Design, synthesis and biological activity of novel asymmetric C66 analogs as anti-inflammatory agents for the treatment of acute lung injury

Accepted Manuscript Design, synthesis and biological activity of novel asymmetric C66 analogs as antiinflammatory agents for the treatment of acute lung injury Pengtian Yu , Lili Dong , Yali Zhang , Wenbo Chen , Shanmei Xu , Zhe Wang , Xiaoou Shan , Jianmin Zhou , Professor, Zhiguo Liu , Ph.D, Associate Professor, Guang Liang PII:

S0223-5234(14)01096-4

DOI:

10.1016/j.ejmech.2014.11.054

Reference:

EJMECH 7547

To appear in:

European Journal of Medicinal Chemistry

Received Date: 23 June 2014 Revised Date:

24 November 2014

Accepted Date: 26 November 2014

Please cite this article as: P. Yu, L. Dong, Y. Zhang, W. Chen, S. Xu, Z. Wang, X. Shan, J. Zhou, Z. Liu, G. Liang, Design, synthesis and biological activity of novel asymmetric C66 analogs as anti-inflammatory agents for the treatment of acute lung injury, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.11.054. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Design, synthesis and biological activity of novel asymmetric C66 analogs as

anti-inflammatory agents for the treatment of acute lung injury Pengtian Yu 1, #, Lili Dong

2, #

, Yali Zhang 1, Wenbo Chen 1, Shanmei Xu 1, Zhe Wang 1, Xiaoou Shan 2,

1

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Jianmin Zhou 1,*, Zhiguo Liu 1, 3*, Guang Liang1

Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University,

1210 University Town, Wenzhou, Zhejiang 325035, China 2

Department of Pediatrics, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou,

Wenzhou Undersun Biotchnology Co., Ltd., Wenzhou, Zhejiang, China

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3

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Zhejiang 325035, China

Graphical abstract:

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Novel C66 analogs were synthesized and evaluated for anti-inflammatory activities in the treatment of ALI.

Keywords: Curcumin, Acute lung injury, Drug design, Chemical stability, Cytokines.

ACCEPTED MANUSCRIPT

Design, synthesis and biological activity of novel asymmetric C66 analogs as anti-inflammatory agents for the treatment of acute lung injury Pengtian Yu 1, #, Lili Dong 2, #, Yali Zhang 1, Wenbo Chen 1, Shanmei Xu 1, Zhe Wang 1, Xiaoou Shan 2, Jianmin Zhou 1,*, Zhiguo Liu 1, 3*, Guang Liang1 1

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These authors contribute equally to this work.

* Corresponding author: Jianmin Zhou, Professor

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#

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Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University, 1210 University Town, Wenzhou, Zhejiang 325035, China 2 Department of Pediatrics, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China 3 Wenzhou Undersun Biotchnology Co., Ltd., Wenzhou, Zhejiang, China

Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University. 1210 University Town, Wenzhou, Zhejiang 325035, China Tel: (+86)-577-86689819; Fax: (+86)-577-86689819

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E-mail: [email protected]

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

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E-mail: [email protected]

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Abstract

Acute lung injury (ALI) is a leading cause of morbidity and mortality in critically-ill patients. Previously, we reported that a symmetric mono-carbonyl analog of curcumin, (C66), exhibits enhanced stability and was found to have efficacy and be involved in potential cytokines inhibition. In the present study, a series of novel asymmetric C66 analogs were designed and synthesized. A majority of them effectively inhibited the LPS-induced expression of TNF-α and IL-6. Significantly, compound 4b2 was found to effectively reduce LPS-induced pulmonary inflammation, as reflected by reductions in concentration of total protein, inflammatory cell count as well as the lung W/D ratio in bronchoalveolar lavage (BAL) fluid. Furthermore, in vivo administration of 4b2 resulted in remarkable improvement in histopathological changes of lung in rats.

Keywords: Curcumin, Acute lung injury, Drug design, Chemical stability, Cytokines. 1

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1. Introduction Acute lung injury (ALI) is characterized by the accumulation of neutrophils in the lungs, accompanied by the development of interstitial edema and a pulmonary inflammatory response [1-3]. Clinically, ALI has proven to be a major cause of acute respiratory failure in critically-ill patients; however, there is currently no effective treatment [4]. A number of clinical studies have documented that numerous inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and

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interleukin-6 (IL-6), play a major role in mediating, amplifying, and perpetuating ALI processes [3, 5, 6]. These observations suggested that the inflammatory process results in lung injury and emphasized that effective therapy must be given early in the ALI developing process. Therefore, the development of anti-inflammatory candidates targeting proinflammatory cytokines or

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inhibiting the over-expression of cytokines has been a valid approach to ALI therapy, providing a new direction in anti-inflammatory drug development.

Curcumin, (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), is a yellow

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substance extracted from the rhizome of the traditional herb Curcuma Longa. Recent advances in pharmacological research reveals that curcumin is a pleiotropic molecule possibly capable of interacting with molecular targets involved in inflammation [7-9], cancer [10], cystic fibrosis and Alzheimer's disease [11-13]. In both phase I and phase II trials, it was determined that the oral intake of curcumin may act as an effective therapeutic measure in the treatment of various inflammatory conditions. As such, significant interest has emerged regarding the potential of curcumin for the prevention and treatment of ALI [14, 15].

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Despite multiple therapeutic effects, there are some disadvantages that limit the development of curcumin as a potential therapeutic agent, including low bioavailability and instability after oral administration due to rapid elimination from the body [16, 17]. With oral administration of several grams of curcumin per day in a phase I trial, pharmacokinetic studies showed that only nanomolar

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concentrations of curcumin could be detected in plasma and target tissues [18]. Therefore, the weakness of a pharmacokinetic profile for curcumin in both humans and animals has raised several concerns that significantly limit its clinical application.

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Considering that the presence of the methylene group and β-diketone moiety contributes to

the chemical instability of curcumin under physiological conditions [19, 20], we previously designed a series of symmetric mono-carbonyl analogues of curcumin (MACs) through the modification

of

its

structural

motif.

Amongst

these

analogues,

(2E,

6E)-2,6-bis(2-(trifluoromethyl)benzylidene) cyclohexanone (C66) showed enhanced stability and a potential inhibitory effect on the release of cytokines such as TNF-α and IL-6 both in vitro and in vivo [21, 22]. Moreover, preliminary SAR studies revealed that the introduction of an ortho-position on the benzene ring by electron withdrawing groups, such as a trifluoromethyl moiety, generally resulted in a dramatically improved inhibitory effect. As a part of our ongoing drug discovery and development activities for targeted ALI therapies, we initiated a search for synthesis and anti-inflammatory evaluation of a series of novel asymmetric C66 analogs, which 2

ACCEPTED MANUSCRIPT possess the trifluoromethyl or nitryl moiety, and found enhanced stability in vitro and greatly improved pharmacokinetic profiles in vivo. Active compounds exhibited significant therapeutic effects in a mouse model, suggesting the potential of C66 analogs for development as a new anti-inflammatory agent in treating ALI.

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Please insert Figure 1

2. Chemistry

The synthesis of asymmetric C66 analogs containing the 2-CF3 or 2-NO2 moiety is shown in

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Scheme 1, along with their chemical structures in Table 1. Stork reaction of commercially available cyclopentanone (1a) and cyclohexanone (1b) with morpholine were carried out in the presence of p-Toluenesulfonic acid (PTSA) to give morpholine enamines 2a and 2b, respectively.

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Aldol condensation of the obtained enamines with 2-CF3 or 2-NO2 substituted benzaldehyde in ethanolic NaOH solution, followed by an acidation step with hydrochloric acid, resulted in different α,β-unsaturated ketones 3a-d with satisfactory yields and high purities. With the key intermediate 3a-d in hand, we introduced various aromatic rings to the molecules via Aldol condensation between the ketones 3a-d and various aromatic aldehydes in a catalyst of 20% NaOH or hydrogen chloride gases, which furnished target asymmetric C66 analogs 4a1-a17 and 4b1-b14 at 20-80% yield. All of the new products were isolated by conventional work-up with

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satisfactory yields. Analytical and spectral data of all synthesized compounds are in full agreement with the proposed structures.

Please insert Table 1

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Please insert Scheme 1

3. Results and discussion 3.1 Inhibitory screening against LPS-induced TNF-a and IL-6 release Lipopolysaccharide, a major cell wall component of Gram-negative bacteria, induces

activation of monocytes and macrophages. Activated macrophages produce an excess of several pro-inflammatory cytokines including TNF-α, IL-6, and IL-12, which leads to serious systemic disorders with a high mortality rate [23]. There is considerable experimental and clinical evidence that pro-inflammatory cytokines play a major role in the pathogenesis of inflammatory-induced ALI. Newly synthesized MACs (asymmetric C66 analogs) were evaluated for their efficacy in ALI treatment via inhibition of TNF-α and IL-6 release in LPS stimulated mouse RAW264.7 macrophages. C66 was used as a positive control. The macrophages were pre-treated with 10 µM 3

ACCEPTED MANUSCRIPT compounds for 30 min and then incubated with 0.5 µg/ml LPS for 24 h. The amounts of TNF-α and IL-6 in culture medium were determined by enzyme-linked immunosorbent assay (ELISA) and normalized to the total protein amounts of the viable cell pellets. The ability (IC50 values) of the tested compounds to reduce pro-inflammatory cytokines IL-6 and TNF-α are depicted in Table 2. The results demonstrated that the majority of synthetic MACs were effective in inhibiting the expression of LPS-induced IL-6 and TNF-α. Their inhibitory

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abilities were comparable to or more pronounced than that of the leading compound, C66. Among these tested compounds, 4b2, 4b3 and 4b8 exhibited the highest inhibitory abilities against both LPS-induced IL-6 and TNF-α expression. In particular, compound 4b2, with a substituted 2-NO2 on the A ring and 3′-OH, 4-OMe on the B ring, showed the strongest inhibitory potency amongst

respectively.

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Please insert Table 2

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the tested analogs, and had the lowest IC50 values; 1.74 µM (IL-6) and 1.89 µM (TNF-α),

3.2 Preliminary structure-activity relationship (SAR)

Chemical modifications as well as the synthesis of the curcumin analogs have been implemented by different groups to find out the SAR conclusion and better leads for the treatment of inflammatory diseases. Many reports [24-26], as in our recent previous publications [8, 27],

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suggest the position and the quantity of the phenyl substituents play an important role in anti-inflammatory activities. Generally, smaller cyclo-ketone linker seems to provide better activities and di-subtituted derivatives are generally more active. Furthermore, exchange of the benzene ring by furan, indole, methyl thiophene, as well as other nitrogen heterocycles, are also

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favorable in increasing the inhibition of IL-6 and TNF-α. Based off these considerations, structure-based design of analogs centered around C66 yielded a focused library of compounds with -CF3 and -NO2 substituted motifs at the C-2 position on the

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left-hand phenyl unit of a MAC skeleton. All 2-CF3 substituted compounds (series A), except compounds 4a1, 4a5 and 4a9, dose-dependently inhibited IL-6 or TNF-α with IC50 values ranging from 5.62 to 27.6 µM, with

most showing better activity than C66. Incorporation of various

alkyl groups at the 4′-hydroxy position yielded compounds that were much more potent in inhibition of IL-6 secretion. For example, an addition of a methy or butyl group in the 4′-hydroxy position gave compounds 4a2-4 (IC50 = 5.62, 13.78 and 8.50 µM) and 4a10-12 (IC50 = 8.48, 12.52 and 9.50 µM) a moderate effect but with lower potency than C66 (IC50 = 13.81 µM). Compounds 4a5, 4a6, 4a13, 4a16-17, 4a8 and 4a14 replaced an alkoxy moiety at the C-4′ position with various groups (-C(Me)3, -OH, -N(Et)2, –CF3, -F or alkyl groups) causing the compounds to have no activity. This result indicates that the alkoxy group at the C-4′ position is critical for the inhibition of IL-6. Meanwhile, it can be seen from Table 2 that the majority of compounds 4

ACCEPTED MANUSCRIPT exhibited equal potency to C66 therefore it was thus difficult to analyze the SAR of series A related TNF-α inhibition. Replacement of a 2-CF3 group with 2-NO2 motifs created asymmetric analogs 4b1-14 (series B), in which these exhibited a dramatic increase in their inhibitory activities against IL-6 and TNF-α compared with that of series A. To further clarify, two examples from each series were presented; 4b2 (IC50(IL-6) = 1.74 µM, IC50(TNF-α) = 1.89 µM), 4b3 (IC50(IL-6) = 1.69 µM,

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IC50(TNF-α) = 1.91 µM), and 4a4 (IC50(IL-6) = 8.50 µM, IC50(TNF-α) = 10.98 µM), 4a7 (IC50(IL-6) = 19.14 µM, IC50(TNF-α) = 22.34 µM), in which showed a 10-fold increase in the activities of series B. Meanwhile, Mono, or tri-substitution of a right-hand phenyl unit with electron-withdrawing or donating groups, such as compounds 4b1, 4b4-5, 4b7, 4b9, and 4b14, dramatically diminished the compounds’ potency in either IL-6 or TNF-α. In comparison,

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di-substituted compounds of 4b2, 4b3 and 4b10-12, displayed greater potency than C66 with IC50 values ranging from 1.69 to 5.64 µM and 1.89-6.78 µM, regarding inhibition of IL-6 and TNF-α,

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respectively. Meanwhile, this observation was in accord with our previous result. Additionally, when thiophene or 2-methylthiophene was introduced on a right-hand phenyl unit, good activity (4b8 and 4b13, IC50(IL-6) and IC50(TNF-α) < 7.50 µM ) was consistently achieved. This suggests that the heterocyclic ring might play a role in the activity.

3.3 Assessment of chemical stability of representative compounds

Curcumin has shown to be a promising therapeutic utility for many diseases and as an

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anti-inflammation agent; however, its clinical application is severely limited because of its poor stability under physiological conditions [16, 28]. In the present study, we designed and synthesized novel MACs through modification of the C66 structural motif to improve the chemical stability of curcumin. The chemical stability of representative compounds, 4b2, 4b3 and

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4b8 was detected and measured by UV/Vis absorption in phosphate buffer (pH 7.4). As shown in Figure 2A, the UV-visible absorption spectrum of curcumin displayed an intense optical absorption region with an absorption at maximum of close to 425 nm. Unfortunately, the curcumin

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was found to be decreased by more than 45% of its original intensity after 25 min of incubation in phosphate buffer. Of interest, three C66 analogs 4b2, 4b3, and 4b8 degraded much less than curcumin, particularly 4b2 and 4b3, which demonstrated remarkable stability under the same condition (Figure 2B-D). This result indicates that these modified MACs are much more stable than curcumin in vitro.

Please insert Figure 2

3.4 4b2 ameliorated histopathological changes of lung in LPS- stimulated rats. LPS-induced ALI is characterized by capillary leakage, increased neutrophils and macrophages, and edema. To investigate the possible mechanism underlying the protective effect 5

ACCEPTED MANUSCRIPT of C66 analogs, we further determined the concentration of total protein, inflammatory cell count, and lung W/D ratio to evaluate whether the representative analogs were able to relieve lung injury in the BALF of LPS-induced ALI rats. Compound 4b2, which demonstrated the highest activities and low degradation, was selected for this study. As shown in Figure 3A-C, the concentration of total protein, total cell count, and lung W/D ratio were significantly increased after LPS challenge compared with the control group. Whereas pretreatment with 4b2 (20 mg/kg) effectively reduced

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the total protein content, total cells, and lung W/D ratio levels. To evaluate the potential role of 4b2 in the histopathology changes of lung in LPS-induced ALI rats, lung tissues was obtained after 6 h injection of LPS with or without treatment and were subjected to histological assessment. As shown in Figure 3D, lung tissues from the control showed a normal structure with no histopathologic changes under light microscope. In the LPS group, the

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lung showed marked pathologic changes, such as inflammatory cell infiltration, interalveolar septal thickening, and interstitial edema. In contrast, administration of 4b2 effectively reduced the airspace inflammation at a concentration of 20 mg/kg. Taken together; these results showed that

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4b2 has a remarkable protective effect on lung injury in a rat model of ALI.

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3.5 Attenuation of acute LPS-induced pulmonary inflammation by 4b2

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Pro-inflammatory cytokines appear in the early phase of an inflammatory response, play a critical role in ALI, and contribute to the severity of lung injury. TNF-α is a crucial cytokine in ALI. Elevated levels of TNF-α has been found in BALF from patients with ALI. Therefore, to further characterize the inhibitory effect of 4b2 on cytokines production, the level of TNF-α were

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measured in BALF collected from animals. As shown in Figure 4A-C, cytokine levels were found to be elevated after LPS challenge compared with naive animals. However, we found that administration of 4b2 could significantly down-regulate the levels of TNF-α in BALF of LPS

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induced ALI rats. Therefore, these results indicated that the protective effects of 4b2 on LPS-induced pulmonary inflammation may be attributed to the inhibition of inflammatory cytokines. To confirm these findings, we further performed immunohistochemistry analysis of CD68, a macrophage marker. As shown in Figure 4D, there was an increased CD68-immunostained positive macrophage observed in the lung sections, whereas there was no significant difference in the number of CD68-stained macrophages between 4b2 pretreatment and control groups. Our results showed that administration of 4b2 resulted in a significant therapeutic effect on LPS-induced pulmonary inflammation.

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ACCEPTED MANUSCRIPT 3.6 4b2 suppressed LPS-induced proinflammatory gene expression in Beas-2B To further confirm the anti-inflammatory actions of compound 4b2, we next evaluated the potency of 4b2 on the inhibition of inflammatory gene expression at the mRNA level. Beas-2B cells were treated with LPS (1.0 µg/mL) for 6 h and examined for the expression of proinflammatory genes in the presence or absence of 4b2 by RT-qPCR study. The results in Figure

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5 shows that LPS induced a significant increase in the mRNA expression of pro-inflammatory cytokines, including TNF-α (A), IL-6 (B), IL-1β (C) and COX-2(D), while 4b2 markedly decreased the LPS-induced increase in mRNA expression of pro-inflammatory cytokines (P <

inflammatory genes in Beas-2B.

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0.01). These data indicate that 4b2 is a potent inhibitor of LPS-induced mRNA overexpression of

4. Conclusion

In conclusion, we synthesized a number of symmetric mono-carbonyl analogs of curcumin in a practical strategy based on C66 as the lead molecule. Among new synthetic compounds, a -CF3 or -NO2 moiety attached to the C-2 position of a MAC scaffold was elucidated and reflected a therapeutic effect on LPS-induced ALI in vitro and in vivo. Bioassay results demonstrated that the

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2-nitryl series of compounds possessed higher inhibitory activities against LPS-induced TNF-α and IL-6 release in RAW 264.7 macrophages. Furthermore, the promising compounds 4b2, 4b3 and 4b8 were found to display excellent anti-inflammatory activity and chemical stability in vitro, compared with C66 and curcumin, respectively. More importantly, compound 4b2 was found to be

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extremely active in inhibiting the expression of TNF-α, IL-6, IL-1β and COX-2 in Beas-2B, acting at the mRNA level. Meanwhile, compound 4b2 showed a remarkable protective effect on pulmonary inflammation and a therapeutic action in LPS-induced acute lung injury. Taken

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together, these results could be particularly useful for further pharmaceutical development to treat LPS-induced ALI.

5. Experimental section 5.1 Chemistry 5.1.1 General Solvents were distilled under positive pressure of dry argon before use and dried by standard methods. Unless otherwise noted, chemicals were obtained from local suppliers and were used without further purification. All reactions were monitored by thin-layer chromatography (250  silica gel 60 F254 glass plates). Column chromatography was carried out on a column Merck silica gel 60 (230-400 mesh ASTM) (Merck KGaA, Darmstadt, Germany). Melting points were 7

ACCEPTED MANUSCRIPT determined on a Fisher-Johns melting apparatus and are uncorrected. Proton nuclear magnetic resonance (1H NMR and 13C NMR) spectra were recorded on Bruker 600 MHz instruments. The chemical shifts were presented in terms of parts per million with TMS as the internal reference. Electron-spray ionization mass spectra in positive mode (ESI-MS) data were recorded on a Bruker Esquire 3000t spectrometer.

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5.1.2 Synthesis of morpholine enamines 2a and 2b To a solution of morpholine (0.12 mol) in dry toluene (70 mL), cyclopentanone or cyclohexanone (0.1 mol) was added dropwise, followed by p-toluenesulfonic acid (1.16 mmol) at room temperature. The mixture solution was then refluxed for 5h. The resulting solution was

5.1.3 Synthesis of α,β-unsaturated ketones 3a-3d

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concentrated in vacuo to give morpholine enamines 2a and 2b with yields of 60-85%.

A solution of morpholine enamines 2a or 2b (3.0 equiv) in Ethanol (15 ml) was added

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2-trifluorobenzaldehyde or 2-nitrobenzaldehyde (1.0 equiv) portionwise. After refluxing for 4-5 h, the resulting mixture was acidated to a pH of 5-6 by a solution of 5% HCl, diluted with brine and extracted with EtOAc (3×10 ml). The combined organic layers were washed with brine and dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was further purified by chromatography on silica gel to give the desired ketones 3a-3d.

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5.1.4 Synthesis of asymmetric MACs 4a1-4a17 and 4b1-b14

To a stirring solution of 3a-3d (0.37 mmol) and various aromatic aldehydes (0.37 mmol) in EtOH (10 ml), 20% NaOH was added dropwise. The reaction mixture was stirred at room temperature for 10 h, then quenched with saturated aqueous NH4Cl solution (10 mL) and extracted

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with EtOAc (3×10 mL). The combined organic layers were washed with brine (15 mL) and dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by recrystallization or chromatography on silica gel to furnish target MACs 4a1-a17 and

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4b1-b14.

5.1.4.1 (2E,5E)-2-(Naphthalen-2-ylmethylene)-5-[2-(trifluoromethyl)benzylidene]cyclopentanon (4a1)： ：

Yellow powder, 62.3% yield, m.p: 137.2-139.0 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.06 (1H, s, H-β), 7.88 (1H, s, H-1'), 7.87 (2H, d, J = 9.0 Hz, H-5', H-8'), 7.85 (1H, d, J = 7.8 Hz, H-4'), 7.79 (1H, s, H-β'), 7.75 (1H, d, J =7.8 Hz, H-3), 7.70 (1H, d, J = 8.4 Hz, H-6), 7.61 (1H, d, J = 7.2 Hz, H-3'), 7.60 (1H, t, J = 7.2 Hz, H-5), 7.53 (2H, t, J = 3.6 Hz, H-6', H-7'), 7.47 (1H, t, J = 6.6 Hz, H-4), 3.19 (2H, t, J = 2.0 Hz, Cyclopentanone-CH2CH2-), 3.01 (2H, t, J = 2.0 Hz, Cyclopentanone-CH2CH2-). ESI-MS m/z: 378.39 (M+H)+.

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ACCEPTED MANUSCRIPT 5.1.4.2 (2E,5E)-2-[2-(Trifluoromethyl)benzylidene]-5-(2,4,5-trimethoxybenzylidene)cyclopentanone (4a2)： ： Yellow powder, 61.9% yield, m.p: 120.2-122.6 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 8.03 (1H, s, H-β), 7.81 (1H, s, H-β'), 7.73 (1H, d, J = 8.4 Hz, H-3), 7.58 (1H, t, J = 2.4 Hz, H-5), 7.58 (1H, d, J = 2.4 Hz, H-6), 7.44 (1H, t, J = 7.8 Hz, H-4), 7.10 (1H, s, H-6'), 6.54 (1H, s, H-3'), 3.949 (3H, s, 4'-OCH3), 3.89 (3H, s, 2'-OCH3), 3.87 (3H, s, 5'-OCH3), 3.03 (2H, t, J = 6.6 Hz,

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Cyclopentanone-CH2CH2-), 2.94 (2H, t, J = 7.2 Hz, Cyclopentanone-CH2CH2-). ESI-MS m/z: 419.3(M+H)+.

5.1.4.3

(2E,5E)-2-(4-Butoxybenzylidene)-5-[2-(trifluoromethyl)benzylidene]cyclopentanone

(4a3)： ：

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Yellow oil, 35.6% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.83 (1H, s, H-β), 7.74 (1H, d, J = 7.8 Hz, H-3), 7.59 (1H, s, H-β'), 7.58 (1H, t, J = 9.0 Hz, H-5), 7. 58 (1H, d, J = 7.8 Hz, H-6), 7.55

4.02

(2H,

t,

J

=

6.6

Hz,

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(2H, d, J = 8.4 Hz, H-2', H-6'), 7.45 (1H, t, J = 7.8 Hz, H-4), 6.95 (2H, d, J = 8.0 Hz, H-3', H-5'), Ar-OCH2CH2CH2CH3),

3.04

(2H,

t,

J

=

4.2

Hz,

Cyclopentanone-CH2CH2-), 2.98 (2H, t, J = 4.2 Hz, Cyclopentanone-CH2CH2-), 1.77-1.81 (2H, m, Ar-OCH2CH2CH2CH3), 1.48-1.52 (2H, m, Ar-OCH2CH2CH2CH3), 0.99 (3H, t, J = 7.2 Hz, Ar-OCH2CH2CH2CH3). ESI-MS m/z: 423.8 (M+Na)+.

cyclopentanone (4a4)： ：

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5.1.4.4 (2E,5E)-2-(3-Hydroxy-4-methoxybenzylidene)-5-[2-(trifluoromethyl)benzylidene] Yellow powder, 50.2% yield, m.p: 143.4-145.6 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.83 (1H, s, H-β), 7.74 (1H, d, J = 7.8 Hz, H-3), 7.59 (1H, s, H-β'), 7.59 (1H, d, J = 7.2 Hz, H-6), 7.55 (1H, t, J = 2.4 Hz, H-5), 7.45 (1H, t, J = 7.2 Hz, H-4), 7.22 (1H, s, H-2'), 7.14 (1H, d, J = 8.4 Hz, H-6'),

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6.92 (1H, d, J = 8.4 Hz, H-5'), 5.67 (1H, s, 3'-OH), 3.95 (3H, s, 4'-OCH3), 3.04 (2H, t, J = 6.0 Hz, Cyclopentanone-CH2CH2-), 2.98 (2H, t, J = 6.0 Hz, Cyclopentanone-CH2CH2-). ESI-MS m/z:

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375.4 (M+H)+.

5.1.4.5 (2E,5E)-2-[4-(tert-butyl)Benzylidene]-5-[2-(trifluoromethyl)benzylidene]cyclopentanone (4a5)： ：

Yellow powder, 34.3% yield, m.p 98.6-100.1oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.84 (1H, s, H-β), 7.74 (1H, d, J = 7.8 Hz, H-3), 7.63 (1H, t, J = 3.0 Hz, H-5), 7.59 (1H, s, H-β'), 7.59 (1H, d, J = 7.2 Hz, H-6), 7.55 (2H, d, J = 8.4 Hz, H-2', H-6'), 7.47 (2H, d, J = 8.4 Hz, H-3', H-5'), 7.45 (1H, t, J = 6.0 Hz, H-4), 3.07 (2H, t, J = 5.4 Hz, Cyclopentanone-CH2CH2-), 2.97 (2H, t, J = 5.4 Hz, Cyclopentanone-CH2CH2-), 1.38 [9H, s, Ar-(CH3)3]. ESI-MS m/z: 385.8 (M+H)+. 5.1.4.6 (2E,5E)-2-(2,4-Dihydroxybenzylidene)-5-[2-(trifluoromethyl)benzylidene]cyclopentanone (4a6)： ： 9

ACCEPTED MANUSCRIPT Red oil, 30.9% yield. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.17 (1H, s, -OH), 9.99 (1H, s, -OH), 7.85 (1H, d, J = 9.0 Hz, H-6'), 7.85 (1H, s, H-β), 7.84 (1H, s, H-β'), 7.77 (1H, t, J = 7.2 Hz, H-5), 7.71 (1H, d, J = 7.8 Hz, H-3), 7.61 (1H, d, J = 7.8 Hz, H-6),7.60 (1H, t, J =7.8 Hz, H-4), 6.42 (1H, d, J = 2.4 Hz, H-5'), 6.37 (1H, s, H-3'), 2.99 (2H, t, J = 2.4 Hz, Cyclopentanone-CH2CH2-), 2.95 (2H, t, J = 3.0 Hz, Cyclopentanone-CH2CH2-). ESI-MS m/z:

RI PT

361.2 (M)+.

5.1.4.7 (2E,5E)-2-(2-Fluoro-5-methoxybenzylidene)-5-[2-(trifluoromethyl)benzylidene] cyclopentanone (4a7)： ：

Yellow oil, 46.7% yield. 1H NMR (600 MHz, CDCl3) δ (ppm) 7.86 (1H, s, H-β), 7.77 (1H, s, H-β'), 7.79 (1H, d, J = 7.8 Hz, H-3), 7.59 (1H, t, J = 3.0 Hz, H-5), 7.59 (1H, s, H-6'), 7.47 (1H, t, J = 7.8

SC

Hz, H-4), 7.05 (2H, d, J = 9.0 Hz, H-6, H-3'), 6.89 (1H, d, J = 9.0 Hz, H-4'), 3.82 (3H, s, 5'-OCH3), 3.01 (2H, t, J = 3.0 Hz, Cyclopentanone-CH2CH2-), 2.97 (2H, t, J = 3.0 Hz,

M AN U

Cyclopentanone-CH2CH2-). ESI-MS m/z: 377.1 (M+H)+.

5.1.4.8 (2E,5E)-2-(2,5-Dimethylbenzylidene)-5-[2-(trifluoromethyl)benzylidene]cyclopentanone (4a8):

Yellow oil, 40.3% yield. 1H NMR (600 MHz, CDCl3) δ (ppm) 7.86 (1H, s, H-β), 7.82 (1H, t, J = 3.0 Hz, H-5 ), 7.75 (1H, d, J = 7.8 Hz, H-3), 7.59 (1H, d, J = 7.8 Hz, H-6), 7.59 (1H, s, H-β'), 7.46 (1H, t, J = 4.2 Hz, H-4), 7.28 (1H, s, H-6'), 7.15 (1H, d, J = 7.8 Hz, H-3'), 7.09 (1H, d, J = 8.4

TE D

Hz,H-4'), 2.99 (2H, t, J = 4.2 Hz, Cyclopentanone-CH2CH2-), 2.93 (2H, t, J = 4.2 Hz, Cyclopentanone-CH2CH2-), 2.54 (3H, s, 5'-CH3), 2.14 (3H, s, 2'-CH3). ESI-MS m/z: 357.1(M+H)+.

EP

5.1.4.9 (2E,6E)-2-(Naphthalen-2-ylmethylene)-6-[2-(trifluoromethyl)benzylidene]cyclohexanone (4a9):

Yellow powder, 66.7% yield, m.p: 131.4-133.2 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.98 (1H,

AC C

s, H-1'), 7.96 (1H, s, H-β ), 7.95 (1H, s, H-β' ), 7.87 (1H, d, J = 7.8 Hz, H-8'), 7.84 (1H, t, J = 8.4 Hz, H-6'), 7.72 (1H, d, J = 7.8 Hz, H-5'), 7.72 (1H, t, J = 7.8 Hz, H-7'), 7.58 (1H, d, J = 9.0 Hz, H-4'), 7.55 (1H, d, J = 4.8 Hz, H-3' ), 7.52 (1H, d, J = 4.8 Hz, H-3 ), 7.51 (1H, d, J = 3.6 Hz, H-6), 7.44 (1H, t, J = 9.0 Hz, H-5), 7.33 (1H, t, J = 3.6 Hz, H-4), 2.62 (4H, t, J = 5.4 Hz, Cyclohexanone-CH2CH2CH2-), 1.68-1.72 (2H, m, Cyclohexanone-CH2CH2CH H2-). ESI-MS m/z: 393.4 (M+H)+.

5.1.4.10 (2E,6E)-2-[2-(Trifluoromethyl)benzylidene]-6-(2,4,5-trimethoxybenzylidene)cyclohexanone (4a10): Yellow powder, 52.7% yield, m.p: 137.2-139.7 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.04 (1H, s, H-β), 7.92 (1H, s, H-β'), 7.71 (1H, d, J = 7.8 Hz, H-3), 7.54 (1H, t, J = 7.8 Hz, H-5), 7.42 (1H, t, 10

ACCEPTED MANUSCRIPT J = 7.8 Hz, H-4), 7.31 (1H, d, J = 7.2 Hz, H-6), 6.93 (1H, s, H-6'), 6.54 (1H, s, H-3'), 3.87 [9H, s, 2',4',5'-(OCH3)], 2.87 (2H, t, J = 5.4 Hz, Cyclohexanone-CH2CH2CH2-), 2.62 (2H, t, J = 5.4 Hz, Cyclohexanone-CH2CH2CH2-), 1.73-1.76 (2H, m, Cyclohexanone-CH2CH2CH2-). ESI-MS m/z: 433.3 (M+H)+.

5.1.4.11 (2E,6E)-2-(4-Butoxybenzylidene)-6-[2-(trifluoromethyl)benzylidene]cyclo-

RI PT

hexanone (4a11): Yellow oil, 53.2% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.83 (2H, d, J = 9.0 Hz, H-2', H-6'), 7.76 (1H, s, H-β), 7.44 (2H, d, J = 8.4 Hz, H-3, H-6), 7.43 (1H, s, H-β'), 7.32 (1H, t, J = 7.2 Hz, H-5), 6.98 (1H, t, J = 9.0 Hz, H-4), 6.93 (1H, d, J = 4.2 Hz, H-3'), 6.92 (1H, d, J = 3.6 Hz, H-5'), (2H,

t,

J

=

6.6

Hz,

Cyclohexanone-CH2CH2CH2-),

Ar-OCH2CH2CH2CH3), 1.79-1.82

(2H,

2.91 m,

(4H,

t,

J

=

6.0

Hz,

Ar-OCH2CH2CH2CH3,

SC

4.00

Cyclohexanone-CH2CH2CH2-), 1.51-1.53 (2H, m, Ar-OCH2CH2CH2CH3 ), 0.99 (3H, t, J = 3.6 Hz,

M AN U

Ar-OCH2CH2CH2CH3). ESI-MS m/z: 415.7 (M+H)+.

5.1.4.12 (2E,6E)-2-(3-Hydroxy-4-methoxybenzylidene)-6-[2-(trifluoromethyl)benzylidene]cyclohexanone (4a12) ：

Yellow powder, 41.7% yield, m.p: 96.8-98.9 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.92 (1H, s, H-β), 7.77 (1H, s, H-β' ), 7.71 (1H, d, J = 7.8 Hz, H-3), 7.54 (1H, t, J = 7.2 Hz, H-5), 7.44 (1H, s, H-2'), 7.43 (1H, d, J = 1.8 Hz, H-6), 7.42 (1H, t, J = 4.2 Hz, H-4), 7.32 (1H, d, J = 7.8 Hz, H-6'),

TE D

7.12 (1H, d, J = 2.4 Hz, H-5'), 5.64 (1H, s, -OH), 3.94 (3H, s, -OCH3), 2.93 (2H, t, J = 6.0 Hz, Cyclohexanone-CH2CH2CH2-), 2.62 (2H, t, J = 5.4 Hz, Cyclohexanone-CH2CH2CH2-), 1.73-1.77 (2H, m, Cyclohexanone-CH2CH2CH2-). ESI-MS m/z: 389.4 (M)+.

EP

5.1.4.13 (2E,6E)-2-[4-(Diethylamino)benzylidene]-6-[2-(trifluoromethyl)benzylidene]cyclohexanone (4a13):

Red oil, 41.9% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.945 (1H, s, H-β), 7.72 (1H, d, J =

AC C

7.8 Hz, H-2'), 7.69 (1H, d, J = 7.2 Hz, H-6'), 7.65 (1H, d, J =7.8 Hz, H-3), 7.54 (2H, d, J = 7.2 Hz, H-3', H-5'), 7.523 (1H, s, H-β'),7.49 (1H, t, J = 7.2 Hz, H-5), 7.39 (1H, d, J = 7.8 Hz, H-6), 7.32 (1H, t, J = 8.4 Hz, H-4), 3.72 [4H, q, J = 6.6 Hz, 4'-N(CH2CH3)2], 2.62 (4H, t, J = 6.6 Hz, Cyclohexanone-CH2CH2CH2-), 1.69-1.71 (2H, m, Cyclohexanone-CH2CH2CH2), 1.24 [6H, t, J = 6.6 Hz, 4'-N(CH2CH3)2]. ESI-MS m/z: 414.5 (M+H)+. 5.1.4.14 (2E,6E)-2-(4-Isopropylbenzylidene)-6-[2-(trifluoromethyl)benzylidene]cyclohexanone (4a14): Yellow oil, 32.9% yield. 1H NMR (600 MHz,CDCl3) δ (ppm): 7.95 (1H, s, H-β), 7.72 (1H, d, J = 7.8 Hz, H-3), 7.69 (2H, d, J = 7.8 Hz, H-2', H-6'), 7.65 (2H, d, J = 7.8 Hz, H-3', H-5'), 7.53 (1H, s, H-β'), 7.49 (1H, t, J = 7.8 Hz, H-5), 7.43 (1H, d, J = 5.4 Hz, H-6), 7.32 (1H, t, J = 8.4 Hz, H-4), 11

ACCEPTED MANUSCRIPT 2.76-2.78 [1H, m, Ar-CH(CH3)2], 2.61 (4H, t, J = 6.6 Hz, Cyclohexanone-CH2CH2CH2-), 1.87-1.89 (2H, m, Cyclohexanone-CH2CH2CH2), 1.58 [6H, s, Ar-CH(CH3)2]. ESI-MS m/z: 385.4 (M+H)+.

5.1.4.15 (2E,6E)-2-(2-Fluorobenzylidene)-6-[2-(trifluoromethyl)benzylidene]cyclohexanone (4a15):

RI PT

Yellow oil, 58.2% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.69 (1H, d, J = 7.8 Hz, H-3), 7.65 (1H, d, J = 7.8 Hz, H-6'), 7.54 (1H, t, J = 6.6 Hz, H-4'), 7.53 (1H, s, H-β), 7.49 (1H, t, J = 7.2 Hz, H-5), 7.42 (1H, d, J = 7.8 Hz, H-6), 7.37 (1H, d, J = 7.2 Hz, H-3'), 7.31 (1H, t, J = 8.4 Hz, H-4), 7.28 (1H, t, J = 6.6 Hz, H-5'), 6.88 (1H, s, H-β'), 2.77 (2H, t, J = 6.0 Hz, Cyclohexanone-CH2CH2CH2-), 2.61 (2H, t, J = 5.4Hz, Cyclohexanone-CH2CH2CH2-), 1.86-1.89

SC

(2H, m, cyclohexanone-CH2CH2CH2-). ESI-MS m/z: 360.2(M)+.

5.1.4.16 (2E,6E)-2-[2,4-bis(Trifluoromethyl)benzylidene]-6-[2-(trifluoromethyl)benzylidene]-

M AN U

cyclohexanone (4a16):

Yellow oil, 54.6% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.97 (1H, s, H-3'), 7.96 (1H, s, H-β), 7.90 (1H, s, H-β' ), 7.82 (1H, d, J = 7.8 Hz, H-3), 7.73 (1H, d, J = 7.8 Hz, H-5'), 7.56 (1H, t, J = 7.8 Hz, H-5), 7.46 (1H, t, J = 9.6 Hz, H-4), 7.44 (1H, d, J = 7.2 Hz, H-6'), 7.33 (1H, d, J = 7.8 Hz, H-6), 2.64 (2H, t, J = 6.0 Hz, Cyclohexanone-CH2CH2CH2-), 2.60 (2H, t, J = 6.0 Hz, Cyclohexanone-CH2CH2CH2-), 1.69-1.72 (2H, m, Cyclohexanone-CH2CH2CH2-). ESI-MS m/z:

TE D

479.2 (M+H)+.

5.1.4.17 (2E,6E)-2-[4-Fluoro-2-(trifluoromethyl)benzylidene]-6-[2-(trifluoromethyl)benzylidene]cyclohexanone (4a17):

(4H,

t,

J

EP

Yellow oil, 57.5% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.9475'), 7.31 (1H, s, H-3' ), 2.62 =

6.0

Hz,

Cyclohexanone-CH2CH2CH2-),

1.25-1.27

(2H,

m,

+

AC C

Cyclohexanone-CH2CH2CH2-). ESI-MS m/z: 429.5 (M+H) . 5.1.4.18 (2E,5E)-2-(Naphthalen-2-ylmethylene)-5-(2-nitrobenzylidene)cyclopentanone (4b1): Yellow powder, 60.0% yield, m.p 180.3-182.6 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.09 (1H, d, J = 8.4 Hz, H-3), 8.070 (1H, s, H-β), 7.88-7.91 (2H, m, H-4, H-5), 7.85-7.86 (2H, m, H-β', H-6), 7.80 (1H, s, H-1'), 7.70 (1H, d, J = 9.0 Hz, H-5'), 7.67 (1H, d, J = 7.2 Hz, H-8'), 7.60 (1H, d, J = 8.4 Hz, H-3'), 7.52-7.54 (3H, m, H-4', H-6', H-7'), 3.20 (2H, t, J = 3.6 Hz, Cyclopentanone-CH2-CH2-), 2.97 (2H, t, J = 3.6 Hz, Cyclopentanone-CH2-CH2-). ESI-MS m/z: 356.1 (M+H)+.

5.1.4.19 (2E,5E)-2-(3-Hydroxy-4-methoxybenzylidene)-5-(2-nitrobenzylidene)cyclopentanone (4b2): 12

ACCEPTED MANUSCRIPT Yellow powder, 59.5% yield, m.p 196.3-198.2 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 9.30 (1H, s, Ar-OH), 8.12 (1H, d, J = 7.8 Hz, H-3), 7.82-7.83 (2H, m, H-4, H-6), 7.65-7.68 (1H, m, H-5), 7.55 (1H, s, H-β), 7.38 (1H, s, H-β'), 7.16 (1H, s, H-2'), 7.16 (1H, d, J = 6.0 Hz, H-6'), 7.04 (1H, d, J = 9.0 Hz, H-5'), 3.83 (3H, s, Ar-OCH3), 2.99 (4H, s, J Cyclopentanone-CH2-CH2-). 13C NMR (600 MHz, DMSO-d6) δ(ppm): 194.6, 149.6, 148.9, 146.6, 141.5, 134.7, 134.2, 133.5, 130.6, 130.1, 130.0, 128.1, 126.3, 124.7, 124.1, 117.0, 112.2, 55.6, 26.0, 25.2. ESI-MS m/z: 352.2

5.1.4.20

RI PT

(M+H)+.

(2E,5E)-2-(2-Fluoro-5-methoxybenzylidene)-5-(2-nitrobenzylidene)cyclopentanone

(4b3):

Yellow powder, 77.9% yield, m.p 180.5-182.4 oC. 1H NMR (600 MHz,CDCl3) δ(ppm): 8.08 (1H,

SC

d, J = 7.8 Hz, H-3), 7.83 (1H, s, H-β), 7.77 (1H, s, H-β'), 7.67 (1H, t, J = 7.8 Hz, H-4), 7.57 (1H, d, J = 6.0 Hz, H-6), 7.53 (1H, t, J = 7.8 Hz, H-5), 7.07 (1H, d, J = 9.0 Hz, H-3'), 7.04 (1H, s, H-6'), 7.04 (1H, d, J = 7.2 Hz, H-4'), 3.82 (3H, s, 5'-OCH3), 3.01 (2H, t, J = 3.9 Hz,

M AN U

Cyclopentanone-CH2-CH2-), 2.91 (2H, t, J = 3.9 Hz, Cyclopentanone-CH2-CH2-). 13C NMR (600 MHz, CDCl3) δ(ppm): 195.0, 157.3, 155.6, 149.1, 140.6, 139.0, 133.1, 131.5, 130.7, 129.5, 129.2, 126.7, 125.1, 116.6, 116.4, 116.2, 115.2, 56.0, 26.7, 26.0. ESI-MS m/z: 375.9 (M+Na)+.

5.1.4.21 (2E,5E)-2-(3-Bromobenzylidene)-5-(2-nitrobenzylidene)cyclopentanone (4b4): Yellow powder, 43.5% yield, m.p 143.2-144.2 oC. 1H NMR (600 MHz,CDCl3) δ(ppm): 8.09 (1H,

TE D

d, J = 7.8 Hz, H-3), 7.84 (1H, s, H-β), 7.71 (1H, s, H-β'), 7.68 (1H, t, J = 7.5 Hz, H-4), 7.58 (1H, d, J = 7.8 Hz, H-6), 7.53 (1H, t, J = 7.5 Hz, H-5), 7.53 (1H, s, H-2'), 7.52 (1H, d, J = 7.8 Hz, H-4'), 7.49 (1H, d, J = 7.8 Hz, H-6'), 7.31 (1H, t, J = 7.8 Hz, H-5'), 3.06 (2H, t, J = 7.8 Hz, Cyclopentanone-CH2-CH2-), 2.93 (2H, t, J = 7.8 Hz, Cyclopentanone-CH2-CH2-). ESI-MS m/z:

EP

383.8 (M+H)+.

5.1.4.22 (2E,5E)-2-[4-(Allyloxy)benzylidene]-5-(2-nitrobenzylidene)cyclopentanon (4b5):

AC C

Yellow powder, 59.0% yield, m.p 153.6-155.2 oC. 1H NMR (600 MHz,CDCl3) δ(ppm): 8.07 (1H, d, J =7.8 Hz, H-3), 7.798 (1H, s, H-β), 7.66 (1H, t, J = 7.2 Hz, H-4), 7.61 (1H, s, H-β'), 7.59 (1H, d, J = 7.2 Hz, H-6), 7.55 (2H, d, J = 8.4 Hz, H-2', H-6'), 7.51 (1H, t, J = 7.2 Hz, H-5), 6.98 (2H, d, J = 8.4 Hz, H-3', H-5'), 6.03-6.09 (1H, m, Ar-OCH2CH=CH2), 5.44 (2H, d, J = 10.8 Hz, Ar-OCH2CH=CH2), 4.60 (2H, d, J = 5.4 Hz, Ar-OCH2CH=CH2), 3.05 (2H, t, J = 7.2 Hz, Cyclopentanone-CH2-CH2-), 2.92 (2H, t, J = 7.2 Hz, Cyclopentanone-CH2-CH2-). ESI-MS m/z: 361.9 (M+H)+.

5.1.4.23 (2E,5E)-2-[(6-Bromobenzo[d][1,3]dioxol-5-yl)methylene]-5-(2-nitrobenzylidene)cyclo pentanone (4b6)： ： Yellow powder, 30.0% yield, m.p 186.9-188.4 oC. 1H NMR (600 MHz, CDCl3) δ(ppm): 8.08 (1H, 13

ACCEPTED MANUSCRIPT d, J =7.2 Hz, H-3), 7.823 (2H, s, H-β, H-β'), 7.660 (1H, t, J = 7.8 Hz, H-4), 7.56 (1H, d, J = 7.8 Hz, H-6), 7.52 (1H, t, J = 7.8 Hz, H-5), 7.13 (1H, s, H-2'), 7.03 (1H, s, H-5'), 6.04 (2H, s, -OCH2O-), 2.95 (2H, t, J = 5.4 Hz, Cyclopentanone-CH2-CH2-), 2.90 (2H, t, J = 5.4 Hz, Cyclopentanone-CH2-CH2-). ESI-MS m/z: 427.7 (M+H)+. 5.1.4.24

(2E,5E)-2-(2-Nitrobenzylidene)-5-(2,4,6-trimethoxybenzylidene)cyclopentanone

RI PT

(4b7): Yellow powder, 76.3% yield, m.p 166.4-168.7 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.03 (1H, d, J = 7.8 Hz, H-3), 7.74 (1H, s, H-β), 7.71 (1H, s, H-β'), 7.62 (1H, t, J = 7.5 Hz, H-4), 7.54 (1H, d, J = 7.8 Hz, H-6), 7.48 (1H, t, J = 7.8 Hz, H-5), 6.15 (2H, s, H-3', H-5'), 3.86 (3H, s, 4'-OCH3), 3.83 (6H, s, 2',6'-OCH3), 2.76 (2H, t, J = 7.5 Hz, Cyclopentanone-CH2-CH2-), 2.64 (2H, t, J = 7.5

SC

Hz, Cyclopentanone-CH2-CH2-). ESI-MS m/z: 395.9 (M+H)+.

5.1.4.25 (2E,5E)-2-(2-Nitrobenzylidene)-5-(thiophen-2-ylmethylene)cyclopentanon (4b8):

M AN U

Yellow powder, 84.1% yield, m.p 158.0-160.2 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.07 (1H, d, J = 7.8 Hz, H-3), 7.85 (1H, s, H-β), 7.79 (1H, s, H-β'), 7.66 (1H, t, J = 7.2 Hz, H-4), 7.59 (2H, d, J = 6.0 Hz, H-6, H-5'), 7.53 (1H, t, J = 7.2 Hz, H-5), 7.42 (1H, d, J = 3.6 Hz, H-3'), 7.18 (1H, t, J = 4.2 Hz, H-4'), 2.97 (4H, s, Cyclopentanone-CH2-CH2-). 13C NMR (600 MHz, CDCl3) δ (ppm): 194.8, 149.1, 141.5, 140.2, 134.7, 133.3, 133.1, 131.6, 130.9, 130.7, 129.4, 128.4, 127.7, 125.1,

TE D

26.4, 25.5. ESI-MS m/z: 311.8 (M+H)+.

5.1.4.26 (2E,5E)-2-(4-Ethoxybenzylidene)-5-(2-nitrobenzylidene)cyclopentanone (4b9): Yellow powder, 54.6% yield, m.p 147.5-148.9 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.06 (1H, d, J = 8.4 Hz, H-3), 7.79 (1H, s, H-β), 7.66 (1H, t, J = 7.5 Hz, H-4), 7.61 (1H, s, H-β'), 7.59 (1H, d,

EP

J = 7.8 Hz, H-6), 7.55 (2H, d, J = 8.4 Hz, H-2', H-6'), 7.51 (1H, t, J = 7.5 Hz, H-5), 6.95 (2H, d, J = 8.4 Hz, H-3', H-5'), 4.09 (2H, q, J = 6.6 Hz, Ar-OCH2CH3), 3.05 (2H, t, J = 7.2 Hz, Cyclopentanone-CH2-CH2-), 2.92 (2H, t, J = 7.2 Hz, 2H, Cyclopentanone-CH2-CH2-), 1.44 (3H, t,

AC C

J = 6.6 Hz, Ar-OCH2CH3). ESI-MS m/z: 349.9 (M+H)+. 5.1.4.27 (2E,5E)-2-(5-Bromo-2-ethoxybenzylidene)-5-(2-nitrobenzylidene)cyclopentanon (4b10):

Yellow powder, 79.3% yield, m.p 139.4-141.5 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.08 (1H, d, J = 8.4 Hz, H-3), 7.94 (1H, s, H-β), 7.82 (1H, s, H-β'), 7.67 (1H, t, J = 7.2 Hz, H-4),

7.57 (1H,

d, J = 3.0 Hz, H-6), 7.57 (1H, s, H-6'), 7.54 (1H, t, J = 7.2 Hz, H-5), 7.42 (1H, d, J = 9.0 Hz, H-4'), 6.81 (1H, d, J = 9.0 Hz, H-3'), 4.09 (2H, q, J = 6.6 Hz, Ar-OCH2CH3), 3.00 (2H, t, J = 7.5 Hz, Cyclopentanone-CH2-CH2-), 2.89 (2H, t, J = 7.5 Hz, Cyclopentanone-CH2-CH2-), 1.47 (3H, t, J = 6.6 Hz, Ar-OCH2CH3). ESI-MS m/z: 427.9 (M+H)+.

14

ACCEPTED MANUSCRIPT 5.1.4.28 (2E,5E)-2-(4-Hydroxy-3-methoxybenzylidene)-5-(2-nitrobenzylidene)cyclopentanone (4b11): Yellow powder, 40.0% yield, m.p 212.9-214.1 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 9.77 (1H, s, Ar-OH), 8.11 (1H, d, J = 7.8 Hz, H-3), 7.82-7.83 (2H, m, H-4, H-6), 7.66 (1H, t, J = 7.5 Hz, H-5), 7.54 (1H, s, H-β), 7.45 (1H, s, H-β'), 7.27 (1H, s, H-2'), 7.19 (1H, d, J = 8.4 Hz, H-6'), 6.90 (1H, d, J = 8.4 Hz, H-5'), 3.84 (3H, s, Ar-OCH3), 3.04 (2H, t, J = 4.2 Hz,

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Cyclopentanone-CH2-CH2-), 2.99 (2H, t, J = 4.2 Hz, Cyclopentanone-CH2-CH2-). ESI-MS m/z: 352.1 (M+H)+.

5.1.4.29

(2E,5E)-2-(2,5-Dimethoxybenzylidene)-5-(2-nitrobenzylidene)cyclopentanone

(4b12):

SC

Yellow powder, 57.4% yield, m.p 174.6-177.8 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 8.13 (1H, d, J = 7.8 Hz, H-3), 7.81-7.84 (2H, m, H-4, H-6), 7.77 (1H, s, H-β), 7.67 (1H, t, J = 7.2 Hz, H-5), 7.58 (1H, s, H-β'), 7.12 (1H, s, H-6'), 7.06 (1H, d, J = 9.0 Hz, H-3'), 7.04 (1H, d, J = 9.0 Hz,

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H-4'), 3.83 (3H, s, 2'-OCH3), 3.76 (3H, s, 5'-OCH3), 3.03 (2H, t, J = 7.5 Hz, Cyclopentanone-CH2-CH2-), 2.96 (2H, t, J = 7.5 Hz, Cyclopentanone-CH2-CH2-). ESI-MS m/z: 366.0 (M+H)+.

5.1.4.30 (2E,5E)-2-[(5-Methylthiophen-2-yl)methylene]-5-(2-nitrobenzylidene)cyclopentanone (4b13):

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Yellow powder, 77.0% yield, m.p 145.7-148.3 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.06 (1H, d, J = 8.4 Hz, H-3), 7.77 (2H, s, H-β, H-β'), 7.66 (1H, t, J = 7.5 Hz, H-4), 7.59 (1H, d, J = 6.6 Hz, H-6), 7.51 (1H, t, J = 7.5 Hz, H-5), 7.24 (1H, d, J = 3.6 Hz, H-3'), 6.84 (1H, d, J = 3.6 Hz, H-4'),

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2.93 (4H, s, Cyclopentanone-CH2-CH2-), 2.56 (3H, s, 5'-CH3). ESI-MS m/z: 325.9 (M)+. 5.1.4.31 (2E,6E)-2-(2-Nitrobenzylidene)-6-(2,4,6-trimethoxybenzylidene)cyclohexanone (4b14)： ：

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Yellow powder, 80.7% yield, m.p 184.2-186.6 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.01 (1H, d, J = 8.4 Hz, H-3), 7.93 (1H, s, H-β), 7.71 (1H, s, H-β'), 7.62 (1H, t, J = 7.2 Hz, H-4), 7.48 (1H, t, J = 7.2 Hz, H-5), 7.37 (1H, d, J = 7.8 Hz, H-6), 6.15 (2H, s, H-3', H-5'), 3.85 (3H, s, 4'-OCH3), 3.82 (6H, s, 2'-OCH3, 6'-OCH3), 2.59 (2H, t, J = 5.4 Hz, Cyclohexanone-CH2CH2CH2-), 2.46 (2H, t, J = 5.4 Hz, Cyclohexanone-CH2CH2CH2-), 1.66-1.70 (2H, m, Cyclohexanone-CH2CH2CH2-). ESI-MS m/z: 409.9 (M+H)+.

5.2 Animals. The present study was approved by Wenzhou Medical College Animal Policy and Welfare Committee (approval documents: 2009/ APWC/0031). Male C57BL/6 mice weighing 18-22 g were purchased from the Animal Center of Wenzhou Medical University (Wenzhou, China). 15

ACCEPTED MANUSCRIPT Animals involved in this experiment were treated in accordance with the Guide for Care and Use of Laboratory Animals of National Institutes of Health. Animals were housed in standard conditions and maintained in a 12:12-h light/dark cycle with food and water ad libitum.

5.3 Cells and reagents Lipopolysaccharide (LPS) were purchased from Sigma (St Louis, MO, USA). Saline was

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prepared as 0.9% NaCl solution. In addition, eBioscience, Inc. (San Diego, CA, USA) was the source of the mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kit and mouse TNF-α ELISA kit. Mouse RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC, U.S.). RAW 264.7 macrophages were incubated in DMEM medium (Gibco,

and 100 mg/ml streptomycin at 37°C with 5% CO2. 5.4 Anti-inflammatory evaluation of synthetic MACs

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Eggenstein, Germany) supplemented with 10% FBS (Hyclone, Logan, UT), 100 U/ml penicillin,

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The anti-inflammatory effects of 31 synthetic C66 analogs were evaluated by inhibition of TNF-α and IL-6 release using in LPS stimulated mouse RAW264.7 macrophages. After treatment of cells with indicated compounds and LPS, the TNF-α and IL-6 levels in medium were determined with an ELISA kit (eBioScience, San Diego, CA) according to the manufacturer’s instructions. Briefly, cells were pretreated with 10 µM of C66 or prepared MACs for 30 min, then treated with LPS (0.5 µg/ml) for 24 h. After treatment, the culture media and cells were collected

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separately. The levels of TNF-α and IL-6 in the media were determined by ELISA. The total protein in cultural plates was collected and the concentrations were determined using Bio-Rad protein assay reagents. The total amount of the inflammatory factor in the media was normalized

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to the total protein amount of the viable cell pellets.

UV-visible absorption spectra of curcumin and its analogs Absorbance readings were taken from 250 to 600 nm using a spectrum Max M5 (Molecular

AC C

Devices, USA). A stock solution of 1 mM curcumin or C66 analogs was prepared and diluted by phosphate buffer (pH 7.4) to a final concentration of 20 mM. In the experiments where degradation of curcumin was recorded, the absorption spectra were collected for over 25 min at 5 min intervals. The UV-visible absorbance spectrum was measured at 25oC at varying time interval in a 1 cm path-length quartz cuvette.

5.5 Real-time quantitative PCR Cells were homogenized in TRIZOL kit (Invitrogen, Carlsbad, CA) for extraction of RNA according to each manufacturer’s protocol. Both reverse transcription and quantitative PCR were carried out using a two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Carlsbad, CA). Eppendorf Mastercycler ep realplex detection system (Eppendorf, 16

ACCEPTED MANUSCRIPT Hamburg, Germany) was used for q-PCR analysis. The primers of genes including TNF-α, IL-6, IL-1β, and β-actin were synthesized by Invitrogen. PCR primers were designed using Primer Premier Version 5.0 software (Premier Biosoft, Palo Alto, CA, USA) and sequences were as follows (Invitrogen): TNF-α sense: 5’-TGGAACTGGCAGAAGAGG-3’; antisense: 5’-AGACAGAAGAGCGTGGTG-3’;

antisense: 5’-AAGTGCATCATCGTTGTTCATACA-3’; IL-1β sense: 5’-ACTCCTTAGTCCTCGGCCA-3’; antisense: 5’-CCATCAGAGGCAAGGAGGAA-3’;

antisense: 5’-TAAAACGCAGCTCAGTAACAGTCCG-3’.

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β-actin sense: 5’-TGGAATCCTGTGGCATCCATGAAAC-3’;

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IL-6 sense: 5’-GAGGATACCACTCCCAACAGACC-3’;

5.6 Histological Analysis

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The amount of each gene was determined and normalized by the amount of β-actin.

Lung tissues were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and cut into 5 µmthick sections. Tissues were stained with hematoxylin and eosin (H&E) and observed with a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan). The severity of microscopic injury was graded from 0 (normal) to 4 (severe) based on the following categories: neutrophil infiltration, interstitial edema, hemorrhage, and hyalinemembrane. The sum of all scores was combined to

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calculate a composite score as described previously [29].

5.7 Wet-to-Dry Weight Ratio

The lung weight-to-dry (W/D) ratio was calculated as a parameter of lung edema. At the end

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of the experiment, animals were sacrificed with an overdose of sodium pentobarbital of 60 mg/kg. The lungs were removed, weighed, and then dried in an oven at 80°C for 48 h to obtain lung W/D

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ratio.

5.8 Protein Concentration in Bronchoalveolar Lavage Fluid (BALF) BALF was obtained at the end of the experimental by irrigating the left lung with saline (3 ×

1.5 mL). This fluid was centrifuged at 1000 rpm for 10 min, and the protein concentration in the supernatant was determined using a BCA protein assay (Pierce, Rockford, IL, USA).

5.9 Statistical analysis Data are expressed as the mean ± standard error of the mean (SEM). Student’s t-test was employed to analyze the differences between sets of data. Statistics were performed using GraphPad Pro (GraphPad, San Diego, CA). P values less than 0.05 (p <0.05) were considered indicative of significance. All experiments were repeated at least three times. 17

ACCEPTED MANUSCRIPT

Acknowledgement Financial support was provided by the National Natural Science Funding of China (21202124, 21272179, 21472142), Project of Zhejiang Provincial Key Constructive Subject (Traditional Chinese Medicine, 2012-XK-A28), Qianjiang Talent Project of Zhejiang Province (2013R10020), Zhejiang Medical&Health Science and Technology Project (2013KYB168), Zhejiang Natural

Reference

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Science Funding (LQ12H30002), and the Project-sponsored by SRF for ROCS, SEM.

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ACCEPTED MANUSCRIPT Inhibition of high glucose-induced inflammatory response and macrophage infiltration by a novel curcumin derivative prevents renal injury in diabetic rats, Br. J. Pharmacol. 166 (2012) 1169-1182. [23] M. Rossol, H. Heine, U. Meusch, D. Quandt, C. Klein, M.J. Sweet, S. Hauschildt, LPS-induced cytokine production in human monocytes and macrophages, Crit. Rev. Immunol. 31 (2011) 379-446.

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biological evaluation of asymmetric indole curcumin analogs as potential anti-inflammatory and antioxidant agents, J. Enzyme. Inhib. Med. Chem. 9 (2014) 7-11.

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mono-carbonyl analogues of curcumin, J. Cancer Therapy. 4 (2013) 113-123. [27] Y. Zhang, C. Zhao, W. He, Z. Wang, Q. Fang, B. Xiao, Z. Liu, G. Liang, S. Yang, Discovery and evaluation of asymmetrical monocarbonyl analogs of curcumin as anti-inflammatory agents, Drug. Des. Devel. Ther. 4 (2014) 373-382.

[28] S. Chakraborti, G. Dhar, V. Dwivedi, A. Das, A. Poddar, i G. Chakrabort, G. Basu, P. Chakrabarti, A. Surolia, B. Bhattacharyya, Stable and potent analogues derived from the

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modification of the dicarbonyl moiety of curcumin, Biochemistry. 52 (2013) 7449-7460. [29] Z. Guo, Q. Li, Y. Han, Y. Liang, Z. Xu, T. Ren, Prevention of LPS-Induced Acute Lung Injury

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in Mice by Progranulin, Mediators Inflamm. 2012 (2012) 540794.

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Figure Legends Table 1. Chemical structures of all synthetic compounds. Table 2. The IC50 values (µM) of synthetic C66 analogs against LPS induced IL-6 and TNF-α. Figure 1. Structural design of asymmetric C66 analogs.

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Figure 2. Three active MACs dose-dependently inhibited LPS-induced TNF-α and IL -6 secretion in RAW 264.7 macrophages. Macrophages were plated at a density of 4.0×105/plate overnight in 37°C and 5% CO2. Cells were pretreated with active compounds in a series concentration of 1µM, 5 µM, and 10 µM for 30 minutes and subsequently incubated with or without LPS (0.5 µg/mL) for

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24 hours. (A) IL-6 and (B) TNF-α levels in the culture medium were measured by ELISA and were normalized by the total protein. The results were presented as the percent of LPS control. Each bar represents the mean ± SEM of the three independent experiments. Statistical significance

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relative to the LPS group was indicated, **P<0.01.

Figure 3. Ultraviolet-visible absorption spectra of Curcumin, 4b2, 4b3, and 4b8 in phosphate buffer (pH 7.4) containing 5% dimethyl sulfoxide.

Figure 4. 4b2 attenuate the LPS-induced ALI in rats. Rats were intratracheal instillation of LPS. 6 hours later, rats were anaesthetized and killed. Bronchoalveolar lavage fluid and lung tissues were

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collected for further tests. (A) Protein concentration in BALF. (B) Total amount of cells in BALF. (C) Wet/Dry ratio. (D) HE stain.

Figure 5. 4b2 attenuate the LPS-induced lung inflammation in rats. Rats were intratracheal

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instillation of LPS. 6 hours later, rats were anaesthetized and killed. Bronchoalveolar lavage fluid and lung tissues were collected for further tests. (A) The amount of TNF-α in BALF. (B and C)

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The amount of inflammatory gene in lung tissue. (D) Immunohistochemical of CD68 stain.

Figure 6. 4b2 inhibited the inflammatory genes expression induced by LPS in Beas-2B. Cells were plated at a density of 7.0×105/plate overnight in 37°C and 5% CO2. Beas-2B were pretreated with 10 µM 4b2 for 30 minutes and subsequently incubated with LPS (1 µg/mL) for 24 hours. Cells were collected and the total RNA was extracted. The mRNA levels of inflammatory cytokines were detected by QPCR (A-D). The results were presented as the percent of LPS control. Each bar represents the mean ± SEM of the three independent experiments. Statistical significance relative to the LPS group was indicated, *P<0.05; **P<0.01.

Scheme 1. General synthetic route for asymmetric C66 analogs 4a1-a17 and 4b1-b14.

21

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Table 1 R

O

' n

Ar

Comp.

n

Comp.

2

4a11

2

4a12

Ar

'

Ar 4a1-4a17: R = -CF3 4b1-4b14: R = -NO2 n

Comp.

3

4b4

Ar

n Br

OMe

OH

4a2 OMe OMe

4a3

OBu

OMe

4b5

4a13 N(Et)2

OMe

4a14 i-Pr

3

F

4a5 C(Me)3

2

4a15

2

4a16

OH

CF3 CF3

2

4a7

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OH F

4a17

F

OMe Me

4a8 Me

2

4b1

OMe

S

2

3

4b10

OEt

EtO Br

3

2 2

OMe

4b11

2

2 2

4b9

CF3

4a6

MeO

4b8

3

O

2

OMe

4b7 2

O

Br

3

OH

4a4

Oallyl

3 4b6

2

2

RI PT

OBu

SC

4a1

OH

2

MeO

4b12

OMe

2

OH

4a9

3

4b2

OMe

TE D

OMe OMe

3

4b3

AC C

EP

4a10

2

OMe

S

4b13

F

OMe

2

Me

2

OMe

4b14 MeO

OMe

3

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Table 2 Compd.

IC50 (µM)a IL-6 TNF-α

IC50 (µM)a IL-6 TNF-α

Compd.

NAb

NA

4a17

NA

27.6 ±5.1

4a2

5.62 ±1.5

17.15 ±2.5

4b1

NA

NA

4a3

13.78 ±3.3

22.52 ±5.1

4b2

1.74 ±0.1

1.89 ±0.2

4a4

8.50 ±2.3

10.98 ±1.0

4b3

1.69 ±0.1

1.91±0.6

4a5

NA

NA

4b4

10.40 ±3.8

15.64 ±3.5

4a6

NA

18.37 ±1.8

4b5

17.30 ±1.7

19.50 ±2.3

4a7

19.14 ±3.7

22.34 ±2.9

4b6

8.75 ±1.9

7.66 ±1.4

4a8

17.05 ±2.2

19.44 ±3.3

4b7

9.59 ±3.1

11.90 ±3.5

4a9

NA

NA

4b8

4a10

8.48 ±2.2

NA

4b9

4a11

12.52 ±1.9

11.46 ±0.2

4b10

4a12

9.50 ±1.1

19.32±2.0

4b11

3.87 ±0.6

6.07 ±1.8

4a13

16.16 ±3.6

23.55 ±4.6

4b12

4.90 ±1.4

6.78 ±1.2

4a14

NA

24.84 ±2.9

4b13

5.58 ±0.2

7.40 ±2.1

4a15

NA

18.45 ±2.5

4b14

27.14 ±4.9

17.33 ±3.1

4a16

NA

14.70 ±3.6

C66

13.81 ±2.2

20.57 ±3.4

NA mean not activity (IC50 >30 µM).

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SC 2.11 ±0.2

3.03 ±0.6

18.54 ±1.1

16.25 ±1.0

5.64 ±1.7

5.63 ±0.8

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Each value represents mean ± SD of three experiments.

b

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a

AC C

RI PT

4a1

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AC C

EP

TE D

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SC

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Figure 1

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Figure 2 0min 5min 10min 15min 20min 25min

0.12

0.08 0.06

0.15

0.10

0.05

0.04 300

400

500

600

0.00

nm

300

0min 5min 10min 15min 20min 25min

OD

0.10

0.05

0.10

400

500

0min 5min 10min 15min 20min 25min

600

nm

4b8 0min 5min 10min 15min 20min 25min

0.09 0.08

OD

4b3

0.15

D

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C

4b2

0.20

SC

OD

0.10

B

RI PT

CUR

OD

A

0.07

0.00 300

TE D

0.06

400

500

AC C

EP

nm

600

0.05 300

400

nm

500

600

CD68

D CON

LPS

LPS

TNF-α

C

80

60

40

*

20

0 80

4b2+LPS

4b 2+ LP S

N

0 4b 2+ LP S

LP S

6

LP S

O

50

0

4b2+LPS

RI PT C

** Wet/Dry ratio

100

Relative mRNA amount (Compared to LPS group %)

SC

4b 2+ LP S

C

O N

0

LP S

N

B

C

** 100

S

H&E

+L P

B

S

50

4b 2

TNF-α

LP

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CON

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Relative amount of Cells (compared to LPS %)

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Relative mRNA amount (Compared to LPS group %)

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Figure 4

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Protein concentration in BALF 4

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Relative amount of cytokines (compared to LPS group %)

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Figure 3 8

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IL-6

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Scheme 1.

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IL-1β Rative amonts of mRNA

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Rative amonts of mRNA

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Figure 5

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Novel asymmetric C66 analogs were synthesized.

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Highlights


Compound 4b2 exhibited better anti-inflammatory activity than C66.

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Compound 4b2 could be a lead compound as an ALI therapeutic agent.

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Supplementary Data

Design, synthesis and biological activity of novel asymmetric C66

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analogs as anti-inflammatory agents for the treatment of acute lung injury 2, #

, Yali Zhang 1, Wenbo Chen 1, Shanmei Xu 1, Zhe Wang 1, Xiaoou

Shan 2, Jianmin Zhou 1,*, Zhiguo Liu 1, 3*, Guang Liang1

Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical

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Pengtian Yu 1, #, Lili Dong

University, 1210 University Town, Wenzhou, Zhejiang 325035, China 2

Department of Pediatrics, The Second Affiliated Hospital, Wenzhou Medical University,

Wenzhou, Zhejiang 325035, China

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Wenzhou Undersun Biotchnology Co., Ltd., Wenzhou, Zhejiang, China

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Cytotoxic evaluation of active C66 analogs, 4b2, 4b3 and 4b8

Materials and methods A human normally hepatic immortal cell line HL-7702 obtained from the Cell Bank of Type

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Culture Collection of Chinese Academy of Sciences was used in the experiments. The cytotoxic evaluation was carried out according to the method described previously.1 Briefly, HL-7702 cells were seeded in a 96-well plate at a density of 5×103 cells/well. After incubation for 24 h in full medium, the cells were switched to the 1640 media without FBS and were then treated with 4b2,

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4b3 and 4b8 (1.0 µg/mL) for 24 h. Following the treatment, 20 µL of MTT (5 mg/mL in PBS, pH 7.4) was added to each well, and the plates were allowed to incubate for a further 4 h at 37 °C.

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Finally, the medium was removed, and 150 µL of DMSO was added to each well to dissolve the formazan crystal. After being shaken for 10 min at room temperature, the absorbance was then measured at a wave-length of 490 nm using a Microplate Reader (Bio-Rad 680, Hercules, CA, USA).

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Result:

Before the further evaluation, active C66 analogs, 4b2, 4b3 and 4b8, were tested for their cytotoxicity and safety in the human normal hepatic cell line, HL-7702, by MTT after 24-hour treatment of the cells with compounds at a concentration of 10 µM. As shown in Figure S1, all of

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these compounds displayed no toxicity in hepatic cells, indicating that they are relatively safe.

survival rate

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Figure S1. The cytotoxic evaluation of active C66 analogs to HL-7702.

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ACCEPTED MANUSCRIPT Reference： ： 1. X.L. Song, B. Li, K. Xu, J. Liu, W. Ju, J. Wang, X.D. Liu, J. Li, Y.F. Qi, Cytotoxicity of water-soluble mPEG-SH-coated silver nanoparticles in HL-7702 cells, Cell Biol. Toxicol. 28

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(2012) 225-237.

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