Synthesis and NLRP3-Inflammasome Inhibitory Activity of the Naturally Occurring Velutone F and of Its Non-Natural Regioisomeric Chalconoids

Plant-derived remedies rich in chalcone-based compounds have been known for centuries in the treatment of specific diseases, and nowadays, the fascinating chalcone framework is considered a useful and, above all, abundant natural chemotype. Velutone F, a new chalconoid from Millettia velutina, exhibits a potent effect as an NLRP3-inflammasome inhibitor; the search for new natural/non-natural lead compounds as NLRP3 inhibitors is a current topical subject in medicinal chemistry. The details of our work toward the synthesis of velutone F and the unknown non-natural regioisomers are herein reported. We used different synthetic strategies both for the construction of the distinctive benzofuran nucleus (BF) and for the key phenylpropenone system (PhP). Importantly, we have disclosed a facile entry to the velutone F via synthetic routes that can also be useful for preparing non-natural analogs, a prerequisite for extensive SAR studies on the new flavonoid class of NLRP3-inhibitors.

oxide synthase [13,14]. Moreover, it has been shown that both synthetic and natural chalconoids play a healthy role in several diseases by inhibiting the NLRP3-inflammasome formation [15][16][17][18][19]. The NLRP3 inflammasome is a large protein complex controlling the production of caspase-1 and ultimately of pro-inflammatory cytokines (IL-1 and IL-18). In this context, it was reported that velutone F (1) (Figure 1), a retrochalcone [20][21][22] recently identified in the ethanolic extract of the leguminous plant Millettia velutina [23,24], inhibits the formation of the NLRP3 active complex. Among the eight new flavonoids identified in the lipophilic crude residue derived from 10 kg of dry vine stems of Millettia velutina, compound 1 exhibited the most potent inhibitory effect against nigericin-induced IL-1 release in THP-1 cells. Velutone F, featuring the 1-phenyl-2-propen-1-one moiety (PhP) and a substituted benzofuran core (BF), can be classified as a hybrid chalcone. The development of hybrid molecules incorporating different pharmacophores, each with its own molecular target, is an important area of research in medicinal chemistry [25]. Actually, both natural and synthetic benzofuran-derived compounds have potential therapeutic interests ranging from antibacterial, antifungal, anti-inflammatory, analgesic, antidepressant, anticonvulsant, anticancer, anti-HIV, antidiabetic, antituberculosis, and antioxidant [26][27][28][29]. We are currently involved in a multidisciplinary study aimed at identifying new antiinflammatory/anticancer compounds that mimic the MCC950 molecular structure ( Figure  1). It has been demonstrated that the diarylsulfonylurea MCC950 powerfully inhibits the NLRP3 activation selectively [30,31]. In detail, MCC950 would seem to reversibly bind the NLRP3 multi-protein complex making it unable to generate the active complex, namely the NLRP3-inflammasome [32,33]. Because of the profoundly different chemical structures of the synthetic MCC950 compared to the one of velutone F (1), it is reasonable to assume that they can block the activation of the NLRP3-inflammasome by interfering with different bio-chemical targets. We were intrigued by the possibility of disposing of multimilligrams amount of the natural substance 1 for the purpose of undertaking pharmacochemical investigations and mostly to shed some light on the mechanism by which compound 1 inhibits the nigericin-induced IL-1 release. Actually, when we started the search, the chemical identity of 1 was ascertained by spectroscopic means exclusively, primarily 2D NMR [23]. However, very recently [24], the same research teams got proof of the chalconoid structure of compound 1 by its semi-synthesis from Khellin.
The reasons mentioned above prompted us to design/develop synthetic routes for preparing both the natural substance originally extracted from the tropical plant Millettia velutina and analogs to be studied by the biologist team as promising anti-inflammatory agents.

Results and Discussion
A host of synthetic strategies has been developed to create the trans-carbon-carbon double bond of the 1,3-diaryl-2-propen-1-one moiety featuring chalcone compounds, We are currently involved in a multidisciplinary study aimed at identifying new anti-inflammatory/anticancer compounds that mimic the MCC950 molecular structure ( Figure 1). It has been demonstrated that the diarylsulfonylurea MCC950 powerfully inhibits the NLRP3 activation selectively [30,31]. In detail, MCC950 would seem to reversibly bind the NLRP3 multi-protein complex making it unable to generate the active complex, namely the NLRP3-inflammasome [32,33]. Because of the profoundly different chemical structures of the synthetic MCC950 compared to the one of velutone F (1), it is reasonable to assume that they can block the activation of the NLRP3-inflammasome by interfering with different bio-chemical targets. We were intrigued by the possibility of disposing of multi-milligrams amount of the natural substance 1 for the purpose of undertaking pharmacochemical investigations and mostly to shed some light on the mechanism by which compound 1 inhibits the nigericin-induced IL-1 release. Actually, when we started the search, the chemical identity of 1 was ascertained by spectroscopic means exclusively, primarily 2D NMR [23]. However, very recently [24], the same research teams got proof of the chalconoid structure of compound 1 by its semi-synthesis from Khellin.
The reasons mentioned above prompted us to design/develop synthetic routes for preparing both the natural substance originally extracted from the tropical plant Millettia velutina and analogs to be studied by the biologist team as promising anti-inflammatory agents.
Initially, we planned a synthetic strategy to compound 1 based on the introduction of the required alkene by Wittig olefination of 5-formyl-4,7-dimethoxy benzofuran 7 (5-FBF) with the 1-phenyl-2-(triphenylphosphoranylidene)ethanone counterpart 8 [35]. To this end we elaborated two synthetic approaches for the preparation of the key intermediate 5-FBF differing in the way the benzofuran core (BF) could be formed via annellation of the carbocyclic ring system onto a preformed furan, synthetic pathway A (Scheme 1), or alternatively, by creating the furan ring by intramolecular cyclization on a preexistent carbocyclic, synthetic pathway B (Scheme 2).
Initially, we planned a synthetic strategy to compound 1 based on the introduction of the required alkene by Wittig olefination of 5-formyl-4,7-dimethoxy benzofuran 7 (5-FBF) with the 1-phenyl-2-(triphenylphosphoranylidene)ethanone counterpart 8 [35]. To this end we elaborated two synthetic approaches for the preparation of the key intermediate 5-FBF differing in the way the benzofuran core (BF) could be formed via annellation of the carbocyclic ring system onto a preformed furan, synthetic pathway A (Scheme 1), or alternatively, by creating the furan ring by intramolecular cyclization on a preexistent carbocyclic, synthetic pathway B (Scheme 2).

Synthetic Pathway A for Target Compound 1
The first route starts by treating the 3-furoic acid with excess LDA to produce the corresponding C-2 lithiated carboxylate lithium salt, which, by reacting with succinic anhydride, gave the dicarboxylic acid 2. The desired acyl derivative 3 could be isolated in a modest yield after esterification of 2 [36,37]. The subsequent carbonyl group protection as dimethyl ketal 4 opened the way to the creation of the annellated six-membered carbocyclic ring. Thus, the Dieckmann cyclization, performed with potassium tert-butoxide at −78 • C, occurred with simultaneous elimination of MeOH from the dimethyl ketal group yielding the aromatic derivative 5, which was taken to the benzofuran derivative 6 by etherification. Subsequently, the methoxycarbonyl group was converted to the required formyl group by a two-step process entailing reduction with LiAlH 4 and oxidation of the resulting primary alcohol with pyridinium chlorochromate (PCC). The resulting 5-FBF was eventually reacted with the stabilized phosphorous ylide 8, in turn, prepared according to the literature [35]. The Wittig olefination under microwave irradiation provided velutone F (1) in 70% yield after chromatographic purification. As expected, NMR spectroscopic data for the synthesized velutone F were superimposable to the ones originally reported for the retrochalcone isolated from Millettia velutina.

Synthetic Pathway A for Target Compound 1
The first route starts by treating the 3-furoic acid with excess LDA to produce the corresponding C-2 lithiated carboxylate lithium salt, which, by reacting with succinic anhydride, gave the dicarboxylic acid 2. The desired acyl derivative 3 could be isolated in a modest yield after esterification of 2 [36,37]. The subsequent carbonyl group protection as dimethyl ketal 4 opened the way to the creation of the annellated six-membered carbocyclic ring. Thus, the Dieckmann cyclization, performed with potassium tert-butoxide at −78 °C, occurred with simultaneous elimination of MeOH from the dimethyl ketal group yielding the aromatic derivative 5, which was taken to the benzofuran derivative 6 by etherification. Subsequently, the methoxycarbonyl group was converted to the required formyl group by a two-step process entailing reduction with LiAlH4 and oxidation of the resulting primary alcohol with pyridinium chlorochromate (PCC). The resulting 5-FBF was eventually reacted with the stabilized phosphorous ylide 8, in turn, prepared according to the literature [35]. The Wittig olefination under microwave irradiation provided velutone F (1) in 70% yield after chromatographic purification. As expected, NMR spectroscopic data for the synthesized velutone F were superimposable to the ones originally reported for the retrochalcone isolated from Millettia velutina.

Synthetic Pathway B for Target Compound 1
The starting move of the alternative synthetic approach to 7 entailing the creation of the furan ring by intramolecular cyclization of the bromobenzene derivative 11 (Scheme 2) was the Dakin-like oxidation of the cheap 2,5-dimethoxybenzaldehyde by using the H 2 O 2 /cat. SeO 2 system in tert-BuOH [38]. Methanolysis of the resulting arylformate promptly furnished the 2,5-dimethoxyphenol 9, which underwent regioselective bromination with NBS affording 10. The exclusive C-4 bromination accounted for the marked para orienting effect of the phenolic hydroxyl group [39].
At this stage, with the aim of creating the annellated 2,3-unsubstituted furan ring, we needed to introduce an O-tethered functionalized two carbon fragment. Thus, compound 10 was easily converted to the aryl ether 11 by treatment with bromoacetaldehyde dimethyl acetal and KOH in dimethylacetamide (DMA) [40]. The anticipated intramolecular electrophilic aromatic substitution was carried out with polyphosphoric acid (PPA), providing the benzofuran derivative 12 in a 50% yield. Transformation of the 5-bromo benzofuran derivative 12 into 5-formyl-4,7-dimethoxy benzofuran 7 (5-FBF) was achieved through halogen-metal exchange with BuLi followed by reaction with DMF [41].
In order to make the critical furan ring annellation step more efficient, we turned our attention to a recently reported protocol for the synthesis of 6-hydroxybenzofuran based on an unusual cycloaddition-cycloreversion sequence [42]. To this end, we prepared the penta-substituted benzene derivative 16 starting from the tri-substituted phenol derivative 9 (Scheme 2), exploiting the well-known ability of the OTHP as an ortho-directing group for the metalation [43]. Accordingly, compound 9 was promptly transformed into the corresponding tetrahydropyranyl ether 14 that was at first ortho-lithiated by treatment with BuLi and later reacted with DMF to give the tetra-substituted phenyl derivative 15. The subsequent bromination para to the phenol group [44] afforded the desired pentasubstituted benzene 16, to which the O-ethoxycarbonylmethylene fragment was easily inserted by standard etherification reaction. In previous saponification, compound 17 was treated with the Ac 2 O-AcONa system with heating to give the required 5-bromo benzofuran derivative 12 in an appreciable 57% yield. The mechanism proposed for the interesting cyclization reaction entails dehydration of the carboxyl group to give an unstable ketene intermediate that is trapped intramolecularly by the formyl group. The thermal [2 + 2] heterocycloaddition reaction is followed by a cycloreversion with the expulsion of CO 2 and production of the 2,3-unsubstituted benzofuran derivative 12 [42].

Synthetic Pathways Providing the Non-Natural Regioisomers of Velutone F
With the aim of learning about stereo-electronic properties of the hybrid benzofuranretrochalcone scaffold, we decided to prepare the non-natural compounds 22, 23, and 28 featuring the PhP moiety attached, respectively, at C-2, C-6, and C-3 of the 4,7-dimethoxy benzofuran core (BF). The non-natural regioisomers of velutone F are previously unknown compounds.

Synthetic Pathways to the Isomers 22 and 23
The direct formylation of electron-rich arenes can be conveniently accomplished via the Vilsmeier-Haack (V-H) reaction. Indeed, the benzo[b]furan nucleus is reported to yield the 2-formyl derivative by reaction with the V-H electrophilic species [47]. We anticipated that the regioselectivity of the V-H reaction could change if electron-donating groups were present on the phenyl ring of the benzo-fused system. In line with our hypothesis, we decided exploring the behavior of 4,7-dimethoxy benzo[b]furan 19 under V-H reaction conditions (Scheme 3). We planned to build the substituted benzo[b]furan 19 from 9 by creating the annellated 2,3-unsubstituted furan ring according to our previously sound synthetic pathway B for target compound 1 (Scheme 2). Thus, once etherified the phenolic group of 9 with the functionalized two carbon fragment, the resulting compound 18 was cyclized to 19 under the action of PPA (Sn-β zeolite also showed to efficiently promote this transformation [48]). As expected, we found the subsequent electrophilic aromatic substitution reaction was poorly regioselective: all but one of the regioisomeric formyl benzofuran derivatives 2-FBF, 5-FBF, and 6-FBF were formed. In detail, chromatographic purification of the residue from the V-H reaction led us to isolate compounds 20 (2-FBF) together with 7 (5-FBF) in 37% yield ( 1 H NMR and HPLC analysis showed the isomers were in a 3.5:6.5 ratio), and compound 21 (6-FBF) in 30% yield. At this stage, we submitted the separated fractions to the Wittig olefination with the stabilized phosphorous ylide 8. We obtained chalcone 23 from 6-FBF, while in the same manner, the inseparable mixture of 2-FBF and 5-FBF furnished chalcones 22 and 1, which, gratifyingly, could be easily separated by column chromatography.
tographic purification of the residue from the V-H reaction led us to isolate compounds 20 (2-FBF) together with 7 (5-FBF) in 37% yield ( 1 H NMR and HPLC analysis showed the isomers were in a 3.5:6.5 ratio), and compound 21 (6-FBF) in 30% yield. At this stage, we submitted the separated fractions to the Wittig olefination with the stabilized phosphorous ylide 8. We obtained chalcone 23 from 6-FBF, while in the same manner, the inseparable mixture of 2-FBF and 5-FBF furnished chalcones 22 and 1, which, gratifyingly, could be easily separated by column chromatography.

Synthetic Pathway to the Isomer 28
We envisaged setting up the PhP moiety of 28 by reaction of 3-formyl benzofuran derivative 27 (3-FBF) with the ylide 8 according to the previously tested protocol entailing a classical Wittig olefination. About 3-FBF preparation, we selected compound 25 as the direct precursor having in mind a controlled oxidation of its C-3 methyl substituent in order to derive the pivotal electrophilic functional group [49,50] (Scheme 4). Thus, we devised preparing compound 25 in two steps from phenol 9, namely: etherification with bromoacetone followed by acid-promoted cyclization of the resulting aryloxy acetone derivative 24. The planned SeO2-oxidation of the C-3 methyl residue of compound 25 furnished a 1:1 mixture of the primary alcohol 26 and the aldehyde 27, which were separable by chromatography. However, we found it very easy to carry out the oxidation of 26 to 3-FBF by using the Corey-Suggs reagent (PCC). Eventually, the microwave-promoted

Synthetic Pathway to the Isomer 28
We envisaged setting up the PhP moiety of 28 by reaction of 3-formyl benzofuran derivative 27 (3-FBF) with the ylide 8 according to the previously tested protocol entailing a classical Wittig olefination. About 3-FBF preparation, we selected compound 25 as the direct precursor having in mind a controlled oxidation of its C-3 methyl substituent in order to derive the pivotal electrophilic functional group [49,50] (Scheme 4). Thus, we devised preparing compound 25 in two steps from phenol 9, namely: etherification with bromoacetone followed by acid-promoted cyclization of the resulting aryloxy acetone derivative 24. The planned SeO 2 -oxidation of the C-3 methyl residue of compound 25 furnished a 1:1 mixture of the primary alcohol 26 and the aldehyde 27, which were separable by chromatography. However, we found it very easy to carry out the oxidation of 26 to 3-FBF by using the Corey-Suggs reagent (PCC). Eventually, the microwave-promoted Wittig reaction between the aryl aldehyde 27 (3-FBF) and the phosphorous ylide 8 provided the aimed chalconoid 28 in good yield.

Inhibition of IL-1β Release In Vitro and In Vivo
Among the inflammasomes, NLRP3 is the most studied and characterized due to its implication in the pathogenesis of different human diseases [51]. The activation of NLRP3 inflammasomes consists of caspase-1 activation, which in turn induces secretion of the inflammatory cytokine IL-1β. Hence, IL-1β release is the most used read-out for NLRP3 inflammasome activation. In this study, IL-1β release was determined by ELISA assay to assess the inhibitory effects of synthesized compounds on NLRP3 inflammasome activation both in mouse bone marrow-derived macrophages (BMDMs) and in human PMA-differentiated lipopolysaccharide (LPS)-primed THP-1 macrophages (Figure 2). Velutone F (1) and the non-natural compounds 22, 23, and 28 demonstrated a high inhibitory capacity on the release of IL-1β.

Inhibition of IL-1 Release In Vitro and In Vivo
Among the inflammasomes, NLRP3 is the most studied and characterized due to its implication in the pathogenesis of different human diseases [51]. The activation of NLRP3 inflammasomes consists of caspase-1 activation, which in turn induces secretion of the inflammatory cytokine IL-1β. Hence, IL-1β release is the most used read-out for NLRP3 inflammasome activation. In this study, IL-1β release was determined by ELISA assay to assess the inhibitory effects of synthesized compounds on NLRP3 inflammasome activation both in mouse bone marrow-derived macrophages (BMDMs) and in human PMAdifferentiated lipopolysaccharide (LPS)-primed THP-1 macrophages (Figure 2). Velutone F (1) and the non-natural compounds 22, 23, and 28 demonstrated a high inhibitory capacity on the release of IL-1β.  To confirm the anti-inflammatory activity of these compounds in vivo, we used an LPS-induced inflammation treatment. Mice were intraperitoneally (IP) pre-injected with velutone F (1) and the non-natural isomers 22, 23, and 28 or vehicle at 25 mg/kg and then were IP injected with LPS (1 mg/kg). After 4 h, plasma and peritoneal exudate were collected from mice and analyzed for evaluation of IL-1 release. The mice that received pretreatment with our compounds displayed a dramatic reduction in IL-1 production both in peritoneal exudate and blood samples (Figure 3). To confirm the anti-inflammatory activity of these compounds in vivo, we used an LPS-induced inflammation treatment. Mice were intraperitoneally (IP) pre-injected with velutone F (1) and the non-natural isomers 22, 23, and 28 or vehicle at 25 mg/kg and then were IP injected with LPS (1 mg/kg). After 4 h, plasma and peritoneal exudate were collected from mice and analyzed for evaluation of IL-1β release. The mice that received pre-treatment with our compounds displayed a dramatic reduction in IL-1 production both in peritoneal exudate and blood samples (Figure 3). natants were analyzed with ELISA test for the evaluation of IL-1 release (n = 3, mean ± SEM). Statistics differences were analyzed using one-way ANOVA: ** p < 0.01, * p < 0.05.
To confirm the anti-inflammatory activity of these compounds in vivo, we used an LPS-induced inflammation treatment. Mice were intraperitoneally (IP) pre-injected with velutone F (1) and the non-natural isomers 22, 23, and 28 or vehicle at 25 mg/kg and then were IP injected with LPS (1 mg/kg). After 4 h, plasma and peritoneal exudate were collected from mice and analyzed for evaluation of IL-1 release. The mice that received pretreatment with our compounds displayed a dramatic reduction in IL-1 production both in peritoneal exudate and blood samples (Figure 3).  Taken together, these results revealed that synthesized velutone F (1), as well as its regioisomers 22, 23, and 28, exerted a strong inhibition on the NLRP3 inflammasome activation both in vitro and in vivo.

Chemistry: Materials and General
All reagents and solvents that were commercially purchased were directly used without prior treatment. Reaction temperatures were recorded using a regular thermometer without correction. Melting points were determined on the Reichert Termovar apparatus and are uncorrected. Reactions were monitored by analytical thin-layer chromatography

Methyl 3-(4-Methoxy-4-oxobutanoyl)furan-2-carboxylate (3)
A solution of 3-Furoic acid (1 g, 8.92 mmol, 1 equiv.) in anhydrous THF (6 mL) was added dropwise, under argon atmosphere, to a cooled (−78 • C) solution of LDA, which was previously prepared from n-BuLi (12.5 mL, 1.6 M in hexane, 19.96 mmol, 2.2 equiv.) and i-Pr 2 NH (2.9 mL, 19.96 mmol, 2.2 equiv.) in THF (25 mL). The reaction mixture was stirred at −78 • C for 2 h, then a solution of succinic anhydride (1 g, 9.99 mmol, 1.1 equiv.) in anhydrous THF (8 mL) was added to the reaction flask. The cooling bath was removed, and aqueous 2 M HCl (30 mL) was added at room temperature. The mixture was extracted with chloroform, the organic phase was dried over Na 2 SO 4, and solvent was evaporated. The solid residue was dissolved in MeOH (50 mL), cooled at 0 • C, and H 2 SO 4 (0.45 mL, 95%) was added dropwise. The mixture was stirred at room temperature for 24 h, then a saturated NaHCO 3 solution was added, and the mixture was extracted with AcOEt. The organic phase, dried over Na 2 SO 4 , was evaporated under reduced pressure to give a crude that was purified by silica gel column chromatography with A/P 1:9, v/v as a solvent system. Title compound (3) was isolated as a colorless oil. Yield 32% over two steps. 1

Methyl 4-Hydroxy-7-methoxybenzofuran-5-carboxylate (5)
tert-BuOK (0.61 g, 5.45 mmol, 2.2 equiv.) was poured into anhydrous THF (65 mL) under an argon atmosphere, and the suspension was cooled to −78 • C. A solution of compound 4 (0.71 g, 2.48 mmol, 1 equiv.) in anhydrous THF (2 mL) was added dropwise, and the mixture turned orange. After 1.5 h of stirring at −78 • C, anhydrous HCl (4 M in 1,4-dioxane) was added. The resulting transparent yellow reaction mixture was allowed to room temperature and stirred for 1 h. The precipitate was filtered through a Gooch filter, and the solvent was evaporated under reduced pressure. Chromatographic purification of the residue with A/P 1:9 v/v as the eluent phase provided title compound 5 as a white solid. Melting point: 125-127 • C. Yield 62%. 1  A mixture of compound 5 (0.21 g, 0.95 mmol, 1 equiv.), Cs 2 CO 3 (1.55 g, 5.75 mmol, 5 equiv.), and CH 3 I (0.29 mL, 4.75 mmol, 5 equiv.) in anhydrous DMF (6 mL) was stirred at room temperature for 16 h under an argon atmosphere. After complete conversion of starting material (checked by TLC A/P9 1:9), the reaction was partitioned between HCl 2 M and AcOEt; the organic phase was washed with HCl 2 M (×2) and brine, dried over Na 2 SO 4, and the solvent was evaporated to dryness. Purification of the crude by column chromatography with A/P 1:4 as the eluent phase afforded title compound 6 as a colorless oil. Yield 96%. 1 (7) A solution of compound 6 (0.2 g, 0.84 mmol, 1 equiv.) in dry Et 2 O (4 mL) was added dropwise to a stirred to a cooled (0 • C) suspension of LiAlH 4 (0.08 g, 2.1 mmol, 2.5 equiv.) in anhydrous Et 2 O (5 mL) under argon atmosphere. The reaction mixture was stirred at room temperature overnight, then it was cooled to 0 • C, and aqueous NaOH solution (5 mL, 2 M) was added. After 30 min, the mixture was filtered on a celite pad, and the phases were separated. The aqueous phase was extracted with Et 2 O. The combined organics were dried over Na 2 SO 4 , and the solvent was evaporated to give (4,7-dimethoxybenzofuran-5yl)methanol as a white crystalline solid, which was used in the next oxidative step without further purification.

Cell Cultures and Stimulation
Bone marrow-derived macrophages (BMDMs) were isolated from C57BL/6 mice as described [52] and differentiated for 7 days in Iscove s Modified Dulbecco s Medium supplemented with 15% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S) and 10 ng/mL M-CSF. THP-1 cells were grown in RPMI medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. THP-1 cells were stimulated by 100 ng/mL PMA overnight to differentiate into macrophages. All cells were grown in a 5% CO 2 incubator at 37 • C. BMDMs were seeded at 5 × 10 5 in 24 well plates. After 12 h, the medium was removed, and cells were treated with LPS from Escherichia coli 055:B5 (1 µg/mL) in fresh Iscove s Modified Dulbecco s Medium for 2 h. After that, the medium was removed and replaced with a serum-free medium containing DMSO or compounds (10 µM) for 30 min. Cells were then stimulated with Nigericin (10 µM) for 1 h. Human THP-1 cells were seeded at 3 × 10 5 cells per well in 24 well plates. The following day, the overnight medium was replaced, and cells were stimulated with LPS (1 µg/mL) for 3 h. The medium was removed and replaced with a serum-free medium containing DMSO or compounds (10 µM) for 30 min. Cells were then stimulated with Nigericin (10 µM) for 1 h.
3.1.30. In Vivo LPS Challenge C57BL/6 mice were IP injected with compounds (25 mg/kg) or vehicle control (DMSO) 30 min before IP injection of LPS 1 mg/kg (4 h) and then were euthanized, and blood and peritoneal exudate were isolated. Mouse plasma was collected after blood centrifugation (1000× g, 15 min at 4 • C). ELISA for IL-1β was performed according to the manufacturer's instructions (R&D Systems).

ELISA
Supernatants from BMDMs and THP-1 cell culture were assayed for mouse or human IL-1β, respectively, by ELISA according to the manufacturer's instructions (R&D Systems).

Conclusions
We succeeded in synthesizing the bioactive compound velutone F (1), the chalconoid contained in Millettia velutina stem, together with other 21 flavonoids. Our chemical total synthesis of velutone F is advantageous compare to the recently reported [24] seven steps semi-synthesis requiring the naturally occurring furanochromone derivative Khellin as the costly starting material (25 G > 800 USD). We could develop synthetic routes A and B; the first one establishes the benzofuran nucleus by creating the annellated carbocyclic ring onto a furan ring, while the second one proceeds exactly the other way. Instead, the Wittig olefination and the Heck coupling reaction are the featuring steps allowing for the assemblage of the 1,3-diaryl enone scaffold. Both the multi-step synthetic routes A and B establish the enone moiety at position C-5 of the 4,7-dimethoxybenzofuran thus, slightly modified synthetic approaches were designed in order to achieve the non-natural chalconoids 22, 23, and 28, which formally are the C-2, C-3, and C-6 regioisomers of velutone F (1). The anti-inflammatory effects of the newly synthesized compounds are also reported.