Monoterpenoid Indole Alkaloids from Kopsia officinalis and the

Feb 20, 2017 - Asymmetric Total Synthesis of Pentacyclic Indole Alkaloid Andranginine and Absolute Configuration of Natural Product Isolated from Kops...
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Monoterpenoid Indole Alkaloids from Kopsia of f icinalis and the Immunosuppressive Activity of Rhazinilam Ting Zeng,† Xiu-Yin Wu,† Shu-Xia Yang, Wei-Chun Lai, Shun-Dong Shi, Qiang Zou, Yang Liu,* and Li-Mei Li* Research Center, Chengdu Medical College, Xindu Avenue 783, Chengdu 610500, People’s Republic of China S Supporting Information *

ABSTRACT: Six new monoterpenoid indole alkaloids, kopsinidines C−E (1−3), 11,12-methylenedioxychanofruticosinic acid (4), 12-methoxychanofruticosinic acid (5), and N(4)-methylkopsininate (7), as well as chanofruticosinic acid (6, as a natural product) and 23 known alkaloids, were obtained from the twigs and leaves of Kopsia off icinalis. Their structures were characterized by physical data analysis. All isolated compounds were evaluated for their immunosuppressive activity on human T cell proliferation. Rhazinilam (29) significantly inhibited human T cell proliferation activated by anti-CD3/ anti-CD28 antibodies (IC50 = 1.0 μM) and alloantigen stimulation (IC50 = ̈ human T cells and peripheral 1.1 μM) without obvious cytotoxicity for naive blood mononuclear cells (0−320 μM). Although it did not affect T cell activation, it induced T cell cycle arrest in the G2/M phase and inhibited proinflammatory cytokine production in activated T cells.

T

325.1891 [M + H]+ (calcd for C20H25N2O2, 325.1916) in the HRESIMS, indicative of 10 indices of hydrogen deficiency. The following 1H and 13C NMR signals suggested the presence of a dihydroindole moiety with an unsubstituted benzene moiety: δH 7.33 (br d, J = 7.5 Hz, H-9), 7.04 (t, J = 7.5 Hz, H-11), 6.72 (td, J = 7.5, 0.8 Hz, H-10), and 6.67 (d, J = 7.5 Hz, H-12) (Table 1) and δC 153.1 (C-13), 131.8 (C-8), 129.8 (C-11), 123.6 (C-9), 120.2 (C-10), and 112.4 (C-12) (Table 2). The UV spectrum showed absorption maxima at 206, 245, and 296 nm, confirming the above deduction. Five indices of hydrogen deficiency were attributable to five rings, and the other five were attributed to a dihydroindole unit in the structure. On the basis of the types of alkaloids from the genus Kopsia,1 compound 1 was likely a kopsine-type alkaloid. The presence of the C-6 methine resonance at δC 51.3 was supported by the HMBC cross-peak of H-6 (δH 2.57)/C-8 (δC 131.8) (Figure 1). One oxygenated methine group (δC 73.4) was assigned to C-22 from the HMBC cross-peaks between H-22 (δH 4.50, dd, J = 9.5, 1.5 Hz) and C-17 (δC 38.7), C-5 (δC 48.8), and C-16 (δC 80.1) (Figure 1). Thus, C-6 and C-16 were linked through C-22, a characteristic of a kopsine-type alkaloid, similar to demethoxycarbonylkopsin (10).18 Comparing the NMR data with those of compound 10 showed that the carbonyl group in compound 10 (δC 212.0) was replaced by an oxygenated methine group (δH 4.50, δC 73.4) in 1. The 2D structure of compound 1 was defined by the key HMBC connectivities shown in Figure 1. On the basis of the caged skeleton as displayed by the molecular model (Figure 2), HO-22 is on the

he genus Kopsia (Apocynaceae) is known for its structurally diverse and cytotoxic monoterpenoid indole alkaloids.1,2 Kopsia off icinalis, another name for Kopsia arborea Blume,3 is native to south Yunnan Province of China, and it has been used to treat rheumatoid arthritis, tonsillitis, pharyngitis, and edema as a folk medicine.4 So far, 47 indole alkaloids, including 12 different types, have been isolated and identified from K. off icinalis.4−14 Some of them have antimanic,5 hepatoprotective,15 cytotoxic,4,16 muscle relaxant, and hypotensive activities.17 In the search for immunosuppressive agents from medicinal plants, six new indole alkaloids, kopsinidines C−E (1−3), 11,12-methylenedioxychanofruticosinic acid (4), 12-methoxychanofruticosinic acid (5), and N(4)-methylkopsininate (7), as well as chanofruticosinic acid (6, as a natural product) and 23 known compounds, were isolated from the twigs and leaves of K. off icinalis. The 1H and 13C NMR data of chanofruticosinic acid (6), kopsinine methochloride (8), and demethoxycarbonylkopsin (10) are reported for the first time. Immunosuppressive screening showed that rhazinilam (29) inhibited the proliferation of human T cells and peripheral blood mononuclear cells (PBMCs) significantly, with IC50 values of 1.0 and 1.1 μM, respectively. At concentrations up ̈ to 320 μM, rhazinilam did not cause cytotoxic effects on naive human T cells and PBMCs under the experimental conditions. Rhazinilam did not inhibit T cell activation but induced T cell cycle arrest in the G2/M phase. Furthermore, it inhibited the production of proinflammatory cytokines in activated T cells.

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RESULTS AND DISCUSSION

The molecular formula of kopsinidine C (1) was determined to be C20H24N2O2 from its protonated molecular ion at m/z of © 2017 American Chemical Society and American Society of Pharmacognosy

Received: July 27, 2016 Published: February 20, 2017 864

DOI: 10.1021/acs.jnatprod.6b00697 J. Nat. Prod. 2017, 80, 864−871

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Chart 1

monoterpenoid indole alkaloid. The HMBC cross-peaks (Figure 1) from OMe (δH 3.93) to NCO2Me (δC 154.2) revealed the presence of an N-methoxycarbonyl group. Moreover, the chemical shift of C-22 in 4 (δC 174.1) was deshielded compared to that of compound 13 (δC 170.9), suggesting that the carboxyl group was substituted at C-2 (δC 77.7) in compound 4. The relative configuration of compound 4 was identical to that of compound 13 on the basis of the similarity of the NMR data and the NOESY correlations from H-15α, H-18α, and H-19α to H-21. Compound 5 was obtained as an amorphous solid, and its molecular formula was determined to be C23H26N2O6 from its protonated molecular ion at m/z of 427.1871 [M + H]+ (calcd 427.1864) in the positive HRESIMS. The 1H and 13C NMR data of compound 5 resembled those of compound 4 (Tables 1 and 2, respectively), except for the different substituent pattern of the benzene moiety. A methoxy group (δH 3.71 and δC 56.6) was present in compound 5 instead of the methylenedioxy group in compound 4. The HMBC cross-peaks from OMe (δH 3.71) to C-12 (δC 149.9) and H-9 (δH 7.20) to C-7 (δC 61.0) suggested that the OMe group was located at C-12. Considering the NOESY spectrum and biosynthesis considerations, the relative configuration of compound 5 was the same as that of compound 4. Therefore, the structure of compound 5 was identified as 12-methoxychanofruticosinic acid. N(4)-Methylkopsininate (7) was purified as an amorphous solid. The HRESIMS data displayed a protonated molecular ion at m/z of 339.2073 [M + H]+, consistent with the molecular formula C21H26N2O2. The UV spectrum gave absorption maxima at 206, 263, and 317 nm (log ε 4.66, 3.79, and 3.13, respectively), which is typical of a dihydroindole chromophore. The 1H and 13C NMR data (Tables 1 and 2, respectively), assigned with the aid of HSQC, suggested that compound 7 was an aspidofractinine-type alkaloid, closely related to

right, which was supported by the NOESY interactions from HO-22 to H-17 and H-5 in DMSO-d6 (Figure 2). Taking biosynthetic considerations into account, the relative configuration of compound 1 was found to be the same as that of compound 10, which was also supported by the NOESY correlations from H-21 to H-15α, H-18α, and H-19α (Figure 2). The 1H and 13C NMR data of compounds 2 and 3 are closely related to those of compound 1 (Tables 1 and 2, respectively). The differences were restricted to the substituents on the benzene moiety. The 1D NMR data of compound 2 showed the signals of a methoxy group at δH 3.86 (3H, s) and δC 56.1. The HMBC interactions from OMe (δH 3.86) to C-12 (δC 147.9) and H-9 (δH 7.07) to C-7 (δC 64.3) suggested that the OMe group is located at C-12. The characteristic data of a methylenedioxy group (δH 5.93 and 5.90; δC 102.3) and a pair of aromatic AB doublets at δH 6.94 (d, J = 7.8 Hz) and 6.35 (d, J = 7.8 Hz) suggested that the benzene ring was substituted by a methylenedioxy group in compound 3. The HMBC crosspeaks from OCH2O (δH 5.93 and 5.90) to C-11 (δC 150.2) and C-12 (δC 133.7) as well as H-9 (δH 6.94) to C-7 (δC 63.6) indicated that the OCH2O group was connected to C-11 and C-12. The full and unambiguous 1H and 13C NMR data and the relative configuration assignments for compounds 2 and 3 (Tables 1 and 2) were confirmed using a combination of HSQC, HMBC, and NOESY experiments, and the compounds were named kopsinidines D and E, respectively. 11,12-Methylenedioxychanofruticosinic acid (4) has a molecular formula of C23H24N2O7, as deduced from its protonated molecular ion at m/z of 441.1675 [M + H]+ (calcd 441.1656 for C23H25N2O7) in the HRESIMS. The 1H and 13C NMR data of 4 (Tables 1 and 2, respectively) resembled those of methyl 11,12-methylenedioxychanofruticosinate (13),19 except that one of the methoxy groups was absent in compound 4. This indicated that compound 4 was a fruticosine-type 865

DOI: 10.1021/acs.jnatprod.6b00697 J. Nat. Prod. 2017, 80, 864−871

866

a

1.15, 2.75, 1.46, 2.00, 1.37, 1.59, 3.84, 4.50,

d (15.1) dd (15.2, 3.8) m m m m s dd (9.5, 1.5)

2

3

d (15.1) dd (15.1, 3.5) m m m m s d (9.4)

3.86, s

1.23, 2.82, 1.50, 2.08, 1.43, 1.65, 3.90, 4.59,

d (15.1) dd (15.1, 3.5) m m m m s d (9.3)

5.93, s 5.90, s

1.23, 2.82, 1.55, 2.11, 1.45, 1.66, 3.87, 4.59,

d (15.1) m m m

1.84, 2.19, 1.53, 1.67,

1.84, 2.19, 1.53, 1.67,

d (15.1) m m m

6.94, d (7.8) 6.35, d (7.8)

3.53, m 3.90, dd (13.0, 6.8) 2.65, m

3.50, 2H, m

7.07, d (7.6) 6.81, t (7.6) 6.84, d (7.6)

3.50, overlapped 3.91, m 2.65, m

3.50, 2H, overlapped

4

d (20.3) d (20.3) m m m m s

d (14.0) m m m

5.98, s 5.90, s

3.93, s

2.26, 2.99, 2.66, 3.73, 1.35, 1.52, 2.61,

1.15, 1.89, 1.21, 1.40,

7.02, d (7.8) 6.66, d (7.8)

2.96, m 3.80, m 3.52, d (5.8)

2.92, 2H, m

5 m m m m d (5.6)

d (18.3) d (18.3) m m overlapped m s

m m m overlapped

3.71, s

3.92, s

2.21, 3.04, 2.43, 3.84, 1.37, 1.52, 2.73,

1.16, 1.92, 1.16, 1.37,

7.20, d (7.9) 7.08, t (7.9) 6.89, d (7.9)

2.83, 2.94, 3.03, 3.88, 3.80,

6

d (19.2) d (19.2) m d (16.4) m overlapped s

d (7.6) t (7.6) t (7.6) d (7.6) m m 2H, overlapped

m m m dd (11.3, 6.4) d (6.1)

3.84, s

2.35, 2.92, 2.95, 3.25, 1.17, 1.46, 2.44,

7.54, 7.09, 7.37, 8.20, 1.12, 1.86, 1.46,

2.83, 2.90, 2.91, 3.79, 3.38,

7 m m m m m overlapped br d (7.5) td (7.5, 3.0) t (7.5) dd (7.5, 3.0) m m m m m overlapped m 2H, m

3.40, s

1.31, m 1.54, m 3.87, s

3.43, 3.74, 3.41, 3.89, 1.57, 1.93, 7.31, 6.81, 7.06, 6.76, 1.80, 2.00, 1.41, 3.03, 3.23, 1.93, 2.40, 1.52,

8 overlapped m overlapped m m m d (7.5) t (7.5) t (7.5) d (7.5) m m m m m m ddd (15.5, 11.5, 4.0) m m m m s

3.52, s

3.84, s

3.54, 3.85, 3.54, 3.98, 1.53, 2.04, 7.37, 6.84, 7.13, 6.80, 1.91, 2.14, 1.70, 3.15, 3.38, 2.00, 2.50, 1.47, 1.64, 1.40, 1.69, 3.95,

Compounds 1, 5−8, and 10 were recorded at 400 MHz, and compounds 2-4 at 600 MHz. Chemical shifts (δ) are expressed in ppm, and J values in Hz.

12-OMe

21 22 CO2Me NCO2Me NMe OCH2O

19

18

16 17

15

br d (7.5) td (7.5, 0.8) t (7.5) d (7.5) d (15.3) m m m

7.33, 6.72, 7.04, 6.67, 1.77, 2.14, 1.45, 1.61,

9 10 11 12 14

6

3.43, overlapped 3.83, m 2.57, m

5

1

3.43, 2H, overlapped

3

position

Table 1. 1H NMR Spectroscopic Data for Compounds 1−3, 7, 8, 10 (in Methanol-d4), and 4−6 (in Pyridine-d5)a 10

1.59, 2.45, 1.66, 2.06, 1.54, 1.72, 4.19,

7.47, 6.76, 7.09, 6.70, 1.79, 2.10, 1.52, 1.62,

m m m m m m s

d (7.6) t (7.6) t (7.6) d (7.6) m m m m

3.62, m 4.01, dd (12.7, 11.7) 2.65, dd (11.3, 6.3)

3.48, 2H, m

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DOI: 10.1021/acs.jnatprod.6b00697 J. Nat. Prod. 2017, 80, 864−871

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Table 2. 13C NMR Spectroscopic Data for Compounds 1−3, 7, 8, 10 (in Methanol-d4), and 4−6 (in Pyridine-d5)a position 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CO2Me NCO2Me NCO2Me NMe OMe OCH2O a

1 δC, type

2 δC, type

3 δC, type

4 δC, type

5 δC, type

6 δC, type

7 δC, type

8 δC, type

10 δC, type

73.6, 48.7, 48.8, 51.3, 63.8, 131.8, 123.6, 120.2, 129.8, 112.4, 153.1, 15.8, 32.3, 80.1, 38.7, 21.8, 37.2, 32.8, 70.4, 73.4,

73.9, 48.7, 48.8, 51.3, 64.3, 132.4, 116.0, 121.4, 111.9, 147.9, 141.4, 15.8, 32.2, 80.1, 38.7, 22.0, 37.2, 32.8, 70.4, 73.6,

74.7, 48.7, 48.8, 51.4, 63.6, 128.6, 116.5, 100.7, 150.2, 133.7, 135.1, 15.8, 32.2, 80.0, 38.7, 22.0, 37.2, 32.9, 70.6, 73.5,

77.7, 47.2, 53.4, 56.4, 59.4, 131.7, 118.1, 103.7, 149.8, 135.2, 126.3, 18.4, 35.3, 207.7, 43.6, 24.2, 36.3, 36.7, 69.9, 174.1,

78.6, 47.2, 53.5, 56.0, 61.0, 140.4, 117.6, 125.3, 113.1, 149.9, 131.9, 18.3, 35.2, 208.8, 44.0, 24.5, 36.8, 37.3, 70.0, 174.2,

75.8, 47.2, 52.9, 57.0, 58.5, 135.4, 125.9, 123.4, 128.9, 115.4, 143.6, 18.2, 35.7, 207.4, 43.6, 25.0, 36.0, 36.2, 69.6, 173.9,

67.2, 61.4, 62.0, 32.3, 60.3, 136.6, 123.6, 122.1, 130.1, 113.8, 150.2, 17.8, 32.4, 41.6, 31.8, 25.1, 35.4, 33.9, 80.4, 176.7,

67.0, 61.4, 62.0, 32.2, 60.3, 136.1, 123.4, 121.0, 130.0, 113.0, 151.6, 17.8, 32.3, 42.1, 31.9, 25.2, 35.6, 33.9, 80.5, 176.0, 53.1,

71.6, 48.8, 53.3, 53.3, 62.3, 131.6, 124.1, 120.7, 130.3, 112.6, 152.8, 15.6, 31.6, 82.1, 42.6, 20.5, 36.8, 33.9, 70.0, 212.0,

C CH2 CH2 CH C C CH CH CH CH C CH2 CH2 C CH2 CH2 CH2 C CH CH

C CH2 CH2 CH C C CH CH CH C C CH2 CH2 C CH2 CH2 CH2 C CH CH

C CH2 CH2 CH C C CH CH C C C CH2 CH2 C CH2 CH2 CH2 C CH CH

C CH2 CH2 CH C C CH CH C C C CH2 CH2 C CH2 CH2 CH2 C CH C

154.2, C 53.3, CH3

C CH2 CH2 CH C C CH CH CH C C CH2 CH2 C CH2 CH2 CH2 C CH C

154.5, C 53.3, CH3

C CH2 CH2 CH C C CH CH CH CH C CH2 CH2 C CH2 CH2 CH2 C CH C

C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH2 CH CH2 CH2 CH2 C CH C

C CH2 CH2 CH C C CH CH CH CH C CH2 CH2 C CH2 CH2 CH2 C CH C

155.7, C 53.2, CH3 57.2, CH3

56.1, CH3

C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH2 CH CH2 CH2 CH2 C CH C CH3

57.2, CH3

56.6, CH3 102.3, CH2

101.6, CH2

Compounds 1, 5−8, and 10 were recorded at 100 MHz, and compounds 2−4 at 150 MHz. Chemical shifts (δ) are expressed in ppm.

group. Taking into account the molecular formula and stability, the presence of an anionic acid (C-22) was essential for forming an inner salt with the quaternary N atom.21 Finally, the structure and relative configuration were confirmed by HMBC and NOESY experiments and biosynthesis considerations. Chanofruticosinic acid (6) exhibited similar 1H and 13C NMR data to those of compounds 4 and 5 (Tables 1 and 2, respectively), except for the aromatic region. The presence of four aromatic hydrogens of an indole moiety (δH 7.09−8.20) in the 1 H NMR spectrum (Table 1) suggested that there was no substituent on the benzene moiety in compound 6. The structure of compound 6 was eventually confirmed by its HRESIMS and 2D NMR data. This is the first time that it has been found to occur naturally, and its NMR data are reported here for the first time. Other known alkaloids were identified, including kopsinine methochloride (8),20 Na-demethoxycarbonyl-12-methoxykopsine (9), 2c demethoxycarbonylkopsin (10), 18 methyl demethoxycarbonylchanofruticosinate (11),10 methyl chanofruticosinate (12),10 methyl 11,12-methylenedioxychanofruticosinate (13),19 methyl 12-methoxychanofruticosinate (14),22 methyl 11,12-methylenedioxy-N 1 -decarbomethoxychanofruticosinate (15),19 kopsininic acid (16),23 (−)-11,12-methylenedioxykopsinaline (17),6 kopsinine (18),13 (−)-N-methoxycarbonyl-11,12-methylenedioxykopsinaline (19),6 5-oxokopsinic acid (20),2c kopsinilam (21),24 (−)-N-methoxycarbonyl12-methoxykopsinaline (22),6 N-carbomethoxy-11-hydroxy-12methoxykopsinaline (23),11 kopsinoline (24),25 (−)-12-methoxykopsinaline (25),6 11,12-methylenedioxykopsinaline N(4)oxide (26),26 kopsinine B (27),27 5,22-dioxokopsane (28),24 rhazinilam (29),28 and pleiocarpamine methochloride (30).29 The isolated compounds from K. off icinalis were screened for their immunosuppressive activity on human T cell proliferation.

Figure 1. HMBC (↶) correlations of compounds 1, 4, and 7.

Figure 2. Key NOESY (↔) correlations of compound 1.

kopsinine methochloride (8).20 The NMR signals arising from the OCH3 group (δH 3.84, δC 53.1) in 8 were absent in the spectra of compound 7. The N4-methyl group in compound 7 was deduced from the HMBC cross-peaks of N4-CH3 (δH 3.40) with C-21 (δC 80.4), C-5 (δC 62.0), and C-3 (δC 61.4), respectively (Figure 1). The 10 indices of hydrogen deficiency indicated by the molecular formula of compound 7 accounted for one benzene moiety, five other rings, and a carboxylate 867

DOI: 10.1021/acs.jnatprod.6b00697 J. Nat. Prod. 2017, 80, 864−871

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Figure 3. Rhazinilam (29) inhibited human T cell proliferation without cytotoxicity. The effect of rhazinilam (29) on human T cell proliferation activated by the anti-CD3/anti-CD28 antibodies (A, B) or allogeneic PBMCs (C) for 72 h was assessed by flow cytometry. The cells without stimulation or drug served as the negative control (0%), while the cells with the stimulation but without the drug served as the positive control ̈ T cells (D) and PBMCs (E) were treated with rhazinilam (29) (20, 40, 80, 160, and 320 μM) or vehicle for 72 h. A CCK-8 (100%). Human naive assay kit was used to assess the viability of cells. The cells without the drug served as control (100%). The results are presented as the mean ± SEM (n = 5 per group) from three independent experiments.

12-Methoxychanofruticosinic acid (5), N(4)-methylkopsininate (7), demethoxycarbonylkopsin (10), and rhazinilam (29) were found to suppress human T cell proliferation with IC50 values of 27.8, 21.6, 25.4, and 1.0 μM, respectively, activated by antiCD3/anti-CD28 antibodies. Rhazinilam (29) had the most potent inhibition on human T cell proliferation. Human T cell proliferation was inhibited by rhazinilam (29) after anti-CD3/ anti-CD28 stimulation with an IC50 value of 1.0 μM (Figure 3A,B) and alloantigen stimulation with an IC50 value of 1.1 μM (Figure 3C). T cell proliferation was inhibited by many compounds via cytotoxicity, but not through immunosuppressive activity. The results showed that rhazinilam (29) had no signif̈ T cells and icant impact on the relative viability of human naive PBMCs for 72 h (Figure 3D,E), implying that rhazinilam (29) exhibited significant immunosuppressive activity but not cytotoxicity. CD25 and CD69 expression were induced by activated T cells.30 Rhazinilam (29) had no effect on the expression of CD25 and CD69 in T cells at all of the concentrations tested (Figure 4A). It increased the percentage of cells in the G2/M phase of T cells, similar to Taxol treatment, indicating that rhazinilam (29) induced arrest in the G2/M phase of the T cell cycle (Figure 4B). Proinflammatory cytokines, including IL-6 and IL-17, play important roles in autoimmune disease pathogenesis.31 Furthermore, IL-6 is crucial for Th17 differentiation.32 Rhazinilam (29) reduced the level of IL-6 and IL-17 in activated T cells (Figure 5), indicating that it might preferably affect Th17 cells in terms of differentiation and function.

Previous studies of rhazinilam focused on its anticancer activity through its antitubulin properties.4,33 The significant immunosuppressive and anti-inflammatory activities of rhazinilam on activated human T cells were probed. The abnormal T cell activation and the production of proinflammatory cytokines play key roles in the development of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis.34 This may explain why K. off icinalis has a mitigation effect in treating rheumatoid arthritis.35 The structure of rhazinilam (29) is totally different from the immunosuppressants used in the clinic or in trials. Because the target and function are determined by structure,36 it may be a potential lead compound for the treatment of autoimmune diseases in the design and development of new drugs.

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured with a PerkinElmer 341 digital polarimeter. The UV spectra were acquired with a PerkinElmer Lambda 35 UV/vis spectrometer. The NMR spectra were acquired on Bruker Avance III 400 MHz and Avance 600 MHz NMR spectrometers. The HRESIMS data were recorded with a BioTOF-Q mass spectrometer. Column chromatography (CC) was done on silica gel (200−300 mesh, Qingdao Marine Chemistry Co., Ltd., Qingdao, People’s Republic of China), reversedphase silica gel (ODS, 50 μm, YMC Co., Ltd., Japan), macroporous resin D-101 (ChengDu KeLong Chemical Co., Ltd., Chengdu, People’s Republic of China), Sephadex LH-20 (40−70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), and amino silica gel (MB 100-40/75, Fuji Silysia Chemical, Ltd., Japan). Semipreparative HPLC and HPLC-PDA analysis were performed on a Waters 2695 apparatus coupled to a Waters 2996 photodiode array detector, which was supported by Waters Empower 2 software. A Waters XTerra RP18 868

DOI: 10.1021/acs.jnatprod.6b00697 J. Nat. Prod. 2017, 80, 864−871

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Figure 4. Rhazinilam (29) did not inhibit T cell activation but induced T cell cycle arrest in the G2/M phase. Human T cells were treated with rhazinilam (29) (0.2, 1, and 5 μM), FK506 (0.1 μM), or vehicle and activated with the anti-CD3/anti-CD28 antibodies for 24 h. The expression of CD25 and CD69 was analyzed on a flow cytometer (A). Human T cells were treated with rhazinilam (29) (0.2, 1, and 5 μM), Taxol (0.1 μM), or vehicle and activated with the anti-CD3/anti-CD28 antibodies for 72 h. Cell cycle progression was analyzed by flow cytometry (B). The results of flow cytometry are representative histograms from three independent experiments. was supplied by Solarbio (Beijing, People’s Republic of China). A human Pan T cell isolation kit II was purchased from Miltenyl Biotec (Bergisch Gladbach, Germany). A CCK-8 assay kit was used to measure cell viability (Dojindo, Kumamoto, Japan). Plant Material. The twigs and leaves of K. of f icinalis were collected in October 2009 from Xishuangbanna Tropical Botanical Garden (XTBG) of the Chinese Academy of Sciences (CAS) in Menglun, Mengla, Yuannan Province, People’s Republic of China. The plant was authenticated by Associate Professor Zhi Na of XTBG, CAS, and a voucher specimen (LMKO0901) was deposited in Chengdu Medical College. Extraction and Isolation. The CHCl3 and n-BuOH extraction of the total alkaloids from the plant material (8.5 kg) has been described previously.37 The CHCl3 extract (160.0 g) was divided into three fractions (A−C) by Sephadex LH-20 CC (MeOH). Fraction A (3.6 g) was subjected to RP-18 CC using a MeOH/H2O gradient (10:90−80:20) to obtain four fractions (A1−A4). Fractions A1 and A3 were isolated using Sephadex LH-20 CC (MeOH) and further purified by semipreparative HPLC to afford compound 7 (13 mg, MeOH/H2O + 0.1% trifluoroacetic acid (TFA), 10:90) at 14 min and compound 6 (25 mg, MeOH/H2O + 0.1% TFA, 15:85) at 15 min. Fraction A2 was purified by semipreparative HPLC to give compound 16 (125 mg, MeOH/ H2O + 0.1% TFA, 15:85) at 15 min. Compounds 6 (27 mg) and 4 (6 mg) were isolated from fraction A4 via semipreparative HPLC (MeOH/H2O + 0.1% TFA, 10:90), and the two compounds eluted at 19 and 15 min, respectively. Compound 5 (10 mg) was also obtained by HPLC, and it eluted at 10 min (MeOH/H2O + 0.1% TFA, 20:80) from fraction A4. Fraction B (143.5 g) was subjected to purification using macroporous resin D-101 CC with MeOH/H2O (40:60−95:5) to produce four fractions (B1−B4). Fraction B1 (9.5 g) was purified on a Sephadex LH-20 column using MeOH as the eluent. It was further purified on an NH-gel column and eluted with a gradient of CHCl3/MeOH (50:1). Finally, it was purified by HPLC to afford 11 (128 mg) (MeCN/H2O + 0.1% TFA, 5:95) at 17 min, 17 (46 mg)

Figure 5. Rhazinilam (29) inhibited proinflammatory cytokine production in activated T cells. Human T cells were treated with rhazinilam (29) (0.2, 1, 5 μM), LY-294002 (50 μM), or vehicle and activated with the anti-CD3/anti-CD28 antibodies for 48 h. The supernatants were collected, and the levels of IL-6 (A) and IL-17 (B) were measured by ELISA. The results are presented as the mean ± SEM (n = 5 per group) from three independent experiments. *P < 0.05 vs the group of imiquimod-induced without drug. column (10 μm, 250 × 10 mm) and a Kromasil 100-5C18 (5 μm, 250 × 4.6 mm) column were used for semipreparative (3 mL/min) and analytical (1 mL/min) HPLC, respectively, with detection at 210 and 230 nm. Silica gel GF254 glass plates (Qingdao Marine Chemistry Co., Ltd., Qingdao, People’s Republic of China) were used for TLC analysis. Spots were visualized under UV light and by spraying with Wagner’s reagent. PBMCs were collected from healthy individuals. RPMI 1640 and DMEM were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was supplied by Invitrogen (Carlsbad, CA, USA). Human lymphocyte separating medium was purchased from Axis-Shield PoC (Oslo, Norway). PBS (1×) 869

DOI: 10.1021/acs.jnatprod.6b00697 J. Nat. Prod. 2017, 80, 864−871

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and 13C NMR, Table 2; HRESIMS m/z 397.1771 [M + H]+ (calcd for C22H25N2O5, 397.1758). N(4)-Methylkopsininate (7): amorphous solid; [α]20 D +21 (MeOH, c 0.1); UV (MeOH) λmax (log ε) 206 (4.66), 263 (3.79), 317 (3.13); 1 H NMR, Table 1, and 13C NMR, Table 2; HRESIMS m/z 339.2073 [M + H]+ (calcd for C21H27N2O2, 339.2067). Kopsinine methochloride (8): white solid; [α]20 D +9 (MeOH, c 0.1); UV (MeOH) λmax (log ε) 207 (4.67), 260 (3.86), 315 (3.24); 1H NMR, Table 1, and 13C NMR, Table 2; HRESIMS m/z 353.2211 [M − Cl]+ (calcd for C22H29N2O2, 353.2224). Demethoxycarbonylkopsin (10): brown paste; 1H NMR, Table 1, and 13C NMR, Table 2; HRESIMS m/z 323.1747 [M + H]+ (calcd for C20H23N2O2, 323.1754). T Cell Preparation. PBMCs were isolated from three healthy donors by density-gradient centrifugation using Lymphoprep. The cells were cultured in RPMI 1640 supplemented with 10% FBS. The T cells were isolated using the Pan T cell isolation kit II human with negative selection. The T cells were stained using the PE-anti-CD3 antibody (BD PharMingen, San Diego, CA, USA), and the number of stained cells was determined using flow cytometry (Acurri C6, Becton Dickinson, San Jose, CA, USA). The T cells were at 95% purity during the following experiments. Cell Proliferation Assay. Flow cytometry was used to probe T cell proliferation using 5-carboxyfluorescein diacetate succinimide ester (CFSE, Molecular Probes, Eugene, OR, USA) labeling. Briefly, ̈ T cells or PBMCs (106 cells/mL) were stained with human naive 2.5 μM CFSE at 37 °C for 10 min, washed with PBS twice, and resuspended in RPMI 1640 medium containing 10% FBS. The labeled ̈ T cells (106 cells/mL) were activated by plate-bound anti-CD3 naive (2 μg/mL, HIT3a clone) and soluble anti-CD28 (1 μg/mL, CD28.2 clone, BD PharMingen). The labeled PBMCs (106 cells/mL) were activated with equal numbers of PBMCs irradiated with 3000 rad from another person. Subsequently, the proliferation of the T cells activated by the anti-CD3/anti-CD28 antibodies or alloantigen was measured by flow cytometry after stimulation for 72 h incubated with or without different concentrations of rhazinilam (29). The cells without stimulation or drug served as the negative control, and the positive control was the cells with the stimulation but without the drug. ̈ T cells (106 cells/mL) and Cell Viability Assay. Human naive PBMCs (106 cells/mL) were treated with different concentrations of rhazinilam (29) or vehicle for 72 h. The CCK-8 assay kit was used to assess the viability of cells by reading the OD values at 450 nm in a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Expression of CD25 and CD69. Human T cells (106 cells/mL) were collected after 24 h of stimulation with the anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) antibodies in the presence of rhazinilam (29) (0.2, 1, and 5 μM), FK506 (0.1 μM), or vehicle. The cells were stained with PE-anti-CD25 or APC-anti-CD69 (BD PharMingen) at 4 °C for 30 min. The cells were washed with PBS and analyzed on a flow cytometer. Cell Cycle Progression Assay. Human T cells (106 cells/mL) were treated with rhazinilam (29) (0.2, 1, and 5 μM), Taxol (0.1 μM, Sigma-Aldrich), or vehicle and activated with or without the anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) antibodies for 72 h. Subsequently, the cells were collected, washed, and analyzed on a flow cytometer using the Cycletest Plus DNA reagent kit (BD PharMingen) following the manufacturer’s protocol. Determination of Cytokines by ELISA. Human T cells (106 cells/mL) were treated with rhazinilam (29) (0.2, 1, and 5 μM), LY-294002 (50 μM, Promega, Madison, WI, USA), or vehicle and activated using the anti-CD3 antibody (2 μg/mL) and the soluble antiCD28 antibody (1 μg/mL) for 24 or 48 h. The supernatants were harvested, and the levels of IL-6 and IL-17 were determined by ELISA kits (eBioscience, San Diego, CA, USA) in accordance with the standard curves of the recombinant cytokines. Statistical Analysis. The inhibitory concentrations of the compounds that reduced cell proliferation by 50% (IC50) were calculated using GraphPad Prism 6 (GraphPad, San Diego, CA, USA). One-way analysis of variance (ANOVA) with Dunnett comparisons on

(MeOH/H2O + 0.1% TFA, 30:70) at 15 min, 24 (24 mg) (MeOH/ H2O + 0.1% TFA, 20:80) at 18 min, 25 (130 mg) (MeOH/H2O + 0.1% TFA, 30:70) at 12 min, 8 (25 mg) (MeOH/H2O + 0.1% TFA, 10:90) at 17 min, 26 (43 mg) (MeOH/H2O + 0.1% TFA, 25:75) at 30 min, and 7 (57 mg) (MeOH/H2O + 0.1% TFA, 40:60) at 6 min. Fraction B2 (92 g) was separated on silica gel CC using a stepwise gradient of petroleum ether/acetone (10:1−1:1) to provide three fractions (B21−B23). Fraction B21 was identified as 18 (3.1 g). After compounds 11 (10.1 g) and 12 (1.1 g) were precipitated from fraction B22 in MeOH, the mother liquor was fractionated using RP-18 CC and eluted with MeOH/H2O (10:90−80:20) to obtain 29 (40 mg), 18 (0.9 g), 11 (1.2 g), 12 (0.3 g), 13 (58 mg), and 14 (92 mg). The last part of B22 was fractionated using semipreparative HPLC with MeOH/H2O + 0.1% TFA at 10:90 to yield 15 (19 mg) at 15 min, 27 (36 mg) at 17 min, 10 (19 mg) at 19 min, and 9 (18 mg) at 22 min. Compound 11 (4.7 g) was crystallized from B23 using MeOH. The mother liquor was fractionated by RP-18 CC and eluted with MeOH/H2O (20:80−80:20), which was followed by semipreparative HPLC to afford 20 (0.11 g) at 12 min, 21 (10 mg) at 16 min, 28 (0.35 g) at 18 min (MeOH/H2O + 0.1% TFA, 46:54), 22 (10 mg, MeCN/H2O + 0.1% TFA, 28:72) at 25 min, and 23 (9 mg, MeOH/H2O + 0.1% TFA, 40:60) at 10 min. Fraction B3 (4.5 g) was subjected to CC over NH-silica gel (CHCl3/MeOH, 30:1−1:1), Sephadex LH-20 (MeOH), and semipreparative HPLC to yield 24 (0.6 g, MeCN/H2O + 0.1% TFA, 20:80) at 23 min and 8 (31 mg, MeOH/H2O + 0.1% TFA, 15:85) at 20 min. Compound 19 (0.6 g) was precipitated from fraction B4 in MeOH. The mother liquor was fractioned on an NH-gel column with CHCl3/MeOH (50:1) to afford 12 (0.7 g). The solution was further purified using HPLC (MeOH/ H2O + 0.1% TFA, 25:75) to afford 24 (29 mg) at 20 min. Fraction C (9.6 g) was fractionated using RP-18 CC and eluted with MeOH/H2O (10:90−20:80) to afford three fractions (C1−C3). Fraction C1 was isolated by semipreparative HPLC (MeOH/H2O + 0.1% TFA, 10:90) to afford 1 (7 mg) at 19 min. Fraction C2 was subjected to a Sephadex LH-20 column (MeOH) and purified by semipreparative HPLC (MeCN/H2O + 0.1% TFA, 8:92) to afford 2 (17 mg) and 3 (11 mg), which eluted at 21 and 25 min, respectively. Fraction C3 was purified by recrystallization from MeOH to obtain 16 (2.7 g). The n-BuOH part (28.3 g) was fractionated by RP-18 CC and eluted with MeOH/H2O (10:90−20:80). The 10% MeOH/H2O fraction was purified by a Sephadex LH-20 column (MeOH) and further purified using HPLC to afford 9 (139 mg, MeOH/H2O + 0.1% TFA, 20:80) at 15 min. Using the same procedure, the separation of the 40−60% MeOH/H2O fraction was performed by Sephadex LH-20 CC (MeOH) and HPLC (MeOH/H2O + 0.1% TFA, 40:60), successively, to afford 30 (856.9 mg) at 11 min. Kopsinidine C (1): amorphous, white solid; [α]20 D +7 (MeOH, c 0.1); UV (MeOH) λmax (log ε) 206 (3.23), 245 (2.72), 296 (2.30); 1 H NMR, Table 1, and 13C NMR, Table 2; HRESIMS m/z 325.1891 [M + H]+ (calcd for C20H25N2O2, 325.1916). Kopsinidine D (2): amorphous solid; [α]20 D +22 (MeOH, c 0.1); UV (MeOH) λmax (log ε) 213 (4.34), 247 (3.72), 292 (3.25); 1H NMR, Table 1, and 13C NMR, Table 2; HRESIMS m/z 355.2023 [M + H]+ (calcd for C21H27N2O3, 355.2016). Kopsinidine E (3): white, amorphous solid; [α]20 D +15 (MeOH, c 0.1); UV (MeOH) λmax (log ε) 198 (3.83), 222 (4.19); 1H NMR, Table 1, and 13C NMR, Table 2; HRESIMS m/z 369.1794 [M + H]+ (calcd for C21H25N2O4, 369.1809). 11,12-Methylenedioxychanofruticosinic acid (4): amorphous, white solid; [α]20 D +41 (MeOH, c 0.1); UV (MeOH) λmax (log ε) 203 (4.19), 227 (4.23); 1H NMR, Table 1, and 13C NMR, Table 2; HRESIMS m/z 441.1675 [M + H]+ (calcd for C23H25N2O7, 441.1656). 12-Methoxychanofruticosinic acid (5): amorphous solid; [α]20 D +21 (MeOH, c 0.1); UV (MeOH) λmax (log ε) 214 (4.33), 246 (3.80), 288 (3.29); 1H NMR, Table 1, and 13C NMR, Table 2; HRESIMS m/z 427.1871 [M + H]+ (calcd for C23H27N2O6, 427.1864). Chanofruticosinic acid (6): white solid; [α]20 D +76 (MeOH, c 0.1); UV (MeOH) λmax (log ε) 206 (4.19), 240 (3.79); 1H NMR, Table 1, 870

DOI: 10.1021/acs.jnatprod.6b00697 J. Nat. Prod. 2017, 80, 864−871

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post-tests was used to analyze data and compare groups. The results are expressed as the mean ± SEM. P < 0.05 was considered to be statistically significant.

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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.6b00697. NMR and HRESIMS spectra of compounds 1−5 and 7 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86-28-62739161. E-mail: [email protected] (Y. Liu). *Tel/Fax: +86-28-62739161. E-mail: [email protected] (L.-M. Li). ORCID

Li-Mei Li: 0000-0002-6681-7414 Author Contributions †

T. Zeng and X.-Y. Wu contributed to this work equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (21172024, 81302786, and 81273530), the Sichuan Youth Science and Technology Fund (2014JQ0049), the Key Project of Chinese Ministry of Education (212151), and the Innovative Research Team Fund of Chengdu Medical College (CYTD15-01).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.6b00697 J. Nat. Prod. 2017, 80, 864−871