Securinega Alkaloids from the Twigs of Securinega suffruticosa and

Article pubs.acs.org/jnp

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

Securinega Alkaloids from the Twigs of Securinega suf fruticosa and Their Biological Activities Kyoung Jin Park,† Chung Sub Kim,†,# Zahra Khan,‡,§ Joonseok Oh,⊥,∥ Sun Yeou Kim,‡,§ Sang Un Choi,∇ and Kang Ro Lee*,† †

Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 21936, Republic of Korea § College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea ⊥ Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States ∥ Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States ∇ Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea

J. Nat. Prod. Downloaded from pubs.acs.org by STATE UNIV NEW YORK PLATTSBURGH on 05/13/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: Seven new Securinega alkaloids, securingines A−G (1−7), together with seven known analogues (8−14), were isolated from the twigs of Securinega suf f ruticosa. Their chemical structures were elucidated by a combined approach of spectroscopic analysis, chemical methods, ECD calculations, and DP4+ probability analysis. The full NMR assignments and the absolute configuration of compound 8 are also reported. In addition, all the isolated phytochemicals (1−14) were assessed for their cytotoxic, anti-inflammatory, and potential neuroprotective activities. Compound 4 showed cytotoxic activity (IC50 values of 1.5−6.8 μM) against four human cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15). Compounds 3, 10, 12, and 13 showed potent inhibitory effects on nitric oxide production (IC50 values of 12.6, 12.1, 1.1, and 7.7 μM, respectively) in lipopolysaccharide-stimulated murine microglia BV-2 cells. Compound 5 exhibited a nerve growth factor production effect (172.6 ± 1.2%) in C6 glioma cells at 20 μg/mL. In a continuing search from Korean medicinal plants for bioactive secondary metabolites, the chemical investigation of S. suf f ruticosa twigs has resulted in the isolation and identification of seven new Securinega alkaloids (1−7), along with seven known compounds (8−14). The chemical structures of 1−8 were elucidated through analysis of spectroscopic data (1D and 2D NMR), HRMS data, electronic circular dichroism (ECD) calculations, chemical methods, and DP4+ probability analysis. Compound 8 was isolated for the first time from a natural source, and the full NMR assignments and the absolute configuration of this compound are reported in this study as well. All the isolates (1−14) were tested for

Securinega alkaloids are a class of plant secondary metabolites with a tetracyclic backbone characterized by a butenolide moiety.1 They have been found only in some plant species such as the Securinega, Flueggea, and Phyllanthus genera (Euphorbiaceae and Phyllanthaceae),1−3 and these species have shown antimalarial, antitumor, and antibacterial activities as well as antineurodegenerative activity.3−5 Securinega suf f ruticosa (Pall.) Rehder (Euphorbiaceae) is a dioecious shrub that can be found commonly in mountainous areas of Korea, Mongolia, mainland China, and Russia. This plant has been used in Chinese traditional medicine to treat infantile paralysis, impotence, rheumatic disease, quadriplegia, and blood circulation disorders.6,7 Previous phytochemical studies related to S. suff ruticosa have shown the occurrence of flavonoids, other phenolic compounds, fatty acids, alkaloids, and diterpenoids.5,7 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 14, 2019

A

DOI: 10.1021/acs.jnatprod.9b00142 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

Table 1.

13

C and 1H NMR Spectroscopic Data for Compounds 1−4 2b,d

1 position

δCa,c

2

65.8

3a

27.8

3b

δH

71.2

5a

27.9

5b 6a

49.3

6b

(J in Hz)

δH (J in Hz)

δCb,c

δHb,c (J in Hz)

δC

δH (J in Hz)

δC

2.35, d (13.1)

67.1

65.5

4.05, brs

86.3

28.9

21.0

49.9

3.09, d (4.7)

51.3

3.01, d (4.5)

2.73, d (4.5)

72.2

3.59, m

72.7

3.75, brdd (12.4, 1.8) 1.71, ddd (13.9, 5.4, 3.1) 1.53, ddd (13.9, 2.5, 1.6) 3.63, brt (2.7)

59.7

1.95, m

2.51, dd (12.1, 2.4) 2.12, dd (13.8, 2.7) 1.77, m

49.3

3.28, t (4.0)

49.6

3.34, t (4.2)

3.16, t (4.2)

28.1

2.07, m

23.5

24.2

2.09, m

23.2

2.29, m

49.5

1.73, m 3.22, ddd (8.9, 4.3, 2.6) 2.75, overlap

48.4

42.3

2.86, m

40.6

2.16, m 2.93, m

2.05, ddd (14.4, 13.0, 5.2) 1.92, m 2.66, m

3.54, brt (2.6) 2.13, dd (13.8, 2.5) 1.57, m 3.08, m 2.52, m

7

62.7

4.07, brs

64.2

4.43, brs

137.0

8a

36.8

2.35, brd (13.4) 1.63, dd (13.4, 10.0)

37.6

2.42, dd (13.5, 4.6) 1.88, dd (13.5, 9.1)

43.0

8b

4d b

1.46, m

4

3b,d

a,c

9 11 12a 12b 13

93.1 174.0 41.1

14

125.0

5.78, d (10.1)

126.9

15

135.6

5.95, d (10.1)

134.6

OCH3-4 OH-7 OH-2

55.2

3.23, s 5.30, brs

56.0

2.88, d (18.8) 2.78, d (18.8)

80.5

93.5 174.4 42.2

3.06, d (18.7) 2.76, d (18.7)

80.9

82.3 172.0 107.5

1.93, tdd (13.8, 4.5, 2.6) 1.51, m 3.27, td (14.0, 3.4) 3.19, dd (14.0, 4.3) 6.35, ddd (9.3, 3.9, 3.1) 3.06, ddd (18.3, 3.9, 1.8) 2.59, dt (18.3, 2.8)

5.77 (s)

166.4 5.93, dd (10.1, 1.7) 6.11, dd (10.1, 2.6) 3.34, s

δH (J in Hz)

δC b

2.80, m

2.82, m

58.8

3.88, t (4.8)

56.3

43.3

2.66, dd (9.9, 4.8) 1.91, d (9.9)

40.5

90.3 172.6 109.2 167.8

69.7

5.12, d (5.8)

123.8

121.3

5.86, m

147.9

56.1

3.33 (s)

5.83, s 6.79, dd (9.1, 1.0) 6.85, dd (9.1, 5.2)

91.4 172.1 110.1

3.92, brt (4.8) 2.89, dd (10.0, 4.8) 1.90, d (10.0)

δHa (J in Hz)

2.55, ddd (13.0, 9.4, 3.8) 3.69, brt (4.8) 2.66, dd (9.6, 4.7) 1.65, d (9.6)

5.90, s

5.92, s

6.76, dd (9.0, 1.2) 6.90, dd (9.0, 5.2)

6.67, dd (9.0, 1.2) 6.85, dd (9.0, 5.2)

164.0 123.7 147.4

5.49, brs

a

Measured in DMSO-d6. bMeasured in chloroform-d. cMeasured at 850 (δH) and 212.5 (δC) MHz. dMeasured at 700 (δH) and 175 (δC) MHz.

their cytotoxic, antineuroinflammatory, and potential neuroprotective activities.

fraction showed significant cytotoxicity and thus was further separated and purified by repeated column chromatography, which resulted in the chemical identification of seven new Securinega alkaloids (1−7) and seven known analogues (8− 14). Securingine A (1), obtained as a colorless gum, gave a molecular formula of C14H19NO5 from the HRESIMS protonated molecular ion peak [M + H]+ at m/z 282.1348

■

RESULTS AND DISCUSSION A methanol extract of the twigs of S. suf f ruticosa was partitioned into different fractions using n-hexane, CHCl3, EtOAc, and n-BuOH. Each fractionated extract was evaluated for its cytotoxic activity. Among them, the CHCl3-soluble B



Article

Figure 1. Key COSY, HMBC, and NOESY correlations of 1−8.

Figure 2. Determination of the relative configuration of 1. (A) Chemical shifts, splitting patterns, and coupling constants of H-2 and H-4 and their relative configurations. (B) 3D structures of the major conformers of 1a and 1b with axial−equatorial positions analysis of H-2 and H-4 and calculated chemical shifts of H-2. (C) DP4+ analysis results for 1a and 1c. (D) Δd values (dS− dR) in ppm of the two MTPA esters (1s and 1r).

respectively, instead of an olefinic group at C-12 and C-13 in 9.8 Further spectroscopic data analysis of the COSY, HSQC, and HMBC spectra showed two partial structures (units 1A and 1B, Figure 1). The HMBC correlation of OCH3-4/C-4 and the COSY correlations of H2-3/H-4, H-4/H2-5, and H2-5/ H2-6 of 1 supported the structure of unit 1A. The unit 1B structure was confirmed through the HMBC cross-peaks of H2-12/C-9, C-11, C-13, and C-14, OH-7/C-8, C-7, and C-15, and H-14/C-9 and the COSY correlations of H2-8/H-7, H-15, and H-14. Observation of an HMBC cross-peak from H2-8 to C-2 indicated a linkage between the two units 1A and 1B through the C-2−C-9 bond, and the remaining index of hydrogen deficiency suggested the presence of a N-1−O-13 bond that resulted in an additional ring formation (Figure 1). Therefore, the planar structure of 1, with an unusual B ring, was confirmed. Among the five stereogenic centers (C-2, C-4, C-7, C-9, and C-13) in 1, the relative configurations of C-2 and C-4 in the A ring were examined initially (Figure 2A). The 1H NMR signal of H-2 showed a doublet with a relatively large coupling constant (13.1 Hz), indicating that H-2 is in an axial position, whereas that of H-4 exhibited a broad triplet with a relatively small coupling constant (2.6 Hz), suggesting that H-4 is in an equatorial position (Figure 2A). Therefore, the configurations of H-2 and H-4 were assigned tentatively as α and β,

(calcd for C14H20NO5, 282.1341), indicating six indices of hydrogen deficiency. The 1H NMR data of 1 established resonances characteristic for two olefinic protons [δH5.95 (1H, d, J = 10.1 Hz, H-15) and 5.78 (1H, d, J = 10.1 Hz, H-14)], two oxygenated methines [δH4.07 (1H, brs, H-7) and 3.54 (1H, brt, J = 2.6 Hz, H-4)], a nitrogenated methylene [δH3.08 (1H, m, H-6a) and 2.52 (1H, m, H-6b)], a nitrogenated methine [δH2.35 (1H, d, J = 13.1 Hz, H-2)], four methylenes [δH2.88 (1H, d, J = 18.8 Hz, H-12a), 2.78 (1H, d, J = 18.8 Hz, H-12b), 2.35 (1H, brd, J = 13.4 Hz, H-8a), 2.13 (1H, dd, J = 13.8, 2.5 Hz, H-5a), 1.95 (1H, m, H-3a), 1.63 (1H, dd, J = 13.4, 10.0 Hz, H-8b), 1.57 (1H, m, H-5b), and 1.46 (1H, m, H-3b)], a methoxy group [δH3.23 (3H, s, OCH3-4)], and a hydroxy group [δH5.30 (1H, brs, OH-7)]. The 13C NMR data of 1 showed the occurrence of 14 carbon resonances, which were confirmed as an ester carbonyl carbon (δC 174.0), two olefinic carbons (δC 135.6 and 125.0), four oxygenated carbons (δC 93.1, 80.5, 71.2, and 62.7), two nitrogenated carbons (δC 65.8 and 49.3), four methylene carbons (δC 41.1, 36.8, 27.9, and 27.8), and a methoxy carbon (δC 55.2). These 1D NMR data (Table 1) resembled those of secu’amamine D (9), isolated from S. suf fruticosa var. amamiensis, indicating that 1 is a Securinega-type alkaloid.8 A comparison of the 1D NMR spectrum of 1 with those of 9 suggested the presence in 1 of a methylene and an oxygenated carbon at C-12 and C-13, C



Article

Figure 3. Experimental and calculated ECD spectra of 1−5 and 7.

data (Table 1) suggested that 2 is similar structurally to 9,8 with the notable difference of the location of the D ring (Figure 1). Analysis of the 2D NMR spectra of 2 also indicated two partial structures (units 2A and 2B, Figure 1). The structure of unit 2A was confirmed as being identical to that of unit 1A by comparing the spectroscopic data of 2 with those of 1. The HMBC cross-peaks of H-7/C-9, H-12/C-14, C-9, and C-11, and H-14/C-9 and C-13 and the COSY correlations of H-14/H-15, H-15/H-7, and H-7/H-8 led to the structural assignment of unit 2B. The HMBC cross-peak of H-8/C-2 was used to establish a linkage between units 2A and 2B. The connection of N-1−O-14 to form the B ring was confirmed by considering the remaining index of hydrogen deficiency. The relative configuration of 2 was established by analyzing the NOESY data. Cross-peaks between H-2/H-8a and H-14/H-12 suggested that H-2 and the C ring in 2 are cofacial in the molecule (Figure 1). On comparison of the experimental and calculated ECD spectra, the absolute configuration of 2 was determined as 2S,4S,8S,12S, which was conducted in a similar manner to that described for 1 (Figure 3). Securingine C (3), a yellowish gum, possesses a molecular formula established as C 13 H 13 NO 3 (HRESIMS m/z 232.0972[M + H]+; calcd for C13H14NO3, 232.0974). The NMR data of 3 were similar to those of 11.10 The major difference was in the presence of an epoxide resonance [δC49.9; δH 3.09 (1H, d, J = 4.7 Hz) and δC 49.3; δH 3.28 (1H, m)] in compound 3, instead of a methoxy group signal [δC 56.0; δH 3.25 (3H, s)] at C-4 in 11 (Table 1). The analysis of the 2D NMR data combined with the eight indices of hydrogen deficiency implied the presence of an epoxide group. The location of the epoxide group at C-3 and C-4 was confirmed through the HMBC correlations from H-2 and H-5 to C-3 and C-4 and the COSY correlations of H-2/H-3, H-3/ H-4, and H-4/H-5, respectively (Figure 1). The relative and absolute configuration of 3 were confirmed, in turn, by NOESY and ECD calculations. The NOESY spectrum of 3 exhibited cross-peaks of H-2/H-6a and H-8a, H-3/H-6b, and H-4/H-6b, indicating that H-2, the epoxide at C-3 and C-4, and C-8 are oriented in the same direction (Figure 1). The

respectively (Figure 2A). Although there are eight (23 = 8) theoretically possible structures from the three unassigned stereogenic centers (C-7, C-9, and C-13), only four of them proved reasonable, since the B, C, and D rings are connected at C-9 and C-13. The 3D structures of the four possible diastereomers (1a−1d) were determined (Figure S59, Supporting Information). All conformers of 1a and 1c possessing a β-oriented C ring showed the same axial− equatorial characters for H-2 and H-4, with those deduced by coupling constants as described above, while those of 1b and 1d did not (Figures 2B and S60, Supporting Information). In addition, the calculated 1H NMR chemical shifts of H-2 in 1a (δH 2.82) and 1c (δH 2.83) were closer to the experimental value (δH 2.51, measured in chloroform-d) than those in 1b (δH 3.47) and 1d (δH 4.42) (Figures 2B and S60, Supporting Information), which also supported the β-orientation of the C ring. To confirm the relative configuration of C-7, the statistical parameter DP4+ was applied. This method was developed by Grimblat et al. to improve the statistical accuracy for stereochemical assignment of organic molecules using GIAO NMR chemical shift calculations.9 Simulated and experimental 1H and 13C NMR chemical shifts of 1a and 1c were used for DP4+ probability analysis. The results indicated the structural equivalence of 1 to 1a (100% probability) with a β-configured hydroxy group (Figures 2C and S61, Supporting Information). Accordingly, the relative configuration of 1 was determined as shown in Figure 2. The absolute configuration of 1 was confirmed with the guidance of the ECD data. As shown in Figure 3, the experimental ECD spectrum of 1 displayed a negative Cotton effect at 247 nm and a positive Cotton effect at 213 nm, and the calculated ECD spectrum of 1 was in accordance with the experimental spectrum in showing a negative Cotton effect at 263 nm (Figure 3). Thus, the absolute configuration of 1 was established as 2S,4S,7R,9S,13R. The 7R configuration was corroborated through the modified Mosher’s method (Figure 2D). Securingine B (2), characterized as a colorless gum, gave a molecular formula of C14H17NO4(HRESIMS m/z 264.1237 [M + H]+; calcd for C14H18NO4, 264.1236). Its spectroscopic D


Journal of Natural Products Table 2.

13

Article

C (175 MHz) and 1H (700 MHz) NMR Spectroscopic Data for Compounds 5−8 in Chloroform-d 5

position

δC

2 3a 3b 4a 4b 5 6a 6b 7a 7b 8a 8b 9 11 12 13 14 15

96.7 36.2 46.2

δH (J in Hz) 5.20, 2.72, 2.41, 3.54, 3.24,

brs td (16.6, 6.9) m ddd (11.3, 7.0, 1.7) m

191.8 162.6

6 δC 65.1 31.3 63.5 32.0 50.0

57.5

4.27, td (4.7, 1.5)

71.1

44.4

2.94, dd (9.8, 4.7) 2.25, d (9.8)

40.8

88.4 172.3 109.0 162.0 123.3 139.3

5.82, s 6.70, dd (9.2, 1.6) 6.72, dd (9.2, 4.8)

82.7 172.0 113.6 164.4 126.5 134.6

7

δH (J in Hz)

δC

δH (J in Hz)

8 δC

δH (J in Hz)

3.34, 1.87, 1.22, 4.09,

dd (12.0, 2.3) d (13.8) m brd (1.6)

156.7 121.1

7.35, d (8.0)

156.8 121.2

7.38, dt (8.0, 0.9)

137.4

7.72, td (8.0, 1.8)

137.0

7.71, td (8.0, 1.8)

1.81, 3.06, 3.02, 4.77,

m m m dt (5.7, 2.9)

124.1 149.1

7.30, overlap 8.57, m

123.6 149.0

7.29, overlap 8.60, ddd (4.8, 1.8, 0.9)

66.9

4.61, brs

25.2

43.3

3.59, dd (11.6, 5.6) 2.05, dd (11.6, 9.6)

32.5

2.49, 2.24, 3.16, 2.14,

2.59, dd (11.4, 3.2) 2.08, dd (11.4, 2.2)

5.87, s 6.90, d (9.4) 6.33, dd (9.4, 5.7)

88.0 172.1 112.9 165.0 121.0 142.4

6.02, s 6.70, dd (10.0, 2.3) 6.25, brd (10.0)

87.1 172.3 111.7 165.5 120.9 139.7

dt (19.6, 5.3) m dd (12,3, 5.3) m

5.92, s 6.69, dd (9.8, 2.6) 6.24, ddd (9.8, 5.3, 2.6)

similar to the known isolate 9,8 except for the chemical shift of C-4 (δC‑4 63.5 for 6; δC‑4 72.2 for 9) and the absence of a methoxy group [δC 56.0; δH 3.31 (3H, s)] in 9, which suggested the presence of a hydroxy functionality at C-4 in 6. The planar structure of 6 was verified from the additional 2D NMR (COSY, HSQC, and HMBC) spectroscopic data (Figure 1). The relative configuration of 6 was determined based on the observed NOESY and 1H NMR data (Figure 1 and Table 2). The small coupling constant of H-4 (δH‑4 4.09, brd, J = 1.6 Hz for 6; δH‑4 3.45, brt, J = 2.7 Hz for 9) and the NOESY cross-peak of H-2/H-8 indicated the relative configuration of 6 to be in agreement with that of 9.8 The absolute configuration of 6 was assigned as 2S, 4S, 7R, 9S by the observation of two negative Cotton effects at 275 and 210 nm and a positive Cotton effect at 230 nm in the ECD spectrum (Figure S46, Supporting Information).8 The molecular formula of securingine G (7, colorless gum) was confirmed as C13H11NO3 from the protonated HRESIMS ion peak at m/z 230.0816 (calcd for [M + H]+, 230.0817) and the 13C NMR data. Inspection of the 1D NMR data of 7 (Table 2) showed similarities to the data of 7a-(pyridin-2-yl)7,7a-dihydrobenzofuran-2(6H)-one (8),11 but the signal for an oxygenated methine proton [δC 66.9; δH 4.61 (1H, brs)] in 7 was replaced by an aliphatic methylene [δC 25.2; δH 2.49 (1H, dt, J = 19.6, 5.3 Hz) and 2.24 (1H, m)] in 8 (Table 2). With the guidance of HMBC cross-peaks of H-14/C-7 and H-8/C7, the configuration of the hydroxy group was determined to be at C-7 (Figure 1). The relative configurations at C-7 and C-9 of 7 were confirmed by their coupling constants and by DP4+ statistical analysis. Comparison of the coupling constants between H-7 and H-8a, H-8b of 7 with the calculated values of two possible epimers, 7a and 7b (Figure 4A), showed that the calculated coupling constants of 7a [9.8 (3JH‑7/H‑8b) and 6.8 (3JH‑7/H‑8a) Hz] were more similar to those of the experimental values [9.6 (3JH‑7/H‑8b) and 5.6 (3JH‑7/H‑8a) Hz] than those of 7b [5.5 (3JH‑7/H‑8b) and 1.4 (3JH‑7/H‑8a) Hz]. In addition, the calculated 1H and 13C NMR chemical shifts of 7a and 7b were analyzed through DP4+ analysis by the application of experimental values, and this resulted in 7a as the correct form for 7 with 100% probability (Figures 4B and S62,

experimental ECD spectrum of 3 showed a negative Cotton effect at 298 nm and a positive Cotton effect at 212 nm (Figure 3). This result matched with the calculated ECD spectrum of 3, leading the absolute configuration of 3 to be established as 2S, 3R, 4S, 7S, 9S. Securingine D (4) was yielded as a white gum, and its molecular formula was determined as C13H13NO4 from the HRESIMS, displaying a protonated positive-ion peak [M + H]+ at m/z 248.0921 (calcd for C13H14NO4, 248.0923). The 1D NMR spectra of 4 resembled those of 3, except for the presence of an oxygenated carbon signal (δC 86.3) in 4, instead of methine signals [δC 59.7; δH 4.05 (1H, brs)] in 3 (Table 1). In the HMBC spectrum, the heteronuclear correlations from H-3, H-6, H-7, and H-8 to C-2 showed the hydroxy group to be located at C-2. The relative configuration of 4 was assigned by the NOESY cross-peaks of OH-2/H-3, H-5b, and H-6a, H3/H-6a, and H-6b/H-8b (Figure 1). A negative Cotton effect at 306 nm and a positive Cotton effect at 211 nm were observed in the experimental ECD spectrum (Figure 3), indicating that the absolute configuration of 4 is 2S, 3S, 4S, 7S, 9S by comparison with its calculated ECD spectrum. Securingine E (5) was identified as a colorless gum, and C13H11NO4 was found to be the molecular formula based on the protonated molecular ion at m/z 246.0770 (calcd for [M + H]+, 246.0766) in the HRESIMS. The 1D NMR data of 5 (Table 2) revealed similarities to those of compound 11,10 except for the presence of two carbonyl carbon signals (δC 191.8 and 162.6) in 5, instead of a methoxy signal [δC 56.0; δH 3.25 (3H, s)] as found in 11 (Table 2). The locations of these two carbonyl carbons were established at C-5 and C-6 based on the HMBC cross-peaks from H-3 to C-5 and from H-4 and H-7 to C-6. The NOESY cross-peak between H-2 and H-8a suggested that H-2 and C-8 are α-oriented (Figure 1). Likewise for the previous isolates, the absolute configuration of 5 was determined as 2S, 7S, 9S by observing the correlation of its experimental and simulated ECD spectra (Figure 3). Securingine F (6) was purified as a white gum with a molecular formula of C13H15NO4, as deduced by the positiveion peak [M + H]+ at m/z 250.1069 (calcd for C13H16NO4, 250.1079) in the HRESIMS. The NMR spectra of 6 were E



Article

the (S)- and (R)-MTPA esters, 7s and 7r, respectively. The assigned 1H NMR signals of the two MTPA esters were calculated as the Δδ values (δS − δR), and the results indicated a 7R configuration (Figure 4C). Furthermore, the similarity of the experimental and simulated ECD of 7 corroborated the structure as 7R,9S (Figure 3). Compound 8 was elucidated as 7a-(pyridin-2-yl)-7,7adihydrobenzofuran-2(6H)-one by analysis of its 2D NMR data and was reported previously as an intermediate product in a synthetic pathway without determination of the absolute configuration.11 In the present study, the full NMR assignments and the absolute configuration of 8 are reported (Table 2). The configuration of 8 was defined as 9S through the correlation of the experimental and calculated ECD spectra (Figure 3). The other isolated compounds were identified as secu’amamine D (9),8 (+)-phyllanthidine (10),12 securitinine (11),10 securinine (12),13 4-epiphyllanthine (13),10 and menisdaurilide (14)14 by NMR data comparison with the reported spectroscopic data in the references. The biosynthetic pathway of the Securinega alkaloids has been studied through isotope-labeling and degradation experiments, revealing that L-lysine and L-tyrosine are two precursors for securinine (12), the most abundant compound among the Securinega alkaloids.15−17 L-Lysine is transaminated, decarboxylated, and cyclized to form 1-piperideine, which is subsequently combined with 4-hydroxyphenylpyruvic acid originating from transamination of L-tyrosine.18 After a few reaction steps, securinine (12), possessing a securinane skeletone, is biosynthesized through the intermediate X1. The intermediate X1 would be oxidized to generate an intermediate X2 with a nitrone group, which has been proposed to be a key functionality for biosynthesis of the other Securinega alkaloids, virosaines A and B.19 Compound 1 would be formed through cyclization, reduction, and hydroxylation/methylation from the nitrone intermediate X2. A proposed biosynthetic route to 2 from securinine (12) may involve generation of another nitrone intermediate X3, which might be further rearranged20 and hydroxylated/methylated. The formation of the core structure of 6, 9, and 10 may be

Figure 4. Determination of relative and absolute configuration of 7. (A) 3D structures of the major conformers of 7a and 7b (top) and their Newman projections from C-7 to C-8 and the calculated coupling constants between H-7 and H-8a, H-8b (bottom). (B) DP4+ analysis result. (C) Δd values (dS− dR) in ppm of the two MTPA esters (7s and 7r).

Supporting Information). To determine the absolute configuration of C-7, the modified Mosher’s method was carried out, with the esterification of 7 with (R)- and (S)-MTPA-Cl giving

Figure 5. Hypothetical biosynthetic pathways of 1−14. F



Article

explained by one more rearrangement from the intermediate X4 formed from X3 via rearrangement.20 Compounds 2, 6, 9, and 10 also may be generated directly from the intermediate X2. When the A ring of the intermediate X1 is aromatized, 7 and 8 would be generated, depending on further dehydration and reduction. Menisdaurilide (14) is believed to be biosynthesized directly from 4-hydroxyphenylpyruvic acid.18 Compounds 1−14 were evaluated for their cytotoxicity against A549 (non-small-cell lung adenocarcinoma), SK-OV-3 (malignant ovarian ascites), SK-MEL-2 (skin melanoma), and HCT-15 (colon adenocarcinoma) cell lines using the sulforhodamine B (SRB) bioassay. Compound 4 showed cytotoxic activities against all four human cancer cell lines with IC50 values of 6.8, 3.4, 1.9, and 1.5 μM, while the positive control (etoposide) showed IC50 values of 0.7, 1.8, 0.3, and 0.3 μM, respectively. Interestingly, while compounds 3 and 4 showed overall structural similarity, only compound 4 displayed potent activities against all the tested lines. This suggests that the presence of the hydroxy group at C-2 may play a critical role in modulating cytotoxicity against the cancer cell lines used. The other compounds did not show any activity (IC50 > 10.0 μM). To test for anti-inflammatory activities, compounds 1−14 were investigated for their effects on nitric oxide (NO) production levels in lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells (Table 3). Compounds 3, 10,

Table 4. Effects of Selected Compounds on NGF Secretion in C6 Cells compound 3 5 12 6-shogaolc

IC50 (μM)a

cell viability (%)b

3 9 10 11 12 13 c L-NMMA

12.6 23.9 12.1 31.5 1.1 7.7 17.0

92.8 ± 7.67 86.5 ± 2.44 96.7 ± 7.88 88.7 ± 4.37 101.2 ± 7.65 107.6 ± 2.48 99.7 ± 2.18

145.3 172.7 141.1 138.7

± ± ± ±

1.04 1.24 4.88 7.43

cell viabilityb (%) 101.4 101.1 101.8 111.6

± ± ± ±

3.51 1.23 0.44 1.78

a C6 cells were treated with 20 μM of each compound. After 24 h, the content of NGF secreted into the C6-conditioned medium was measured by ELISA. The level of secreted NGF is expressed as the percentage of the untreated control (set as 100%). bThe cell viability after treatment with 20 μM of each compound was determined by an MTT assay and is expressed as a percentage (%). Results are the means of three independent experiments, and the data are expressed as means ± SD. cPositive control substance.

exhibiting values of 145.3 ± 1.0% and 141.1 ± 4.9%, respectively.

■

EXPERIMENTAL SECTION

General Experimental Procedures. The experimental procedures were conducted as described previously.21,22 Plant Material. The twigs of S. suff ruticosa were obtained in November 2013 from Goesan, Korea, and were authenticated by one of the authors (K.R.L.). A voucher specimen (SKKU-NPL 1420) of the plant is stored at the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea. Extraction and Isolation. Twigs of S. suff ruticosa (8.0 kg) were extracted three times with 80% aqueous MeOH at room temperature and filtered. The residue was concentrated in vacuo to obtain a crude methanol extract (480 g). This was then dissolved with deionized water and partitioned with different solvents of n-hexane, CHCl3, EtOAc, and n-BuOH to give extracts of 36, 16, 51, and 100 g, respectively. The CHCl3-soluble partition (16 g) was further fractionated by passage of a silica gel column (CHCl3−MeOH, 110:1 → 1:1), to produce eight main fractions (C1−C8). Fraction C1 (1.4 g) was subjected to a silica gel column chromatography (nhexane−acetone, 3.5:1 → 1:1) to give seven subfractions (C1A− C1G). Subfraction C1B (495 mg) was subjected to passage over an RP-C18 silica gel column, eluting with 55% aqueous MeOH, to acquire three subfractions (C1B1−C1B3). Compound 10 (5 mg, tR = 43.3 min) was obtained by purifying subfraction C1B2 (231 mg) using semipreparative HPLC (42% aqueous MeOH). Subfraction C1C (160 mg) was fractionated over a Lobar-A RP-18 column with a solvent system of 50% aqueous MeOH and further purified by semipreparative HPLC (40% aqueous MeOH), to yield compounds 2 (2 mg, tR = 42.1 min) and 8 (2 mg, tR = 28.3 min). Fraction C2 (580 mg) was fractionated over a silica gel column (n-hexane−acetone, 3.5:1 → 1:1) to give nine subfractions (C2A−C2I). Subfraction C2C (92 mg) was separated over a Lobar-A RP-18 column with a solvent system of 50% aqueous MeOH, followed by semipreparative HPLC (70% aqueous MeOH), to obtain compound 12 (27 mg, tR = 16.9 min). Subfraction C2E (111 mg) was purified by semipreparative HPLC (50% aqueous MeOH) to afford compound 13 (24 mg, tR = 16.1 min). Fraction C3 (2.2 g) was separated over an RP-C18 silica gel column (55% aqueous MeOH) to yield five subfractions (C3A− C3E). Subfraction C3A (1.2 g) was subjected to silica gel column chromatography (n-hexane−acetone, 3:1 → 1:1), which gave seven subfractions (C3A1−C3A7). Subfraction C3A1 (10 mg) was isolated by semipreparative HPLC (34% aqueous MeOH) to obtain compound 9 (2 mg, tR = 46.1 min). Subfraction C3A4 (20 mg) was purified by semipreparative HPLC (40% aqueous MeOH) to give compounds 1 (3 mg, tR = 31.5 min), 6 (4 mg, tR = 20.3 min), 7 (3 mg, tR = 19.0 min), and 14 (3 mg, tR = 11.5 min). Compounds 4 (4 mg, tR = 20.5 min) and 11 (43 mg, tR = 23.4 min) were acquired by purification of fractions C3A5 (20 mg) and C3A6 (762 mg), respectively, using semipreparative HPLC (40% aqueous MeOH).

Table 3. Inhibitory Effects of Selected Compounds on NO Production Induced by LPS in BV-2 Cells compound

NGF secretiona (%)

a The IC50 value of each compound was defined as the concentration (μM) that caused 50% inhibition of NO production in LPS-activated BV-2 cells. bThe cell viability after treatment with 20 μM of each compound was measured using the MTT assay and is expressed as a percentage (%). Results are the means of three independent experiments, and data are expressed as the means ± SD. cPositive control substance.

12, and 13 exhibited inhibitory effects on NO production with IC50 values of 12.6, 12.1, 1.1, and 7.7 μM, respectively, whereas compounds 9 and 11 displayed moderate effects (IC50 23.9 and 31.5 μM, respectively). The MTT cell viability test suggested that all compounds had no cytotoxic effect on BV-2 cell survival, except for compound 4, which demonstrated cytotoxicity at 20 μM. This implies that compound 4 may have cytotoxicity on BV-2 cells as well as the aforementioned four human cancer cell lines. Furthermore, compounds 1−14 were tested for their neuroprotection activity by measuring the secretion of nerve growth factor (NGF) from C6 cells into the medium (Table 4). Compound 5 stimulated NGF release with a stimulation level of 172.7 ± 4.9% (cf. 138.7 ± 7.4% for 6-shogaol, positive control) without cell toxicity at a concentration of 20 μM. Compounds 3 and 12 were also more potent than 6-shogaol, G



Article

7-(S)-MTPA ester (7s): 1H NMR (pyridine-d5, 700 MHz) δ 6.36 (H-7), 3.93 (H-8a), 2.33 (H-8b), 6.27 (H-12), 6.74 (H-14), 6.14 (H15). 7-(R)-MTPA ester (7r): 1H NMR (pyridine-d5, 700 MHz) δ 6.34 (H-7), 3.90 (H-8a), 2.18 (H-8b), 6.28 (H-12), 6.80 (H-14), 6.26 (H15). Computational Analysis. Conformational searches and subsequent geometry optimization were performed by the same method as described in a previously reported study.22 Excitation energies and rotatory strengths for ECD spectra (in MeOH solvent) and gaugeinvariant atomic orbital (GIAO) shielding constants for chemical shifts (in chloroform solvent) were calculated at the B3LYP/631+G(d,p) level for those conformers with more than 5% population based on their respective Boltzmann populations. ECD spectra were Boltzmann-weighted and generated using the SpecDis program.23 Chemical shifts were calculated from the GIAO shielding tensors via the equation below24 (δxcalc: calculated 1H/13C NMR chemical shift for nucleus x, σo: shielding tensor for the proton/carbon nucleus in tetramethylsilane, σx: shielding tensor for the proton/carbon nucleus x):

Subfraction C3A7 (34 mg) was purified by semipreparative HPLC (30% aqueous MeOH) to yield compounds 3 (4 mg, tR = 20.1 min) and 5 (3 mg, tR = 18.5 min). Securinigine A (1): colorless gum; [α]D25 +36.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 311 (sh), 258 (3.60), 211 (4.84) nm; ECD (MeOH) λmax (Δε) 247 (−0.62), 213 (+2.67) nm; IR (KBr) νmax 3703, 3375, 2972, 2868, 2356, 1659, 1537, 1340, 1055 cm−1; 1H (850 MHz) and 13C (212.5 MHz) NMR data in DMSO-d6 and 1H (700 MHz) and 13C (175 MHz) NMR data in chloroform-d, see Table 1; HRESIMS (positive-ion mode) m/z 282.1348 [M + H]+ (calcd for C14H20NO5, 282.1341). Securinigine B (2): colorless gum; [α]D25 −13.6 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 264 (sh), 213 (3.64) nm; ECD (MeOH) λmax (Δε) 238 (−5.51), 211 (+3.25) nm; IR (KBr) νmax 3692, 3334, 2946, 2835, 2074, 1658, 1456, 1021 cm−1; 1H (700 MHz) and 13C (175 MHz) NMR data in chloroform-d, see Table 1; HRESIMS (positive-ion mode) m/z 264.1237 [M + H]+ (calcd for C14H18NO4, 264.1236). Securinigine C (3): yellowish gum; [α]D25 −243.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 255 (2.37), 207 (3.39) nm; ECD (MeOH) λmax (Δε) 298 (−5.95), 212 (+3.11) nm; IR (KBr) νmax 3673, 3344, 2943, 2832, 1758, 1660, 1453, 1024 cm−1; 1H (700 MHz) and 13C (175 MHz) NMR data in chloroform-d, see Table 1; HRESIMS (positive-ion mode) m/z 232.0972 [M + H]+ (calcd for C13H14NO3, 232.0974). Securinigine D (4): white gum; [α]D25 −202.7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 309 (sh), 258 (3.73), 211 (4.66) nm; ECD (MeOH) λmax (Δε) 306 (−4.90), 211 (+3.21) nm; IR (KBr) νmax 3703, 3346, 2945, 2868, 2075, 1666, 1406, 1022 cm−1; 1H (700 MHz) and 13C (175 MHz) NMR data in chloroform-d and 1H (700 MHz) NMR data in DMSO-d6, see Table 1; HRESIMS (positive-ion mode) m/z 248.0921 [M + H]+ (calcd for C13H14NO4, 248.0923). Securinigine E (5): colorless gum; [α]D25 −166.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 321 (1.21), 262 (1.48), 206 (3.37) nm; ECD (MeOH) λmax (Δε) 351 (−5.55), 259 (+3.42), 225 (−0.49) nm; IR (KBr) νmax 3703, 3345, 2944, 2868, 2074, 1659, 1405, 1022 cm−1; 1H (700 MHz) and 13C (175 MHz) NMR data in chloroformd, see Table 2; HRESIMS (positive-ion mode) m/z 246.0770 [M + H]+ (calcd for C13H12NO4, 246.0766). Securinigine F (6): white gum; [α]D25 −167.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 256 (3.48), 207 (4.28) nm; ECD (MeOH) λmax (Δε) 275 (−6.94), 230 (−0.06), 210 (−0.77) nm; IR (KBr) νmax 3703, 3335, 2945, 2868, 2074, 1658, 1406, 1022 cm−1; 1H (700 MHz) and 13C (175 MHz) NMR data in chloroform-d, see Table 2; HRESIMS (positive-ion mode) m/z 250.1069 [M + H]+ (calcd for C13H16NO4, 250.1079). Securinigine G (7): colorless gum; [α]D25 −25.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 257 (3.75), 211 (4.04) nm; ECD (MeOH) λmax (Δε) 274 (−2.44), 219 (+1.82) nm; IR (KBr) νmax 3703, 3335, 2944, 2833, 2075, 1661, 1455, 1023 cm−1; 1H (700 MHz) and 13C (175 MHz) NMR data in chloroform-d, see Table 2; HRESIMS (positiveion mode) m/z 230.0816 [M + H]+ (calcd for C13H12NO3, 230.0817). 7a-(Pyridin-2-yl)-7,7a-dihydrobenzofuran-2(6H)-one (8): colorless gum; [α]D25 −216.6 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 258 (3.77), 205 (4.60) nm; ECD (MeOH) λmax (Δε) 282 (−7.03), 232 (+1.70), 214 (−0.57) nm; IR (KBr) νmax 3703, 3357, 2945, 2868, 1658, 1406, 1053, 1023 cm−1; 1H (700 MHz) and 13C (175 MHz) NMR data in chloroform-d, see Table 2; EIMS (positive-ion mode) m/z 282.1 [M + H]+. Preparation of Mosher Ester Derivatives of 1 and 7 (1s, 1r, 7s, and 7r). The modified Mosher esterification was conducted using the method described previously.21 1-(S)-MTPA ester (1s): 1H NMR (pyridine-d5, 700 MHz) δ 5.82 (H-7), 2.53 (H-8a), 2.43 (H-8b), 3.04 (H-12a), 2.54 (H-12b), 6.13 (H-14), 6.17 (H-15). 1-(R)-MTPA ester (1r): 1H NMR (pyridine-d5, 700 MHz) δ 5.80 (H-7), 2.62 (H-8a), 2.24 (H-8b), 3.20 (H-12a), 2.98 (H-12b), 6.20 (H-14), 6.22 (H-15).

x δcalc =

σo − σx 1 − σ o/106

The calculated 1H and 13C NMR chemical shifts were averaged as described above and used for calculations of DP4+ probability using an Excel sheet.9 The coupling constants were calculated from the major conformer using Macromodel (version 2016-2, Schrodinger LLC) software. Cytotoxicity Assessment, NO Production, Viability in LPSStressed BV-2 Cells, and NGF and Cell Viability Assays. The bioactivity assays for the isolates were conducted using methods described previously.22,25−27

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00142.

■

HRESIMS and NMR data of compounds 1−8 (PDF)

AUTHOR INFORMATION

Corresponding Author

*(K. R. Lee) Tel: +82-31-290-7710. Fax: +82-31-290-7730. Email: [email protected]. ORCID

Chung Sub Kim: 0000-0001-9961-4093 Kang Ro Lee: 0000-0002-3725-5192 Present Address #

Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States, and Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2016R1A2B2008380). We are grateful to the Korea Basic Science Institute (KBSI) for the mass spectrometric analysis. H



■

Article

REFERENCES

(1) Komlaga, G.; Genta-Jouve, G.; Cojean, S.; Dickson, R. A.; Mensah, M. L. K.; Loiseau, P. M.; Champy, P.; Beniddir, M. A. Tetrahedron Lett. 2017, 58, 3754−3756. (2) Zhang, H.; Zhu, K.-K.; Han, Y.-S.; Luo, C.; Wainberg, M. A.; Yue, J.-M. Org. Lett. 2015, 17, 6274−6277. (3) Wang, G.-C.; Wang, Y.; Zhang, X.-Q.; Li, Y.-L.; Yao, X.-S.; Ye, W.-C. Chem. Pharm. Bull. 2010, 58, 390−393. (4) Bardaji, G. G.; Canto, M.; Alibes, R.; Bayon, P.; Busque, F.; de March, P.; Figueredo, M.; Font, J. J. Org. Chem. 2008, 73, 7657−7662. (5) Raj, D.; Luczkiewicz, M. Fitoterapia 2008, 79, 419−427. (6) Raj, D.; Kokotkiewicz, A.; Luczkiewicz, M. Appl. Biochem. Biotechnol. 2015, 175, 1576−1587. (7) Yuan, W.; Lu, Z.; Liu, Y.; Meng, C.; Cheng, K.-D.; Zhu, P. Chem. Pharm. Bull. 2005, 53, 1610−1612. (8) Ohsaki, A.; Kobayashi, Y.; Yoneda, K.; Kishida, A.; Ishiyama, H. J. Nat. Prod. 2007, 70, 2003−2005. (9) Grimblat, N.; Zanardi, M. M.; Sarotti, A. M. J. Org. Chem. 2015, 80, 12526−12534. (10) Arbain, D.; Birkbeck, A. A.; Byrne, L. T.; Sargent, M. V.; Skelton, B. W.; White, A. H. J. Chem. Soc., Perkin Trans. 1 1991, 1863−1869. (11) Honda, T.; Namiki, H.; Kudoh, M.; Nagase, H.; Mizutani, H. Heterocycles 2003, 59, 169−187. (12) Moraes, L. S.; Donza, M. R. H.; Rodrigues, A. P. D.; Silva, B. J. M.; Brasil, D. S. B.; Zoghbi, M.D. G.; Andrade, E. H. A.; Guilhon, G. M. S. P.; Silva, E. O. Molecules 2015, 20, 22157−22169. (13) Alibes, R.; Ballbe, M.; Busque, F.; de March, P.; Elias, L.; Figueredo, M.; Font, J. Org. Lett. 2004, 6, 1813−1816. (14) Elo Manga, S. S.; Messanga, B. B.; Sondengam, B. L. Fitoterapia 2001, 72, 706−708. (15) Parry, R. J. Tetrahedron Lett. 1974, 4, 307−310. (16) Sankawa, U.; Yamasaki, K.; Ebizuka, Y. Tetrahedron Lett. 1974, 21, 1867−1868. (17) Sankawa, U.; Ebizuka, Y.; Yamasaki, K. Phytochemistry 1977, 16, 561−563. (18) Chirkin, E.; Atkatlian, W.; Porée, F.-H. In The Alkaloids; Knoölker, H.-J., Ed.; Academic Press: London, 2015; Chapter 1, pp 1−120. (19) Zhao, B.-X.; Wang, Y.; Zhang, D.-M.; Huang, X.-J.; Bai, L.-L.; Yan, Y.; Chen, J.-M.; Lu, T.-B.; Wang, Y.-T.; Zhang, Q.-W.; Ye, W.-C. Org. Lett. 2012, 14, 3096−3099. (20) Li, J.-Y.; Zhao, B.-X.; Zhang, W.; Li, C.; Huang, X.-J.; Wang, Y.; Sun, P.-H.; Ye, W.-C.; Chen, W.-M. Tetrahedron 2012, 68, 3972− 3979. (21) Kim, C. S.; Subedi, L.; Kim, S. Y.; Choi, S. U.; Kim, K. H.; Lee, K. R. J. Nat. Prod. 2016, 79, 387−394. (22) Kim, C. S.; Oh, J.; Subedi, L.; Kim, S. Y.; Choi, S. U.; Lee, K. R. J. Nat. Prod. 2018, 81, 1795−1802. (23) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Pescitelli, G. SpecDis, Version 1.70; Berlin, Germany, 2017. (24) Kim, C. S.; Oh, J.; Subedi, L.; Kim, S. Y.; Choi, S. U.; Lee, K. R. Sci. Rep. 2017, 7, 43646. (25) Kim, C. S.; Bae, M.; Oh, J.; Subedi, L.; Suh, W. S.; Choi, S. Z.; Son, M. W.; Kim, S. Y.; Choi, S. U.; Oh, D.-C.; Lee, K. R. J. Nat. Prod. 2017, 80, 471−478. (26) Kim, C. S.; Subedi, L.; Oh, J.; Kim, S. Y.; Choi, S. U.; Lee, K. R. J. Nat. Prod. 2017, 80, 1134−1140. (27) Suh, W. S.; Kim, C. S.; Subedi, L.; Kim, S. Y.; Choi, S. U.; Lee, K. R. J. Nat. Prod. 2017, 80, 2502−2508.

I


Securinega Alkaloids from the Twigs of Securinega suffruticosa and

Recommend Documents