Microsomal Prostaglandin E2

Article pubs.acs.org/jnp

Evaluation of Dual 5‑Lipoxygenase/Microsomal Prostaglandin E2 Synthase‑1 Inhibitory Effect of Natural and Synthetic AcronychiaType Isoprenylated Acetophenones Alexandra Svouraki,† Ulrike Garscha,‡ Eirini Kouloura,† Simona Pace,‡ Carlo Pergola,‡ Verena Krauth,‡ Antonietta Rossi,§ Lidia Sautebin,§ Maria Halabalaki,† Oliver Werz,*,‡ Nicolas Gaboriaud-Kolar,† and Alexios-Leandros Skaltsounis*,† †

Department of Pharmacognosy and Natural Products Chemistry, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, GR-15771, Athens, Greece ‡ Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, 07743 Jena, Germany § Department of Pharmacy, University of Naples Federico II, Naples, Italy S Supporting Information *

ABSTRACT: Among the pathways responsible for the development of inflammatory responses, the cyclooxygenase and lipoxygenase pathways are among the most important ones. Two key enzymes, namely, 5-LO and mPGES1, are involved in the biosynthesis of leukotrienes and prostaglandins, respectively, which are considered attractive therapeutic targets, so their dual inhibition might be an effective strategy to control inflammatory deregulation. Several natural products have been identified as 5-LO inhibitors, with some also being dual 5-LO/mPGES-1 inhibitors. Here, some prenylated acetophenone dimers from Acronychia pedunculata have been identified for their dual inhibitory potency toward 5-LO and mPGES-1. To gain insight into the SAR of this family of natural products, the synthesis and biological evaluation of analogues are presented. The results show the ability of the natural and synthetic molecules to potently inhibit 5-LO and mPEGS-1 in vitro. The potency of the most active compound (10) has been evaluated in vivo in an acute inflammatory mouse model and displayed potent anti-inflammatory activity comparable in potency to the drug zileuton used as a positive control.

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treatment.6 Despite continuous efforts for developing 5-LO inhibitors,7 no LT biosynthesis inhibitor other than zileuton has been marketed due to toxic side effects or to lack of efficacy in vivo. Recent data suggest that anti-inflammatory drugs with dual or multiple targets possess higher efficacy accompanied by reduced severity of side effects.2 In this context, initially, dual inhibition of 5-LO and COX led to greater anti-inflammatory efficiency and lower gastric toxicity.8 More recently, dual inhibitors of 5-LO and microsomal prostaglandin E2 synthase-1 (mPGES-1) were proposed as a promising class of antiinflammatory agents that hamper cardiovascular toxicity that was observed previously.9 Among the identified 5-LO inhibitors, several classes of natural products such as flavonoids (epi-gallocatechin, Figure 1), phenols (curcumin, Figure 1), coumarins, and terpenoids have been highlighted as promising molecules.10 Several of these natural inhibitors possess unsaturated aliphatic chains

nflammation is the immune response of the human body when faced with any tissue injury. Deregulation of the inflammatory process may lead to severe chronic inflammationrelated diseases such as asthma, rheumatism, atherosclerosis, or even cancer. There are several pro-inflammatory mediators that include eicosanoids, which are lipid mediators produced from arachidonic acid (AA).1 Two main enzymatic routes are involved in eicosanoid biosynthesis, namely, the cyclooxygenase (COX) pathway and the 5-lipoxygenase (5-LO) pathway.2 While the first one triggers the biosynthesis of prostaglandins (PGs) including PGE2 linked with pain, fever, and arthritis, the 5-LO pathway is involved with the biosynthesis of leukotrienes (LTs). LTs have potent pro-inflammatory properties, and elevated concentrations of LTB4 and cysteinyl-leukotrienes (Cys-LTs) have been detected in asthmatic subjects.3,4 5-LO is a nonheme iron-containing enzyme that catalyzes the first two steps in the biosynthesis of LTs from AA.5 Suppression of LT production through inhibition of 5-LO is thus considered an attractive strategy for the development of therapeutics, highlighting 5-LO as a drug target. Zileuton, a hydroxyurea derivative with a benzothiophene core, is the only 5-LO inhibitor introduced to the market and is prescribed for asthma © 2017 American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of Phil Crews Received: November 4, 2016 Published: February 27, 2017 699

DOI: 10.1021/acs.jnatprod.6b01008 J. Nat. Prod. 2017, 80, 699−706

Journal of Natural Products

Article

Figure 1. Natural product inhibitors of 5-LO and mPEGS-1/5-LO.

Figure 2. Acronychia-type acetophenones from A. pedunculata.

on SAR studies with enhanced potency as dual mPGES-1 and 5-LO inhibitors,17 such as benzoquinones with aliphatic chains,18 prenylated chalcones, and flavonoids.12,15 Overall, most of the identified natural products acting as 5-LO inhibitors are phenolic substructures or phloroglucinol-based molecules possessing prenylated or modified prenylated chains with possible aliphatic connections between each phloroglucinol moiety, clearly indicating a structural feature for the development of this type of inhibition. Within the context of a continuation of work aimed at discovering novel natural dual mPGES-1/5-LO inhibitors, the trunk bark of Acronychia pedunculata (L.) Miq., a tree growing in Southeast Asia, was selected to be investigated.19 The genus

(prenyl, geranyl, or isoprenyl), such as hyperforin or garcinol (Figure 1). More recently, indirubin was highlighted as a new chemotype for inhibition of 5-LO through unique binding in the ATP pocket of the enzyme.11 Finally, phloroglucinolderived molecules were shown to inhibit 5-LO as well, such as myrtucommulone A isolated from Myrtus communis, hyperforin isolated from Hypericum perforatum,12,13 and arzanol isolated from Helichrysum italicum (Figure 1).6,14,15 Interestingly, myrtucommulone A, hyperforin,12 and arzanol also inhibit mPGES-1,15,16 and myrtucommulone A was characterized as the first natural product that inhibits both mPGES-1 and 5LO.1 To date, a number of natural compounds and synthetic analogues have indicated advantageous structural features based 700



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Scheme 1. Synthesis of Phloroglucinol Monomersa

Acronychia, belonging to the Rutaceae family, is composed of 44 species, some of which have a long traditional use in Asian ethnomedicine, notably for treating microbial or viral infections.18 Moreover, the aerial parts of certain species are used as food ingredients, whereas the leaves and flowers are incorporated in cosmetic preparations.18 Regarding A. pedunculata, there are limited data in the literature regarding its beneficial health effects, but some reports refer to its traditional use against chronic inflammatory diseases such as asthma and rheumatism.20,21 Alkaloids, triterpenoids, and acetophenones are the characteristic chemical classes of the genus Acronychia. Particularly, acetophenones encompass certain structural features typical of this genus, and they are named commonly Acronychia-type acetophenones, while most of them have been recently reported as new natural products from A. pedunculata (Figure 2).19 Structurewise, this class of compounds can be divided into monomers (one phloroglucinol unit)22,23 and fully substituted dimers (two phloroglucinol units).19,20 The occurrence of isoprenyl and modified isoprenyl chains represents an integral part of this chemical group. They commonly incorporate an additional ring biosynthetically originated from the ring closure of an isoprenyl side chain (Figure 2). Depending on the orientation of the fusion relative to the isobutyl chain, Acronychia-type acetophenones occur as A-type (ring closure on the phenol ortho to the chain) and B-type (ring closure on the phenol para to the chain). Taken together, based on the occurrence of this structural motif with the long traditional use of A. pedunculata for the treatment of inflammation-related diseases, it becomes clear that Acronychia-type acetophenones could serve as a promising scaffold for 5-LO or dual 5-LO and mPEGS-1 inhibition. In order to gain better insight into the structure−activity relationships (SAR) of Acronychia-type acetophenones, various mono- and dimeric analogues of these were synthesized. Among them, the first synthesis of demethylacrovestone was carried out. All synthetic analogues obtained, together with the isolated natural Acronychia-type acetophenones, were evaluated for mPGES-1 and 5-LO inhibition. Finally, the potency of the most active molecule (10) has been evaluated in vivo using a mouse model of acute inflammation (i.e., zymosan-induced peritonitis).

a Reagents and conditions: (a) BF3-Et2O, 1,4-dioxane, rt, 12 h; (b) geranyl bromide, K2CO3, acetone, rt, 18 h; (c) K2CO3, acetone, 40 °C, 20 h; (d) CH2Cl2, 40 °C, 24 h.

In the case of monomers possessing additional rings such as a furan or pyran moiety, numerous methodologies have been developed for obtaining these naturally widespread heterocycles. The furan moiety has been introduced on the acetophenone skeleton (11, 22% yield, Scheme 1) following a procedure that successfully provided furan-containing acronycine analogues.24 For the synthesis of a pyran-ringcontaining monomer, a versatile methodology involving 3methyl-2-butenal25 has been preferred and led successfully to compound 12 with moderate yield (34%) with the main byproduct possessing two rings also being produced.25 Synthesis of Phloroglucinol Dimers. The naturally occurring Acronychia-type acetophenone dimers can be further subdivided into homo- and heterodimers, with the latter far exceeding the former in number.19,20,26 The only homodimer isolated so far is demethylacrovestone (14), which lacks a methoxy group compared to the model compound acrovestone (1), constituting a totally symmetrical dimer.26 Having no SAR data concerning the biological activity of compound 14 and with this being the only representative of a homodimer produced by A. pedunculata, the chemical development of such molecules was considered challenging. In addition, guided by the goal to generate a library of Acronychia-type acetophenones analogues, the synthesis of demethylacrovestone along with a new type of homodimers was undertaken. The presence of both natural monomethylated and nonmethylated monomers possessing 3,3-dimethylpropanone patterns22 led to the hypothesis of putative biosynthetic formation of the dimers eventually through the connection of the aforementioned monomers with an acylphloroglucinol unit, thus representing the starting point of the synthetic strategy. Acronychia-type acetophenones could be disconnected in the isobutyl chain to give the two monomers and an isobutyl carbocation. A work reporting the synthesis of an electron-rich dirayl alkane using a Friedel−Craft reaction with aldehydes27 allowed envisaging the access of the desired dimers following this approach. As a proof of concept, the acetophenone 8 has been engaged in a Friedel− Craft reaction with isovaleraldehyde in the presence of aluminum chloride in refluxing anhydrous toluene (Scheme 2). The nonsubstituted homodimer 13 connected with an isobutyl chain has been successfully obtained although with

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RESULTS AND DISCUSSION Synthesis of Phloroglucinol Monomers. The first goal was the synthesis of phloroglucinol monomers, not only in order to evaluate their activity but also to serve as building blocks for the synthesis of dimers. In total, five monomers have been produced with different substitution patterns on the core of acetyl phloroglucinol, i.e., prenyl, geranyl, pyran, and furan (Scheme 1). However, due to the difficulty in achieving high percentages of stereoselectivity as a result of the equivalent reactivity of each carbon in the phloroglucinol skeleton, the yields obtained were low to moderate. Nevertheless, the synthesis of the monoprenylated acetophenone 9 was achieved in satisfactory yield using a procedure involving 2-methyl-3buten-2-ol and BF3-Et2O (Scheme 1). This method represents a practical alternative to the traditional strategy involving prenyl bromide in the presence of potassium carbonate, which generally leads to several products including C-diprenylated and O-prenylated phloroglucinols. Compound 10 has been obtained following a classical approach involving the use of geranyl bromide (Scheme 1). 701



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Scheme 2. Synthesis of Phloroglucinol Homodimers

moderate yield (Scheme 2). On the basis of this strategy, a small assembly of homodimers has been obtained including demethylacrovestone (14), synthesized here for the first time, as well as different analogues possessing geranyl (15), pyran (16), or furan (17) rings (Scheme 2). Compound 14 and its analogues were evaluated for their activity against 5-LO and mPGES-1. Evaluation of 5-LO and mPEGS-1 Inhibition. Inhibition of 5-LO activity was first evaluated by means of a cell-free assay using isolated human recombinant 5-LO, which allows the identification of direct interference of the test compound with the target enzyme, and IC50 values have been calculated for both natural and synthetic compounds displaying 5-LO activity below 50% at 10 μM. Zileuton was used as a positive control. The results are summarized in Table 1.

Figure 3. Characteristics of 5-LO inhibition by compounds 1 and 10. (A) Inhibition of 5-LO and mPGES1 activity by compounds 1 and 10. (B) Effects of compound 10 on the activity of human recombinant 5LO at different AA concentrations. (C) Inhibition of 5-LO by compound 10 is reversible (wash-out experiments). Zil = positive control, zileuton. Data in (A) and (C) are given as means ± SEM, n = 3−4. Data in (B) are means of a representative experiment out of three independent experiments. **, p < 0.01; ***, p < 0.001; versus 100% control.

Table 1. Evaluation of Natural and Synthetic AcronychiaType Acetophenones toward 5-LOa 5-LO

mPEGS-1

compound

cell -free IC50 (μM)

intact cells IC50 (μM)

cell-free IC50 (μM)

1 2 3 4 5 6 7 9 10 13 14 15 16 17 zileuton MK886

2.7 ± 0.2 6 ± 0.6 7.3 ± 1.8 2 ± 0.5 >10 5.3 ± 1.3 2.5 ± 0.3 n.d.b 1.0 ± 0.1 1.7 ± 0.1 >10 3.6 ± 0.4 >10 2.4 ± 1 0.8 ± 0.1 n.d.b

2.3 ± 0.6 >10 5.3 ± 0.7 5.0 ± 0.6 4.5 ± 1 7.3 ± 0.7 6.3 ± 2 n.d.b 1.4 ± 0.3 6.3 ± 0.3 >10 >10 5±3 >10 1.7 ± 0.7 n.d.b

1.1 ± 0.02 4.2 ± 0.2 2.7 ± 0.1 1.9 ± 0.2 1.1 ± 0.1 1.3 ± 0.2 1.1 ± 0.1 >10 3.5 ± 0.1 1.0 ± 0.1 >10 5.34 ± 1.6 >10 1.2 ± 0.1 n.db 2.5 ± 0.5

a

poor membrane permeation into the cell, but other mechanisms may contribute as well. All the natural products were found to be also good inhibitors of mPEGS-1 in a cell-free assay (Table 1), supporting the Acronychia-type acetophenones as a new class of dual 5-LO/mPEGS-1 inhibitors. The synthetic dimers 13−17 presented good inhibitory activities toward 5LO, particularly in the cell-free assays. However, as for the natural heterodimers, their activity in the cell-based assays was decreased, except for compound 15. Surprisingly, demethylacrovestone (14) was found to be inactive despite several repetitions of the assay for unexplained reasons, while the unsubstituted dimer 13 was found to be potent in both the cellfree assay and the cell-based assay. Concerning the inhibition of mPEGS-1, monomers 10 and 11 and dimers 13 and 16 displayed good inhibitory activity, suggesting that they also act as dual 5-LO/mPEGS-1 inhibitors. Overall, these results may reflect a similar mode of action between the natural Acronychiatype acetophenones and the synthetic homodimers. The position and nature of additional rings (3−6) had a considerable impact on the inhibitory efficiency. The pyrancontaining products (3 and 4) were more active, and A-type Acronychia-type acetophenones improved the potency of the molecules (4 and 6) compared to B-type (3 and 5). Demethylacronyline (9) was inactive in cell-free assays. However, introduction of an extended unsaturated chain, such as a geranyl moiety, led to potent inhibition of 5-LO in the cell-free assay, as exemplified by compound 10 (IC50 = 1.0 μM) (Figure 3A). Compound 10 has recently been identified as a soybean 15lipoxygenase inhibitor.28 The soybean 15-LO assay is used as a preliminary study of human 15-LO, due to the similarities in their structure and mechanism.29 Additionally, human 5-LO and 15-LO possess a high level of similarity, especially in the area of the catalytic site.30,31 The main differences rely on the orientation of helix α-2 affecting the position of Phe177 and Tyr181 (for 5-LO) and Leu181 (for 15-LO-1) residues,30

Data are given as means ± SEM n = 3−4. bn.d.: not determined.

All the natural compounds displayed a medium to high inhibition of 5-LO in cell-free assays. Specifically, acrovestone (1) (Figure 3A), acropyranol A (4), and acrovestenol (7) inhibited 5-LO potently, with an IC50 around 2 μM, comparable to the positive control zileuton. Apart from acrovestone (1), all the natural metabolites showed no 5-LO inhibitory activity in the cell-based assays used (Table 1). These cell-based assays were performed using activated human neutrophils, which allows the analysis of the interference of the test compounds with 5-LO in a cellular (biological) environment. The loss of potency of the majority of compounds in the cell-based assay might be explained by 702



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and mPEGS-1 in the development of chronic inflammatory diseases and the traditional use of A. pedunculata to treat such diseases connect traditional medicine, plant metabolites, and biological targets. Furthermore, synthetic compounds derived from the natural metabolites and specifically homodimers were found to possess a similar activity profile compared to natural products, while the access to monomers revealed their 5-LO inhibitory activity, highlighting interesting structural features. Among the derivatives investigated, demethylacrovestone (14) was synthesized for the first time, whereas a simple synthetic route to the homodimers was proposed. Using a mouse model of acute inflammation, one of the synthetic compounds, geranylphloroglucinol (10), displayed a potent anti-inflammatory response comparable to the known 5-LO inhibitor zileuton, a drug used for asthma treatment. Acronychia-type acetophenones and acrovestone (as an active natural product) represent a new class of 5-LO inhibitors and a new chemotype for the development of more potent agents. Further studies should be carried out for the evaluation of additional acetophenones of this type including both monomers and dimers, as well as for the investigation of additional mechanisms of action.

which may direct the carbon of AA to be oxidized (C-5, C-12, or C-15). In silico studies have revealed previously the interaction of a geranyl acylphloroglucinol derivative with soybean 15-LO;28 the molecule interacts deeply inside the catalytic cavity with His518, His513, and Glu716, three important residues coordinating the active-site iron. In this case, Phe177 and Leu181 were directed outward, opening a large access allowing the phloroglucinol derivatives to enter the active site. In the present case, in the absence of a cocrystal structure and the low reliability of docking calculations (only a modified structure of 5-LO affecting the size of catalytic site is available30), few positive conclusions could be proposed. Nevertheless, in the case of 5-LO, Phe177, and Tyr181 in being more inward and in closing off the access to the catalytic site,30 interactions of compound 10 deeply inside the cavity could be discarded. Therefore, the hypothesis of π−π interactions of the phloroglucinol moiety with one of the aforementioned residues at the entrance of the active site and insertion of the geranyl chain inside the binding cavity could be proposed. To support this hypothesis, a competition assay was performed between compound 10 and the 5-LO substrate AA at different concentrations (Figure 3B). This revealed that 10 is not a true competitor of AA binding, but based on wash-out experiments (10-fold dilution), 10 inhibits 5-LO in a reversible manner (Figure 3C), partly confirming the hypothesis. Up to now, the precise mode of inhibition of 5-LO by homo- or heteroacetophenone dimers remained elusive. Nevertheless, the extension of the unsaturated chain on acylphloroglucinol appears as a vector to develop new chemical entities, and the evaluation of such compounds would certainly provide new insights in terms of both SAR and mechanism of action. In Vivo Evaluation in a Mouse Model. Next, the effects of the most interesting compound, 10, was evaluated in vivo using a mouse model of acute inflammation, namely, zymosaninduced peritonitis. Zileuton and indomethacin were used as controls. The ip pretreatment of mice with 10 (10 mg/kg, 30 min before zymosan injection) inhibited the LTC4 levels (38%) and cell infiltration (32%) in the peritoneal exudates, 30 min and 4 h after zymosan injection, respectively. No repression of PGE2 production was observed (Table 2), indicating that compound 10 preferentially acted in vivo as a 5-LO inhibitor rather than a dual 5-LO/mPEGS-1 inhibitor as suggested by the in vitro assays. The study has revealed that Acronychia-type acetophenones are natural inhibitors of 5-LO, and some also displayed dual 5LO/mPEGS-1 inhibition in vitro. The involvement of 5-LO

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Natural Acetophenones. All seven natural acronychia-type acetophenones reported in this study (Figure 1) have been previously isolated from the trunk bark of Acronychia pedunculata and unambiguously identified by spectroscopic methods.19 The purity of all compounds was determined by HPLC-DAD and 1H NMR spectroscopy and determined to be >95%. Chemistry. All chemicals were purchased from Aldrich Chemical Co. Microwave-assisted reactions were performed in a multimode Milestone Start E apparatus (Milestone, Italy). Melting points were recorded on a Büchi apparatus and are uncorrected. FT-IR spectra were recorded on a PerkinElmer RX1 FT-IR spectrometer. UV spectra were recorded on a Shimadzu UV-160A UV−visible recording spectrophotometer. NMR spectra were recorded on Bruker Avance 600 spectrometer (Karlsruhe, Germany) (1H 600 MHz, 13C 150 MHz); chemical shifts are expressed in ppm downfield from tetramethylsilane. HRMS spectra were recorded on an LTQ-Orbitrap spectrometer (ThermoScientific, Bremen, Germany). Column chromatography was conducted using flash silica gel 60 (40−63 μm) from Merck. Purity of the compounds has been determined by HPLC-DAD (Thermo Fisher Scientific) and was above 95%. 1-(2,4,6-Trihydroxy-3-(3-methylbut-2-en-1-yl)phenyl)ethanone (9). 2,4,6-Trihydroxyacetophenone (8) (100 mg, 0.54 mmol) was dissolved in 5 mL of 1,4-dioxane. To this solution were added 61.75 μL of 2-methyl-3-buten-2-ol (0.54 mmol) and 17 μL of BF3-Et2O (0.14 mmol), and the mixture was stirred for 12 h at room temperature. The solvent was evaporated, and the resulting residue was purified using silica gel column chromatography with CH2Cl2/ EtOAc (94:6) as solvent system. This led to 26.9 mg of 9 being collected as a yellow solid (20% yield): 1H NMR (CDCl3, 600 MHz) δ 5.91 (1H, s, H-5), 5.23 (1H, t, J = 6.0 Hz, H-2), 3.42 (2H, d, J = 6.0 Hz, H-1′), 2.68 (3H, s, CH3CO), 1.37 (6H, s, H-4′, H-5′); 13C NMR (CDCl3, 150 MHz) δ 203.9 (C, CH3CO−Ar), 162.2 (C, C-6), 161.3 (C, C-4), 161.1 (C, C-2), 136.2 (C, C-3′), 121.4 (CH, C-2′), 105.2 (C, C-3), 103.3 (C, C-1), 96.5 (CH, C-5), 32.9 (CH3, CH3CO-Ar), 21.7 (CH3, C-4′, C-5′), 21.4 (CH2, C-1′); (+)-HRESIMS m/z 259.1102 [M + Na]+ (calcd for C13H16O4Na, 259.0946).32 (E)-1-(3-(3,7-Dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxyphenyl)ethanone (10). 2,4,6-Trihydroxyacetophenone (8) (50 mg, 0.17 mmol) was dissolved in 10 mL of acetone, and then were added 82.9 mg (0.37 mmol) of K2CO3 and 57 μL (0.17 mmol) of geranyl bromide. After 18 h at room temperature, the reaction was quenched with the addition of 2 N HCl. The solvent was evaporated and the residue was extracted with EtOAc and water. The organic fraction was

Table 2. Effect of Compound 10 on the Production of Proinflammatory Metabolites and Cell Infiltration treatmenta vehicle (2% DMSO) 10 (10 mg/kg) zileuton (10 mg/kg) indomethacin (5 mg/kg)

LTC4 (ng/ mL)b

inflammatory cells (× 106)c

PGE2 (ng/ mL)c

92.2 ± 12.8

7.3 ± 0.72

10.5 ± 1.34

57.3 ± 7.1 65 ± 2.41

5.00 ± 0.69e n.d.d

11.3 ± 1.5 n.d.d

n.dd

2.08 ± 1.02f

3.52 ± 1.06f

EXPERIMENTAL SECTION

a Male mice (n = 6−8, each group) were treated ip 30 min before ip injection of zymosan. bLTC4 and PGE2 levels were evaluated 30 min after injection with zymosan. cCell infiltration was evaluated 4 h after zymosan injection. dn.d.: not determined. ep < 0.05 versus vehicle (Student’s t test). fp < 0.01 versus vehicle (Student’s t test).

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dried on Na2SO4, filtered, and evaporated. The crude product was purified with silica gel column chromatography using CH2Cl2/EtOAc (92:8). In total, 35.1 mg of 10 as a yellow solid was collected (68% yield): 1H NMR (CDCl3, 600 MHz) δ 5.91 (1H, s, H-5), 5.32 (1H, t, J = 6.3 Hz, H-2′), 5.06 (1H, t, J = 6.6 Hz, H-7′), 3.51 (4H, d, J = 7.2 Hz, H-1′, H-6′), 2.81 (3H, s, CH3CO), 1.34 (5H, s, H-4′, H-5′), 1.33 (6H, s, H-9′, H-10′); 13C NMR (CDCl3, 150 MHz) δ 204.2 (C, CH3CO− Ar), 160.8 (C, C-6), 159.1 (C, C-4), 158.2 (C, C-2), 136.6 (C, C-3′), 135.9 (C, C-8′), 123.3 (CH, C-7′), 121.5 (CH, C-2′), 108.7 (CH, C5), 106.5 (C, C-3), 104.7 (C, C-1), 30.8 (CH3, CH3CO-Ar), 28.5 (CH2, C-5′), 28.40 (CH3, C-4′), 21.3 (CH2, C-1′), 21.2 (CH2, C-6′), 20.3 (CH3, C-9′, C-10′); (+)-HRESIMS m/z 327.1727 [M + Na]+ (calcd for C18H24O4Na, 327.1572).33 1-(4,6-Dihydroxy-2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran-5yl)ethanone (11). 2,4,6-Trihydroxyacetophenone (8) (50 mg, 0.27 mmol) was dissolved in 3 mL of acetone, and 74 mg (0.54 mmol) of K2CO3 and 60.7 μL (0.41 mmol) of 1,4-dibromo-2-methylbut-2-ene34 were added. The mixture was heated at 40 °C for 20 h. The solvent was evaporated, and the residue was extracted with EtOAc and water. After gathering, the organic phase was dried on Na2SO4, filtered, and evaporated. Compound 11 was purified with a silica gel column using as solvent system CH2Cl2/EtOAc (96:4). Altogether, 14.1 mg of 11 was collected as a yellow solid (22% yield): 1H NMR (CDCl3, 600 MHz) δ 5.92 (1H, s, H-5), 5.62 (1H, brs, H-2′), 5.03 (2H, s, H-5′), 3.40 (2H, brs, H-1′), 2.74 (3H, s, H-4′), 2.68 (3H, s, CH3CO); 13C NMR (CDCl3, 150 MHz) δ 203,1 (C, CH3CO−Ar), 161.3 (C, C-6), 159.9 (C, C-4), 152.5 (C, C-2), 144.5 (C, C-3′), 120.8 (CH, C-2′), 111.8 (CH2, C-5′), 109.2 (CH, C-5), 106.5 (C, C-3), 101.1 (C, C-1), 32.8 (CH3, C-4′), 32.5 (CH3, CH3CO−Ar), 20.6 (CH2, C-1′); (−)-HRESIMS m/z 233.1133 [M − H]− (calcd for C13H13O4, 233.0892).34 1-(5,7-Dihydroxy-2,2-dimethyl-2H-chromen-8-yl)ethanone (12). 2,4,6-Trihydroxyacetophenone (8) (50 mg, 0.27 mmol) was dissolved in 5 mL of CH2Cl2, and 16.4 μL of 3-methyl-2-butenal (0.17 mmol) was added. The reaction mixture was heated to 40 °C for 24 h. After completion of the reaction, the solvent was evaporated and the residue was extracted with EtOAc and water. The organic fractions were dried on Na2SO4, filtered, and evaporated. The crude product was subjected to purification on a silica gel column, and 13.4 mg of pure compound 12 was collected as a yellow solid (34% yield): 1H NMR (CDCl3, 600 MHz) δ 6.64 (1H, d, J = 10.02 Hz, H-1′), 5.92 (1H, s, H-5), 5.43 (1H, d, J = 10.02 Hz, H-2′), 2.67 (3H, s, CH3CO), 1.41 (6H, brs, H-4′, H5′); 13C NMR (CDCl3, 150 MHz) δ 204.1 (C, CH3CO−Ar), 160.1 (C, C-6), 159.8 (C, C-4), 152,5 (C, C-2), 124.5 (CH, C-2′), 116.8 (CH, C-1′), 109.1 (CH, C-5), 104.5 (C, C-1), 103.8 (C, C-2), 102.6 (C, C-3), 77.1 (C, C-3′), 32.4 (CH3, CH3CO−Ar), 28.4 (CH3, C-4′, C-5′); (−)-HRESIMS m/z 233.1132 [M − H]− (calcd for C13H13O4, 233.0892).25 1,1′-((3-Methylbutane-1,1-diyl)bis(2,4,6-trihydroxy-3,1phenylene))diethanone (13). 2,4,6-Trihydroxyacetophenone (8) (1.0 g, 5.4 mmol) was dissolved in 15 mL of toluene, and 72 mg (0.54 mmol) of anhydrous AlCl3 was added. The reaction mixture was stirred for 15 min at room temperature. Then isovaleraldehyde (250 μL, 2.7 mmol) was added, and the mixture was heated under reflux for 48 h. The solvent was evaporated, the residue was extracted with CH2Cl2 and a saturated aqueous solution of NaCl, and the organic phase was dried over Na2SO4, filtered, and evaporated. The crude product was purified with a silica gel column (CH2Cl2/MeOH, 97:3). Altogether, 287 mg of compound 13 was collected as an orange solid (13% yield): mp 149−150 °C; UV (MeOH) λmax (log ε) 289 (3.92), 236 (4.52) nm; IR (Nujol) νmax 3320, 2919, 2720, 1619, 1520, 1455, 1382, 1268, 1155, 988, 935, 850 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.91 (2H, brs, H-3), 4.64 (1H, t, J = 7.5 Hz, H-1′), 2.68 (6H, s, CH3CO), 2.18 (2H, brs, H-2′), 1.41 (1H, m, H-3′), 0.87 (6H, d, J = 7.1 Hz, H-4′, H-5′); 13C NMR (CDCl3, 150 MHz) δ 203.8 (C, CH3CO−Ar), 163.1 (C, C-4), 162.6 (C, C-6), 159.1 (C, C-2), 109.0 (C, C-5), 104.5 (C, C-1), 96.3 (CH, C-3), 38.9 (CH2, C-2′), 32.1 (CH3, CH3CO−Ar), 27.4 (CH, C-1′), 26.5 (CH, C-3′), 22.1 (CH3, C4′, C-5′); (−)-HRESIMS m/z 403.1390 [M − H]− (calcd for C21H23O8, 403.1398).

Demethylacrovestone (14). 2,4,6-Trihydroxy-3-prenylacetophenone (9) (30 mg, 0.12 mmol) was dissolved in 5 mL of toluene. A 1.7 mg amount of anhydrous AlCl3 (0.012 mmol) and 6.8 μL of isovaleraldehyde (0.06 mmol) were then added. The reaction was irradiated at 300 W (T = 111 °C) for 1.5 h. The solvent was evaporated, and the residue was extracted with CH2Cl2 and a saturated aqueous solution of NaCl. The organic phases were gathered, dried over NaSO4, filtered, and evaporated. The crude product was purified on a silica gel column (CH2Cl2/cyclohexane, 1:1). In total, 3.1 mg of compound 14 was collected as a yellow solid (5% yield): mp 149−150 °C; UV (MeOH) λmax (log ε) 347 (1.55), 303 (4.89), 244 (6.19), 209 (4.93) nm; IR (Nujol) νmax 3320, 3137, 2919, 2724, 1610, 1519, 1458, 1376, 1318, 1242, 1173, 988, 940, 845 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.28 (2H, t, J = 6.9 Hz, H-2′), 4.69 (1H, brs, H-1″), 3.47 (4H, d, J = 6.0 Hz, H-1′), 2.78 (6H, s, CH3CO), 2.15 (2H, s, H-2″), 1.65 (1H, brs, H-3″), 1.34 (12H, s, H-4′, H-5′), 0.87 (6H, brs, H-4″, H-5″); 13 C NMR (CDCl3, 150 MHz) δ 201.1 (C, CH3CO−Ar), 161.3 (C, C6), 161.0 (C, C-4), 160.5 (C, C-2), 136.6 (C, C-3′), 121.5 (CH, C-2′), 109.1 (C, C-5), 107.2 (C, C-3), 103.4 (C, C-1), 37.1 (CH2, C-2″), 30.4 (CH3, CH3CO-Ar), 27.8 (CH, C-1″), 25.5 (CH, C-3″), 21.7 (CH2, C-1′), 20.9 (CH3, C-4′, C-5′), 20.5 (CH3, C-4″,C-5″); (+)-HRESIMS m/z 563.2599 [M + Na]+ (calcd for C31H40O8Na, 563.2621). 1-(3-(1-(3-Acetyl-5-((E)-3,7-dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxyphenyl)-3-methylbutyl)-5-((Z)-3,7-dimethylocta-2,6-dien-1yl)-2,4,6-trihydroxyphenyl)ethanone (15). 2,4,6-Trihydroxy-3geranylacetophenone (10) (182 mg, 0.60 mmol) was dissolved in 5 mL of toluene, and after 15 min of stirring, 23 μL of isovaleraldehyde (0.30 mmol) and 8 mg of anhydrous AlCl3 (0.06 mmol) were added. The reaction mixture was heated under reflux for 48 h. The solvent was evaporated, and the residue was extracted with CH2Cl2 and saturated aqueous NaCl solution. The organic phase was dried over NaSO4, filtered, and evaporated. The crude product was purified on a silica gel column (CH2Cl2/cyclohexane, 6:4). In total, 15 mg of compound 15 was collected as a yellow solid (11% yield): mp 147− 148 °C; UV (MeOH) λmax (log ε) 346 (1.42), 303 (4.27), 245 (4.83) nm; IR (Nujol) νmax 3246, 2919, 2850, 1619, 1594, 1420, 1360, 1281, 1165, 957, 841 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.32 (2H, t, J = 6.6 Hz, H-2′), 5.06 (2H, t, J = 6.6 Hz, H-7′), 4.66 (1H, brs, H-1″), 3.51 (8H, d, J = 7.2 Hz, H-1′, H-6′), 2.81 (6H, s, CH3CO), 2.17 (2H, brs, H-2″), 1.78 (1H, m, H-3″), 1.34 (12H, s, H-9′, H-10′), 1.33 (10H, s, H-4′, H-5′), 0.88 (6H, brs, H-4″, H-5″); 13C NMR (CDCl3, 150 MHz) δ 204.2 (C, CH3CO−Ar), 160.8 (C, C-6), 159.1 (C, C-4), 158.2 (C, C-2), 136.6 (C, C-3′), 135.9 (C, C-8′), 123.3 (CH, C-7′), 121.55 (CH, C-2′), 108.7 (C, C-5), 106.1 (C, C-3), 104.7 (C, C-1), 38.8 (CH2, C-2″), 30.7 (CH3, CH3CO−Ar), 29.2 (C, C-3″), 28.5 (CH2, C-5′), 28.4 (CH3, C-4′), 27.6 (CH, C-1′), 26.2 (CH, C-3″), 21.3 (CH2, C-1′), 21.2 (CH2, C-6′), 21.6 (CH3, C-4″, C-5″), 20.4 (CH3, C-9′, C-10′); (+)-HRESIMS m/z 699.3865 [M + Na]+ (calcd for C41H56O8Na, 699.3873). 1,1′-(5,5′-(3-Methylbutane-1,1-diyl)bis(4,6-dihydroxy-2-(prop-1en-2-yl)-2,3-dihydrobenzofuran-7,5-diyl))diethanone (16). 2-Isoprenyl-5-acetyl-4,6-dihydroxy-2,3-dihydrobenzofuran (11) (250 mg, 1.07 mmol) was dissolved in 10 mL of toluene, and 43.5 μL of isovaleraldehyde (0.53 mmol) and 14.3 mg of anhydrous AlCl3 (0.11 mmol) were added. The mixture was heated under reflux for 48 h. The solvent was evaporated, and the residue was extracted with CH2Cl2 and saturated aqueous NaCl solution. The organic phase was dried over Na2SO4, filtered, and evaporated. The crude product was purified with column chromatography (CH2Cl2/cyclohexane, 6:4). A 15 mg amount of compound 16 was obtained as a yellow oil (10% yield): mp 150−151 °C; UV (MeOH) λmax (log ε) 293 (3.91), 237 (4.45), 204 (4.75) nm; IR (Nujol) νmax 3340, 2960, 2919, 1610, 1584, 1450, 1370, 1255, 1203, 1158, 970, 891, 810 cm−1; 1H NMR (CDCl3, 600 MHz) δ 0.88 (6H, d, J = 7.1 Hz, H-4″, H-5″), 1.42 (1H, brs, H-3″); 2.49 (2H, brs, H-2″), 2.68 (6H, s, CH3CO), 2.72 (6H, s, H-4′), 3.40 (4H, brs, H1′), 4.70 (1H, brs, H-1″); 5.03 (4H, s, H-5′), 5.62 (2H, brs, H-2′); 13C NMR (CDCl3, 150 MHz) δ 202.1 (C, CH3CO−Ar), 161.3 (C, C-6), 159.9 (C, C-4), 152.5 (C, C-2), 144.5 (C, C-3′), 120.8 (CH, C-2′), 111.8 (CH2, C-5′), 109.2 (C, C-5), 106.5 (C, C-3), 101.1 (C, C-1), 704



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38.1 (CH2, C-2″), 32.8 (CH3, C-4′), 32.5 (CH3, CH3CO-Ar), 27.4 (CH, C-1″), 26.2 (CH, C-3″), 22.1 (CH3, C-4″, C-5″), 20.6 (CH2, C1′); (−)-HRESIMS m/z 535.2329 [M − H]− (calcd for C31H35O8, 535.2337). 1,1′-(6,6′-(3-Methylbutane-1,1-diyl)bis(5,7-dihydroxy-2,2-dimethyl-2H-chromene-8,6-diyl))diethanone (17). (5,5′-Isobutyl)diacetophenone (13) (35 mg, 0.087 mmol) was dissolved in dichloromethane, 16.7 μL of 3-methyl-2-butenal (0.174 mmol) was added, and the mixture was heated under reflux for 24 h. The solvent was evaporated, and the residue was purified using silica gel column chromatography (CH2Cl2/cyclohexane, 95:5); 5 mg of 17 was collected as a yellow solid (12% yield): mp 149−150 °C; UV (MeOH) λmax (log ε) 292 (3.32), 249 (3.29) nm; IR (Nujol) νmax 3320, 2967, 2921, 1616, 1582, 1443, 1366, 1256, 1209, 1152, 964, 877, 807 cm−1; 1H NMR (CDCl3, 600 MHz) δ 6.64 (2H, d, J = 10.02 Hz, H-2′), 5.44 (2H, d, J = 10.02 Hz, H-1′), 4.67 (1H, t, J = 7.0 Hz, H-1″), 2.67 (6H, s, CH3CO), 2.30 (2H, m, H-2″), 1.41 (12H, s, H-4′, H-5′), 1.22 (1H, brs, H-3″), 0.84 (6H, d, J = 7.1 Hz, H-4″, H-5″); 13C NMR (CDCl3, 150 MHz) δ 203.7 (C, CH3CO−Ar), 160.1 (C, C-6), 159.8 (C, C-4), 152.7 (C, C-2), 124.8 (CH, C-2′), 117.3 (CH, C-1′), 109.1 (C, C-5), 104.5 (C, C-1), 102.6 (C, C-3), 77.1 (C, C-3′), 33.9 (CH2, C-2″), 32.4 (CH3, CH3CO−Ar), 28.4 (CH3, C-4′, C-5′), 27.6 (CH, C1″), 27.4 (CH, C-3″), 20.6 (CH3, C-4″, C-5″); (−)-HRESIMS m/z 535.2339 [M − H]− (calcd for C31H35O8, 535.2337). Cells and Cell Isolation. Human peripheral blood was taken from fasted (12 h) healthy donors that had not taken any anti-inflammatory drugs during the last 10 days, by venipuncture in heparinized tubes (16 IE heparin/mL blood). The blood was centrifuged at 4000g for 20 min at room temperature for preparation of leukocyte concentrates (University Hospital, Jena, Germany). The protocols for experiments with human leukocytes were approved by the ethical commission of the Friedrich-Schiller-University Jena (No. 4025-02/14). All methods were performed in accordance with the relevant guidelines and regulations. Leukocyte concentrates were subjected to dextran sedimentation and centrifugation on Nycoprep cushions (PAA Laboratories, Linz, Austria). Contaminating erythrocytes of pelleted neutrophils were lysed by hypotonic lysis, and neutrophils were washed twice in ice-cold PBS and finally resuspended in PBS pH 7.4 containing 1 mg/mL glucose and 1 mM CaCl2 (PGC buffer) (purity >96−97%). For analysis of acute cytotoxicity of test compounds during preincubation periods (routinely 15 min), the viability of neutrophils was analyzed by light microscopy and trypan blue exclusion. None of the compounds (up to 10 μM) caused a cytotoxic effect within 30 min of neutrophil incubation (not shown). Determination of 5-LO Activity in Intact Cells. For determination of 5-LO products in intact neutrophils, 5 × 106 cells were resuspended in 1 mL of PGC buffer, preincubated for 15 min at 37 °C with a test compound or vehicle (0.3% DMSO), and incubated for 10 min at 37 °C with Ca2+-ionophore A23187 (2.5 μM) with 20 μM AA. After 10 min at 37 °C, the reaction was stopped on ice by addition of 1 mL of methanol. A 30 μL portion of 1 N HCL, 500 μL of PBS, and 200 ng of prostaglandin B1 were added, and the samples were subjected to solid-phase extraction on C18 columns (100 mg, UCT, Bristol, PA, USA). 5-LO products (LTB4, trans-isomers, 5H(p)ETE) were quantified by HPLC, and quantities calculated on the basis of the internal standard, prostanglandin B1. Cysteinylleukotrienes C4, D4, and E4 were not detected, since their amounts were below the detection limit, and oxidation products of LTB4 were not determined. Expression, Purification, and Cell-Free Activity Determination of 5-LO. Escherichia coli MV1190 was transformed with pT3-5LO plasmid, and recombinant 5-LO protein was expressed at 27 °C as described previously.35 Cells were lysed in 50 mM triethanolamine/ HCl at pH 8.0, 5 mM EDTA, soybean trypsin inhibitor (60 μg/mL), 1 mM phenylmethanesulfonyl fluoride, and lysozyme (500 μg/mL), homogenized by sonication (3 × 15 s), and centrifuged at 40000g for 20 min at 4 °C. The 40000g supernatant (S40) was applied to an ATPagarose column to partially purify 5-LO, as described previously.35 Aliquots of semipurified 5-LO were diluted with ice-cold PBS containing 1 mM EDTA, and 1 mM ATP was added. Samples were

preincubated with each test compound as indicated. After 10 min at 4 °C, samples were prewarmed for 30 s at 37 °C, and 2 mM CaCl2 plus 20 μM AA were added to start 5-LO product formation. The reaction was stopped after 10 min at 37 °C by addition of 1 mL of ice-cold methanol, and the formed metabolites were analyzed by RP-HPLC as described before.35 5-LO products include the all-trans isomers of LTB4 and 5-H(p)ETE. Preparation of mPGES-1 and Cell-Free mPGES-1 Activity Assay. Preparations of A549 cells and the determination of mPGES-1 activity were performed as described previously.36 In brief, cells were treated with 1 ng/mL interleukin-1β for 48 h at 37 °C, 5% CO2. Cells were harvested and sonicated, and the homogenate was subjected to differential centrifugation at 10000g for 10 min and 174000g for 1 h at 4 °C. The pellet (microsomal fraction) was resuspended in 1 mL of homogenization buffer (0.1 M potassium phosphate buffer, pH 7.4, 1 mM phenylmethanesulfonyl fluoride, 60 μg/mL soybean trypsin inhibitor, 1 μg/mL leupeptin, 2.5 mM glutathione, and 250 mM sucrose), and the total protein concentration was determined. Microsomal membranes were diluted in potassium phosphate buffer (0.1 M, pH 7.4) containing 2.5 mM glutathione. Test compounds or vehicle was added, and after 15 min at 4 °C, a reaction (100 μL total volume) was initiated by addition of 20 μM prostaglandin H2. After 1 min at 4 °C, the reaction was terminated using stop solution (100 μL; 40 mM FeCl2, 80 mM citric acid, and 10 μM 11β-prostagladin E2 as internal standard). Prostaglandin E2 was separated by solid-phase extraction and analyzed by RP-HPLC, as described previously.36 Animals and Zymosan-Induced Peritonitis. Male CD-1 mice (8−9 weeks old, Charles River, Calco, Italy) were housed in a controlled environment (21 ± 2 °C) and provided with standard rodent chow and water. All animals were allowed to acclimate for 4 days prior to experiments and were subjected to a 12 h light−12 h dark schedule. Experiments were conducted during the light phase. The experimental protocol was approved by the Animal Care Committee of the University of Naples Federico II, in compliance with Italian regulations on protection of animals used for experimental and other scientific purpose (Ministerial Decree 116/92) as well as with the European Economic Community regulations (Of ficial Journal of European Community L 358/1 12/18/1986). Compound 10, zileuton, or indomethacin at the indicated dose or vehicle (0.5 mL of 0.9% saline solution containing 2% DMSO) was given ip 30 min before zymosan ip injection (0.5 mL of suspension of 2 mg/mL in 0.9% w/v saline). Mice were sacrificed by inhalation of CO2 at the indicated time, followed by a peritoneal lavage with 3 mL of cold PBS. Exudates were collected, the cells in the exudates were counted, and leukotrienes C4 and prostaglandin E2 were measured by enzyme immunoassay (Cayman Chemical).37 Statistics. Data are expressed as means ± SE. IC50 values were graphically calculated from averaged measurements at four or five different concentrations of the compounds using SigmaPlot 9.0 (Systat Software Inc., San Jose, CA, USA). Statistical evaluation of the data was performed by one-way ANOVA, followed by a Bonferroni or Tukey−Kramer post hoc test for multiple comparisons, respectively. A p value < 0.05 (*) was considered significant.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01008. NMR spectra of the synthesized compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: (+49) 3641949801. Fax: (+49) 3641949802. E-mail: [email protected] (O. Werz). *Tel: (+30) 2107274598. Fax: (+30) 2107274594. E-mail: [email protected] (A.-L. Skaltsounis). 705



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(23) Kozaki, S.; Takenaka, Y.; Mizushina, Y.; Yamaura, T.; Tanahashi, T. J. Nat. Med. 2014, 68, 421−426. (24) Boutefnouchet, S.; Gaboriaud-Kolar, N.; Minh, N. T.; Depauw, S.; David-Cordonnier, M.-H.; Pfeiffer, B.; Léonce, S.; Pierré, A.; Tillequin, F.; Lallemand, M.-C.; Michel, S. J. Med. Chem. 2008, 51, 7287−7297. (25) Adler, M. J.; Baldwin, S. W. Tetrahedron Lett. 2009, 50, 5075− 5079. (26) De Silva, L. B.; Herath, W. M.; Liyanage, C.; Kumar, V.; Uddin Ahmad, V.; Sultana, A. Phytochemistry 1991, 30, 1709−1710. (27) Jaratjaroonphong, J.; Sathalalai, S.; Techasauvapak, P.; Reutrakul, V. Tetrahedron Lett. 2009, 50, 6012−6015. (28) Ng, C. H.; Rullah, K.; Aluwi, M. F.; Abas, F.; Lam, K. W.; Ismail, I. S.; Narayanaswamy, R.; Jamaludin, F.; Shaari, K. Molecules 2014, 19, 11645−11659. (29) Wecksler, A. T.; Garcia, N. K.; Holman, T. R. Bioorg. Med. Chem. 2009, 17, 6534−6539. (30) Gilbert, N. C.; Bartlett, S. G.; Waight, M. T.; Neau, D. B.; Boeglin, W. E.; Brash, A. R.; Newcomer, M. E. Science 2011, 331, 217− 219. (31) Gilbert, N. C.; Rui, Z.; Neau, D. B.; Waight, M. T.; Bartlett, S. G.; Boeglin, W. E.; Brash, A. R.; Newcomer, M. E. FASEB J. 2012, 26, 3222−3229. (32) Diller, R. A.; Riepl, H. M.; Rose, O.; Frias, C.; Henze, G.; Prokop, A. Chem. Biodiversity 2005, 2, 1331−1337. (33) Jung, D. H.; Lee, Y. R.; Kim, S. H. Helv. Chim. Acta 2010, 93, 635−647. (34) Bigi, F.; Casiraghi, G.; Casnati, G.; Sartori, G. Tetrahedron 1983, 39, 169−174. (35) Fischer, L.; Szellas, D.; Rådmark, O.; Steinhilber, D.; Werz, O. FASEB J. 2003, 17, 949−951. (36) Koeberle, A.; Siemoneit, U.; Bühring, U.; Northoff, H.; Laufer, S.; Albrecht, W.; Werz, O. J. Pharmacol. Exp. Ther. 2008, 326, 975− 982. (37) Rossi, A.; Pergola, C.; Pace, S.; Rådmark, O.; Werz, O.; Sautebin, L. Pharmacol. Res. 2014, 87, 1−7.

Nicolas Gaboriaud-Kolar: 0000-0001-9307-1921 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by project NATPROT (No. 3207) funded by the Greek Secreteriat for Research and Technology and action ARISTEIA II. We thank Dr. M. Litaudon (Institut de Chimie des Substances Naturelles, CNRS) and Dr. K. Awang and Prof. H. A. Hadi (both of the University of Malaya) for the plant collection. The authors also thank the Deutsche Forschungsgemeinschaft (DFG) within the SFB1127 (Chemical Mediators in Complex Biosystems) for funding.

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DEDICATION Dedicated to Professor Phil Crews, of the University of California, Santa Cruz, for his pioneering work on bioactive natural products.

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REFERENCES

(1) Medzhitov, R. Nature 2008, 454, 428−435. (2) Koeberle, A.; Werz, O. Drug Discovery Today 2014, 19, 1871− 1882. (3) Peters-Golden, M.; Henderson, W. R. N. Engl. J. Med. 2007, 357, 1841−1854. (4) Montuschi, P. Mini-Rev. Med. Chem. 2008, 8, 647−656. (5) Rådmark, O.; Werz, O.; Steinhilber, D.; Samuelsson, B. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2015, 1851, 331−339. (6) Pergola, C.; Werz, O. Expert Opin. Ther. Pat. 2010, 20, 355−375. (7) Steinhilber, D.; Hofmann, B. Basic Clin. Pharmacol. Toxicol. 2014, 114, 70−77. (8) Laufer, S. A.; Augustin, J.; Dannhardt, G.; Kiefer, W. J. Med. Chem. 1994, 37, 1894−1897. (9) Koeberle, A.; Zettl, H.; Greiner, C.; Wurglics, M.; SchubertZsilavecz, M.; Werz, O. J. Med. Chem. 2008, 51, 8068−8076. (10) Werz, O. Planta Med. 2007, 73, 1331−1357. (11) Pergola, C.; Gaboriaud-Kolar, N.; Jestädt, N.; König, S.; Kritsanida, M.; Schaible, A. M.; Li, H.; Garscha, U.; Weinigel, C.; Barz, D.; Albring, K. F.; Huber, O.; Skaltsounis, A. L.; Werz, O. J. Med. Chem. 2014, 57, 3715−3723. (12) Koeberle, A.; Rossi, A.; Bauer, J.; Dehm, F.; Verotta, L.; Northoff, H.; Sautebin, L.; Werz, O. Front. Pharmacol. 2011, 2.10.3389/fphar.2011.00007 (13) Albert, D.; Zündorf, I.; Dingermann, T.; Müller, W. E.; Steinhilber, D.; Werz, O. Biochem. Pharmacol. 2002, 64, 1767−1775. (14) Feißt, C.; Franke, L.; Appendino, G.; Werz, O. J. Pharmacol. Exp. Ther. 2005, 315, 389−396. (15) Bauer, J.; Koeberle, A.; Dehm, F.; Pollastro, F.; Appendino, G.; Northoff, H.; Rossi, A.; Sautebin, L.; Werz, O. Biochem. Pharmacol. 2011, 81, 259−268. (16) Koeberle, A.; Pollastro, F.; Northoff, H.; Werz, O. Br. J. Pharmacol. 2009, 156, 952−961. (17) Koeberle, A.; Werz, O. Biochem. Pharmacol. 2015, 98, 1−15. (18) Epifano, F.; Fiorito, S.; Genovese, S. Phytochemistry 2013, 95, 12−18. (19) Kouloura, E.; Halabalaki, M.; Lallemand, M.-C.; Nam, S.; Jove, R.; Litaudon, M.; Awang, K.; Hadi, H. A.; Skaltsounis, A.-L. J. Nat. Prod. 2012, 75, 1270−1276. (20) Wu, T.-S.; Wang, M.-L.; Jong, T.-T.; McPhail, A. T.; McPhail, D. R.; Lee, K.-H. J. Nat. Prod. 1989, 52, 1284−1289. (21) Lesueur, D.; De Rocca Serra, D.; Bighelli, A.; Minh Hoi, T.; Huy Thai, T.; Casanova, J. Nat. Prod. Res. 2008, 22, 393−398. (22) Su, C.-R.; Kuo, P.-C.; Wang, M.-L.; Liou, M.-J.; Damu, A. G.; Wu, T.-S. J. Nat. Prod. 2003, 66, 990−993. 706


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