Design and synthesis novel di-carbonyl analogs of curcumin (DACs) act as potent anti-inflammatory agents against LPS-induced acute lung injury (ALI)

Article abstract of DOI:10.1016/j.ejmech.2019.02.042

A novel series of di-carbonyl analogs of curcumin (DACs) were prepared and evaluated for their anti-inflammatory properties. Preliminary results showed that a vast majority of compounds tested in this study could effectively suppress LPS-induced production of tumor necrosis factor (TNF)-α and interleukin (IL)-6. Structure-activity relationships of the compounds were discussed. Compounds 5a27 and 5a28 showed the most potent anti-inflammatory activities and had higher structural stability and orally bioavailability than curcumin in vitro. Mechanistically, they inhibited the activation of macrophages via the blockade of mitogen-activated protein kinase (MAPK) signaling and nuclear translocation of NF-κB. In vivo, 5a27 and 5a28 markedly alleviated lipopolysaccharides (LPS)-induced acute lung injury (ALI). The wet/dry ratio of lungs was significantly normalized by the active compounds, which was consistent with the suppression of neutrophil infiltration and production of proinflammatory cytokines. Collectively, these results present a new series of curcumin analogs as promising anti-inflammatory agents for treatment of ALI.

Full text of DOI:10.1016/j.ejmech.2019.02.042

Accepted Manuscript
Design and synthesis novel di-carbonyl analogs of curcumin (DACs) act as potent
anti-inflammatory agents against LPS-induced acute lung injury (ALI)
Jianchang Qian, Xianxin Chen, Sheng Shu, Wenxin Zhang, Bo Fang, Xiaojing Chen,
Yunjie Zhao, Zhiguo Liu, Guang Liang
PII:
S0223-5234(19)30156-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.042
EJMECH 11134
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 November 2018
Revised Date: 11 February 2019
Accepted Date: 11 February 2019
Please cite this article as: J. Qian, X. Chen, S. Shu, W. Zhang, B. Fang, X. Chen, Y. Zhao, Z. Liu,
G. Liang, Design and synthesis novel di-carbonyl analogs of curcumin (DACs) act as potent anti-
inflammatory agents against LPS-induced acute lung injury (ALI), European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.042.
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ACCEPTED MANUSCRIPT
Design and synthesis novel di-carbonyl analogs of curcumin (DACs)
act as potent anti-inflammatory agents against LPS-induced acute
lung injury (ALI)
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Jianchang Qian 1,#, Xianxin Chen 1,#, Sheng Shu 1,#, Wenxin Zhang , Bo Fang , Xiaojing Chen , Yunjie
Zhao 1,*, Zhiguo Liu 1,*, Guang Liang 1
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Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University,
1210 University Town, Wenzhou, Zhejiang 325035, China
Graphical abstract:
Novel di-carbonyl analogs of curcumin (DACs) were designed, synthesized and evaluated for
anti-inflammatory activities for the treatment of ALI.

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Design and synthesis novel di-carbonyl analogs of curcumin
(DACs) act as potent anti-inflammatory agents against
LPS-induced acute lung injury (ALI)
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Jianchang Qian 1,#, Xianxin Chen 1,#, Sheng Shu 1,#, Wenxin Zhang 1, Bo Fang 1, Xiaojing Chen 1,
Yunjie Zhao 1,*, Zhiguo Liu 1,*, Guang Liang 1
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Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical
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University, 1210 University Town, Wenzhou, Zhejiang 325035, China
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# These authors contribute equally to this work.
* Corresponding author:
Yunjie Zhao, 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: aabye1100@aliyun.com
and Zhiguo Liu, 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: lzgcnu@163.com
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Abstract
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A novel series of di-carbonyl analogs of curcumin (DACs) were prepared and evaluated for
their anti-inflammatory properties. Preliminary results showed that a vast majority of compounds
tested in this study could effectively suppress LPS-induced overproduction of tumor necrosis
factor (TNF)-α and interleukin (IL)-6. Structure-activity relationships of the compounds were
discussed. Compounds 5a27 and 5a28 showed the most potent anti-inflammatory activities and
had higher structural stability and orally bioavailability than curcumin in vitro. Mechanistically,
they inhibited the activation of macrophages via the blockade of mitogen-activated protein kinase
(MAPK) signaling and nuclear translocation of NF-κB. In vivo, 5a27 and 5a28 markedly
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alleviated lipopolysaccharides (LPS)-induced acute lung injury (ALI). The wet/dry ratio of lungs
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was significantly normalized by the active compounds, which was consistent with the suppression
of neutrophil infiltration and production of proinflammatory cytokines. Collectively, these results
present a new series of curcumin analogs as promising anti-inflammatory agents for treatment of
ALI.
16 Key words: Di-carbonyl analogs of curcumin; Structural stability; Acute lung injury;
Anti-inflammation.
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1. Introduction
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Acute lung injury (ALI) is a severe clinical syndrome of hypoxemic respiratory failure with
substantial morbidity and mortality [1]. Even survivors of ALI have their long-term quality of life
compromised owing to a lack of effective therapeutic approaches [2, 3]. Recent advances in
unveiling the pathogenesis of ALI have identified the blockade of excessively inflammatory
cascades as an effective strategy to attenuate lung injury [4]. Despite some encouraging preclinical
evidence for potential pharmacological treatments, the phase III clinical trials of most of the drug
candidates, such as glucocorticoids, surfactants and procysteine, were not successful. There is thus
an unmet need for effective anti-inflammatory drugs to treat patients with ALI [5].
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Bacterial infection is one of the most common causes of ALI [6]. Release of endotoxins,
especially lipopolysaccharide (LPS), is the leading contributor. This is a potent activator of
toll-like receptor 4 (TLR4), which triggers the nuclear translocation of the nuclear factor κB
(NF-κB), thereby promoting the transduction of proinflammatory molecules, such as cytokines
interleukin 6 (IL-6), IL-1β and tumor necrosis factor α (TNF-α) [7]. Therefore, suppression of
inflammatory responses by blocking activation of NF-κB could be a potential strategy of treating
ALI.
Natural products and their plant-derived analogs are expected to play a crucial role in the
design
of
novel
anti-inflammatory
agents
[8,
9].
Curcumin,
[(1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)], is
a
phenolic compound
originally isolated from the rhizome of Curcuma longa. Its anti-inflammatory [10-12], antioxidant
[13] and antitumor activities [14, 15] have attracted much attention along with its favorable
toxicity profile. However, the clinical development of curcumin is limited by its instability and
poor solubility in water, resulting in limited oral bioavailability which fails to reach the minimum
effective therapeutic concentration [16, 17]. Previous studies have demonstrated that the active
methylene group of the β-diketone moiety in the structure of curcumin could be related to its
instability under physiological conditions, inducing rapid degradation and metabolism [18-20]. In
another study, curcumin was incubated in buffer solutions of pH 7.2 at 37 °C, and about 90%
degraded within 30 min. Trans-6-(4'-hydroxy-3'-methoxyphenyl)-2,4-dioxo-5-hexenal was
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predicted as the key degradation product and vanillin, ferulic acid and feruloyl methane were
identified as minor degradation products [21] (Fig. 1). Numerous curcumin analogs have been
designed and synthesized in an attempt to overcome these limitations and improve the chemical
instability.
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Recently, our research group has synthesized a panel of mono-carbonyl analogues of curcumin
(MACs), by modifying the central β-diketone moiety to a mono ketone group. These MACs
exhibited greater metabolic stability and bioactivity than the parent molecule [22, 23]. For
example, we incorporated the active methylene group into an α, β-unsaturated cyclohexanone, and
changed the substituted benzene rings into 3,4,5-trimethoxyphenyl rings, to furnish a structurally
symmetrical MAC C03. Pleasingly, a higher anti-inflammatory activity and stability were
observed with C03 than that of curcumin. Additionally, in our ongoing search for similar
functional groups that are able to replace the unstable β-diketone moiety in curcumin, we
investigated a new chemical entity containing an imide moiety. Isolated from the plant long
pepper (Piper longum), piperlongumine (PL) constitutes two α, β-unsaturated imides and exhibits
highly selective anticancer activities and remarkable anti-inflammatory properties [24, 25].
Interestingly, the structure of PL also contains an active 3,4,5-trimethoxyphenyl group.
Encouraged by these findings, we introduced piperidin-2-one and 3,4,5-trimethoxyphenyl moieties
into the curcumin skeleton to afford a series of novel di-carbonyl analogs of curcumin (DACs)
whose biological activities were then evaluated.
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Please insert the Figure 1
24 2. Results and discussion
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2.1. Chemistry
The syntheses and structures of the DACs (6a01–6a29) are illustrated in Scheme 1. Briefly,
treatment of commercially available or previously synthesized cinnamic acids (1a01–24, 1a26–27,
1a29) with oxalyl chloride and a catalytic amount of DMF provided the corresponding cinnamoyl
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chlorides 2a01–24, 2a26–27 and 2a29. After simple work-up to remove most of the solvent, the
crude products were used directly for the next step without further purification. Alternatively, the
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other key intermediate (E)-3-(3,4,5-trimethoxybenzylidene) piperidin-2-one (4) was prepared from
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available
3,4,5-trimethoxybenzaldehyde
(3)
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tert-butyl
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2-oxopiperidine-1-carboxylate in the presence of sodium hydride. The title amides 5a01–5a24,
5a26–5a27 and 5a29 were furnished in satisfactory yields via coupling of intermediate 4 and the
previously synthesized cinnamoyl chlorides. Furthermore, after catalytic removal of the acetyl
protecting group in 20% NaHCO3, 5a24 and 5a27 were successfully converted into desired
compounds 5a25 and 5a28, respectively. The structures of all new DACs were fully characterized
by proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic resonance (13C NMR)
and electrospray ionization mass spectrometry (ESI-MS).
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Please insert the Scheme 1
2.2 Structure-activity relationship (SAR) and cytotoxic evaluation
Lipopolysaccharide (LPS) is a major biologically active component of Gram-negative bacteria
and plays an important role in the development of systemic inflammation responses, especially in
the expression of several inflammatory cytokines, such as TNF-α and IL-6 [26, 27]. Furthermore,
these two cytokines activate an inflammatory-induced ALI reaction process [28]. To explore the
inhibitory effects of the new DACs, LPS induced production of TNF-α and IL-6 was determined
in mouse primary macrophages (MPMs). Following an established procedure [29], all the newly
synthesized compounds were tested for in vitro anti-inflammatory activity through an
enzyme-linked immunosorbant assay (ELISA) at a single concentration of 10 µM (Fig.2A and B).
Our initial biological screening indicated that the majority of DACs exhibited effective
inhibition (39.6–99.5% inhibitory rates) against the LPS-stimulated production of both TNF-α and
IL-6. Compounds 5a01–5a07, with mono-halogen substituents in the phenyl ring, had weak
potency in inhibiting release of either IL-6 or TNF-α (inhibitory rates <85.3%), relative to the
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positive control of curcumin or PL. However, the DAC 5a08 with a strong electron-withdrawing
nitro group at the para-position of the phenyl ring, demonstrated marked inhibitory activities (94.1%
and 38.6% for IL-6 and TNF-α, respectively). For further enhancement of anti-inflammatory
activity, di-substituted compounds 5a09–5a16, bearing di-halogens or 2-fluoro atoms at the
phenyl ring, were evaluated. 3,4-Difluoro, 2,6-dichloro and 2-fluoro-4-methoxy substituted
compounds 5a09, 5a12 and 5a16, showed comparable in vitro activities to that of curcumin.
Conversely, introduction of 2,5-difluoro (5a10), 2,4-dichloro (5a11), 2-fluoro-6-chloro (5a13),
2-chloro-4-fluoro (5a14) and 2-fluoro-5-methoxy (5a15) substituents to the phenyl ring afforded
significantly reduced potencies, with inhibition rates ranging from 38.5% to 53.4% (for TNF-α)
and 46.8% to 68.2% (for IL-6).
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Incorporation of a methoxy group at different positions of the phenyl ring yielded compounds
5a17–5a22. Except compound 5a20, which had a 2,3-dimethoxyphenyl ring, these compounds
exhibited significant inhibition on the production of IL-6 and TNF-α. Changing from the two
fluoro groups in 5a10 to the methyl groups in 5a23 gave a similar potency with slightly increased
inhibition of TNF-α production. With the phenyl ring in place, we next incorporated hydroxy
groups or acetoxy groups at the ortho-, meta- or para- position, yielding compounds 5a24–5a29
which provided higher IL-6 inhibition than that of curcumin. Notably, greater activity occurred
when the methoxy group was located at the meta-position (5a18, 5a22, 5a27 and 5a28), indicating
that the position of the methoxy group might play an important role in the activity. Importantly,
compounds 5a27 and 5a28 with a hydroxyl or acetoxy substituent at the para- position of the
phenyl ring displayed the greatest effects in reducing LPS-induced TNF-α and IL-6 production,
with their inhibitory rates reaching 90.9–92.5% (for TNF-α) and 99.3–99.7% (for IL-6),
respectively. These findings support the anti-inflammatory effects of the novel di-carbonyl
analogs of curcumin.
Please insert the Figure 2
Before further studies, the cytotoxicity and safety of these synthetic DACs were evaluated in
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MPMs by MTT assays at concentration of 10 µM. As shown in Fig. 2C, almost all of the
compounds exhibited no significant effects on cell proliferation, supporting their reasonable safety.
What’s more, no significant differences were observed while comparing DACs to PL and
curcumin.
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2.3 Dose-dependent inhibition of LPS-induced release of cytokines
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DACs effectively inhibited the production of proinflammatory cytokines without significant
cytotoxicity. To further investigate the anti-inflammatory activities of the active compounds, two
of the most potent compounds, 5a27 and 5a28, were selected. As shown in Fig.3A and B, 5a27
and 5a28 exhibited dose-dependent inhibition of LPS-induced release of TNF-α and IL-6 in active
RAW 264.7 mouse macrophages, within the dose range of 2.5 to 20 µM. The potency of DACs in
suppressing TNF-α and IL-6 production is much higher than curcumin’s. The IC50 values of 5a27
and 5a28 were at the low µM levels (5a27: 2.33 ± 0.78 µM and 2.40 ± 0.44 µM for TNF-α and
IL-6 respectively; 5a28: 3.69 ± 1.34 µM and 3.68 ± 0.59 µM for TNF-α and IL-6 respectively),
rendering these compounds promising anti-inflammatory agents.
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2.4 Chemical stability and orally bioavailability
In view of the limited clinical application of curcumin resulting from its instability and poor
metabolism, we designed and synthesized these chemically modified DACs. Among them, the
most active analogs 5a27 and 5a28 were chosen for stability test in phosphate buffer (pH 7.4)
using ultraviolet-visible (UV-visible) spectroscopy. As shown in Fig. 4A, the optical density (OD)
values of the maximal absorption peak of curcumin decreased quickly within 25 min, consistent
with those of previous reports. Of interest, the two DACs 5a27 and 5a28 only slightly
decomposed during incubation, demonstrating remarkable structural stability under the same
condition (Fig.4B and C). This result suggests that these modified active DACs are chemically
more stable than curcumin in vitro.
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Poor bioavailability is another dramatical problem that hinder the application of curcumin.
Therefore, the most active DAC, 5a27, was chosen to determine the orally bioavailability. As Fig.
4D shown, there were two absorption peaks after intragastric administration. So, it’s probably
following liver and intestinal circulation. The pharmacokinetic parameters were determined and
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shown in Table 1. According to formula F = ꢀꢁꢂ ꢃ.ꢄ ×ꢅꢄꢆꢇ ꢈ.ꢉ
ꢀꢁꢂ ꢈ.ꢉ ×ꢅꢄꢆꢇ ꢃ.ꢄ, orally bioavailability of 5a27 was
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figured out, which is 26.62 %. However, the bioavailability of curcumin was reported to be 1 %
previously [30]. The data proved that the bioavailability of DACs was dramatically improved
compared to Curcumin.
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Please insert the Figure 4 and Table 1
2.5 Mechanism of suppression of inflammatory responses
Having shown that 5a27 and 5a28 potently inhibited LPS-induced macrophage activation and
presented higher stability than curcumin, we explored the underlying mechanism of the
anti-inflammatory effects by studying the MAPK signal pathway. Mitogen-activated protein
kinases (MAPKs), a family of serine/threonine protein kinases, are widely known to mediate
fundamental biological processes of inflammatory responses [31]. By activation of the MAPK
signaling pathway, especially p38 and ERK, inflammation mediators can be regulated at the
transcription and translation levels. Thus, the MAPK signaling pathway carries potential targets
for anti-inflammatory therapeutics. Furthermore, that the pathway has been recognized as the
mechanistic target of curcumin lends further support to the notion. Therefore, we evaluated the
effects of 5a27 and 5a28 on the MAPK signaling pathway. RAW 264.7 mouse macrophages were
pretreated with the compounds for half of an hour, followed by stimulation with LPS for 30
minutes. The cell lysates were harvested and immunoblotted. As shown in Fig. 5A, 5a27 and 5a28
markedly inhibited P-P38 and P-ERK, indicating the suppression of MAPK signaling. However,
the potency of inhibiting P-P38 by 5a28 was lower compared to 5a27 and curcumin.
Nuclear factor κB (NF-κB) is a protein complex that controls the transcription of cytokines,
e.g. TNF-α and ILs, and is regulated downstream by MAPK. Consistent with the inhibition of
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MAPK signaling cascades, IκB (inhibitor of NF-κB) was significantly down-regulated by 5a27
and 5a28 (Fig.5A). Meanwhile, the translocation of P65, a component of NF-κB activation, was
significantly attenuated by treatment with the active compounds (Fig. 5B). Given that inactivation
of NF-κB would block transcription of proinflammatory cytokines, we further determined the
effects of the active DACs on the RNA level of proinflammatory factors. As shown in Fig. 5C–G,
5a27 and 5a28 significantly inhibited the transcription of TNF-α, IL-6, IL-1β, ICAM-1 and
VCAM-1 compared with LPS alone. These data solidly indicated that 5a27 and 5a28 potently
inhibited inflammation through blocking MAPK signal transduction and inactivation of NF-κB.
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2.6 Alleviation of LPS-induced acute lung injury
The active compounds were evaluated in a ALI mouse model over a week. Thus 5a27, 56a28
and curcumin were administered to C57/BL6 mice at 10 mg/kg intraperitoneally and sacrificed 6
hours later. ALI is characterized by rapid-onset respiratory failure following direct and indirect
insults to the lung parenchyma and vasculature. We therefore investigated the wet/dry weight ratio
of the mice lungs, which was dramatically increased by LPS and significantly reduced by 5a27
and 5a28 (Fig. 6A). These results indicated that cell infiltration in lung tissues was suppressed
after treatment with the DACs. Examination of bronchoalveolar lavage fluid (BALF) revealed that
the DACs normalized total cell count and neutrophil count in BALF (Fig. 6B-C). Histological
evaluation of the mice lungs revealed typical histopathological alterations in ALI. In the LPS
group, interstitial edema, pulmonary congestion, thickened alveolar septa, inflammatory
infiltration and lung tissue destruction were observed (Fig. 6D). 5a27 and 5a28 however,
dramatically improved the pathological lesions compared with the LPS group. Besides, positive
staining of biomarkers of lymphocytes and macrophages was significantly attenuated after
treatment (Fig.6D).
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Proinflammatory factors, TNF-α and IL-6 play a critical role in driving inflammation in the
early phase of ALI. As shown in Fig. 6E–H, release of TNF-α and IL-6 was obviously inhibited
compared with the LPS group either in serum or BALF. Moreover, challenge of LPS increased the
mRNA expression of inflammatory cytokines and adhesion factors, such as TNF-α, IL-6, IL-1β,
VACM-1 and ICAM-1. However, their expressions were markedly suppressed after treatment with
DACs (Fig.7 A–E). Collectively, these results indicated that 5a27 and 5a28 could potently protect
against pulmonary inflammation in vivo, and this novel chemical scaffold could potentially be
used for treating ALI and other inflammatory injures.
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15 3. Conclusion
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In summary, we have synthesized a series di-carbonyl analogs of curcumin (DACs) and their
anti-inflammatory activity was evaluated. The majority of synthetic DACs were effective in
inhibiting LPS-induced production of TNF-α and IL-6 in MPMs. Key preliminary SAR findings
included that 1) mono-halogen substituent in the phenyl ring decreased the anti-inflammation, but
a strong electron-withdrawing (such as nitro group) at the para-position of the phenyl ring,
resulted in a significant increase in activities; 2) The position and number of methoxy group in
phenyl ring was crucial for the inhibitory activity. The most potent DACs 5a27 and 5a28 showed
higher chemical stability than curcumin in the biological medium. Mechanistical study found that
DACs significantly inhibit inflammation via blocking MAPK signaling pathway and activation of
NF-κB. Furthermore, pretreatment with these two active compounds attenuated LPS-induced ALI
in mice via reducing the production of inflammatory cytokines, the W/D ratio, and inflammatory
cell infiltration into lung tissue. Overall, this study supports the notion that anti-inflammatory
DACs could be a promising strategy for the treatment of ALI. Continued medicinal chemistry
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efforts should be made to obtain potent and drug-like DACs for potential use in clinic.
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4. Experimental
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4.1 General methods
All reagents and solvents of analytical grade were purchased from local suppliers and were
used without further purification while following special instructions. All reactions were
performed in a dry nitrogen atmosphere unless otherwise noted. The progress of reactions was
monitored by silica gel thin layer chromatography (250 µ silica gel 60 F254 glass plates) and the
spots were detected under UV light (254 nm). Column chromatography was performed using
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Merck silica gel 60 (200–300 mesh ASTM) (Merck KGaA, Darmstadt, Germany). H and 13C
NMR spectra were recorded by a Bruker instrument (Brucker AVANCE DRX-500) with
tetramethylsilane (TMS) as an internal standard (500 MHz for H, 125 MHz for 13C). The H or
13C NMR spectra of target anti-inflammatory compounds were provided in the Supplementary
data. Melting points were obtained on a Fisher-Johns melting apparatus and were uncorrected.
Electron-spray ionization mass spectroscopy (ESI-MS) data were collected in positive mode using
a Bruker Esquire 3000t spectrometer.
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4.2 General procedures for the synthesis of cinnamoyl chlorides (2a01-24, 2a26-27, 2a29)
To a solution of cinnamic acid (1a01-24, 1a26-27, 1a29) (1.0 mmol) in dry CH2Cl2 was added
oxalyl chloride (5.0 equiv) and a catalytic amount of DMF (0.01 equiv). The reaction mixture was
stirred at room temperature for 5 h before the solvent was removed. The residue was dried under
high vacuum and used in the next step without further purification.
4.2 Synthesis of (E)-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one (4)
A solution of NaH (0.376 g, 12.55 mmol) in dry THF (2 mL) was stirred at 0 °C for 10 min, and
then a solution of tert-butyl 2-oxopiperidine-1-carboxylate (0.50 g, 2.51 mmol) and
3,4,5-trimethoxybenzaldehyde (3) (0.54 g, 2.76 mmol) in dry THF (2 mL) was added slowly. The
reaction mixture was stirred at room temperature for 4 h. The resulting solution was then
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quenched with saturated NH4Cl solution (40 mL) and extracted with EtOAc (3×50 mL). The
combined organic layers were washed with water (100 mL), brine (100 mL), dried over anhydrous
MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography
on silica gel providing the desired compound 4 (0.20 g, 28.7%) as a white powder. 1H NMR (500
MHz, CDCl3) δ (ppm): 7.72 (s, 1H), 6.89 (s, 1H), 6.62 (s, 2H), 3.85 (s, 10H), 3.43 (s, 2H), 2.83 (t,
J = 5.5 Hz, 2H), 1.95 – 1.80 (m, 2H).
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4.3 General procedures for the synthesis of DACs 5a01–5a29
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A 1.6 M solution of n-BuLi in hexane (0.26 mL, 0.43 mmol) was added dropwise to a solution
of (E)-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one (4) (0.36 mmol) in anhydrous THF (4 mL)
at −78 °C under nitrogen. After 2 h, a solution of cinnamoyl chlorides (2a01–24, 2a26–27, 2a29)
(1.0 mmol) in dry THF (2 mL) wad added dropwise. The reaction solution was stirred for 10 min
and then allowed to warm up to room temperature for 2 h. The resulting mixture was quenched
with saturated NH4Cl solution (10 mL), and the mixture was extracted with AcOEt (3×25 mL).
The organic layers were dried over MgSO4 and concentrated at reduced pressure. The residue was
purified by column chromatography on silica gel providing the target compounds 5a01–5a29.
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4.3.1 (E)-1-[(E)-3-(2-Fluorophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a01).
Yellow powder, 36.3% yield, m.p: 135.8-138.3 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.86 (d,
J = 16.1 Hz, 2H), 7.68 – 7.63 (m, 2H), 7.34 (dd, J = 13.3, 6.8 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H),
7.12 – 7.06 (m, 1H), 6.69 (s, 2H), 3.92 – 3.89 (m, 11H), 2.90 (t, J = 5.6 Hz, 2H), 2.00 – 1.94 (m,
2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.7, 167.8, 153.1, 139.5, 139.0, 135.1, 131.2, 130.8,
129.6, 128.9, 124.7, 124.2, 116.1, 115.9, 107.7, 60.9, 56.2, 44.1, 26.4, 22.4. ESI-MS m/z: 426.26
(M)+.
4.3.2 (E)-1-[(E)-3-(3-Fluorophenyl)acryloyl)]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
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(5a02). Yellow powder, 38.7% yield, m.p: 97.1-98.0 C. H NMR (500 MHz, CDCl3) δ (ppm):
7.87 ( s, 1H), 7.65 ( d, J = 15.52 Hz, 1H), 7.58 ( d, J = 15.58 Hz, 1H), 7.35 (s, 1H), 7.34 (d, J =
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11.32 Hz, 1H), 7.30 ( d, J = 10.06 Hz, 1H), 7.06 ( t, J = 6.29 Hz, 1H), 6.69 (s, 2H), 3.93-3.91 ( m,
2H), 3.89 (s, 3H), 3.88 ( s, 6H), 2.93 – 2.88 ( m, 2H), 2.01 – 1.94 ( m, 2H). 13C NMR (125 MHz,
CDCl3) δ (ppm): 169.5, 167.9, 153.1, 141.3, 139.6, 130.8, 130.2, 130.2, 129.6, 124.2, 123.8, 116.7,
116.6, 114.4, 114.2, 107.9, 60.9, 56.2, 44.0, 26.4, 22.4. ESI-MS m/z: 426.13 (M)+.
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4.3.3 (E)-1-[(E)-3-(4-Fluorophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a03).
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Yellow powder, 38.5% yield, m.p: 131.4-133.1 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.87 (s,
1H), 7.69 (d, J = 15.6 Hz, 1H), 7.59 (dd, J = 8.3, 5.6 Hz, 2H), 7.54 (d, J = 15.6 Hz, 1H), 7.07 (t, J
= 8.5 Hz, 2H), 6.69 (s, 2H), 3.92 – 3.89 (m, 11H), 2.90 (t, J = 5.7 Hz, 2H), 2.00 – 1.94 (m, 2H).
13C NMR (125 MHz, CDCl3) δ (ppm): 169.7, 167.9, 153.1, 141.7, 139.5, 139.1, 130.8, 130.1,
129.7, 122.1, 115.9, 115.7, 107.7, 60.9, 56.2, 44.0, 26.4, 22.4. ESI-MS m/z: 426.19 (M)+.
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4.3.4 (E)-1-[(E)-3-(2-Chlorophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a04).
Yellow powder, 41.5% yield, m.p: 134.1-135.7 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.10 ( d,
J = 15.6 Hz, 1H), 7.86 (s, 1H), 7.74 ( d, J = 7.00 Hz, 1H), 7.58 ( d, J = 15.5 Hz, 1H), 7.40 ( d, J =
7.19 Hz, 1H), 7.30 – 7.27 ( m, 2H), 6.68 (s, 2H), 3.92 ( t, J = 5.82 Hz, 2H), 3.89 ( s, 3H), 3.88 (s,
6H), 2.90 ( t, J = 5.67 Hz, 2H), 1.99 – 1.94 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.4,
167.9, 153.1, 139.6, 139.1, 138.4, 135.0, 133.5, 130.8, 130.6, 130.0, 129.6, 127.9, 126.9, 124.9,
107.7, 60.9, 56.2, 44.0, 26.4, 22.4. ESI-MS m/z: 442.29 (M)+.
4.3.5 (E)-1-[(E)-3-(3-Bromophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a05).
Yellow powder, 42.3% yield, m.p: 113.8-116.4 oC.1H NMR (500 MHz, CDCl3) δ (ppm): 7.87 ( s,
1H), 7.75 ( s, 1H), 7.62 ( d, J = 15.61 Hz, 1H), 7.57 ( d, J = 15.61 Hz, 1H), 7.49 ( t, J = 7.66 Hz,
2H), 7.24 ( d, J = 7.81 Hz, 1H), 6.99 ( s, 2H), 3.92 – 3.90 ( m, 2H), 3.90 (s, 3H), 3.88 ( s, 6H),
2.90 ( t, J = 7.32 Hz, 2H), 1.99 – 1.94 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.5,
167.9, 153.1, 141.0, 139.7, 139.2, 137.4, 132.7, 130.8, 130.6, 130.2, 129.6, 127.0, 123.9, 122.9,
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107.9, 60.9, 56.3, 44.1, 26.4, 22.5. ESI-MS m/z: 486.19 (M)+.
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4.3.6 (E)-1-[(E)-3-(4-Bromophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a06).
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Yellow powder, 45.8% yield, m.p: 126.9-128.5 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.87 (s,
1H), 7.65 ( d, J = 15.63 Hz, 1H), 7.59 ( d, J = 15.62 Hz, 1H), 7.51 ( d, J = 8.54 Hz, 1H), 7.46 ( d,
J = 8.53 Hz, 1H), 6.69 ( s, 2H), 3.92-3.90 ( m, 2H), 3.90 ( s, 3H), 3.89 ( s, 6H), 2.91 ( t, J = 7.69
Hz, 2H), 2.00-1.94 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.6, 167.9, 153.1, 141.5,
139.6, 139.1, 134.2, 131.9, 130.8, 129.6, 124.1, 123.0, 107.7, 106.8, 60.98, 56.2, 44.1, 26.4, 22.4.
ESI-MS m/z: 486.19 (M)+.
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4.3.7
(E)-1-{(E)-3-[2-(Trifluoromethyl)phenyl]acryloyl}-3-(3,4,5-trimethoxybenzylidene)
piperidin-2-one (5a07).
Yellow powder, 34.8% yield, m.p: 101.1-102.9 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.05 (dd,
J = 15.4, 2.1 Hz, 1H), 7.87 (s, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.58 – 7.54
(m, 2H), 7.46 (t, J = 7.6 Hz, 1H), 6.68 (s, 2H), 3.94 – 3.89 (m, 11H), 2.93 – 2.88 (m, 2H), 1.97 (dt,
J = 12.3, 6.2 Hz, 2H).13C NMR (125 MHz, CDCl3) δ (ppm): 169.1, 167.9, 153.1, 139.6 (s), 139.1
(s), 137.7 (s), 134.2, 131.9, 130.8, 129.5, 129.1, 128.2, 126.5, 126.0, 107.7, 60.9, 56.2, 44.0, 26.4,
22.4. ESI-MS m/z: 476.31 (M)+.
4.3.8 (E)-1-[(E)-3-(4-Nnitrophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a08).
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Yellow powder, 34.5% yield, m.p: 180.1-182.3 C. H NMR (500 MHz, CDCl3) δ (ppm): H
NMR (500 MHz, CDCl3) δ 8.22 (d, J = 8.7 Hz, 2H), 7.86 (s, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.66 (s,
2H), 6.67 (s, 2H), 3.92 – 3.89 (m, 2H), 3.88 (s, 3H), 3.87 (s, 6H), 2.90 (t, J = 5.5 Hz, 2H), 2.00 –
1.93 (m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.1, 168.0, 153.1, 148.2, 141.5, 140.0,
139.1, 130.6, 129.3, 128.7, 126.7, 124.0, 107.7, 60.9, 56.2, 44.2, 26.4, 22.4. ESI-MS m/z: 453.21
(M)+.
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4.3.9
(E)-1-[(E)-3-(3,4-Difluorophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a09).
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Yellow powder, 39.8% yield, m.p: 149.8-151.0 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.85 ( s,
1H), 7.79 ( d, J = 15.97 Hz, 1H), 7.68 ( d, J = 16.00 Hz, 1H), 7.35 ( d, J = 8.07 Hz, 1H), 7.35 ( s,
1H), 7.18 ( t, J = 8.04 Hz, 1H), 6.67 ( s, 2H), 3.94 ( t, J = 5.80 Hz, 2H), 3.89 ( s, 3H), 3.88 ( s, 6H),
2.91 ( t, J = 7.23 Hz, 2H), 2.00 – 1.94 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.2,
167.7, 153.0, 139.6, 139.0, 136.6, 135.9, 135.1, 132.8, 130.8, 130.5, 129.5, 129.4, 128.7, 107.7,
60.9, 56.2, 44.1, 26.4, 22.4. ESI-MS m/z: 444.19 (M)+.
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4.3.10
(E)-1-[(E)-3-(2,5-Difluorophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a10).
Yellow powder, 36.2% yield, m.p: 134.8-136.4 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.88 ( s,
1H), 7.77 ( d, J = 15.80 Hz, 1H), 7.60 ( d, J = 15.77 Hz, 1H), 7.36 – 7.32 ( m, 1H), 7.09 – 6.99 ( m,
2H), 6.69 ( s, 2H), 3.94 – 3.91 (m, 2H), 3.90 ( s, 3H), 3.89 ( s, 6H), 2.91 ( t, J = 7.37 Hz, 2H), 2.00
– 1.94 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.4, 167.9, 153.1, 139.8, 133.5, 130.8,
129.5, 125.9, 125.8, 117.7, 117.5, 117.2, 117.0, 114.5, 114.3, 107.8, 60.9, 56.2, 44.1, 26.4, 22.4.
ESI-MS m/z: 444.19 (M)+.
4.3.11
(E)-1-[(E)-3-(2,4-Dichlorophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)
piperidin-2-one (5a11).
Yellow powder, 43.5% yield, m.p: 161.4-163.1 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.00 (d,
J = 15.6 Hz, 1H), 7.86 ( s, 1H), 7.67 ( d, J = 8.48 Hz, 1H), 7.55 (d, J = 15.60 Hz, 1H), 7.43 ( s,
1H), 7.25 ( t, J = 6.61 Hz, 1H), 6.68 ( s, 2H), 3.91 ( t, J = 5.82 Hz, 2H), 3.89 ( s, 3H), 3.88 (s, 6H),
2.90 (t, J = 5.63 Hz, 2H), 1.99 – 1.94 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.2,
167.9, 153.1, 139.7, 139.2, 137.0, 135.8, 135.5, 132.1, 130.7, 129.8, 129.5, 128.6, 127.4, 125.3,
107.8, 60.9, 56.2, 44.1, 26.4, 22.4. ESI-MS m/z: 476.25 (M)+.
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4.3.12
(E)-1-[(E)-3-(2,6-Dichlorophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)
piperidin-2-one (5a12).
3
Yellow powder, 46.3% yield, m.p: 124.0-125.1 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.85 ( s,
1H), 7.79 ( d, J = 15.97 Hz, 1H), 7.68 (d, J = 16.02 Hz, 1H), 7.35 ( d, J = 8.15 Hz, 2H), 7.17 ( t, J
= 7.85 Hz, 1H), 6.67 ( s, 2H), 3.94 ( t, J = 5.32 Hz, 2H), 3.89 ( s, 3H), 3.88 (s, 6H,), 2.90 ( t, J =
6.16 Hz, 2H), 2.00 – 1.94 ( m,2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.2, 167.7, 153.1,
139.6, 139.1, 136.5, 135.9, 135.1, 132.8, 130.8, 130.5, 129.5, 129.4, 128.7, 107.8, 60.9, 56.2, 44.1,
26.4, 22.4. ESI-MS m/z: 476.18 (M)+.
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4.3.13
(E)-1-[(E)-3-(2-Chloro-6-fluorophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)
piperidin-2-one (5a13).
Yellow powder, 45.6% yield, m.p: 129.8-131.9 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.88 ( s,
1H), 7.86 ( d, J = 15.71 Hz, 1H), 7.76 ( d, J = 15.99 Hz, 1H), 7.26 – 7.20 ( m, 2H), 7.06 – 7.01 ( m,
1H), 6.68 ( s, 2H), 3.93 ( t, J = 5.84 Hz, 2H), 3.90 ( s, 3H), 3.88 ( s, 6H), 2.91 ( t, J = 7.58 Hz, 2H),
2.00 – 1.94 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.9, 167.7, 153.0, 139.6, 139.0,
132.2, 130.8, 130.2, 130.1, 129.5, 129.4, 129.3, 125.8, 114.8, 114.7, 107.7, 60.9, 56.2, 44.1, 26.4,
22.4. ESI-MS m/z: 460.22 (M)+.
4.3.14
(E)-1-[(E)-3-(2-Chloro-4-fluorophenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)
piperidin-2-one (5a14).
Yellow powder, 46.4% yield, m.p: 148.7-150.8 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.03 ( d,
J = 15.62 Hz, 1H), 7.86 ( s, 1H), 7.76 – 7.72 (m, 1H), 7.53 ( d, J = 15.59 Hz, 1H), 7.17 ( d, J =
8.40 Hz, 1H), 7.02 – 6.98 ( m, 1H), 6.68 ( s, 2H), 3.93 – 3.91 ( m, 2H), 3.90 ( s, 3H), 3.89 ( s, 6H),
2.91 ( t, J = 7.41 Hz, 2H), 2.00 – 1.92 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 169.3,
167.9, 162.0, 153.1, 139.6, 137.2, 130.8, 129.6, 129.2, 129.2, 124.8, 117.4, 117.2, 114.7, 114.5,
107.8, 60.9, 56.2, 44.1, 26.4, 22.4. ESI-MS m/z: 460.01 (M)+.
4.3.15
(E)-1-[(E)-3-(2-Fluoro-5-methoxyphenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)
piperidin-2-one (5a15).
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Yellow powder, 37.5% yield, m.p: 105.7-107.4 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.87 ( s,
1H), 7.81 ( d, J = 15.77 Hz, 1H), 7.62 (d, J = 15.77 Hz, 1H), 7.12– 7.09 ( m, 1H), 7.02 ( t, J = 9.33
Hz, 1H), 7.88 – 7.84 ( m, 1H), 6.69 ( s, 2H), 3.92-3.90 ( m, 2H), 3.90 ( s, 3H), 3.89 ( s, 6H), 3.82.
13C NMR (125 MHz, CDCl3) δ (ppm): 169.7, 167.8, 157.0, 155.7, 155.0, 153.1, 139.5, 135.2,
130.9, 129.7, 124.8, 117.0, 116.7, 116.5, 112.8, 107.8, 60.9, 56.2, 55.9, 44.1, 26.4, 22.51. ESI-MS
m/z: 456.23 (M)+.
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4.3.16
(E)-1-[(E)-3-(2-Fluoro-4-methoxyphenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)
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piperidin-2-one (5a16).
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Yellow powder, 37.2% yield, m.p: 100.1-101.5 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.87 ( s,
1H), 7.83 ( d, J = 15.76 Hz, 1H), 7.58 ( d, J = 7.67 Hz,1H), 7.57 ( d, J = 15.89 Hz, 1H), 6.68 ( s,
2H), 6.71 ( d, J = 8.72 Hz, 1H), 6.64 (d, J = 12.35 Hz, 1H), 3.92 – 3.90 ( m, 2H), 3.90 ( s, 3H),
3.89 ( s, 6H), 3.83 ( s, 3H), 2.90 ( t, J = 7.50 Hz, 2H), 1.98 – 1.93 ( m, 2H). 13C NMR (125 MHz,
CDCl3) δ (ppm): 169.9, 167.8, 163.4, 162.3, 153.1, 139.3, 135.6, 130.9, 129.8, 122.0, 122.0, 116.0,
110.7, 107.8, 101.8, 101.6, 60.9, 56.2, 55.7, 44.0, 26.5, 22.5. ESI-MS m/z: 456.23 (M)+.
4.3.17
(E)-1-[(E)-3-(2-Methoxyphenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a17).
Yellow powder, 36.2% yield, m.p: 130.1-132.9 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.10 (d,
J = 15.79 Hz, 1H), 7.86 ( s, 1H), 7.64 ( d, J = 15.76 Hz, 1H), 7.64 ( d, J = 7.76 Hz, 1H), 7.34 ( t,
J = 8.61 Hz, 1H), 6.96 ( t, J = 7.49 Hz, 1H), 6.91 ( d, J = 8.28 Hz, 1H), 6.68 ( s, 2H), 3.92 – 3.90
( m, 2H), 3.90 ( s, 6H), 3.88 ( s, 6H), 2.90 ( t, J = 7.57 Hz, 2H), 1.98 – 1.92 ( m, 2H). 13C NMR
(125 MHz, CDCl3) δ (ppm): 170.2, 167.8, 158.3, 153.1, 139.1, 139.0, 138.4, 131.1, 131.0, 130.0,
128.5, 124.3, 122.5, 120.6, 111.1, 107.8, 60.9, 56.2, 55.5, 44.0, 26.5, 22.5. ESI-MS m/z: 438.17
(M)+.
4.3.18
(E)-1-[(E)-3-(3-Methoxyphenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
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(5a18).
Yellow powder, 35.3% yield, m.p: 137.6-139.0 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.87 (s,
1H), 7.70 ( d, J = 15.59 Hz, 1H), 7.59 ( d, J = 15.59 Hz, 1H), 7.30 ( t, J = 7.87 Hz, 1H), 7.12 ( s,
1H), 6.92 ( d, J = 8.13 Hz, 1H), 6.69 ( s, 2H), 3.92 – 3.90 ( m, 2H), 3.90 ( s, 3H), 3.89 (s, 6H),
3.84 ( s, 3H), 2.90 ( t, J = 7.31 Hz, 2H), 1.98 – 1.93 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ
(ppm): 169.8, 167.8, 159.9, 153.1, 143.0, 139.4, 139.2, 136.6, 130.9, 129.8, 129.7, 122.7, 120.9,
115.8, 113.3, 107.8, 60.9, 56.2, 55.3, 44.0, 26.4, 22.5. ESI-MS m/z: 438.24 (M)+.
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4.3.19
(E)-1-[(E)-3-(2,4-Dimethoxyphenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)
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piperidin-2-one (5a19).
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Yellow powder, 32.5% yield, m.p: 143.3-145.1 C. H NMR (500 MHz, CDCl3) δ (ppm): H
NMR (500 MHz, CDCl3 ) δ (ppm): 8.05 (d, J = 15.7 Hz, 1H), 7.85 (s, 1H), 7.58 (s, 1H), 7.56 (d, J
= 7.1 Hz, 1H), 6.68 (s, 2H), 6.49 (dd, J = 8.5, 2.1 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 3.88 (t, J =
5.1 Hz, 15H), 3.83 (s, 2H), 2.88 (t, J = 5.4 Hz, 2H), 2.03 – 1.86 (m, 2H). 13C NMR (125 MHz,
CDCl3 ) δ (ppm): 165.2, 162.5, 157.3, 154.6, 147.8, 133.9, 133.3, 125.8, 124.8, 114.6, 112.2,
102.4, 99.9, 93.0, 55.7, 50.9, 50.2, 38.7, 21.2, 17.3. ESI-MS m/z: 468.33 (M)+.
4.3.20 (E)-1-[(E)-3-(2,3-Dimethoxyphenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)
piperidin-2-one (5a20).
Yellow powder, 31.3% yield, m.p: 118.9-120.3 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.05 (d,
J = 15.8 Hz, 1H), 7.86 (s, 1H), 7.63 (d, J = 15.7 Hz, 1H), 7.30 – 7.26 (m, 1H), 7.05 (t, J = 8.0 Hz,
1H), 6.93 (d, J = 8.1 Hz, 1H), 6.68 (s, 2H), 3.94 – 3.83 (m, 17H), 2.89 (t, J = 6.2 Hz, 2H), 2.00 –
1.92 (m, 2H). 13C NMR ( 125 MHz, CDCl3 ) δ (ppm): 170.0, 167.8, 153.1, 148.6, 139.3, 139.1,
137.6, 130.9, 129.9, 129.5, 124.0, 123.5, 119.5, 113.7, 107.8, 61.4, 60.9, 56.2, 55.9, 44.0, 26.5,
22.5. ESI-MS m/z: 468.26 (M)+.
4.3.21
(E)-3-(3,4,5-Trimethoxybenzylidene)-1-[(E)-3-(2,4,5-trimethoxyphenyl)acryloyl]
piperidin-2-one (5a21).
Yellow powder, 38.3% yield, m.p: 158.4-159.6 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.11 (d,
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J = 15.7 Hz, 1H), 7.85 (s, 1H), 7.54 (d, J = 15.7 Hz, 1H), 7.13 (s, 1H), 6.68 (s, 2H), 6.50 (s, 1H),
3.93 (s, 3H), 3.91 – 3.87 (m, 17H), 2.91 – 2.87 (m, 2H), 1.99 – 1.92 (m, 2H). 13C NMR (125 MHz,
CDCl3 ) δ (ppm): 170.3, 167.8, 154.1, 153.0, 152.1, 143.3, 139.0, 138.5, 131.0, 130.1, 119.7,
116.0, 111.0, 107.7, 97.0, 60.9, 56.5, 56.2, 56.0, 44.0, 26.5, 22.5. ESI-MS m/z: 498.22 (M)+.
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4.3.22
(E)-3-(3,4,5-Trimethoxybenzylidene)-1-[(E)-3-(3,4,5-trimethoxyphenyl)acryloyl]
7
piperidin-2-one (5a22).
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Yellow powder, 32.3% yield, m.p: 176.4-178.1 C. H NMR (500 MHz, DMSO-d6) δ (ppm): δ
7.76 (s, 1H), 7.54 (d, J = 15.6 Hz, 1H), 7.39 (d, J = 15.6 Hz, 1H), 6.99 (s, 2H), 6.84 (s, 2H), 3.82
(d, J = 3.3 Hz, 12H), 3.79 – 3.77 (m, 2H), 3.70 (d, J = 2.8 Hz, 6H), 2.89 (t, J = 5.5 Hz, 2H), 1.90 –
1.84 (m, 2H). 13C NMR (125 MHz, CDCl3 ) δ (ppm): 169.7, 167.9, 153.4, 153.1, 143.4, 140.1,
139.4, 139.1, 130.8, 130.8, 129.8, 121.6, 107.8, 105.6, 60.9, 56.2, 44.0, 26.4, 22.5. ESI-MS m/z:
498.22 (M)+.
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4.3.23
(E)-1-[(E)-3-(2,5-Dimethylphenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a23).
Yellow powder, 32.6% yield, m.p: 118.1-119.9 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.01 (d,
J = 15.5 Hz, 1H), 7.88 (s, 1H), 7.51 (d, J = 15.5 Hz, 1H), 7.49 (s, 1H), 7.08 (s, 2H), 6.69 (s, 2H),
3.89 (d, J = 5.8 Hz, 9H), 2.92 – 2.88 (m, 2H), 2.42 (s, 3H), 2.33 (s, 3H), 1.97 (dt, J = 12.2, 6.3 Hz,
2H). 13C NMR (125 MHz, CDCl3 ) δ (ppm): 170.0, 167.9, 153.1, 141.0, 139.4, 139.0, 135.6, 134.8,
133.8, 130.9, 130.6, 129.8, 127.1, 122.9, 107.7, 60.9, 56.2, 44.0, 26.5, 22.5, 20.9, 19.3. ESI-MS
m/z: 436.27 (M)+.
4.3.24
2-{(E)-3-Oxo-3-[(E)-2-oxo-3-(3,4,5-trimethoxybenzylidene)piperidin-1-yl]prop-1-en-1-yl}phe
nyl acetate (5a24).
Yellow powder, 31.4% yield, m.p: 134.8-136.4 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.86 ( s,
1H), 7.78 (d, J = 15.73 Hz, 1H), 7.74 (d, J = 7.77 Hz, 1H), 7.64 (d, J = 15.71 Hz, 1H), 7.39 ( t, J =
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8.20 Hz, 1H), 7.26 ( t, J =7.32 Hz, 1H), 7.13 (d, J = 8.12 Hz,1H), 6.68 (s, 2H), 3.92 – 3.90 (m,
2H), 3.90 (s, 3H), 3.89 (s, 6H), 2.90 (t, J = 7.22 Hz, 2H), 2.41 ( s, 3H), 1.99 – 1.93 (m, 2H). 13C
NMR (125 MHz, CDCl3) δ (ppm): 169.5, 169.2, 167.8, 153.1, 149.4, 139.5, 139.2, 136.4, 130.8,
130.6, 129.7, 128.2, 127.9, 126.2, 124.4, 123.0, 107.8, 60.9, 56.2, 44.0, 26.4, 22.4, 21.0. ESI-MS
m/z: 466.24 (M)+.
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4.3.25
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(E)-1-[(E)-3-(2-Hydroxyphenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)piperidin-2-one
(5a25).
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Yellow powder, 32.5% yield, m.p: 134.8-136.4 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.94 ( d,
J = 14.76 Hz, 1H), 7.87 (s, 1H), 7.60 ( d, J = 15.14 Hz, 1H), 7.50 ( s, 1H), 7.20 ( s, 1H), 6.90 ( s,
2H), 6.67 ( s, 2H), 3.93 – 3.90 (m, 2H), 3.89 ( s, 3H), 3.87 (s, 6H), 2.93 – 2.88 ( m, 2H), 2.01 –
1.94 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 170.5, 168.0, 155.3, 153.1, 139.7, 139.1,
138.3, 131.0, 130.8, 129.6, 129.3, 123.6, 122.5, 120.6, 116.6, 107.9, 60.9, 56.3, 44.1, 26.5, 22.5.
ESI-MS m/z: 424.24 (M)+.
4.3.26
3-{(E)-3-oxo-3-[(E)-2-oxo-3-(3,4,5-trimethoxybenzylidene)piperidin-1-yl]prop-1-en-1-yl}phen
yl acetate (5a26)
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Yellow powder, 47.6% yield, m.p: 129.4.-131.6 C. H NMR (500 MHz, DMSO-d6) δ (ppm):
7.82 (s, 1H), 7.66 (d, J = 12.5 Hz, 1H), 7.61 – 7.47 (m, 3H), 7.08 (s, 2H), 6.65 (s, 2H), 3.85 (s,
12H), 3.79 (s, 1H), 2.86 (s, 2H), 2.27 (s, 3H), 1.93 (s, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm):
169.6, 167.8, 153.1, 151.8, 141.8, 139.4, 132.9, 130.8, 129.7, 129.2, 122.6, 121.9, 107.9, 60.8,
56.2, 44.0, 26.4, 22.4, 21.0. ESI-MS m/z: 465.57 (M)+.
4.3.27
2-Methoxy-4-{(E)-3-oxo-3-[(E)-2-oxo-3-(3,4,5-trimethoxybenzylidene)piperidin-1-yl]prop-1-e
n-1-yl}phenyl acetate (5a27).
Yellow powder, 38.4% yield, m.p: 134.8-136.4 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.86 ( s,
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1H), 7.69 ( d, J = 15.52 Hz, 1H), 7.52 ( d, J = 15.58 Hz, 1H), 7.21 ( d, J = 7.98 Hz, 1H), 7.17 ( s,
1H), 7.05 ( d, J = 8.13 Hz, 1H), 6.69 (s, 2H), 3.93 – 3.90 ( m, 2H), 3.90 ( s, 3H), 3.88 ( s, 9H),
2.90 (t, 2H), 2.32 ( s, 3H), 1.99 – 1.93 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ (ppm): 164.4,
163.5, 162.6, 147.8, 146.0, 137.2, 135.9, 134.2, 133.8, 129.0, 125.6, 124.5, 117.8, 117.3, 116.0,
106.4, 102.5, 55.7, 51.0, 50.7, 38.8, 21.2, 17.2, 15.4. ESI-MS m/z: 496.27 (M)+.
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4.3.28 (E)-1-[(E)-3-(4-Hydroxy-3-methoxyphenyl)acryloyl]-3-(3,4,5-trimethoxybenzylidene)
piperidin-2-one (5a28).
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Yellow powder, 31.6% yield, m.p: 134.8-136.4 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.86 ( s,
1H), 7.70 ( d, J = 15.51 Hz,1H), 7.50 ( d, J = 15.50 Hz, 1H), 7.16 ( d, J = 8.23 Hz, 1H), 7.10 ( s,
1H), 6.92 ( d, J = 8.19 Hz, 1H), 6.69 ( s, 2H), 5.86 ( s, 1H), 3.94 ( s, 3H), 3.92 – 3.90 (m, 2H),
3.90 (s, 3H), 3.89 ( s, 6H), 2.89 ( t, J = 5.89 Hz, 2H), 1.99 – 1.93 ( m, 2H). 13C NMR (125 MHz,
CDCl3) δ (ppm): 169.9, 167.9, 153.1, 147.8, 146.7, 143.8, 139.2, 130.9, 130.0, 128.1, 127.9, 123.2,
119.8, 114.6, 109.8, 107.9, 60.9, 56.2, 56.0, 44.0, 26.4, 22.5. ESI-MS m/z: 454.20 (M)+.
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4.3.29
2-Methoxy-5-{(E)-3-oxo-3-[(E)-2-oxo-3-(3,4,5-trimethoxybenzylidene)piperidin-1-yl]prop-1-e
n-1-yl}phenyl acetate (5a29).
Yellow powder, 38.3% yield, m.p: 134.8-136.4 oC. 1H NMR (500 MHz, CDCl3) δ (ppm): 7.86 (s,
1H), 7.68 (d, J = 15.51 Hz, 1H), 7.51 ( d, J = 15.53 Hz, 1H), 7.43 (d, J = 8.32 Hz, 1H), 7.35 ( s,
1H), 6.95 ( d, J = 8.44 Hz, 1H), 6.69 ( s, 2H), 3.91 – 3.89 ( m, 2H), 3.90 ( s, 3H), 3.89 ( s, 6H),
3.86 (s, 3H), 2.89 (s, 2H), 2.32 ( s, 3H), 1.99 – 1.92 ( m, 2H). 13C NMR (125 MHz, CDCl3) δ
(ppm): 169.6, 168.6, 167.8, 153.1, 142.3, 142.2, 140.1, 139.3, 130.9, 129.9, 128.5, 127.9, 122.0,
120.9, 112.2, 107.9, 60.9, 56.2, 55.9, 44.0, 26.4, 22.5, 20.5. ESI-MS m/z: 496.27 (M)+.
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4.4 Cells and Reagents
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The final concentration of DMSO in assays did not exceed 0.1%. All other reagents not
mentioned were obtained from Sigma unless otherwise specified. Mouse peritoneal macrophages
(MPMs) were prepared as described previously [31]. Briefly, ICR mice of 20–25 g were
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intraperitoneally injected with 6% thioglycollate solution [beef extract (0.3 g), tryptone (1 g),
sodium chloride (0.5 g) and soluble starch (6 g) in 100 mL water]. 3 days later, the mice were
sacrificed. The peritoneal cavity was washed with 8 mL of PBS, and centrifuged at 4 °C, 1000
rpm. The supernatant was discarded, the precipitate resuspended with RPMI-1640 (Gibco/BRL
life Technologies, Eggenstein, Germany) containing 10% (v/v) FBS (Hyclone, Logan, UT, USA),
100 U/mL penicillin G and 100 mg/mL streptomycin. Cells were incubated at 37 °C under a 5%
CO2 atmosphere.
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4.5 Immunofluorescence and immunoblotting
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Raw 264.7 cells were seeded into 6-well plates and pretreated with compounds for 0.5 hour,
followed by incubation with LPS for another 40 minutes. Thereafter, cells were fixed and
permeabilized with 4% paraformaldehyde and 100% methanol, respectively. Cells were washed
twice with PBS containing 1% BSA, then incubated with primary antibodies of P65 (1:200) at
4 °C overnight. Samples were subsequently incubated with PE-conjugated secondary antibodies
(1:200) for 2 hours. Finally, cells were counterstained with DAPI, and viewed with a Nikon
fluorescence microscope (Nikon, Japan).
For immunoblotting assays, cells or lung tissues (weight of 30–50 mg) were lysed (Boster
Biological Technology, USA). Protein concentration was measured using a Bio-Rad protein assay
kit (Bio-rad, USA). Samples were loaded and separated in 10% or 12% SDS-PAGE gels. The gel
was electro-transferred to a nitrocellulose membrane, and blocked in Tris-buffered saline at pH 7.6
containing 0.05% Tween 20 and 5% non-fat milk. Specific antibodies were incubated for
biomarkers. Immuno-reactive bands were detected by incubating with secondary antibodies
conjugated with horseradish peroxidase, and visualized using enhanced chemiluminescence
reagents (Bio-Rad, Hercules, CA).
P-P38, P38, P-ERK, ERK, P65, IκBα and GAPDH antibodies were purchased from Cell
Signaling Technology (Danvers, MA, USA). TNF-α antibodies were obtained from Abcam
(Abcam, USA). Secondary antibodies were purchased from Yeasen Biotechnology (Shanghai,
China).
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4.6 Determination of concentrations of TNF-α and IL-6
TNF-α and IL-6 concentrations in culture medium or animal samples were determined by
ELISA according to the manufacturer’s instructions (Bioscience, San Diego, CA). The amount of
TNF-α or IL-6 was normalized to the protein concentration of each sample.
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4.7 Real-time quantitative PCR
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Total RNA was extracted using TRIZOL (Life Technologies, Carlsbad, CA). Reverse
transcription and quantitative PCR (RT-qPCR) were set up using M-MLV Platinum RT-qPCR Kit
(Life Technologies). Real-time qPCR was carried out on an Eppendorf Realplex 4 instrument
(Eppendorf, Hamburg, Germany). Primers for genes (i.e. TNF-α, IL-6 and β-actin) were obtained
from Life Technologies. The primer sequences used are shown in Table 2. The relative amount of
each gene was normalized to β-actin.
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Table 2. Primers used for real-time qPCR assay
Gene
TNF-α
IL-6
Species
Mouse
Mouse
Mouse
Forward (5’-3’)
Reverse (5’-3’)
TGATCCGCGACGTGGAA
ACCGCCTGGAGTTCTGGAA
AAGTGCATCATCGTTGTTCATACA
TACGACCAGAGGCATACAG
GAGGATACCACTCCCAACAGACC
CCGTGAAAAGATGACCCAGA
β-actin
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4.8 Mouse models
Male C57BL/6 mice of 20–25 g were obtained from the Animal Centre of Wenzhou Medical
University (Wenzhou, China). Mice were housed at constant room temperature with a 12:12 h
light–dark cycle, fed a standard rodent diet and water, and acclimatized to the laboratory for at
least 3 days before use. All animal care and experimental procedures were approved by the
Wenzhou Medical College Animal Policy and Welfare Committee.
LPS-induced ALI. The mice were randomly divided into groups as follows: control (eight mice
received vehicle of 0.9% saline), LPS (eight mice received LPS alone), curcumin, 5a27 or 5a28
(each of the three compounds was administered to a group of eight mice at 10 mg/kg). The active
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compounds were given daily via intraperitoneal injection consecutively for one week. The mice
were euthanized with chloral hydrate 6 hours after intratracheal injection of LPS at 5 mg/kg.
Broncho alveolar lavage fluid (BALF), blood and lung tissues were collected for further analysis.
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4.9 Histomorphological and immunohistochemical examination
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Lung tissues were routinely fixed and embedded in paraffin. Paraffin blocks were sectioned
into slices of 5 µm thick. Thereafter, the slices were stained with Hematoxylin and Eosin (H&E
assay kit, Beyotime, Shanghai, China).
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For immunohistochemistry, rehydrated slices were subjected to antigen retrieval in 0.01 mol/L
citrate buffer (pH 6.0) by microwaving, and then placing in 3% hydrogen peroxide in methanol at
room temperature for 30 min. Sections were incubated with primary antibodies at 4 °C overnight,
followed by secondary antibodies (1:200; Santa Cruz, USA). The slices were visualized by
3,3-diaminobenzidine (DAB) (ZSGB-Bio, Beijing, China), counterstained with hematoxylin, and
viewed under the microscope (Nikon, Japan).
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4.10 Pharmacokinetic study of 5a27
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Male SD rats, weight 250 ± 20 g, were starved overnight before the experiment. Tap water
was available ad libitum. Intragastric or intravenously administration of 5a27 with the dose of 20
mg/kg, 5 mg/kg respectively (n = 5). Blood samples from tail were collected 0.083, 0.25, 0.5, 1, 2,
4, 6, 9, 12, 24 hours after dosing. They were centrifuged at 12000 rpm for 10 minutes. Remove
supernatants to 1.5 mL tube and add 2-fold volume of acetonitrile. The mixtures were vortexed
and centrifuged at 12000 rpm for 10 minutes. The supernatants were analyzed by UHPLC-MS/MS.
The pharmacokinetic parameters were determined by using DAS software (Version 3.0).
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4.11 Statistical analysis
All data were shown as meansꢀ±ꢀSEM, by averaging values from three independent
experiments. Statistical analyses were performed using GraphPad Pro. Prism 5.0 (GraphPad, San
Diego, CA). One-way ANOVA followed by multiple comparisons test with Bonferroni correction
were used to analyze the differences between sets of data.
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Acknowledgement
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Financial support was provided by the National Natural Science Funding of China (81773579,
81803600), Zhejiang Natural Science Funding (LY17B020008), and the Wenzhou Public Welfare
Science and Technology Project (2018Y0465). We also thank Amy Tong from Liwen Bianji,
Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this
manuscript.
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