Biomimetic synthesis of the natural product salviadione and its hybrids: discovery of tissue-specific anti-inflammatory agents for acute lung injury

Biomimetic synthesis of the natural product salviadione and its hybrids was achieved leading to tissue-specific anti-inflammatory agents for acute lung injury.


Introduction
Acute lung injury (ALI) is an unmet medical need and currently has no effective therapies. 1 It is believed that the inammatory storm is a critical contributor to the occurrence of ALI, 2 and many released pro-inammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) and IL-1b, are key players in the progression of ALI. 3 As such, blocking the excessive production of these cytokines by anti-inammatory agents including corticosteroids represents a promising strategy to prevent and treat this disease. 4 However, most of these agents have no favorable benets to ALI patients due to low efficacy and severe side effects. 5 Therefore, novel anti-inammatory agents with high efficacy and better safety are highly needed for ALI treatment.
Natural products are generally valuable starting points for drug discovery. Tanshinones, such as tanshinone I (1), tanshinone IIA (2), cryptotanshinone (3), and miltirone (4), represent a group of lipophilic quinoidal diterpenes from the traditional Chinese anti-inammatory medicine Salvia miltiorrhiza ("Danshen") ( Fig. 1). 6 The anti-inammatory effect of 2 with therapeutic potential for ALI has been well documented. 7 Nevertheless, further clinical investigation of 2 to treat ALI is seriously impaired by its modest potency and poor pharmacokinetic (PK) properties likely arising from the ortho-quinone moiety. 8 Since ortho-quinone is a major structural determinant for the bioactivity of tanshinones, attempts to replace or mask this moiety oen counteract its bioactivity. 9 Therefore, development of novel chemical approaches to modify the ortho-quinone of 2 both to improve PK properties and to retain or improve the anti-inammatory activity still remains an unsolved challenge.
Salviadione (5) is another lipophilic ingredient also isolated from "Danshen" but lacking the ortho-quinone moiety (Fig. 1). 10 Though 5 was barely studied due to its extremely low natural abundance, its unique structure bearing a benzo[def]carbazole-3,5-dione (BCD) core attracted our attention. Considering the same origin as that of 2, we propose that hybridization of 5 with 2 would deliver a new compound series 6 bearing the furanfused BCD core ( Fig. 1) that may retain or improve the anti-inammatory activity of 2. Although the tetracyclic alkaloid 5 was rst synthesized in 2011 by Söderberg and co-workers with a palladium-catalyzed CO-mediated reductive Nheterocyclization as the key step (Fig. 2a), 11 long reaction steps and harsh reaction conditions make this approach unfeasible for the construction of 5 on a large scale. Since both compounds 5 and 4 were isolated from the same plant, it is likely that 5 is biogenetically derived from 4 under an ammonia atmosphere (Fig. 2b). As such, hybrid 6 might be accessed from 2 through a similar reaction pathway (Fig. 2c). In this report, we rst describe our efforts to establish a biomimetic synthesis of 5 from 4, and then apply this strategy to synthesize 6 from 2 (or its analogues). Subsequently, the anti-inammatory activity of these compounds potentially for treating ALI is investigated as well.

Results and discussion
Biomimetic synthesis of natural product 5 Inspired by our recent success in the C sp 3 -H acyloxylation or aryloxylation of 2, 12 we attempted to convert 4 to 5 through amination with various amines by using TEMPO as the oxidant (Scheme 1). Initially, with ammonia (7.0 M solution in MeOH) or BocNH 2 as the amino source, the reaction failed to give the desired BCD products. Gratifyingly, when benzyl amine was used, the desired N-benzyl product 12 was obtained in 60% yield. Aer optimization of the reaction conditions (Table S1 †), treating the mixture of 4, benzyl amine, and TEMPO in toluene at 120 C was identied as the optimal condition. Unfortunately, subsequent N-deprotection of the benzyl group catalyzed by Pd/C (10%) under a H 2 atmosphere resulted in a complex mixture rather than the expected 5. Alternatively, 4aminophenyl acetate was selected as the amination reagent. Pleasingly, the corresponding benzo[def]carbazole 13 was achieved in 62% yield under the optimal conditions. Subsequent hydrolysis with K 2 CO 3 followed by treatment with PhI(OAc) 2 successfully delivered the natural product 5 in 90% yield. The spectroscopic data of the synthetic 5 were in complete agreement with those of the isolated alkaloid 10,11 (Table S2 †). Thus, by means of a one-pot N-heterocyclization reaction, the biomimetic synthesis of 5 was accomplished from 4 in 56% overall yield over 2 steps.

Synthesis of hybrid derivatives of the natural product 2 with 5
To incorporate the unique structural feature of alkaloid 5 into 2, we designed compound series 6 bearing the benzo[def]carbazole core. As shown in Scheme 2, treating 2 with various anilines under the same reaction conditions as those for 12 provided the N-aryl products 6a-n with furan-fused BCDs in 38-85% yields (Scheme 2). Among them, aniline exhibited the highest yield (85% for 6a). para-Halogenated anilines were found to give high yields (73% for 6b and 80% for 6c). The structure of 6b was unambiguously conrmed by single X-ray crystallographic analysis (Fig. S1 †). Nevertheless, the aniline substrate with the para-triuoromethyl group led to a dramatically decreased yield (38% for 6d). Anilines with electron-donating groups at the ortho-, meta-, and para-positions were well tolerated to provide 6e-j in 50-75% yields. In the case of N-(4-aminophenyl)acetamide, in addition to the major product 6e (50%), a side product 14 with the 2-ene functionality was obtained in 10% yield. 3,4-Di-substituted anilines were also compatible with the reaction and the corresponding products 6k-n were obtained in 51-72% yields.
In addition to anilines, various alkyl amines were found to take part in the reaction as well providing the N-alkyl products 15a-h and 15j-k in relatively lower yields (mostly 35-53% except 15d and 15k), likely due to the high nucleophilic properties of alkyl amines that might undergo other side reactions (Scheme 2). For example, in the case of butan-1amine, byproducts 15i and 15l were isolated in 16% and 20% yields, respectively, together with the major product 15h (38%). Other tanshinone analogues including 3 were also suitable as the substrate to generate the corresponding products 17a-e in 50-75% yields under the standard reaction conditions. In the case of the 17-hydroxyl substrate, the expected product 17b was obtained in 65% yield, together with the 2-ene byproduct 18 in 15% yield.
The isolated byproducts 11a-b, 14, 15i, 15l, and 18 indicated that the reaction may occur through a unique tandem Nheterocyclization process including condensation of 11-ketone of 2 with amines followed by successive imine-enamine tautomerism, intramolecular 1,6-aza-conjugate addition, and dehydrogenative aromatization to generate a key intermediate 11, such as 11a-b and 15i. Further allylic oxidation of 11 with TEMPO provides the nal products. Alternatively, allylic hydroxylation of 11 followed by elimination led to side products 2-enes, such as 14, 15l and 18 (Fig. S2 in the ESI †). Indeed, in our control experiments, treatment of 2 with 4-chloroaniline or benzylamine in an argon atmosphere without oxidants yielded 11a-b in 70% and 65% yields, respectively, which were further transferred to corresponding 6b and 15a by TEMPO, respectively (Scheme S1 †).

In vitro bioactivity of selected hybrid derivatives
Since TNF-a and IL-6 are two well-known pro-inammatory cytokines that mainly contribute to inammation-induced ALI, we rst tested the inhibitory effects of compounds 2, 5, 6a-n, 15a-k and 17a-e at 10 mM against their release induced by LPS in mouse primary peritoneal macrophages. L6H21 (19) 4b was used as the positive control. Most of the synthesized hybrids show markedly improved inhibitory effects (inhibition rates >50%) than the parent compounds 2 and 5 ( Fig. S3 †), while lacking signicant cytotoxic effects on the cell viability at 10 mM (Fig. S4 †), indicating that their anti-inammatory activity was not ascribed to the cell death or injury. Compounds 6b-c, 15a-b, 15f, and 15h suppressed LPS-induced production of IL-6 and TNF-a in a dose-dependent manner (Fig. S5 †), further validating their signicant anti-inammatory effects. Particularly, 15a exhibited the most potent activity against the release of IL-6 and TNF-a with IC 50 values of 1.62 mM and 6.37 mM, respectively. Compound 15a was further found exhibiting negligible hERG inhibition with an IC 50 value greater than 40 mM, indicating its low risk of cardiac toxicity (Table S3 †).

Metabolic and pharmacokinetic properties of 15a
Compare to 2, compound 15a possessed about 4-to 7-fold enhanced metabolic stability in both human and rat microsomes (Table 1). Also, it exhibited markedly improved overall pharmacokinetic (PK) properties with a half-life (T 1/2 ) of 4.05 h and an oral bioavailability (F) of 30.2% (Table S4 †) when compared to those of 2 (T 1/2 ¼ 0.34 h; F < 3.5% (ref. 8a)). A tissue distribution study showed that 15a was mainly distributed in Scheme 2 Synthesis of compounds 6a-n, 15a-k, and 17a-e. the lung with time extending. At 12 h, the lung drug concentration is about 11 to 300 times higher than that in other tissues (Table 2), indicating the selective lung accumulation of 15a suitably for treating ALI.

In vivo anti-ALI effect of 15a
Encouraged by the potent in vitro anti-inammatory activity as well as the promising drug-like properties, the in vivo anti-ALI effect of 15a was further evaluated. As shown in Fig. 3A, the lung wet/dry weight ratio, an index of lung edema, was signicantly increased aer LPS-stimulation, compared with the control group. Nevertheless, pretreatment with 15a at 5 mg kg À1 effectively reduced the lung wet/dry ratio, indicating that 15a suppressed LPS-induced lung edema. The protein concentration in mice bronchial alveolar lavage uid (BALF) is an important indicator for the structural integrity of the alveolar wall. It was found that the protein concentration of mice BALF was markedly increased aer LPS instillation, whereas the increase was signicantly inhibited by pretreatment with 15a (Fig. 3B). Recruitment of neutrophils into the pulmonary compartment is also an important characteristic of ALI. The LPS challenge led to a strong increase in the number of neutrophils in the BALF (Fig. 3D), which was also signicantly decreased by pretreatment with 15a. In addition, the effects of 15a on the histopathological features of ALI mice lungs were also tested ( Fig. 3C and E). Compared to the normal structure of mice lung tissues, the LPS treatment remarkably increased the alveolar wall thickness, hemorrhage, alveolar collapse, and inammatory inltration in the lungs (Fig. 3E, middle panel). As a comparison, the group pretreated with 5 mg kg À1 of 15a displayed very little histopathological changes, when compared to the normal lung structure. In Fig. 3C, the injury score of lung tissues is well correlated with the histopathological changes as shown in Fig. 3E. All the results indicate that 15a effectively attenuates LPS-induced ALI in mice with optimal treatment potential for ALI. Considering that pro-inammatory cytokines play critical roles in the progression of ALI, the production levels of TNFa and IL-6 in the BALF and serum from ALI mice were then examined as biomarkers of the in vivo anti-ALI effects of 15a. As shown in Fig. 4A and B, the levels of TNF-a and IL-6 in the BALF of LPS-induced ALI mice were distinctly suppressed by pretreatment with 15a at 5 mg kg À1 ; in contrast, no signicant inhibition was observed in the serum ( Fig. 4C and D) probably due to the low distribution of 15a in the plasma, demonstrating that 15a possesses a unique anti-inammatory activity selectively in lung tissue, thus probably avoiding potential side effects arising from systematic inhibition of pro-inammatory cytokines.
Besides, 15a markedly suppressed the mRNA expression of pro-inammatory cytokines, including TNF-a, IL-6, and IL-1b, and the adhesion molecule intercellular cell adhesion a Six rats were assigned to each group.  molecule-1 (ICAM-1), in the LPS-treated lung tissues (Fig. S6 †).
To validate the inhibitory effect of 15a against macrophage inltration into the lung, immunohistochemistry analysis of CD68, a macrophage marker, was performed in the lung tissue. Pretreatment with 15a at 5 mg kg À1 signicantly attenuated LPS-induced lung macrophage inltration into the lung, as evidenced by CD68-immunostaining in Fig. 5. These data indicate that 15a improves ALI therapy via its anti-inammatory actions including decreasing inammatory gene expression and inhibiting macrophage inltration.

Conclusions
In summary, we have developed a biomimetic synthetic approach featuring one-pot tandem N-heterocyclization that allows for convenient assembly of the natural product salviadione in 56% overall yield over 2 steps as well as a series of unique hybrids of 5 with 2 bearing a furan-fused BCD core. Most of the resulting compounds exhibited markedly improved in vitro anti-inammatory effects. Particularly, 15a was identied as the most potent with an IC 50 value of 1.62 mM against the release of IL-6 induced by LPS in macrophages. In addition to its negligible cellular and cardiovascular toxicity, when compared to the natural product 2, the derivative 15a exhibited signicantly improved drug-like properties including metabolic stability (4-to 7-fold enhancement), pharmacokinetic properties (T 1/2 ¼ 4.05 h; F ¼ 30.2%) and preferable tissue distribution in the lung (11-to 300-fold selectivity). More importantly, 15a effectively attenuates LPS-induced ALI in mice via lung tissue-specic anti-inammatory actions, thus conrming the furan-fused BCD core as a unique chemotype with ALI therapeutic potential.

Ethical statements
Protocols involving the use of animals were approved by the Wenzhou Medical University Animal Policy and Welfare Committee, and all experiments were performed in compliance with the Animal Ethics Procedures and Guidelines of the People's Republic of China.

Conflicts of interest
There are no conicts of interest to declare.