New lathyrane diterpenoid hybrids have anti-inflammatory activity through the NF- κ B signaling pathway and autophagy

The lathyrane diterpenoid/3-hydroxyflavone hybrid 8d1 shows potent anti-inflammatory activities, which could serve as a promising anti-inflammatory agent. ABSTRACT In our ongoing work on the identification of potent anti-inflammatory agents, we designed and synthesized three series of lathyrane diterpenoid hybrids in which the lathyrane diterpenoid skeleton was hybridized with other anti-inflammatory pharmacophores. Unexpectedly, lathyrane diterpenoid/3-hydroxyflavone hybrids showed more potent anti-inflammatory activity in RAW264.7 cells than did the corresponding parent compounds. Compound 8d1 exhibited potent anti-inflammatory activity with low cytotoxicity (IC 50 = 1.55 ± 0.68 μM), and downregulated LPS-induced expression of iNOS and COX-2, as well as I κ B α phosphorylation. This compound also inhibited the expression and nuclear translocation of NF- κ B, and stimulated autophagy induction. Thus, 8d1 ’s anti-inflammatory mechanism is associated with inhibition of the NF- κ B signaling pathway and increasing autophagy. This compound may serve as a promising anti-inflammatory agent.


In brief
The lathyrane diterpenoid/3-hydroxyflavone hybrid 8d1 shows potent anti-inflammatory activities, which could serve as a promising anti-inflammatory agent.

INTRODUCTION
Inflammation is a complex response of the body to defense harmful stimuli [1]. After immune cells are stimulated, the NF-κB pathway, together with associated pathways such as the MAPK, ERK1/2 and JNK pathways, activates target genes and releases inflammatory mediators [2,3]. During the process, excess inflammatory mediators can lead to chronic or acute inflammatory diseases, thus posing a serious threat to human health [4].
Autophagy is a complex evolutionarily conserved process involving the degradation of damaged organelles, misfolded proteins and pathogens in cells in response to various stress reactions [5]. Autophagy can be classified as macroautophagy, microautophagy or chaperone-mediated autophagy according to how intracellular substrates enter lysosomes. Beyond its role in maintaining biological homeostasis, autophagy is involved in various diseases, such as inflammatory diseases, infectious diseases and diabetes [6,7]. Whereas excessive autophagy may lead to a persistent inflammatory state that exacerbates disease [8], appropriate autophagy alleviates the inflammatory response by inhibiting the assembly and activation of NLRP3 inflammasomes [9,10], and inducing the transformation of macrophages into M2-type cells with anti-inflammatory effects [11].
Active natural products are important lead-compound sources for the development of therapeutic drugs [12,13]. The terpenes are among the most promising medicinal natural products, owing to their diverse structure and abundant sources. Terpenes are often used as lead compounds in the synthesis of drugs based on natural compounds. Some macrocyclic or polycyclic diterpenoids have various biological activities, such as anticancer [14], multidrug resistance reversal [15] and antiviral [16] effects. In recent years, some lathyrane diterpenoids have been found to display favorable anti-inflammatory activity (Figure 1). Researchers have explored these compounds' anti-inflammation mechanisms and performed in vivo experiments to verify their effectiveness [17][18][19][20][21][22][23].
Hybridization of two of the same or different pharmacophore types into a new molecule can enhance bioactivity or decrease adverse effects, because hybrid molecules may have new mechanisms of action. Many hybrid molecules have been found to exhibit improved bioactivity over the original pharmacophores, thus revealing that hybridization is a useful strategy to develop novel drugs [24,25]. Hybridizing a lathyrane diterpenoid skeleton with other anti-inflammatory pharmacophores has the potential to provide novel anti-inflammatory candidates [26].
Herein, we designed and synthesized three series of epoxylathyrol and lathyrol hybrids in which the lathyrane diterpenoid skeleton was hybridized with other anti-inflammatory pharmacophores. The inhibitory activity toward lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells and the mechanisms of these hybrids were investigated. These findings encouraged us to investigate appropriate methods for modifying lathyrane diterpenoids.
To maintain the high anti-inflammatory activity of derivatives, the esterification of C-5-hydroxyl was selected as the initial modification method (Scheme 1) on the basis of our previous work [18]. Lathyrol (and epoxylathyrol) was prepared according to a previously described method [36], and this was followed by the esterification of C-5 hydroxyl with chloroacetic acid to obtain compound 1. The chlorine atom of 1 was substituted by an azide group via reacted with sodium azide to obtain compound 2. We selected several anti-inflammatory pharmacophores, compounds 5a-g (aspirin [37], isatin [26], flavonoid [38], cinnamic acid [39] and chalcone [40], respectively) with active hydrogen and reacted them with propargyl bromide to obtain

Evaluation of anti-inflammatory activity and structure activity relationships
Nitric oxide, an inflammatory mediator, is produced in response to pathogen-associated molecular patterns [42]. The excessive NO levels produced during acute or chronic inflammation are responsible for tissue injury, either directly or indirectly [43]. All prepared compounds were tested for their inhibitory activity toward LPSinduced NO production in RAW264.7 cells. The anti-inflammatory activity of series 1 is described as the IC 50 values of NO inhibition rates in Table 1. Among the tested compounds, 8d showed the strongest activity against LPS-stimulated NO release (IC 50 value: 0.91 ± 1.38 μM).
The preliminary structure activity relationships (SARs) indicated that our strategy was successful: the anti-inflammatory effects were improved through combination of lathyrane diterpenoid with other anti-inflammatory pharmacophores through 1,2,3-triazole. Approximately three-quarters of the lathyrol hybrids showed better inhibitory activity than Euphorbia factor L 3 . In contrast, only one-quarter of the epoxylathyrol hybrids showed more potent inhibitory activity than Euphorbia factor L 1 . Meanwhile, two 3-hydroxyflavone derivatives clearly exhibited potent anti-inflammatory efficacy, in sharp contrast to aspirin and acetaminophen. However, the anti-inflammatory activity of 3-hydroxyflavone was not strong (IC 50 >20 μM). To further improve the anti-inflammatory activity of lathyrane diterpenoid/3-hydroxyflavone hybrids, we synthesized series 2 and 3, in which the linker and flavonoid was changed, respectively.
The general synthetic route of series 2 is provided in Scheme 2. In this series, the 1,2,3-triazole linker was replaced by a linear linker with a different length. Moreover, the effects of chemical bonds between linkers and pharmacophores or lathyrane diterpenoids on the activity of hybrids were also investigated. In series 3, the 3-hydroxyflavone of 8d was replaced by quercetin, kaempferol and farrerol (Scheme 3). The mono-substituted intermediates 8h-j were obtained by controlling the equivalent of bromopropyne and potassium carbonate. The attachment position of the propynyl group was determined on the basis of heteronuclear multiple bond correlations.

Toxicity
We evaluated the cytotoxicity of compounds 8d and 8d1 toward RAW264.7 cells. As shown in Figure 4, compound 8d showed clear cytotoxicity at a 12.5 mM concentration. In contrast, compound 8d1 showed lower cytotoxicity than 8d at the same concentration.
Our results revealed that 1,2,3-triazole enhanced the cytotoxicity of compounds while increasing their activity. Compound 8d1 was selected for further pharmacological study.

Compound 8d1 suppresses LPS-induced iNOS and COX-2 expression
We next explored the anti-inflammatory mechanism of compound 8d1. In the inflammatory process, increases in iNOS and COX-2 lead to the production of various cytokines and inflammatory mediators, such as NO, TNFα, IL-1β and IL-6. INOS, which catalyzes the expression of NO, is usually upregulated and expressed by macrophages in response to inflammatory stimuli. COX-2 was a major target for the discovery of nonsteroidal anti-inflammatory drugs, and it participates in the arachidonic acid cascade [44]. Thus, we analyzed the inhibitory effects of 8d1 on LPS-mediated expression of iNOS and COX-2 by using western blotting ( Figure 5A). As expected, LPS stimulation markedly increased iNOS and COX-2 protein expression, and compound 8d1 significantly and completely inhibited the high expression of iNOS ( Figure 5B) and decreased the expression of COX-2 ( Figure 5C) induced by LPS.

Compound 8d1 inhibits the NF-κB pathway in LPS-induced RAW264.7 cells
NF-κB is a member of a transcription factor family that controls the expression of genes associated with inflammatory, apoptosis and immune responses [45]. NF-κB and the members of its signaling pathway play essential roles in many stages of inflammatory diseases [46]. In the cytoplasm, IκB binds NF-κB, thus masking the nuclear localization signal and inhibiting the activity of NF-κB [47,48]. However, the phosphorylation of IκBα activates, and leads to the nuclear translocation of, NF-κB [49]. To further assess whether 8d1 exerted an anti-inflammatory effect through the NF-κB signaling pathway, we pretreated cells with different concentrations of 8d1 before induction with LPS. The ratio of p-NF-κB/NF-κB and p-IκBα/IκBα significantly decreased with 8d1 preprotection, thus indicating the inhibition of NF-κB signaling pathway (Figure 6A-C). The translocation of NF-κB in LPS-stimulated RAW264.7 cells was detected by western blotting and immunofluorescence. After treatment with LPS, NF-κB translocated into the nucleus, but NF-κB nuclear translocation was blocked by 8d1 at 10 μM ( Figure 6D-F). These data demonstrated that 8d1 negatively regulated NF-κB and blocked the nuclear translocation of NF-κB in RAW264.7 cells induced with LPS.

Compound 8d1 activates autophagy in LPS-induced RAW264.7 cells
Autophagy is regulated by various autophagy proteins, such as LC3B, Beclin 1 and P62. P62 affects autophagy by participating in autophagy-lysosomal protein degradation [50]. LC3B plays an important role in autophagosome bilayer membrane elongation and substrate recognition [51]. Moreover, the expression of LC3B is closely associated with the number of autophagosomes; therefore, the ratio of LC3B II/LC3B I is often used to represent the level of autophagy.
To examine the effect of 8d1 on autophagy in LPSinduced RAW264.7 cells, we incubated cells with different concentrations of 8d1 (1.25, 2.5, 5 and 10 μM). The changes in autophagy proteins levels were observed through western blotting. Compound 8d1 increased the ratio of LC3B II/LC3B I, and decreased the level of P62 in a concentration-dependent manner ( Figure 7A). Simultaneously, the autophagy inhibitor chloroquine (CQ) was used to block the binding of autophagosomes and lysosomes to observe the changes in autophagosomes. After treatment with 10 μM 8d1, the number of autophagosomes significantly increased ( Figure 7B). Immunofluorescence experiments also confirmed the above results ( Figure 7C).

CONCLUSION
In summary, we designed and synthesized three series of epoxylathyrol and lathyrol hybrids, in which the lathyrane diterpenoid skeleton was hybridized with other anti-inflammatory pharmacophores. Many compounds displayed favorable inhibitory activity toward LPS-induced NO production in RAW264.7 cells. The preliminary SARs illustrated that 3-hydroxyflavone significantly enhanced the anti-inflammatory activity of two lathyrane diterpenoids, although its own activity was not strong. Meanwhile, different linkers and the chemical bond between the linker and lathyrane diterpenoid influenced the activity of hybrids.
Among all hybrids, compound 8d1 exhibited potent anti-inflammatory activity with low cytotoxicity. Further studies revealed that 8d1 exerted anti-inflammatory effects by decreasing COX-2 and iNOS production, inhibiting the activation of NF-κB and inducing autophagy. These findings indicated that compound 8d1 may serve

Chemistry
All starting materials and solvents used in the synthesis were obtained from commercial sources and used without further purification. Reactions were monitored through thin-layer chromatography on silica gel plates (GF 254 , Qingdao Haiyang Chemical Co. Ltd. China) and visualized under ultraviolet light. 1 H NMR and 13 C NMR spectra were obtained on a Bruker AVANCE 400 or 600 spectrometer (Bruker Instruments Inc. Germany). Chemical shifts are expressed in δ values (ppm) relative to TMS, and coupling constants are reported in Hertz.

Synthesis of lathyrol and epoxylathyrol.
Lathyrol and epoxylathyrol were synthesized according to our previous method [36].

Synthesis of compounds 1 and 3.
Lathyrol (0.60 mmol) was dissolved in DCM (3 mL), and then chloroacetic acid (0.72 mmol), EDCI (0.72 mmol) and DMAP (0.06 mmol) were successively added to the solution. The reaction was stirred for approximately 8-10 h at room temperature, quenched with saturated NH 4 Cl solution and extracted with DCM. The combined organic layers were dried over Na 2 SO 4 , filtered and evaporated, thus yielding the crude product of compound 1 as a white solid (72% yield). This procedure was also applied to the preparation of compound 3 (by epoxylathyrol, white solid, 70% yield).

Synthesis of compounds 2 and 4.
To a solution of compound 1 (1.0 eq) in DMF, NaN 3 (3 eq) was added at room temperature. The reaction was heated to 60°C and stirred for approximately 6 h. After completion of the reaction, EtOAc was added to the mixture. The organic layer was washed with brine three to five times, then dried over Na 2 SO 4 , filtered, concentrated under reduced pressure and purified by CC, thus yielding compound 2 as a white solid. This procedure was also applied to the preparation of compound 4 (from compound 3).

Synthesis of compounds 9 and 10.
Compounds 9 and 10 were synthesized according to our previous method [36].

Synthesis of compounds 11 and 12.
Compounds 11 and 12 were synthesized according to our previous method [18].

Synthesis of compounds 6d1 and 6d2.
Compound 6d1 was synthesized from 5d and methyl bromoacetate according to the synthetic method for compounds 6a-g.
The crude product of 6d1 (0.8 mmol, 1.0 eq) was dissolved in THF (30 mL), and LiOH·H 2 O aqueous solution (10 mL, 1.6 mmol, 2.0 eq) was then added to the solution. The mixture was stirred for 3-4 h at room temperature. After completion of the reaction, THF was removed by reduced pressure. The residue was diluted with water, and the pH was slowly adjusted to 2-3 with 1 M HCI. The resulting solid was filtered and washed with water, then dried in an oven, thus yielding 6d2 as a white solid (75% for two steps).

Synthesis of compounds 6d3 and 6d4.
Compound 6d3 was synthesized from 5d and 1,3-dibromopropane according to the synthetic method for compounds 6a-g. Compound 6d4 was synthesized from 6d3 according to the synthetic method for compound 2.

Synthesis of compounds 6d5 and 6d6.
Compound 6d5 was synthesized from 5d and N-Boc-glycine according to the synthetic method for compound 1.
Compound 6d5 (0.34 mmol) was dissolved in DCM (2 mL), and the reaction was placed in an ice bath. TFA (2 mL) was added to the solution after 15 min. The reaction was stirred for 30 min. After completion of the reaction, THF and DCM were removed by reduced pressure. The crude product (6d6's TFA salt) was used in the following reactions without further purification (95% yield).

Synthesis of compounds 7d1 and 8d1.
Compound 7d1 (or 8d1) was synthesized from 9 (or 10) and 3-hydroxyflavone according to the synthetic method for compound 1.