Total Synthesis and Anti-Inflammatory Evaluation of Osajin, Scandenone and Analogues

In this study, the total synthesis of osajin, scandenone and their analogues have been accomplished. The key synthetic steps include aldol/intramolecular iodoetherification/elimination sequence reactions and a Suzuki coupling reaction to assemble the tricyclic core, chemoselective propargylation and Claisen rearrangement reactions to obtain natural compounds. In addition, we also designed and synthesized twenty-five natural product analogues. All synthetic compounds were screened for anti-inflammatory activity against tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Collectively, Compound 39e and 39d were considered as promising lead compounds for further development.


Introduction
Flavonoids are naturally occurring secondary metabolites predominantly originating from fruits, herbs, fungi and vegetables which are characterized by a 2-phenyl-4H-chromene structure [1,2].Flavonoids, a class of natural polyphenolics, are classified into flavanols, flavanones, flavones, isoflavones, flavonols and anthocyanidins [1].Modern pharmacological evaluations and animal studies have demonstrated their anticancer, anti-inflammatory, antioxidant, antimicrobial and antiviral activities [3][4][5][6].In the last decades, various synthetic and natural flavonoid derivatives have been actively investigated as drugs used in treating human disease [7,8].Despite the fact that a large number of flavonoids have been isolated and identified, there is still need for further exploitation of bioactive lead compounds with novel structures.
Osajin and scandenone are natural flavonoid compounds that exist in various plants and Chinese herbal medicines such as Derris scandens, Flemingia philippinensis and Millettia pulchra (Figure 1) [9][10][11].These compounds were elucidated based on detailed analysis of NMR and HRMS data [12].Previous studies have shown that these natural products impede the growth of various cancerous cells and possess anti-inflammatory activities [13,14].Due to their interesting biological activities, further research and development are necessary for the discovery of lead compounds.Herein, the total synthesis of osajin and scandenone is described.In addition, we synthesized and characterized twenty-five natural product analogues through different synthetic routes.All the newly synthesized compounds were tested for their anti-inflammatory activities against tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.
The key intermediate 10 was prepared via coupling of compounds 6 and 7 based on the aldol reaction, which may suffer from poor functional group tolerance to exposed hydroxyl groups.For this reason, the free hydroxyl group of substance 6 was protected using a methyl group to give 9 with satisfactory yield, and the methyl-protected compound 9 was reacted with 7 to afford 10 in 88% yield.Compound 10 was further reacted to obtain 11 via addition reaction and we detected the low yield (from 20% to 36%) by trying different solvents and temperatures at the beginning.We introduced one equivalent pyridine into the reaction system, which improved the yield to 78%.The coupling of compounds 11 and 8 resulted in compound 12 via the Suzuki coupling reaction, which was deeply explored under different reaction systems.We were only afforded compound 12 with a 40% yield under the conditions of Pd(OAc)2/MeOH/Na2CO3 (Table 1, entry 1) [17].The yield of the reaction has not been significantly improved by changing the solvent and base (entries 2 and 3).The yield was increased up to 60% when the temperature of the reaction system reached 50 °C (entry 4).Following this, we selected Pd(PPh3)4 as catalyst for further investigation [18].After the systematic research of the reaction conditions, we found that increasing the temperature of this reaction system obviously improved the yield of 12 (entries 5-7).When the temperature was further increased, the yield was not remarkably improved (entries 7 and 8).The optimal condition was obtained by using PdCl2(dppf) as a catalyst and 1,4-Dioxane/H2O as a solvent at 50 °C (entry 9) [19].The coupled product 12 proceeded smoothly in the presence of a 40% aqueous solution of HBr in refluxing water to provide the tricyclic core 3. Chemoselective propargylation of the C7-hydroxyl of compound 3 provided 13 under the conditions of KI/K2CO3/CuI/ in 72% yield, which suffered an aromatic Claisen rearrangement, resulting in cyclization compound 14 (52% yield) and 15 (41% yield) under an elevated temperature condition of 250 °C (Scheme 2) [20].

Results and Discussion
The isolated compounds are characterized by a C6-C3-C6 skeleton structure with a 2″,2″-dimethylpyran ring and a linear side chain.The retrosynthetic analysis of target molecules is depicted in Scheme 1. Osajin and scandenone were envisaged to be obtained via chemoselective propargylation, intramolecular cyclization and Claisen rearrangement sequence reactions of compound 3 [15].The key intermediate 3 could be derived from the materials 1-(2,4,6-trihydroxyphenyl)ethan-1-one ( 6), 1,1-dimethoxy-N,N-dimethylmethanamine ( 7) and (4-hydroxyphenyl)boronic acid (8) through aldol/intramolecular iodoetherification/elimination/Suzuki coupling sequential chemical reactions (Scheme 1) [16].The key intermediate 10 was prepared via coupling of compounds 6 and 7 based on the aldol reaction, which may suffer from poor functional group tolerance to exposed hydroxyl groups.For this reason, the free hydroxyl group of substance 6 was protected using a methyl group to give 9 with satisfactory yield, and the methyl-protected compound 9 was reacted with 7 to afford 10 in 88% yield.Compound 10 was further reacted to obtain 11 via addition reaction and we detected the low yield (from 20% to 36%) by trying different solvents and temperatures at the beginning.We introduced one equivalent pyridine into the reaction system, which improved the yield to 78%.The coupling of compounds 11 and 8 resulted in compound 12 via the Suzuki coupling reaction, which was deeply explored under different reaction systems.We were only afforded compound 12 with a 40% yield under the conditions of Pd(OAc)2/MeOH/Na2CO3 (Table 1, entry 1) [17].The yield of the reaction has not been significantly improved by changing the solvent and base (entries 2 and 3).The yield was increased up to 60% when the temperature of the reaction system reached 50 °C (entry 4).Following this, we selected Pd(PPh3)4 as catalyst for further investigation [18].After the systematic research of the reaction conditions, we found that increasing the temperature of this reaction system obviously improved the yield of 12 (entries 5-7).When the temperature was further increased, the yield was not remarkably improved (entries 7 and 8).The optimal condition was obtained by using PdCl2(dppf) as a catalyst and 1,4-Dioxane/H2O as a solvent at 50 °C (entry 9) [19].The coupled product 12 proceeded smoothly in the presence of a 40% aqueous solution of HBr in refluxing water to provide the tricyclic core 3. Chemoselective propargylation of the C7-hydroxyl of compound 3 provided 13 under the conditions of KI/K2CO3/CuI/ in 72% yield, which suffered an aromatic Claisen rearrangement, resulting in cyclization compound 14 (52% yield) and 15 (41% yield) under an elevated temperature condition of 250 °C (Scheme 2) [20].The key intermediate 10 was prepared via coupling of compounds 6 and 7 based on the aldol reaction, which may suffer from poor functional group tolerance to exposed hydroxyl groups.For this reason, the free hydroxyl group of substance 6 was protected using a methyl group to give 9 with satisfactory yield, and the methyl-protected compound 9 was reacted with 7 to afford 10 in 88% yield.Compound 10 was further reacted to obtain 11 via addition reaction and we detected the low yield (from 20% to 36%) by trying different solvents and temperatures at the beginning.We introduced one equivalent pyridine into the reaction system, which improved the yield to 78%.The coupling of compounds 11 and 8 resulted in compound 12 via the Suzuki coupling reaction, which was deeply explored under different reaction systems.We were only afforded compound 12 with a 40% yield under the conditions of Pd(OAc) 2 /MeOH/Na 2 CO 3 (Table 1, entry 1) [17].The yield of the reaction has not been significantly improved by changing the solvent and base (entries 2 and 3).The yield was increased up to 60% when the temperature of the reaction system reached 50 • C (entry 4).Following this, we selected Pd(PPh 3 ) 4 as catalyst for further investigation [18].After the systematic research of the reaction conditions, we found that increasing the temperature of this reaction system obviously improved the yield of 12 (entries 5-7).When the temperature was further increased, the yield was not remarkably improved (entries 7 and 8).The optimal condition was obtained by using PdCl 2 (dppf) as a catalyst and 1,4-Dioxane/H 2 O as a solvent at 50 • C (entry 9) [19].The coupled product 12 proceeded smoothly in the presence of a 40% aqueous solution of HBr in refluxing water to provide the tricyclic core 3. Chemoselective propargylation of the C7-hydroxyl of compound 3 provided 13 under the conditions of KI/K 2 CO 3 /CuI/ in 72% yield, which suffered an aromatic Claisen rearrangement, resulting in cyclization compound 14 (52% yield) and 15 (41% yield) under an elevated temperature condition of 250 • C (Scheme 2) [20].Having prepared the critical intermediate 14 successfully, we sought to establish skeletons of natural products.The C4′-hydroxy group of intermediate 14 was protected using a tert-butyldimethylsilyl (TBS) group to give 16, which was further reacted to obtain 17 via a nucleophilic substitution reaction (64% yield in two steps).Compound 17 was subjected to Claisen rearrangement reaction to obtain 18 in 65% yield (along with 25% isopentenyl exfoliation product 16 as a byproduct).Deprotection of the TBS group of 18 was smoothly accomplished upon treatment with a large excess of tetrabutylammonium fluoride (TBAF) in THF to afford 2 in 90% yield at room temperature.Based on the above, we successfully synthesized the natural product osajin (2) (5.82% overall yield).NMR spectroscopic data of the synthetic osajin (2) were well matched with those reported in the Having prepared the critical intermediate 14 successfully, we sought to establish skeletons of natural products.The C4 ′ -hydroxy group of intermediate 14 was protected using a tert-butyldimethylsilyl (TBS) group to give 16, which was further reacted to obtain 17 via a nucleophilic substitution reaction (64% yield in two steps).Compound 17 was subjected to Claisen rearrangement reaction to obtain 18 in 65% yield (along with 25% isopentenyl exfoliation product 16 as a byproduct).Deprotection of the TBS group of 18 was smoothly accomplished upon treatment with a large excess of tetrabutylammonium fluoride (TBAF) in THF to afford 2 in 90% yield at room temperature.Based on the above, we successfully synthesized the natural product osajin (2) (5.82% overall yield).NMR spectroscopic data of the synthetic osajin (2) were well matched with those reported in the literature (Supplementary Table S2) [12].Flavonoids containing the hydroxyisoprenyl structure usually exhibit multiple biological activities.The Schenck ene reaction of the natural product 2 with the photosensitizer yielded secondary allylic alcohol 22.Beyond that, we synthesized natural product analogues 19-21 through etherification, esterification and hydrolysis reactions (Scheme 3).The TBS-protected compound 23 was prenylated with 3,3-dimethylallyl bromide 5 to afford 24 (74% yield), which was further reacted in the presence of Eu(fod) 3 to obtain the desired rearrangement product 25 (76% yield).Subsequent deprotection of the 4 ′ -OH of 25 with TBAF afforded the natural products 1 in 93% yield (7.07% overall yield).NMR spectroscopic data of the synthetic scandenone (1) were well matched with those reported in the literature (Supplementary Table S1) [12].In previous synthesis, the conversion of compound 3 to 41 is accompanied by the production of byproduct 41 and 42 (Supplementary Scheme S1), which limited the yield of this reaction [21].We synthesized natural compounds through a series of reactions and the new synthesis methods provide approaches for the structural modification of flavonoids.In the same way, we synthesized a new series of analogues 26-30 via acetylation or methylation of the free hydroxy groups and the Schenck ene reaction (Scheme 4) [22].
same way, we synthesized a new series of analogues 26-30 via acetylation or methylation of the free hydroxy groups and the Schenck ene reaction (Scheme 4) [22].In previous work, we explored the new synthetic routes for the structural modification of flavonoids.Herein, we synthesized and characterized a series of natural product analogues and tested their anti-inflammatory activity.Compound 31 was converted to 33 in two steps via a nucleophilic substitution reaction with allyl bromide followed by Claisen rearrangement at a high temperature (65% yield in two steps).Compound 33 was treated with available reagents under alkene metathesis reaction conditions, leading to 34a-34g (yield ranging from 85% to 92%) (Scheme 5A).Compound 35 was subjected to Claisen rearrangement/methylation/aldol sequence reactions to establish the chalcone In previous work, we explored the new synthetic routes for the structural modification of flavonoids.Herein, we synthesized and characterized a series of natural product analogues and tested their anti-inflammatory activity.Compound 31 was converted to 33 in two steps via a nucleophilic substitution reaction with allyl bromide followed by Claisen rearrangement at a high temperature (65% yield in two steps).Compound 33 was treated with available reagents under alkene metathesis reaction conditions, leading to 34a-34g (yield ranging from 85% to 92%) (Scheme 5A).Compound 35 was subjected to Claisen rearrangement/methylation/aldol sequence reactions to establish the chalcone In previous work, we explored the new synthetic routes for the structural modification of flavonoids.Herein, we synthesized and characterized a series of natural product analogues and tested their anti-inflammatory activity.Compound 31 was converted to 33 in two steps via a nucleophilic substitution reaction with allyl bromide followed by Claisen rearrangement at a high temperature (65% yield in two steps).Compound 33 was treated with available reagents under alkene metathesis reaction conditions, leading to 34a-34g (yield ranging from 85% to 92%) (Scheme 5A).Compound 35 was subjected to Claisen rearrangement/methylation/aldol sequence reactions to establish the chalcone skeleton 38a-38f [23].We introduced different substituents on the benzene ring of compound 38 to increase the structural diversity.Subsequently, we synthesized a range of analogues 39a-3 that possess a hydroxyisoprenyl structure based on the Schenck ene reaction (overall yield ranging from 29% to 47%) (Scheme 5B) [22].
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 5 of 12 skeleton 38a-38f [23].We introduced different substituents on the benzene ring of compound 38 to increase the structural diversity.Subsequently, we synthesized a range of analogues 39a-3 that possess a hydroxyisoprenyl structure based on the Schenck ene reaction (overall yield ranging from 29% to 47%) (Scheme 5B) [22].We attempted to evaluate the anti-inflammatory effects of the synthetic compounds by measuring the levels of TNF-α and IL-6 in LPS-stimulated RAW264.7 macrophages with dexamethasone as a reference control [24].Most of the compounds suppressed various degrees of cytokine liberation contrasted with the LPS control.The activity of the compounds was improved by introducing different side chains (Scheme 5A, 34a-34f).Subsequently, we constructed the structure of chalcone and removed the 2″,2″-dimethylpyran ring.Compounds 38a-38f yielded secondary allylic alcohol 39a-39f with the photosensitizer via the Schenck ene reaction.Compounds containing a chalcone skeleton and the hydroxyisoprenyl side chain exhibited remarkable anti-inflammatory activity, and compounds 39e and 39d exhibited the highest inhibitory activity among them, respectively (Figure 2a,b).Meanwhile, we verified the excellent anti-inflammatory activity in vivo with a biological evaluation of mice.Compounds 39d and 39e presented much stronger inhibitory effects against the LPS-induced inflammatory response compared to the reference control dexamethasone (Figure 3).These compounds were considered as promising lead compounds for further development to discover new therapeutic agents with anti-inflammatory properties.We attempted to evaluate the anti-inflammatory effects of the synthetic compounds by measuring the levels of TNF-α and IL-6 in LPS-stimulated RAW264.7 macrophages with dexamethasone as a reference control [24].Most of the compounds suppressed various degrees of cytokine liberation contrasted with the LPS control.The activity of the compounds was improved by introducing different side chains (Scheme 5A, 34a-34f).Subsequently, we constructed the structure of chalcone and removed the 2 ′′ ,2 ′′ -dimethylpyran ring.Compounds 38a-38f yielded secondary allylic alcohol 39a-39f with the photosensitizer via the Schenck ene reaction.Compounds containing a chalcone skeleton and the hydroxyisoprenyl side chain exhibited remarkable anti-inflammatory activity, and compounds 39e and 39d exhibited the highest inhibitory activity among them, respectively (Figure 2a,b).Meanwhile, we verified the excellent anti-inflammatory activity in vivo with a biological evaluation of mice.Compounds 39d and 39e presented much stronger inhibitory effects against the LPS-induced inflammatory response compared to the reference control dexamethasone (Figure 3).These compounds were considered as promising lead compounds for further development to discover new therapeutic agents with anti-inflammatory properties.

General Information
All reactions were carried out under an inert nitrogen atmosphere with dry solvents under anhydrous conditions unless otherwise stated.Flash chromatography was performed using silica gel (200-400 mesh).Thin layer chromatography (TLC) was performed using Silica gel 60 F254 plates and visualized using UV light.

Materials
All commercially available chemicals and solvents were used as received without further purification unless otherwise stated.

Procedure for the Synthesis of Osajin and Scandenone
To a stirred solution of 1-(2,4,6-trihydroxyphenyl)ethan-1-one (5.00 g, 29.73 mmol) and K2CO3 (9.04 g, 65.44 mmol) in anhydrous acetone (80 mL), dimethyl sulfate (5.78 mL, 60.95 mmol) was slowly added at 60 °C for 4 h.After cooling down to room temperature, the reaction mixture was filtered and washed with acetone.The filtrate was extracted with

General Information
All reactions were carried out under an inert nitrogen atmosphere with dry solvents under anhydrous conditions unless otherwise stated.Flash chromatography was performed using silica gel (200-400 mesh).Thin layer chromatography (TLC) was performed using Silica gel 60 F254 plates and visualized using UV light.

Materials
All commercially available chemicals and solvents were used as received without further purification unless otherwise stated.

Procedure for the Synthesis of Osajin and Scandenone
To a stirred solution of 1-(2,4,6-trihydroxyphenyl)ethan-1-one (5.00 g, 29.73 mmol) and K2CO3 (9.04 g, 65.44 mmol) in anhydrous acetone (80 mL), dimethyl sulfate (5.78 mL, 60.95 mmol) was slowly added at 60 °C for 4 h.After cooling down to room temperature, the reaction mixture was filtered and washed with acetone.The filtrate was extracted with

General Information
All reactions were carried out under an inert nitrogen atmosphere with dry solvents under anhydrous conditions unless otherwise stated.Flash chromatography was performed using silica gel (200-400 mesh).Thin layer chromatography (TLC) was performed using Silica gel 60 F254 plates and visualized using UV light.

Materials
All commercially available chemicals and solvents were used as received without further purification unless otherwise stated.

Conclusions
In conclusion, we have accomplished the total synthesis of osajin, scandenone and analogues from commercially available starting materials.The key reactions for the preparation of these compounds involve the aldol reaction, Claisen rearrangement, Schenck ene reaction and Suzuki coupling reaction.Additionally, we have designed and synthesized twenty-five natural product analogues, which were screened for anti-inflammatory activity against TNF-α and IL-6 in LPS-stimulated RAW264.7 macrophages.From this series of compounds, compounds 39e and 39d emerged as promising lead candidates for the development of novel anti-inflammatory drugs.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph17010086/s1,Table S1: Comparison of 1 H NMR Spectral Data.Table S2: Comparison of 1 H NMR Spectral Data.Scheme S1: Previous synthesis of osajin and scandenone.Figure S1: Cell viability of RAW264.7 macrophages induced with synthetic compounds after 24 h via CCK-8 assay.Experimental procedures and characterization data ( 1 H NMR, 13 C NMR and HRMS n ) can be found in the Supplementary Materials.
Author Contributions: R.W. conceptualization, methodology, software, formal analysis, investigation, data curation, writing-original draft and writing-review and editing; R.M. investigation, visualization, conceptualization and writing-review and editing; K.F. offered much help in the process of biological experiments; H.L. offered much help in the process of chemical experiments; W.Z. funding acquisition, conceptualization, supervision and writing-review and editing; H.J. supervision and writing-review and editing.All authors have read and agreed to the published version of the manuscript.

Figure 2 .
Figure 2. (a) The synthetic compounds suppress the production of TNF-α.(b) The synthetic compounds suppress the production of IL-6.Effects of analogues on the production of serum TNF-α and IL-6 induced via LPS.The results are shown as means ± SD (n = 3) of at least three independent experiments.The results are shown as *** p < 0.001 versus LPS and ### p < 0.001 versus LPS + Dex.

Figure 3 .
Figure 3. (A) Schematic of the mouse sepsis model and administration methods.(B) The effect of 39d/39e on the survival rate of septic mice ( n = 5).

Figure 2 . 12 Figure 2 .
Figure 2. (a) The synthetic compounds suppress the production of TNF-α.(b) The synthetic compounds suppress the production of IL-6.Effects of analogues on the production of serum TNF-α and IL-6 induced via LPS.The results are shown as means ± SD (n = 3) of at least three independent experiments.The results are shown as *** p < 0.001 versus LPS and ### p < 0.001 versus LPS + Dex.

Figure 3 .
Figure 3. (A) Schematic of the mouse sepsis model and administration methods.(B) The effect of 39d/39e on the survival rate of septic mice ( n = 5).

Figure 3 .
Figure 3. (A) Schematic of the mouse sepsis model and administration methods.(B) The effect of 39d/39e on the survival rate of septic mice (n = 5).

Table 1 .
Optimization of the Suzuki coupling reactions a .

Table 1 .
Optimization of the Suzuki coupling reactions a .
a Standard conditions: 11 (0.05 mmol) and palladium catalyst in solvent (5 mL) under argon.b rt: room temperature.c Yield of the isolated product.d The ratio of 1,4-Dioxane to H2O is 3:1.