenzylidene)-tetrahydropyran-4-one (8): Kelly solid, 44.2% yield,
o
1
mp﹥200.0 C. H-NMR (600MHz, Acetone-d6), δ: 8.93 (s, 2H, Ar-CH=C×2), 7.65 (s, 2H,
4-OH×2), 7.31 (d, J = 8.4 Hz, 4H, Ar-H2×2, Ar-H6×2), 6.94 (d, J = 8.4 Hz, 4H, Ar-H3×2, Ar-H5×2),
4.92 (s, 4H, CH2-O-CH2). ESI-MS m/z: 309.1 (M+H)+, calcd for C19H16O4: 308.1.
4.2.9. (3E,5E)-3,5-Bis(3-hydroxybenzylidene)-tetrahydropyran-4-one (9): Kelly solid, 40.2% yield,
o
1
mp 200.0 C Decompose. H-NMR (600MHz, CDCl3), δ: 8.61 (s, 2H, Ar-CH=C×2), 7.66 (s, 2H,
Ar-H2×2), 7.31 (t, 2H, Ar-H6×2), 6.90~6.92 (m, 4H, Ar-H4×2, Ar-H5×2), 4.94 (s, 4H, CH2-O-CH2).
13C-NMR (DMSO-d6): 184.9, 157.5, 138.1, 135.3, 135.2, 133.4, 129.8, 128.7, 121.6, 121.5, 121.2,
116.9, 116.8, 116.7, 116.6, 67.7. ESI-MS m/z: 306.7 (M-H)-, calcd for C19H16O4: 308.1.
4.2.10. (3E,5E)-3,5-Bis(2,4,6-trimethoxybenzylidene)-tetrahydropyran-4-one (10): Orange yellow
solid, 58.4% yield, mp 208.4~210.4 oC. 1H-NMR (600MHz, CDCl3), δ: 7.77 (s, 2H, Ar-CH=C×2),
6.12 (s, 4H, CH2-O-CH2), 4.42 (d, J = 1.2 Hz, 4H, Ar-H3×2, Ar-H5×2), 3.84 (s, 6H, 4-OCH3×2),
3.81 (s, 12H, 2-OCH3×2, 6-OCH3×2). ESI-MS m/z: 457.2 (M+H)+, calcd for C25H28O8: 456.18.
4.2.11. (3E,5E)-3,5-Bis(2,4-dimethoxybenzylidene)-tetrahydropyran-4-one (11): Yellow solid,
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1
47.0% yield, mp 129.4~132.7 C. H-NMR (600MHz, CDCl3), δ: 8.05 (s, 2H, Ar-CH=C×2), 6.99
(d, J = 8.4 Hz, 2H, Ar-H6×2), 6.50 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 2H, Ar-H5×2), 6.47 (d, J = 2.4 Hz,
2H, Ar-H3×2), 4.81 (d, J = 1.8 Hz, 4H, CH2-O-CH2), 3.88 (s, 6H, 2-OCH3×2), 3.85 (s, 6H,
6-OCH3×2). ESI-MS m/z: 397.3 (M+H)+, calcd for C23H24O6: 396.16.
4.2.12. (3E,5E)-3,5-Bis(2-methoxybenzylidene)-tetrahydropyran-4-one (12): Yellow solid, 47.4%

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yield, mp 138.5~141.1 C. H-NMR (600MHz, CDCl3), δ: 8.09 (s, 2H, Ar-CH=C×2), 7.37 (d, J =
8.0 Hz, 2H, Ar-H6×2), 7.07 (d, J = 8.0 Hz, 2H, Ar-H4×2), 6.98 (t, J = 8.0 Hz, 2H, Ar-H5×2), 7.93 (d,
J = 8.0 Hz, 2H, Ar-H3×2), 4.82 (d, J = 8.5 Hz, 4H, CH2-O-CH2), 3.87 (s, 6H, 2-OCH3×2). ESI-MS
m/z: 337.2 (M+H)+, calcd for C21H20O4: 336.14.
4.2.13. (3E,5E)-3,5-Bis(2,3-dimethoxybenzylidene)-tetrahydropyran-4-one (13): Yellow solid,
74.6% yield, mp 99.0~101.0 oC. 1H-NMR (600MHz, CDCl3), δ: 8.04 (s, 2H, Ar-CH=C×2), 7.07 (t,
J = 8.4 Hz, 2H, Ar-H5×2), 6.96 (dd, J1 = 0.6 Hz, J2 = 8.4 Hz, 2H, Ar-H6×2), 6.72 (dd, J1 = 0.6 Hz,
J2 = 8.4 Hz, 2H, Ar-H4×2), 4.83 (s, 4H, CH2-O-CH2), 3.87 (s, 6H, 3-OCH3×2), 3.85 (s, 6H,
2-OCH3×2). ESI-MS m/z: 397.3 (M+H)+, calcd for C23H24O6: 396.16.
4.2.14. (3E,5E)-3,5-Bis(2-(trifluoromethyl)benzylidene)-tetrahydropyran-4-one (14): Yellow solid,
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1
84.8% yield, mp 178.7~181.1 C. H-NMR (600MHz, CDCl3), δ: 8.07 (s, 2H, Ar-CH=C×2), 7.75
(d, J = 7.8 Hz, 2H, Ar-H3×2), 7.57 (t, J = 7.8 Hz, 2H, Ar-H5×2), 7.48 (t, J = 7.8 Hz, 2H, Ar-H4×2),
7.18 (d, J = 7.8 Hz, 2H, Ar-H6×2), 4.66 (s, 4H, CH2-O-CH2). ESI-MS m/z: 413.1 (M+H)+, calcd for
C21H14F6O2: 412.09.
4.2.15. (3E,5E)-3,5-Bis(3,4-dihydroxybenzylidene)-tetrahydropyran-4-one (15): Dark green solid,
75.2% yield, mp 253.0 oC (Black charring) [>300.0 oC, Lit [22]]. 1H-NMR (600MHz, DMSO-d6), δ:
9.56 (brs, 2H, 4-OH×2), 9.24 (brs, 2H, 3-OH×2), 7.46 (s, 2H, Ar-CH=C×2), 6.81 (d, J = 7.8 Hz, 2H,
Ar-H6×2), 6.81 (s, 2H, Ar-H2×2), 6.76 (d, J = 8.4 Hz, 2H, Ar-H5×2), 4.84 (s, 4H, CH2-O-CH2).
ESI-MS m/z: 339.1 (M-H)-, calcd for C19H16O6: 340.09.
4.2.16. (3E,5E)-3,5-Bis(2-bromobenzylidene)-tetrahydropyran-4-one (16): Yellow solid, 24.8%
yield, mp 212.6 oC (Zoom). 1H-NMR (600MHz, CDCl3), δ: 7.99 (s, 2H, Ar-CH=C×2), 7.66 (d, J =
7.8 Hz, 2H, Ar-H3×2), 7.34 (t, J = 7.8 Hz, 2H, Ar-H5×2), 7.22~7.25 (m, 2H, Ar-H4×2), 7.11 (d, J =
7.2 Hz, 2H, Ar-H6×2), 4.75 ( d, J = 1.8 Hz, 4H, CH2-O-CH2). ESI-MS m/z: 433.1 (M+H)+, calcd
for C19H14Br2O2: 431.94.
4.2.17. (3E,5E)-3,5-Bis(3,4,5-trimethoxybenzylidene)-tetrahydropyran-4-one (17): Yellow solid,
40.6% yield, mp 188.1~189.9 oC. 1H-NMR (600MHz, CDCl3), δ: 7.76 (s, 2H, Ar-CH=C×2), 6.55 (s,
4H, Ar-H2×2, Ar-H6×2), 4.97 (s, 4H, CH2-O-CH2), 3.89 (s, 6H, 4-OCH3×2), 3.87 (s, 12H,
13
3-OCH3×2, 5-OCH3×2). C-NMR (CDCl3): 184.6, 152.7, 139.1, 136.1, 131.8, 129.7, 107.6, 68.1,
60.5, 55.8. ESI-MS m/z: 457.4 (M+H)+, calcd for C25H28O8: 456.18.
4.2.18. (3E,5E)-3,5-Bis(2,3-dimethoxybenzylidene)-1-methylpiperidin-4-one (18): Light Yellow

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solid, 38.1% yield, mp 126.6~128.4 oC. 1H-NMR (600MHz, CDCl3), δ: 7.17 (s, 2H, Ar-CH=C×2),
7.05 (d, J = 7.8 Hz, 2H, Ar-H6×2), 7.03 (t, J = 7.8 Hz, 2H, Ar-H4×2), 7.00 (d, J = 7.8 Hz, 2H,
Ar-H5×2), 3.89 (s, 4H, CH2-N-CH2), 3.85-3.88 (m, 12H, 2-OCH3×2, 3-OCH3×2), 2.50 (s, 3H,
N-CH3). ESI-MS m/z: 410.2 (M+H)+, calcd for C24H27NO5: 409.19.
4.2.19. (3E,5E)-3,5-Bis(4-hydroxy-3-methoxybenzylidene)-tetrahydropyran-4-one (19): Kelly solid,
53.0% yield, mp 196.5~198.7 oC. 1H-NMR (600MHz, DMSO-d6), δ: 9.68 (s, 2H, 4-OH×2), 7.60 (s,
2H, Ar-CH=C×2), 7.02 (s, 2H, Ar-H2×2), 6.89 (d, J = 8.4 Hz, 2H, Ar-H6×2), 6.87 (d, J = 8.4 Hz, 2H,
Ar-H5×2), 4.91 (s, 4H, CH2-O-CH2), 3.83 (d, J = 8.4 Hz, 6H, 3-OCH3×2). ESI-MS m/z: 369.3
(M+H)+, calcd for C21H20O6: 368.13.
4.2.20. (3E,5E)-3,5-Bis(3,4-dimethoxybenzylidene)-tetrahydropyran-4-one (20): Yellow solid,
42.7% yield, mp 178.2~180.0 oC [180.0-181.0 oC, Lit [23]]. 1H-NMR (600MHz, CDCl3), δ: 7.78 (s,
2H, Ar-CH=C×2), 6.93 (d, J = 8.4 Hz, 2H, Ar-H6×2), 6.91 (s, 2H, Ar-H2×2), 6.88 (d, J = 8.4 Hz,
Ar-H5×2), 4.96 (s, 4H, CH2-O-CH2), 3.95 (s, 6H, 3-OCH3×2), 3.932 (s, 6H, 4-OCH3×2). ESI-MS
m/z: 397.3 (M+H)+, calcd for C23H24O6: 396.16.
4.2.21. (3Z,5Z)-3,5-Bis(2-chlorobenzylidene)-tetrahydrothiopyran-4-one (21): Light Yellow solid,
49.8% yield, mp 128.7-130.8 oC. 1H-NMR (600MHz, CDCl3), δ: 7.85 (s, 2H, Ar-CH=C×2), 7.46 (t,
J = 9.0 Hz, 2H, Ar-H3×2), 7.29-7.31 (m, 2H, Ar-H6×2), 7.29-7.31 (m, 2H, Ar-H5×2), 7.29-7.31 (m,
2H, Ar-H4×2), 3.75 (s, 4H, CH2-S-CH2). ESI-MS m/z: 361.2 (M+H)+, calcd for C19H14Cl2OS:
360.01.
4.2.22. (3Z,5Z)-3,5-Bis(4-methoxybenzylidene)-tetrahydrothiopyran-4-one (22): Yellow solid,
39.7% yield, mp 177.9~180.0 oC [174.0-176.0 oC, Lit [25]]. 1H-NMR (600MHz, CDCl3), δ: 7.75 (s,
2H, Ar-CH=C×2), 7.38 (d, J = 8.4 Hz, 4H, Ar-H2×2, Ar-H6×2), 6.94 (d, J = 8.4 Hz, 4H, Ar-H3×2,
Ar-H5×2), 3.93 (s, 4H, CH2-S-CH2), 3.85 (s, 6H, 4-OCH3×2). ESI-MS m/z: 353.3 (M+H)+, calcd
for C21H20O3S: 352.11.
4.2.23. (3Z,5Z)-3,5-Bis(2,4-dimethoxybenzylidene)-tetrahydrothiopyran-4-one (23): Yellow solid,
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21.2% yield, mp 164.0~165.4 C. H-NMR (600MHz, CDCl3), δ: 7.89 (s, 2H, Ar-CH=C×2), 7.19
(d, J = 8.4 Hz, 2H, Ar-H6×2 ), 6.50 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 2H, Ar-H3×2), 6.48 (d, J = 2.4 Hz,
Ar-H5×2), 3.84 (s, 6H, 2-OCH3×2), 3.83 (s, 6H, 4-OCH3×2), 3.77 (s, 4H, CH2-S-CH2). ESI-MS
m/z: 413.3 (M+H)+, calcd for C23H24O5S: 412.13.
4.2.24. (3E,5E)-3,5-Bis(2-fluorobenzylidene)-tetrahydropyran-4-one(24): Yellow solid, 72.7% yield,

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mp 127.6~128.5 C [157.0-158.0 C, Lit [26]]. H-NMR (600MHz, CDCl3), δ: 7.92 (s, 2H,
Ar-CH=C×2), 7.37~7.39 (m, 2H, Ar-H6×2), 7.18~7.21 (m, 4H, Ar-H3×2, Ar-H4×2), 7.13~7.17 (m,
2H, Ar-H5×2), 4.787 (s, 4H, CH2-O-CH2). ESI-MS m/z: 313.3 (M+H)+, calcd for C19H14F2O2:
312.1.
4.2.25. (3Z,5Z)-3,5-Bis(2,3-dimethoxybenzylidene)-tetrahydrothiopyran-4-one (25): Yellow solid,
79.5% yield, mp 88.1~90.2 oC. 1H-NMR (600MHz, CDCl3), δ: 7.86 (s, 2H, Ar-CH=C×2), 7.07 (t, J
= 7.8 Hz, 2H, Ar-H5×2), 6.94 (d, J = 7.8 Hz, 2H, Ar-H6×2), 6.86 (d, J = 7.8 Hz, 2H, Ar-H4×2), 3.93
(s, 6H, 2-OCH3×2), 3.84 (s, 6H, 3-OCH3×2), 3.76 (s, 4H, CH2-S-CH2). ESI-MS m/z: 413.3 (M+H)+,
calcd for C23H24O5S: 412.13.
4.2.26. (3E,5E)-3,5-Bis(2-bromobenzylidene)-1-methylpiperidin-4-one (26): Light yellow solid,
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85.4% yield, mp 145.4~147.0 C. H-NMR (600MHz, CDCl3), δ: 7.99 (s, 2H, Ar-CH=C×2), 7.68
(d, J = 7.8 Hz, 2H, Ar-H2×2), 7.38 (t, J = 7.8 Hz, 2H, Ar-H5×2), 7.24~7.29 (m, 4H, Ar-H4×2,
Ar-H6×2), 3.65 (s, 4H, CH2-N-CH2), 2.41 (s, 3H, N-CH3). 13C-NMR (CDCl3): 185.6, 135.9, 135.0,
133.5, 132.7, 129.8, 129.6, 126.6, 124.7, 56.1, 45.0. ESI-MS m/z: 446.0 (M+H)+, calcd for
C20H17Br2NO: 444.97.
4.2.27. (3E,5E)-3,5-Bis(3,4-dichlorobenzylidene)-1-methylpiperidin-4-one (27): Light yellow solid,
64.3% yield, mp 161.9-163.7 oC. 1H-NMR (600MHz, CDCl3), δ: 7.67 (s, 2H, Ar-CH=C×2), 7.50 (d,
J = 8.4 Hz, 2H, Ar-H5×2), 7.46 (s, 2H, Ar-H2×2), 7.21 (d, J = 7.8 Hz, 2H, Ar-H6×2), 3.71 (s, 4H,
CH2-N-CH2), 2.50 (s, 3H, N-CH3). ESI-MS m/z: 426.0 (M+H)+, calcd for C20H15Cl4NO: 424.99.
4.2.28. (3E,5E)-3,5-Bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidin-4-one (28): Yellow solid,
16.9% yield, mp 139.6~142.4 oC [151.0-152.0 oC, Lit[27]]. 1H-NMR (600MHz, CDCl3), δ: 7.79 (s,
2H, Ar-CH=C×2), 6.65 (s, 4H, Ar-H2×2, Ar-H6×2), 3.96 (s, 6H, 4-OCH3×2), 3.916 (s, 12H,
3-OCH3×2, 5-OCH3×2), 3.85 (s, 4H, CH2-N-CH2), 2.51 (s, 3H, N-CH3). 13C-NMR (CDCl3): 186.1,
152.6, 138.7, 136.2, 131.9, 130.3, 107.5, 60.5, 56.5, 55.8, 45.1. ESI-MS m/z: 470.0 (M+H)+, calcd
for C26H31NO7: 469.21.
4.2.29. (3E,5E)-3,5-Bis(4-fluorobenzylidene)-1-ethylpiperidin-4-one (29): Light yellow solid,
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1
57.7% yield, mp 139.0-140.5 C. H-NMR (600MHz, CDCl3), δ: 7.79 (s, 2H, Ar-CH=C×2), 7.38
(dd, J1 = 6.0 Hz, J2 = 7.8 Hz, 4H, Ar-H2×2, Ar-H6×2), 7.12 (t, J = 8.4 Hz, 4H, Ar-H3×2, Ar-H5×2),
3.82 (s, 4H, CH2-N-CH2), 2.64 (q, J = 7.2 Hz, 2H, N-CH2), 1.07 (t, J = 7.2 Hz, 3H, CH3). ESI-MS
m/z: 340.2 (M+H)+, calcd for C21H19F2NO: 339.14.

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4.2.30. (3E,5E)-3,5-Bis(2,3-dimethoxybenzylidene)-1-ethylpiperidin-4-one (30): Yellow solid,
68.3% yield, mp 68.6~71.0 oC. 1H-NMR (600MHz, CDCl3), δ: 8.03 (s, 2H, Ar-CH=C×2), 7.08 (dd,
J1 = 8.4 Hz, J2 = 7.8 Hz, 2H, Ar-H6×2), 6.95 (d, J = 8.4 Hz, 2H, Ar-H5×2), 6.85 (d, J = 7.8 Hz, 2H,
Ar-H4×2), 3.88 (s, 6H, 3-OCH3×2), 3.84 (s, 6H, 2-OCH3×2), 2.54 (q, J = 7.2 Hz, 2H, N-CH2), 0.99
(t, J = 7.2 Hz, 3H, CH3). ESI-MS m/z: 424.3 (M+H)+, calcd for C25H29NO5: 423.2.
4.2.31. (3E,5E)-3,5-Bis(4-hydroxybenzylidene)-1-propylpiperidin-4-one (31): Yellow solid, 65.3%
o
o
1
yield, mp 209.0-211.4 C [198.0-200.0 C, Lit [21]]. H-NMR (600MHz, CDCl3), δ: 7.79 (s, 2H,
Ar-CH=C×2), 7.43 (d, J = 8.0 Hz, 4H, Ar-H2×2, Ar-H6×2), 6.93 (d, J = 8.0 Hz, 4H, Ar-H3×2,
Ar-H5×2), 4.56 ( s, 4H, N-CH2×2), 3.23 (s, 2H, N-CH2-), 1.68 (s, 2H, N-CH2-CH2), 0.99 (t, J = 7.2
Hz, 3H, CH3). ESI-MS m/z: 350.0 (M+H)+, calcd for C22H23NO3: 349.17.
4.2.32. (3E,5E)-3,5-Bis(4-fluorobenzylidene)-tetrahydropyran-4-one (32): Yellow solid, 52.0%
yield, mp 188.5-190.8 oC. 1H-NMR (600MHz, CDCl3), δ: 7.79 (s, 2H, Ar-CH=C×2), 7.31 (dd, J1 =
5.4 Hz, J2 = 8.4 Hz, 4H, Ar-H2×2, Ar-H6×2), 7.10~7.14 (m, 4H, Ar-H3×2, Ar-H5×2), 4.90 (d, J = 1.8
Hz, 4H, CH2-O-CH2). ESI-MS m/z: 313.1 (M+H)+, calcd for C19H14F2O2: 312.1.
4.2.33. (3E,5E)-3,5-Bis(2,4-dichlorobenzylidene)-tetrahydropyran-4-one (33): Yellow solid, 75.0%
yield, mp 202.1 oC (Zoom). 1H-NMR (600MHz, CDCl3), δ: 7.96 (s, 2H, Ar-CH=C×2), 7.50 (d, J =
8.5 Hz, 2H, Ar-H6×2), 7.30 (s, 2H, Ar-H3×2), 7.05 (d, J = 8.5 Hz, 2H, Ar-H5×2), 4.75 (s, 4H,
CH2-O-CH2). ESI-MS m/z: 413.4 (M+H)+, calcd for C19H12Cl4O2: 411.96.
4.2.34. (3Z,5Z)-3,5-Bis(2,4,6-trimethoxybenzylidene)-tetrahydrothiopyran-4-one (34): Yellow solid,
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60.5% yield, mp 212.5 C (Charring). H-NMR (600MHz, CDCl3), δ: 7.64 (s, 2H, Ar-CH=C×2),
6.14 (s, 4H, Ar-H3×2, Ar-H5×2), 3.84 (s, 6H, 4-OCH3×2), 3.80 (s, 12H, 6-OCH3×2, 2-OCH3×2),
13
3.50 (s, 4H, CH2-S-CH2). C-NMR (CDCl3): 187.8, 162.7, 158.7, 134.8, 128.8, 106.0, 90.0, 55.2,
54.9, 31.2. ESI-MS m/z: 473.2 (M+H)+, calcd for C25H28O7S: 472.16.
4.2.35. (3Z,5Z)-3,5-Bis(2-bromobenzylidene)-tetrahydrothiopyran-4-one (35): Yellow solid, 59.0%
yield, mp 169.2-170.9 oC. 1H-NMR (600MHz, CDCl3), δ: 7.80 (s, 2H, Ar-CH=C×2), 7.66 (dd, J1 =
1.2 Hz, J2 = 7.8 Hz, 2H, Ar-H3×2), 7.35 (dt, J1 = 1.2 Hz, J2 = 7.8 Hz, 2H, Ar-H5×2), 7.27 (dd, J1 =
1.2Hz, J2 = 7.8 Hz, 2H, Ar-H6×2), 7.22 (dt, J1 = 1.2, J2 = 7.8 Hz, 2H, Ar-H4×2), 3.73 (s, 4H,
13
CH2-S-CH2). C-NMR (CDCl3): 188.0, 135.8, 135.1, 134.6, 132.7, 129.9, 129.6, 126.7, 124.3,
29.2. ESI-MS m/z: 449.0 (M+H)+, calcd for C19H14Br2OS: 447. 91.

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4.3. Animal care and maintenance
Male C57BL/6 mice (n=64) weighing 18-22 g and Sprague Dawley (SD) rats (n=28) were
obtained from the SLAC Laboratory Animal Center (Shanghai, China). All animals were housed at
a constant room temperature with a 12:12 hr light-dark cycle and fed with a standard rodent diet
and water. The animals were acclimatized to the laboratory at least for 7 days before used. The
animal care and experimental procedures were approved by the Wenzhou Medical University
Animal Policy and Welfare Committee, and received humane care according to the National
Institutes of Health (USA) guidelines. Reports of studies involving animals are in accordance with
the ARRIVE guidelines for reporting experiments involving animals [28, 29].
4.4. Cell culture
Human Umbilical Vein Endothelial Cells (HUVECs) and RAW 264.7 macrophages were
obtained from the Shanghai Institute of Biosciences and Cell Resources Center (Chinese Academy
of Sciences, Shanghai, China) and cultured in DMEM medium supplemented with 10% FBS, 100
U/mL penicillin, and 100 mg/mL streptomycin at 37 °C with 5% CO2. Primary peritoneal
macrophages (MPMs) were prepared as previously described [13]. Briefly, C57BL/6 mice were
stimulated by intraperitoneal (i.p.) injection of 1.5 mL of thioglycollate solution per mouse and kept
in pathogen-free conditions for 3 days before peritoneal macrophage isolation. MPMs were
harvested by washing the peritoneal cavity with 8 mL RPMI-1640 medium per mouse, centrifuged,
and the pellet was re-suspended in RPMI-1640 medium with 10% FBS, 100 U/mL penicillin, and
100 mg/mL streptomycin. Non-adherent cells were removed by washing with medium 4 hr after
seeding. Experiments were undertaken after MPMs adhered firmly to the culture plates.
4.5. Detection of TNF-α and IL-6 secretion
MPMs were pretreated with DMSO or MACs for 30 min, followed by treatment with LPS (0.5
µg/mL) for 24 hr. The conditioned media and the MPMs were separately collected. TNF-α and IL-6
in media were determined with an ELISA kit according to the manufacturer’s instructions. The total
amount of cytokines in the medium was normalized to total protein determined from MPMs. The
amount of TNF-α in bronchoalveolar lavage fluid (BALF) and serum were also detected by ELISA
assay.

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4.6. Real-time quantitative PCR
The mRNA from MPMs and lung tissue was determined by standard real-time qPCR. MPMs
were pretreated with DMSO or MACs for 30 min, followed by treatment with LPS (0.5 µg/mL) for
6 hr. Total RNA was isolated from cells and lung tissues (20-30 mg) using Trizol-reagent and
quantified by UV absorption at 260 and 280 nm. Both reverse transcription and qPCR 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, Hamburg,
Germany) was used for qPCR analysis. The primers of genes were synthesized by Invitrogen
(Invitrogen, Shanghai, China). The mRNA values were normalized to β-actin mRNA.
4.7. Western Blot Assay
MPMs were pretreated with DMSO, MAC 17 or 28 at different concentrations (2.5, 5.0 or 10.0
µM) for 30 min, followed by treatment with LPS (0.5 µg/mL) for 20 min. MPMs were collected,
lysed, subjected to 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF)
membrane (Bio-Rad Laboratories). The membranes were blocked for 1.5 hr at room temperature
with 5% nonfat dry milk in TBST (Tris-buffered saline containing 0.05% Tween 20) and incubated
with specific primary antibodies overnight at 4 °C. The membranes were washed in TBST,
incubated with a 1:3000 dilution of HRP-conjugated secondary antibody for 1 hr at room
temperature, and visualized using enhanced chemiluminescence reagents (Beyotime Biotech,
Nantong, China).
4.8. Nuclear translocation of NF-κB
MPMs were pretreated with DMSO, MAC 17 or 28 at different concentrations (2.5, 5.0 or 10.0
µM) for 30 min, followed by treatment with LPS (0.5 µg/mL) for 1 hr. Nuclear and cytoplasmic
proteins were extracted from MPMs using a nuclear and cytoplasmic protein extraction kit
(Beyotime Biotech, Nantong, China) following manufacturer’s instructions. The protein
concentration in the nuclear and cytoplasmic fractions was determined using the Bio-Rad protein
assay reagent extracts, and subjected to Western immunoblot analysis.

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4.9. Electrophoretic mobility shift assay (EMSA)
MPMs were pretreated with DMSO, MAC 17 or 28 (10.0 µM for 30 min), followed by
treatment with LPS (0.5 µg/mL) for 1 hr. Nuclear extracts were prepared using the Nuclear and
Cytoplasmic Protein Extraction Kit (Beyotime Biotech, Nantong, China) and protein concentrations
were determined. EMSA was performed with a non-radioactive (biotin label) gel shift assay
according to the manufacturer's protocol. Briefly, 5 µg of nuclear extract was incubated with 2
nM of NF-κB probe in binding buffer for 30 min at room temperature. The reaction mixtures were
separated in a 4 % nondenaturing polyacrylamide gel in 0.5 × TBE at 100 V for 1 hr, the
DNA/protein complex was transferred to nylon membrane, conjugated with Streptavidin-HRP, and
visualized with enhanced chemiluminescence reagents (Beyotime Biotech, Nantong, China). The
NF-κB
consensus
oligonucleotides
(double
stranded
DNA)
were:
5’-AGTTGAGGGGACTTTCCCAGGC-3’.
4.10. NF-κB-Luciferase reporter assay
RAW 264.7 cells with stable expression of NF-κB luciferase reporter construct were seeded in
6-well plates (2x105 cells/well) and incubated for 24 hr. Cells were washed, incubated in fresh
culture medium, pretreated with MAC 17 or 28 (2.5, 5.0 or 10.0 µM), stimulated with LPS for 6 hr.
Cells were extracted in lysis buffer and luciferase activities measured using the luciferase assay
system (Promega, Madison, WI, USA) according to the manufacturer's instructions.
4.11. LPS-induced animal models of ALI and sepsis
ALI: Male SD rats (n=28) were randomly divided into four weight-matched groups: control,
LPS, 17+LPS and 28+LPS. Rats were administered orally with MAC 17 or 28 (20 mg/kg/day) for
one week prior to intratracheal challenge with LPS. Thirty min after the last dosing of MACs, the
rats were anesthetized by ether, and their necks dissected to expose the trachea for injection with
LPS (5 mg/kg) or sterile water (50-60 µL) using a 100 µL syringe. After 6 hr of intracheal injection
of LPS, the rats were euthanized with ketamine, thorax opened to expose the lungs for collection of
BALF.
Sepsis: MACs 17 and 28 were dissolved with macrogol 15 hydroxystearate (a nonionic
solubilizer for injection) (BASF, Ludwigshafen, Germany) with or without medium chain

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triglycerides (MCT) (BASF, Ludwigshafen, Germany) in water bath at 37 ºC at a final
concentration of 2 mg/mL. The final solubilizer concentration ranged 5–10%, and MCT 0.5–2%.
For vehicle, a mixture of solubilizer and MCT was prepared at 10% and 2%, respectively. Male
C57BL/6 mice weighing 18–22 g were pretreated with MAC 17 or 28 (10 mg/kg) in a water
solution by intravenous (IV) injection 15 min before the IV injection of LPS (20 mg/kg). Control
animals received a similar volume (100 µL) of vehicle. The mice mortality was recorded for 7
days.
4.12. BALF analysis
The collected BALF was centrifuged at 1000 rpm for 10 min at 4 °C, the supernatant was used
for protein concentration detection and cytokine determinations. The cell pellet was re-suspended in
50 µL of physiological saline, and total number of cells counted. Cytospin preparations were made
for evaluation of cell differentials stained with Wright-Giemsa staining (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China). Neutrophils were counted from a total of 200 cells under
a light microscopy.
4.13. Myeloperoxidase activity
Tissue myeloperoxidase activity (MPO) was determined as index of neutrophil activity in lungs
using a MPO Detection Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing China). Lung
tissue was homogenized in 1 mL of 50 mM potassium PBS (pH 6.0) containing 0.5%
hexadecyltrimethylammonium hydroxide and centrifuged at 12,000 rpm at 4 °C for 20 min. The
supernatant (10 µL) was transferred into PBS (pH 6.0) containing 0.17 mg/mL
3,3’-dimethoxybenzidine and 0.0005% H2O2. MPO activity was determined by measuring
absorbance values at 460 nm in a spectrophotometer, and presented as units per gram of total
protein (U/g). Total protein content in the samples was determined by Bio-Rad protein assay.
4.14. Lung wet/dry ratio
Pulmonary edema was evaluated by the lung wet/dry weight ratio. The middle lobe of the right
lung was collected, the wet weight was recorded, heated in a thermostatic oven at 65 °C for 72 hr,
and weight recorded. The ratio of wet/dry lung weight was reported.

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4.15. Histopathological evaluation
The superior lobe of the right lung was collected, fixed in 4% paraformaldehyde, embedded in
paraffin and cut into 5 µm sections. The sections were stained with hematoxylin and eosin using
standard protocol for light microscopy examination.
4.16. Immunohistochemistry
Paraffin tissue sections (5 µm) were deparaffinized in xylene, and hydrated using an ethanol
gradient. Pressure-cooker was used for antigen retrieval. After treatment with 30% H2O2, the
sections were blocked in 5% bovine serum albumin and incubated with primary anti-CD68
antibody overnight at 4 °C. The slides were incubated with HRP-labeled secondary antibody for 10
min, stained with 3,3-diaminobenzidine tetrahydrochloride (DAB), and evaluated by light
microscopy.
4.17. Binding affinity of MAC 17 or 28 with MD2
The binding affinity of 17 or 28 with rhMD2 and MD2-ArgR90A proteins was determined by
surface plasmon resonance (SPR) analysis using a ProteOn XPR36 Protein Interaction Array
system (Bio-Rad Laboratories, Hercules, CA) with a HTE sensor chip (ProteOn™, #176-5033).
The protein was loaded to the sensors which was activated with 10 mM NiSO4. MAC 17 or 28
samples (at 6.25, 12.5, 25, 50, 100 and 200 µM) were prepared with running buffer (PBS, 0.1%
SDS, 5% DMSO). Sensor and sample plates were placed on the instrument, and MAC 17 or 28
samples were captured in flow cell one, leaving the second flow cell as blank. The 6 concentrations
were injected simultaneously at a flow rate of 30 µM/min for 120 s of association phase, followed
with 120 s of dissociation phase at 25 °C. The final graphs were obtained by subtracting blank
sensorgrams from the duplex or quadruplex sensorgrams, and data were analyzed with ProteOn
manager software. The KD was calculated by global fitting of the kinetic data from various
concentrations of 17 or 28 using 1:1 Langmuir binding model.
4.18. Competition displacement of bis-ANS by MACs
The ability of MAC 17 or 28 to compete off the fluorescent probe bis-ANS bound to human

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recombinant MD2 (rhMD2) was determined. Bis-ANS has the structural motif similar to lipid A of
LPS and binds within the hydrophobic binding pocket of MD2[17]. RhMD2 protein (5 nM) and
1,1׳-Bis(anilino)-4,4׳-bis(naphthalene)-8,8׳-disulfonate (bis-ANS, 5 µM) were mixed in PBS (pH
7.4) and incubated to reach stable relative fluorescence units (RFUs) emitted at 430–590 nm
(excitation at 385 nm) at 25 °C in a 1 cm path-length quartz cuvette measured with a spectraMax
M5 (Molecular Devices, Sunnyvale, CA). MAC 17 or 28 (2.5-30 µM) was added, and fluorescence
units (RFUs) determined (emission at 430-590 nm).
4.19. Competition with LPS bound on MD2
The ability of MAC 17 or 28 to compete off biotin-labeled LPS (biotin-LPS) bound to rhMD2
and MD2-ArgR90A was determined by ELISA. Anti-rhMD2 antibody was coated onto 96-well
dishes at 4 °C overnight, and rhMD2 (4 µg/mL) in 10 mM Tris-HCl buffer was added to the
pre-coated plates for 1.5 hr at room temperature. The plates were washed with PBST, and
biotin-labeled LPS was added to the plate with or without MAC 17 or 28 (1 µM). Absorbance was
determined at 450 nm.
4.20. Competition with cell surface-bound LPS
The ability of MAC 17 or 28 to compete off LPS bound to the cell surface of HUVECs was
determined. HUVECs were serum starved 3 hr, and were next incubated for 30 min with or without
FITC-labeled LPS (FITC-LPS) (50 µg/mL) in the presence or absence of MAC 17 or 28 (0.1, 1 and
10 µM). HUVECs were fixed with paraformaldehyde for 10 min at 4 °C and washed with PBS for
analysis by flow cytometry.
4.21. Evaluation of TLR4-MD2 complex dimerization by co-IP
The effects of MAC 17 or 28 on the formation of the activating dimerization of the TLR4-MD2
complex were evaluated in RAW 264.7 macrophages and lung tissues. The cells and lung tissues
were lysed by extraction buffer (containing mammalian protein extraction reagent supplemented
with protease and phosphatase inhibitor cocktails) and centrifuged at 12,000 rpm for 10 min at 4 °C.
Sufficient amount of MD2 antibody was added to 400 µg proteins and gently rotated at 4 °C
overnight. The immunocomplex was collected with protein A+G agarose (Beyotime Biotech,

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Nantong, China), the precipitates were washed 4x with ice-cold PBS, and proteins were released by
boiling in sample buffer. The proteins were prepared for Western blot analysis using the
precipitating MD2 antibody, and blotting for TLR4.
4.22. Docking simulation of MAC 17 or 28 with MD2
The binding mode of MAC 17 or 28 with human MD2 protein (PDB ID: 2E56) was performed
using molecular modeling packages in Sybyl-2.0 (Tripos, St. Louis, MO). Energy minimization
of modeled structures was performed using the Tripos force field with Gasteiger- Hückel charges, a
fixed dielectric constant of 80 and a non-bonded cutoff radius of 8Å. Minimization was carried
out for a maximum f 5,000 iterations subject to a termination gradient of 0.05 kcal/(mol·Å). The
ligand-binding groove on MD2 protein was kept rigid, whereas all torsible bonds of MAC 17 or 28
were set free to allow flexible docking to produce more than 100 structures. Final docked
conformations were clustered within the tolerance of 1 Å root-mean-square deviation.
4.23. Statistical analysis
The results are presented as means ± SEM. One-way ANOVA was employed to analyze
differences between sets of data. Statistics were performed using GraphPad Software (GraphPad,
San Diego, CA). P values less than 0.05 (p <0.05) were considered significant, and all experiments
were repeated at least three times.
Acknowledgements
This work was financially supported by the National Key Science Project of MOST
(2017YFA0506000), National Natural Science Funding of China (81503123 to Y.L., 81622043 to
G.L. and 81773579 to Z.L.), Zhejiang Natural Science Funding (LR16H310001 to G.L.
LY17B020008 to Z.L. and LY18H310011 to Y.L.).
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