The three-dimensional structural motifs of natural products [
1] are increasingly recognized for their significance in chemical biology [
2,
3] and drug discovery [
4]. Polycyclic natural products containing a bicyclo[3.2.2]nonane motif (highlighted in red in
Fig. 1) [
5–
7], such as goupiolone B [
5], phleghenrine A [
6], and acremoxanthone A [
7] (
1–
3,
Fig. 1), present a formidable challenge to chemical synthesis. A variety of methods have been developed to construct such a structural motif in natural products and related compounds [
8–
17]. However, many of them involve multi-step sequences [
8–
13]. Strategically, the intermolecular Diels–Alder cycloaddition of tropone derivatives and olefins (highlighted in blue in
Fig. 1) offers a desirable approach for assembling bicyclo[3.2.2]nonanes [
15–
17]. Benzo[2,3]tropones [
18] would be advantageous substrates for the [4 + 2] cycloaddition [
19,
20] because they are not prone to undesired [6 + 4] or [8 + 2] cycloaddition [
21,
22] compared to standard tropones. However, most reported examples are indeed formal [4 + 2] reactions [
5,
23–
25] proceeding
via enolate intermediates, which limit the applicability [
5,
24] and sometimes result in unsatisfactory efficiency [
25]. To our knowledge, the inverse-electron-demand Diels–Alder (IEDDA) reaction [
26,
27] involving benzo[2,3]tropones remains largely unexplored. Following our continued interest in the Diels–Alder cycloaddition for complex molecule synthesis [
28–
36], we initiated an investigation into the IEDDA reaction of benzo[2,3]tropone-type dienes with electron-rich dienophiles and explored its application in the synthesis of polycyclic natural products and biologically active molecules, particularly autophagy regulators [
37,
38] and cell cycle inhibitors [
39,
40]. Herein, we report the assembly of densely substituted bicyclo[3.2.2]nonanes
via such a cycloaddition reaction, which leads to the discovery of an autophagy suppressor.
We first explored the cycloaddition of 6-methoxycarbonylbenzo[2,3]tropone (
4) and cyclopentadiene (
5) (
Table 1). Notably, Ghosh and co-workers demonstrated that benzo[2,3]tropone acted as a dienophile in a normal-electron-demand Diels–Alder (NEDDA) reaction with
5 [
41]. Introducing the methoxycarbonyl group would not only provide a functional handle for further derivatization of the product(s), but also enhance the dienic reactivity of benzo[2,3]tropone in the IEDDA reaction. To our delight, thermal treatment of
4 and
5 at 80 ℃ afforded the desired cycloadducts (
6a and
6b) in a
ca. 18:1 ratio (entry 1), with a strong preference for the
endo product. The position of the C═C bond within the cyclopentene ring of each product was determined by X-ray crystallographic analysis (
Table 1). The excellent
endo selectivity may be attributable to secondary orbital interactions [
42]. Despite complete suppression of the NEDDA reaction (
Table 1), a substantial portion of
4 was recovered, which resulted in reduced efficiency of the IEDDA reaction (entry 1). Thus, we turned to Lewis acids to lower the LUMO energy of
4. As shown in
Table 1 (entries 2–4), BF
3·OEt
2, Me
2AlCl, and Bi(OTf)
3 significantly increased the dienic reactivity of
4 but concomitantly compromised the
endo/
exo selectivity. Our experience with lanthanide-mediated Diels–Alder cycloaddition [
29,
32,
36] prompted us to examine europium-based promoters for this reaction. Eu(OTf)
3 improved both efficiency and selectivity (entry 5), while Eu(fod)
3 rendered the optimal results (92% combined yield,
endo: exo > 20:1) (entry 6). Switching the solvent from CH
2Cl
2 to THF markedly reduced the yield of the desired products (entry 7).
Having established the optimal conditions, we investigated the substrate scope of the IEDDA reaction (
Table 2). As shown in entries 1–4, the cycloaddition of
4 with various cyclic dienes demonstrated high
endo selectivity. 6,6-Dimethylfulvene (
7) performed well in this reaction; cycloadducts
8a and
8b (
ca. 10:1) were isolated in 82% combined yield (entry 1). Despite steric hindrance, TMS-cyclopentadiene (
9) reacted smoothly with
4 to afford compound
10 as the sole detectable diastereomer in 67% yield (entry 2). Retention of the TMS group in
10 supported a concerted rather than a stepwise mechanism. 1,2,3,4-Tetramethyl-1,3-cyclopentadiene (
11) could equilibrate
in situ to its positional isomer through a [1,5]-H shift [
32,
43], which subsequently underwent cycloaddition to form
12, a remarkably crowded tetracyclic compound, in 44% yield (entry 3). The IEDDA reaction between 1-siloxycyclohexadiene
13 and
4 afforded
14a and
14b (
ca. 6:1) in 90% combined yield (entry 4). Due to the lower reactivity of mono-alkenes, the stronger promoter Bi(OTf)
3 was required for their cycloaddition with
4 (entries 5 and 6). For cyclopentene (
15), the
exo product (
16) was obtained as the single detectable diastereomer in 66% yield (entry 5). For norbornene (
17), a mixture of the
exo and
endo products (
18a and
18b;
ca. 2:1) was isolated in 68% yield (entry 6). Notably, two analogs of
4 (
19 and
20), bearing different substituents on the benzene ring, underwent smooth cycloaddition with
5 to give
endo products
21 and
22, respectively, as single diastereoisomers (entries 7 and 8). The structures of
12, 16, 18a, and a derivative of
14a were confirmed by X-ray crystallographic analysis (
Fig. 2).
We further derivatized the obtained bicyclo[3.2.2]nonane-containing compounds to identify biologically active candidates. A representative example is illustrated in
Scheme 1. Selective epoxidation of
6a with
m-CPBA in the presence of NaHCO
3 afforded compound
23 in 72% yield. Acidic hydrolysis of the epoxide, followed by oxidative cleavage of the resulting diol with aqueous NaIO
4, provided bis-aldehyde
24, which underwent double reductive amination [BnNH
2, NaBH
3CN, HOAc] to furnish tetracyclic amine
25 in 74% overall yield.
In light of our sustained interest in modulating autophagy with small molecules [
37,
38], we evaluated the effect of the above compounds on autophagic flux. The autophagy marker microtubule-associated protein light chain 3B (LC3B) [
44,
45] was analyzed in human lung carcinoma cells (A549) by immunoblotting. As shown in
Fig. 3A, compound
25 induced a dose-dependent increase in the levels of LC3B-II, the phosphatidylethanolamine-conjugated form of LC3B, which pointed to this compound as a potential regulator of autophagy. To investigate whether compound
25 stimulates autophagy upstream or inhibits it downstream, we compared LC3B-II levels in A549 cells treated with
25 alone
versus those co-treated with
25 and the autophagy inducer rapamycin (RAPA) [
44,
45]. The co-treatment significantly increased LC3B-II levels (
Fig. 3B), which suggested a suppressive role for this compound in downstream autophagy. Autophagic flux was further assessed by using mCherry–eGFP–LC3B [
46], a tandem fluorescent protein-tagged LC3 reporter. In A549 cells expressing this reporter, the fluorescence pattern of LC3B-positive puncta revealed that treatment with
25 led to an accumulation of autophagosomes and a reduction in autolysosomes (
Fig. 3C), which indicated that this compound inhibits autophagy at a downstream stage. These findings highlight this compound as a promising chemical scaffold for developing autophagy modulators [
47].
In conclusion, we developed a facile IEDDA approach to densely substituted bicyclo[3.2.2]nonanes, which enabled the discovery of an autophagy regulator. Exploration of the synthesis of bicyclo[3.2.2]nonane-containing natural products via this method is currently underway in our laboratories.
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