Protoilludane, Illudalane, and Botryane Sesquiterpenoids from the

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Protoilludane, Illudalane, and Botryane Sesquiterpenoids from the Endophytic Fungus Phomopsis sp. TJ507A Shuangshuang Xie,†,⊥ Ye Wu,‡,⊥ Yuben Qiao,† Yi Guo,† Jianping Wang,† Zhengxi Hu,† Qing Zhang,† Xiaonian Li,§ Jinfeng Huang,† Qun Zhou,† Zengwei Luo,† Junjun Liu,† Hucheng Zhu,† Yongbo Xue,*,† and Yonghui Zhang*,† †

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation and Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China S Supporting Information *

ABSTRACT: To explore the chemical diversity of metabolites from endophytic fungi, the strain Phomopsis sp. TJ507A, isolated from the medicinal plant Phyllanthus glaucus, was investigated. A 2,3-seco-protoilludane-type sesquiterpenoid (1), eight protoilludane-type sesquiterpenoids (2−9), four illudalane-type sesquiterpenoids (10a/10b, 11, and 12), and a botryane-type sesquiterpenoid (13) in addition to seven known sesquiterpenoids (14−20) were identified from the liquid culture of the fungus. Structures of the isolated compounds, including their absolute configurations, were elucidated based on extensive spectroscopic analyses, a modified Mosher analysis, electronic circular dichroism (ECD) calculations, and [Rh2(OCOCF3)4]-induced ECD spectra as well as X-ray crystallographic analyses. Compound 1 represents the first example of a naturally occurring sesquiterpenoid containing the unusual 2,3-seco-protoilludane scaffold. Compounds 1 (p < 0.001); 2−6, 15, and 18 (p < 0.01); and 7, 9, and 20 (p < 0.05) displayed β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activities ranging from 19.4% to 43.8% at the concentration of 40 μM. LY2811376 was used as the positive control with an inhibitory activity of 38.6% (p < 0.01). Furthermore, none of these compounds showed obvious hepatotoxicity at concentration of 40 μM.

E

conditions are satisfied.11 Using multiple culture conditions has been shown to be an effective approach to obtaining diverse metabolites.12 As part of our continuous commitment to discovering structurally intriguing and biologically active natural products from medicinal plants13 and endophytic fungi,14 in 2016, we identified a series of enantiomeric lignans and neolignans with antioxidant activity from the medical plant Phyllanthus glaucus.15 Subsequently, two unusual ergostane-type steroids with potent anti-inflammatory activities were identified from the solid-state cultures of Phomopsis sp. TJ507A, an endophytic fungus isolated from P. glaucus.16 Most recently, in an attempt to investigate the structurally diverse metabolites from the plant-derived fungus, Phomopsis sp. TJ507A was further investigated by culturing in liquid media. This work led to the isolation of 14 new sesquiterpenoids including a 2,3-seco-

ndophytic fungi are relatively unexplored compared to soil isolates and are a potential source of secondary metabolites with diverse structures and biological activities.1 Structurally diverse metabolites produced by endophytic fungi not only provide protection and ultimately aid in the survival of their host plants but also have great potential in medicine, agriculture, and modern industry.2 Taking Taxomyces andreanae for example, this endophytic strain was found to be capable of producing paclitaxel, a well-known natural therapeutic agent for breast and ovarian cancers.3 Plant-derived fungi of the genus Phomopsis are reported to be producers of various secondary metabolites including terpenoids,4 oblongolides,5 steroids,6 and cytochalasans,7 which have been shown to be, for example, immunosuppressive,4 cytotoxic,8 antimicrobial,9 and phytotoxic.10 Generally, the biosynthesis of secondary metabolites in microorganisms is highly regulated and modulated by genes and pathway-specific regulators; however, gene clusters often remain inactive until suitable physiological and environmental © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 25, 2017

A

DOI: 10.1021/acs.jnatprod.7b00889 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

Table 1. 1H NMR (400 MHz) Data for Compounds 1−6 (δ in ppm, J in Hz) 1a

no. 1a 1b 2a 2b 7 9a 9b 10 12 13a 13b 14 15 a

1.97, m 4.55, 4.41, 6.45, 1.82, 1.70, 3.19, 1.73, 1.10,

m m d (2.6) dd (12.9, 8.0) dd (12.9, 4.0) td (8.4, 2.6) s s

1.20, s 1.08, s

2a 2.69, 1.74, 3.06, 2.76, 6.70, 3.96,

m td (10.7, 9.9, 2.7) m m s s

1.64, s 1.17, s 1.09, s 1.30, s

3a 1.97, m 3.06, 2.78, 6.47, 1.93, 1.62, 3.40, 1.67, 3.61, 3.48, 1.07, 1.20,

m ddd (16.2, 8.0, 3.2) d (2.7) m dd (13.0, 8.1) td (8.4, 2.7) s d (10.7) d (10.8) s s

4b

5b

6a

2.41, dd (10.4, 8.6) 1.88, dd (10.5, 7.6) 5.10, td (8.9, 7.6, 1.6)

2.42, dd (10.4, 8.6) 1.89, dd (10.4, 7.6) 5.11, td (8.9, 7.6, 1.6)

2.46, dd (10.7, 8.4) 1.86, m 5.15, t (7.3)

6.39, 1.59, 2.00, 3.40, 1.81, 1.15,

6.50, 1.95, 1.63, 3.40, 1.81, 3.52, 3.41, 1.07, 1.12,

6.47, 1.63, 1.76, 3.34, 1.85, 1.17,

d (2.6) dd (13.5, 7.9) dd (13.6, 8.6) m d (1.5) s

3.39, s 1.11, s

d (2.6) dd (13.1, 8.6) dd (13.1, 8.2) m d (1.6) d (10.9) m s s

d (2.6) dd (13.0, 8.6) dd (13.0, 8.0) td (8.3, 2.6) d (1.6) s

1.06, s 1.08, s

Recorded at 400 MHz in chloroform-d. bRecorded at 400 MHz in methanol-d4.

■

RESULTS AND DISCUSSION Phomophyllin A (1) was obtained as a colorless oil. Its molecular formula was confirmed to be C15H20O2 by the 13C NMR data and high-resolution electrospray ionization mass spectrometric (HRESIMS) data. The formula indicates six indices of hydrogen deficiency. The 1H NMR data (Table 1) of 1 displayed four methyl groups (δH 1.73, H3-12; δH 1.10, H313; δH 1.20, H3-14; and δH 1.08, H3-15), one olefinic proton (δH 6.45, H-7), and one oxygenated methylene (δH 4.41 and 4.55, H2-2). The 13C NMR (Table 3) and DEPT spectroscopic data of 1 showed the presence of four methyl groups, three methylenes (one oxygenated), two methines, a carbonyl, and two quaternary and four olefinic carbon atoms. Apart from the two double bonds (δC 182.0, C-3; δC 108.1, C-4; δC 137.3, C-6; and δC 145.7, C-7) and carbonyl group (δC 186.8, C-5), the

protoilludane, phomophyllin A (1); eight protoilludane sesquiterpenoids, phomophyllins B−I (2−9); four illudalane sesquiterpenoids, phomophyllins J−M (10a/10b, 11, and 12); and a botryane sesquiterpenoid, phomophyllin N (13), in addition to seven known sesquiterpenoids that were identified as granulone B (14),17 radulone B (15),18 2-(2,2,4,6tetramethylindan-5-yl)ethanol (16),19 pterosin Z (17),20 onitin (18), 21 dehydrobotrydienol (19), 22 and 7-hydroxy-10oxodehydrodihydrobotrydial (20).23 Notably, all the compounds isolated were reported from Phomopsis species for the first time. Compound 1 is the first naturally occurring sesquiterpenoid containing the unusual 2,3-seco-protoilludane carbon scaffold. Herein, the isolation, structural elucidation, and biological activity evaluation of these metabolites are described. B



Article

Table 2. 1H NMR (400 MHz) Data for Compounds 7−13 (δ in ppm, J in Hz) 7a

no. 1a 1b 2a 2b 4 6 7a 7b 9a 9b 10 11 12 13 14 15 OMe COMe a

8a

2.07, m 2.80, ddd (16.2, 7.5, 3.7) 3.10, m

2.42, m 1.69, m 5.15, t (7.8)

9a

10b

11a

1.26, m

3.56, t (8.2)

4.12, t (8.0)

3.59, m

1.96, ddd (12.3, 9.0, 2.9) 1.72, m 3.09, q (6.4)

2.92, m

3.00, t (8.0)

2.99, m

1.85, 1.51, 1.82, 1.56, 2.41,

m m m m dd (13.2, 7.6)

2.74, d (15.5) 2.50, d (15.5) 4.24, s

3.12, dd (8.3, 2.6)

m m m m m m

1.66, 0.99, 1.16, 1.28,

1.79, 1.05, 1.01, 1.19,

s s s s

1.12, 1.16, 1.07, 1.19,

d (6.4) s s s

2.21, 1.26, 0.99, 2.26, 3.40,

3.99, d (8.3)

d (1.1) s s s

13a

6.55, s

2.52, 1.75, 1.45, 1.41, 1.31, 2.21,

6.49, d (2.6)

12b

2.49, d (15.3) 2.77, d (15.3) 4.58, s

s s s s s

2.25, 1.25, 1.01, 2.35,

s s s s

4.99, s

2.31, 1.06, 1.17, 2.55,

s s s s

2.25, m 1.69, d (13.2)

4.69, 2.23, 1.24, 1.37, 1.24, 3.67,

m s s s s m

2.07, s


Table 3. 13C NMR (100 MHz) Data for Compounds 1−13 (δ in ppm)

a

no.

1a

2b

3a

4a

5b

6a

7a

8a

9a

10b

11a

12b

13a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OCH3 COCH3 COCH3

38.1, t 71.9, t 182.0, s 108.1, s 186.8, s 137.3, s 145.7, d 45.8, s 39.7, t 52.0, d 45.4, s 7.8, q 27.7, q 28.8, q 19.2, q

24.9, t 28.7, t 169.9, s 126.5, s 185.7, s 138.7, s 151.4, d 47.1, s 78.0, d 84.2, s 50.9, s 9.6, q 26.5, q 23.0, q 23.1, q

32.8, t 28.9, t 170.9, s 126.9, s 187.3, s 142.4, s 142.1, d 52.1, s 34.2, t 53.7, d 47.7, s 9.7, q 70.3, t 22.6, q 19.5, q

44.8, t 71.1, d 173.7, s 127.5, s 189.9, s 142.8, s 144.5, d 53.2, s 35.4, t 57.6, d 39.8, s 9.8, q 23.9, q 70.3, t 20.0, q

44.9, t 71.2, d 173.8, s 127.4, s 189.8, s 141.8, s 145.1, d 53.2, s 34.8, t 56.0, d 39.9, s 9.8, q 22.9, q 70.2, t 20.3, q

44.4, t 70.6, d 170.8, s 126.8, s 188.6, s 138.5, s 147.4, d 46.0, s 39.3, t 55.3, d 38.6, s 9.8, q 28.7, q 27.7, q 20.1, q

33.1, t 29.4, t 169.7, s 127.1, s 186.6, s 136.0, s 145.6, d 47.1, s 81.3, d 59.4, d 45.9, s 9.8, q 19.6, q 26.1, q 20.4, q

45.1, t 71.9, d 169.8, s 125.1, s 202.5, s 49.9, d 40.6, t 36.9, s 42.0, t 57.0, d 39.7, s 9.7, q 31.9, q 31.9, q 17.4, q

24.0, t 28.2, t 82.1, s 45.1, d 215.5, s 85.6, s 54.5, t 40.1, s 46.4, t 58.0, d 45.8, s 6.9, q 27.1, q 29.7, q 20.7, q

62.1, t 34.2, t 135.5, s 125.3, s 150.4, s 128.7, s 43.7, t 44.1, s 93.1, d 140.8, s 127.0, s 12.3, q 23.1, q 29.2, q 15.7, q 58.2, q

63.3, t 29.2, t 134.3, s 123.4, s 148.6, s 126.5, s 41.1, t 43.2, s 83.2, d 142.2, s 126.7, s 12.0, q 22.7, q 28.2, q 15.1, q

61.7, t 33.5, t 139.9, s 132.6, s 152.7, s 136.9, s 76.6, d 52.6, s 211.8, s 131.1, s 129.4, s 12.5, q 20.6, q 23.6, q 13.6, q

136.6, s 124.6, s 156.3, s 110.0, d 153.2, s 42.1, s 54.7, t 51.0, s 136.8, s 59.0, t 11.4, q 32.7, q 26.9, q 31.7, q 71.6, t

21.2, q 171.3, s


Figure 1. Key 2D NMR correlations of compounds 1, 4, 5, and 8.

remaining three indices of hydrogen deficiency in the molecule implied that 1 was likely a tricyclic sesquiterpenoid. The absorption band at 1632 cm−1 in the IR spectrum of 1 indicated

the presence of a carbonyl group, while the characteristic absorption band at 1130 cm−1 revealed the presence of an ether functionality. All proton resonances were assigned to their C



Article

seco-protoilludane carbon scaffold. A possible biosynthetic pathway of 1 was proposed (Scheme 1, Supporting Information). Phomophyllin B (2) was initially isolated as a white powder, and its molecular formula was determined to be C15H20O3 by HRESIMS. Analysis of the NMR data showed that compound 2 had similar structural features to those of compound 14, and it was therefore assigned as a protoilludane sesquiterpenoid derivative. The main difference between 2 and 14 was the presence of an additional oxidized methine signal (δC 78.0, C9) and the absence of a methylene signal in the 13C NMR spectrum of 2. The proton−proton coupling system of H2-1 (δH 1.74 and 2.69) with H2-2 (δH 2.76 and 3.06) of 2 and the HMBC cross-peaks (Figure S15, Supporting Information) of H-9 (δH 3.96) with C-8, C-11, C-13, and C-14 and H-1a (δH 2.69) with C-3, C-10, C-11, and C-15 confirmed that the two oxygenated carbons were located at C-9 and C-10, respectively. The NOESY interactions between H-9 and H3-13 and H3-15 of 2 (Figure S16, Supporting Information) suggested these protons were orientated in the same direction. A suitable crystal of 2 was subsequently subjected to a single-crystal X-ray diffraction experiment with Cu Kα radiation [CCDC 1536435, Flack parameter 0.12(6)],26 which unambiguously established the absolute structure of 2 as 9R,10S,11S (Figure 3). Phomophyllin C (3), obtained as a colorless oil, has the molecular formula C15H20O2. Through detailed analysis of the NMR data, compound 3 was assigned to be a protoilludane sesquiterpenoid derivative similar to compound 14. The main difference between 14 and 3 was the position of the hydroxy group in the molecules. These conclusions were confirmed by analysis of the NMR data. The 1H−1H COSY correlations (Figure S25, Supporting Information) of H2-1 (δH 1.97) with H2-2 (δH 2.78 and 3.06) and H2-9 (δH 1.62 and 1.93) with H10 (δH 3.40) of 3, in addition to the HMBC correlations (Figure S24, Supporting Information) of H2-13 with C-7, C-8, C-9, and C-14, confirmed the hydroxy substitution at C-13. H10, H-9b, and H3-14 of 3 were arbitrarily assigned as β-oriented by the key NOESY correlations of H-10 with H3-14 and H3-14 with H-9b. H3-15, H-9a, and H2-13 were assigned to be αoriented based on the correlations of H3-15 with H-9a and H9a with H2-13 (Figure S26, Supporting Information). 3 was assigned as 8R,10R,11R since the calculated ECD curve of 3 was in good agreement with the experimental ECD curve (Figure 4). Phomophyllin D (4) was isolated as a colorless oil and had a molecular formula of C15H20O3 based on the HRESIMS data. The 1H NMR and 13C NMR data of 4 resembled those of 3; however, a methylene group (δC 28.9, C-2) in 3 was

respective carbons with the help of the HSQC data. Based on the key correlations of H2-1 (δH 1.97) with H2-2 and of H2-9 with H-10 in the 1H−1H COSY spectrum of 1, as well as the key correlations (Figure 1) between H2-2 and C-1, C-3, and C11; H-7 and C-5, C-6, C-8, C-9, and C-10; H-12 and C-3, C-4, and C-5; H3-13 and C-7, C-9, and C-14; and H-15 and C-1, C3, C-10, and C-11 observed in the HMBC spectrum of 1, the planar structure of 1, containing a furan, was established as shown (Figure 1). To determine the relative configuration of 1, assuming H-10 was β-oriented, the NOESY correlations (Figure 1) of H-10 with H2-1 and H3-14 indicated that these protons were on the same side, also indicating beta-orientations. The cross-peak of H3-13 with H3-15 revealed they were cofacial, and they were assigned to be α-oriented. Therefore, the relative configuration of 1 was elucidated as 10R,11R. Subsequently, to determine the absolute configurations of the chiral centers of 1, a timedependent density functional theory (TDDFT) analysis at the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level of theory with IEFPCM in MeOH was performed for 1 (10R,11R) and ent-1 (10S,11S). The experimental electronic circular dichroism (ECD) curve of 1 was in good agreement with the calculated ECD curve of 1, which allowed the absolute configuration of 1 to be assigned as 10R*,11R* (Figure 2).

Figure 2. Experimental and calculated ECD spectra in MeOH of compound 1.

Briefly, protoilludane-type sesquiterpenoids, which feature the distinctive tricyclic perhydro-1H-cyclobuta[e]indene scaffold that is generated by various higher fungi through a humulene cyclization pathway,24 have been synthesized.25 In this case, it is worth noting that compound 1 is the first naturally occurring sesquiterpenoid isolated that features a 2,3-

Figure 3. ORTEP drawings of compounds 2, 9, and 10. D



Article

Figure 4. Calculated ECD spectra of 3 (left) and experimental ECD spectra of 2−7 (right) in MeOH.

oxygenated to a methine (δC 71.1, C-2) in 4. This conclusion was further confirmed by the HMBC correlations (Figure 1) of H-2 (δH 5.10) with C-1, C-3, and C-4. The key NOESY (Figure 1) correlations of H-2/H3-15, H3-15/H-9a, and H-9a/H3-13 (δH 1.15) revealed that these protons were located on the same side of the molecule, and they were assigned to be α-oriented. The correlations of H-10 (δH 3.40) with H-9b and H-9b with H3-14 (δH 3.39) suggested that these protons were on the opposite face of the structure, and they were assigned as βoriented. By treating 4 with Rh2(OCOCF3)4 in anhydrous CH2Cl2, a negative Cotton effect at 359 nm in the ECD spectrum (Figure 5) was observed for the complex. Therefore,

2.46) with H-2 and the correlation of H2-9 (δH 1.63 and 1.76) with H-10 (δH 3.34) in the 1H−1H COSY experiment (Figure S55, Supporting Information), and the key correlations from H2 to C-1, C-3, and C-4 in the HMBC spectrum (Figure S54, Supporting Information) suggested that the oxidized carbon in 6 was C-2. The relative configuration of 6 was established by the key NOESY correlation (Figure S56, Supporting Information) of H-2 with H-1a and H3-15 and of the correlation of H-10 with H-1b and H3-14. The absolute configuration of C-2 in 6 was confirmed using a modified Mosher’s analysis.28 By treating 6 with (R)- and (S)-αmethoxy-α-trifluoromethylphenylacetyl chloride (MTPA)-Cl, S- (6a) and R-MTPA (6b) esters were produced, respectively, and the differences in the chemical shifts (Δδ = δS − δR) of the esters were recorded (Figure 6). Consequently, the absolute configuration of C-2 in 6 was confirmed to be R.

Figure 5. ECD spectra of the [Rh2(OCOCF3)4] complexes of compounds 4, 5, and 11 with the intrinsic ECD spectrum subtracted.

Figure 6. ΔδH(S−R) values (in chloroform-d) of the MTPA esters of 6 and 7.

successful implementation of the bulkiness rule allowed C-2 to be assigned as R.27 Hence, the absolute configuration of 4 was assigned as 2R,8S,10R,11R. A step-by-step comparison of the 1D and 2D NMR data of phomophyllin E (5) to those of 4 revealed that 5 and 4 shared the same planar structure. The relative configuration of 5 was determined by analyzing its NOESY data and comparing its proton chemical shifts with those of 4. In the NOESY spectrum (Figure 1) of 5, distinct correlations between H3-15 and H-2 (δH 5.11) and H-9α, between H-9α and H2-13 (δH 3.41 and 3.52), and between H3-14 (δH 1.07) and H-10 (δH 3.40) and H-9b were observed, which suggested that the orientation of the oxidized methylene at C-8 in 5 is opposite that of 4, meaning 5 and 4 are C-8 epimers. The 2R configuration of 5 was determined by a negative Cotton effect at 366 nm in the Rh2(OCOCF3)4-induced ECD spectrum (Figure 5). Thus, the absolute configuration of 5 was assigned as 2R,8R,10R,11R. Phomophyllin F (6) has the same molecular formula (C15H20O2) as 3 according to the HRESIMS data. The main difference between 6 and 3 was the position of the oxidized carbon. The combination of the obvious downfield chemical shift of H-2 (δH 5.15), the correlation of H2-1 (δH 1.86 and

Phomophyllin G (7) was obtained as a colorless oil, and the molecular formula (C15H20O2) was determined based on the HRESIMS and 1D NMR data. The 1D NMR data of 7 were similar to those of 6. The correlations of H2-1 (δH 2.07) with H2-2 (δH 2.80 and 3.10) and H-9 (δH 3.99) with H-10 (δH 3.12) in the 1H−1H COSY spectrum (Figure S65, Supporting Information) coupled with the HMBC cross-peaks (Figure S64, Supporting Information) of H-9 with C-8, C-10, C-11, C-13, and C-14 of 7 indicated the hydroxy group was located at C-9. The relative configuration of 7 was subsequently deduced by the NOESY correlations (Figure S66, Supporting Information) of H-9 with H3-13 and H3-15 and the correlation of H-10 with H3-14. Subsequently, the absolute configuration of 7 was confirmed using a modified Mosher’s analysis similar to the method used for 6, and the differences in the chemical shifts (Δδ = δS − δR) of the corresponding esters (7a, and 7b) were recorded (Figure 6). Therefore, the absolute configuration at C-9 in 7 was assigned as S. Finally, the absolute configuration of 7 was determined as 9S,10R,11R by comparison of the experimental ECD curve of 7 with those of 2−6 (Figure 4). E



Article

Figure 7. Experimental and calculated ECD spectra of 8 and 13 in MeOH.

Figure 8. Chiral HPLC chromatograms of 10 and the ECD spectra (in MeOH).

cm−1) characteristic absorptions suggested the presence of a benzene ring and a hydroxy functionality. Inspection of its 1H and 13C NMR spectra confirmed 10 was an illudalane-type sesquiterpenoid derivative that resembled the known compound coprinol.29 The main differences between 10 and coprinol were the presence of a methoxy group (δH 3.40, δC 58.2) and an additional methine signal and the absence of a methylene group in 10 compared to coprinol. This observation was supported by the HMBC correlations of H-9 (δH 4.24) with C-8, C-10, C-14, and C-16 and the correlation of H3-16 (δH 3.40) with C-9 in the HMBC spectrum of 10 (Figure S94, Supporting Information). A suitable crystal of 10 was obtained in MeOH and was subsequently subjected to single-crystal Xray crystallography (Figure 3). Consequently, 10 was found to be a pair of enantiomers based on the single-crystal data. Subsequently, chiral HPLC was successfully used to separate 10, giving 10a and 10b (phomophyllins J and K) in a ratio of approximately 1.1:1, and the enantiomers displayed mirror image ECD curves (Figure 8). The orientation of 10-OMe of 10a was defined to be α-oriented based on similarity to the calculated experimental ECD curves of 10a; thus, 10-OMe of 10b was β-oriented (Figure 8). The molecular formula of phomophyllin L (11), C17H24O4, was determined based on HRESIMS and 13C NMR data. Comparison of the 1D NMR and HRESIMS data of 11 with coprinol29 revealed that 11 is also an illudalane-type sesquiterpenoid, but the molecular mass of 11 is 58 amu higher, which was deduced to be from a hydroxy and an acetyl group. The key correlations of H-9 (δH 4.58) with C-6, C-7, and C-10 and that of H-1 with C-2, C-16, and C-17 in the HMBC spectrum (Figure S104, Supporting Information) of 11 suggested that the hydroxy group was located at C-9 and the acetyl group was located at C-1. The configuration at C-9 of 11 was determined to be S by a positive Cotton effect at 346 nm in the Rh2(OCOCF3)4-induced ECD spectrum (Figure 5). Phomophyllin M (12) was isolated as a white powder, and its molecular formula was determined to be C15H20O4 based on

Phomophyllin H (8) was also obtained as a colorless oil, and its HRESIMS data corresponded to a molecular formula of C15H22O2. By comparing the NMR data of 8 with those of 6, compound 8 was found to have the same carbon backbone. The main difference was that the Δ6,7 double bond of 6 was reduced in 8. This conclusion was verified by the HMBC correlations (Figure 1) of H-6 (δH 2.52) with C-5, C-7, and C10 and of H2-7 (δH 1.45 and 1.75) with C-8, C-9, and C-14. H2, H-6, H3-13, and H3-15 were assigned to be α-oriented based on the NOESY correlations (Figure 1) of H-2/H3-15, H3-15/ H-6, and H-6/H3-13, while H-10 and H3-14 were assigned to be β-oriented due to the cross-peak of H-10/H3-14. The absolute configuration of 8 was established to be 2R,6S,10S,11R by comparing its experimental and calculated ECD curves (Figure 7). Phomophyllin I (9) was initially isolated as a white powder, and its molecular formula was established to be C15H24O3 based on its HRESIMS and 13C NMR spectra. According to the 1D NMR data of 9, it is also a protoilludane-type sesquiterpenoid, and it contains two oxygenated aliphatic quaternary carbons (δC 82.1, C-3 and δC 85.6, C-6) and one carbonyl carbon (δC 215.5, C-5). The positions of the oxygenated carbons were confirmed via the 1H−1H COSY correlations (Figure S85, Supporting Information) of H2-1 (δH 1.26) with H2-2 (δH 1.72 and 1.96) and the correlation of H2-9 (δH 1.56 and 1.82) with H-10 (δH 2.41), along with the HMBC correlations (Figure S84, Supporting Information) of H3-12 with C-3, C-4, and C-5 and H-10 with C-1, C-3, C-6, C-11, and C-15. The multiple chiral centers of 9 prompted us to attempt to confirm its absolute configuration using crystallography. The absolute configuration of 9 was established to be 3R,4R,6S,10R,11R via a single-crystal X-ray diffraction experiment with Cu Kα radiation (Figure 3). Compound 10 was initially obtained as a white powder; its HRESIMS spectrum allowed us to deduce a molecular formula of C16H24O3, implying four degrees of unsaturation. In addition, the UV (λmax 206, 287 nm) and IR (νmax 3447 F



Article

DUO diffractometer equipped with graphite-monochromatized Cu Kα radiation source (λ = 1.541 78 Å). Silica gel (200−300 mesh, Qingdao Marine Chemical, Inc., Qingdao, People’s Republic of China) and Lichroprep RP-C18 gel (40−63 μm, Merck, Darmstadt, Germany) were used for column chromatography. Thin-layer chromatography analyses were carried out on precoated plates (200−250 μm thickness, silica gel 60 F254, Qingdao Marine Chemical, Inc.) and detected with 5% H2SO4 in EtOH followed by heating. Fungus Material. The fungus Phomopsis sp. TJ507A was isolated from the leaves of Phyllanthus glaucus collected from Lushan Mountain in Jiangxi Province, People’s Republic of China, in September 2014. The fungus was identified by one of the authors (J.W.). The sequence data for this fungal strain have been submitted to GenBank with accession no. KY039149. A voucher specimen (M2016651) has been deposited in the China Center for Type Culture Collection in Wuhan University, Hubei Province, China. Extraction and Isolation. The fungus Phomopsis sp. TJ507A was cultured on potato dextrose agar at 28 °C for 5 days as seed cultures before subculturing in 1 L Erlenmeyer flasks with liquid medium (g/L: yeast 3, peptone 5, glucose 10, and maltose 3); the total amount of production medium was 150 L. The cultures were incubated in a rotary shaker incubator at 28 °C at 150 rpm for 10 days. Then, the mycelia were extracted with 75% EtOH at room temperature. The crude extract was partitioned with EtOAc, and then the solvent was evaporated under reduced pressure. The comparison of the liquid-state culture and solid-state culture was performed on the HPLC at 210 nm (Figure S133, Supporting Information). The obtained residue (150.0 g) was subjected to silica gel chromatography (100−200 mesh) and eluted with petroleum ether−EtOAc (50:1−0:1, gradient system) to give nine fractions (A−I). Fr. C (10.0 g) was repurified on Sephadex LH-20 (MeOH) to give two fractions, CA (3.5 g) and CB (2.3 g). The subsequent purification of CA using an ODS RP-C18 column (MeOH−H2O, 50:50−0:100) and semipreparative HPLC afforded 1 (5.5 mg, tR 14.5 min, MeOH−H2O, 80:20), 6 (15.6 mg, tR 13.5 min, MeOH−H2O, 50:50), and 17 (6.4 mg, tR 15.2 min, MeOH−H2O, 90:10). After separation on Sephadex LH-20 (MeOH), Fr. D (18.6 g) was purified by alternating column chromatography (CC) on silica gel using a gradient of petroleum ether−acetone (50:1−0:1) and on an ODS RP-C 18 column (MeOH−H2O, 40:60−0:100) prior to subjecting the sample to semipreparative HPLC to afford compounds 9 (2.0 mg, tR 13.3 min, CH3CN−H2O, 60:40) and 16 (7.8 mg, tR 14.2 min, MeOH−H2O, 50:50). Fr. E (10.3 g) was separated into three fractions (EA, EB, and EC) by Sephadex LH-20 (MeOH). Subsequently, Fr. EB was further purified with an ODS RP-C18 column (MeOH−H2O, 40:60−0:100) and semipreparative HPLC to afford 7 (5.6 mg, tR 15.3 min, MeOH−H2O, 70:30) and 8 (1.2 mg, tR 14.0 min, MeOH−H2O, 80:20). Fr. F (16.6 g) was subjected to Sephadex LH-20 chromatography (MeOH) and then further purified on an ODS RP-C18 column (MeOH−H2O, 40:60−0:100) to yield five fractions (FA−FE); compounds 2 (20.1 mg, tR 14.5 min, MeOH− H2O, 70:30), 3 (3.6 mg, tR 11.5 min, MeOH−H2O, 80:20), and 20 (8.1 mg, tR 23.5 min, CH3CN−H2O, 50:50) were ultimately isolated from FB (4.5 g) by semipreparative HPLC, and 18 (13.4 mg, tR 13.5 min, CH3CN−H2O, 50:50) was obtained from FC (2.0 g). Fr. I (15.2 g) was separated into four fractions (IA−ID) by using Sephadex LH20 (MeOH) and silica gel columns with petroleum ether−EtOAc (50:1−0:1). Fr. IB (5.3 g) was subjected to an ODS RP-C18 column (MeOH−H2O, 40:60−0:100) and further purified by semipreparative HPLC to afford 4 (8.6 mg, tR 15.5 min, MeOH−H2O, 65:35), 10 (2.5 mg, tR 10.5 min, MeOH−H2O, 85:15), 11 (2.4 mg, tR 14.7 min, MeOH−H2O, 70:30), and 15 (15.2 mg, tR 15.5 min, CH3CN−H2O, 40:60). Compounds 5 (8.2 mg, tR 13.7 min, MeOH−H2O, 50:50), 12 (4.7 mg, tR 8.5 min, MeOH−H2O, 85:15), 13 (3.1 mg, tR 18.2 min, MeOH−H2O, 60:40), 14 (20 mg, tR 14.5 min, MeOH−H2O, 75:25), and 19 (27 mg, tR 17.8 min, MeOH−H2O, 60:40) were isolated from Fr. H (18.7 g) after it was separated on a Sephadex LH-20 column (MeOH), a silica gel column with petroleum ether−EtOAc (50:1− 0:1), an ODS RP-C18 column (MeOH−H2O, 40:60−0:100), and semipreparative HPLC.

the HRESIMS and 1D NMR data. By detailed analysis of the 1D and 2D NMR spectroscopic data, 12 was elucidated to be an analogue of 10 with the major difference being the appearance of a carbonyl carbon signal and the absence of a methylene and a methoxy signal in the 13C NMR spectrum. The HMBC correlations of H-7 (δH 4.99) with C-5, C-6, C-9, C-13, and C-14 and the correlation of both H3-13 and H3-14 with C-9 (δC 211.8) confirmed that the hydroxy group was located at C-7 and the carbonyl was at C-9. The absolute configuration of C-7 was assigned to be R by comparison of the ECD effect at approximately 330 nm with that of (3R)-pterosin D.30 Phomophyllin N (13) was isolated as a white powder. Its molecular formula (C15H22O3) was determined based on the HRESIMS and 13C NMR spectra. The 1H NMR data of 13 displayed one singlet at δH 6.55, and the 13C NMR spectrum showed six aromatic carbon signals and two oxidized methylene signals, confirming that 13 was an analogue of dehydrobotrydienol31 with an additional hydroxyl substituent at C-3. This prediction was further supported by the key HMBC (Figure S124, Supporting Information) correlations of H-4 (δH 6.55) with C-2, C-3, C-5, C-6, and C-9 and H3-11 with C-3. The absolute configuration of C-8 was assigned as R due to the agreement between the experimental and calculated ECD curves of 13 (Figure 7). In recent years, our research group has focused on the discovery of lead compounds for use against Alzheimer disease (AD).32 β-Site amyloid precursor protein cleaving enzyme 1 (BACE1, β-secretase) is considered a prime therapeutic target of AD, and BACE1 inhibitors are being intensely pursued.33 Although hundreds of inhibitors have been discovered in recent years, the toxicities of these compounds have been a major obstacle to therapies for AD. For example, Eli Lilly and Co.’s phase II trial of a promising BACE1 inhibitor was unfortunately suspended due to the possibility of liver toxicity.34 In our study, the selected compounds were evaluated for their BACE1 inhibitory activities. Consequently, compounds 1−7, 9, 15, 18, and 20 displayed BACE1 inhibitory activities ranging from 19.4% to 43.8% at the concentration of 40 μM (Figure S131, Supporting Information), which exhibited statistically significant differences compared to the activity observed in the absence of inhibitor. LY2811376 was used as the positive control with an inhibitory activity of 38.6% (p < 0.01). None of the tested compounds showed obvious hepatotoxicity to normal human liver L-02 cells at a concentration of 40 μM (Figure S132, Supporting Information).

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a PerkinElmer PE-341 polarimeter (PerkinElmer, Waltham, MA, USA). UV spectra were obtained on a Varian Cary 50 UV/vis spectrophotometer (Varian, Salt Lake City, UT, USA). Experimental ECD spectra were measured on a JASCO-810 spectrometer. IR spectra were acquired on a Bruker Vertex 70 FTIR spectrophotometer (Bruker, Karlsruhe, Germany). NMR spectra were recorded on a Bruker AM-400 NMR spectrometer (Bruker, Germany). All chemical shifts (δ) were referenced to residual solvent signals. HRESIMS data were recorded on a Thermo Fisher LTQ XL LC/MS (Thermo Fisher, Palo Alto, CA, USA). Semipreparative HPLC separations were performed on a Dionex HPLC system equipped with an Ultimate 3000 DAD (Thermo Fisher, Scientific, Germany). A reversed-phase (RP) C18 column (10 × 250 mm) and a CHIRALPAK IC column (4.6 × 250 mm) were used for the separation. The crystallographic data were obtained on a Bruker APEX G



Article

Phomophyllin A (1): colorless oil; [α]20 D −182 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 203 (3.96), 261 (3.75), 298 (3.99) nm; IR (KBr) νmax 3450, 2957, 1632, 1461, 1388, 1209, 1130 cm−1; ECD (MeOH) λmax (Δε) 252 (+1.9), 296 (−1.0), 297 (−0.9), 330 (−3.1) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 3; HRESIMS ([M + H]+ m/z 233.1556, calcd for C15H21O2, 233.1542). Phomophyllin B (2): white crystal; [α]20 D −48 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 202 (3.89), 256 (3.85), 278 (3.81) nm; IR (KBr) νmax 3417, 2958, 2870, 1708, 1644, 1460, 1375, 1108 cm−1; ECD (MeOH) λmax (Δε) 243 (−4.2), 288 (+3.7), 356 (−1.4) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 3; HRESIMS ([M + H]+ m/z 249.1486, calcd for C15H21O3, 249.1491). Phomophyllin C (3): colorless oil; [α]20 D −93 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (3.77), 256 (3.72), 278 (3.69) nm; IR (KBr) νmax 3451, 2956, 2850, 1642, 1016 cm−1; ECD (MeOH) λmax (Δε) 228 (+5.3), 287 (+1.9), 353 (−1.8) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 3; HRESIMS ([M − H]− m/z 231.1394, calcd for C15H19O2, 231.1385). Phomophyllin D (4): colorless oil; [α]20 D −121 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 202 (3.78), 260 (3.71), 289 (3.68) nm; IR (KBr) νmax 3437, 2960, 2929, 1626, 1459, 1239, 885 cm−1; ECD (MeOH) λmax (Δε) 222 (−9.5), 264 (+3.3), 358 (−1.8) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 3; HRESIMS ([M + Na]+ m/z 271.1328, calcd for C15H20O3Na, 271.1310). Phomophyllin E (5): white powder; [α]20 D −116 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (3.78), 262 (3.72), 288 (3.71) nm; IR (KBr) νmax 3494, 2960, 2929, 1626, 1459, 1239, 885 cm−1; ECD (MeOH) λmax (Δε) 230 (−4.7), 287 (+2.8), 358 (−0.9) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 3; HRESIMS ([M + Na]+ m/z 271.1317, calcd for C15H20O3Na, 271.1310). Phomophyllin F (6): colorless oil; [α]20 D −158 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 202 (3.72), 258 (3.64), 289 (3.49) nm; IR (KBr) νmax 3406, 2956, 2867, 1753, 1645, 1616, 1460, 1374, 1300, 1243, 1120, 1029 cm−1; ECD (MeOH) λmax (Δε) 226 (−7.4), 265 (+3.5), 355 (−1.6) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 3; HRESIMS ([M + H]+ m/z 233.1522, calcd for C15H21O2, 233.1542). Phomophyllin G (7): colorless oil; [α]20 D −123 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 202 (3.69), 260 (3.58), 288 (3.55) nm; IR (KBr) νmax 3423, 2958, 2867, 1642, 1608, 1087 cm−1; ECD (MeOH) λmax (Δε) 235 (−8.0), 269 (+2.5), 355 (−1.9) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 2 and 3; HRESIMS ([M + H]+ m/z 233.1518, calcd for C15H21O2, 233.1542). Phomophyllin H (8): colorless oil; [α]20 D −7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.31), 248 (3.27) nm; IR (KBr) νmax 3421, 2951, 2867, 1726, 1649, 1365, 1345, 1122 cm−1; ECD (MeOH) λmax (Δε) 213 (−13.5), 252 (−10.2), 327 (−1.4) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 2 and 3; HRESIMS ([M + Na]+ m/z 257.1519, calcd for C15H22O2Na, 257.1517). Phomophyllin I (9): white crystal; [α]20 D −95 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 203 (3.55) nm; IR (KBr) νmax 3450, 2953, 1708, 1279, 1047, 959 cm−1; ECD (MeOH) λmax (Δε) 286 (−5.5) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 2 and 3; HRESIMS ([M + Na]+ m/z 275.1540, calcd for C15H24O3Na, 275.1623). Phomophyllins J (10a) and K (10b): white crystals; 10a: [α]20 D +5.9 (c 0.1, MeOH), 10b: [α]20 D −8.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.62), 287 (3.27) nm; IR (KBr) νmax 3447, 2959, 2928, 1642, 1638, 1460, 1384, 1086 cm−1; 10a: ECD (MeOH) λmax (Δε) 206 (−0.1), 222 (+1.2), 236 (+0.7), 251 (+0.9) nm, 10b: ECD (MeOH) λmax (Δε) 207 (+4.0), 222 (+1.7), 236 (+1.5), 258 (+1.1) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 2 and 3; HRESIMS ([M + Na]+ m/z 287.1625, calcd for C16H24O3Na, 287.1623). Phomophyllin L (11): colorless oil; [α]20 D −40 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 206 (4.45) nm; IR (KBr) νmax 3453, 2957, 1719, 1640, 1436, 1247, 663 cm−1; ECD (MeOH) λmax (Δε) 201 (+0.13),

203 (−0.48), 207 (+0.05), 209 (−0.29), 211 (+0.21), 219 (−0.10), 225 (−0.04), 232 (−0.09), 245 (+0.05) nm; 1H NMR (400 MHz) and 13 C NMR (100 MHz) data see Tables 2 and 3; HRESIMS ([M + Na]+ m/z 315.1606, calcd for C17H24O4Na, 315.1572). Phomophyllin M (12): white powder; [α]20 D −18.5 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (3.93), 261 (3.99) nm; IR (KBr) νmax 3414, 2965, 2927, 1628, 1571, 1458, 1090, 1027, 720 cm−1; ECD (MeOH) λmax (Δε) 229 (−0.2), 240 (−0.5), 267 (+7.4), 292 (−3.5) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 2 and 3; HRESIMS ([M + Na]+ m/z 287.1265, calcd for C15H20O4Na, 287.1259). Phomophyllin N (13): white powder; [α]20 D −5.7 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (4.21) nm; IR (KBr) νmax 3445, 2921, 2849, 1640, 1469, 1384, 1017 cm−1; ECD (MeOH) λmax (Δε) 204 (−0.2), 213 (+0.1), 233 (+0.03) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 2 and 3; HRESIMS ([M + Na]+ m/ z 273.1484, calcd for C15H22O3Na, 273.1467). Preparation of the (R)- and (S)-MTPA Esters of Compounds 6 and 7. Compound 6 (1.0 mg) was dissolved in 2 mL of anhydrous CH2Cl2. Subsequently, dimethylaminopyridine (40 mg), triethylamine, and (R)-MTPA-Cl (20 μL) were added sequentially. The reaction was stirred at room temperature, and after 2 h, the reaction was quenched by the addition of 1 mL of MeOH. The residue, obtained after concentrating the reaction mixture under reduced pressure, was subjected to a small silica gel column to isolate the (S)-MTPA ester of 6 (1.0 mg). The (R)-MTPA ester (0.8 mg) was prepared with (S)MTPA-Cl and purified in the same way. 7 was treated with (R)- and (S)-MTPA-Cl in the same manner as 6 to yield the corresponding (S)and (R)-MTPA esters, respectively. (R)-MTPA derivative of 6: 1H NMR (CDCl3, 400 MHz) δH 1.995 (dd, J = 11.1, 7.3 Hz, H-1a), 2.607 (dd, J = 11.1, 8.0 Hz, H-1b), 1.648 (m, H-9a), 1.758 (dd, J = 13.0, 8.0 Hz, H-9b), 3.374 (td, J = 8.2, 2.5 Hz, H-10), 1.174 (s, H-15), 6.511 (d, J = 2.6 Hz, H-7), 1.669 (d, J = 1.6 Hz, H-12). (S)-MTPA derivative of 6: 1H NMR (CDCl3, 400 MHz) δH 2.058 (dd, J = 11.1, 7.4 Hz, H-1a), 2.615 (dd, J = 11.0, 8.1 Hz, H-1b), 1.642 (dd, J = 13.3, 8.6 Hz, H-9b), 1.775 (dd, J = 13.0, 8.0 Hz, H-9), 3.411 (td, J = 8.4, 2.6 Hz, H-10), 1.177 (s, H-15), 6.507 (d, J = 2.7 Hz, H-7), 1.492 (d, J = 1.7 Hz, H-12). (R)-MTPA derivative of 7: 1H NMR (CDCl3, 400 MHz) δH 1.871 (m, H-1a), 1.986 (td, J = 10.2, 3.1 Hz, H-1b), 2.751 (ddd, J = 16.7, 7.9, 2.5 Hz, H-2a), 3.060 (m, H-2b), 6.456 (d, J = 2.5 Hz, H-7), 3.348 (dd, J = 7.6, 2.5 Hz, H-10), 1.665 (m, H-12), 1.397 (s, H-15), 1.188 (s, H13), 0.923 (s, H-14). (S)-MTPA derivative of 7: 1H NMR (CDCl3, 400 MHz) δH 1.856 (m, H-1a), 1.988 (td, J = 10.1, 2.6 Hz, H-1b), 2.736 (ddd, J = 16.1, 8.0, 2.0 Hz, H-2a), 3.045 (m, H-2b), 6.444 (d, J = 2.5 Hz, H-7), 3.246 (dd, J = 7.6, 2.6 Hz, H-10), 1.651 (m, H-12), 1.392 (s, H-15), 1.226 (s, H13), 0.970 (s, H-14). X-ray Crystallographic Analysis. Crystals of 2, 9, and 10 were obtained from methanol and H2O. The intensity data for 2, 9, and 10 were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97,35 expanded using difference Fourier techniques, and refined by the program with full-matrix least-squares calculations. The non-hydrogen atoms were refined anisotropically, and the hydrogen atoms were fixed at calculated positions. Crystallographic data (excluding structure factor tables) for the reported structures have been deposited with the Cambridge Crystallographic Data Center (CCDC) as supplementary publications numbers CCDC 1536435 for 2, CCDC 1536436 for 9, and CCDC 1539966 for 10. Crystal data for compound 2: C15H20O3, M = 248.31, a = 7.9251(3) Å, b = 11.9865(4) Å, c = 14.2732(5) Å, α = 90°, β = 90°, γ = 90°, V = 1355.87(8) Å3, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.671 mm−1, 7747 reflections measured, 2429 independent reflections (Rint = 0.0386). The final R1 values were 0.0513 (I > 2σ(I)). The final wR(F2) values were 0.1681 (I > 2σ(I)). The final R1 values were 0.0515 (all data). The final wR(F2) values H



Article

were 0.1684 (all data). The goodness of fit on F2 was 1.087. Flack parameter = 0.12(6).26 Crystal data for compound 9: C15H24O3, M = 252.34, a = 11.3606(3) Å, b = 13.5941(4) Å, c = 9.1502(2) Å, α = 90°, β = 90°, γ = 90°, V = 1413.13(6) Å3, T = 100(2) K, space group P21212, Z = 4, μ(Cu Kα) = 0.644 mm−1, 8664 reflections measured, 2566 independent reflections (Rint = 0.0285). The final R1 values were 0.0303 (I > 2σ(I)). The final wR(F2) values were 0.0848 (I > 2σ(I)). The final R1 values were 0.0304 (all data). The final wR(F2) values were 0.0849 (all data). The goodness of fit on F2 was 1.151. Flack parameter = 0.04(4).26 Crystal data for compound 10: C16H24O3, M = 264.35, a = 8.7757(12) Å, b = 9.1560(12) Å, c = 9.8895(13) Å, α = 73.107(4)°, β = 85.312(6)°, γ = 82.044(5)°, V = 752.29(17) Å3, T = 100(2) K, space group P1̅, Z = 2, μ(Cu Kα) = 0.629 mm−1, 8340 reflections measured, 2513 independent reflections (Rint = 0.0672). The final R1 values were 0.1889 (I > 2σ(I)). The final wR(F2) values were 0.4334 (I > 2σ(I)). The final R1 values were 0.1933 (all data). The final wR(F2) values were 0.4464 (all data). The goodness of fit on F2 was 2.079. Computational Methods. To determine the absolute configurations of 1, 3, 8, and 13, conformational analyses were carried out by using the BALLOON program.36 The quantum chemical computations were conducted with Gaussian 09 software.37 The input geometries of 1, 3, 8, and 13 and their enantiomers were optimized at the B3LYP/6-31G(d) level of theory in MeOH with the IEFPCM solvation model using Gaussian 09. The harmonic vibrational frequencies were calculated to confirm the stability of the final conformers. The ECD spectra were calculated for each conformer using TDDFT methodology at the B3LYP/6-311++G(d,p)//B3LYP/ 6-31G(d) level of theory with MeOH as solvent by the IEFPCM solvation model implemented in the Gaussian 09 program. The ECD spectra for each conformer were simulated using a Gaussian function with a bandwidth σ of 0.4 eV. BACE1 Inhibition Assay. The activities of the test compounds were assessed by a BACE1 FRET (fluorescence resonance energy transfer) assay kit purchased from Sigma-Aldrich (Catalogue No. CS0010). The enzyme inhibition assay was carried out according to the manufacturer’s instructions: the fluorescent assay buffer and BACE1 substrate solution were added to 96-well plates and mixed well by gentle pipetting. BACE1 enzyme solution (2 μL, 0.3 unit/μL) was added in the plates and diluted to a final volume of 100 μL as the controls (absence of inhibitor). A mixture of the test compound (2 μL, 40 μM, final concentration), BACE1 substrate (2 μL, 50 μM), and BACE1 enzyme (20 μL, 0.3 unit/μL) dissolved in the assay buffer was incubated for 2 h at 37 °C in the well plates. LY2811376 (Selleck Chemical, USA) was used as a positive control. The final concentration of DMSO was less than 4% (v/v). All reactions were performed in triplicate. Fluorescence intensities of the mixtures were measured using an EnSpire multimode plate reader (PerkinElmer, Singapore) with excitation and emission wavelengths of 320 and 405 nm, respectively. The fluorescence intensity of the blank was subtracted from all fluorescence readings. The inhibition rates of the test compounds were calculated by the following formula: [(IFo − IFi)/IFo] × 100%, where IFi and IFo are the fluorescence intensities of BACE1 in the presence and absence of the compounds, respectively. Cell Culture and Treatment of Compounds. L-02 cells were obtained from the Boster Biological Technology Co., Ltd. (Wuhan, China) and maintained in DMEM containing 10% fetal bovine serum (Gibco BRL Co., Grand Island, NY, USA) at 37 °C in a humidified incubator containing 5% CO2. All tested compounds were dissolved in DMSO. The final treatment concentration was 40 μM (DMSO concentration was less than 0.4% in the assay). Hepatotoxicity Assay. L-02 cells were seeded at a density of 5 × 103 cells/wells in 96-well plates and incubated for over 12 h. They were than treated with the test compounds at concentrations of 40 μM. After 24 h of incubation, cell viability was evaluated by the MTT method. Briefly, 20 μL of MTT (5 mg/mL) was added to 100 μL of cell culture medium, and the plates were incubated for another 4 h. The formed formazan crystals were dissolved in 150 μL of DMSO, and

the absorbance of each well was recorded at 570 nm with a microplate reader (BioTek Instruments, Inc., USA).

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00889. 1D and 2D NMR, HRESIMS, UV, IR, CD spectra of 1− 13 (PDF) X-ray crystallographic data of 2 (CIF) X-ray crystallographic data of 9 (CIF) X-ray crystallographic data of 10 (CIF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (Y. Xue). *E-mail: [email protected]. (Y. Zhang). ORCID

Yuben Qiao: 0000-0002-8881-1232 Zhengxi Hu: 0000-0002-1247-5615 Junjun Liu: 0000-0001-9953-8633 Yongbo Xue: 0000-0001-9133-6439 Yonghui Zhang: 0000-0002-7222-2142 Author Contributions ⊥

S. Xie and Y. Wu contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the Analytical and Testing Center at Huazhong University of Science and Technology for assistance in conducting CD, IR, and UV analyses. This work was financially supported by the Program for Changjiang Scholars of Ministry of Education of the People’s Republic of China (No. T2016088); National Natural Science Foundation for Distinguished Young Scholars (No. 81725021); Innovative Research Groups of the National Natural Science Foundation of China (81721005); the National Natural Science Foundation of China (Nos. 31200258, 31370372, 31270395, 81573316, 81641129, 21702067, and 31770379); the Academic Frontier Youth Team of HUST; and the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College (HUST). We thank the Analytical and Testing Center at Huazhong University of Science and Technology for assistance in testing of ECD, UV, and IR analyses.

■

REFERENCES

(1) (a) Tan, R.; Zou, W. Nat. Prod. Rep. 2001, 18, 448−459. (b) Hemtasin, C.; Kanokmedhakul, S.; Kanokmedhakul, K.; Hahnvajanawong, C.; Soytong, K.; Prabpai, S.; Kongsaeree, P. J. Nat. Prod. 2011, 74, 609−613. (c) Siriwardane, A. M.; Kumar, N. S.; Jayasinghe, L.; Fujimoto, Y. Nat. Prod. Res. 2015, 29, 1−4. (2) Strobel, G.; Daisy, B.; Castillo, U.; Harper, J. J. Nat. Prod. 2004, 67, 257−268. (3) Stierle, A.; Strobel, G.; Stierle, D. Science 1993, 260, 214−216. (4) Wei, W.; Jian, G.; Shen, Y.; Chu, Y. L.; Xu, Q.; Tan, R. Eur. J. Org. Chem. 2014, 2014, 5728−5734. (5) Dai, J.; Krohn, K.; Gehle, D.; Kock, I.; Flörke, U.; Aust, H. J.; Draeger, S.; Schulz, B.; Rheinheimer, J. Eur. J. Org. Chem. 2005, 2005, 4009−4016.

I



Article

(6) Wu, S.; Huang, R.; Miao, C.; Chen, Y. Chem. Biodiversity 2013, 10, 1276−1283. (7) Yan, B.; Wang, W.; Hu, D.; Sun, X.; Kong, L.; Li, X.; Du, X.; Luo, S.; Liu, Y.; Li, Y. Org. Lett. 2016, 18, 1108. (8) Yang, H.; Gao, Y.; Niu, D.; Yang, L.; Gao, X.; Du, G.; Hu, Q. Angew. Chem., Int. Ed. 2014, 45, 189−193. (9) Goddard, M. L.; Mottier, N.; Jeanneretgris, J.; Christen, D.; Tabacchi, R.; Aboumansour, E. J. Agric. Food Chem. 2014, 62, 8602− 8607. (10) Cimmino, A.; Andolfi, A.; Zonno, M. C.; Troise, C.; Santini, A.; Tuzi, A.; Vurro, M.; Ash, G.; Evidente, A. J. Nat. Prod. 2012, 75, 1130−1137. (11) Shao, Z.; Rao, G.; Li, C.; Abil, Z.; Luo, Y.; Zhao, H. ACS Synth. Biol. 2013, 2, 662−669. (12) (a) Bode, H. B.; Bethe, B.; Höfs, R.; Zeeck, A. ChemBioChem 2002, 3, 619−627. (b) Liu, Y.; Li, X. M.; Meng, L. H.; Jiang, W. Li.; Xu, G. M.; Huang, C. G.; Wang, B. G. J. Nat. Prod. 2015, 78, 1294− 1299. (c) Masi, M.; Meyer, S.; Clement, S.; Pescitelli, G.; Cimmino, A.; Cristofaro, M.; Evidente, A. J. Nat. Prod. 2017, 80, 2771−2777. (13) (a) Lai, Y.; Xue, Y.; Zhang, M.; Zhang, J.; Tang, W.; Liu, J.; Lei, L.; Yan, J.; Luo, Z.; Zuo, J.; Li, Y.; Yao, G.; Zhang, Y. Phytochemistry 2013, 96, 378−388. (b) Zhu, H.; Chen, C.; Yang, J.; Li, X. N.; Liu, J.; Sun, B.; Huang, S.; Li, D.; Yao, G.; Luo, Z.; Li, Y.; Zhang, J.; Xue, Y.; Zhang, Y. Org. Lett. 2014, 16, 6322−6325. (c) Hu, Z.; Lai, Y.; Zhang, J.; Wu, Y.; Luo, Z.; Yao, G.; Xue, Y.; Zhang, Y. Biochem. Syst. Ecol. 2014, 56, 60−64. (d) Xue, Y.; Wu, Y.; Zhu, H.; Li, X. N.; Qian, J. F.; Lai, Y.; Chen, C.; Yao, G.; Luo, Z.; Li, Y.; Zhang, Y. Fitoterapia 2014, 99, 204−210. (e) Lu, Y.; Xue, Y.; Liu, J.; Yao, G.; Li, D.; Sun, B.; Zhang, J.; Liu, Y.; Qi, C.; Xiang, M.; Luo, Z.; Du, G.; Zhang, Y. J. Nat. Prod. 2015, 78, 2205−2214. (f) Zhu, H.; Chen, C.; Liu, J.; Sun, B.; Wei, G.; Li, Y.; Zhang, J.; Yao, G.; Luo, Z.; Xue, Y.; Zhang, Y. Phytochemistry 2015, 115, 222−230. (g) Xue, Y.; Li, X.; Du, X.; Li, X.; Wang, W.; Yang, J.; Chen, J.; Pu, J.; Sun, H. Phytochemistry 2015, 116, 253−261. (14) (a) Hu, Z.; Wang, J.; Bi, X.; Zhang, J.; Xue, Y.; Yang, Y.; Luo, Z.; Yao, G.; Zhang, Y. Tetrahedron Lett. 2014, 55, 6093−6095. (b) Tang, Y.; Xue, Y.; Du, G.; Wang, J.; Liu, J.; Sun, B.; Li, X. N.; Yao, G.; Luo, Z.; Zhang, Y. Angew. Chem. 2016, 47, 4137−4141. (15) Wu, Z.; Lai, Y.; Zhou, L.; Wu, Y.; Zhu, H.; Hu, Z.; Yang, J.; Zhang, J.; Wang, J.; Luo, Z.; Xue, Y.; Zhang, Y. Sci. Rep. 2016, 6, 24809. (16) Hu, Z.; Wu, Y.; Xie, S.; Sun, W.; Guo, Y.; Li, X.; Liu, J.; Li, H.; Wang, J.; Luo, Z.; Xue, Y.; Zhang, Y. Org. Lett. 2017, 19, 258−261. (17) Nord, C. L.; Menkis, A.; Lendel, C.; Vasaitis, R.; Broberg, A. Phytochemistry 2014, 102, 197−204. (18) Fabian, K.; Lorenzen, K.; Anke, T.; Johansson, M.; Sterner, O. Z. Naturforsch., C: J. Biosci. 2001, 53c, 939−945. (19) Finkielsztein, L. M.; Bruno, A. M.; Renou, S. G.; Moltrasio Iglesias, G. Y. Bioorg. Med. Chem. 2006, 14, 1863−1870. (20) Fukuoka, M.; Yoshihira, K.; Natori, S.; Mihashi, K.; Nishi, M. Chem. Pharm. Bull. 1983, 31, 3113−3128. (21) Syrchina, A. I.; Gorenysheva, O. N.; Semenov, A. A.; Biyushkin, V. N.; Malinovskii, T. I. Chem. Nat. Compd. 1978, 14, 432−436. (22) Duránpatrón, R.; Hernándezgalán, R.; Collado, I. G. J. Nat. Prod. 2000, 63, 182−184. (23) Krohn, K.; Dai, J. U.; Aust, H. J.; Drager, S.; Schulz, B. J. Nat. Prod. 2005, 68, 400−405. (24) Siengalewicz, P.; Mulzer, J.; Rinner, U. Eur. J. Org. Chem. 2011, 2011, 7041. (25) (a) Johnson, E. P.; Vollhardt, K. P. C. J. Am. Chem. Soc. 1991, 113, 381−382. (b) Schwartz, B. D.; Matoušová, E.; White, R.; Banwell, M. G.; Willis, A. C. Org. Lett. 2013, 15, 1934−1937. (c) A Pitaval, A.; Leboeuf, D.; Ceccon, J.; Echavarren, A. M. Org. Lett. 2013, 15, 4580− 4583. (d) Siengalewicz, P.; Mulzer, J.; Rinner, U. J. Org. Chem. 2016, 81, 2078−2086. (26) Flack, H. D.; Bernardinelli, G. Acta Crystallogr., Sect. A: Found. Crystallogr. 1999, 55, 908−91. (27) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. Angew. Chem., Int. Ed. 1991, 56, 4092−4096.

(28) (a) Gerards, M.; Snatzke, G. Tetrahedron: Asymmetry 1990, 1, 221−236. (b) Xiong, L.; Zhu, C.; Li, Y.; Tian, Y.; Lin, S.; Yuan, S.; Hu, J.; Hou, Q.; Chen, N.; Yang, Y.; Shi, J. J. Nat. Prod. 2011, 74, 1188− 1200. (29) Pettit, G. R.; Meng, Y. H.; Pettit, R. K.; Herald, D. L.; Cichacz, Z. A.; Doubek, D. L.; Richert, L. J. Nat. Prod. 2010, 73, 388−392. (30) Kuroyanagi, M.; Fukuoka, M.; Yoshihira, K.; Natori, S. Chem. Pharm. Bull. 1979, 27, 731−741. (31) Duránpatrón, R.; Hernándezgalán, R.; Collado, I. G. J. Nat. Prod. 2000, 63, 182−184. (32) (a) Qi, C.; Bao, J.; Wang, J.; Zhu, H.; Xue, Y.; Wang, X.; Li, H.; Sun, W.; Gao, W.; Lai, Y.; Chen, J.; Zhang, Y. Chem. Sci. 2016, 7, 6563−6572. (b) Guo, Y.; Zhang, N.; Chen, C.; Huang, J.; Li, N.; Liu, J.; Zhu, H.; Tong, Q.; Zhang, J.; Luo, Z.; Xue, Y.; Zhang, Y. J. Nat. Prod. 2017, 80, 1493−1504. (33) Ellis, C. R.; Shen, J. J. Am. Chem. Soc. 2015, 137, 9543. (34) Lahiri, D. K.; Maloney, B.; Long, J. M.; Greig, N. H. Alzheimer's Dementia 2014, 10, 411−419. (35) Sheldrick, G. M.; Schneider, T. R. Methods Enzymol. 1997, 277, 319−343. (36) (a) Vainio, M. J.; Johnson, M. S. J. Chem. Inf. Model. 2007, 47, 2462−2474. (b) Puranen, J. S.; Vainio, M. J.; Johnson, M. S. J. Comput. Chem. 2010, 31, 1722−1732. (37) Frisch, G. W. T. M. J.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. GAUSSIAN 09, Revision D.01; Gaussian, Inc.: Wallingford, CT, 2009.

J


Protoilludane, Illudalane, and Botryane Sesquiterpenoids from the

Recommend Documents