Total Syntheses and Anti-Inflammatory Studies of Three Natural Coumarins: Glycycoumarin, Glycyrin, and 3-O-Methylglycyrol

Licorice (Glycyrrhiza uralensis Fisch), a significant traditional Chinese herbal medicine, has been extensively utilized in China to treat various ailments. Natural bioactive coumarins, glycycoumarin, glycyrin, and 3-O-methylglycyrol, were isolated from licorice, and they exhibited various pharmacological properties. In this report, we have accomplished the total synthesis of glycycoumarin, glycyrin, and 3-O-methylglycyrol in 5–7 linear steps from commercially available 2,4,6-trihydroxybenzaldehyde with yields of 12.3–21.2%. Additionally, their anti-inflammatory activities were studied and compared. Glycycoumarin, glycyrin, and 3-O-methylglycyrol exhibited different levels of anti-inflammatory activities, with glycyrin being the most potent. Mechanistic studies indicated that glycyrin exerted its anti-inflammatory properties by inhibiting the activation of TNF-α, IL-6, and IL-1β, making it a potential anti-inflammatory lead compound for further optimization and discovery of new agents.


Introduction
Licorice (Glycyrrhiza uralensis Fisch), a significant traditional Chinese herbal medicine listed in the pharmacopoeia of the People's Republic of China, has been extensively utilized in China to treat various ailments, including spleen and stomach disorders, palpitations, shortness of breath, coughs, influenza infection, and liver disease [1,2].Licorice is also recognized for producing a variety of bioactive natural products, such as glycosides, flavonoids, and coumarins [3,4].Glycycoumarin (Figure 1, compound 1) is a naturally occurring coumarin that was initially extracted from licorice by Zhu et al. [5].It has exhibited several beneficial pharmacological properties, including anti-inflammatory, antioxidant, and hepatoprotective effects [6].Among these properties, its anti-inflammatory activity is particularly noteworthy.Glycycoumarin (1) has been shown to inhibit the production of nitric oxide (NO), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) in LPS-induced macrophages [7].Glycycoumarin (1) ameliorates alcohol-induced hepatic injury by activating Nrf2 and autophagy [8].Furthermore, glycycoumarin (1) protects mice against acetaminophen-induced liver injury primarily by activating sustained autophagy [9].
As illustrated in Figure 1, the natural coumarins glycyrin (2) [10] and 3-O-methylglycyrol (3) were also isolated from licorice [11].They possess chemical structures closely related to glycycoumarin (1).Glycyrin (2) has displayed therapeutic activities against metabolic syndrome [12] and exhibited antimicrobial and antiviral properties [13].However, no data on anti-inflammatory activity for either compound 2 or 3 have been reported in the literature.Given the promising anti-inflammatory activity associated with the structurally similar natural coumarin 1, obtaining robust and easy access to these natural products is of significant interest.However, the isolation of compounds 1-3 from licorice plants has been achieved with low yield.Therefore, establishing a synthetic method to obtain these compounds for further pharmacological investigation is highly desirable.As illustrated in Figure 1, the natural coumarins glycyrin (2) [10] and 3-O-methylglycyrol (3) were also isolated from licorice [11].They possess chemical structures closely related to glycycoumarin (1).Glycyrin (2) has displayed therapeutic activities against metabolic syndrome [12] and exhibited antimicrobial and antiviral properties [13].However, no data on anti-inflammatory activity for either compound 2 or 3 have been reported in the literature.Given the promising anti-inflammatory activity associated with the structurally similar natural coumarin 1, obtaining robust and easy access to these natural products is of significant interest.However, the isolation of compounds 1-3 from licorice plants has been achieved with low yield.Therefore, establishing a synthetic method to obtain these compounds for further pharmacological investigation is highly desirable.
To the best of our knowledge, the first total synthesis of compound 1 was recently reported by Song et al. [14], while the syntheses of natural coumarins 2 and 3 remain undescribed.Our ongoing interest in discovering new anti-inflammatory agents from natural products prompted us to develop a reliable and efficient synthetic route for natural compounds 1-3 [15][16][17].Herein, we present systematic studies toward the syntheses of natural compounds 1-3, with compounds 2 and 3 being synthesized for the first time.Additionally, their anti-inflammatory activities were studied and compared.

Synthesis of Natural Coumarins 1-3
Scheme 1 outlines the retrosynthetic methodology aimed at synthesizing the desired compounds 1-3.We envisaged that compound 3 could be generated through an intramolecular cross dehydrogenative C-O coupling reaction of compound 2, and both target compounds 1 and 2 could be readily obtained through Perkin condensation between 2-(2,4dihydroxyphenyl)acetic acid (8) and the corresponding 2-hydroxybenzaldehydes (15 or 19).Scheme 1. Retrosynthetic analysis of compounds 1-3.To the best of our knowledge, the first total synthesis of compound 1 was recently reported by Song et al. [14], while the syntheses of natural coumarins 2 and 3 remain undescribed.Our ongoing interest in discovering new anti-inflammatory agents from natural products prompted us to develop a reliable and efficient synthetic route for natural compounds 1-3 [15][16][17].Herein, we present systematic studies toward the syntheses of natural compounds 1-3, with compounds 2 and 3 being synthesized for the first time.Additionally, their anti-inflammatory activities were studied and compared.

Synthesis of Natural Coumarins 1-3
Scheme 1 outlines the retrosynthetic methodology aimed at synthesizing the desired compounds 1-3.We envisaged that compound 3 could be generated through an intramolecular cross dehydrogenative C-O coupling reaction of compound 2, and both target compounds 1 and 2 could be readily obtained through Perkin condensation between 2-(2,4-dihydroxyphenyl)acetic acid (8) and the corresponding 2-hydroxybenzaldehydes (15 or 19).As illustrated in Figure 1, the natural coumarins glycyrin (2) [10] and 3-O-methylglycyrol (3) were also isolated from licorice [11].They possess chemical structures closely related to glycycoumarin (1).Glycyrin (2) has displayed therapeutic activities against metabolic syndrome [12] and exhibited antimicrobial and antiviral properties [13].However, no data on anti-inflammatory activity for either compound 2 or 3 have been reported in the literature.Given the promising anti-inflammatory activity associated with the structurally similar natural coumarin 1, obtaining robust and easy access to these natural products is of significant interest.However, the isolation of compounds 1-3 from licorice plants has been achieved with low yield.Therefore, establishing a synthetic method to obtain these compounds for further pharmacological investigation is highly desirable.
To the best of our knowledge, the first total synthesis of compound 1 was recently reported by Song et al. [14], while the syntheses of natural coumarins 2 and 3 remain undescribed.Our ongoing interest in discovering new anti-inflammatory agents from natural products prompted us to develop a reliable and efficient synthetic route for natural compounds 1-3 [15][16][17].Herein, we present systematic studies toward the syntheses of natural compounds 1-3, with compounds 2 and 3 being synthesized for the first time.Additionally, their anti-inflammatory activities were studied and compared.
As shown in Scheme 2, the synthesis of key intermediate 8 commenced with readily available 3-bromophenol (4).Treatment of compound 4 with glyoxylic acid (5) under basic conditions yielded compound 6 in 68%.Subsequently, the ɑ-OH group of compound 6 was reduced using SnCl2/HCl to give 2-bromo-4-hydroxyphenylacetic acid (7), which was then hydroxylated in the presence of oxine-copper/NaOH to generate the desired intermediate, 2-(2,4-dihydroxyphenyl)acetic acid (8), with a 92% yield [18].Meanwhile, the 2-and 4-phenol groups of 2,4,6-trihydroxybenzaldehyde (9) were selectively protected, yielding compound 10 (66%) through treatment with bromo(methoxy)methane (MOMBr) in the presence of diisopropylethylamine (DIPEA) (Scheme 3A) [19].We anticipated that the selective O-methylation after the coumarin scaffold formation would be challenging.Hence, the methoxyl group was introduced early, resulting in compound 11 (81%).Due to the hydrogen bond between the 2-phenol group and the formyl group (-CHO), the MOM group at the 2-phenol position can be selectively deprotected using a 3M HCl solution in MeOH (1:10), resulting in compound 12 with a 60% yield.Prenylation of compound 12 furnished prenyl ether 14 with a yield of 79% [20].Compound 14 was then subjected to a Claisen/Cope rearrangement to give the 5-prenylbenzaldehyde 15 in N,N-dimethylaniline as a solvent [20].Upon heating prenyl ether 14 in N,N-dimethylaniline at reflux for 1 h, we found that two products, 15 and 15′ (Scheme 3B), could be isolated [21].The formation of the byproduct 15′ has been rationalized by assuming a dearomatized intermediate A that results from a Claisen rearrangement (step 1), and from intermediate A, the byproduct 15′ is formed through aromatization.Intermediate A may undergo a second [3,3]-sigmatropic rearrangement (step 2, Cope-rearrangement) to cyclohexadienone intermediate B, from which the desired para-product 15 is formed after aromatization.After understanding the reaction mechanism, we extended the reaction time to 5 h, successfully obtaining product 15 with a yield of 77% without the byproduct 15′.
With intermediates 2-(2,4-dihydroxyphenyl)acetic acid (8) and 5-prenylbenzaldehyde 15 in our hands, these two fragments were then coupled under Perkin condensation conditions, using CH3COOK in refluxing acetic anhydride (Ac2O), to give acetylated glycycoumarin (16) with a 61% yield [18,[22][23][24].Under these reaction conditions, the MOM protecting group at the C-7 position was concurrently transformed into the acetyl (-Ac) group.Global deacetylation with KOH in MeOH at 0 °C afforded the target compound glycycoumarin (1) with an 87% yield [23].The NMR data of the synthetic sample were consistent with reported data [25] for natural glycycoumarin (1) (Table 1).Meanwhile, the 2-and 4-phenol groups of 2,4,6-trihydroxybenzaldehyde (9) were selectively protected, yielding compound 10 (66%) through treatment with bromo(methoxy)methane (MOMBr) in the presence of diisopropylethylamine (DIPEA) (Scheme 3A) [19].We anticipated that the selective O-methylation after the coumarin scaffold formation would be challenging.Hence, the methoxyl group was introduced early, resulting in compound 11 (81%).Due to the hydrogen bond between the 2-phenol group and the formyl group (-CHO), the MOM group at the 2-phenol position can be selectively deprotected using a 3M HCl solution in MeOH (1:10), resulting in compound 12 with a 60% yield.Prenylation of compound 12 furnished prenyl ether 14 with a yield of 79% [20].Compound 14 was then subjected to a Claisen/Cope rearrangement to give the 5-prenylbenzaldehyde 15 in N,N-dimethylaniline as a solvent [20].Upon heating prenyl ether 14 in N,N-dimethylaniline at reflux for 1 h, we found that two products, 15 and 15 ′ (Scheme 3B), could be isolated [21].The formation of the byproduct 15 ′ has been rationalized by assuming a dearomatized intermediate A that results from a Claisen rearrangement (step 1), and from intermediate A, the byproduct 15 ′ is formed through aromatization.Intermediate A may undergo a second [3,3]-sigmatropic rearrangement (step 2, Cope-rearrangement) to cyclohexadienone intermediate B, from which the desired para-product 15 is formed after aromatization.After understanding the reaction mechanism, we extended the reaction time to 5 h, successfully obtaining product 15 with a yield of 77% without the byproduct 15 ′ .
No. Natural Product (δ1)  Following the successful synthesis of glycycoumarin (1), the syntheses of coumarins 2 and 3 were then pursued (Scheme 4).The prenylbenzaldehyde fragment of compound 2 was prepared from commercially available 2,4,6-trihydroxybenzaldehyde (9), which underwent a selective methylation to give 2-hydroxybenzaldehyde 17.Compound 17 was prenylated, and the resulting prenyl ether 18 was treated with N,N-dimethylaniline at 195 • C to furnish 19 via a Claisen/Cope rearrangement.Subsequently, Perkin condensation of prenylbenzaldehyde 19 and 2-(2,4-dihydroxyphenyl)acetic acid ( 8) gave acetylated coumarin 20, which was deacetylated to give target molecule 2 with an 83% yield.The synthetic compound 2 was characterized by 1 H NMR and 13 C NMR, and the data were identical with those of the natural product ( Following the successful synthesis of glycycoumarin (1), the syntheses of coumarins 2 and 3 were then pursued (Scheme 4).The prenylbenzaldehyde fragment of compound 2 was prepared from commercially available 2,4,6-trihydroxybenzaldehyde ( 9), which underwent a selective methylation to give 2-hydroxybenzaldehyde 17.Compound 17 was prenylated, and the resulting prenyl ether 18 was treated with N,N-dimethylaniline at 195 °C to furnish 19 via a Claisen/Cope rearrangement.Subsequently, Perkin condensation of prenylbenzaldehyde 19 and 2-(2,4-dihydroxyphenyl)acetic acid (8) gave acetylated coumarin 20, which was deacetylated to give target molecule 2 with an 83% yield.The synthetic compound 2 was characterized by 1 H NMR and 13 C NMR, and the data were identical with those of the natural product (Table 2) [3].

Anti-Inflammatory Activity of Natural Coumarins 1-3
Since nitric oxide (NO), a vital gas signaling molecule, is a unique transmitter of acute or chronic inflammation [27], the anti-inflammatory activities and cytotoxic effects of compounds 1-3 were evaluated by monitoring cell viability and NO inhibition in LPS-induced RAW264.7 cells, respectively.The effects of compounds 1-3 on cell viability were first evaluated using a CCK8 assay.RAW264.7 cells were exposed to different doses of the tested compound for 24 h.Meanwhile, 0.1% DMSO was used as the control.Significant position was unstable under these harsh conditions, leading to the production of numerous by-products.Therefore, a 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU)-promoted intramolecular dehydrogenation/oxa-Michael reaction was used to successfully obtain 3 in a moderate yield (58%) under milder conditions (DBU/H2O at 50 °C and then HCl at 50 °C) [18].The NMR data of the synthetic compound 3 were consistent with the data reported for natural 3-O-methylglycyrol (3) (Table 2) [26].

Anti-Inflammatory Activity of Natural Coumarins 1-3
Since nitric oxide (NO), a vital gas signaling molecule, is a unique transmitter of acute or chronic inflammation [27], the anti-inflammatory activities and cytotoxic effects of compounds 1-3 were evaluated by monitoring cell viability and NO inhibition in LPS-induced RAW264.7 cells, respectively.The effects of compounds 1-3 on cell viability were first evaluated using a CCK8 assay.RAW264.7 cells were exposed to different doses of the tested compound for 24 h.Meanwhile, 0.1% DMSO was used as the control.Significant

Anti-Inflammatory Activity of Natural Coumarins 1-3
Since nitric oxide (NO), a vital gas signaling molecule, is a unique transmitter of acute or chronic inflammation [27], the anti-inflammatory activities and cytotoxic effects of compounds 1-3 were evaluated by monitoring cell viability and NO inhibition in LPSinduced RAW264.7 cells, respectively.The effects of compounds 1-3 on cell viability were first evaluated using a CCK8 assay.RAW264.7 cells were exposed to different doses of the tested compound for 24 h.Meanwhile, 0.1% DMSO was used as the control.Significant cytotoxic effects were observed on the growth of RAW264.7 cells incubated with 1 and 2 at a dose of 100 µM (Figure 2).Cell survival was restored to that of the normal control group at reduced doses of 0.01, 0.1, 1, 10, 25, and 50 µM.The same concentration ranges were also used in cell-based studies to determine their anti-inflammatory activities.
To determine the anti-inflammatory activity of compounds 1-3, the mouse macrophagelike cell line RAW264.7 was used.Upon treatment with LPS (0.5 µg/mL), RAW264.7 cells produced an increased amount of NO.After treatment with different concentrations of coumarins 1-3, NO secretion was strongly inhibited (Figure 3).Compound 1 exhibited greater activity in inhibiting NO secretion at a dose of 25 µM and 50 µM, and the concentration of 50 µM with the inhibition rate exceeding 50%.Compound 2 showed strong inhibition of NO secretion only at 50 µM.Compound 3 inhibited NO secretion in a concentration-dependent manner.cytotoxic effects were observed on the growth of RAW264.7 cells incubated with 1 and 2 at a dose of 100 µM (Figure 2).Cell survival was restored to that of the normal control group at reduced doses of 0.01, 0.1, 1, 10, 25, and 50 µM.The same concentration ranges were also used in cell-based studies to determine their anti-inflammatory activities.To determine the anti-inflammatory activity of compounds 1-3, the mouse macrophage-like cell line RAW264.7 was used.Upon treatment with LPS (0.5 µg/mL), RAW264.7 cells produced an increased amount of NO.After treatment with different concentrations of coumarins 1-3, NO secretion was strongly inhibited (Figure 3).Compound 1 exhibited greater activity in inhibiting NO secretion at a dose of 25 µM and 50 µM, and the concentration of 50 µM with the inhibition rate exceeding 50%.Compound 2 showed strong inhibition of NO secretion only at 50 µM.Compound 3 inhibited NO secretion in a concentration-dependent manner.To confirm the function of compounds 1-3 in the LPS-induced production of proinflammatory cytokines, the expression levels of three critical pro-inflammatory cytokines, TNF-α, IL-6, and IL-1β mRNA, were analyzed by qRT-PCR, respectively [28].As shown in Figure 4   To determine the anti-inflammatory activity of compounds 1-3, the mouse macrophage-like cell line RAW264.7 was used.Upon treatment with LPS (0.5 µg/mL), RAW264.7 cells produced an increased amount of NO.After treatment with different concentrations of coumarins 1-3, NO secretion was strongly inhibited (Figure 3).Compound 1 exhibited greater activity in inhibiting NO secretion at a dose of 25 µM and 50 µM, and the concentration of 50 µM with the inhibition rate exceeding 50%.Compound 2 showed strong inhibition of NO secretion only at 50 µM.Compound 3 inhibited NO secretion in a concentration-dependent manner.To confirm the function of compounds 1-3 in the LPS-induced production of proinflammatory cytokines, the expression levels of three critical pro-inflammatory cytokines, TNF-α, IL-6, and IL-1β mRNA, were analyzed by qRT-PCR, respectively [28].As shown in Figure 4  To confirm the function of compounds 1-3 in the LPS-induced production of proinflammatory cytokines, the expression levels of three critical pro-inflammatory cytokines, TNF-α, IL-6, and IL-1β mRNA, were analyzed by qRT-PCR, respectively [28].As shown in Figure 4
3.2.19.NO Determination RAW264.7 cells were seeded in 96-well plate at a density of 1 × 10 5 cells/well.The cells were treated with different concentrations of the test compounds; after 1 h, the cells were stimulated with LPS (0.5 µg/mL) and then incubated for 24 h.The nitrite accumulated in the culture medium was measured as an indicator of NO production based on a diazotisation reaction using the Griess reagent system as previously described [32].

Real-Time Quantitative PCR
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was using to extract total cellular RNA according to the manufacturer's instructions.RNA concentration was determined by examining the absorbance at 260 nm using a Varioskan Flash Multimode Reader (Thermo Fisher Scientific).Total RNA (2 µg) was reverse-transcribed using TransScript ® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Cat No: AT341-01, TransGen Biotech, Beijing, China).Equal amounts of cDNA were subjected to RT-qPCR with TransStart ® Top Green qPCR SuperMix (Cat No: AQ131-02, TransGen Biotech, Beijing, China) using a Chromo4 detection system (Bio-Rad, California, USA).GAPDH served as reference genes to eliminate differences in the number of cells.Quantitative analyses were performed using the threshold cycle number (Ct), where the signal was detected above the background and was in the exponential phase.Relative RNA expression was analyzed by 2 −△△C(t) , and DMSO was used as a control.The sequences of primers used are showed in Table 3.

Conclusions
In summary, the total synthesis of glycycoumarin (1), glycyrin (2), and 3-O-methylglycyrol (3) has been accomplished in 5-7 steps from commercially available 2,4,6trihydroxybenzaldehyde (9) with yields of 13.5%, 21.2%, and 12.3%, respectively.Notably, glycyrin (2) and 3-O-methylglycyrol (3) were synthesized for the first time.Our synthetic strategy features a Perkin condensation to establish the 3-phenyl-2H-chromen-2-one framework and a Claisen/Cope rearrangement to introduce the isopentene group to the coumarin core.Furthermore, the anti-inflammatory potencies of the synthetic natural products 1-3 were investigated using various in vitro systems, including the inhibition of NO production in LPS-induced RAW264.7 cells and the inhibition of three critical pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β).Among compounds 1-3, the anti-inflammatory activities of glycyrin (2) and 3-O-methylglycyrol (3) were first reported.Generally, compounds 1-3 exhibited different levels of anti-inflammatory activities, with compound 2 being the most potent.Mechanistic studies indicated that compound 2 exerted its anti-inflammatory property by inhibiting the activation of TNF-α, IL-6, and IL-1β.Hence, compound 2 could be a potential anti-inflammatory lead compound for further optimization and discovery of new agents.

Figure 3 .
Figure 3. Effects of the tested compounds 1 (a), 2 (b) and 3 (c) on LPS-stimulated NO production in RAW264.7 cells; 0.1% DMSO was used as the control.Graphic data were run in triplicate and are presented as the mean ± SD (compared with LPS, * p < 0.05, *** p < 0.001).
, LPS significantly induced the mRNA expressions of TNF-α, IL-6, and IL-1β compared with the normal control.Compound 1 demonstrated significant inhibition of IL-6 and TNF-α expression at concentrations of 25 and 50 µM but had no effect on IL-1β.Compound 2 exhibited significant inhibition of IL-6 and IL-1β expression at concentrations of 25 and 50 µM.It also significantly inhibited TNF-α at 50 µM.Compound 3 exhibited concentration-dependent inhibition of IL-6 and IL-1β, and it inhibited TNF-α only at a concentration of 50 µM.

Figure 3 .
Figure 3. Effects of the tested compounds 1 (a), 2 (b) and 3 (c) on LPS-stimulated NO production in RAW264.7 cells; 0.1% DMSO was used as the control.Graphic data were run in triplicate and are presented as the mean ± SD (compared with LPS, * p < 0.05, *** p < 0.001).
, LPS significantly induced the mRNA expressions of TNF-α, IL-6, and IL-1β compared with the normal control.Compound 1 demonstrated significant inhibition of IL-6 and TNF-α expression at concentrations of 25 and 50 µM but had no effect on IL-1β.Compound 2 exhibited significant inhibition of IL-6 and IL-1β expression at concentrations of 25 and 50 µM.It also significantly inhibited TNF-α at 50 µM.Compound 3 exhibited concentration-dependent inhibition of IL-6 and IL-1β, and it inhibited TNF-α only at a concentration of 50 µM.

Figure 3 .
Figure 3. Effects of the tested compounds 1 (a), 2 (b) and 3 (c) on LPS-stimulated NO production in RAW264.7 cells; 0.1% DMSO was used as the control.Graphic data were run in triplicate and are presented as the mean ± SD (compared with LPS, * p < 0.05, *** p < 0.001).

Table 1 .
Comparison of the
1The NMR data were recorded in DMSO-d 6.

Table 2 .
Comparison of the