Design, Synthesis, and Anti-Inflammatory Activities of 12-Dehydropyxinol Derivatives

Pyxinol skeleton is a promising framework of anti-inflammatory agents formed in the human liver from 20S-protopanaxadiol, the main active aglycone of ginsenosides. In the present study, a new series of amino acid-containing derivatives were produced from 12-dehydropyxinol, a pyxinol oxidation metabolite, and its anti-inflammatory activity was assessed using an NO inhibition assay. Interestingly, the dehydrogenation at C-12 of pyxinol derivatives improved their potency greatly. Furthermore, half of the derivatives exhibited better NO inhibitory activity than hydrocortisone sodium succinate, a glucocorticoid drug. The structure–activity relationship analysis indicated that the kinds of amino acid residues and their hydrophilicity influenced the activity to a great extent, as did R/S stereochemistry at C-24. Of the various derivatives, 5c with an N-Boc-protected phenylalanine residue showed the highest NO inhibitory activity and relatively low cytotoxicity. Moreover, derivative 5c could dose-dependently suppress iNOS, IL-1β, and TNF-α via the MAPK and NF-κB pathways, but not the GR pathway. Overall, pyxinol derivatives hold potential for application as anti-inflammatory agents.


Introduction
Inflammation is the physiological defense response against infection and injury. Excessive and prolonged inflammation can cause damage and result in various chronic diseases, including respiratory diseases, neurodegenerative diseases, and cancer [1,2]. Glucocorticoids (GCs) are the most effective drugs that inhibit inflammation in clinical settings; however, their side effects are extremely serious [3]. Nonetheless, owing to a lack of other effective drugs, GCs continue to be widely used to treat diseases such as COVID-19 [4]. Therefore, there is an urgent need to develop safer and more effective anti-inflammatory agents.
Ginsenosides are the primary pharmacological ingredients of ginseng, the well-known orally administered herb that strengthens vitality, extends life, and supports a healthy heart [5]. To date, ginsenosides are known to possess antitumor, memory preserving, anti-inflammatory, and cardioprotective activities [5][6][7][8]. Especially, the anti-inflammatory effects are remarkable because their aglycones exhibit structural similarity with GCs [9,10]. 20S-Protopanoxadiol (PPD, Figure 1) is one such aglycone and has been reported to be the main intestinal metabolite of protopanoxadiol-type ginsenosides that exhibit superior anti-inflammatory activities [9,11]. Recently, pyxinol ( Figure 1) has been identified as the main PPD metabolite in the human liver and has attracted special attention in drug development [12][13][14]. Pyxinol and its derivatives have been demonstrated to exert cardioprotective, Previous studies have reported that the 24R-epimer of pyxinol is selectively metabolized by CYP3A4 in the human liver to produce oxidation metabolites [14], such as 3-dehydropyxinol or 12-dehydropyxinol [12]. As the modification at C-3 could substantially improve the anti-inflammatory activities of pyxinol derivatives and the structural diversity of amino acids, 24 new 3-amino acid-12-dehydropyxinol derivatives were prepared in this study, and their anti-inflammatory effects in lipopolysaccharide (LPS)-induced macrophages were assessed. Of these, an N-Boc-protected phenylalanine derivative of 12dehydropyxinol (5c) was identified to be the most potent compound and was used for mechanistic studies.

Chemistry
As described previously [16,29], pyxinol (1) and 24S-pyxinol (2) were obtained via the one step epoxidation of commercial 20S-PPD, which is shown in Scheme 1. 3,12-Didehydropyxinol was then synthesized via Dess-Martin oxidation and was subsequently selectively reduced in NaBH4/isopropanol solution to obtain 12-dehydropyxinol with a yield of >90%. Amino acid-modified 12-dehydropyxinol derivatives (5a-5f) were next produced via DMAP-mediated esterification. Later, trifluoroacetic acid was utilized for N-Boc deprotection to obtain derivatives 6a-6f. The 24S-epimers (9a-9f, 10a-10f) were similarly synthesized from 24S-pyxinol. Previous studies have reported that the 24R-epimer of pyxinol is selectively metabolized by CYP3A4 in the human liver to produce oxidation metabolites [14], such as 3-dehydropyxinol or 12-dehydropyxinol [12]. As the modification at C-3 could substantially improve the anti-inflammatory activities of pyxinol derivatives and the structural diversity of amino acids, 24 new 3-amino acid-12-dehydropyxinol derivatives were prepared in this study, and their anti-inflammatory effects in lipopolysaccharide (LPS)-induced macrophages were assessed. Of these, an N-Boc-protected phenylalanine derivative of 12-dehydropyxinol (5c) was identified to be the most potent compound and was used for mechanistic studies.

NO-Inhibition of 12-Dehydropyxinol Derivatives
The inhibitory effect of specific compounds on LPS-triggered NO production is closely related to their anti-inflammatory activity [30,31]. Herein, the NO inhibition effects of all synthesized 12-dehydropyxinol derivatives were assessed in RAW264.7 macrophages using a Griess assay with HSS as the positive control. The cytotoxicity assay showed that most of the 12-dehydropyxinol derivatives did not display any evident toxic effects on RAW264.7 cells, except 5f, 6a, 6f, and 10d when used at a concentration of 20 μM (Figure 2A). Then, these derivatives with no evident cytotoxicity were used for the NO inhibition assay. The treatment of RAW264.7 cells with 12-dehydropyxinol Scheme 1. Synthesis of 12-dehydropyxinol derivatives.

NO-Inhibition of 12-Dehydropyxinol Derivatives
The inhibitory effect of specific compounds on LPS-triggered NO production is closely related to their anti-inflammatory activity [30,31]. Herein, the NO inhibition effects of all synthesized 12-dehydropyxinol derivatives were assessed in RAW264.7 macrophages using a Griess assay with HSS as the positive control. The cytotoxicity assay showed that most of the 12-dehydropyxinol derivatives did not display any evident toxic effects Molecules 2023, 28, 1307 4 of 16 on RAW264.7 cells, except 5f, 6a, 6f, and 10d when used at a concentration of 20 µM (Figure 2A). Then, these derivatives with no evident cytotoxicity were used for the NO inhibition assay. The treatment of RAW264.7 cells with 12-dehydropyxinol derivatives significantly suppressed the LPS-triggered NO production. Furthermore, most of the derivatives exhibited better NO inhibition activity than the parental compounds (1, 4, 7, and 8) ( Figure 2B), which agrees with previous results that modification at C-3 immensely improves anti-inflammatory activities [26,27]. Interestingly, most of the 12-dehydropyxinol derivatives also exhibited better NO inhibition activity than HSS, the positive control. Several of the 12-dehydropyxinol derivatives (5a, 5b, 5c, 5d, 6e, 10b, and 10c) exhibited even better NO inhibition activity than Y13, which has the highest potency among the known pyxinol derivatives with a hydroxy group at C-12. This observation suggests that dehydrogenation at C-12 of pyxinol can largely improve anti-inflammatory activity. Derivatives (5a-5f and 6e) with a 24R configuration exhibited better potency than the corresponding 24S derivatives (9a-9f and 10e), which is consistent with our previous findings that R/S stereochemistry at C-24 affects anti-inflammatory activity, with the 24Rconfiguration being preferred [26,27]. The modification types of amino acid residues at C-3 also largely affect their potency, with N-Boc-protected aromatic amino acids being preferred. This finding was quite different from pyxinol derivatives, in which N-Boc-protected neutral aliphatic amino acids were preferred, which suggests that the dehydrogenation at C-12 greatly influences the effect of the C-3 modification pattern on the improvement of anti-inflammatory activities. In most cases, the deprotection of Boc-protected amines reduced their anti-inflammatory effects, but not for the derivatives with an aromatic group. Based on the NO-inhibition activities of 12-dehydropyxinol derivatives, the structureactivity relationship (SAR) is summarized in Figure 3. Derivative 5c with N-Boc-protected phenylalanine was identified to possess the highest NO inhibition activity and was selected for further studies.
Molecules 2023, 28, x FOR PEER REVIEW 4 of 17 derivatives significantly suppressed the LPS-triggered NO production. Furthermore, most of the derivatives exhibited better NO inhibition activity than the parental compounds (1, 4, 7, and 8) ( Figure 2B), which agrees with previous results that modification at C-3 immensely improves anti-inflammatory activities [26,27]. Interestingly, most of the 12-dehydropyxinol derivatives also exhibited better NO inhibition activity than HSS, the positive control. Several of the 12-dehydropyxinol derivatives (5a, 5b, 5c, 5d, 6e, 10b, and 10c) exhibited even better NO inhibition activity than Y13, which has the highest potency among the known pyxinol derivatives with a hydroxy group at C-12. This observation suggests that dehydrogenation at C-12 of pyxinol can largely improve anti-inflammatory activity. Derivatives (5a-5f and 6e) with a 24R configuration exhibited better potency than the corresponding 24S derivatives (9a-9f and 10e), which is consistent with our previous findings that R/S stereochemistry at C-24 affects anti-inflammatory activity, with the 24Rconfiguration being preferred [26,27]. The modification types of amino acid residues at C-3 also largely affect their potency, with N-Boc-protected aromatic amino acids being preferred. This finding was quite different from pyxinol derivatives, in which N-Boc-protected neutral aliphatic amino acids were preferred, which suggests that the dehydrogenation at C-12 greatly influences the effect of the C-3 modification pattern on the improvement of anti-inflammatory activities. In most cases, the deprotection of Boc-protected amines reduced their anti-inflammatory effects, but not for the derivatives with an aromatic group. Based on the NO-inhibition activities of 12-dehydropyxinol derivatives, the structure-activity relationship (SAR) is summarized in Figure 3. Derivative 5c with N-Boc-protected phenylalanine was identified to possess the highest NO inhibition activity and was selected for further studies.

Inhibition of LPS-Mediated Cytokines Production Using 5c
To fully confirm the anti-inflammatory effects of 5c, the concentration dependent NO-inhibition assay was first assessed. As expected, the derivative 5c could exert a NOinhibitory effect in a concentration-dependent manner and even exhibit a significant NOinhibitory effect as low as 1 μM, while the derivative 5c exhibited no evident cytotoxicity at as high as 80 μM concentration ( Figure 4A,B). Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are the other known proinflammatory cytokines that are rapidly yielded in inflammation and accelerated inflammatory progression [32]. We thus next evaluated its ability to inhibit IL-1β and TNF-α production in RAW264.7 cells. LPS-triggered cells were treated with 5c (5, 10, and 20 μM), after which the IL-1β and TNF-α levels were measured using an enzyme-linked immunosorbent assay (ELISA), which indicated a marked upregulation in the levels of IL-1β and TNF-α after LPS-induction, whereas the derivative 5c concentration dependently suppressed this upregulation, which was even better than that of HSS ( Figure 4C,D). These data further confirm the robust anti-inflammatory activities of derivative 5c.

Inhibition of LPS-Mediated Cytokines Production Using 5c
To fully confirm the anti-inflammatory effects of 5c, the concentration dependent NO-inhibition assay was first assessed. As expected, the derivative 5c could exert a NOinhibitory effect in a concentration-dependent manner and even exhibit a significant NOinhibitory effect as low as 1 µM, while the derivative 5c exhibited no evident cytotoxicity at as high as 80 µM concentration ( Figure 4A,B). Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are the other known proinflammatory cytokines that are rapidly yielded in inflammation and accelerated inflammatory progression [32]. We thus next evaluated its ability to inhibit IL-1β and TNF-α production in RAW264.7 cells. LPS-triggered cells were treated with 5c (5, 10, and 20 µM), after which the IL-1β and TNF-α levels were measured using an enzyme-linked immunosorbent assay (ELISA), which indicated a marked upregulation in the levels of IL-1β and TNF-α after LPS-induction, whereas the derivative 5c concentration dependently suppressed this upregulation, which was even better than that of HSS ( Figure 4C,D). These data further confirm the robust anti-inflammatory activities of derivative 5c.

Inhibition of LPS-Mediated Cytokines Production Using 5c
To fully confirm the anti-inflammatory effects of 5c, the concentration dependent NO-inhibition assay was first assessed. As expected, the derivative 5c could exert a NOinhibitory effect in a concentration-dependent manner and even exhibit a significant NOinhibitory effect as low as 1 μM, while the derivative 5c exhibited no evident cytotoxicity at as high as 80 μM concentration ( Figure 4A,B). Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are the other known proinflammatory cytokines that are rapidly yielded in inflammation and accelerated inflammatory progression [32]. We thus next evaluated its ability to inhibit IL-1β and TNF-α production in RAW264.7 cells. LPS-triggered cells were treated with 5c (5, 10, and 20 μM), after which the IL-1β and TNF-α levels were measured using an enzyme-linked immunosorbent assay (ELISA), which indicated a marked upregulation in the levels of IL-1β and TNF-α after LPS-induction, whereas the derivative 5c concentration dependently suppressed this upregulation, which was even better than that of HSS ( Figure 4C,D). These data further confirm the robust anti-inflammatory activities of derivative 5c.

Inhibition of LPS-Mediated iNOS Upregulation and NF-κB and MAPK Activation Using 5c
Inducible nitric oxide synthase (iNOS) is the key regulator of NO synthesis in response to inflammatory stimuli and aggravates inflammatory diseases [30,33]. We thus assessed the ability of 5c to regulate LPS-triggered iNOS expression via the 24-h treatment of RAW264.7 cells with 5c (5, 10, and 20 µM) and LPS (1 µg/mL). The resulting iNOS level was analyzed using Western blotting. In line with the promotion of NO production, LPS induced a significant upregulation of iNOS, whereas 5c suppressed this upregulation in a concentration-dependent manner ( Figure 5). iNOS, IL-1β, and TNF-α are positively regulated in inflammation by the NF-κB pathway [34]. Both IκB and NF-κB p65 phosphorylation are key steps in the activated NF-κB pathway, which lead to the upregulation of the above inflammation-related proteins. Subsequently, the impacts of 5c on LPS-triggered NF-κB activation were assessed. Compound 5c inhibited LPS-triggered IκB and NF-κB p65 phosphorylation in a concentration-dependent manner, and these inhibitory effects of 5c were better than that of HSS ( Figure 6A). PPD and other ginsenosides have been confirmed to exert anti-inflammatory effects by inhibiting the NF-κB pathway, albeit weakly [9,28]. Our previous study has indicated that pyxinol might be the predominant active form of these ginsenosides that is responsible for the anti-inflammatory activity via the NF-κB pathway [27]. 24R-epimer of pyxinol is preferentially metabolized in the human liver via oxidation, with dehydrogenation being one of the main forms of oxidation [12,13]. Together, these results, therefore, suggest that 12-dehydropyxinol derivatives may be the active forms of ginsenosides in humans, which exert anti-inflammatory effects and prevent the upregulation of inflammatory proteins by inhibiting NF-κB activation.

Inhibition of LPS-Mediated iNOS Upregulation and NF-κB and MAPK Activation U 5c
Inducible nitric oxide synthase (iNOS) is the key regulator of NO synthesi sponse to inflammatory stimuli and aggravates inflammatory diseases [30,33]. W assessed the ability of 5c to regulate LPS-triggered iNOS expression via the 24-h tre of RAW264.7 cells with 5c (5, 10, and 20 μM) and LPS (1 μg/mL). The resulting iNO was analyzed using Western blotting. In line with the promotion of NO productio induced a significant upregulation of iNOS, whereas 5c suppressed this upregulat concentration-dependent manner ( Figure 5). iNOS, IL-1β, and TNF-α are positivel lated in inflammation by the NF-κB pathway [34]. Both IκB and NF-κB p65 phosp tion are key steps in the activated NF-κB pathway, which lead to the upregulation above inflammation-related proteins. Subsequently, the impacts of 5c on LPS-tri NF-κB activation were assessed. Compound 5c inhibited LPS-triggered IκB and p65 phosphorylation in a concentration-dependent manner, and these inhibitory of 5c were better than that of HSS ( Figure 6A). PPD and other ginsenosides hav confirmed to exert anti-inflammatory effects by inhibiting the NF-κB pathway weakly [9,28]. Our previous study has indicated that pyxinol might be the predo active form of these ginsenosides that is responsible for the anti-inflammatory activ the NF-κB pathway [27]. 24R-epimer of pyxinol is preferentially metabolized in man liver via oxidation, with dehydrogenation being one of the main forms of ox [12,13]. Together, these results, therefore, suggest that 12-dehydropyxinol derivativ be the active forms of ginsenosides in humans, which exert anti-inflammatory effe prevent the upregulation of inflammatory proteins by inhibiting NF-κB activation The MAPK pathway is another major pathway that is activated to upregulate matory proteins, such as iNOS and IL-1β, in inflammatory stimuli [35]. c-Jun N-te kinase (JNK), extracellular regulated protein kinases (ERK1/2), and p38 are the m naling intermediaries in the MAPK pathway and are involved in the anti-inflam activity of pyxinol [26]. The impact of 5c on the LPS-activated MAPK pathway w assessed. LPS was observed to activate the MAPK pathway via a marked upregula JNK, ERK1/2, and p38 phosphorylation. On the contrary, 5c inhibited this upregula JNK and p38 phosphorylation in a concentration-dependent manner. However, 5c inhibit the upregulation of ERK1/2 phosphorylation ( Figure 6B). Our previous st dicated that pyxinol derivatives could only inhibit the LPS-induced upregula ERK1/2 and p38 phosphorylation [26]. Other ginsenosides have also been repo The MAPK pathway is another major pathway that is activated to upregulate inflammatory proteins, such as iNOS and IL-1β, in inflammatory stimuli [35]. c-Jun N-terminal kinase (JNK), extracellular regulated protein kinases (ERK1/2), and p38 are the main signaling intermediaries in the MAPK pathway and are involved in the anti-inflammatory activity of pyxinol [26]. The impact of 5c on the LPS-activated MAPK pathway was then assessed. LPS was observed to activate the MAPK pathway via a marked upregulation of JNK, ERK1/2, and p38 phosphorylation. On the contrary, 5c inhibited this upregulation of JNK and p38 phosphorylation in a concentration-dependent manner. However, 5c did not inhibit the upregulation of ERK1/2 phosphorylation ( Figure 6B). Our previous study indicated that pyxinol derivatives could only inhibit the LPS-induced upregulation of ERK1/2 and p38 phosphorylation [26]. Other ginsenosides have also been reported to selectively modulate JNK phosphorylation to suppress inflammation [6,36]. The data suggest that these signaling intermediaries of MAPK may interact with these compounds and that the dehydrogenation at C-12 may greatly affect this interaction. This issue needs to be resolved in the future. In summary, compound 5c exhibited its anti-inflammatory activity via the inhibition of JNK and p38 activation.
Molecules 2023, 28, x FOR PEER REVIEW 7 of 17 selectively modulate JNK phosphorylation to suppress inflammation [6,36]. The data suggest that these signaling intermediaries of MAPK may interact with these compounds and that the dehydrogenation at C-12 may greatly affect this interaction. This issue needs to be resolved in the future. In summary, compound 5c exhibited its anti-inflammatory activity via the inhibition of JNK and p38 activation.

The GR-Independent Effect of 5c on Its Anti-Inflammatory Activity
GCs are well-known anti-inflammatory drugs in clinical settings that regulate antiinflammation by activating the GC receptor (GR). Previous studies have reported that the activation of GR signaling is involved in the anti-inflammatory activity of ginsenosides [10,37]. Whether the anti-inflammatory activity of 5c was related to the activation of GR signaling was assessed next. As expected, the inhibitory effect of HSS on LPS-triggered NO production was completely blocked by RU-486, a specific GR antagonist, whereas the NO inhibition effect of 5c was not impacted by it ( Figure 7A). Similar results were observed for other 12-dehydropyxinol derivatives. To further confirm whether an in situ interaction existed between GR and 5c, the cellular thermal shift assay (CETSA) was performed. CETSA is a powerful label-free method used to analyze protein-compound interactions in physiological environments. Such interactions in situ can increase/decrease a protein's overall resistance to thermal denaturation, thereby resulting in the amount change of the protein in the soluble lysate fraction at a high temperature [38,39]. Briefly, RAW264.7 cells were incubated with either 5c (20 μM) or HSS (20 μM), followed by thermal denaturation at indicated temperatures. Then, the cells were lysed with freeze-thaw cycles, and soluble fractions were collected and evaluated using Western blotting. Compared with DMSO-treatment, HSS-treatment stabilized GR at high temperatures, which proves the expected interactions between GR and HSS ( Figure 7B). In contrast, 5c-treatment did not exhibit any detectable differences from DMSO-treatment at the same conditions. Molecular docking analysis also suggested that HSS had a strong binding affinity for GR; however, 5c did not have this affinity (data not shown). These results strongly imply that 5c exerts its anti-inflammatory activity in GR-independent ways.

The GR-Independent Effect of 5c on Its Anti-Inflammatory Activity
GCs are well-known anti-inflammatory drugs in clinical settings that regulate antiinflammation by activating the GC receptor (GR). Previous studies have reported that the activation of GR signaling is involved in the anti-inflammatory activity of ginsenosides [10,37]. Whether the anti-inflammatory activity of 5c was related to the activation of GR signaling was assessed next. As expected, the inhibitory effect of HSS on LPS-triggered NO production was completely blocked by RU-486, a specific GR antagonist, whereas the NO inhibition effect of 5c was not impacted by it ( Figure 7A). Similar results were observed for other 12-dehydropyxinol derivatives. To further confirm whether an in situ interaction existed between GR and 5c, the cellular thermal shift assay (CETSA) was performed. CETSA is a powerful label-free method used to analyze protein-compound interactions in physiological environments. Such interactions in situ can increase/decrease a protein's overall resistance to thermal denaturation, thereby resulting in the amount change of the protein in the soluble lysate fraction at a high temperature [38,39]. Briefly, RAW264.7 cells were incubated with either 5c (20 µM) or HSS (20 µM), followed by thermal denaturation at indicated temperatures. Then, the cells were lysed with freeze-thaw cycles, and soluble fractions were collected and evaluated using Western blotting. Compared with DMSO-treatment, HSS-treatment stabilized GR at high temperatures, which proves the expected interactions between GR and HSS ( Figure 7B). In contrast, 5c-treatment did not exhibit any detectable differences from DMSO-treatment at the same conditions. Molecular docking analysis also suggested that HSS had a strong binding affinity for GR; however, 5c did not have this affinity (data not shown). These results strongly imply that 5c exerts its anti-inflammatory activity in GR-independent ways.
To to stirring for 1 d at rt, water was added to the reaction mixture and extracted with CH2Cl2, dried over Na2SO4, and purified using column chromatography (V petroleum ether: V ethyl acetate) to produce 5a-5f and 9a-9f.

Cellular Thermal Shift Assay (CETSA)
Protein-compound interactions in physiological environments were analyzed using CETSA [38,39]. RAW264.7 cells (2 × 10 7 cells/mL) were seeded for 24 h. After 2 h treatment with 5c (20 µM), HSS (20 µM), or DMSO, the cells were split into eight equal groups. The groups were heated for 3 min at 40, 43, 46, 49, 52, 55, 58, and 61 • C, followed by incubation at RT for 3 min. The cells then underwent five freeze-thaw cycles by placing in liquid nitrogen for 15 s followed by heating in a heating block at 30 • C for 3 min and brief vortexing. The lysates were centrifuged (12,000 rpm, 10 min) and the GR concentrations measured using Western blotting used GAPDH as the control.

Statistical Analyses
The data are represented as the means ± SD and, when compared using Student's ttest, p < 0.05 values were statistically significant. All experiments were repeated in triplicate.

Conclusions
Pyxinol, the key pharmacophore of the liver metabolites of ginsenosides [12][13][14], was selected as the core skeleton for drug development in several groups. The effects of the dehydrogenation of pyxinol at C-12 on its potency have been rarely studied. In this study, 24 amino acid residue-conjugated 12-dehydropyinol derivatives were produced and their inhibiting effects on LPS-triggered NO production were assessed. Compared with Y13, which was identified to be the most potent derivative of pyxinol, half of the 12-dehydropyinol derivatives exhibited comparable potencies and several of them even showed much higher potencies than Y13. These results indicate that dehydrogenation at C-12 largely promotes the anti-inflammatory activity of C-3-modified derivatives. The SAR study further alluded that modification of 12-dehydropyxinol at C-3 with an N-Bocprotected aromatic amino acid led to an obvious increase in its anti-inflammatory activity, with 24R being the most preferred. Derivative 5c was then identified as exhibiting the most potent activity and relatively low cytotoxicity. Further studies have indicated that 5c exerted robust GR-independent anti-inflammatory activity by inhibiting the activation of NF-κB and MAPK to suppress IL-1β, TNF-α, and iNOS upregulation. The selective interaction of 5c with JNK is the possible mechanism for inhibiting MAPK activation. These data highlight that 12-dehydropyinol derivatives hold immense drug-development potential owing to their anti-inflammatory activity.