Isolation, Structure Elucidition, and Immunosuppressive Activity of

Aug 7, 2017 - Six new (1–3 and 6–8) and seven known diterpenoids were isolated from the whole plant of Ligularia fischeri. Compound 1 is a new 15 ...
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Isolation, Structure Elucidition, and Immunosuppressive Activity of Diterpenoids from Ligularia fischeri Fekadu-Roge Gobu,† Jian-Jun Chen,*,† Jun Zeng,† Wen-Jun Wei,† Wei-Feng Wang,‡ Chang-Jun Lin,‡ and Kun Gao*,† †

State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, and ‡School of Life Sciences, Lanzhou University, Lanzhou 730000, People’s Republic of China S Supporting Information *

ABSTRACT: Six new (1−3 and 6−8) and seven known diterpenoids were isolated from the whole plant of Ligularia f ischeri. Compound 1 is a new 15,16-dinorerythroxylane-type diterpenoid possessing a C18 skeleton, and 2 is the first example of a 6/6/ 6/6/5/5-fused hexacyclic ent-kaurane diterpenoid with 19,20-olide and 11,16-epoxy moieties. The structures of the new compounds were elucidated by spectroscopic analysis and chemical methods. The absolute configurations of 1 and 7 were determined by single-crystal X-ray diffraction. Compounds 1−13 were evaluated for their immunosuppressive activity, and 4, 7, and 13 showed moderate inhibitory activities against human B lymphoblast HMy2.CIR cells with IC50 values of 56.3 ± 2.2, 13.3 ± 0.8, and 31.4 ± 0.9 μM, respectively.

T

scavenging power places L. f ischeri in the category of edible plants that serve as a dietary source for humans and act as an antioxidant against toxic chemicals that cause cell death.6,7 In previous studies, many eremophilane-type sesquiterpenoids and diterpenoids have been isolated from L. f ischeri.8 As part of an ongoing effort to find new bioactive compounds from Ligularia,9 the whole plants of L. f ischeri were targeted and afforded six new (1−3, 6−8) and seven known (4, 5, 9−13) diterpenoids with three different skeletons. Compounds 4, 7, and 13 showed immunosuppressive activity against human B lymphoblast HMy2.CIR cells in a dose-dependent manner. Herein, the isolation, structural elucidation, and biological evaluation of these compounds are described.

axonomically Ligularia species have been placed under the Compositae family of the tribe Senecioneae. Among about 150 species of this family, more than 100 are distributed in China. Out of these, about 30 species have been utilized in Chinese folk remedies for centuries due to their potential healing power for tumor, bacterial infections, rheumatism, asthma, hepatitis, and hemoptysis.1,2 Phytochemical studies revealed that Ligularia species contain a variety of secondary metabolites with fascinating structures and interesting biological activities.3,4 The characteristic components that have been isolated from this genus include pyrrolizidine alkaloids and sesqui-, di-, and triterpenoids.3 They have played an important role in drug discovery because of their varying core backbones and interesting bioactivities such as antibacterial, insecticidal, cytotoxic, protein tyrosine phosphatase inhibitory, antihepatotoxic, antioxidative, antithrombotic, and anticoagulating activities.3,4 Therefore, these results encourage natural product researchers to target this genus for the discovery of new and medicinally significant secondary metabolites. Ligularia f ischeri (Ledeb.) Turcz (Compositae), a perennial leafy edible plant distributed in wet shady regions of Europe and Asia, has been used to treat jaundice, hepatic problems, scarlet fever, coughs, and hepatic diseases.5 Its high radical© XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The whole plants of L. f ischeri were extracted with MeOH, and the extract was partitioned with EtOAc and H2O. The EtOAc extract was fractionated by column chromatography (CC) over silica gel, Sephadex LH-20, and semipreparative HPLC to afford a new 15,16-dinorerythroxylane diterpenoid (1), five new Received: March 10, 2017

A

DOI: 10.1021/acs.jnatprod.7b00198 J. Nat. Prod. XXXX, XXX, XXX−XXX

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dihydroxykaur-20-oic acid (12),15 and ent-2β-hydroxymanool (13).16 Fischericin A (1) was obtained as colorless crystalline needles, mp 135−136 °C, [α]27 D = +45 (c 0.2, MeOH), isolated by semipreparative HPLC using a H2O/MeOH (35:65) solvent system at a flow rate of 2 mL/min. Its molecular formula was assigned as C18H30O2 by the HRESIMS ion at m/z 301.2137 [M + Na]+ (calcd 301.2138) and 13C NMR spectroscopic data, indicating four indices of hydrogen deficiency. The IR spectrum displayed absorption bands at 3388 and 1632 cm−1 for hydroxy and olefinic groups, respectively. Its 1H NMR spectrum displayed resonances for three methyl groups at δH 0.85 (s, H3-20), 1.24 (s, H3-17), and 1.26 (s, H3-19), an oxygenated methine at δH 4.33 (1 H, t, J = 3.0 Hz, H-3), and two olefinic methylene protons at δH 4.84 (d, J = 1.2 Hz, H-18a) and 4.78 (d, J = 1.2 Hz, H-18b) (Table 1). The 13C NMR and DEPT spectra of 1 displayed 18 carbon signals, including three methyl, eight methylene (one olefinic), three methine (one oxygenated), and three quaternary carbons (one olefinic), as well as one oxygenated tertiary carbon (Table 2). The data indicated that compound 1 should be a nor-diterpenoid carrying a terminal olefin and two hydroxy groups.17 The scaffold of 1 was elucidated by HMBC and 1H−1H COSY experiments. The HMBC cross-peaks of H2-18/C-3, C-4, C-5; H3-19/C-4, C-5, C-6, C-10; H3-20/C-8, C-9, C-10, C-11; H3-17/C-12, C-13, C-

highly oxygenated ent-kauranoids (2, 3, and 6−8), and seven known diterpenoids (4, 5, and 9−13). By comparing their NMR data with literature data, the structures of the known compounds were assigned as tripterifordin (4),10 tripterinin (5),11 ent-16α-hydroxykaur-20-oic acid (9),12 kauranol (10),13 ent-3α,16α,17-trihydroxykaur-19-oic acid (11),14 ent-16α,17-

Table 1. 1H NMR Data of Compounds 1−3 and 6−8 (δ in ppm, J in Hz) 1a,c

position 1a 1b 2a 2b 3a 3b 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14a 14b 15a 15b 16 17a 17b 18a 18b 19 20a 20b

1.79, 1.52, 2.01, 1.56, 4.33,

m overlapped m overlapped t (3.0)

1.73, 1.50, 1.51, 1.25, 1.10,

m m overlapped m tt (12.0, 3.0)

0.87, 1.68, 0.90, 1.68, 1.52,

dd (12.0, 1.8) overlapped dt (13.2, 4.2) overlapped overlapped

2b,c

3b,d

2.17, dt (13.2, 2.8) 1.51, br d (13.2) 1.72, m

2.01, m 1.85, m 1.80, m

1.93, 1.55, 1.29, 1.82, 1.21, 1.85, 1.29,

1.81, 1.59, 2.15, 2.01, 1.53, 3.76,

br d (12.0) br d (12.0) m m m m m

6b,c

m m dd (13.6, 3.2) m dt (13.6, 1.6) dd (3.6, 1.6)

1.71, br s

1.48, br t (12.6) 1.29, dt (12.6, 2.4)

4.47, br s 1.50, m 2.29, 1.95, 1.33, 1.58, 1.40,

t (6.4) dd (12.0, 3.6) br d (12.0) d (11.2) d (11.2)

1.99, 1.34, 1.46, 1.69, 1.93, 1.95,

m m m m m m

2.53, d (14.8) 1.65, d (14.8)

2.60, 0.58, 1.40, 1.30, 1.35, 1.17, 1.20, 1.82, 1.77, 1.74, 1.64,

br d (12.8) br t (8.4) m m m m m m m m m

8b,c

2.58, br d (12.8) 0.58, td (12.8, 4.0) 1.41, m 1.35, 1.19, 1.24, 1.83,

m m m m

2.63, 0.79, 2.33, 1.66, 1.41, 1.18, 1.01, 1.50,

br d (12.8) m m m m m br d (12.0) m

1.80, overlapped 1.67, d (12.4)

1.73, m 1.54, m

1.35, m

1.36, m

1.31, br d (8.4)

1.83, 1.52, 1.36, 1.22, 1.78, 1.82, 1.31, 1.59,

1.87, 1.52, 1.38, 1.21, 1.97, 1.84, 1.33, 1.60, 1.49,

2.06, m

m m m m m m m overlapped

1.24, s

1.27, s

1.39, s

1.32, s

4.84, 4.78, 1.26, 0.85,

1.20, s

1.19, s

0.92, s

5.10, dd (11.6, 2.4) 4.12, d (11.6)

5.36, dd, (12.8, 2.4) 4.29, d, (12.8)

d (1.2) d (1.2) s s

7b,c

0.75, s 10.41, s

m m m m m m m d (14.8) d (14.8)

3.74, d (11.2) 3.61, d (11.2) 0.91, s 0.75, s 10.39, s

1.46, m 2.01, 2.15, 1.57, 1.66, 1.57,

m br d (12.4) m d (13.6) d (13.6)

4.41, d (11.6) 4.34, d (11.6) 0.92, s 0.84, s

a

Recorded at 600 MHz. bRecorded at 400 MHz. cRecorded in CDCl3. dRecorded in methanol-d4/CDCl3 (v/v = 20:1). Fer in 8: 7.07 (1H, br s, H2′), 6.92 (1H, d, J = 8.0, H-5′), 7.09 (1H, br d, J = 8.0, H-6′), 7.64 (1H, d, J = 16.0, H-7′), 6.34 (1H, d, J = 16.0, H-8′), 3.93 (3H, s, OCH3). B

DOI: 10.1021/acs.jnatprod.7b00198 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Thus, the structure of compound 1, fischericin A, is defined as the new erythroxylane-type diterpenoid with a C18 skeleton. Compound 2 had a molecular formula of C20H28O3, as deduced from HRESIMS (found m/z 339.1930 [M + Na]+, calcd 339.1931) and 13C NMR data, indicating seven indices of hydrogen deficiency. The IR spectrum showed absorption at 1721 cm−1, consistent with the presence of a δ-lactone group.19 The 13C NMR and DEPT data (Table 2) showed 20 carbon signals, including two methyl, nine methylene (one oxygenated), four methine (one oxygenated), one oxygenated tertiary, and three quaternary carbons, as well as one carbonyl carbon. A comparison of the carbon resonances of 2 with those of the ent-kaurane-type diterpenoid tripterifordin (4) indicated that 2 differed from 4 by the replacement of a methylene (δC 17.5) by an oxygenated methine (δC 75.9) and that C-16 was deshielded from δC 79.0 to 86.0 in 2. These results suggested that compound 2 is an ent-kaur-19,20-olide derivative. This skeletal type was further supported by analysis of the 2D NMR spectroscopic data (Figure 1), where the oxygenated and carbonyl resonances at δC 75.3, 75.9, 86.0, and 176.2 were assigned to C-20, C-11, C-16, and C-19, respectively. According to the seven indices of hydrogen deficiency of 2, apart from a carbonyl group and five rings (including a lactone moiety), the remaining index of hydrogen deficiency was assumed to be an oxygen-containing ring bridging C-11 and C-16.20 HMBC correlation from H-11 to C-16 further confirmed the above conclusion. Thus, the 2D structure of 2 was deduced as shown in Figure 1. The NOESY correlation of H-11/H-1α together with the coupling pattern of H-11 (br s) showed that H-11 occupies an α-axial position (Figure 2). Considering the rigidity of the oxolane ring and literature data,20 Me-17 should be in an α-axial position, although no direct NOESY correlation was evident. Therefore, the structure of 2, fischericin B, was defined as 11β,16β-epoxy-ent-kaur-19,20-olide. Compound 2 is the first example of a 6/6/6/6/5/5-fused hexacyclic ent-kaurane diterpenoid with 19,20-olide and 11,16-epoxy moieties. Fischericin C (3) possessed the molecular formula C20H30O5 as shown by the HRESIMS (found m/z 373.1981 [M + Na]+, calcd 373.1985) and 13C NMR data. Its IR absorption bands at 3429 and 1702 cm−1 indicated the presence of hydroxy and carbonyl groups. The 13C NMR (DEPT) spectra showed signals for two methyl, nine methylene (one oxygenated), three methine (one oxygenated), two oxygenated tertiary, and three quaternary carbons, as well as one carbonyl carbon, suggesting an ent-kauranoid-20,4-lactone similar to tripterifordin (4). The difference between 4 and 3 was that an oxygenated methine carbon (δC 78.3, C-7) and an oxygenated tertiary carbon (δC 77.0, C-9) in 3 replaced the methylene (δC 39.7) and methine (δC 50.3) carbons in 4, respectively. Furthermore, the C-6, C-8, C-10, and C-11 resonances of 3 were deshielded to δC 30.7 (Δ 8.3 ppm), 53.3 (Δ 8.5 ppm), 44.0 (Δ 5.3 ppm), and 28.4 (Δ

Table 2. 13C NMR Data of Compounds 1−3 and 6−8 (δ in ppm) position

1a,c

2b,c

3b,d

6b,c

7b,c

8b,c

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

16.5 34.8 74.7 160.8 40.4 38.8 25.5 45.1 37.2 56.2 37.5 36.1 71.4 43.0

40.2 20.9 39.8 43.3 51.3 21.6 40.3 44.5 54.5 36.2 75.9 35.8 45.3 43.5 57.1 86.0 23.2 23.2 176.2 75.3

35.5 21.5 41.4 43.8 39.9 30.7 78.3 53.3 77.0 44.0 28.4 26.0 47.7 38.8 49.8 79.9 24.1 23.6 179.3 76.7

35.1 19.4 42.0 33.9 55.8 20.0 40.4 45.4 58.8 54.4 17.9 24.7 49.0 41.3 56.3 79.7 24.3 32.2 21.0 208.1

34.9 19.2 41.7 33.7 55.5 19.8 41.0 44.5 58.3 54.2 17.9 23.9 45.3 39.8 51.5 82.1 65.8 32.0 20.8 207.9

39.5 20.6 42.3 34.0 56.5 20.4 41.4 45.0 54.9 48.5 18.3 24.6 45.7 35.5 53.4 80.1 68.5 33.1 22.4 182.0

26.9 109.2 23.7 12.7

a

Recorded at 150 MHz. bRecorded at 100 MHz. cRecorded in CDCl3. Recorded in methanol-d4/CDCl3 (v/v = 20:1). Fer in 8: 126.7 (C1′), 109.4 (C-2′), 146.8 (C-3′), 148.1 (C-4′), 114.8 (C-5′), 123.2 (C6′), 145.6 (C-7′), 114.8 (C-8′), 167.6 (C-9′), 55.9 (OCH3).

d

14; H2-6/C-5, C-8, C-10; H2-7/C-5, C-8, C-9, C-14; H-8/C-7, C-11, C-13, C-14, C-20; H-10/C-1, C-2, C-4, C-5, C-19, C-20; and H2-14/C-8, C-9, C-12, C-13, C-17, together with the 1 H−1H COSY couplings as shown in Figure 1, indicated the presence of the 6/6/6 fused rings A, B, and C with the terminal olefinic group located at C-4 and three methyl groups located at C-5, C-9, and C-13, respectively. Collectively, 1 should be a 15,16-dinorerythroxylane diterpenoid derivative.18 In addition, the HMBC correlations from H2-18 (δH 4.78 and 4.84) and H21 (δH 1.52 and 1.79) to C-3 (δC 74.7, d) and from H2-11 (δH 0.90 and 1.68), H-8 (δH 1.10), and H3-17 (δH 1.24) to C-13 (δC 71.4, s) implied hydroxy groups at C-3 and C-13, respectively. Hence, the C18 structural backbone was deduced as shown in 1. Rings A, B, and C of the erythroxylane-type diterpenoid were trans-fused with H-8 and H-10 in β-axial positions and Me-19 and Me-20 in α-axial positions. The 3J2,3 values (t, J = 3.0 Hz) showed that H-3 had a β-axial orientation.18 The NOESY correlation between H3-17 and H-8 indicated that H3-17 was in a β-orientation (Figure 2). The structure was further confirmed by single-crystal X-ray diffraction analysis using Cu Kα radiation at 273.77(10) K (Figure 3), and the absolute configuration of 1 was assigned as (3R, 5R, 8S, 9S, 10R, 13S) based on a Flack absolute structure parameter of −0.27(15).

Figure 1. 1H−1H COSY (bold) and selected HMBC (arrows) correlations of 1−3 and 6. C

DOI: 10.1021/acs.jnatprod.7b00198 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. Key NOESY correlations of 1−3.

HMBC correlations of H2-17/C-13, C-15, C-16 and H2-15/C17. Thus, the structure of 7, fischericin E, was assigned as 16α,17-dihydroxy-ent-kaur-20-al. Its structure was further confirmed by single-crystal X-ray diffraction analysis using Cu Kα radiation at 297.16(10) K (Figure 4). The value of the Flack parameter −0.01(15) permitted unambiguous assignment of the (5R,8S,9R,10S,13R,16R) absolute configuration as shown in 7.

Figure 3. ORTEP drawing of 1.

10.9 ppm), respectively, indicating the presence of hydroxy groups at C-7 and C-9. This deduction was supported by the 2D NMR correlations, especially the HMBC cross-peaks of H5, H-6, H-14, H-15/C-7 and H-1, H-5, H-7, H-14, H-15, H-20/ C-9 (Figure 1). The NOESY correlation of CH3-17/H-12β indicated that 16-OH had an α-axial orientation. Meanwhile, 9OH had a β-orientation based on the NOESY correlations of H-20β/H-11α (Figure 2). The 3J6,7 values (dd, J = 3.6, 1.6 Hz) of H-7 and the NOESY correlations of H-7/H-14, 15α indicated that 7-OH was β-oriented. Thus, the structure of compound 3, fischericin C, was established as 7β,9β,16αtrihydroxy-ent-kaur-19,20-olide. Fischericin D (6) was assigned a molecular formula of C20H32O2 by HRESIMS (found m/z 305.2471 [M + Na]+, calcd 305.2475) and 13C NMR data. Its 1H NMR spectrum showed three methyl singlets (δH 0.75, 0.92, and 1.32) and a formyl proton singlet (δH 10.40). The 13C NMR spectrum of compound 6 was similar to that of 16α-hydroxy-ent-kaur-20-oic acid (9) except for the chemical shifts of C-10 and C-20 [6: δC 54.4 (C-10), 208.1 (C-20); 9: δC 48.8 (C-10), 179.1 (C-20)].12 This difference indicated that a C-10 formyl group in 6 replaced the hydroxycarbonyl group in 9. The HMBC cross-peaks of H20/C-10, C-1 and H-5, H-9/C-20 confirmed this conclusion (Figure 1). Hence, the structure of compound 6, fischericin D, was defined as 16α-hydroxy-ent-kaur-20-al. The molecular formula of compound 7 was determined to be C20H32O3 based on its HRESIMS and 13C NMR data. The 1H and 13C NMR spectra of 7 displayed typical signals for an entkaur-20-al, similar to those of 6. Significant differences included the presence of an oxygenated methylene group [δH 3.61, (d, J = 11.2), 3.74, (d, J = 11.2), δC 65.8] and the absence of CH3-17 in 7. In addition, C-16 was deshielded from δC 79.7 to 82.1. These differences indicated that in compound 7 CH3-17 was replaced by a hydroxymethyl group. This was confirmed by

Figure 4. ORTEP drawing of 7.

Compound 8 was obtained as a colorless solid, which displayed a sodium adduct ion at m/z 535.2670, [M + Na]+, in accordance with a molecular formula of C30H40O7. Its IR spectrum displayed absorptions for hydroxy (3395 cm−1), carbonyl (1697 cm−1), and aromatic (1458, 1514, 1594 cm−1) functionalities. The NMR spectra showed resonances similar to those of 16α,17-dihydroxy-ent-kaur-20-oic acid (12).15 Significant differences involved the presence of a trans-feruloyl moiety [δH 3.93 (3H, s), 6.34 (1H, d, J = 16.0), 6.92 (1H, d, J = 8.0), 7.07 (1H, br s), 7.09 (1H, br d, J = 8.0), 7.64 (1H, d, J = 16.0); δC 55.9, 109.4, 114.8 × 2, 123.2, 126.7, 145.6, 146.8, 148.1, 167.6]21 and that H2-17 was deshielded in 8 (δH 4.11 and 4.19) compared to 12 (δH 4.34 and 4.41). These results indicated that in compound 8 the C-17 hydroxy group was replaced by a trans-feruloyloxy group, which was confirmed by deacylation of 8 to afford 12.9f Hence, the structure of compound 8, fischericin F, was assigned as 17-O-E-feruloyl-16α-hydroxyent-kaur-20-oic acid. As an important component of the humoral immune system, B lymphocytes play a key role in the process of tumor formation and metastasis. Recent studies have found that the elimination of B cells and concurrent vaccination with cellular vaccines may greatly enhance the activity of anticancer vaccine therapy in patients due to the antitumor immunity inhibition of B cells.22,23 Furthermore, some kaurane and abietane diterpenoids were reported to exhibit potent immunosuppressive activity.24 The immunosuppressive activities of compounds D

DOI: 10.1021/acs.jnatprod.7b00198 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. Growth Prohibition of Human B Lymphoblastoid Cells after Treatment with 4, 7, and 13a

a

compd

control

2.5 μM

5 μM

10 μM

20 μM

40 μM

IC50b

4 7 13

1 ± 0.039 1 ± 0.039 1 ± 0.039

0.849 ± 0.175 0.791 ± 0.188 0.934 ± 0.149

0.809 ± 0.134 0.685 ± 0.041 0.812 ± 0.291

0.762 ± 0.062 0.525 ± 0.077 0.761 ± 0.212

0.683 ± 0.086 0.455 ± 0.207 0.648 ± 0.192

0.600 ± 0.096 0.301 ± 0.186 0.390 ± 0.019

56.3 ± 2.2 μM 13.3 ± 0.8 μM 31.4 ± 0.9 μM

Results indicated are mean ± SD (n = 3). bConcentration needed for 50% inhibition (IC50). (15 g) eluting with petroleum ether/acetone (10:1−1:1, gradient system). Application of silica gel CC (9 g) to separate D.5.3 (0.5 g) using petroleum ether/acetone as mobile phases afforded compound 9 (100 mg). Likewise, the 90% MeOH sample (19.1 g) was subjected to silica gel CC (1000 g) using petroleum ether/acetone (15:1−5:1, gradient system) as mobile phases to give eight fractions (A−H) based on the TLC results. Next, C (0.51 g) was subjected to silica gel CC (8 g) using CHCl3/EtOAc (10:1−1:1, gradient system) as mobile phases to afford fractions C.1−C.3. Fraction C.2 (0.21 g) was further separated using Sephadex LH-20 CC (MeOH/CHCl3, 1:1) to give four fractions, C.2.1−C.2.4. Using semipreparative HPLC (eluted with 60% MeOH/H2O, YMC-Pack ODS-A, 5 μm, 250 × 10 mm column, flow rate 2.0 mL/min, 50 min), C.2.2 and C.2.3 were further separated to afford compounds 5 (9 mg) and 8 (6 mg). Four fractions, D.1−D.4, were obtained upon subjecting D (0.7 g) to silica gel CC (15 g) using petroleum ether/acetone (10:1−1:1, gradient system). Fraction D.2.1 (0.31 g) was subjected to Sephadex LH-20 CC (MeOH/CHCl3, 1:1) followed by CC eluting with petroleum ether/EtOAc (20:1−1:1, gradient system) to give compounds 2 (7 mg) and 6 (5 mg). Fraction G (0.4 g) was subjected to silica gel CC (10 g) by eluting with petroleum ether/acetone (10:1−1:1, gradient system) to afford fractions G.1−G.4. Fractions G.2.1−G.2.3 were obtained upon subjecting G.2 (100 mg) to silica gel CC (10 g) eluting with CHCl3/EtOAc (10:1−1:1, gradient system). Compounds 3 (8 mg) and 10 (6 mg) were obtained upon separating G.2.2 (30 mg) by using CC. Compound 11 (8 mg) was obtained by RP silica gel column chromatography of G.2.3 (95 mg). Fischericin A (1): colorless needles (acetone); mp 135−136 °C; [α]27 D = +45 (c 0.2, MeOH); IR (KBr) νmax 3388, 2925, 2858, 1451, 1378, 1149 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z [M + Na]+ calcd for C18H30O2Na, 301.2137; found, 301.2138. Fischericin B (2): colorless solid; [α]27 D = −43 (c 0.1, MeOH); IR (KBr) νmax 3336, 2927, 2870, 1721, 1125 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z [M + Na]+ calcd for C20H28O3Na, 339.1930; found, 339.1931. Fischericin C (3): colorless solid; [α]27 D = −18 (c 0.2, MeOH); IR (KBr) νmax 3509, 3429, 3321, 1702, 1189 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z [M + Na]+ calcd for C20H30O5Na, 373.1981; found, 373.1985. Fischericin D (6): colorless needles, [α]27 D = −23 (c 0.2, CHCl3); IR (KBr) νmax 3328, 2926, 1703, 1459, 1261 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z [M + H]+ calcd for C20H33O2, 305.2471; found, 305.2475. Fischericin E (7): colorless solid; [α]27 D = −40 (c 0.2, MeOH); IR (KBr) νmax 3381, 2936, 1698, 1455, 1027 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z [M + Na]+ calcd for C20H32O3Na, 343.2237; found, 343.2244. Fischericin E (8): yellowish solid; [α]27 D = −25 (c 0.2, MeOH); IR (KBr) νmax 3395, 2933, 1698, 1594, 1514, 1458, 1266, 739 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z [M + Na]+ calcd for C30H40O7Na, 535.2670; found, 535.2666. X-ray Diffraction Analysis of Fischericin A (1). A colorless needle with dimensions 0.18 × 0.16 × 0.14 mm was obtained from CHCl3 and used for X-ray analysis (CCDC 1524475). A suitable crystal of 1 was mounted on a SuperNova, Dual, Cu at zero, Eos diffractometer and kept at 273.77(10) K during data collection. The crystal at the specified temperature was observed to belong to the monoclinic space group P212121, a = 10.4703(8) Å, b = 8.0849(10) Å, c = 33.511(2) Å, α = γ = 90°, β = 92.726(6)°, V = 2833.5(4) Å3, Z = 6, μ(Cu Kα) = 0.560 mm−1, Dcalc = 1.092 g/cm3, λ = 1.541 84 Å, and

1−13 were evaluated in vitro against human B lymphoblast HMy2.CIR cells using the sulforhodamine B (SRB) method. The result showed that compounds 4, 7, and 13 inhibited the proliferation of human B lymphoblast HMy2.CIR cells at concentrations of 2.5−40 μM in a dose-dependent manner (Table 3). Compounds 1 and 5 promoted the cell proliferation in a dose-dependent manner, and other compounds showed no activity. The stems and leaves of L. f ischeri are used for eating and medicinal purposes, indicating that they have low toxicity to humans. Herein, the active compounds have the potential to be exploited as active immunomodulatory lead compounds in tumor immunotherapy.

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EXPERIMENTAL SECTION

General Experimental Procedures. A Perkin−Elmer 341 polarimeter was used for measuring optical rotations. A Nicolet NEXUS 670 FT-IR spectrometer was used to measure infrared absorptions. Melting points were determined by an X-4 digital display micro melting point apparatus and are uncorrected. 1H, 13C, and 2D NMR spectroscopic data were acquired on a Varian Mercury-600BB or Bruker Avance III-400 instrument. A SuperNova, Dual, Eos diffractometer was applied to collect X-ray diffraction data; the structure was solved with the Superflip program using charge flipping and refined with the ShelXL program (using graphite-monochromated Cu Kα radiation). A Bruker APEXII mass spectrometer was used to acquire HRESIMS data. Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and column chromatography with silica gel (200−300 mesh, Qingdao Marine Chemical Factory, China) were used. Semipreparative HPLC using a Waters 1525 series instrument equipped with a YMC-Pack ODS-A column (250 × 10 mm, 5 μm) was used for sample isolation. TLC was carried out on GF254 plates. Plant Material. The leaves and roots of L. f ischeri were collected in Baoji, Shanxi Province, China, in August 2013 and identified by Professor Zhang Guoliang, Lanzhou University. A voucher specimen (no. 20130816-2) was deposited at the Natural Product Laboratory of State Key Laboratory of Applied Organic Chemistry, Lanzhou University. Extraction and Isolation. The leaves and roots of L. f ischeri (10 kg) were chipped and extracted with MeOH (3 × 5 L, 7 days each) at room temperature. After evaporation under reduced pressure, an extract (630 g) was obtained and partitioned with EtOAc and nBuOH. The EtOAc-soluble portion (122.8 g) was subjected to microporous resin (1000 g) eluting with H2O/MeOH (1:0−0:1, gradient system). Six fractions, A−G, were collected based on the percentage of the solvents used, i.e., 100% water sample, 30, 50, 80, 90, 100% MeOH sample, and 100% acetone sample. Then, the 80% MeOH sample (17.2 g) was applied to silica gel CC (206 g) to give five fractions, D.1−D.5, eluting with petroleum ether/acetone (50:1− 1:1, gradient systems). Next, D.4 (1.7 g) was subjected to Sephadex LH-20 CC (MeOH/CHCl3, 1:1) followed by CC eluting with CHCl3/EtOAc (20:1−1:1, gradient system) to afford four fractions, D.4.1−D.4.4. Using semipreparative HPLC (eluted with 60% MeOH/ H2O, YMC-Pack ODS-A, 5 μm, 250 × 10 mm column, flow rate 2.0 mL/min, 60 min) D.4.2 and D.4.3 were separated to yield compounds 1 (5 mg) and 13 (7 mg). Fraction D.5 (2.3 g) was applied to silica gel CC (75 g) using petroleum ether/acetone as the mobile phases, followed by Sephadex LH-20 CC (MeOH/CHCl3, 1:1), to yield four fractions, D.5.1−D.5.4. Compounds 4 (20 mg), 7 (10 mg), and 12 (40 mg) were obtained by separating D.5.2 (120 mg) using silica gel CC E

DOI: 10.1021/acs.jnatprod.7b00198 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

F(000) = 1032.219 27 reflections collected in the range of 3.96° ≤ θ ≤ 69.91°, yielded 8985 unique reflections. The structure was determined by direct methods and was refined using full-matrix least-squares on F2 values for 2720 I > 2σ(I). Hydrogen atoms were fixed at calculated positions, and non-hydrogen atoms were refined anisotropically. The final indices gave the following values: R = 0.0454, Rw = 0.0960. Goodness of fit = 0.906. The scattering factors were referred to the International Tables for X-ray Crystallograpy.25 X-ray Diffraction Analysis of Fischericin D (7). A colorless crystalline solid with dimensions 0.33 × 0.26 × 0.23 mm was obtained from acetone and used for X-ray analysis (CCDC 1523372). A crystal of 7 was mounted on a SuperNova, Dual, Cu at zero, Eos diffractometer. The crystal, at 297.16(10) K, was observed to belong to the monoclinic space group P212121, with a = 6.3439(2) Å, b = 42.2604(13) Å, c = 7.7062(2) Å, α = γ = 90°, β = 113.463(4)°, V = 1895.17(12) Å3, Z = 4, μ(Cu Kα) = 0.641 mm−1, Dcalc = 1.186 g/cm3, λ = 1.541 84 Å, and F(000) = 744.100 62 reflections collected in the range of 4.19° ≤ θ ≤ 68.25°, yielded 5575 unique reflections. The structure was determined by direct methods and was refined using fullmatrix least-squares on F2 values for 5061 I > 2σ(I). Hydrogen atoms were fixed at calculated positions, and non-hydrogen atoms were refined anisotropically. The final indices gave the following values: R = 0.0483, Rw = 0.1200, goodness of fit = 1.083. The scattering factors were referred to the International Tables for X-ray Crystallograpy.25 Chemical Transformation of Fischericin F (8). Fischericin F (8, 1.5 mg, 0.0029 mmol) was added to 4 mL of K2CO3/MeOH (5%), and the reaction mixture was stirred for 10 h at room temperature. The MeOH was removed under vacuum, and the residue was diluted with EtOAc. The organic phase was washed with NaHCO3 and brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified via silica gel CC (CHCl3/ EtOAc, 5:1) to give 12 (0.4 mg). Immunosuppressive Assay. The immunosuppressive activity of compounds 1−13 toward human B lymphoblast HMy2.CIR cells was determined in 96-well microtiter plates by the SRB method. Briefly, cells were seeded in 96-well plates in medium with 5% fetal bovine serum. After 24 h, cells were fixed with 10% TCA (trichloroacetic acid) to represent cell population at the time of drug addition (T0). After additional incubation of DMSO or the compound for 48 h, cells were fixed with 10% TCA and SRB at 0.4% (w/v) in 1% HOAc to stain cells. Unbound SRB was washed out by 1% HOAc, and SRB-bound cells were solubilized with 10 mM Trizma base. The absorbance was read at a wavelength of 540 nm. Using the following absorbance measurements, such as time zero (T0), control growth (C), and cell growth in the presence of the compound (Tx), the percentage growth was calculated at each of the compound concentration levels. Percentage growth inhibition was calculated as [1 − (Tx − T0)/(C − T0)] × 100%.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the National Natural Science Foundation of China (No. 31470421).

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00198. NMR, IR, and HRESIMS spectra for compounds 1−3 and 6−8 (PDF) Crystallographic data (CIF) Crystallographic data (CIF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.-J. Chen). *E-mail: [email protected]. (K. Gao). ORCID

Jian-Jun Chen: 0000-0001-5937-5569 Kun Gao: 0000-0002-3856-3672 F

DOI: 10.1021/acs.jnatprod.7b00198 J. Nat. Prod. XXXX, XXX, XXX−XXX