Identification of Ingol and Rhamnofolane Diterpenoids from Euphorbia

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. 2018, 81, 1209−1218

Identification of Ingol and Rhamnofolane Diterpenoids from Euphorbia resinifera and Their Abilities to Induce Lysosomal Biosynthesis Ning-Dong Zhao,†,‡,§,⊥ Xiao Ding,‡,⊥ Yu Song,‡ Dong-Qiong Yang,‡ Hai-Li Yu,‡ Tiwalade Adegoke Adelakun,‡ Wen-Dan Qian,‡ Yu Zhang,‡ Ying-Tong Di,‡ Fang Gao,*,† Xiao-Jiang Hao,*,‡ and Shun-Lin Li*,‡ †

College of Chemistry and Chemical Engineering, Chongqing University, Chongqing, 400044, 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 § College of Chemistry and Engineering, Wenshan University, Wenshan 663000, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: The phytochemical study of Euphorbia resinifera afforded 18 structurally diverse diterpenoids, including 14 new ingol-type diterpenoids, euphorblins A−N (1−14), a new rhamnofolane diterpenoid, euphorblin O (15), and three known analogues (16−18). The structures of these compounds were deduced using 2D NMR spectroscopy and NOE experiments. The structure of compound 1 was confirmed by single-crystal X-ray crystallography. The abilities of the compounds to enhance lysosomal biosynthesis were evaluated through LysoTracker Red staining. Among the 10 active compounds, compounds 2, 4, and 18 showed remarkable immunofluorescence strength, and their LysoTracker staining intensities were 155.9%, 143.5%, and 140.7%, respectively, greater than that of the control. A series of lysosomal genes were also found to be upregulated by these compounds, which further confirms their ability to induce lysosome biosynthesis and suggests that these diterpenoids have potential as lead compounds for the development of drugs for the treatment of lysosome-related diseases.

which mimics Alzheimer disease. Therefore, it is important to identify additional diterpenoids in Euphorbia species that can induce lysosomal biosynthesis. The drug euphorbium is the air-dried latex of Euphorbia resinifera Berg., and it is usually applied to cavities or nerves to suppress chronic pain, mitigate toothache, and treat articular tuberculosis.13 The fresh latex of cultivated E. resinifera Berg. is a convenient source of the daphnane diterpenoid resiniferatoxin.14 Euphorbium also contains ingenol, 12-deoxyphorbol ester, ingol-type diterpenoids, triterpenoids, bisnorsesquiterpenoids, and other constituents.14−20 However, phytochemical and pharmacological studies on ingol-type diterpenoids from euphorbium are inadequate, and sufficient in-depth studies have not been conducted. In the present research, 18 diterpenoids were isolated from Euphorbia resinifera Berg. including 14 new ingol-type diterpenoids, euphorblins A−N

Euphorbia is the largest genus in the Euphorbiaceae family, and over 2000 species in this family have been identified throughout the world, including approximately 80 species distributed in China.1 Euphorbia species have attracted considerable attention recently because of the diverse structures and intriguing biological activities of their constituents. Previous studies on the chemical components of the plants from the genus Euphorbia have resulted in a large array of macrocyclic diterpenoids, such as ingenane, tigliane, daphnane, jatrophane, lathyrane, myrsinane, and premyrsinane. Some of them exhibited potent properties such as vasorelaxant, antitumor, and antimultidrug resistance activities.2−11 We previously reported an ingenol-type diterpenoid, HEP-14 (20-deoxyingenol-5β-O-angelate), isolated from Euphorbia peplus, which induces lysosomal biosynthesis.12 The data indicated that HEP-14 regulates lysosomal biosynthesis in a protein kinase C (PKC)-dependent and mTORC1-independent manner. HEP14 promotes clearance of lipid droplets in HepG2 cells and limits amyloid plaque formation in the brain of APP/PS1 mice, © 2018 American Chemical Society and American Society of Pharmacognosy

Received: November 19, 2017 Published: April 18, 2018 1209

DOI: 10.1021/acs.jnatprod.7b00981 J. Nat. Prod. 2018, 81, 1209−1218

Journal of Natural Products

Article

Chart 1

Table 1. 1H NMR Spectroscopic Data (δ) for Compounds 1−9a position 1α

1

3 2.76 2.04

b

4

5

6

8

9

2.80, dd (15.0, 9.0) 1.70, d (15.0) 2.50, m 5.32, d (8.6) 5.58, br s 5.18, d (1.4) 4.48, dd (10.9, 2.0) 0.85, dd (10.9, 9.0) 0.98b

2.80, dd (15.0, 9.1)

2.72, dd (14.9, 9.2) 1.60, d (14.9)

2.78, dd (15.0, 9.1) 1.66, d (15.0)

2.77, dd (15.0, 9.1)

0.98b

1.05b

2.51, m 5.00, d (8.5) 5.29, br s 5.14, d (1.8) 4.54, dd (10.6, 1.8) 1.13, dd (10.5, 9.2) 1.06b

2.50, 5.17, 5.40, 5.12, 4.53,

0.86 dd (11.0, 9.0)

2.36, m 4.07, d (8.4) 5.39, br s 5.28, d (1.1) 4.53, dd (10.1, 1.7) 1.07b

4.80, dd (11.1, 4.0)

4.82, dd (10.6, 4.0) 2.85, m 1.02, d (7.5) 2.05, d (1.5) 1.04, s 0.81, s 1.03, d (6.8)

4.83, dd (11.0, 3.9) 2.86, m 0.89, d (7.5) 2.04, d (0.95) 1.07, s 0.83, s 1.04, d (7.3) 2.09, s 2.05, s 1.98, s 3.64, 3.60, AB q (14.8) 7.12, d (8.5)

4.83, dd (9.2, 3.9)

9

2.48, m 4.72, d (8.4) 4.78, br s 5.10, s 4.51, dd (10.3, 1.7) 0.99b

11

0.97b

12 13 16 17 18 19 20 3-Ac 8-Ac 12-Ac 2′

4.79, dd (10.5, 4.0) 2.79, m 0.84, d (7.4) 2.01, s 1.08, s 0.83, s 1.00, d (7.5) 2.07, s 2.05, s 2.01, s 5.28, s

4′/8′

7.36−7.38, m

7.27, d (8.7)

7.22, d (8.6)

7.28, d (8.8)

7.36, d (8.7)

5′/ 7′ 6′ 2′−OH/ 5′−OH 6′-OMe/ OH 2-OH

7.32−7.34, m 7.32−7.34, m 3.60, br s/

6.88, d (8.7)

6.79, d (8.6)

6.88, d (8.6)

6.87, d (8.7)

7.00, d (2.1)/6.96, dd (8.3, 2.1) / 6.82, d (8.3)

3.45, d (4.3)/ 3.83, s/

3.51, d (5.0)/ /6.14, br s

3.34 d (5.2)/ 3.84, s/

3.79, d (4.1)/ 3.78, s/

3.75, d (4.3)/5.65, br s 3.86/

2 3 5 7 8

2.75, dd (14.9, 9.3) 1.62, d (14.9) 2.53, m 4.68, d (8.4) 4.87, br s 5.23b 4.55, dd (10.4, 1.7) 1.03b

b

2.71, dd (14.9, 9.1) 1.62, d (14.9) 2.50, m 4.78, d (8.5) 4.82, br s 5.08, d (1.6) 4.53, dd (10.4, 1.6) 0.85, dd (10.4, 9.0) 0.98, dd (10.7, 9.0) 4.80, dd (10.7, 4.0) 2.80, m 0.86, d (7.2) 2.04, d (1.1) 1.09, s 0.83, s 1.02, d (7.3) 2.08, s 2.03, s 2.08, s 5.23, d (4.3)

1β

2.68, dd (14.9, 9.1) 1.60, d (14.9)

2

1.06b

4.56, s 4.75, br s 5.12, s 4.54, dd (10.6, 1.8) 1.08, dd (10.6, 9.1) 1.06b

4.82, dd (9.7, 4.1) 2.80, m 0.81, d (7.5) 2.02, d (1.4) 1.10, s 0.83, s 1.02, d (7.3) 2.08, s 2.09, s 2.03, s 5.22b

4.81, dd (10.8, 3.9) 2.77b 1.11, s 2.02, s 1.13, s 0.85, s 1.01, d (7.3) 2.13, s 2.06, s 2.09, s 5.27, d (5.2)

4.79, dd (11.1, 4.0) 2.88, m 0.97, d (7.5) 2.07, s 0.90, s 0.75, s 1.04, d (7.3) 2.13, s 1.88, s 2.07, s 5.25, d (4.1)

1.70, d (15.0) 2.50, 5.34, 5.59, 5.19, 4.49,

2.89, 0.99, 2.08, 0.90, 0.76, 1.04, 2.13, 2.07, 1.94, 5.20,

m d (8.6) br s s dd (10.9, 2.0)

m d (7.5) d (1.4) s s d (7.4) s s s d (4.3)

7

1.95, s 2.09, s 3.72, 3.70, AB q (14.5) 7.27−7.30, m 7.30−7.35, m 7.25−7.29, m

6.76, d (8.5)

1.67, d (15.0) m d (8.5) br s d (1.2) dd (10.7, 1.8)

1.13, dd (10.7, 9.1) 1.03b

2.88, m 0.92, d (7.5) 2.06, d (1.4) 1.05, s 0.82, s 1.04, d (7.3) 2.06, s 2.09, s 1.99, s 3.62, 3.59, AB q (15.1) 6.84, d (2.1)/ 6.72 dd (8.2, 2.1) /6.78 d (8.2) /5.83 br s

/5.75, br s

3.87, s/

3.33, br s

a Data (δ) were measured in CDCl3 for 1−3 and 5−9 at 500 MHz and for 4 at 600 MHz. Coupling constants (J) in Hz are given in parentheses. Assignments of 1H NMR data were based on HSQC and HMBC experiments. bOverlapping signals.

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Article

Table 2. 1H NMR Spectroscopic Data (δ) for Compounds 10−17a position

10

11

12

13

14

1α

2.84b

2.81b

1β 2 3

2.05b

2.04, d (14.9)

4.89, s

5 6 7

5.33, br s 5.17, d (1.7)

5.14, d (1.7)

5.38, d (1.9)

4.97, d (2.0)

5.68, d (2.1)

8

4.53, dd (10.6, 1.8)

9

1.13, dd (10.6, 9.0)

4.72, dd (10.8, 1.9) 1.14, dd (10.3, 9.4) 1.02b

3.50, ddd (10.3, 6.7, 2.3) 1.20 dd (10.4, 9.2) 1.02b

3.77, ddd (10.4, 5.2, 2.1) 1.12 dd (10.3, 9.1) 0.97b

4.85, dd (11.0, 3.8) 3.22, m

4.87, dd (11.2, 4.0) 2.92, m

4.85, dd (11.3, 3.9) 3.32, m

4.90, s

2.79, dd (15.0, 9.0) 1.70, d (15.0) 2.50, m 5.17, d (8.5)

2.77, dd (15.0, 9.0) 1.69, d (15.0) 2.43, m 5.37, d (8.6)

2.83, dd (15.1, 9.1) 1.71, d (15.1) 2.54, m 5.34, d (8.6)

5.33, br s

5.54, br s

5.55, br s

5.87, br s

11

1.05

12

4.83, dd (10.9, 3.9)

13

2.83b

4.53, dd 1.7) 1.12, dd 8.9) 1.05, dd 9.1) 4.82, dd 3.9) 2.83b

16

1.16, s

1.15, s

0.93, d (7.7)

0.99, d (7.5)

0.95, d (7.5)

17

2.04, d (1.1)

2.03, d (1.4)

2.05, d (1.4)

5.41, s

18 19 20 1-Ac 3-Ac 7-Ac 8-Ac 12-Ac 13-Ac 2′

1.07, s 0.82, s 1.03, d (7.4)

1.06, s 0.82, s 1.04, d (7.4)

4.66 d (12.6) 4.37 d (12.6) 1.07, s 0.83, s 1.01, d (7.2)

1.11, s 1.07, s 1.02, d (7.4)

1.10, s 1.05, s 1.02, d (7.2)

2.10, s

2.09, s

2.03, s

2.11, s 2.14, s

2.03, s 2.14, s

2.07, s

2.11, s

b

(10.7, (10.7, (10.9, (10.9,

14

4′/8′ 5′/7′ 6′ 2-OH/6′OMe 11-OH/17OMe

2.08, s 1.99, s

15 5.33, d (7.4)

2.46, m α 2.68, d (15.2, 9.1) β 1.82, d (15.2) 3.59, m 4.98, dd (9.3, 3.7) 2.32, m 3.11, d (4.5)

5.13, d (2.9) 5.21, dd (12.3, 2.9) 2.40, dd (12.3, 4.6) a 4.65, s b 4.29, s 1.64, s 1.61, 0.95, 0.93, 2.12,

s d (7.4) d (6.8) s

16

17

2.81, dd (15.0, 9.2) 1.67, d (15.0) 2.56, m 5.22, d (8.5)

2.73, dd (14.9, 9.2) 1.59, d (14.9) 2.35, m 4.07, d (8.5)

5.79, br s

5.38, br s

4.22, s

5.26, d (1.2)

4.54, dd (10.7, 1.3) 1.38 dd (10.7, 9.3) 1.09 dd (11.1, 9.3) 4.85, dd (11.1, 3.9) 2.88, m

4.54, dd (10.3, 1.9) 1.06b

4.83, dd (10.6, 4.0) 2.86, m

0.90, d (7.5)

1.01, d (7.5)

2.04, d (1.05)

2.05, d (1.5)

1.07, s 0.85, s 1.06, d (7.3)

1.04, s 0.82, s 1.03, d (7.3)

1.05b

2.09, s 1.88, s

1.97, s 2.09, s

1.97, s 2.08, s

3.72, 3.69, AB q (14.7) 7.26−7.27, m 7.30−7.33, m 7.27−7.29, m 3.18, br s /

3.66, 3.61, AB q 3.66, s (14.8) 7.17, d (8.7) 7.18, d (8.7) 6.84, d (8.7) 6.85, d (8.6)

1.76, s 3.72, 3.64, AB q (15.2) 7.21, d (8.7) 6.85, d (8.7)

3.65, 3.63, AB q (14.6) 7.20, d (8.7) 6.86, d (8.7)

3.15, br s/3.79, s /3.80, s

/3.78, s

/3.78, s

/3.34, s 3.52, s

2.10, s 2.08, s

1.95, s 2.08, s

2.61, br s/

a

Data (δ) were measured in CDCl3 for 10−17 at 500 MHz. Coupling constants (J) in Hz are given in parentheses. Assignments of 1H NMR data were based on HSQC and HMBC experiments. bOverlapping signals.

[α]25D +25 (c 0.1, MeOH). HRESIMS analysis [m/z 649.2640 [M + Na]+ (calcd 649.2619)] revealed a molecular formula (C34H42O11) that implied 14 indices of hydrogen deficiency. The IR absorption bands at 3437, 1738, 1632, and 1242 cm−1 indicated the presence of hydroxy, carbonyl, aromatic, and olefinic groups, respectively. Both 1H and 13C NMR data analyses (Tables 1 and 3) revealed the presence of three acetoxy groups (δH 2.01, 2.05, 2.07, each 3H; δC 170.4, 170.4, 170.6) and a 2-hydroxy-2-phenylacetyl unit [δH 7.32−7.38 (5H, m), 5.28 (1H, s), and 3.60 (br s); δC 172.1, 128.9, 2 × 126.5, 2 × 128.7, 138.0, 73.1]. In addition to these signals, the 13C NMR spectrum displayed signals for the remaining 20 skeletal carbons, including five methyls (two tertiary, two secondary, one olefinic), a methylene, nine methines (an olefinic and four oxygenated carbons), two oxygenated tertiary carbons, two

(1−14), a new rhamnofolane diterpenoid, euphorblin O (15), and three known analogues (16−18). The physical data of euphorblins P and Q (16 and 17) are being reported for the first time. In this report, the isolation, structure elucidation, and evaluation of the lysosomal biosynthesis induction activity of the above compounds are described. Ten compounds caused statistically significant increases in lysosome biosynthesis. Thus, three compounds were selected for further evaluation, and all of them showed notable abilities to induce lysosome biosynthesis at different doses and concentrations, which suggests additional treatment options for lysosome-related diseases.

■

RESULTS AND DISCUSSION Ingol-Type Diterpenoids. Compound 1 was purified as colorless crystals with a melting point of 212−214 °C and 1211



Article

Table 3. 13C NMR Spectroscopic Data (δ) for Compounds 1−9a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3-Ac 8-Ac 12-Ac 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 6′-OMe

1

2

3

4

5

6

7

8

9

31.4, CH2 29.5, CH 77.3, CH 73.0, C 116 9, CH 138.5, C 78.4, CH 71.2, CH 24.5, CH 19.3, C 30.6, CH 70.4, CH 43.0, CH 207.4, C 70.9, C 16.8, CH3 17.3, CH3 29.1, CH3 16.0, CH3 13.3, CH3 170.6, C 21.0, CH3 170.4, C 21.0, CH3 170.4, C 20.5, CH3 172.1, C 73.1, CH 138.0, C 126.5, CH 128.7, CH 128.9, CH 128.7, CH 126.5, CH

31.5, CH2 29.6, CH 77.2, CH 73.0, C 116 9, CH 138.6, C 78.5, CH 71.2, CH 24.5, CH 19.3, C 30.6, CH 70.4, CH 43.0, CH 207.4, C 71.0, C 16.8, CH3 17.3, CH3 29.2, CH3 16.1, CH3 13.3, CH3 170.6, C 21.0, CH3 170.4, C 21.0, CH3 170.4, C 20.5, CH3 172.3, C 72.7, CH 130.0, C 127.8, CH 114.2, CH 159.9, C 114.2, CH 127.8, CH 55.3, CH3

31.5, CH2 29.5, CH 78.0, CH 72.9, C 117.1, CH 138.3, C 77.8, CH 71.4, CH 24.4, CH 19.2, C 30.6, CH 70.3, CH 43.2, CH 207.4, C 71.1, C 16.8, CH3 17.4, CH3 29.1, CH3 16.0, CH3 13.3, CH3 171.8, C 21.6, CH3 170.5, C 21.0, CH3 170.4, C 21.0, CH3 172.5, C 72.5, CH 130.0, C 127.8, CH 116.1, CH 156.4, C 116.1, CH 127.8, CH

38.7, CH2 75.5, C 86.3, CH 69.5, C 116.3, CH 139.1, C 78.4, CH 71.1, CH 24.5, CH 19.4, C 30.5, CH 70.3, CH 43.0, CH 206.0, C 69.3, C 26.1, CH3 17.3, CH3 29.2, CH3 16.0, CH3 13.3, CH3 173.1, C 20.4, CH3 170.4, C 21.0, CH3 170.3, C 20.9, CH3 172.4, C 72.4, CH 129.9, C 127.5, CH 114.2, CH 160.0, C 114.2, CH 127.5, CH 55.3, CH3

31.4, CH2 29.4, CH 76.7, CH 73.3, C 117.9, CH 138.3, C 77.4, CH 71.5, CH 24.5, CH 19.1, C 30.6, CH 70.5, CH 43.0, CH 207.5, C 71.3, C 17.0, CH3 17.4, CH3 29.0, CH3 15.9, CH3 13.4, CH3 171.5, C 20.8, CH3 170.3, C 20.8, CH3 170.4, C 21.0, CH3 172.7, C 72.6, CH 130.1, C 127.9, CH 114.0, CH 160.0, C 114.0, CH 127.9, CH 55.3, CH3

31.4, CH2 29.4, CH 76.8, CH 73.3, C 117.9, CH 138.3, C 77.4, CH 71.5, CH 24.5, CH 19.1, C 30.7, CH 70.5, CH 42.9, CH 207.5, C 71.3, C 17.0, CH3 17.4, CH3 28.8, CH3 15.9, CH3 13.4, CH3 171.6, C 21.0, CH3 170.4, C 20.8, CH3 170.4, C 20.8, CH3 172.5, C 72.5, CH 131.1, C 112.8, CH 145.7, C 146.8, C 110.4, CH 118.5, CH 55.9, CH3

31.7, CH2 32.0, CH 76.0, CH 75.6, C 117.7, CH 138.8, C 76.5, CH 71.6, CH 24.5, CH 19.1, C 30.6, CH 70.6, CH 43.0, CH 207.7, C 72.8, C 16.0, CH3 17.6, CH3 29.1, CH3 16.0, CH3 13.4, CH3

31.5, CH2 29.4, CH 77.3, CH 73.2, C 117.0, CH 139.2, C 76.7, CH 71.5, CH 24.6, CH 19.3, C 30.6, CH 70.6, CH 43.1, CH 207.6, C 71.1, C 16.9, CH3 17.5, CH3 29.1, CH3 16.1, CH3 13.4, CH3 171.3, C 20.6, CH3 170.4, C 21.0, CH3 170.5, C 21.0, CH3 170.5, C 40.8, CH2 125.8, C 130.4, CH 115.8, CH 155.1, C 115.8, CH 130.4, CH

31.5, CH2 29.5, CH 76.9, CH 73.3, C 117.0, CH 139.4, C 76.9, CH 71.4, CH 24.6, CH 19.3, C 30.6, CH 70.6, CH 43.0, CH 207.6, C 71.1, C 17.0, CH3 17.5, CH3 29.0, CH3 16.1, CH3 13.4, CH3 171.0, C 20.6, CH3 170.4, C 21.0, CH3 170.4, C 20.9, CH3 170.4, C 40.9, CH2 126.8, C 115.6, CH 145.7, C 146.0, C 110.8, CH 120.8, CH 55.9, CH3

170.3, C 20.9, CH3 170.4, C 21.0, CH3 170.0, C 41.8, CH2 133.9, C 129.2, CH 128.7, CH 127.3, CH 128.7, CH 129.2, CH

a

Data (δ) were measured in CDCl3 for 1−3 and 5−9 at 125 MHz and for 4 at 150 MHz. Coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments.

quaternary carbons, and a ketocarbonyl carbon. The spectroscopic data indicated that compound 1 had different acyl groups at C-3, C-7, and C-8 by comparison with the NMR data of 12-O-acetylingol-3,8-ditiglate.21 The COSY experiment (Figure 1) showed a cross-peak of OH-2′ with H-2′ and the HMBC cross-peaks from H-7 (δH 5.10) and H-2′ (δH 5.28) to C-1′ (δC 172.1) and from H-2′ (δH 5.28) to C-4′ (δC 126.5), C8′ (δC 126.5), C-3′ (δC 138.0), and C-1′ (δC 172.1), indicating that C-2′ was hydroxylated and the 2-hydroxy-2-phenylacetoxy

moiety was located at C-7. Consequently, the remaining three acetoxy groups must be attached at C-3, C-8, and C-12 based on the HMBC correlations from H-3 (δH 4.72, d, J = 8.4 Hz), H-8 (δH 4.51, dd, J = 10.3, 1.7 Hz), and H-12 (δH 4.79, dd, J = 10.5, 4.0 Hz) to the three acetyl carbonyls at δC 170.6, 170.4, and 170.4. The relative configuration of 1 was determined by a ROESY experiment (Figure 2) and was identical to that 12-Oacetylingol-3,8-ditiglate.21 The assignment of OH-2′ as being

Figure 1. Key COSY (bold) and HMBC (→) correlations of 1 and 15.

Figure 2. Selected ROESY correlations of 1 and 15. 1212



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β-oriented was verified by X-ray diffraction (Cu Kα radiation) (Figure 3), which indicated a (2′S) absolute configuration. In

br s) to C-1 (δC 38.7) and C-2 (δC 75.5). The key ROESY correlations of CH3-16 with H-1β and of OH-2 with H-3 indicated that OH-2 was α-oriented. Thus, the structure of euphorblin D (4) was defined as shown. Compound 5 has the same molecular formula as compound 2 (C35H44O12). Comparison of the 1H and 13C NMR data (Tables 1 and 3) of 5 and 2 suggested that they were isomers differing in the C-2′ configuration. For compound 5, H-3 and H-5 were shifted by ΔδH + 0.54 and 0.76, respectively, due to the anisotropic deshielding by the benzene ring. Thus, C-2′ of compound 5 was R-configured. This assignment was verified by basic hydrolysis of compound 5, which afforded (R)-4methoxymandelic acid (Figure S157, Supporting Information), and the specific rotation ([α]25D −166) of (R)-4-methoxymandelic acid confirmed the (2′R) absolute configuration.23 Therefore, the structure of euphorblin E (5) was determined as illustrated. HRESIMS data analysis of compound 6 (m/z 695.2657 [M + Na]+, calcd 695.2674) revealed a molecular formula of C35H44O13. A comparison of the NMR data (Tables 1 and 3) of compound 6 with those of compound 5 suggested that C-5′ in 6 carried a hydroxy group. This deduction was verified by the presence of an ABX spin system involving H-7′ (δH 6.82, d, J = 8.3 Hz), H-8′ (δH 6.96, dd, J = 8.3, 2.1 Hz), and H-4′ (δH 7.00, d, J = 2.1 Hz) in the 1H NMR spectra of compound 6 and by the HMBC cross-peaks between 6′-OCH3 (δH 3.86, s) and C6′ (δC 146.8) and between OH-5′ (δH 5.65, br s) and C-4′ (δC 112.8). Therefore, the structure of euphorblin F (6) was assigned as shown. HRESIMS data analysis of compound 7 (m/z 591.2584 [M + Na]+, calcd 591.2565) revealed a molecular formula of C32H40O9. A comparison of the 1H and 13C NMR data (Tables 1 and 3) of compounds 7 and 1 revealed that the H-3 resonance was shielded (ΔδH −0.65), suggesting that a hydroxy group was located at C-3 in compound 7. This was confirmed by the HMBC correlations from H-3 (δH 4.07, d, J = 8.4 Hz) to C-2 (δC 32.0), C-4 (δC 75.6), and C-16 (δC 16.0). In addition, a phenylacetoxy group was present at C-7 instead of the (S)-2hydroxy-2-phenylacetoxy group in 1 by the observation of an HMBC correlation between H-7 (δH 5.28, d, J = 1.1 Hz) and C-1′ (δC 170.0) (CO of the phenylacetyl group). Hence, the structure of euphorblin G (7) was determined as illustrated. The molecular formula of compound 8 was deduced as C34H42O11 from its HRESIMS data (m/z 665.2372 [M + K]+, calcd 665.2359). NMR data (Tables 1 and 3) revealed that compound 8 was similar to 18 but with the methoxy substituent at C-6′ replaced by a hydroxy substituent in compound 8, which was confirmed by the HMBC correlations between OH-6′ (δH 5.75, br s) and C-5′ (δC 115.8), C-7′ (δC 115.8), and C-6′ (δC 155.1). Thus, the structure of euphorblin H (8) was defined as shown. HRESIMS data analysis of compound 9 (m/z 679.2742 [M + Na]+, calcd 679.2725) revealed a molecular formula of C35H44O12. The 1H and 13C NMR data (Tables 1 and 3) of compound 9 closely resembled those of compound 18. The only difference was the presence of a hydroxy group at C-5′ in compound 9, which was confirmed by the 1H NMR signals for an ABX spin system involving H-7′ (δH 6.78 d, J = 8.2 Hz), H8′ (δH 6.72, dd, J = 8.2, 2.1 Hz), and H-4′ (δH 6.84, d, J = 2.1 Hz) and by the HMBC cross-peaks between OCH3-6′ (δH 3.87, s) and C-6′ (δC 146.0) and between OH-5′ (δH 5.83, br s) and C-4′ (δC 115.6) and C-5′ (δC 145.7). Hence, the structure of euphorblin I (9) was defined as shown.

Figure 3. ORTEP drawing of the X-ray structure of 1.

evaluation of the absolute configuration of the stereocenters of the medium-sized ring and considering the anisotropic effect of the (S)-2-hydroxy-2-phenylacetyl moiety, the observation of H3 and H-5 (Table1) being shifted dramatically upfield indicates that they are anisotropically shielded by the benzene ring.22 Hence, the structure of euphorblin A (1) was unambiguously assigned as shown. Analysis of the 1H and 13C NMR spectra of compounds 2 and 3 showed that their structures were similar to compound 1, with differences involving the substituents of the benzene moiety. Compound 2 was isolated as a white, amorphous powder ([α]25D +14, c, 0.1, MeOH). Its molecular formula was established to be C35H44O12 by HRESIMS data analysis (m/z 679.2732 [M + Na]+ calcd 679.2725). Its 1H NMR data (Table 1) showed an aromatic methoxy group (δH 3.83, s) and four aromatic protons (δH 7.27 and 6.88, 2H, d, J = 8.7 Hz each) of an AA′BB′ spin system of a p-disubstituted benzene ring, suggesting the para position must be methoxylated. Furthermore, the HMBC correlations between the methoxy protons (δH 3.83, s) and the quaternary sp2 carbon (δC 159.9) confirmed the above assignment. HRESIMS data analysis of compound 3 (m/z 665.2548 [M + Na]+, calcd 665.2568) revealed a molecular formula of C34H42O12. A hydroxy group was located at C-6′ in 3 instead of the methoxy group in compound 2 (NMR data, Tables 1 and 3), and this structural change caused the signal of C-6′ to be upshifted by ΔδC −3.5 compared to that of compound 2. The structural assignment was confirmed by the weak HMBC correlations from OH-6′ (δH 6.14, br s) to C-5′ (δC 116.1) and C-7′ (δC 116.1). The relative configurations of euphorblins B (2) and C (3) were proposed to be the same as compound 1 based on their ROESY spectra. HRESIMS data analysis of compound 4 revealed a molecular formula of C35H44O13 (m/z 695.2689 [M + Na]+, calcd 695.2674). Both the 1H and 13C NMR data (Tables 1 and 3) indicated that the structure of compound 4 closely resembled compound 2, the difference being the presence of a hydroxy group at C-2 in 4, which was verified by the presence of an oxygenated sp3 C-2 (δC 75.5) in 4 instead of the methine (δC 29.6) in 2 and by the HMBC correlations from OH-2 (δH 3.33, 1213



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Table 4. 13C NMR Spectroscopic Data (δ) for Compounds 10−17a position

10

11

12

13

14

15

16

17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1-Ac

38.8, CH2 75.5, C 85.9, CH 69.7, C 116.5, CH 139.8, C 76.8, CH 71.4, CH 24.6, CH 19.3, C 30.6, CH 70.4, CH 43.1, CH 206.3, C 69.2, C 26.3, CH3 17.5, CH3 29.1, CH3 16.1, CH3 13.3, CH3

40.2, CH2 76.9, C 87.3, CH 71.2, C 117.8, CH 141.3, C 78.2, CH 72.8, CH 26.0, CH 20.7, C 32.0, CH 71.8, CH 44.5, CH 207.7, C 70.6, C 27.6, CH3 18.9, CH3 30.5, CH3 17.5, CH3 14.7, CH3



31.3, CH2 29.4, CH 76.6, CH 72.7, C 121.6, CH 140.2, C 73.3, CH 71.6, CH 27.5, CH 18.7, C 31.0, CH 71.2, CH 43.0, CH 207.4, C 71.2, C 16.9, CH3 102.1, CH 29.5, CH3 16.5, CH3 13.5, CH3

79.7, CH 29.2, CH 30.2, CH2 70.4, C 204.5, C 39.5, CH 70.3, CH 43.6, CH 44.0, CH 73.9, C 72.4, C 76.6, CH 67.5, CH 46.1, CH 138.4, C 111.6, CH2 23.8, CH3 26.0, CH3 17.0, CH3 11.8, CH3 170.4, C 21.0, CH3



3-Ac

173.0, C 21.0, CH3

174.3, C 21.9, CH3

170.6, C 20.5, CH3

170.7, C 21.1, CH3 170.3, C 21.1, CH3

170.3, C 20.5, CH3 169.9, C 21.3, CH3

7-Ac 8-Ac 12-Ac

170.3, C 20.6, CH3 170.4, C 20.9, CH3

171.7, C 22.3, CH3 171.8, C 22.4, CH3

170.7, C 21.0, CH3 170.3, C 21.0, CH3

170.8, C 20.6, CH3

170.1, C 41.7, CH2 133.8, C 129.2, CH 128.7, CH 127.5, CH 128.7, CH 129.2, CH

171.8, C 42.2, CH2 127.1, C 131.6, CH 115.5, CH 160.2, C 115.5, CH 131.6, CH 56.7, CH3

170.1, C 20.8, CH3 170.4, C 21.0, CH3 170.5, C 21.2, CH3

170.6, C 21.1, CH3

13-Ac 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 6′-OMe 17-OMe

170.7, C 20.7, CH3

170.0, C 20.5, CH3 171.1, C 40.5, CH2 126.0, C 130.3, CH 114.0, CH 158.7, C 114.0, CH 130.3, CH 55.3, CH3


170.3, C 20.9, CH3 170.3, C 21.0, CH3


54.6, CH3 56.6, CH3

a

Data (δ) were measured in CDCl3 for 10−17 at 125 MHz. Coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments.

comparison of the NMR data (Tables 2 and 4) of compounds 11 and 10. This conclusion was verified by the presence of an AA′BB′ spin system of a p-disubstituted benzene unit (δH 7.17 and 6.84 (2H, d, J = 8.7 Hz each); δC 160.2, 127.1, 2 × 131.6, 2 × 115.5) in the 1H and 13C NMR spectra and the HMBC correlation from the methoxy protons (δH 3.79, s) to the sp2 quaternary C-6′ (δC 160.2). Thus, the structure of euphorblin K (11) was defined as shown. HRESIMS data analysis of compound 12 (m/z 679.2744 [M + Na]+, calcd 679.2725) revealed a molecular formula of C35H44O12. The 1H and 13C NMR data (Tables 2 and 4) showed similarities to those of compound 18 except for the presence of a 6-hydroxymethyl group (δH 4.66, 4.37, δC 60.7) in 12 rather than the C-6 methyl (δH 2.06, δC 17.5) in 18. This

HRESIMS data analysis of compound 10 revealed a molecular formula of C34H42O11. Compounds 10 and 7 were analogues except for the presence of a hydroxy group at C-2 in compound 10, which was confirmed by the HMBC cross-peaks from OH-2 (δH 3.18, s) to the corresponding carbons [C-1 (δC 38.8) and C-2 (δC 75.5)]. An acetoxy group was attached to C3 based on the HMBC correlation between H-3 (δH 4.89, s) and the acetyl carbonyl (δC 173.0). The ROESY correlations of CH3-16 with H-1β and of OH-2 with H-3 confirmed that the OH-2 group was α-oriented. Hence, the structure of euphorblin J (10) was defined as illustrated. Compound 11 had a molecular formula of C35H44O12 based on the HRESIMS ion at m/z 679.2736 [M + Na]+ (calcd 679.2725). A methoxy group was located at C-6′ by 1214



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Figure 4. Identification of compounds 2, 4, and 18 that induce lysosomal biosynthesis. (A) Compounds 1−18 were tested in HeLa cells by LysoTracker Red staining. HeLa cells were treated for 3 h with compounds 1−18 and HEP-14 (40 μM) and stained with LysoTracker Red. Quantification of compound-induced lysosomes (fold induction of LysoTracker Red staining) is shown in A. Dimethyl sulfoxide (DMSO) treatment was used as the negative control, whereas HEP-14 was used as the positive control. Independent triplicates were repeated. (B) Representative images of HeLa cells stained with LysoTracker Red that were treated with compound 2, 4, 18, and positive control HEP-14 (40 μM). Scale bars represented 100 μm in all images. (C) Quantification of compounds 2, 4, and 18 induced lysosomes in a concentration-dependent manner (fold induction of LysoTracker Red staining). Three independent experiments were repeated. (D) Quantification of compounds 2, 4, and 18 induced lysosomes in a time-dependent manner (fold induction of LysoTracker Red staining). n = 3 independent experiments. (E) Compounds 2, 4, and 18 induce the expression of lysosomal genes. HeLa cells were treated with the compounds (40 μM, 3 h) and subjected to qRT-PCR analysis. Data are presented from data collected from three independent experiments. Bar graph represents mean ± SD. Comparisons were made between DMSO and compound treatment. Student’s t test was used to check the statistical differences. p < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Compound 14 gave a molecular formula of C28H40O11 based on the HRESIMS ion at m/z 575.2471 [M + Na]+ (calcd 575.2463). Its 1H and 13C NMR spectroscopic data (Tables 2 and 4) are closely related to those of compound 13 with the exception of two methoxy groups at C-17 in 14, which was verified by the presence of a dioxygenated methine carbon (δC 102.1) in 14 rather than the C-6 methyl signal (δC 17.7) in 13, and by the HMBC correlations from H-17 (δH 5.41, s) to the two methoxy carbons (δC 54.6, 56.6). Hence, the structure of euphorblin N (14) was determined as illustrated. Euphorblin Q (17) had a molecular formula of C33H42O10. Analysis of both MS and NMR data (Tables 2 and 4) of 17 and 7 revealed that the only difference was the presence of a 5′methoxy group in 17, which was confirmed by an AA′BB′ spin system of a p-disubstituted benzene moiety (δH 7.20 and 6.86 (2H, d, J = 8.7 Hz each); δC 158.8, 125.9, 2 × 130.2, 2 × 114.0) in the 1H NMR spectrum and by the HMBC correlation from

was confirmed by the HMBC correlations of CH2-17 to C-5, C6, and C-7 and of H-5 to C-17, C-6, and C-7. Thus, the structure of euphorblin L (12) was defined as shown. Compounds 13 and 16 shared the same molecular formula of C26H36O9. Both compounds contained three acetoxy groups by comparison of their NMR data (Tables 2 and 4). The HMBC data of compound 13 showed correlations between H-3 (δH 5.37, d, J = 8.6 Hz), H-7 (δH 4.97, d, J = 2.0 Hz), and H-12 (δH 4.87, dd, J = 11.2, 4.0 Hz) with their respective acetoxy carbons (δC 170.7, 170.3, and 170.8); thus the acetoxy groups were assigned to C-3, C-7, and C-12. For compound 16, the three acetoxy groups were placed at C-3, C-8, and C-12 based on the HMBC cross-peaks between H-3 (δH 5.22, d, J = 8.5 Hz), H-8 (δH 4.54, dd, J = 10.7, 1.3 Hz), and H-12 (δH 4.85, dd, J = 11.1, 3.9 Hz) and their respective acetoxy carbons (δC 170.7, 170.4, 170.5). Thus, the structures of euphorblins M (13) and P (16) were defined as shown. 1215



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the methoxy protons (δH 3.78, s) to the sp2 quaternary C-6′ (δC 158.8). A Rhamnofolane Diterpenoid. HRESIMS data analysis of 15 (m/z 695.2460 [M + K]+, calcd 695.2464) revealed a molecular formula of C35H44O12 that implied 14 indices of hydrogen deficiency. The NMR spectra of 15 revealed the presence of three acetoxy groups, a p-methoxyphenylacetyl moiety, an isolated carbonyl, and an isopropenyl fragment (Tables 2 and 4). A mutually coupled system at H-1/H-2/H-3 was suggested by the 1H−1H COSY data. The HMBC correlations (Figure 1) from H-3β to C-4 and C-10 revealed that 15 contained a five-membered ring. Likewise, the sevenmembered ring, which contained a C-5 carbonyl group, was confirmed by the 1H−1H COSY correlations between H-6/H7/H-8/H-9 as well as the HMBC correlations between H-9 and C-1 and C-4. The six-membered ring was linked at C-8 and C-9 based on the HMBC correlations between Me-18 and C-9, H12 and C-9, and H-14 and C-9 and C-8. These data indicated that 15 was a derivative of curcusone A.24 The three acetoxy groups, the p-methoxyphenylacetoxy moiety, and the isopropenyl fragment were assigned at C-1, C-7, C-13, C-12, and C-14 based on the HMBC correlations from H-1 (δH 5.33, d, J = 7.4 Hz), H-7 (δH 4.98, dd, J = 9.3, 3.7 Hz), and H-13 (δH 5.21, dd, J = 12.3, 2.9 Hz) to three acetoxy carbonyls (δC 170.4, 170.1, and 170.0); H-12 (δH 5.13, d, J = 2.9 Hz) to the pmethoxyphenylacetyl carbonyl (δC 171.1); and H-16a (δH 4.65, s) and H-16b (δH 4.29, s) to C-14 (δC 46.1), respectively. In addition, the 11-hydroxy group was supported by the crosspeaks between OH-11 (δH 2.61, br s) and C-9 (δC 44.0), C-12 (δC 76.6), and C-11 (δC 72.4). The relative configuration of 15 was deduced by a ROESY experiment (Figure 2). The ROESY cross-peaks of H-3β/Me19, H-1/H-2, and H-1/H-9 and the coupling constant of J1,2 = 7.4 Hz indicated that H-1 and H-2 were α-oriented.25 The ROESY cross-peaks of H-9/HO-11 and H-9/H-14 indicated that these protons were in close proximity, and they were assigned to be α-oriented as well. The relative configuration at C-8 was deduced from the coupling constant of J8,9 = 4.5 Hz, which indicated that H-8 was α-oriented.26 The ROESY crosspeaks of H-6/H-18, H-7/H-13, H-12/H-18, and H-13/H-18 suggested H-6, H-7, H-12, and H-13 were β-oriented. Therefore, the structure of euphorblin O (15) was defined as shown. The known diterpenoid euphorbia factor RL 4 (18)17,18 was identified by comparing its observed and reported NMR data. Lysosomal Biosynthesis Induction Activity. To investigate whether these compounds can induce lysosomal biosynthesis, LysoTracker Red staining was used to monitor the induction of lysosome. Ten of the compounds increased the LysoTracker staining intensity as shown in Figure 4A. The cells were treated for 3 h with compounds 2, 4, and 18 at 40 μM, and these compounds increased the LysoTracker staining intensity by 155.9%, 143.5%, and 140.7%, respectively (Figure 4A and B). It was further tested whether the lysosome induction activities of these compounds are time- and concentration-dependent. As shown in Figure 4C, HeLa cells were treated for 3 h with 10, 20, 40, and 60 μM solutions of the test compounds. Induction of lysosome was increased in a concentration-dependent manner, with the greatest increase observed at 60 μM. Treating HeLa cells with a 40 μM solution of the test compounds for five different time intervals showed that the compounds exert their lysosomal biosynthesis induction activities in a time-dependent manner (Figure 4D).

Many lysosomal genes were regulated by these compounds during lysosome biosynthesis. To confirm that compounds 2, 4, and 18 induce lysosomal biosynthesis, the expression of a set of lysosomal genes was monitored, including lysosomal-associated membrane protein 1 (LAMP1), cathepsin B (CTSB), cathepsin A (CTSA), and ATPase H+ transporting V0 subunit E1 (ATP6 V0E1). As shown in Figure 4E, all these genes were upregulated at mRNA levels 3 h after treatment with compounds 2, 4, and 18. These data further demonstrated that compounds 2, 4, and 18 can induce lysosomal biosynthesis. The structures of ingol diterpenoids 1−14 and 16−18 differ in their substitution patterns, making them a good set of homologous compounds for evaluating structure−activity relationships, and the major structure−activity relationships were the following. (i) The p-methoxyphenylacetoxy moiety at C-7 is a key substituent responsible for the ability to induce lysosomal biosynthesis based on comparison of 18 vs 16; 18 vs 8 and 9; and 2 vs 1 and 3. (ii) A (2′S) absolute configuration in 2 was more beneficial than the (2′R) absolute configuration in 5. (iii) The acetylation of the OH group at C-3 appears to be responsible for the difference in the activities of compounds 18 and 17. (iv) By comparing compounds 18 and 12, it can be inferred that the hydroxy group at C-17 is not required to induce lysosomal biosynthesis.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were obtained using a Mettler Toledo MP50 micro melting point apparatus. Optical rotations were determined on a Jasco P-1020 polarimeter. UV spectra were measured with a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained using a Tenor 27 spectrophotometer. 1D and 2D NMR spectra were determined on a Bruker spectrometer with tetramethylsilane as the internal standard. HRESIMS data were collected on a triple quadrupole mass spectrometer. X-ray data were collected using a Bruker APEX DUO instrument. Preparative HPLC separations were performed on an Agilent 1200 liquid chromatograph with a Waters X-Select CSH Prep RP C18 (19 × 150 mm) column, and semipreparative HPLC separations were carried out using an Agilent 1100 liquid chromatograph with a Waters X-Bridge Prep Shield RP C18 (10 × 150 mm) column. Silica gel (100−200 mesh and 300−400 mesh, Qingdao Marine Chemical, Inc., Qingdao, P. R. China) and Sephadex LH-20 (40−70 mm, Amersham Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography. Plant Material. Euphorbium, the dried latex of E. resinifera Berg., was bought from a medicinal market (Urumqi, P. R. China) in July 2016 and identified by Prof. Mammat Nurahmat (Xinjiang Uygur Medical School). A voucher specimen (KUN No. 1010725) was deposited at the State Key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany, Chinese Academy of Science (CAS). Extraction and Isolation. Euphorbium (10 kg) was extracted with 95% MeOH (3 × 50 L) under reflux (4, 3, and 3 h, respectively). The MeOH extracts were combined and concentrated under vacuum to obtain the crude extract. This was suspended in water and partitioned with petroleum ether and EtOAc successively. The EtOAc portion (2.3 kg) was subjected to normal-phase silica gel column chromatography eluted with a gradient of petroleum ether−EtOAc (from 1:0 to 0:1) to yield seven major fractions (1−7). Fr.3 (130 g) was subjected to a reverse-phase separation (CH3OH−H2O, 4:6 to 9:1) and afforded 12 fractions (3A−3L), and compound 18 (0.56 g) was crystallized from Fr.3C using MeOH. Fr.4 (175 g) was fractionated using a reversephase separation (CH3OH−H2O, 3:7 to 9:1) to get 22 subfractions (4A−4V). Fr.4D (6 g) was separated using normal-phase column chromatography and petroleum ether−acetone (8:1 to 6:1) as mobile phase, which gave seven subfractions (4D1−4D7). Fr.4D3 (1.2 g) was fractionated by preparative HPLC (50% CH3CN in water, 10 mL/ min), and five fractions were obtained (4D3a−4D3e). Fr.4D3a (260 1216



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mg) was subjected to a normal-phase gel column eluted with CH2Cl2− MeOH (200:1 to 50:1) and afforded 11 (20 mg), 2 (71 mg), and 17 (12 mg). Fr.4D3b (220 mg) was separated by CC over normal-phase silica gel eluted with CH2Cl2−MeOH (250:1 to 50:1) and afforded 5 (170 mg). Fr.4D3c (80 mg) was purified by CC over normal-phase silica gel eluted with CHCl2−MeOH (200:1 to 30:1) to give 10 (6 mg), 1 (30 mg), and 7 (8 mg). Fr.4D3e (18 mg) was further fractionated using semipreparative HPLC (50% CH3CN in water) to yield 12 (8 mg, tR = 28.9 min). 8 (39 mg, tR = 47.0 min, 57% CH3CN in water) was obtained using the same method from Fr.4D6 (120 mg). Fr.4B (3.6 g) was subjected to a normal-phase column chromatography and petroleum ether−EtOAc (10:1) as mobile phase, followed by semiprepative HPLC (40% CH3CN in water) to obtain 9 (20 mg, tR = 28.5 min). Fr.4C (1.4 g) was subjected to a Sephadex LH-20 column (MeOH) and then further purified by semipreparative HPLC (40% CH3CN in water) to give 16 (32 mg, tR = 29.5 min). Fr.4E (1.6 g) was subjected to normal-phase CC eluted with CH2Cl2−MeOH (200:1 to 20:1) to give six fractions (4E1−4E6). Fr.4E3 (0.74 g) was purified by semipreparative HPLC (50% CH3CN in water) and provided 15 (7 mg, tR = 38.5 min). Fr.5 (210 g) was separated by a C18 silica gel column (MeOH−H2O from 3:7 to 10:0) and afforded 23 subfractions (5A−5W). Fr.5D (3.5 g) was fractionated on a Sephadex LH-20 column (MeOH) and gave two fractions (5D1 and 5D2). Fr.5D1 was treated similarly and purified on a normal-phase silica gel column eluted with CH2Cl2−MeOH (200:1 to 20:1), and 10 subfractions (5D1a−5D1j) were obtained. Fr.5D1c (0.2 g) was subjected to purification using semipreparative HPLC (32% CH3CN in water) and afforded 14 (3 mg, tR = 50.0 min). Fr.5E (520 mg) was subjected to Sephadex LH-20 chromatography eluted with MeOH and then Sephadex LH-20 chromatography eluted with acetone to afford two subfractions (5E1 and 5E2). Fr.5E1 (1.7 g) was separated on a normal-phase silica gel column eluted with CH2Cl2−MeOH (200:1 to 50:1) and afforded 11 subfractions (5E1a−5E1k). Fr.5E1b (36 mg) was subjected to semipreparative HPLC (45% CH3CN in water) to give 13 (5 mg). 4 (1.5 mg, tR = 38.5 min) and 6 (2 mg, tR = 53.5 min) were obtained using the same method from Fr.5E1c (120 mg). Following separation by semipreparative HPLC (45% CH3CN in water), Fr.5D1g (30 mg) yielded 3 (10 mg, tR = 21.5 min). Euphorblin A (1): colorless crystals from methanol; mp 212−214 °C; [α]25D +25 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.18) nm; IR (KBr) νmax 3437, 1738, 1632, 1242 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 3; HRESIMS m/z 649.2640 [M + Na]+ (calcd for C34H42O11Na 649.2619). Euphorblin B (2): white, amorphous powder; [α]25D +14 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.33), 230 (3.99), 273 (4.33) nm; IR (KBr) νmax 3435, 2937, 1739, 1513, 1374, 1243, 1023 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 3; HRESIMS m/z 679.2732 [M + Na]+ (calcd for C35H44O12Na 679.2725). Euphorblin C (3): white, amorphous powder; [α]25D +15 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (4.31), 227 (4.02), 276 (4.32) nm; IR (KBr) νmax 3449, 2956, 2938, 1739, 1614, 1515, 1446, 1374, 1240, 1022, 967 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 3; HRESIMS m/z 665.2548 [M + Na]+ (calcd for C34H42O12Na 665.2568). Euphorblin D (4): white, amorphous powder; [α]25D −38 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.33), 227 (4.06), 273 (3.23) nm; IR (KBr) νmax 3461, 2926, 1739, 1513, 1458, 1374, 1242, 1026, 967 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 3; HRESIMS m/z 695.2689 [M + Na]+ (calcd for C35H44O13Na 695.2674). Euphorblin E (5): white, amorphous powder; [α]25D −29 (c 0.2, CHCl3); UV (CHCl3) λmax (log ε) 240 (3.83) 275 (3.38) nm; IR (KBr) νmax 3447, 2937, 1742, 1512, 1372, 1249, 1025 cm−1; 1H and 13 C NMR (CDCl3) data, see Tables 1 and 3; HRESIMS m/z 679.2740 [M + Na]+ (calcd for C35H44O12Na 679.2725). Euphorblin F (6): white, amorphous powder; [α]25D −15 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.50), 227 (3.96), 281 (3.51) nm; IR (KBr) νmax 3442, 2929, 1738, 1512, 1443, 1374, 1242, 1024 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 3; HRESIMS m/z 695.2657 [M + Na]+ (calcd for C35H44O13Na 695.2674).

Euphorblin G (7): white, amorphous powder; [α]25D +15 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.25) nm; IR (KBr) νmax 3434, 2930, 1738, 1632, 1374, 1246, 1024 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 3; HRESIMS m/z 591.2584 [M + Na]+ (calcd for C32H40O9Na 591.2565). Euphorblin H (8): white, amorphous powder; [α]25D −11 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (3.13), 224 (3.89), 278 (4.15) nm; IR (KBr) νmax 3440, 2936, 1739, 1618, 1516, 1375, 1242, 1022 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 3; HRESIMS m/z 665.2372 [M + K]+ (calcd for C34H42O11K 665.2359). Euphorblin I (9): white, amorphous powder; [α]25D −7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.46), 222 (3.94), 281 (3.39) nm; IR (KBr) νmax 3437, 2928, 1738, 1632, 1244 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 1 and 3; HRESIMS m/z 679.2742 [M + Na]+ (calcd for C35H44O12Na 679.2725) Euphorblin J (10): white, amorphous powder; [α]25D −38 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 204 (4.42) nm; IR (KBr) νmax 3440, 2937, 1740, 1632, 1373, 1240, 1024 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 4; HRESIMS m/z 649.2635 [M + Na]+ (calcd for C34H42O11Na 649.2619). Euphorblin K (11): white, amorphous powder; [α]25D −52 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.39), 223 (4.19), 275 (3.3) nm; IR (KBr) νmax 3446, 2939, 1740, 1514, 1372, 1241, 1033 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 4; HRESIMS m/z 679.2736 [M + Na]+ (calcd C35H44O12Na 679.2725). Euphorblin L (12): white, amorphous powder; [α]25D −32 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.36), 221 (4.23), 275 (3.30) nm; IR (KBr) νmax 3439, 2937, 1739, 1514, 1373, 1246, 1024 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 4; HRESIMS m/z 679.2744 [M + Na]+ (calcd C35H44O12Na 679.2725). Euphorblin M (13): white, amorphous powder; [α]25D −25 (c, 0.3, MeOH); UV (MeOH) λmax (log ε) 204 (3.97) nm; IR (KBr) νmax 3457, 2930, 1738, 1450, 1372, 1242, 1050, 1021 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 4; HRESIMS m/z 515.2240 [M + Na]+ (calcd C26H36O9Na 515.2252). Euphorblin N (14): white, amorphous powder; [α]25D −29 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.01) nm; IR (KBr) νmax 3453, 2936, 1740, 1446, 1372, 1243, 1075; 1H and 13C NMR (CDCl3) data, see Tables 2 and 4; HRESIMS m/z 575.2471 [M + Na]+ (calcd C28H40O11Na 575.2463). Euphorblin O (15): white, amorphous powder; [α]25D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 201 (4.03), 223 (3.84), 275 (4.03) nm; IR (KBr) νmax 3457, 2939, 1744, 1513, 1373, 1247, 1034; 1H and 13 C NMR (CDCl3) data, see Tables 2 and 4; HRESIMS m/z 695.2460 [M + K]+ (calcd C35H44O12K 695.2464). Euphorblin P (16): white, amorphous powder; [α]25D +50 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 204 (4.03), 227 (3.37) nm; IR (KBr) νmax 3445, 2938, 1738, 1373, 1243, 1021 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 4; HRESIMS m/z 531.1997 [M + K]+ (calcd C26H36O9K 531.1991). Euphorblin Q (17): white, amorphous powder; [α]25D +3 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (4.11), 225 (3.88), 276 (3.02) nm; IR (KBr) νmax 3437, 2935, 1738, 1514, 1374, 1247, 1024 cm−1; 1H and 13C NMR (CDCl3) data, see Tables 2 and 4; HRESIMS m/z 637.2409 [M + K]+ (calcd for C33H42O10K 637.2410). X-ray Crystallographic Data for 1. Formula: C34H42O11; M = 626.67; orthorhombic, a = 14.5146(6) Å, b = 15.1172(6) Å, c = 15.1784(6) Å, α = 90°, β = 90°, γ = 90°, V = 3330.4(2) Å3, Z = 4, μ(Cu Kα) = 0.772 mm−1, 16 492 reflections measured, 5872 independent reflections (Rint = 0.0478). The final R1 values were 0.0582 (I > 2σ(I)). The final wR(F2) values were 0.1677 (I > 2σ(I)). The final R1 values were 0.0584 (all data). The final wR(F2) values were 0.1685 (all data). The goodness of fit on F2 was 1.136 [Flack parameter = 0.06(5)]. The X-ray data of compound 1 are available free of charge in the database of the Cambridge Crystallographic Data Center (CCDC 1584687) www.ccdc.cam.uk/conts/retrieving.html. Cell Culture. The HeLa cell line was used in this experiment. Detailed methods for the cell culture experiment can be found in the Supporting Information. (Figure S163, Supporting Information) 1217



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Screening for Compounds That Induce Lysosomal Biosynthesis. Different concentrations of compounds 1−18 were screened for activity at different time intervals. Detailed methods for the lysosomal biosynthesis experiment can be found in the Supporting Information (Figure S164). Quantitative Real-Time PCR with Reverse Transcription (qRT-PCR). Gene expression was quantified with 7900HT Fast (Applied Biosystems). Detailed methods for the qPCR experiment can be found in the Supporting Information (Figure S165). Statistics and Reproducibility. Data analyses were carried out using Prism 5, and Student’s t tests were employed for statistical analyses with a level of significance of p < 0.05.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00981. IR, HRESIMS, and 1D and 2D NMR spectra of compounds 1−18 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel and Fax: +86-23-65106479. E-mail: fanggao1971@gmail. com (F. Gao). *Tel: +86-871-65223263. Fax: +86-871-65223070. E-mail: [email protected] (X.-J. Hao). *Tel: +86-871-65223263. Fax: +86-871-65223070. E-mail: [email protected] (S.-L. Li). ORCID

Yu Zhang: 0000-0001-8562-344X Xiao-Jiang Hao: 0000-0001-9496-2152 Author Contributions ⊥

N.-D. Zhao and X. Ding contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported financially by the National Natural Science Foundation of China (nos. 21432010, 31470427, and 81703393) and the Central Asian Drug Discovery and Development Center of the Chinese Academy of Sciences (20150526).

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REFERENCES

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