Unusual Bicyclo[3.2.1]Octanoid Neolignans from Leaves of Piper crocatum and Their Effect on Pyruvate Dehydrogenase Activity

Three undescribed bicyclo[3.2.1]octanoid neolignan glucosides, along with a known neolignan, were isolated from the leaves of Piper crocatum Ruiz & Pav. Their chemical structures were elucidated using extensive spectroscopic analyses including 1D and 2D NMR experiments and HR-ESI-MS analysis, as well as through comparison with previously reported data. Two compounds were assessed for their inhibitory effect against pyruvate dehydrogenase E1α S300 phosphorylation. The fluorescent image suggested that both compounds (60 µM) revealed a stronger inhibition effect than the positive control (dichloroacetate, DCA 5 mM), with IC50 values of 99.82 µM and 80.25 µM, respectively.


Introduction
The genus Piper belongs to the Piperaceae family, considered as one of the largest genera among angiosperms and one of the most abundant species in tropical forests [1]. Several species have high economic value, such as Piper nigrum (black pepper), the representative species of the Piperaceae family used worldwide as a condiment having medicinal properties. Piper species have been widely investigated, and a number of physiological activity components have been isolated, which can be characterized into the typical classes of alkaloids, amides, chalcones, dihydrochalcones, flavanones, lignans, neolignans, propenylphenols, steroids, and terpenes [1]. However, only a few chemical investigations of P. crocatum have been reported, which were limited to sterols, lignans, and neolignans [2][3][4]. In China, people have used the dried stem of Piper kadsura (Choisy) Ohwi as a traditional Chinese medicine to treat asthma and rheumatism for hundreds of years. The species of P. crocatum is commonly used in folk medicine in Indonesia to treat various diseases [2]. Many species of the Piperaceae family have been extensively studied for their potential antitumor, antimicrobial, and antifungal properties [2,5]. Therefore, the chemical constitution of P. crocatum still needs to be investigated because it is a source of novel natural products with potential activities. The metabolic characteristics of neoplastic and non-neoplastic cells are considerably different. However, non-neoplastic cells predominantly depend on ATP/energy produced by pyruvate oxidation in the mitochondria, and each molecular glucose oxidized generates 36 ATPs; whereas proliferating cancer cells predominantly rely on aerobic glycolysis in the cytoplasm, and up to four ATPs are produced for each glucose molecule [6]. Pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase (PDK) are key mitochondrial enzymes in the metabolic pathway of glucose, and their interaction regulates the proportion between aerobic respiration and the Warburg effect [7,8]. Because all tumors rely on metabolic alterations for growth, metastasis, and survival, atypical pathways may be potential targets of antineoplastic drugs.
lignans, propenylphenols, steroids, and terpenes [1]. However, only a few chemical investigations of P. crocatum have been reported, which were limited to sterols, lignans, and neolignans [2][3][4]. In China, people have used the dried stem of Piper kadsura (Choisy) Ohwi as a traditional Chinese medicine to treat asthma and rheumatism for hundreds of years. The species of P. crocatum is commonly used in folk medicine in Indonesia to treat various diseases [2]. Many species of the Piperaceae family have been extensively studied for their potential antitumor, antimicrobial, and antifungal properties [2,5]. Therefore, the chemical constitution of P. crocatum still needs to be investigated because it is a source of novel natural products with potential activities. The metabolic characteristics of neoplastic and non-neoplastic cells are considerably different. However, non-neoplastic cells predominantly depend on ATP/energy produced by pyruvate oxidation in the mitochondria, and each molecular glucose oxidized generates 36 ATPs; whereas proliferating cancer cells predominantly rely on aerobic glycolysis in the cytoplasm, and up to four ATPs are produced for each glucose molecule [6]. Pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase (PDK) are key mitochondrial enzymes in the metabolic pathway of glucose, and their interaction regulates the proportion between aerobic respiration and the Warburg effect [7,8]. Because all tumors rely on metabolic alterations for growth, metastasis, and survival, atypical pathways may be potential targets of antineoplastic drugs.

Discussion
Compound 1 was isolated as a white amorphous powder. Its molecular formula was established as C27H36O11 using HR-ESI-MS. The 1 H NMR spectrum (Table 1) showed the presence of an ABX system of aromatic protons at  To our knowledge, this study is the first to isolate bicyclo[3.2.1]neolignan glucoside derivative compounds from the natural product and macrophyllin-type neolignans from P. crocatum.
Compound 2 was isolated as a colorless gum. The molecular formula was established as C 28 H 38 O 12 using HR-ESI-MS. The 1 H and 13 C spectroscopic data (Table 1) of compound 2 were determined to be identical to those of compound 4 except for that of the glucose moiety, and the signal was assigned to the anomeric proton of the glucosyl group at δ H 4.66 (d, J = 7.2 Hz, H-1"). Thus, the absolute configuration of the C 3 -bicyclooctane moiety of compound 2 was the same as that of compound 4, and the absolute configurations were confirmed using X-ray [3]. Based on the abovementioned data, the absolute configuration of compound 2 was determined as (1 R,2 R,3 S,7S,8R)-∆ 5 ,8 -2 -hydroxy-3,5,3 ,5tetramethoxyl-4 -oxo-8. Compound 3 was isolated as a colorless gum. The molecular formula was established as C 28 H 38 O 12 using HR-ESI-MS. The 1 H NMR spectrum data (Table 1) , H-7 ). The 1 H NMR chemical shift of the C-8 methyl group at δ H 1.28 (d, J = 7.0 Hz, H-9) and other compounds indicated a transrelationship between the C-7 aryl and C-8 methyl groups. However, the 13 C NMR spectrum showed one quaternary carbon δ C 86.2 (C-1 ) along with olefinic carbons at δ C 139.5 (C-5 ) and 156.5 (C-6 ), which were considerably different from the other three compounds. The HMBC correlations further confirmed these connections among the bicyclooctane moiety through colo1 -OMe (δ H 3.34)/C-1 (δ C 86.2), which indicated the deshielding effect of C-1 (δ C 86.2) compared with the other three compounds. Together with H-7 (δ H 2.92 and 2.99), C-5 (δ C 139.5), C-6 (δ C 156.5), and C-4 (δ C 199.1) confirmed that the allyl group was connected to an olefinic carbon C-5 (δ C 139.5). Subsequently, by comparing with the reported literature [6,11], compound 3 was considered to comprise a macrophyllin-type bicyclo Reports from the literature were compared [12][13][14] for the total synthesis of bicyclo[3.2.1]octanoid neolignans. The speculated biosynthetic pathways are in the Supplementary Materials.

PDH Inhibition Effect
PDH is essential in the pathway of glucose metabolism and ATP production. Suppression of PDH reduces the aerobic oxidation of pyruvate and promotes its transformation to lactate in cytoplasm. Additionally, decreased flux of pyruvate via PDH diminishes the amount of acetyl-CoA entering into the TCA cycle. Accordingly, defects in both PDH and oxidative phosphorylation activities result in lactic acidosis and initiation of the Warburg effect [7]. The activity of PDH is mainly controlled by reversible phosphorylation of the E1α subunit on residue sites serine 293 (Site 1), 300 (Site 2), and 232 (Site 3), which are mediated by PDK and pyruvate dehydrogenase phosphatase (PDP) phosphorylation of E1α by PDKs, which inactivates PDH, whereas PDP exhibits active/reactive catalytic activity by dephosphorylating the complex [15]. Compounds 2 and 3 were evaluated for their inhibitory effect against PDHE1α S300 phosphorylation; we discuss the phosphorylation inhibitory effect of the E1α subunit on residue site serine 300 (Site 2). Fluorescent image results suggested that compounds 2 and 3 inhibited phosphorylation of E1α S300 at 60 µM with better effect than that of dichloroacetate (DCA) at 5 mM concentration; compound 2 showed the strongest effect among all three compounds (Figure 3).
Although previous research on DCA was promising, its application to anticancer treatment is limited by its non-specificity, low potency, and requirement for high doses to exhibit therapeutic effects, which always leads to peripheral neurological toxicity. The position exchange of the allyl group on C-1 and methoxyl group on C-5 resulted in a higher IC 50 value difference between compounds 2 and 3 and revealed a better inhibitory effect of compound 2 than that of compound 3 and DCA in fluorescent imaging. Although compounds 2 and 3 possessed a rather high toxicity, the consumption of these two com-Plants 2021, 10, 1855 6 of 9 pounds was 83 times lower than that of DCA. The described type of neolignan compounds is one of the major components of Piper plants.
tion of the E1α subunit on residue sites serine 293 (Site 1), 300 (Site 2), and 232 (Site 3), which are mediated by PDK and pyruvate dehydrogenase phosphatase (PDP) phosphorylation of E1α by PDKs, which inactivates PDH, whereas PDP exhibits active/reactive catalytic activity by dephosphorylating the complex [15]. Compounds 2 and 3 were evaluated for their inhibitory effect against PDHE1α S300 phosphorylation; we discuss the phosphorylation inhibitory effect of the E1α subunit on residue site serine 300 (Site 2). Fluorescent image results suggested that compounds 2 and 3 inhibited phosphorylation of E1α S300 at 60 µM with better effect than that of dichloroacetate (DCA) at 5 mM concentration; compound 2 showed the strongest effect among all three compounds ( Figure  3).   Although previous research on DCA was promising, its application to anticancer treatment is limited by its non-specificity, low potency, and requirement for high doses to exhibit therapeutic effects, which always leads to peripheral neurological toxicity. The position exchange of the allyl group on C-1′ and methoxyl group on C-5′ resulted in a higher IC50 value difference between compounds 2 and 3 and revealed a better inhibitory effect of compound 2 than that of compound 3 and DCA in fluorescent imaging. Although compounds 2 and 3 possessed a rather high toxicity, the consumption of these two compounds was 83 times lower than that of DCA. The described type of neolignan compounds is one of the major components of Piper plants.

Extraction and Isolation
The dried leaves of P. crocatum (2.6 kg) were reflux extracted with MeOH (5 L × 3 times). The total extraction (400.0 g) of MeOH was suspended in deionized water and partitioned with hexane, and water fraction. Then the water fraction was partitioned sequentially with EtOAc and BuOH, yielding EtOAc (1A, 16.1 g), BuOH (1B, 65.0 g). The EtOAc fraction was subjected to a silica gel column chromatography with a gradient of

Pyruvate Dehydrogenase Complex (PDH) Cellular Activity
Point two percent gelatin was added to a black 96-well plate with a clear bottom and incubated for 1 h. Afterwards, the plate was washed with growth media. Human AD-293