Marine Furanocembranoids-Inspired Macrocycles Enabled by Pd-catalyzed Unactivated C(sp3)-H Insertion Reaction of Donor/Donor Carbenes

Biomimetic modularization and function-oriented synthesis of structurally diversied natural product-like macrocycles in a step-economical fashion is highly desirable. Inspired by marine furanocembranoids, herein, we unprecedentedly synthesized diverse alkenes substituted furan-embedded macrolactams via a modular biomimetic assembly strategy. The success of this assembly is the development of crucial Pd-catalyzed carbene coupling between ene-yne-ketones as donor/donor carbene precursors and unactivated Csp3 ‒ H bonds which represents a great challenge in organic synthesis. Notably, this method not only obviates the use of unstable, explosive, and toxic diazo compounds, but also can be amenable to allenyl ketones carbene precursors. DFT calculations demonstrated that a 1,4-Pd shift could be involved in the mechanism. Moreover, the collected furanocembranoids-like macrolactams showed signicant anti-inammatory activities against TNF-α, IL-6, and IL-1β and the low cytotoxicity is comparable to Dexamethasone.


Introduction
Many natural macrocyclic small-molecules have evolved to interfere with protein-protein interactions, and often have been harnessed as probes for target validation and starting points for lead compounds for drugs discovery. [1][2][3][4][5][6][7] For example, marine cembranoids or furanobutenolide-based cembranoids exhibit a wide range of biological activities, e.g. antitumoral, antimicrobial and anti-in ammatory (Fig. 1A). A deeper examination into their scaffolds re ects that alkene substituted furan is of crucial importance for the biological activities. Despite these valuable function, gene expression limitations of soft corals and di culty of resupply could hamper the sustainability of them. Therefore, the development of new strategies and methods to expeditiously access and enrich diverse natural furanocembranoids-like chemical space is highly desirable. [8][9][10][11][12][13][14] Inspired by natural products or privileged scaffold 15 and our interest in developing new coupling reactions, [16][17][18][19] we set out to create polysubstituted alkene furanembedded macrolactams via a short and modular biomimetic strategy, which simply utilizes either the fundamental building blocks from living organism's endogenous ligands or mimics, such as amino acids or unnatural amino acids. 20-21 A retrosynthetic analysis indicated that a successive and concise Csp 3 -H carbene coupling, and amidation could assemble these readily available building blocks, such as aryl bromides, natural or unnatural amino acids and enynones, faithfully into the target molecules (Fig. 1B).
Over the past decade, transition-metal (e.g., Pd, Rh, Ir)-catalyzed carbene cross-coupling 22 has emerged as a powerful tool for organic chemists to construct polysubstituted alkenes that might be di cult to synthesize by other methods. Concomitant with the rapid growth of this eld is the combination of C-H activation [23][24][25] and carbene cross-coupling due to the remarkable advantage in atom-and step-economy.

Results
Stimulated by the challenges of our synthetic target macrolactams, we rst examined the module assembly by optimizing unactivated Csp 3 -H ole nation. 1-Bromo-2-(tert-butyl)benzene and 3-(4,4dimethylpent-2-yn-1-ylidene)pentane-2,4-dione were selected as the model substrates and a number of reaction parameters such as base, ligand, Pd catalyst, and solvents were screened. After considerable experimentation, we were pleased to discover that a simple cocktail containing [PdCl(allyl)] 2 (5 mol%), t BuXphos (30 mol%), and NaOAc in DMF at 100 °C in 1,4-dioxane established the reaction conditions, affording compound 3aa in 76% isolated yield with high stereoselectivity after 4 h ( Table 1, entry 1). This Csp 3 -H ole nation is distinctive from Martin and co-workers' recent work, 42 in which they described an interesting Pd-catalyzed [4+1] cycloaddition of diazo esters. A series of control experiments were also conducted to validate the role of each parameter. Not surprisingly, the examined parameters were all essential for this transformation. The use of either DIPEA or KOAc did not further improve the yield of the desired product 3aa (Table 1, entries 2 and 3). Notably, the ligand appears to be important, as replacing t BuXphos with Xphos or Brettphos provided 3aa in a much lower yield and no reaction occurred in the absence of ligand (Table 1, entries 6, 7, and 8). In addition, a diminished yield was observed when Pd(MeCN) 4 (OTf) 2 or Pd(OAc) 2 was employed ( Table 1, entries 9 and 10). The in uence of the solvents was also investigated. While similar e ciency was obtained using DMA, only traces of product were obtained in THF and no detectable amount of 3aa could be found in acetonitrile (Table 1, entries 13 and   14).
After determining the optimal reaction conditions, we turned our attention to evaluate the scope of this Pd-catalyzed intermolecular unactivated Csp 3 -H bond insertion reaction with ene-yne-ketones as donor/donor carbene precursors. As shown in Table 2, our Csp 3 -H carbene ole nation method turned out to be widely applicable regardless of the electronic variations at the para and meta positions on the aromatic ring of the aryl bromides (3aa-3ea). Likewise, the naphthyl bromide employed for the synthesis of 3fa served well as a partner in the reaction. Gratifyingly, functional groups on the tertiary alkyls including cyano and ester are compatible (3ga-3ha), although aryl, secondary, and primary alkyls are not reactive probably due to steric hindrance or β-H elimination. [63][64][65][66] Particularly interesting was the observation that the aryl bromide substrate substituted with free amine did not interfere, providing 3ja in a good yield without traces of the N-H bond carbene insertion product being observed. Remarkably, the ene-yne-ketones containing ketone, ester, and heterocyclic ring can be successfully transformed into corresponding products (3ab-3ad) in good to excellent yields (77-91%).
To evaluate the generality of the protocol, alternatively, we investigated this Csp 3 -H carbene ole nation process using allenyl ketones as donor/donor carbene precursors. 47 As illustrated in Table 3, a wide range of allenyl ketones with electron-donating or -withdrawing substituents were well tolerated and a series of alkenes derivatives substituted with dihydrofurans were obtained. Generally, reactions of allenyl ketones with electron-donating substituents attached to a phenyl ring proceeded in higher yields than those having electro-withdrawing groups (5ab, 5ac, 5ad, 5ah). Moreover, the relative con guration of 5ab was unambiguously assigned by the X-ray crystal structure analysis. Particularly, substrates bearing furanyl and thienyl functional groups were also amenable to the standard conditions, which provided the pharmaceutical bis-heterocyclic compounds in decent yields with excellent stereoselectivities.
The identi cation of lead compounds greatly bene ts from fragment-based drug design and the ability to directly modify the privileged scaffolds. Therefore, to highlight the potential application of these Csp 3 -H bond carbene coupling reactions in medicinal chemistry, late-stage cyclization/ole nation of complex and bioactive molecules was subjected to our established protocol. Remarkably, the alkenes substituted with furans and dihydrofurans products derived from Repaglinide, Isoxepac, Mycophenolic acid, Adapalene and Dehydrocholic acid were synthesized in moderate to excellent yields (Table 4). For example, Repaglinide, an antidiabetic drug used to control blood sugar in type 2 diabetes mellitus, had also been installed with 1-bromo-2-(tert-butyl)benzene and subjected into this protocol, gave access to the product 3ka in an excellent 92% yield. Notably, starting from Isoxepac, a non-steroidal anti-in ammatory drug with analgesic activity, which was successfully converted to new furan or dihydrofuran-containing Isoxepac (3la, 5lc) in 84% and 74% yield, respectively.
Once the crucial connection of the aryl bromides and enynone building blocks was successfully established, we next selected different natural or unnatural amino acids and attempted to assemble them to the macrolactams via a short and modular biomimetic strategy. With 6-8 steps, eight novel polysubstituted alkene-embedded macrolactams (6a-6h) were e ciently assembled (Scheme 1). To explore whether these alkene-embedded macrolactams could successfully exhibit pharmacologically relevant features, the macrolactams 6a-6h were investigated for the inhibitory effects on in ammatory mediators by LPS-induced in ammatory responses in RAW 246.7 macrophages. The results showed that 6g exhibited prominent inhibitory effects on the production of TNF-α, IL-6, and IL-1β with IC50 values of 0.45, 1.59, and 0.59 μM, respectively. It should be noted that these pro-in ammatory cytokines are critically involved in the process of in ammation, immunity, cell survival and apoptosis, and metabolic diseases. [67][68][69] Both 6g and 6h were approximately 10 times more potent in the inhibitory activity on IL-6 than the drug Dexamethasone, the widely used corticosteroid medication to relieve in ammation (see the Supporting Information). More importantly, they did not show obvious cytotoxicity at the indicated concentrations compared to Dexamethasone. We further examined the effects of 6g on the levels of phosphorylation of NF-κB and IκB-α induced by LPS in RAW 246.7 cells. As expected, 6g could abrogate the phosphorylation of NF-κB and IκB-α, an NF-κB inhibitory protein, whose phosphorylation results in its degradation and promotes subsequent translocation of NF-κB into nucleus and transcription of in ammatory genes ( Figure 2B). 70 The current preliminary pharmacological results indicated a promising prospect of 6g to be developed as a novel anti-in ammatory agent, with competitive potency and safety advantage.
Apart from the scope of these conversions and intriguing anti-in ammatory activities, we were also interested in the reaction mechanism. Two possible catalytic cycles are shown in Scheme 2. To gain insight into the proposed catalytic cycles and see which cycle is more favorable, we carried out DFT calculations to investigate the detailed mechanism. Although the similar mechanistic pathways have been proposed by others, 42 (Figure 3(b)). Path A involves alkyne-activation cyclization followed by migratory insertion (Cycle A in Scheme 2), while Path B engages protonation rst and then alkyneactivation cyclization (Cycle B in Scheme 2). Clearly, Path A requires to pass through a very high-lying transition state (TS 6-7A ) for the migratory insertion. The high-lying TS 6-7A structure is a result of the unfavorable migration step that involves weakening/breaking of the two strong Pd-C bonds in the 5membered ring of IM6A. Figure 3(c) shows that when the migration insertion occurs on the carbene structure IM7B without a 5membered ring moiety, a very small barrier of 7.5 kcal/mol is calculated. After the migratory insertion, which is highly exergonic, β-hydride elimination occurs easily (almost barrierless), followed by reductive elimination and ligand (aryl bromide) coordination to regenerate the active species A.
The calculation results suggest that Cycle B is favorable. From Figure 3, we can also see that the CMD transition state structure TS 2-3 (Figure 3(a)) and the protonation transition state structure TS 4-5B (Path B in Figure 3(b)) show similar stability, although the latter lies slightly higher than the former. On the basis of the results, TS 4-5B (Path B in Figure 3(b)) is the rate-determining transition state, and therefore, the overall reaction barrier is 34.1 kcal/mol, corresponding to the energy difference between IM1 and TS 4-5B .
The calculated overall energy barrier is moderately high, which is understandable in view of the fact that the reaction temperature is 100 °C. In Figure 3, the series of transformation from IM2 to IM5B corresponds to a 1,4-Pd-shift.
Apart from all of the calculations mentioned above, we also calculated a pathway, which is closely related to Path A but starts from IM4B (instead of IM4) to react with 2a. The calculation results ( Figure   S1) indicate that this pathway is slightly favorable than Path A, but still less favorable than Path B.

Conclusion
In summary, we have developed two types of Pd-catalyzed intermolecular unactivated Csp 3 -H bond insertion reactions by choosing ene-yne-ketones and allenyl ketones as donor/donor carbene precursors, allowing for the construction of a diversity of alkenes substituted with furans and dihydrofurans. These two carbene cross couplings exhibit high e ciency and stereoselectivity, which can be applied to latestage cyclization/ole nation of different therapeutic drugs. DFT mechanistic studies supported that a unique 1,4-Pd shift was involved in the catalytic cycle. Furthermore, alkenes substituted with furans as novel building blocks were successfully assembled via a short and modular biomimetic strategy into macrolactams, which showed signi cant anti-in ammatory activity with less cytotoxicity.

Data availability
All relevant data are available in Supplementary Information, Supplementary Data and from the authors.