Alkaloids with Immunosuppressive Activity from the Bark of

Article Cite This: J. Nat. Prod. 2018, 81, 1841−1849

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Alkaloids with Immunosuppressive Activity from the Bark of Pausinystalia yohimbe Ye Liu,†,‡,# Heng-Yi Yu,†,§,# Hong-Zhe Xu,† Jun-Jun Liu,† Xiang-Gao Meng,⊥ Ming Zhou,† and Han-Li Ruan*,†

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†

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ Medical Science Research Center, Zhongnan Hospital of Wuhan University, Wuhan 430071, People’s Republic of China § Department of Pharmacy, Tongji Hospital Affiliated Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ⊥ College of Chemistry, Central China Normal University, Wuhan 430079, People’s Republic of China S Supporting Information *

ABSTRACT: Ten new alkaloids (1−10), including two pairs of enantiomeric mixtures (5a,b and 6a,b), and 15 known analogues (11−25) were obtained from the bark of Pausinystalia yohimbe. The structures of 1−25 were established by spectroscopic methods, and the absolute configurations of compounds 1−10 were resolved by X-ray diffraction and ECD data analyses. The in vitro immunosuppressive activities of selected isolates were tested. Compounds 11 and 16 exhibited moderate inhibition with IC50 values of 16.8 and 27.6 μM against ConA-induced T lymphocyte proliferation and 13.5 and 40.5 μM against LPS-induced B lymphocyte proliferation, respectively.

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more efficient use of this plant, we performed a phytochemical study on the bark of P. yohimbe and isolated 10 new (1−10) and 15 known (11−25) alkaloids. Selected isolates were investigated for cytotoxicities against human HepG2, MCF7, HT-29, and HeLa cancer cell strains, as well as their immunosuppressive activities against T and B lymphocyte proliferation.

ubiaceae, being composed of 611 genera and about 13 500 species, is an important part of tropical and subtropical flora. Plants in this family, such as Rubia cordifolia, Morinda of f icinalis, Uncaria rhynchophylla, and Cinchona ledgeriana, are widely used in traditional medicine for their remarkable therapeutic functions.1 The major biologically active components of these plants are alkaloids, which have important bioactivities and therapeutic uses.2,3 Some alkaloids derived from Rubiaceae have been widely used in clinical practice, e.g., the antimalarial quinine isolated from Cinchona ledgeriana and the antihypertensive drug rhynchophylline obtained from Uncaria rhynchophylla.1 Pausinystalia yohimbe (Rubiaceae) is mainly distributed in the tropical region of the west coast of Africa.4,5 The bark of P. yohimbe has been used as an aphrodisiac by the local Africans and for the treatment of fever, leprosy, and cough in West Africa.4,5 The main chemical constituents of this plant are alkaloids, especially yohimbine and its isomers, which belong to indole alkaloids with a wide range of biological activities.6 In the process of seeking more bioactive alkaloids and to make © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Compound 1 was obtained as a yellow, amorphous solid that gave a positive Dragendorff’s test. The molecular formula of 1 was assigned as C22H28N2O4 by the HRESIMS ion (m/z 385.2127 [M + H]+, calcd 385.2127) integrated with its NMR information. The 1H NMR data (Table 1) showed the diagnostic signals of an olefinic group (δH 7.52, 1H, s, H-17), an ortho-disubstituted phenyl system (δH 7.43, 1H, dt, J = 7.7, Received: April 25, 2018 Published: July 30, 2018 1841

DOI: 10.1021/acs.jnatprod.8b00324 J. Nat. Prod. 2018, 81, 1841−1849

Journal of Natural Products

Article

Chart 1

1.1 Hz, H-9; δH, 7.30, 1H, dt, J = 8.1, 1.1 Hz, H-12; 7.07, 1H, ddd, J = 8.1, 7.1, 1.1 Hz, H-11; δH 7.00, 1H, ddd, J = 7.7, 7.1, 1.1 Hz, H-10), two methoxy groups (δH 3.85, 3H, brs, OCH324; δH 3.69, 3H, brs, OCH3-23), and an ethyl group (δH 1.44, 1H, m, H-19a; δH 1.14, 1H, m, H-19b; δH 0.87, 3H, t, J = 7.5 Hz, H-18). The 13C NMR and DEPT data (Table 2) displayed 22 carbon signals allocated to three methyls, five methylenes, eight methines, three quaternary carbons, two nitrogenated tertiary sp2 carbons, and an ester carbonyl carbon. Analyses of the 1H−1H COSY spectrum (Figure 1) led to the assignment of fragments −CH−CH2−CH−CH(CH2CH3)−CH2− and −CH2−CH2−. All these spectroscopic characteristics combined with HMBC correlations (Figure 1) suggested 1 to be a tetracyclic yohimbine indole alkaloid structurally similar to corynantheidine (12),7 except that an oxygen atom was attached to N-4 in 1, which was proven by its molecular formula and the deshielded chemical shifts of C-3 (δC 71.0, +9.4 ppm), C-5 (δC 66.4, +12.9 ppm), and C-21 (δC 72.3, + 14.5 ppm) in 1 compared to those of compound 12.8−10 Based upon biosynthetic principles, H-15 is α-oriented in the corynantheine-type alkaloids.11−13 The NOESY signals (Figure 2) of H-3/H-15, H-21b and H-21a/H-18 suggested that H-3 and H-20 were both α-oriented. The H-3 resonance (δH 4.37, 1H, brs) hinted that it possessed an α-equatorial orientation (Figure 2) and the N−O bond was α-oriented.9,14,15 The (3S) configuration was ascertained by the positive Cotton effect at 270 nm in the electronic circular dichroism (ECD) spectrum

(Figure 3).9,14,16−18 Thus, the structure of 1 was defined as (4R)-corynantheidine N4-oxide. Compound 2, a yellow, amorphous solid, has a molecular formula of C22H28N2O4, the same as 1, according to the HRESIMS (m/z 407.1922 [M + Na]+, calcd 407.1947) in combination with the NMR data. Comparison of the 1D and 2D NMR spectra of 2 with those of 1 revealed that the two compounds possessed the same 2D structure. H-3 resonated as a doublet of doublets (δH 4.58, J = 9.2, 3.7 Hz), which indicated that it occupied an α-axial orientation (Figure 2) and the N−O bond was β-oriented.9,14,15 The absolute configurations of C-3, C-15, and C-20 in 2 were in accord with those of 1 through comparison of the NOESY correlations (Figure 2) and experimental ECD spectra (Figure 3). Therefore, the structure of 2 was defined as (4S)-corynantheidine N4-oxide, the N-4 diastereomer of compound 1. Compound 3 was obtained as a yellow, amorphous solid having the molecular formula C22H26N2O4, as ascertained by the HRESIMS (m/z 383.1975 [M + H]+; calcd 383.1971) and NMR data. The 1D NMR data of 3 were similar to those of 1 (Tables 1 and 2), except that the ethyl group attached to C-20 in 1 was replaced by a vinyl group [δC 117.9 (C-18) and 138.2 (C-19)] in 3, proven by the HMBC correlations from H-18 (δH 4.07) to C-19 and C-20 and from H-19 (δH 5.51) to C-20. The 1H NMR chemical shift of H-3 at δH 4.07 (brd, J = 11.9 Hz) indicated that it occupied an α-axial orientation (Figure 2) and the N−O bond was α-oriented.9,14,15 The absolute configurations of C-3, C-15, and C-20 in 3 were established 1842



Article

Table 1. 1H NMR Spectroscopic Data (δH) of Compounds 1−10 (J in Hz)a position

1b

2b

3b 4.07, brd (11.9)

1 3

4.37, brs

5a

3.52, overlap

5b

3.52, overlap

4.58, dd (9.2, 3.7) 3.79, ddd (12.6, 7.7, 5.0) 3.66, brt (5.5)

6a

3.36, overlap

3.25, overlap

6b

2.99, m

14a 14b

2.83, dd (15.6, 4.6) 7.43, dt (7.7, 1.1) 7.00, ddd (7.7, 7.1, 1.1) 7.07, ddd (8.1, 7.1, 1.1) 7.30, dt (8.1, 1.1) 2.78, overlap 2.06, brs

15

9

4b

11.55, s

3.40, m

4.43, dd (12.2, 4.6) 4.10, m

3.40, m

3.33, overlap

3.34, ddd (14.8, 8.4, 2.0) 2.78, overlap

3.36, overlap 2.94, m

7.45, brd (7.5)

7.43, d (8.1)

7.02, td (7.5, 7.0, 1.1) 7.09, ddd (8.1, 7.0, 1.1) 7.34, dt (8.1, 1.1) 2.73, overlap 2.03, dt (13.4, 3.1) 2.85, overlap

6.98, ddd (8.1, 7.0, 1.1) 7.05, ddd (8.1, 7.0, 1.1) 7.25, dt (8.1, 1.1) 2.26, m 2.26, m

2.78, overlap

7.46, dt (7.9, 1.0) 7.02, ddd (7.9, 7.0, 1.0) 7.10, ddd (8.2, 7.0, 1.0) 7.30, dt (8.2, 1.0) 2.54, overlap 2.39, dt (15.3, 8.8) 3.54, overlap

17 18

7.52, s 0.87, t (7.5)

7.36, brs 0.93, t (7.4)

7.46, s 5.04, m

3.07, td (11.7, 4.6) 7.42, s 5.08, m

19a

1.44, m

1.28, m

5.51, ddd (17.2, 10.3, 8.6)

5.50, ddd (17.2, 10.3, 7.9)

19b 20

1.14, m 3.07, overlap

1.28, m 2.54, overlap

3.30, overlap

21a

3.36, overlap

4.28, m

21b

3.02, overlap

3.26, overlap

3.76, ddd (11.9, 8.4, 3.9) 3.14, dd (11.6, 4.2) 2.91, overlap

3.47, m

23 24

3.69, brs 3.85, brs

3.56, s 3.56, s

3.67, s 3.83, s

3.61, s 3.82, s

10 11 12

5c

3.61, overlap

4.64, t (6.90) 4.64, t (6.90) 3.29, t (6.90) 3.29, t (6.90) 7.66, d (7.5) 7.13, t (7.5) 7.28, t (7.5) 7.67, d (7.5)

9.62, s 1.17, t (7.4) 3.12, q (7.4)

8.17, s

6b 11.46, s

3.89, m

7c 10.07, s

7.43, d (6.8)

8c

9.54, s 0.89, t (7.6) 1.80, q (7.5)

3.69, d (2.8)

7.46, d (6.8)

10b

3.44, dd (12.0, 2.8) 3.69, m

3.40, dd (11.7, 2.4) 3.70, d (9.5)

3.61, m

3.63, m

2.49, overlap

2.49, overlap

2.49, overlap

2.49, overlap

9.38, s

3.62, m

3.78, m 3.21, overlap 3.21, overlap 7.67, d (7.6) 7.13, t (7.6) 7.34, t (7.6) 7.67, d (7.6)

9b

3.24, m

8.04, d (7.6)

7.59, d (8.0)

7.46, d (7.4)

7.46, d (7.4)

7.38, t (7.6)

7.17, ddd (8.0, 6.9, 1.1) 7.36, ddd (8.3, 6.9, 1.1) 7.48, d (8.3)

6.73, t (7.4)

6.73, t (7.4)

7.46, dd (8.5, 7.4) 6.89, d (8.5)

7.46, dd (8.6, 7.4) 6.89, d (8.6) 2.60, overlap 2.49, overlap

9.77, s 1.00, t (7.5)

1.06, t (7.4)

2.54, overlap 0.93, brd (13.5) 2.70, td (11.7, 3.4) 7.35, s 5.04, m

7.41, brs 0.84, t (7.5)

1.84, m

1.76, m

5.48, m

1.43, brs

1.54, m 3.64, m

1.69, m 3.45, m

3.64, overlap

1.10, brs 2.90, brs

4.50, dd (13.5, 5.6) 4.35, dd (13.5, 1.8)

3.60, m

7.64, t (7.6) 7.71, d (7.6)

3.46, m 3.88, s

3.48, dd (11.7, 4.2) 3.29, overlap 3.61, s 3.78, s

2.49, overlap

3.63, m 3.13, t (11.8) 3.63, s 3.79, brs

a Data (δ) were measured in methanol-d4 for 1−4, 9, and 10, in DMSO-d6 for 5 and 6, and in CDCl3 for 7 and 8. The assignments were based on DEPT and experimental ECD spectra of compounds and 1H−1H COSY, HSQC, and HMBC experiments. b400 MHz. c600 MHz.

ortho-disubstituted phenyl system (δH 7.67, 1H, d, J = 7.5 Hz, H-12; δH 7.66, 1H, d, J = 7.5 Hz, H-9; δH 7.28, 1H, t, J = 7.5 Hz, H-11; δH 7.13, 1H, t, J = 7.5 Hz, H-10), two methylene groups (δH 4.64, 2H, t, J = 6.9 Hz, H-5; δH 3.29, 2H, t, J = 6.9 Hz, H-6), and an ethyl group (δH 1.17, 3H, t, J = 7.4 Hz, H-18; δH 3.12, 2H, q, J = 7.4 Hz, H-19). The 13C NMR data (Table 2) displayed 20 carbon signals separated by DEPT experiments into a methyl, three methylenes, six methines (including a formyl proton), six quaternary carbons, three nitrogenated tertiary sp2 carbons, and an ester carbonyl carbon. H-5 correlating with H-6 in the 1H−1H COSY spectrum (Figure 1) led to the fragment −CH2−CH2− corresponding to the C-5/ C-6 linkage. The above information combined with the HMBC correlations (Figure 1) indicated that compound 5 resembled the known mitralactonal,20 except for the absence of the 9methoxy group and the presence of a Δ20(21) olefinic bond. Its location was ascertained by the HMBC correlations of H-21 with C-3, C-15, C-20 and of H-5, H-6 with C-21. The structure of 5 was finally established via the X-ray diffraction data using Mo Kα irradiation (Figure 4), which showed that compound 5 comprised a pair of isomers involving the N-4 stereogenic center. Thus, the structures of 5 were defined and named (4R)- and (4S)-mitralactonals B (5a and 5b), respectively.

to be the same as those of 11 via the NOESY correlations of H-15 with H-3, H-18, H-19, H-3 with H-14b, and H-14a with H-20 (Figure 2),19 in combination with the positive Cotton effect at 270 nm in the experimental ECD spectrum (Figure 3). Accordingly, 3 was defined as the N4-oxide of 11 and named (4R)-corynantheine N-oxide. Compound 4, a yellow, amorphous solid, had the same molecule formula (C22H26N2O4) as 3 assigned by the HRESIMS (m/z 407.1771 [M + Na]+, calcd 407.1990) in combination with the NMR data. Comparison of the NMR data of 4 and 3 revealed that the two compounds possessed the same 2D structure. H-3 of 4 resonated as a doublet of doublets (δH 4.43, 1H, dd, J = 12.2, 4.6 Hz) in the 1H NMR spectrum, which implied that 4 was the N-4 diastereomer of 3 based on analysis of their NOESY spectra (Figure 2) and experimental ECD data (Figure 3). Compound 4 was hence determined as (4S)-corynantheine N-oxide. Compound 5 was obtained as reddish-brown needles with green fluorescence. The molecular formula was deduced as C20H16N2O3 by the HRESIMS (m/z 355.1057 [M + Na]+, calcd 355.1059) in combination with the NMR data. The 1H NMR and HSQC data of 5 disclosed the presence of an indolic NH group (δH 11.55, 1H, s, H-1), a formyl group (δH 9.62, 1H, s, H-17), an olefinic proton (δH 8.17, 1H, s, H-21), an 1843


Journal of Natural Products Table 2.

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Article

C NMR Spectroscopic Data (δC, Type) of Compounds 1−10a

position

1b

2b

3b

4b

5c

6b

7c

8c

9b

10b

2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

130.5, C 71.0, CH 66.4, CH2 19.1, CH2 106.2, C 127.9, C 118.9, CH 120.3, CH 122.5, CH 112.4, CH 138.1, C 27.9, CH2 39.0, CH 111.5, C 162.4, CH 11.0, CH3 24.4, CH2 34.8, CH 72.3, CH2 170.2, C 51.8, CH3 62.4, CH3


130.6, C 70.6, CH 66.2, CH2 19.0, CH2 106.2, C 127.8, C 119.0, CH 120.4, CH 122.6, CH 112.6, CH 138.1, C 27.4, CH2 38.4, CH 111.3, C 162.2, CH 117.9, CH2 138.2, CH 39.2, CH 71.9, CH2 170.0, C 51.8, CH3 62.4, CH3


123.4, C 121.1, C 53.9, CH2 19.9, CH2 114.4, C 124.9, C 119.9, CH 120.6, CH 124.8, CH 113.7, CH 139.6, C 138.3, C 141.6, C 93.0, C 182.0, CH 16.2, CH3 25.8, CH2 126.3, C 138.1, CH 171.3, C

123.5, C 139.4, C 50.4, CH2 20.1, CH2 121.2, C 124.5, C 120.9, CH 121.1, CH 127.1, CH 114.2, CH 141.0, C 127.4, C 150.7, C 103.5, C 184.6, CH 8.0, CH3 32.0, CH2 71.0, C 59.2, CH2 169.1, C

127.1, C 127.6, C 130.2, CH 108.3, CH 127.8, C 120.5, C 121.7, CH 121.9, CH 130.1, CH 113.0, CH 142.1, C 126.6, C 140.5, C 99.9, C 184.0, CH 11.0, CH3 24.2, CH2 34.1, CH 56.4, CH2 174.2, C

124.1, C 134.1, C 51.1, CH2 20.9, CH2 118.2, C 124.8, C 120.0, CH 121.0, CH 126.5, CH 112.4, CH 139.3, C 128.1, C 156.3, C 98.1, C 163.8, C 12.0, CH3 23.9, CH2 35.5, CH 52.9, CH2 167.6, C 51.5, CH3



a Data (δ) were measured in methanol-d4 for 1−4, 9, and 10, in DMSO-d6 for 5 and 6, and in CDCl3 for 7 and 8. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments. b100 MHz. c150 MHz.

Figure 1. Key HMBC (→) and 1H−1H COSY (bold lines) correlations of compounds 1 and 5−9.

Compound 6 was isolated as reddish-brown needles with a red fluorescence. Its molecular formula, C20H18N2O4, was resolved by the HRESIMS (m/z 373.1156 [M + Na]+, calcd 373.1164) and 13C NMR analyses. Comparison of its 1D NMR data with those of 5 (Tables 1 and 2) revealed that they possessed similar structures, except that the Δ20(21) double bond in 5 was hydroxylated in 6, as corroborated by the HMBC signals (Figure 1) from H2-21 to C-3, C-15, C-19, C20 and from H3-18 and H2-19 to C-20, and the oxygenated tertiary carbon at δC 71.0 (C-20). Crystals of 6 were analyzed via the X-ray diffraction data acquired with Mo Kα radiation (Figure 5), which showed a pair of C-20 enantiomers. However, we failed to resolve the racemate by chiral-phase column chromatography (CC). Therefore, the structures of the enantiomers were defined as (20S)- and (20R)mitralactonals C (6a and 6b), respectively.

Compound 7 was obtained as a red, amorphous solid with a red fluorescence and had the same molecular formula C20H16N2O3 as 5 based on the HRESIMS (m/z 333.1234 [M + H]+, calcd 333.1239) and 13C NMR data analyses. The NMR data (Tables 1 and 2) of 7 and 5 showed many similarities, suggesting they were analogues, except that the Δ20(21) double bond in 5 was replaced by a Δ5(6) double bond in 7. This was confirmed by the 1H−1H COSY correlations of H3-18/H2-19/H-20/H2-21 and H2-5/H2-6, along with the HMBC correlations from H-5 to C-3, C-7, C-21 and from H-6 to C-2, C-8. The absolute configuration of 7 was assigned by comparison of its experimental and calculated ECD spectra. As shown in Figure 6, the signs of the Cotton effects in the experimental ECD spectrum were identical to those in the calculated ECD spectrum of (20S)-7. Thus, the structure of mitralactonal D (7) was defined as shown. 1844



Article

Figure 2. Key NOESY (↔) correlations of compounds 1−4, 9, and 10.

calcd 399.1920) and 13C NMR data. The 1H NMR data of 9 showed the presence of an ortho-disubstituted phenyl system (δH 7.46, 1H, d, J = 7.4 Hz, H-9; δH 6.73, 1H, t, J = 7.4 Hz, H10; δH 7.46, 1H, dd, J = 8.5, 7.4 Hz, H-11; δH 6.89, 1H, d, J = 8.5 Hz, H-12), four olefinic signals (δH 7.35, 1H, s, H-17; δH 5.48, 1H, m, H-19; δH 5.04, 2H, m, H-18), and two methoxy groups (δH 3.78, 3H, s, OCH3-24; δH 3.61, 3H, s, OCH3-23). The 1H NMR data of 9 closely resembled those of 4, but detailed analysis of their 13C NMR data (Table 2) revealed that the two olefinic carbons belonging to the indole ring in 4 were replaced by a ketocarbonyl (δC 203.6, C-7) and a nitrogenated tertiary carbon (δC 73.4, C-2) in 9, as supported by the HMBC correlations from H-3, H-6, and H-9 to C-7 and from H-6 to C-2. These typical characteristics suggested that 9 possessed a pseudoindoxyl skeleton with an indolone fragment.21,22 The spectroscopic data of 9 were similar to mitragynine pseudoindoxyl,22 with the main differences being the absence of a 9-methoxy group and the replacement of the 20-ethyl group by an ethylene group, which was supported by the 1D NMR, HMBC, and 1H−1H COSY data (Figure 1). In addition, compound 9 was reasonably deduced as an N4-oxide from the typical downfield shifts of the carbon signals at δC 81.0, 68.4, and 68.9 for C-3, C-5, and C-21, respectively. The H-15 was α-oriented based on the biosynthetic pathway.11,12 H-15 correlated with H-3 and H-19 in the NOESY spectrum (Figure 2), suggesting an α-oriented H-3 and a β-oriented H20. The 1H NMR resonance of H-3 (δH 3.44, 1H, dd, J = 12.0, 2.8 Hz) indicated that H-3 occupied an α-axial orientation (Figure 2) and the N−O bond was β-oriented.9 The ECD spectrum of 9 exhibited the same negative Cotton effect at 400 nm (Figure 3) as mitragynine pseudoindoxyl, which established the (2S, 3S) absolute configuration.22 Thus, the structure of 9 was defined as corynantheine pseudoindoxyl N-oxide. Compound 10 was also obtained as a green fluorescent oil with a molecular formula of C22H28N2O5 determined by the HRESIMS (m/z 401.2066 [M + H]+, calcd 401.2076) and 13C NMR data and had two more hydrogen atoms than 9. Comparison of the NMR data of 10 and 9 manifested 10 was similar to 9, except that the ethylene group in 9 was replaced by an ethyl group in 10, as indicated by the 1H−1H COSY and HMBC data. The absolute configuration of 10 was assigned as identical to 9 by the 1H NMR signal of H-3 (δH 3.40, 1H, dd, J

Figure 3. Experimental ECD spectra of compounds 1−4, 9, and 10.

Compound 8, a yellow, amorphous solid with green fluorescence, had the molecular formula of C21H20N2O4 as corroborated by the HRESIMS (m/z 365.1500 [M + H]+, calcd 365.1501) and 13C NMR data. The NMR data of 8 and 7 (Tables 1 and 2) showed many similarities, except that the Δ5(6) double bond in 7 was replaced by a −CH2−CH2− fragment in 8 (Figure 1) and the formyl group (C-17) in 7 was replaced by a methoxycarbonyl group in 8. The experimental ECD pattern of 8 showed some similarity to that of 7 (Figure 7). Thus, the absolute configuration of mitralactonal E (8) was deduced as (20S). Compound 9 was isolated as green oil with a green fluorescence. The molecular formula of 9 was determined as C22H26N2O5 by the HRESIMS (m/z 399.1919 [M + H]+, 1845



Article

Figure 4. X-ray ORTEP drawing of compound 5.

Figure 5. X-ray ORTEP drawing of compound 6.

= 11.7, 2.4 Hz), the NOESY correlations (Figure 2), and similar Cotton effects in the ECD spectrum (Figure 3). Accordingly, the structure of compound 10 was defined as dihydrocorynantheine pseudoindoxyl N-oxide. The known alkaloids were identified as corynantheine (11),7,23 corynantheidine (12),7 4R-dihydrocorynantheine (13),24 (4S)-dihydrocorynantheine (14),25 dihydrocorynantheine N-oxide (15),26 corynanthine (16),25 yohimbine (17),27 corynoxine B (18),28 corynoxeine (19),29 corynoxinic B (20),12 corynoxine (21),30 isocorynoxeine (22),31 corynoxinic (23),12 β-yohimbine oxindole (24),27 and 3,4,5,6tetradehydroyohimbine (25),32 according to the corresponding reported and observed spectroscopic data. Compounds 1−4, 7, 8, 11−16, 18, 19, 21, and 23 were tested for their cytotoxic activities against MCF-7, HepG2, HT-29, and HeLa cancer cell strains by the MTT method in vitro (positive control, doxorubicin hydrochloride). None of the compounds showed significant activities (Table S2, Supporting Information). Additionally, compounds 1, 3, 4, 7, 8, 11, 15, and 16 were tested for their immunosuppressive

Figure 6. Experimental and calculated ECD spectra of 7.

activities in vitro (Table S3, Supporting Information). Compounds 11 and 16 exhibited moderate inhibition effects against concanavalin A (ConA)-induced T lymphocyte proliferation (IC50, 16.8 and 27.6 μM) and showed moderate inhibition against lipopolysaccharide (LPS)-induced B lymphocyte proliferation (IC50, 13.5 and 40.5 μM). Cyclosporin A (CsA) and mycophenolate mofetil (MMF) were used as positive controls.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter. IR spectra were recorded on a PerkinElmer FT-IR spectrometer. UV spectra were obtained using a Varian Cary 50 scan UV/vis spectrophotometer. An

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sequentially by silica gel (petroleum ether/CH2Cl2, 2:1 to 0:1, and CH2Cl2/CH3OH, 50:1 to 20:1), then further separated by MPLC with RP-C18 CC (CH3OH/H2O, 50:50 to 90:10) and HPLC (CH3OH/H2O/Et2NH, 70:30:0.05) to yield compounds 11 (117.3 mg) and 12 (12.0 mg); subfraction C2 was purified by silica gel (petroleum ether/acetone, 3:1 to 1:1) and HPLC (CH3OH/H2O/ Et2NH, 70:30:0.05) to afford compounds 20 (16.5 mg) and 22 (4.6 mg); subfraction C3 was subjected to silica gel (CH2Cl2/CH3OH, 10:1 to 0:1) and HPLC (CH3OH/H2O/Et2NH, 70:30:0.05) to yield compound 25 (6.6 mg). Fr. D was subjected to a silica gel column (petroleum ether/acetone, 6:1 to 1:2) to yield subfractions D1−D4. Subfraction D2 was chromatographed on silica gel (petroleum ether/ acetone, 8:1 to 2:1) and HPLC (CH3OH/H2O/Et2NH, 85:15:0.05) to yield compound 14 (32.7 mg); subfraction D3 was purified under the same condition as D2 to afford compounds 18 (10.7 mg) and 21 (6.5 mg); subfraction D4 was separated successively by silica gel (CH2Cl2/CH3OH, 1:0 to 5:1), MPLC with RP-C18 (CH3OH/H2O, 40:60 to 100:0), and HPLC (CH3OH/H2O/Et2NH, 65:35:0.05) to afford compounds 1 (14.3 mg), 9 (4.6 mg), 10 (1.1 mg), and 17 (2.0 mg). Fr. E was subjected to silica gel (CH2Cl2/CH3OH, 30:1 to 5:1) to obtain subfractions E1 and E2. Subfraction E1 gave colorless cubic crystals of compound 16 (1.3 g) by recrystallization from the sediment in CH2Cl2/CH3OH solvent; subfraction E2 was purified by MPLC with RP-C18 CC (CH3OH/H2O, 40:60 to 100:0) and HPLC (CH3OH/H2O/Et2NH, 60:40:0.05) to afford compounds 2 (7.9 mg), 3 (55.0 mg), 4 (30.5 mg), and 15 (38.6 mg). (4R)-Corynantheidine N-oxide (1): yellow, amorphous solid; [α]20D +64 (c 0.4, CH3OH); UV (CH3OH) λmax (log ε) 224 (3.06), 290 (2.26) nm; ECD (c 0.03, CH3OH) 214 (Δε −25.51), 230 (Δε +27.84), 270 (Δε +12.75) nm; IR (film) νmax 3410, 2943, 2877, 1697, 1634, 1452, 1436, 1382, 1325, 1290, 1247, 1146, 1109, 1007, 955, 747, 719 cm−1; HRESIMS data, [M + H]+ m/z 385.2127 (calcd for C22H29N2O4, 385.2127, Δ 0 ppm); 1H and 13C NMR (methanold4) data, Tables 1 and 2. (4S)-Corynantheidine N-oxide (2): yellow, amorphous solid; [α]20D +21 (c 0.4, CH3OH); UV (CH3OH) λmax (log ε) 223 (3.05), 290 (2.24) nm; ECD (c 0.06, CH3OH) 213 (Δε −17.84), 238 (Δε +14.97), 270 (Δε +13.58) nm; IR (film) νmax 3395, 2940, 2878, 1695, 1631, 1453, 1436, 1383, 1332, 1288, 1247, 1145, 1129, 1086, 1047, 990, 744, 688 cm−1; HRESIMS data, [M + Na]+ m/z 407.1922 (calcd for C22H28N2O4Na, 407.1947, Δ −6.1 ppm); 1H and 13C NMR (methanol-d4) data, Tables 1 and 2. (4R)-Corynantheine N-oxide (3): yellow, amorphous solid; [α]20D +56 (c 0.3, CH3OH); UV (CH3OH) λmax (log ε) 224 (3.36), 290 (2.54) nm; ECD (c 0.03, CH3OH) 214 (Δε −28.32), 230 (Δε +30.83), 269 (Δε +10.74) nm; IR (film) νmax 3404, 3207, 2979, 2945, 2851, 1697, 1638, 1453, 1437, 1326, 1293, 1249, 1190, 1147, 1128, 1025, 992, 960, 940, 879, 818, 776, 747, 681, 627 cm−1; HRESIMS data, [M + H]+ m/z 383.1975 (calcd for C22H27N2O4, 383.1971, Δ +1.0 ppm); 1H and 13C NMR (methanol-d4) data, Tables 1 and 2. (4S)-Corynantheine N-oxide (4): yellow, amorphous solid; [α]20D +65 (c 0.3, CH3OH); UV (CH3OH) λmax (log ε) 224 (3.18), 289 (2.28) nm; ECD (c 0.05, CH3OH) 217 (Δε −32.76), 237 (Δε +38.64), 267 (Δε +13.43) nm; IR (film) νmax 3387, 3233, 2948, 2852, 1696, 1638, 1454, 1437, 1330, 1302, 1286, 1246, 1213, 1189, 1147, 1117, 1028, 989, 934, 863, 844, 773, 746, 683 cm−1; HRESIMS data, [M + Na]+ m/z 405.1771 (calcd for C22H26N2O4Na, 405.1790, Δ −4.7 ppm); 1H and 13C NMR (methanol-d4) data, Tables 1 and 2. Mitralactonal B (5): reddish-brown needles (MeOH); mp >320 °C; HRESIMS data, [M + Na]+ m/z 355.1057 (calcd for C20H16N2O3Na, 355.1059, Δ −0.6 ppm); 1H NMR and 13C NMR (DMSO-d6) data, Tables 1 and 2. Mitralactonal C (6): reddish-brown needles (MeOH); mp >320 °C; HRESIMS data, [M + Na]+ m/z 373.1156 (calcd for C20H18N2O4Na, 373.1164, Δ −2.1 ppm); 1H and 13C NMR (DMSO-d6) data, Tables 1 and 2. Mitralactonal D (7): red, amorphous solid; UV (CH3OH) λmax (log ε) 203 (3.17), 248 (2.96), 289 (2.76), 429 (2.54), 500 (2.41) nm; ECD (c 0.02, CH3OH) 245 (Δε +7.61), 292 (Δε +12.66), 333 (Δε −9.78) nm; IR (film) νmax 3423, 2922, 2851, 1638, 1631, 1475,

Figure 7. Experimental ECD spectra of 7 and 8. LTQ-Orbitrap XL MS (Thermo) was used for HRESIMS spectra performance. A JASCO J-810 spectropolarimeter was used for ECD spectra recording. NMR spectroscopic data were acquired on a Bruker AM-400 or a DRX-600 NMR spectrometer. A Bruker SMART APEXII CCD diffractometer was utilized to acquire the X-ray crystallographic data with Mo Kα radiation (λ = 0.710 73 Å). TLC analysis was performed on silica gel GF254 plates (Yantai, China). CC purification was performed with silica gel (Qingdao, China), Sephadex LH-20 gel (GE Healthcare), or ODS gel (YMC). An EZ Purifier III chromatograph was used for MPLC with Spherical C18ODS columns. HPLC was conducted on Agilent 1260 or 1100 system by ODS-A C18 (YMC) columns. Plant Material. The P. yohimbe bark was provided by Tianjin Jianfeng Natural Product R&D Co., Ltd., China, in April 2010, and authenticated by the licensed pharmacist Mr. Dan Liu in the same company. A voucher specimen (ID 20100401) was deposited in the herbarium of the author’s laboratory. Extraction and Isolation. Air-dried Pausinystalia yohimbe bark (13 kg) was chopped and extracted with 95% EtOH. The crude extract (4.5 kg) was suspended in 2% HCl solution. The acidic part was basified to pH 9−10 with a 2% NaOH solution and was subsequently partitioned with CHCl3 to afford an alkaloidal extract (210 g). The extract was fractionated by a silica gel CC (200−300 mesh, petroleum ether/CH2Cl2, 1:0 to 0:1, and CH2Cl2/CH3OH, 50:1 to 0:1) to afford Frs. A−F. Fr. B was subjected to a silica gel CC (petroleum ether/CH2Cl2, 8:1 to 0:1, and CH2Cl2/CH3OH, 50:1 to 10:1) to give subfractions Frs. B1−B4. Fr. B1 was separated sequentially by Sephadex LH-20 CC (CH2Cl2/CH3OH, 1:1), followed by semipreparative RP-HPLC (CH3OH/H2O/Et2NH, 75:25:0.05), to afford compounds 8 (2.1 mg) and 19 (3.5 mg); subfraction B2 gave reddish-brown needles of compound 5 (7.5 mg) by recrystallization from the sediment in the CH2Cl2/CH3OH solvent. The mother liquor was further purified by Sephadex LH-20 (CH 2 Cl 2 /CH 3 OH, 1:1) and HPLC (CH 3 OH/H 2 O/Et 2 NH, 70:30:0.05) to afford compounds 13 (11.1 mg) and 23 (29.0 mg); subfraction B3 gave reddish-brown needles of compound 6 (5.3 mg) by recrystallization from the sediment in the CH2Cl2/CH3OH solvent; subfraction B4 was sequentially purified by silica gel (CH2Cl2/CH3OH, 30:1 to 10:1) and HPLC (CH3OH/H2O/ Et2NH, 60:40:0.05) to yield compounds 7 (1.1 mg) and 24 (1.3 mg). Fr. C was subjected to a silica gel column (CH2Cl2/CH3OH, 1:0 to 10:1) to give subfractions Frs. C1−C3. Fr. C1 was separated 1847



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1357, 1329, 1220, 1148, 1064, 752, 671 cm−1; HRESIMS data, [M + H]+ m/z 333.1234 (calcd for C20H17N2O3, 333.1239, Δ −1.5 ppm); 1 H and 13C NMR (CDCl3) data, Tables 1 and 2. Mitralactonal E (8): yellow, amorphous solid; [α]20D −30 (c 0.2, CH3OH); UV (CH3OH) λmax (log ε) 206 (2.88), 303 (2.54), 485 (1.66) nm; ECD (c 0.02, CH3OH) 230 (Δε +15.86), 255 (Δε +9.18), 340 (Δε −0.94) nm; IR (film) νmax 3438, 2928, 2853, 1638, 1441, 1377, 1328, 1292, 1188, 1148, 1061, 745, 652 cm−1; HRESIMS data, [M + H]+ m/z 365.1500 (calcd for C21H21N2O4, 365.1501, Δ −0.3 ppm); 1H and 13C NMR (CDCl3) data, Tables 1 and 2. Corynantheine pseudoindoxyl N-oxide (9): green oil; [α]20D −210 (c 0.5, CH3OH); UV (CH3OH) λmax (log ε) 234 (2.96), 400 (1.92) nm; ECD (c 0.06, CH3OH) 209 (Δε −22.43), 247 (Δε +31.96), 316 (Δε +10.82), 400 (Δε −18.27) nm; IR (film) νmax 3406, 2948, 2851, 1691, 1618, 1490, 1466, 1437, 1328, 1304, 1243, 1195, 1147, 1127, 1106, 991, 925, 772, 755, 695 cm−1; HRESIMS data, [M + H]+ m/z 399.1919 (calcd for C22H27N2O5, 399.1920, Δ −0.2 ppm); 1 H and 13C NMR (methanol-d4) data, Tables 1 and 2. Dihydrocorynantheine pseudoindoxyl N-oxide (10): green oil; [α]20D −299 (c 0.1, CH3OH); UV (CH3OH) λmax (log ε) 235 (3.19), 400 (2.14) nm; ECD (c 0.01, CH3OH) 222 (Δε −29.39), 244 (Δε +23.30), 316 (Δε +7.76), 400 (Δε −8.21) nm; IR (film) νmax 3436, 2958, 2851, 1679, 1633, 1621, 1490, 1466, 1437, 1328, 1304, 1251, 1146, 1099, 990, 753 cm−1; HRESIMS data, [M + H]+ m/z 401.2066 (calcd for C22H29N2O5, 401.2076, Δ −2.5 ppm); 1H and 13C NMR (methanol-d4) data, Tables 1 and 2. X-ray Crystal Structure Analysis of Compounds 5 and 6. Suitable crystals of compounds 5 and 6 were both obtained at room temperature from MeOH solutions. The detailed methodology for Xray diffraction analysis has been reported previously.33 The crystallographic data of 5 and 6 were deposited at the CCDC (numbers 1822876 and 1823272, respectively). Crystal data for 5: C20H16N2O3, MW = 332.35, monoclinic, crystal dimension 0.12 × 0.10 × 0.10 mm3, space group Cc, a = 25.670(5) Å, b = 7.0317(13) Å, c = 17.015(3) Å, V = 2982.8(9) Å3, Z = 8, α = γ = 90.00°, β = 103.786(3)°, ρ(calcd) = 1.480 Mg·m−3, μ(Mo Kα) = 0.101 mm−1, F(000) = 1392, independent reflections 9315 (Rint = 0.0635), final R1 indices 0.0610, wR2 = 0.1310 [I > 2σ(I)], goodnessof-fit on F2 0.995. The crystal structure model of compound 5 is pseudo-centrosymmetric and can also be refined in the centrosymmetric space group C2/c. However, the C-5 and C-6 atoms are seriously disordered when the structural model was solved and refined in the C2/c space group, which is not the case in the space group of Cc. Besides, the R and wR factors were poor in C2/c, and only an unwilling structure model could be found. Cc was thus selected in order to refine the structure. The absolute structure could not be determined due to the pseudosymmetry. As a result, a twin refinement for racemic twinning was done and led to a BASF parameter of −0.4(17). CCDC number: 1822876. Crystal data for 6: C20H18N2O4, MW = 350.36, monoclinic, crystal dimension 0.12 × 0.10 × 0.10 mm3, space group P21/n, a = 10.596(3) Å, b = 19.264(6) Å, c = 16.921(5) Å, V = 3315.0(18) Å3, Z = 8, α = γ = 90.00°, β = 106.312(5)°, ρ(calcd) = 1.404 Mg·m−3, μ(Mo Kα) = 0.099 mm−1, F(000) = 1472, independent reflections 5404 (Rint = 0.1051), final R1 indices 0.0583, wR2 = 0.1314 [I > 2σ(I)], goodnessof-fit on F2 1.011. CCDC number: 1823272. ECD Calculations. The detailed methodology for ECD calculation has been reported in a previous publication.34 After Boltzmann weighting, the spectra were combined on the basis of their contributions of population (Tables S1, Figure 105, Supporting Information). Cytotoxicity Assays. The cytotoxic activity assays were done by an MTT method using the cancer cell strains MCF-7, HepG2, HeLa, and HT-29, which were cultured in DMEM or RPMI-1640 medium (Hyclone, USA) replenished with 10% fetal bovine serum (FBS, Sijiqing, China) and penicillin−streptomycin solution in 5% CO2 at 37 °C. The detailed methodology for the cytotoxicity assay has been described in a previous publication.35 The percentage of viable cells was quantified at 490 nm by a Synergy 2 Enzyme immunoassay instrument (BioTek).

Immunosuppressive Activity Assays.36,37 Animals. Male C57BL/6 mice (6−8 weeks old) were obtained from Tongji Experimental Animal Center and were settled in specific conditions (photoperiod 12 h light/12 h dark, temperature 22 ± 1 °C, relative humidity 55 ± 5%). The experimental and husbandry contact with the mice were performed under SPF conditions. Preparation of Spleen Cells from Mice. The cervical dislocation method was used to sacrifice the C57BL/6 mice, and then the spleens were dissected. Single-cell suspensions were obtained by grinding the spleen from a 40 μm filter screen, and erythrocytes were dissociated with lysis buffer. Lymphocytes were washed and suspended in the medium (RPMI 1640, Hyclone, USA) replenished with 10% FBS and penicillin−streptomycin solution. T-Cell and B-Cell Function Assay. The 5 × 105 (180 μL per well) spleen cells, acquired from male C57BL/6 mice, were cultured in 96well plates in 5% CO2 at 37 °C for 24 h. The cultures were stimulated with ConA (5 μg/mL, Sigma, USA) or with LPS (10 μg/mL, Sigma, USA) to induce proliferative responses of T or B cells, respectively. After that, 20 μL of MTT (5 μg/μL) was added, and the mixtures were incubated for 4 h. Then, each 96-well plate was pelleted by centrifugation (1500 rpm, 5 min), and DMSO (100 μL per well) was added after the supernatant was decanted. The percentage of viable cells was quantified at 490 nm. Positive controls were CsA (Sigma, USA) and MMF (Sigma, USA).

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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.8b00324. HRESIMS, NMR, IR, UV, and ECD spectra of compounds 1−10; cytotoxic and immunosuppressive activities of the selected compounds (PDF) Crystallographic data (CIF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-13339986848. E-mail: [email protected]. ORCID

Jun-Jun Liu: 0000-0001-9953-8633 Han-Li Ruan: 0000-0003-0882-1009 Author Contributions #

Y. Liu and H.-Y. Yu contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by the National Natural Science Foundation of China (Nos. 31770380, 21572073, 31270394, and 31500282) and the Fundamental Research Funds for the Central Universities (No. 2016YXMS150). We thank the staff at the Analytical and Testing Center of Huazhong University of Science and Technology for spectroscopic data collection.

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REFERENCES

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Alkaloids with Immunosuppressive Activity from the Bark of

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