Structurally Diverse Sesquiterpenoids from the Endangered

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Cite This: J. Nat. Prod. 2018, 81, 2195−2204

Structurally Diverse Sesquiterpenoids from the Endangered Ornamental Plant Michelia shiluensis Juan Xiong,†,# Li-Jun Wang,†,# Jianchang Qian,‡ Pei-Pei Wang,§ Xue-Jiao Wang,† Guang-Lei Ma,† Huaqiang Zeng,⊥ Jia Li,§ and Jin-Feng Hu*,†

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†

Department of Natural Products Chemistry and ‡Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, People’s Republic of China § State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China ⊥ Institute of Bioengineering and Nanotechnology, The Nanos 138669, Singapore S Supporting Information *

ABSTRACT: A preliminary phytochemical investigation on the MeOH extract of the leaves and twigs of the endangered ornamental plant Michelia shiluensis led to the isolation of 16 sesquiterpenoids. The isolated compounds comprised germacrane- (1−4, 13, 14), guaiane- (5−9, 15), amorphane- (10), and eudesmane-type (11, 12, 16) sesquiterpenoids. The new structures (1−12) were elucidated by spectroscopic and computational methods, and their absolute configurations (except for 9) were assigned by single-crystal X-ray diffraction crystallographic data and/or electronic circular dichroism spectra. Shiluolides (A−D, 1−4) are unprecedented C16 or C17 homogermacranolides, and their putative biosynthetic pathways are briefly discussed. Shiluone D (8) is a rare 1,10-secoguaiane sesquiterpenoid featuring a new ether-containing spirocyclic ring, whereas shiluone E (9) represents the first example of a 1,5−4,5-di-seco-guaiane with a rare 5,11-lactone moiety. Shiluone F (10) is the first amorphane-type sesquiterpenoid possessing an oxetane ring bridging C-1 and C-7. Bioassay evaluations indicated that lipiferolide (13) showed noteworthy cytotoxicities toward human cancer cell lines MCF-7 and A-549, with IC50 values of 1.5 and 7.3 μM, respectively. Shiluone D (8) exerted inhibition against protein tyrosine phosphatase 1B (IC50: 46.3 μM). Michelia is a genus in the Magnolia family (Magnoliaceae) that encompasses around 50 recorded species of evergreen trees and shrubs widespread in tropical and subtropical south and southeast Asian countries. There are approximately 41 species distributed in Southern China.2 Michelia plants not only are useful as timbers and garden plants but also have various medical benefits such as anticancer, anti-inflammation, and antipyretic.3 Previous studies revealed that Michelia species are rich in cytotoxic sesquiterpene lactones.4 Michelia shiluensis Chun et Y. F. Wu is endemic to China.4 This indigenous species earned its name because the plant was first identified in Shilu, a town on Hainan Island.2,5 It is usually found in evergreen broad-leaved forests and can grow up to 18 m or even taller in its native habitat. The population of M. shiluensis is thought to have declined by more than 50% over the past decade.5 It has been regarded as “endangered” by the International Union for Conservation of Nature (IUCN).5 This plant has also been nationally protected in China at the “second-grade”.6 Nowadays, the wild type can be found in fewer than five locations on Hainan Island,5 but people are planting these as ornamentals. Historically, rare and endangered plants are better sources for producing nature-derived drugs.7 As part of an ongoing © 2018 American Chemical Society and American Society of Pharmacognosy

research program on rare and endangered ornamental plants endemic to China,1a−c,f−h a sample (including the leaves and twigs) of M. shiluensis cultivated at a botanical garden was phytochemically investigated. As a result, a number of structurally diverse sesquiterpenoids were isolated and identified (Figure 1). Among them, 12 are new, including four homogermacranolide (1−4), three guaiane (5−7), two seco-guaiane (8, 9), one amorphane (10), and two enteudesmane (11, 12) sesquiterpenoids. Also isolated were lipiferolide (13),8 epitulipinolide diepoxide (14),8 7-epi-11hydroxychabrolidione A (15),9 and carissone (16).10 The cytotoxic and protein tyrosine phosphatase 1B (PTP1B)1c inhibitory activities of these isolates were evaluated.

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RESULTS AND DISCUSSION Shiluolide A (1) was crystallized as colorless needles in nhexane−CH2Cl2 (2:1, v/v). Its molecular formula (C18H26O6) was assigned based on the HRESIMS (m/z 361.1625 [M + Na]+, calcd 361.1622) and 13C NMR data. The IR spectrum Received: May 14, 2018 Published: October 5, 2018 2195

DOI: 10.1021/acs.jnatprod.8b00386 J. Nat. Prod. 2018, 81, 2195−2204

Journal of Natural Products

Article

carbon signals attributable to an acetyl substituent (δ 169.7, 21.3). Among these resonances, a lactone carbonyl (δ 175.4), a trisubstituted double bond (δ 127.7, 131.7), and an oxirane ring (δC 61.9, 67.5) could be distinguished. The above NMR data resembled those of lipiferolide (13), a co-occurring known germacranolide sesquiterpenoid with a regular C15backbone previously isolated from Liriodendron tulipifera and Anthemis cupaniana.8 The major difference between 1 and 13 is that the exomethylene group of the α-methylene-γ-lactone moiety in 13 was replaced by an ethyl and a tertiary hydroxy group at C-11, implying a homogermacrane-type sesquiterpenoid lactone with 16 carbons for 1. This was confirmed by the observation of an isolated ethyl group spin system [δH 1.84/ 1.87 (dq, J = 14.0, 7.0 Hz), CH2−13; δH 0.99 (t-like, J = 7.0 Hz), Me-16] in its COSY spectrum and the HMBC correlations from the terminal Me-16 to C-11 (δ 78.1) and from H2-13 to C-7 (δ 50.5) and C-12 (δ 175.4) (Figure 2). Figure 1. Chemical structures of compounds 1−16.

displayed typical absorptions for a saturated γ-lactone (νmax 1783 cm−1) and an acetate group (νmax 1722 cm−1).11 Its 1H NMR data showed resonances of four methyls [δ 0.99 (t, Me16); 1.36 (s, Me-15); 1.73 (br s, Me-14); 2.05 (s, −MeCO)], three oxymethine protons [δ 2.70 (H-5); 4.56 (H-6); 5.53 (H8)], and one olefinic proton (δ 5.33, H-1) (Table 1). The 13C and DEPT NMR data revealed that the core structure of 1 contains 16 carbons (including three methyls, four methylenes, five methines, two oxygenated tertiary and one quaternary carbons, and one carbonyl carbon), in addition to the two Table 1. 1H and

13

1 1 2 3

4 5 6 7 8 9 10 11 12 13 14 15 16 17 COCH3 COCH3

5.33, br d (11.6) βH: 2.46, m αH: 2.26, m βH: 2.18, br dd (12.7, 6.4) αH: 1.30, ddd (12.7, 12.6, 6.7) 2.70, d (9.4) 4.56, dd (9.4, 9.0) 2.35, br d (9.0) 5.53, br d (6.2) βH: 2.73, dd (14.1, 6.2) αH: 2.16, br. d (14.1)

1.87, 1.84, 1.73, 1.36, 0.99,

dq (14.0, 7.0) dq (14.0, 7.0) br s s t (7.0)

2.05, s

The relative configuration of 1 was determined by interpretation of its ROESY NMR spectrum. The correlations

C NMR Data (CDCl3, δ in ppm, J in Hz) of Compounds 1−4 δH

no.

Figure 2. 1H−1H COSY and key HMBC correlations of compounds 1−4.

2 δC 127.7 24.0 36.5

61.9 67.5 77.2 50.5 69.8 44.3 131.7 78.1 175.4 29.1 20.3 16.8 8.4 21.3 169.7

δH 5.29, br d (11.8) βH: 2.42, m αH: 2.24, m βH: 2.18, br dd (13.0, 6.5) αH: 1.29, ddd (13.0, 12.4, 7.0) 2.77, d (9.2) 4.64, dd (9.2, 9.2) 2.42, br d (9.2) 5.51, br d (6.0) βH: 2.72, dd (14.2, 6.0) αH: 2.10, br d (14.2)

2.87, 2.81, 1.71, 1.35,

d (17.1) d (17.1) br s s

2.29, s 2.05, s

3 δC

δH

127.7 3.02, br d (11.2) 24.0 αH: 2.19, m βH: 1.56, ma 36.5 βH: 2.31, br d (12.9)

4 δC 62.1 23.5 35.5

αH: 1.42, ddd (13.0, 13.0, 6.5) 61.9 67.2 78.2 52.5 69.8 44.3

2.85, d (9.7) 4.65, dd (9.7, 9.5) 2.21, br d (9.5) 5.55, br d (7.0) βH: 2.92, dd (14.8, 7.0) αH: 1.22, br d (14.8)

131.5 76.8 175.0 45.4 1.88, 1.80, 20.3 1.38, 16.8 1.48, 208.4 0.95, 31.8 21.2 2.09, 169.6

dq (14.0, 7.0) dq (14.0, 7.0) s s t (7.0) s

δH 2.97, br d (11.4) αH: 2.19, m βH: 1.57, ma βH: 2.29, dd (13.1, 4.0)

δC 62.4 23.5 35.5

αH: 1.42, ddd (13.1, 13.1, 6.7) 60.8 64.7 76.6 50.9 66.0 44.7

2.91, d (9.3) 4.40, dd (9.6, 9.3) 2.56, br d (9.6) 5.51, br d (6.9) βH: 3.00, dd (15.2, 6.9) αH: 1.27, br d (15.2)

61.0 66.0 75.5 55.5 66.2 43.9

59.0 78.1 174.8 28.8

1.58, 2H, ma

58.5 77.7 175.5 25.4

20.8 16.7 8.3

1.34, s 1.46, s 0.98, t (7.0)

20.6 16.6 7.0

21.2 169.2

2.12, s

21.3 168.8

a

Overlapped with a broad water peak in CDCl3. 2196



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Figure 3. Key ROE correlations and ORTEP drawing of 1.

Figure 4. Key ROE correlations and ORTEP drawing of 2.

NMR data. Analyses of their 1D NMR data (Table 1) implied that they both possessed the same C16 homogermacranolide scaffold as compound 1. The major differences were that resonances for the Δ1(10) double bond in 1 were not observed for 3 and 4. Instead, signals typical of a 1,10-epoxy moiety [δH 3.02/2.97 (1H, H-1), 1.48/1.46 (3H, s, Me-14); δC 62.1/62.4 (C-1), 59.0/58.5 (C-10)] were present. The presence of such a 1,10-epoxy group in 3 and 4 was confirmed by the spin system from H-1 through H2-3 in their COSY spectra and the key HMBC correlations from Me-14 to C-1/C-9/C-10 and from H-8 to C-10. The 1D and 2D NMR data of compounds 3 and 4 implied that they possess the same 2D structure (Figure 2), but different configurations at C-11 (Figure 5). Notable differerces between these two compounds occurred around the lactone ring (ΔδC 1.1, −4.6, 3.4 for C-6, C-7, and C-13, respectively). Consistent with this, the ROE correlations for compounds 3 and 4 were similar, except that the correlation between H-6 and H2-13 could only be found for 4 (Figure 5A), indicating the 11-ethyl group was β-oriented in 4 rather than its α-orientation in 3. The (1R,4R,5S,6S,7R,8R,10S,11R) absolute configuration of 4 was defined via its single-crystal X-ray diffraction data [Flack parameter: 0.1(3)] (Figure 6). As for 3, its electronic circular dichroism (ECD) curve showed a negative Cotton effect (CE) around 230 nm (similar to those of compounds 1 and 2 but opposite that of 4, Figure 5B), thus allowing the definition of an (11S) configuration for 3. Hence, the absolute configuration of 3 was defined as (1R,4R,5S,6S,7R,8R,10S,11S). The four homogermacranolides 1−4 have unprecedented C16 (1, 3, and 4) or C17 (2) carbon skeletons, representing

of H-1/H-5, H-5/H-7, H-7/H-8, H-7/H-9α, H-9β/Me-14, Me-14/Me-15, and Me-15/H-6 indicated that H-5, H-7, and H-8 were α-oriented, whereas Me-15 and H-6 were β-oriented (Figure 3). As for C-11, the hydroxy group was β-oriented based on the strong ROE correlations of H-8/H2-13 and H-7/ Me-16, along with the absence of the correlation of H-6/H213. The (4R,5S,6S,7R,8R,11R) absolute configuration and hence the structure of 1 were unambiguously confirmed by Cu Kα X-ray crystallographic data analysis (Figure 3) [Flack absolute structure parameter: 0.0(2)].12 Shiluolide B (2) was isolated as colorless needles (nhexane−CH2Cl2 1:1, v/v) and had the molecular formula C19H26O7 deduced from the HRESIMS ion at m/z 389.1573 ([M + Na]+). Its NMR data (Table 1) were comparable to those of 1, except for the presence of a second acetyl group (δH 2.29, s; δC 31.8 and 208.4), along with the absence of the methyl triplet signals. This suggested that compound 2 is a new C17 sesquiterpenoid with the introduction of a 13-acetyl group to the germacranolide skeleton. This was confirmed by COSY and HMBC NMR data (Figure 2). In particular, the HMBC cross-peaks of the acetyl methyl (δ 2.29, Me-17) with C-13 (δ 45.4) and the ketocarbonyl carbon (δ 208.4, C-16) and of H2-13 (δ 2.81/2.87) with C-7 (δ 52.5) and C-12 (δ 175.0) were observed. The same relative configuration as that of compound 1 was characterized for 2 by the ROE correlations shown in Figure 4. Its (4R,5S,6S,7R,8R,11R) absolute configuration [Flack parameter: −0.01(6)] was established via its X-ray crystallographic data. Shiluolides C (3) and D (4) had the same elemental composition C18H26O7 deduced by their HRESIMS and 13C 2197



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Figure 5. Key ROE correlations (A) and experimental ECD curves in CH3CN (B) of epimers 3 and 4.

the thioester group to a primary alcohol followed by a cascade process of dehydration, hydroxylation, and oxidation reactions would afford the C17-type homogermacranolide 2. The positive-mode HRESIMS data of shiluone A (5) showed a protonated molecular ion at m/z 253.1791 [M + H]+, in accordance with the elemental composition of C15H24O3 (four indices of hydrogen deficiency). The presence of an enone chromophore was inferred from the IR (1693 and 1644 cm−1) and UV (λmax 237 nm) spectra. The 1H NMR spectrum of 5 revealed the presence of four methyls [three oxygenated secondary methyls at δ 1.21 (Me-12), 1.28 (Me13), 1.44 (Me-15), and one secondary at δ 1.01 (Me-14)] and an isolated methylene group (δ 2.59/2.53, ABq, J = 18.6 Hz). From the 13C and DEPT NMR data, 15 carbon resonances comprising four methyls, four methylenes, two methines, two oxygenated tertiary carbons (δ 73.9, 76.6), two quaternary olefinic carbons (δ 145.9, 174.3), and a ketocarbonyl (δ 204.7) (Table 2) were present in 5. As the double bond and carbonyl group consumed two out of the four degrees of unsaturation, 5 was deduced to be a bicyclic sesquiterpenoid. The HMBC cross-peaks of H2-6 with C-1/C-5/C-8/C-11, of H2-8 with C10/C-11, of H2-9 with C-1, and of Me-14 with C-1/C-9/C-10 allowed the construction of a seven-membered ring (Figure 7), implying 5 possesses a guaiane-type skeleton. The 2D structure was defined by the HMBC correlations shown in Figure 7 and by comparing its 1D NMR data with those of related known structures (e.g., pancherione14). Compared with pancherione, an additional tertiary hydroxy group was present in 5, which was located at C-4 via the HMBC correlations of Me-15 and H2-6 with C-4 (δ 76.6). The relative configuration of 5 was

Figure 6. ORTEP drawing of 4.

insertion of an additional C1 or C2 unit to the normal germacranolide scaffold, respectively. In nature, the C1 methyl building unit is derived from S-adenosylmethionine (SAM), whereas a C2 unit is usually supplied by the acetyl coenzyme A (acetyl-CoA).13 Putative biosynthetic pathways for the four homogermacranolides are proposed in Scheme 1. For the C16type homosesquiterpenoids (1, 3, and 4), SAM may Cmethylate the exocyclic olefinic bond. The resulting carbocation would be quenched with water to introduce the 11-hydroxy group. As for 2, the C2 unit could be introduced by the nucleophilic attack of the acetyl-CoA enolate anion onto the 11,13-epoxy moiety generated by epoxidation of the exocyclic double bond. Subsequently, a two-step reduction of 2198



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Scheme 1. Proposed Biosynthetic Pathways in Detail of Compounds 1−4

Table 2. 1H and

13

C NMR Data (CDCl3, δ in ppm, J in Hz) of Compounds 5−7 5 δH

no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OH

βH: 2.59, d (18.6) αH: 2.53, d (18.6)

βH: 3.01, br d (15.8) αH: 2.13, dd (15.8, 12.3) 1.37, m βH: 1.92, m αH: 1.59, m βH: 1.53, m αH: 1.80, m 2.97, m 1.21, 1.28, 1.01, 1.44,

s s d (6.5) s

6 δC

δH

145.6 204.7 50.9 76.6 174.3 27.3 50.4 27.2 32.6 26.9 73.9 25.0 28.6 17.3 26.6

δC

βH: 2.60, d (18.6) αH: 2.55, d (18.6)

βH: 2.93, br d (15.6) αH: 2.23, dd (15.6, 12.9) 1.29, m βH: 1.97, m αH: 1.64, m βH: 1.50, m αH: 1.80, m 2.96, m 1.26, 1.28, 1.06, 1.49,

s s d (6.8) s

7 δH

145.8 204.2 51.1 76.6 173.9 27.8 50.1 26.7 32.6 26.8 73.6 26.3 27.7 17.1 26.8

δC

βH: 2.62, dd (18.7, 6.4) αH: 2.02, br d (18.7) 2.76, m βH: 2.56, br d (17.4) αH: 2.37, dd (17.4, 11.9) 1.72, m βH: 1.84, m αH: 1.49, m βH: 1.74, m αH: 2.04, m

1.18, 1.24, 1.45, 1.19, 4.71,

144.3 210.6 43.7 38.7 179.2 28.9 46.8 24.1 36.0 71.5 73.2 24.4 29.1 26.6 18.9

s s s d (6.5) s

Me-14 suggested that H-6α, H-8α, and Me-14 assumed α-axial orientations, while Me-15, H-6β, and H-7 were on the opposite side (Figure 8). The absolute configuration of 5 was corroborated by comparing its ECD spectrum with those of

Figure 7. 1H−1H COSY and/or key HMBC correlations of 5−12.

determined via the coupling constants and ROESY data. A large J value (12.3 Hz) between H-6α and H-7 was indicative of their trans diaxial relationship. The ROE correlations of H6β/Me-15, H-6β/H-7, H-6α/Me-14, H-6α/H-8α, and H-8α/

Figure 8. Key ROE correlations of 5−7 and 10−12. 2199



Article

reference substances.15,16 According to the ECD studies on the guiai-1(5)-en-2-one sesquiterpenoids, the signs of CEs induced by the enone moiety depend only on the chirality of C-10, and a pair of C-10 epimers show mirror-image-like ECD curves.15,16 The ECD spectrum of 5 displayed similar CEs [241 nm (Δε +19.5), 319 nm (Δε −8.4)] to those of cyperusols A115 and nardoguaianone I,16 allowing the assignment of the (10R) configuration. Thus, the structure of shiluone A (5) was defined as (4R,7S,10R)-2-oxo-4α,11dihydroxyguaia-1(5)-ene. According to the HRESIMS data of 6, its chemical formula (C15H24O3) was identical to that of 5. Comparsion of their 1D and 2D NMR data suggested that these two sesquiterpenoids shared the same 2D structure. Unlike 5, the ROE corrleations of Me-14 with H-6α and H-8α were absent in compound 6, indicative of a 10β-methyl group (Figure 8). In agreement with this, its ECD spectrum was nearly antipodal to that of 5, with a negative CE at 242 nm (Δε −20.8) and a positive one at 315 nm (Δε +4.6). Thus, shiluone B (6) was identified as the C-10 epimer of 5, and its structure was defined as (4R,7S,10S)-2oxo-4α,11-dihydroxyguaia-1(5)-ene. A sodium adduct ion at m/z 275.1624 [M + Na]+ in its HRESIMS data showed that shiluone C (7) had the same molecular formula (C15H24O3) as 5 and 6. Likewise, the NMR spectroscopic data of 7 were comparable to those of 5 and 6 (Table 2). The most noteworthy difference was that the methyl doublet at δ 1.19 was correlated with C-5 rather than C-1 in the HMBC spectrum of 7, while the methyl singlet at δ 1.45 (s) had a 3J correlation with C-1 (Figure 7). This suggested that the OH group at C-4 in 5 and 6 was located at C-10 in 7. In good agreement with this assumption, an additional D2O-exchangeable signal at δ 4.71 (s) arising from the intramolecular hydrogen bonded hydroxy group (as depicted in Figure 7) was observed in the 1H NMR spectrum. As in 5 and 6, a large J value of 11.9 Hz observed for H-6α is characteristic of a diaxial relationship with H-7. The ROE cross-peaks of Me-15/H-6α, H-4/H-6β, H-6β/H-7, H-6α/H9α, H-9β/Me-14, and Me-14/H-8β (Figure 8) demonstrated the β-orientation of H-4, H-7, and Me-14. The (10R) absolute configuration of 7 was defined by the CEs [233 nm (Δε −4.9), 322 nm (Δε +2.6)] in its ECD spectrum, which were similar to those of 6 and related known structures (e.g., nardoguaianones E and H16), but opposite those of 5. The structure of shiluone C (7) was defined as (4R,7S,10R)-2-oxo-11,14α-dihydroxyguaia-1(5)-ene. The molecular formula C15H24O4 of 8 was established by the sodiated molecular ion at m/z 291.1567 in its HRESIMS, indicative of four indices of hydrogen deficiency. A bicyclic structure was concluded for 8, since its 13C NMR spectrum displayed resonances attributable to two ketocarbonyls (δC 208.3, 216.0) along with 13 sp3 carbons. The latter, interpreted via the DEPT and HSQC data, comprised four methyls, five methylenes, a methine, and three oxygen-bearing tertiary carbons (δC 87.8, 85.3, and 77.9). Three tertiary methyl singlets (δH 1.04, 1.26, 1.31), an acetyl methyl (δH 2.17), and two pairs of geminal protons adjacent to the ketocarbonyl groups [δH 2.38 (dd, J = 17.5, 4.5 Hz)/2.31 (dd, J = 17.5, 12.0 Hz); 2.56 (ddd, J = 17.0, 8.5, 6.0 Hz)/2.44 (ddd, J = 17.0, 9.0, 8.5 Hz)] were observed in the 1H NMR spectrum. The 2D structure of 8 was deduced via its 2D NMR data. Two spin systems as shown in bold in Figure 7, corresponding to H2-2/ H2-3 and H2-6/H-7/H2-8/H2-9, were defined by the 1H−1H COSY spectrum. The 9-acetyl group was evidenced from the

key HMBC correlations of the acetyl methyl (Me-14) with C-9 and C-10. The isopropyl group was positioned at C-7 based on the HMBC cross-peaks from the geminal methyls Me-12 and Me-13 to C-11 (δC 85.3) and C-7 (δC 49.3). The remaining groups (i.e., a ketocarbonyl, two oxygenated tertiary carbons, and an isolated −CH2−CH2− fragment) constructed a cyclopentanone ring, with C-5 being the only possible linkage position to the fragment of C-6−C-14 (Figure 7), on the basis of the HMBC correlations from Me-15 to C-3/C-4/C-5 and from H2-2 to C-1/C-3. The presence of such a cyclopentanone ring in 8 was congruent with a strong IR absorption at 1748 cm−1.17 The remaining index of hydrogen deficiency required the construction of an epoxy moiety in 8 present between C-5 and C-11 based on their relatively downfield carbon shifts (δC 87.8 for C-5 and δC 85.3 for C-11) when compared to C-4 (δC 77.9). This assumption and the relative configuration of 8 were substantiated by analyzing the coupling constants and ROESY data, with the usage of ab initio calculations.1i The magnitude of JH‑7/H‑6b (12.0 Hz) implied that these two protons are both axially oriented. Moreover, in the ROESY spectrum, Me-15 showed cross-peaks with H-3a/H-3b/H-6a but no correlation with H-6b. A six-membered 4,11-epoxy ring could, thus, be excluded, as none of the corresponding diastereoisomers among the stable conformers matched the ROE data. In addition, the 4,11-epoxy structures possessed much higher relative energies (Δ > 10 kcal/mol) than those of the 5,11epoxy isomers, implying the latter structures to be more stable. The proton−proton distances and NMR shifts of the four diastereoisomers with the 5,11-epoxy ring were calculated (Figure S59, Tables S4−S6, Supporting Information). Thus, the ROE cross-peaks of Me-15 with H-3a/H-3b/H-6a (equatorial)/H-7, of Me-12 with H-6b (axial)/H-8a/H-8b/ Me-13, of Me-13 with H-7/H-8a/Me-12, of H-6b with H-8b, and of H-6a with Ha-9/Hb-9 were only compatible with the proposed structure as depicted in Figure 9. This deduction was

Figure 9. Key ROE correlations of compound 8.

confirmed by the 1H and 13C NMR data computations, according to a DP4 analysis of the experimental and calculated NMR data (Tables S5, S6, Supporting Information).18 Biosynthetically, shiluone D (8) may arise from the oxidative cleavage of the C-1/C-10 bond in a guaianol precursor, similar in structure to mandassidione.19 Subsequent intramolecular etherification between C-5 and C-11 would generate the new ether-containing spirocyclic ring for 8. The positive-mode HRESIMS (m/z 291.1570 [M + Na]+) and 13C NMR data of shiluone E (9) established its molecular formula as C15H24O4 (four indices of hydrogen deficiency). Its 1 H NMR spectrum displayed signals for a secondary methyl (δ 1.12, Me-14), two tertiary methyls (δ 1.24, s, Me-12; 1.44, s, Me-13), and an acetyl methyl (2.19, s, Me-15) (Table 3). Its 2200


Journal of Natural Products Table 3. 1H and

13

Article

C NMR Data (δ in ppm, J in Hz) of Compounds 8−10 8 δH

no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OH

2.38, 2.31, 2.11, 1.83,

2.08, 1.87, 1.85, 1.71, 1.47, 2.56, 2.44,

dd (17.5, 4.5) dd (17.5, 12.0) br dd (12.8, 12.0) m

br d (12.1) dd (12.1, 12.0) m ddd (14.5, 6.0, 4.5) ddd (14.5, 8.0, 5.7) ddd (17.0, 8.5, 6.0) ddd (17.0, 9.0, 8.5)

1.04, s 1.26, s 2.17, s 1.31, s not observed

9 δC

10

δH

216.0 32.6 33.1 77.9 87.8 30.9 49.3 24.4 42.8 208.3 85.3 22.5 27.9 30.0 22.0

δC

2.68, 2H, overlapped 2.73, overlapped 2.69, overlapped

212.6 34.8 36.9

2.62, 2.29, 2.17, 1.40, 1.24, 1.65, 1.37, 2.58,

dd (17.0, 8.0) dd (17.0, 12.1) m m m m m m

1.24, 1.44, 1.12, 2.19,

s s d (6.5) s

13

δH

207.2 175.6 35.1 46.0 27.3 31.7 46.3 86.9 21.9 27.7 16.8 30.0

δC

βH: 2.10, ddd (14.5, 4.0, 3.4) αH: 1.82, ddd (β14.5, 13.9, 4.4) αH: 2.01, ddd (14.2, 4.4, 3.4) βH: 1.48, ddd (14.2, 13.9, 4.0)

3.18, s

βH: 2.01, m αH: 1.80, m βH: 1.67, m αH: 1.45, m 1.35, m 2.35, sept (6.5) 0.73, d (6.5) 0.89, d (6.5) 0.85, d (6.8) 1.52, s 4.01, s

79.8 33.1 36.0 76.7 212.2 57.2 77.8 32.1 25.9 33.1 33.1 15.2 16.1 14.2 24.7

isopropyl group, the COSY spectrum also showed the spin systems of H2-2/H2-3 and H2-8/H2-9/H-10/Me-14 (Figure 7). In the HMBC spectrum of 10, the 2J or 3J couplings of H-6 (δ 3.18, s) with C-1, C-2, C-5, C-7, C-8, C-10, and C-11, of Me-12/Me-13 with C-7 and C-10, of Me-14 with C-1, C-9, and C-10, and of Me-15 with C-3, C-4, and C-5 indicated that compound 10 features a cadinan-5-one skeleton with C-1, C-4, and C-7 being oxygenated. Furthermore, the ROE cross-peaks among Me-15, H-6, and H-2α (Figure 8), together with the large vicinal coupling constant (JH‑2α/H‑3β = 13.9 Hz) for H-2α, required these protons to all be axially oriented. Based on this evidence, the epoxy moiety in 10 could only be placed between C-1 and C-7, as the formation of either a 1,4-epoxy or 4,7epoxy ring would create too much strain in ring A, and none of the ROE correlations would be observed among the aforementioned protons. Consistent with this, the hydroxy proton (δ 4.01, s) showed a 3J HMBC correlation with C-5. The ROEs of Me-14/H-2β, Me-14/H-9α, and H-9β/H-10 were evident (Figure 8), whereas those of H-6 with H-8β or H10 or of H-10 with H-2β were absent, suggesting that 10 is a cis-fused amorphane derivative with Me-14 being α-oriented.20 By application of the cyclohexanone octant rule,21 the absolute configuration of 10 was defined as (1R,4S,6R,7R,8R), since a diagnostic negative CE at 288 nm arising from the C-5 carbonyl chromophore was observed in its ECD spectrum. Accordingly, the structure shiluone E (10) was characterized as (1R,4S,6R,7R,8R)-1,7-epoxy-4-hydroxyamorph-5-one. Shiluones F (11) and G (12) had the same elemental composition of C15H26O3 assigned by their HRESIMS and 13C NMR data. Likewise, their 1D NMR data (Table 4) were similar, revealing the presence of four methyls, two oxygenbearing tertiary carbons (11: δC 72.6, 78.8; 12: δC 74.6, 75.3, in CDCl3), and a ketocarbonyl group (11: δC 216.0; 12: δC 216.7, in CDCl3). Collectively, these data implied that these two compounds are bicyclic sesquiterpenoids. The COSY, HSQC, and HMBC spectroscopic data (Figure 7) showed that compounds 11 and 12 share the same 5,11-dihydroxy-1-oxo-

C NMR spectrum exhibited 15 carbon resonances typical of a sesquiterpenoid skeleton. With the aid of DEPT and HSQC experiments, these signals were classified as four methyls, five methylenes, two methines, one oxygenated tertiary carbon (δ 86.9), and three carbonyls (two ketocarbonyl at δ 212.6/207.2 and one esterifed at 175.6). By further inspection of its 2D NMR data (Figure 7), the 2D structure of 9 was constructed. In the COSY spectrum, a long-range spin system of H2-6/H-7/ H2-8/H2-9/H-10/H3-14 was observed. The locations of the two ketocarbonyls were evidenced from the HMBC correlations of Me-14/C-1, H2-3/C-1, H2-2/C-4, Me-15/C-4, and Me-15/C-3. The HMBC cross-peaks of H2-6 with the ester carbonyl and of Me-12/Me-13 with C-7 and C-11 were observed. Collectively, as two ketocarbonyls and an ester group accounted for three indices of hydrogen deficiency, the remaining index necessitated the presence of a γ-lactone ring bridging C-5 and C-11. It is the first 1,5−4,5-di-seco-guaiane sesquiterpenoid containing a rare 5,11-lactone moiety. Its relative and absolute configurations remain to be defined. The molecular formula (C15H24O3) of shiluone E (10) was assigned based on a protonated molecular ion at m/z 253.1793 ([M + Na]+) in its positive-mode HRESIMS and 13C NMR data. From its 1H NMR spectrum, a secondary methyl (δ 0.85, Me-14) and a methyl linked to an oxygenated secondary carbon (δ 1.52, Me-15), an isopropyl [δ 0.73, 0.89 (each d, J = 6.5 Hz); 2.35 (sept., J = 6.5 Hz)], a methine proton (δ 3.18, s), and a D2O-exchangeable proton at δ 4.01 (s) were readily recognized. Fifteen carbon resonances comprising four methyl, four methylene, three methine, three oxygenated tertiary (δC 79.8, 77.8, and 76.7), and a ketocarbonyl (δC 212.2) carbon were observed for 10 (Table 3). From the above data, compound 10 contains, in addition to a ketocarbonyl group, two O atoms but three oxygenated sp3 carbons, suggesting the presence of an epoxy moiety in this bicyclic sesquiterpenoid. The detailed structure of 10, possessing a 6-oxabicyclo[3.1.1]heptane moiety as depicted in Figure 1, was deduced by inspection of its 2D NMR spectroscopic data. Apart from the 2201



Article

Table 4. 1H and 13C NMR Data (δ in ppm, J in Hz) of Compounds 11 and 12 11 δH a

no. 1 2

3 4 5 6

7 8

9

10 11 12 13 14 15

αH: 2.85, ddd (15.0, 10.3, 6.0) βH: 2.58, ddd (15.0, 5.6, 5.6) βH: 2.67, m αH: 1.62, m 2.17, dd, overlapped αH: 2.13, dd (12.9, 12.6) βH: 1.97, dd (12.9, 3.6) 2.39, dddd (12.6, 12.6, 3.6, 3.6) βH: 1.94, m αH: 1.55, m βH: 2.66, ddd (13.1, 13.1, 4.0) αH: 1.86, ddd (13.1, 3.3, 3.3)

1.37, 1.39, 1.30, 1.17,

s s s d (7.6)

configuration for 11.25 As for 12, the chiralities of C-5, C-7, and C-10 were assumed to be consistent with 11 based on biosynthetic considerations. Thus, the structures of sesquiterpenoids 11 and 12 were elucidated to be (4R,5S,7S,10S)5,12-dihydroxyeudesma-1-one and (4S,5S,7S,10S)-5,12-dihydroxyeudesma-1-one, respectively. Considering that the germacrane-type sesqutierpenoids (1− 4, 13, and 14) feature a common lactone ring substituted with an exocyclic alkyl group, being similar to the well-known anticancer agent camptothecin,26 these isolates were first evaluated for their cytotoxities toward the human cancer cell lines MCF-7 and A-549. Of these, only lipiferolide (13) exhibited antiproliferative activities, with IC50 values of 1.5 (MCF-7) and 7.3 μM (A-549). Shiluolide A (1) showed moderate cytotoxicity toward MCF-7 cells (IC50: 45.7 μM). Doxorubicin [(IC50s: 0.0039 μM (MCF-7) and 0.21 μM (A549)] was employed as the positive control (Table 5).

12 δCa

δC b

215.5 34.5

216.0 34.2 αH: 2.71, ddd (15.0, 13.2, 7.5) βH: 2.22, br dd (15.0, 5.6) 28.2 αH: 1.75, m βH: 1.68, m 40.9 2.11, br d (13.2)

28.7 41.3

δH b

δC b 216.7 36.5

29.8 33.7

78.1 33.6

78.8 32.9 αH: 2.09, dd (13.8, 13.2) βH: 1.63, m

75.3 30.2

43.6

43.1 1.61, m

39.9

22.1

21.3 βH: 1.98, br d (15.4) αH: 1.66, m 30.2 βH: 2.14, ddd (13.0, 12.0, 4.2) αH: 1.33, m

19.8

51.3 72.6 27.0 28.1 20.5 17.9

52.1 74.6 29.6 30.5 21.2 14.5

31.1

51.9 71.2 27.5 28.3 20.7 17.7

1.31, 1.36, 1.24, 0.96,

s s s d (7.0)

Table 5. Cytotoxic Activities of Compounds against Cancer Cell Lines MCF-7 and A-549 IC50 (μM)

25.9

a

compound

MCF-7

A-549

1 13 doxorubicina

45.7 ± 3.8 1.5 ± 0.2 0.0039 ± 0.0016

>200 7.3 ± 0.4 0.21 ± 0.04

Doxorubicin: positive control.

Recently, a number of sesquiterpenoids were obtained from Manglietia aromatic, another endangered ornamental plant also belonging to the family Magnoliaceae.1f Among the isolates from this plant, a cadinane-type sesquiterpenoid, (1S,6S,7S)-1hydroxycadin-4,9-dien-8-one, was found to exhibit inhibition against PTP1B,1f a potential therapeutic target for type 2 diabetes mellitus by negatively regulating the insulin signaling pathway.1i Therefore, all the isolated sesquiterpenoids from the title plant were also screened for their PTP1B inhibitory effects. However, only shiluone D (8) showed moderate inhibition, with an IC50 value of 46.3 μM [oleanolic acid (positive control): IC50 = 3.3 μM].1c,i

a

Recorded in pyridine-d5. bRecorded in CDCl3.

eudesmane scaffold. The presence of the carbonyl group at C-1 was also verified by the deshielding of C-10 (1: δC 51.3; 2: δC 52.1, in CDCl3). A literature survey revealed that a similar 2D structure was reported in 1994 for a synthetic sesquiterpenoid, [(4α,4aα,6α,8aα)]-octahydro-4,8α-dimethyl-6-[1-hydroxy-1methylethyl]-1(2H)-naphthalenone.22 However, the reported NMR data are quite different from those of 11 and 12, implying each should have a different relative configuration. Unlike the known structure with a cis-fused bicyclic ring, both 11 and 12 possess a trans-fused ring system, since no pyridinesolvent shifts (11: Δδ = 0.07 ppm; 12: Δδ = 0.00 ppm) were evident for Me-14 in these two compounds (Figures S75 and S81, Supporting Information).23 Compared to the corresponding resonance (δC 33.7) in 12, the C-4 resonance in 11 was shifted downfield by 7.2 ppm, which results from a β-effect of 5-OH on C-4 only when 15-Me and 5-OH are in a 1,2-diaxial relationship.24 Hence, compounds 11 and 12 are concluded to be epimeric at C-4. The relative configurations of the epimers 11 and 12 were assessed by analyzing the key coupling constants (Table 4) and ROE correlations (Figure 8). For both 11 and 12, H-7 was axial, as it exhibited large J values (12.6 Hz) with the vicinal H6α and H-8α. The β-position of H-7 was ascertained by the ROE cross-peaks of Me-14/H-6α and H-6β/H-7. The different orientations of Me-15 in these two epimers were also corroborated by the ROESY data as shown in Figure 8. Finally, the positive CE at 308 nm (Δε +2.9) in the ECD curve of 11 accounted for an atom predominance in the positive octants, which allowed the assignment of a (5S,10S)

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations and ECD data were determined using an Autopol IV automatic and a JASCO810 polarimeter, respectively. UV and IR data were acquired respectively on a Shimadzu UV-2550 and an Avatar 360 ESP FTIR spectrometer. NMR spectra were measured on a Varian Mercury Plus 400 MHz and a Bruker Avance III 600 MHz spectrometer. An AB Sciex TripleTOF 5600 and an Agilent 1100 series mass spectrometer were used to acquire the HR-ESIMS and ESIMS data, respectively. Xray crystallographic data were acquired on a Bruker Apex Duo diffractometer (Cu Kα). HPLC separations were performed on a Waters e2695 instrument equipped with Water 2998 (PDA) and 2424 (ELS) detectors and a Cosmosil C18-MS-II column (250 × 10 mm, 5 μm, flow rate: 3.0 mL/min). Silica gel (Kang-Bi-Nuo Silysia Chemical Ltd.), MCI gel CHP20P (Mitsubishi Chemical Industries Ltd.), and Sephadex LH-20 (Amersham Biosciences) were utilized for column chromatography (CC). Plant Material. A leaf and twig sample of Michelia shiluensis was collected and identified by Mr. Huan Ke at Foshan Botanical Garden, Guangdong, PR China, in 2014. A voucher specimen (No. 20141111) was deposited at our department in Fudan University. Extraction and Isolation. After drying and pulverization, the collected plant materials (1.8 kg) were extracted with MeOH (3 × 3.5 L) at ambient temperature (298 K). The combined extracts were 2202



Article

1082 cm−1; 1H and 13C NMR data, see Table 2; (+)-HRESIMS m/z 253.1794 [M + H]+ (calcd for C15H25O3, 253.1798). Shiluone C (7): colorless, amorphous solid; [α]25D +22 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 236 (5.5) nm; ECD (MeOH) λmax (Δε) 233 (−4.9), 322 (+2.6) nm; IR (dried film) νmax 3441, 2961, 2925, 2853, 1678, 1630, 1462, 1373, 1301, 1187, 1149, 1098 cm−1; 1 H and 13C NMR data, see Table 2; (+)-HRESIMS m/z 275.1624 [M + Na]+ (calcd for C15H24O3Na, 275.1618). Shiluone D (8): white, amorphous powder; [α]25D −7 (c 0.1, CHCl3); IR (dried film) νmax 3444, 2965, 2930, 2853, 1748, 1719, 1462, 1371, 1273, 1205, 1168, 1081 cm−1; 1H and 13C NMR data, see Table 3; (+)-HRESIMS m/z 291.1567 [M + Na]+ (calcd for C15H24O4Na, 291.1567). Shiluone E (9): colorless oil; [α]25D −3 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 224 (6.1) nm; IR (dried film) νmax 3310, 2955, 2918, 2850, 1736, 1711, 1492, 1461, 1377, 1189, 1160, 1082 cm−1; 1 H and 13C NMR data, see Table 3; (+)-HRESIMS m/z 291.1570 [M + Na]+ (calcd for C15H24O4Na, 291.1567). Shiluone F (10): white, amorphous powder; [α]25D −3 (c 0.3, CHCl3); ECD (MeCN) λmax (Δε) 288 (−1.3) nm; IR (dried film) νmax 3386, 2963, 2935, 2876, 1703, 1462, 1374, 1310, 1269, 1171, 1120, 1057 cm−1; 1H and 13C NMR data, see Table 3; (+)-HRESIMS m/z 253.1793 [M + H]+ (calcd for C15H24O3Na, 253.1798). Shiluone G (11): white, amorphous powder; [α]25D +14 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 254 (5.2) nm; ECD (MeOH) λmax (Δε) 233 (−4.9), 308 (+2.9) nm; IR (dried film) νmax 3415, 2960, 2928, 2868, 1699, 1466, 1377, 1348, 1310, 1202, 1138, 1141, 965 cm−1; 1H and 13C NMR data, see Table 4; (+)-HRESIMS m/z 277.1774 [M + Na]+ (calcd for C15H26O3Na, 277.1772). Shiluone H (12): 27 white, amorphous powder; 1H and 13C NMR data, see Table 4; (+)-ESIMS m/z 277.2 [M + Na]+; 531.4 [2 M + Na]+. X-ray Crystallographic Data of Shiluolides A (1), B (2), and D (4). Detailed data are included in the Supporting Information (Tables S1−S3). The crystal structures were solved by direct methods (SHELXS-9728) and refined by full-matrix least-squares calculations on F2.29 CCDC-1818757 (1), CCDC-1818758 (2), and CCDC1818759 (4), containing the supplementary crystallographic data, can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk. Cytotoxicity Assay. The human breast cancer cell line MCF-7 and lung cancer cell line A-549 were purchased from American Type Culture Collection (ATCC). The in vitro cytotoxicities were assessed by the MTS assay by following the manufacturer’s instructions (Promega, G1111).1c,30 The optical density was measured at 492 nm on a microplate reader. IC50 values were calculated using GraphPad Prism 5.0 software. PTP1B Inhibition Assay. For the general procedure, see our previous reports.1i,k

condensed in vacuo to give a crude residue (110.0 g), which was suspended in water (1.0 L) followed by successive partitions with petroleum ether (3 × 1.5 L) and EtOAc (3 × 1.5 L). The EtOAc partition (8.3 g) was fractionated over MCI gel with gradient mixtures of MeOH−H2O (30% → 100%), affording six fractions (Fr. A−Fr. F). Fr. C (0.9 g) was separated on a silica gel column (petroleum ether−EtOAc, 2:1) to afford subfractions C1−C8. The fifth subfraction (0.2 g) was rechromatographed over silica gel (CH2Cl2−EtOAc, 1:1) followed by HPLC separation (MeOH− H2O, 55:45) to afford compound 7 (2.8 mg, tR = 13.5 min). Compounds 5 (6.0 mg, tR = 9.3 min) and 6 (0.9 mg, tR = 12.5 min) were purified from Fr. C7 (0.1 g) by HPLC (MeOH−H2O, 60:40). Fr. D (1.6 g) was separated by silica gel CC with petroleum ether/ EtOAc gradients (6:1 → 4:1 → 3:1 → 1:1) to yield Fr. D1−D8. Fr. D2 (0.2 g) was purified by Sephadex LH-20 (MeOH) and HPLC (MeOH−H2O, 70:30) to give 13 (19.2 mg, tR = 9.0 min). Sesquiterpenoids 8 (0.9 mg, tR = 13.7 min) and 10 (2.8 mg, tR = 9.0 min) were obtained from Fr. D3 (0.1 g) by silica gel CC (CH2Cl2−EtOAc, 4:1) with a final purification on HPLC (MeOH− H2O, 60:40). Compounds 1 (2.2 mg, tR = 19.6 min) and 9 (10.2 mg, tR = 14.5 min) were obtained from Fr. D4 (0.1 g) by chromatographic separation employing Sephadex LH-20 (MeOH) and HPLC (MeOH−H2O, 55:45). Fr. D5 (0.3 g) was subjected to Sephadex LH-20 eluted with MeOH with a further purification by HPLC (MeOH−H2O, 50:50), affording 2 (3.4 mg, tR = 17.5 min), 3 (0.5 mg, tR = 15.0 min), and 14 (6.0 mg, tR = 19.5 min). Compounds 4 (1.3 mg, tR = 17.0 min), 11 (2.5 mg, tR = 12.0 min), and 15 (3.1 mg, tR = 16.2 min) were obtained from Fr. D6 (0.2 g) by repeated separations over silica gel and HPLC (MeCN−H2O, 50:50). Compounds 12 (3.2 mg, tR = 10.5 min) and 16 (1.7 mg, tR = 15.0 min) were obtained from Fr. E (1.2 g) by repeated CC over silica gel and a final purification on HPLC (MeOH−H2O, 60:40). Shiluolide A (1): colorless needles, mp 206.5−207.0; [α]25D −8 (c 0.2, CHCl3); UV (MeCN) λmax (log ε) 206 (6.4), 232 (6.2), 281 (5.5) nm; ECD (MeCN) λmax (Δε) 202 (−4.8), 235 (−0.4) nm; IR (dried film) νmax 3409, 2962, 2921, 2851, 1783, 1722, 1668, 1456, 1374, 1361, 1261, 1134, 1037 cm−1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z 361.1625 [M + Na]+ (calcd for C18H26O6Na, 361.1622). Shiluolide B (2): colorless needles, mp 211.4−211.9; [α]25D −5 (c 0.3, CHCl3); UV (MeCN) λmax (log ε) 206 (6.6), 274 (5.4) nm; ECD (MeCN) λmax (Δε) 203 (−3.0), 227 (−0.4) nm; IR (dried film) νmax 3396, 2963, 2926, 2863, 1785, 1741, 1714, 1440, 1365, 1244, 1134, 1070 cm−1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z 389.1573 [M + Na]+ (calcd for C19H26O7Na, 389.1571). Shiluolide C (3): white, amorphous powder; [α]25D −8 (c 0.04, CHCl3); UV (MeCN) λmax (log ε) 208 (6.3), 224 (6.1), 275 (5.4) nm; ECD (MeCN) λmax (Δε) 209 (+0.5), 235 (−0.8) nm; IR (dried film) νmax 3446, 2964, 2915, 2849, 1775, 1736, 1490, 1457, 1375, 1266, 1141, 1056 cm−1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z 377.1572 [M + Na]+ (calcd for C18H26O7Na, 377.1571). Shiluolide D (4): colorless needles; mp 211.9−212.9; [α]25D −36 (c 0.03, CHCl3); UV (MeCN) λmax (log ε) 202 (6.5), 223 (5.9), 274 (5.1) nm; ECD (MeCN) λmax (Δε) 212 (+1.5), 232 (+0.8) nm; IR (dried film) νmax 3452, 2953, 2915, 2843, 1775, 1747, 1452, 1386, 1227, 1145, 1052 cm−1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z 377.1573 [M + Na]+ (calcd for C18H26O7Na, 377.1571). Shiluone A (5): colorless, amorphous solid; [α]25D −32 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 237 (5.6) nm; ECD (MeOH) λmax (Δε) 212 (−7.7), 241 (+19.5), 319 (−8.4) nm; IR (dried film) νmax 3389, 2963, 2925, 2850, 1693, 1644, 1463, 1378, 1260, 1235, 1190, 1082 cm−1; 1H and 13C NMR data, see Table 2; (+)-HRESIMS m/z 253.1791 [M + H]+ (calcd for C15H25O3, 253.1798). Shiluone B (6): colorless, amorphous solid; [α]25D +5 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 239 (5.6) nm; ECD (MeOH) λmax (Δε) 212 (+11.7), 242 (−20.8), 315 (+4.6) nm; IR (dried film) νmax 3388, 2958, 2918, 2850, 1688, 1644, 1490, 1464, 1377, 1191, 1160,

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00386. 1D and 2D NMR, HRESIMS, and ECD spectra of 1−12 and details of computation for 8 (PDF) X-ray crystallographic data of 1 (CIF) X-ray crystallographic data of 2 (CIF) X-ray crystallographic data of 4 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +86 21 51980172. ORCID

Juan Xiong: 0000-0002-9955-7081 Huaqiang Zeng: 0000-0002-8246-2000 2203



Article

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Jin-Feng Hu: 0000-0002-0367-1454 Author Contributions #

J. Xiong and L.-J. Wang contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by NSFC grants (Nos. 81773599, 21772025, 21472021) and a MOST grant (2018ZX09735008005).

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REFERENCES

(1) Phytochemical and Biological Studies on Rare & Endangered Plants Endemic to China. Part XIII. Recently, J.-F. Hu’s group at Fudan University has launched a special program to systematically identify bioactive novel natural products from rare and endangered (wild or cultivated) plants endemic to China. So far, 12 have been investigated. Presented herein in chronological order, with the most recent work listed first: (a) Part XII (endangered Sinocalycanthus chinensis, wild): Ma, G.-L.; Xiong, J.; Osman, E. E. A.; Huang, T.; Yang, G.-X.; Hu, J.-F. Phytochemistry 2018, 151, 61−68. (b) Part XI (endangered Gmelina hainanensis, cultivated): Xiong, J.; Wu, X.-Y.; Wang, P.-P.; Lau, C.; Fan, H.; Ma, G.-L.; Tang, Y.; Li, J.; Hu, J.-F. Phytochem. Lett. 2018, 25, 17−21. (c) Part X (endangered Camellia crapnelliana, cultivated): Xiong, J.; Wan, J.; Ding, J.; Wang, P.-P.; Ma, G.-L.; Li, J.; Hu, J.-F. J. Nat. Prod. 2017, 80, 2874−2882. (d) Part IX (endangered Podocarpus imbricatus, cultivated): Hu, C.-L.; Xiong, J.; Xiao, C.-X.; Tang, Y.; Ma, G.-L.; Wan, J.; Hu, J.-F. J. Asian Nat. Prod. Res. 2018, 20, 101−108. (e) Part VIII (endangered Pinus kwangtungensis, cultivated): Hu, C.-L.; Xiong, J.; Wang, P.-P.; Ma, G.-L.; Tang, Y.; Yang, G.-X.; Li, J.; Hu, J.-F. Phytochem. Lett. 2017, 20, 239−245. (f) Part VII (endangered Manglietia aromatica, cultivated): Wang, L.-J.; Xiong, J.; Zou, Y.; Mei, Q.-B.; Wang, W.-X.; Hu, J.-F. Phytochem. Lett. 2016, 18, 202−207. (g) Part VI (rare Oresitrophe rupif raga, wild): Wu, X.-Y.; Xiong, J.; Liu, X.-H.; Hu, J.-F. Chem. Biodiversity 2016, 13, 1030−1037. (h) Part V (endangered Ginkgo biloba, cultivated): Ma, G.-L.; Xiong, J.; Yang, G.-X.; Pan, L.-L.; Hu, C.-L.; Wang, W.; Fan, H.; Zhao, Q.-H.; Zhang, H.-Y.; Hu, J.-F. J. Nat. Prod. 2016, 79, 1354−1364. (i) Part IV (endangered Pinus dabeshanensis, wild): Hu, C.-L.; Xiong, J.; Gao, L.-X.; Li, J.; Zeng, H.-Q.; Hu, J.-F. RSC Adv. 2016, 6, 60467−60478. (j) Part III (endangered plant Abies beshanzuensis, wild). Hu, C.-L.; Xiong, J.; Li, J.-Y.; Gao, L.-X.; Wang, W.-X.; Cheng, K.-J.; Yang, G.-X.; Li, J.; Hu, J.F. Eur. J. Org. Chem. 2016, 2016, 1832−1835. (k) Part II-a (rare Chloranthus oldhamii, wild): Xiong, J.; Hong, Z.-L.; Xu, P.; Zou, Y.-K.; Yu, S.-B.; Yang, G.-X.; Hu, J.-F. Org. Biomol. Chem. 2016, 14, 4678− 4689. Part II-b: Xiong, J.; Hong, Z.-L.; Gao, L.-X.; Shen, J.; Liu, S.-T.; Yang, G.-X.; Li, J.; Zeng, H.-Q.; Hu, J.-F. J. Org. Chem. 2015, 80, 11080−11085. (l) Part I (rare Chloranthus sessilifolius, wild): Wang, L.-J.; Xiong, J.; Liu, S.-T.; Pan, L.-L.; Yang, G.-X.; Hu, J.-F. J. Nat. Prod. 2015, 78, 1635−1646. (2) Editorial Committee of the Flora of China, Chinese Academy of Sciences. Flora of China; Science Press: Beijing, 1996; Vol. 30 (1), pp 151−152, 181. (3) Kumar, D.; Kumar, S.; Taprial, S.; Kashyap, D.; Kumar, A.; Prakash, O. Zhongxiyi Jiehe Xuebao 2012, 10, 1336−1340. (4) (a) Ogura, M.; Cordell, G. A.; Farnsworth, N. R. Phytochemistry 1978, 17, 957−961. (b) Cassady, J. M.; Ojima, N.; Chang, C. J.; Mclaughlin, J. L. Phytochemistry 1979, 18, 1569−1570. (5) The IUCN Red List of Threatened Species 2015: e.T191503A1986187; http://dx.doi.org/10.2305/IUCN.UK.2015-2. RLTS.T191503A1986187.en. (6) The State Forestry Administration and the Ministry of Agriculture, List of Wild Plants of National Priority Protection I, 1999, http://www.forestry.gov.cn/yemian/minglu1.htm. (7) (a) Ibrahim, M. A.; Na, M.; Oh, J.; Schinazi, R. F.; McBrayer, T. R.; Whitaker, T.; Doerksen, R. J.; Newman, D. J.; Zachos, L. G.; 2204


Structurally Diverse Sesquiterpenoids from the Endangered

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