Nitric Oxide Inhibitory Activity and Absolute Configurations of

Jan 7, 2016 - ... Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, People's Republic of China...
1 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/jnp

Nitric Oxide Inhibitory Activity and Absolute Configurations of Arylalkenyl α,β-Unsaturated δ/γ-Lactones from Cryptocarya concinna Bing-Yuan Yang, Ling-Yi Kong,* Xiao-Bing Wang, Yang-Mei Zhang, Rui-Jun Li, Ming-Hua Yang, and Jian-Guang Luo* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: During an ongoing exploration of potential anti-inflammatory agents from medicinal plants, eight new arylalkenyl α,β-unsaturated δ-lactones, cryptoconcatones A−H (1−8), and two unusual arylalkenyl α,β-unsaturated γ-lactones, cryptoconcatones I and J (9 and 10), were identified from the leaves and twigs of Cryptocarya concinna. The structures of these compounds were established based on spectroscopic data (MS, 1D/2D NMR), and their absolute configurations were determined with Riguera’s method, the modified Mosher’s method, chemical derivatization, and the Snatzke chirality rule. Compounds 4−6 and 8−10 showed inhibitory activity toward nitric oxide (NO) production in lipopolysaccharide-induced RAW 264.7 macrophages, particularly compounds 4 and 8−10, with IC50 values of 3.2, 4.2, 3.4, and 7.5 μM, respectively.

T

he genus Cryptocarya (Lauraceae) comprises more than 220 species that are widely distributed in the subtropics. Phytochemical investigations of this genus have led to the isolation of various secondary metabolites, such as α-pyrone derivatives,1,2 flavonoids,3,4 and alkaloids.5,6 Cryptocarya concinna Hance is a typical monsoon evergreen broad-leaved tree distributed in lower subtropical mainland China.7 Recently, a chemical investigation of C. concinna was performed on its stems, resulting in the discovery of a series of cytotoxic and antimicrobial flavonoids.8 The extract of the leaves and branches of C. concinna was reported to exhibit potent antiinflammatory activity;9 however, studies are lacking on its chemical constituents, which may pose an obstacle to further developing and utilizing this medicinal plant. During an ongoing search for new anti-inflammatory agents from medicinal plants in China,10−12 we investigated the leaves and twigs of C. concinna, which led to the isolation of 10 new arylalkenyl α,β-unsaturated δ/γ-lactones (1−10). The structures of these lactones were elucidated through physical data (HRESIMS and NMR experiments) and chemical derivatization. The isolated compounds were evaluated for their potential anti-inflammatory activity in the NO production of LPSstimulated RAW 264.7 macrophages, and the results obtained are discussed herein.

C21H26O6Na, 397.1622). The IR absorption bands at 3451 and 1642 cm−1 implied the presence of a hydroxy functional group and a conjugated carbonyl group, respectively. The 1H NMR spectrum (Table 1) displayed a monosubstituted phenyl group [δH 7.40 (2H, d, J = 7.4 Hz), 7.29 (2H, t, J = 7.4 Hz), and 7.20 (1H, t, J = 7.4 Hz)], two trans-olefinic protons at δH 6.59 (1H, d, J = 15.9 Hz) and 6.27 (1H, dd, J = 15.9, 6.2 Hz), two cisolefinic protons at δH 7.01 (1H, m) and 5.96 (1H, dt, J = 9.7, 1.2 Hz), four oxymethine groups [δH 5.33 (1H, m), 4.55 (1H, m), 4.47 (1H, m), and 3.83 (1H, m)], a methylene group [δH 2.40 (2H, m)], an acetoxy group [δH 2.02 (3H, s)], and six aliphatic proton signals between δH 1.68 and 2.07. The 13C NMR spectrum, with the aid of the HSQC experiment, revealed 21 carbon signals, ascribed to two ester carbonyls (δC 173.1 and 166.6), eight aromatic/olefinic carbons (δC 148.3, 138.6, 134.2, 130.7, 129.7, 129.7, 128.6, 127.6, 127.6, and 121.5), four oxymethines (δC 76.7, 70.3, 69.8, and 65.8), four methylenes (δC 46.2, 44.1, 41.2, and 30.6), and one acetoxy methyl carbon (δC 21.2). The aforementioned data were similar to those obtained for the known compound cryptomoscatone E3, which was previously isolated from the branches and stem bark of C. moschata.13 The difference between 1 and cryptomoscatone E3 was the presence of a 2′-O-acetyl rather than a 2′-hydroxy group, which was determined by the HMBC correlations from H-2′ (δH 5.33) to the acetoxy carbonyl (δC 173.1), C-6 (δC 76.7), and C-4′ (δC 65.8). The assignment of

■

RESULTS AND DISCUSSION Cryptoconcatone A (1) was isolated as a yellowish gum. The molecular formula was determined to be C21H26O6 by 13C NMR and HRESIMS data (m/z 397.1624 [M + Na]+, calcd for © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 19, 2015

A

DOI: 10.1021/acs.jnatprod.5b00839 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H NMR Data (500 MHz) of Compounds 1−5 (δ in ppm, J in Hz) position 3 4 5 6 1′a 1′b 2′ 3′a 3′b 4′ 5′a 5′b 6′ 7′ 8′ 2″, 6″ 3″, 5″ 4″ 2′-OC(O)CH3 4′-OC(O)CH3 a

1a

2a

3a

4a

5a

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

5.96, 7.01, 2.40, 4.55, 2.07, 1.96, 5.33, 1.80, 1.70, 3.83, 1.68, 1.68, 4.47, 6.27, 6.59, 7.40, 7.29, 7.20, 2.02,

dt (9.7, 1.2) m m m m m m m m m m m m dd (15.9, 6.2) d (15.9) d (7.4) t (7.4) t (7.4) s

5.99, 7.05, 2.39, 4.72, 1.96, 1.66, 3.94, 1.75, 1.75, 5.33, 1.89, 1.89, 4.30, 6.27, 6.60, 7.42, 7.31, 7.23,

dt (9.7, 1.5) ddd (9.7, 5.8, 2.5) m m m m m m m m m m m dd (15.9, 6.6) d (15.9) d (7.4) t (7.4) t (7.4)

5.98, 7.03, 2.40, 4.53, 2.04, 1.82, 5.23, 1.96, 1.96, 5.18, 1.93, 1.87, 4.25, 6.24, 6.60, 7.42, 7.32, 7.23, 1.99, 2.01,

2.04, s

dt (9.7, 1.2) m m m m m m m m m m m m dd (15.9, 6.8) d (15.9) d (7.5) t (7.5) t (7.5) s s

6.00, 7.06, 2.43, 4.75, 1.93, 1.70, 4.18, 1.61, 1.61, 4.18, 1.74, 1.74, 4.52, 6.31, 6.63, 7.41, 7.31, 7.22,

dd (9.8, 1.8) m m m m m m m m m m m m dd (15.9, 6.2) d (15.9) d (7.4) t (7.4) t (7.4)

6.11, 7.10, 4.09, 4.69, 2.10, 1.77, 4.22, 1.66, 1.66, 4.22, 1.77, 1.77, 4.55, 6.34, 6.64, 7.43, 7.33, 7.24,

d (9.7) dd (9.7. 5.8) dd (5.8, 2.4) dt (10.2, 2.4) m m m m m m m m m dd (15.9, 6.2) d (15.9) d (7.5) t (7.5) t (7.5)

Measured in methanol-d4.

Riguera’s method,17−20 and chemical derivatization. First, the ECD spectra of 1 and 2 (Figure S23, Supporting Information) displayed a positive Cotton effect at 250−272 nm (n−π* transition in the α,β-unsaturated-δ-lactone moiety), indicating that the absolute configurations at C-6 of 1 and 2 were R according to the Snatzke chirality rule.14−16 Subsequently, bis(R)- and bis(S)-MTPA esters (1a and 1b; Figure 2) of 1 were prepared by reacting with (S)-(+)- and (R)-(−)-αmethoxy-α-trifluoromethylphenylacetyl chloride (MTPA-Cl), respectively. After the proton chemical shifts of 1a and 1b were assigned based on 1H NMR and 1H−1H COSY data, the calculation of the ΔδH(S−R) values for 1a and 1b according to the model of anti-1,n Type B (n odd) established the absolute configurations of 4′S and 6′S of 1, respectively (Figure 2). Similarly, for derivatized bis(R)-(−)- and bis(S)-(+)-MTPA esters (2a and 2b) of 2, the ΔδH(S−R) values of H-1′ and H-2′ (Δδ = −0.24, −0.28, and −0.03) were negative, whereas those of H-6′ (Δδ = +0.10) and H-7′ (Δδ = +0.19) were positive (Figure 2), a difference that unequivocally demonstrated that 2 possesses 2′S and 6′S configurations according to the syn-1,n Type D (n odd) model of Riguera’s method. Finally, to establish the absolute configurations of C-2′ in 1 and C-4′ in 2, the hydroxy groups in 1 and 2 were acetylated using Ac2O in pyridine (Scheme 1). The products 1c and 2c had the same retention times in HPLC analysis, identical 1H NMR data (Figures S70 and 71, Supporting Information), and similar Cotton effects in the ECD spectra (Figure S77, Supporting Information). These results suggested that 2 possessed atom arrangements of its stereogenic centers identical to those of 1. Thus, the absolute configurations of 1 and 2 were established as 6R, 2′R, 4′S, 6′S and 6R, 2′S, 4′R, 6′S, respectively. Cryptoconcatone C (3), a yellowish gum, possessed a molecular formula of C23H28O7 according to the 13C NMR and HRESIMS data at m/z 439.1731 [M + Na]+ (calcd for C23H28O7Na, 439.1727). The 1H and 13C NMR data (Tables 1 and 3) were similar to those of 1. However, two acetoxy groups (δH 2.01 and 1.99; δC 172.9, 172.7, 21.3, and 21.2) were

all carbon and proton signals of 1 was confirmed based on a detailed analysis of the 1D and 2D NMR spectra, and the 2D structure of 1 was elucidated as shown in Figure 1.

Figure 1. Chemical structures of compounds 1−10.

Cryptoconcatone B (2) was also purified as a yellowish gum and yielded the same molecular formula as 1 in accordance with its 13C NMR data and HRESIMS spectrum (m/z 397.1625 [M + Na]+, calcd for C21H26O6Na, 397.1622). The 1H and 13C NMR data (Tables 1 and 3) of 2 closely resembled those of 1, suggesting that 2 was also an acetylated derivative of 6-[ωarylalkenyl]-5,6-dihydro-α-pyrone and, hence, an isomer of 1. The HMBC correlations from H-4′ (δH 5.33) to the acetoxy carbonyl (δC 173.4), C-2′ (δC 64.6), and C-6′ (δC 70.4) indicated that the acetoxy group of 2 was located at C-4′ (δC 70.5). Cryptoconcatones A (1) and B (2) consist of three structural units: an α,β-unsaturated δ-lactone, a phenyl ring, and a 2,4,6trisubstituted alkyl chain. The determination of the absolute configuration of the three stereogenic carbon atoms in the 2,4,6-trisubstituted alkyl chain poses great challenges. In this paper, the absolute configurations of 1 and 2 were defined through a combination of the Snatzke chirality rule,14−16 B

DOI: 10.1021/acs.jnatprod.5b00839 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 2. 1H NMR Data (500 MHz) of Compounds 6−10 (δ in ppm, J in Hz) position 3 4 5 6 1′a 1′b 2′ 3′a 3′b 4′ 5′a 5′b 6′ 7′ 8′ 2″, 6″ 3″, 5″ 4″ 2′-OC(O)CH3 4′-OC(O)CH3 a

6a

7a

8b

9a

10a

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

6.08, 7.06, 4.06, 4.50, 2.23, 1.93, 5.39, 1.82, 1.76, 3.93, 1.70, 1.70, 4.48, 6.29, 6.62, 7.42, 7.31, 7.22, 2.04,

d (9.7) dd (9.7, 5.8) dd (5.8, 2.7) dt (10.0, 2.7) m m m m m m m m m dd (15.9, 6.4) d (15.9) d (7.5) t (7.5) t (7.5) s

6.09, 7.08, 4.05, 4.64, 2.08, 1.71, 3.95, 1.75, 1.82, 5.35, 1.94, 1.87, 4.30, 6.27, 6.61, 7.42, 7.32, 7.23,

d (9.7) dd (9.7, 5.8) dd (5.8, 2.6) dt (10.2, 2.6) m m m m m m m m m dd (15.9, 6.6) d (15.9) d (7.4) t (7.4) t (7.4)

6.04, 6.89, 2.36, 4.79, 1.87, 1.87, 4.13, 1.30, 1.93, 4.03, 2.15, 1.76, 4.79, 6.21, 6.59, 7.42, 7.32, 7.23,

dd (9.7, 1.4) ddd (9.7, 5.7, 2.7) m m m m m dd (22.5, 10.5) m m m m m dd (16.4, 4.2) d (16.4) d (7.4) t (7.4) t (7.4)

6.26, d (5.5) 7.64, d (5.5)

6.24, d (5.5) 7.63, d (5.5)

5.49, 2.72, 2.72, 5.26, 1.82, 1.82, 3.91, 1.68, 1.68, 4.47, 6.30, 6.61, 7.41, 7.31, 7.22, 2.01,

5.55, 2.56, 2.56, 3.79, 1.85, 1.85, 5.32, 1.75, 1.75, 4.28, 6.27, 6.59, 7.41, 7.31, 7.23,

t (8.0) m m m m m m m m m dd (15.9, 6.6) d (15.9) d (7.5) t (7.5) t (7.5) s

2.05, s

t (7.7) m m m m m m m m m dd (15.9, 6.7) d (15.9) d (7.5) t (7.5) t (7.5)

2.02, s

b

Measured in methanol-d4. Measured in CDCl3.

Table 3. 13C NMR Data (125 MHz) of Compounds 1−10 (δ in ppm)

a

position

1a

2a

3a

4a

5a

6a

7a

8b

9a

10a

2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″ 2″, 6″ 3″, 5″ 4″ 2′-OC(O)CH3 2′-OC(O)CH3 4′-OC(O)CH3 4′-OC(O)CH3

166.6 121.5 148.3 30.6 76.7 41.2 69.8 44.1 65.8 46.2 70.3 134.2 130.7 138.6 127.6 129.7 128.6 173.1 21.2

167.0 121.5 148.6 31.0 76.8 44.0 64.6 44.5 70.5 43.8 70.4 133.6 131.2 138.6 127.6 129.7 128.7

166.5 121.5 148.2 30.6 76.6 41.2 68.4 40.9 69.3 43.5 70.5 133.4 131.5 138.5 127.6 129.7 128.8 172.9 21.3 172.7 21.2

167.1 121.6 148.6 31.0 77.0 44.4 65.3 46.7 66.4 46.4 70.5 134.3 130.7 138.7 127.6 129.7 128.6

166.2 122.8 147.0 63.3 79.4 39.7 65.1 46.7 66.3 46.2 70.3 134.1 130.5 138.5 127.4 129.5 128.4

165.9 122.9 147.0 63.2 79.3 37.1 70.1 44.3 65.8 46.2 70.3 134.2 130.8 138.6 127.6 129.7 128.6 173.2 21.2

166.3 122.9 147.2 63.4 79.4 39.6 64.6 44.6 70.6 43.8 70.5 133.5 131.2 138.5 127.6 129.7 128.7

164.4 121.8 145.2 30.3 74.8 41.9 65.6 41.8 64.6 38.7 72.7 129.0 131.8 136.8 126.8 128.8 127.9

171.9 120.4 145.7 153.0 112.9 32.8 71.8 43.1 65.7 46.3 70.3 134.1 130.8 138.6 127.6 129.7 128.6 172.8 21.3

172.1 120.1 145.8 152.6 114.6 35.9 68.3 43.8 70.7 43.6 70.5 133.5 131.3 138.5 127.6 129.7 128.7

173.4 21.3

173.4 21.3

173.3 21.3

Measured in methanol-d4. bMeasured in CDCl3.

observed, indicating that 3 was an O-acetyl analogue of 1. The HMBC correlations from H-2′ (δH 5.23) to the acetoxy carbonyl (δC 172.7), C-6 (δC 76.6), and C-4′ (δC 69.3) and from H-4′ (δH 5.18) to the acetoxy carbonyl (δC 172.9), C-3′ (δC 40.9), and C-6′ (δC 70.5) showed that the O-acetyl groups were present at C-2′ and C-4′, respectively. Furthermore, acetylation of 3 yielded product 3c (Scheme 1), whose HPLC retention time, 1H NMR data (Figure S72, Supporting Information), and ECD spectrum (Figure S77, Supporting Information) were in accordance with those of 1c. Thus, the

absolute configuration of 3 was established as 6R, 2′S, 4′S, 6′S, as shown in Figure 1. Cryptoconcatone D (4) was also obtained as a yellowish gum. Its molecular formula C19H24O5 was determined by the 13 C NMR and HRESIMS data at m/z 355.1518 [M + Na]+ (calcd for C19H24O5Na, 355.1516). The 13C NMR data in CDCl3 of 4 were very similar to those of cryptomoscatone E3, the differences being the chemical shifts of C-2′, C-4′, and C-6′ [C-2′ (δC 65.3), C-4′ (δC 67.0), C-6′ (δC 70.9) in 4, in contrast to C-2′ (δC 64.5), C-4′ (δC 70.2), C-6′ (δC 73.8) in C

DOI: 10.1021/acs.jnatprod.5b00839 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 2. Models of Riguera’s method and the ΔδH(S−R) values (ppm) obtained for the bis-MTPA esters (1a, 1b, 2a, 2b, 10a, and 10b).

Scheme 1. Formation of 1c−4c from Compounds 1−4

deduced to exhibit an anti/anti relative configuration (Figure 3), in contrast to that of cryptomoscatone E3 (C-2′/C-4′ and C-4′/C-6′ with an anti/syn relative configuration, shown as C2′S*, C-4′R*, and C-6′R* in ref 13). Similar to 1−3, the absolute configuration of C-6 in 4 was deduced to be R due to the positive ECD Cotton effect (250−272 nm). Finally, the absolute configuration of 4 was determined to be 6R, 2′R, 4′R, 6′S via the acetylation product (4c), which was proven to be identical to 1c by a comparison of the HPLC retention times, 1 H NMR, and ECD spectra (Figures S73 and 77, Supporting Information). The molecular formula of cryptoconcatone E (5) was established as C19H24O6, whereas cryptoconcatones F (6) and G (7) shared the same molecular formula, C21H26O7, as determined from 13C NMR and HRESIMS data. The 1H and 13 C NMR data for 5, 6, and 7 (Tables 2 and 3) suggested that these compounds were oxygenated derivatives of 4, 1, and 2, respectively. In the HMBC spectra of 5−7, the presence of the same correlations from H-5 to C-3, C-4, C-6, and C-1′ placed an additional hydroxy group at C-5 for each of these compounds. Moreover, the syn relationship of H-5/H-6 deduced from the 3J-coupling constant (J = 2.4−2.7 Hz) (Table 2), combined with the ECD spectra of 5−7 (Figure S44, Supporting Information) exhibiting a positive Cotton effect (250−272 nm), defined the (5S, 6S) absolute configuration.16,23 In the 13C NMR data of 1, 2, and 4 (Table 3), the chemical shifts of the γ-carbon (C-4′ for 1; C-2′ and C-6′ for 2) were slightly shielded by 0.1−0.7 ppm when the anti-1,3-diol was monoacetylated (1, 2 vs 4).24 Therefore, Kishi’s method could still be applied to such a monoacetylated anti-1,3-diol

cryptomoscatone E3], suggesting that 4 and cryptomoscatone E3 were diastereoisomers. The relative configurations of C-2′, C-4′, and C-6′ in 4 were defined with Kishi’s method.21,22 Kishi’s method states that a distinctive chemical shift of the central carbon is dependent on the 1,3- and 3,5- relative configuration and independent of the functionalities present outside of the 1,3,5-triol segment and can be used to determine the 1,3- and 3,5- relative configurations. Given the database (database in methanol-d4), the expected chemical shifts of C-3 in a 1,3,5-triol system are within the scope of δC 66.3 ± 0.5 for an anti/anti relative configuration, δC 68.5 ± 0.5 for an anti/syn or syn/anti configuration, and δC 70.7 ± 0.5 for a syn/syn configuration (Figure 3). Because the chemical shift of C-4′ in 4 was at δC 66.4 (data in methanol-d4), C-2′/C-4′ and C-4′/C-6′ were

Figure 3. 13C NMR chemical shift (ppm; measured in methanol-d4) of Kishi’s method and the relative configuration of C-4′/C-2′ and C-4′/ C-6′ in compound 4. D

DOI: 10.1021/acs.jnatprod.5b00839 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

system. According to Kishi’s method, the central carbon (C-4′) at δC 66.3 and 65.8 (Table 3) in 5 and 6, respectively, also suggests that both 5 and 6 had anti/anti configurations for C2′/ C-4′ and C4′/C-6′. A comparison of the NMR spectra of the 2′,4′,6′-trisubstituted alkyl chain in 5, 6, and 7 with those of 4, 1, and 2, respectively, showed similar 1H and 13C NMR data, particularly for C-2′, C-4′, and C-6′. Therefore, in terms of structural homology, the absolute configurations of 5−7 were defined as (5S, 6S, 2′R, 4′R, 6′S), (5S, 6S, 2′R, 4′S, 6′S), and (5S, 6S, 2′R, 4′R, 6′S), respectively. Cryptoconcatone H (8) was isolated as a yellowish gum and assigned a molecular formula of C19H22O4 by 13C NMR data and HRESIMS, showing a sodium adduct ion at m/z 337.1412 [M + Na]+, indicating nine indices of hydrogen deficiency. Analysis of the 1H and 13C NMR data (Tables 2 and 3) confirmed the presence of an α,β-unsaturated-δ-lactone and a styryl functional group. An additional ring was proposed for 8, as eight indices of hydrogen deficiency were for an α,βunsaturated-δ-lactone and a styryl group. The HMBC correlations from H-6′ to C-2′, H-5′ to C-3′, H-4′ to C-2′, and H-3′ to C-1′ suggested the presence of a tetrahydropyran ring spanning C-2′ to C-6′. In addition, the HMBC correlations of H-4′ to C-2′, H-3′ to C-4′, and H-5′ to C-4′ confirmed that a hydroxy group was located at C-4′. The absolute configuration of C-6 was determined to be R because the ECD spectrum displayed a positive Cotton effect at 250−272 nm (Figure S44, Supporting Information). Furthermore, the modified Mosher’s method25 was successfully applied to determine a 4′S configuration as shown in Figure 4. Thus, the absolute configurations of C-2′ and C-6′ were deduced to be 2′S and 6′R, respectively, based on the ROESY correlations of H-2′/H-4′ and H-4′/H-6′.

Figure 5. Key HMBC and ROESY correlations for compound 9.

C-2′ (δC 68.3), C-3′ (δC 43.8), and C-5′ (δC 43.6) indicated that the acetoxy group was located at C-4′ in 10. The Δ5,6 double bond possessed Z-geometry, as indicated by the crosspeaks of H-4/H-6 in the ROESY spectrum. The absolute configurations of the C-2′ and C-6′ stereogenic carbons in 10 were determined with Riguera’s method. The ΔδH(S−R) values of the derivatized bis(S)- and bis(R)-MTPA esters (10a and 10b) of 10 were negative at H-1′ and H-2′ and positive at H-6′ and H-7′ (Figure 2). Thus, the configuration of 10 was determined to be 2′S and 6′S. Similar to compound 1, the chemical shift of C-4′ (δC 65.7) in 9 indicated that C-2′/C4′ and C-4′/C-6′ had an anti/anti relative configuration. Furthermore, the acetylation products (9c and 10c) of 9 and 10 were identical (Figures S75, 76, and 78, Supporting Information); therefore, the absolute configurations of 9 and 10 were determined to be 2′S, 4′S, 6′S and 2′S, 4′R, 6′S, respectively. Strikingly, cryptoconcatones I and J (9 and 10) represent rare examples of arylalkenyl α,β-unsaturated γlactones in nature. Because the extract from the leaves and branches of C. concinna has been reported to exhibit potent anti-inflammatory activity,9 all isolated compounds were tested for their inhibition of NO production induced by LPS in RAW 264.7 macrophages. Cell viability was determined initially with the MTT method to discern whether the inhibition of NO production resulted from the cytotoxicity of the tested compounds. As a result, no cytotoxic effects (over 90% cell survival) of compounds 1−6 and 8−10 at concentrations up to 100 μM on RAW 264.7 cells were observed, while compound 7 exhibited cytotoxicity (less than 15% cell survival) at a concentration of 50 μM. Thus, compounds 1−6 and 8−10 were selected to further assay their NO inhibition of LPS-activated RAW 264.7 cells with the compound N-monomethyl-L-arginine as the positive control (IC50 = 45.0 ± 3.7 μM). As shown in Table 4, compounds 9 and 10 showed significant activity, with IC50 values of 3.4 ± 0.5 and 7.5 ± 1.3 μM, respectively. However, their close analogues, 1 and 2, were inactive (IC50 > 50 μM), indicating that the α,βunsaturated-γ-lactone ring plays a more important role than does the α,β-unsaturated-δ-lactone ring in the NO inhibitory

Figure 4. ΔδH(S−R) values (ppm) obtained for the MTPA esters (8a and 8b).

Cryptoconcatone I (9) was assigned the molecular formula C21H24O6 according to the 13C NMR and HRESIMS data (m/z 395.1464 [M + Na]+, calcd for C21H24O6Na, 395.1465). In addition to having arylalkenyl signals identical to those of 1, the 1 H and 13C NMR data of 9 (Tables 2 and 3) displayed signals of three olefinic protons at δH 5.49 (1H, t, J = 8.0 Hz), 7.64 (1H, d, J = 5.5 Hz), and 6.26 (1H, d, J = 5.5 Hz); four olefinic carbons at δC 145.7, 153.0, 120.4, and 112.9; and two ester carbonyls at δC 171.9 and 172.8. The HMBC correlations from H-3 (δH 6.26) to C-2 (δC 171.9) and C-5 (δC 153.0) and from H-4 (δH 7.64) to C-3 (δC 120.4) and C-5 (δC 153.0) indicated the presence of an α,β-unsaturated-γ-lactone ring. Furthermore, the Δ5,6 conjugated double bond was supported by the HMBC correlations from H-6 (δH 5.49) to C-4 (δC 145.7) and C-2′ (δC 71.8). The ROESY cross-peaks of H-4/H-6 suggested the Z-geometry of the Δ5,6 double bond. Cryptoconcatone J (10) possessed the same molecular formula as compound 9. The only difference between 9 and 10 was the position of the acetoxy group. The HMBC correlations from H-4′ (δH 5.32) to the acetoxy carbonyl carbon (δC 173.3),

Table 4. Inhibition of Compounds 1−10 of the NO Production in LPS-Activated RAW 264.7 Cells compound

IC50 ± SD (μM)b

compound

1 2 3 4 5

>50 >50 >50 3.2 ± 0.2 13.8 ± 2.1

6 8 9 10 a L-NMMA

IC50 ± SD (μM)b 27.6 4.2 3.4 7.5 45.0

± ± ± ± ±

1.3 0.3 0.5 1.3 3.7

a N-Monomethyl-L-arginine was used as a positive control. bValues are represented as the means ± SD based on three independent experiments.

E

DOI: 10.1021/acs.jnatprod.5b00839 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Cryptoconcatone A (1): yellowish gum; [α]25 D +9 (c 0.1, MeOH); UV λmax (log ε) 210 (4.21), 251 (4.16), 282 (3.17), 291 (3.01) nm; ECD (c 2.2 × 10−4 g mL−1, MeOH) λ (Δε) 200 (+9.84), 234 (+2.13), 254 (+3.77) nm; IR (KBr) νmax 3451, 1714, 1642, 1384, 1257, 1050, 817, 752, 697 cm−1; 1H NMR and 13C NMR spectroscopic data (see Tables 1 and 3); HRESIMS m/z 397.1642 [M + Na]+ (calcd for C21H26NaO6, 397.1622). Cryptoconcatone B (2): yellowish gum; [α]25 D +28 (c 0.1, MeOH); UV λmax (log ε) 213 (4.08), 250 (4.09), 282 (3.14), 291 (3.01) nm; ECD (c 2.4 × 10−4 g mL−1, MeOH) λ (Δε) 200 (+11.12), 231 (+2.81), 251 (+4.74) nm; IR (KBr) νmax 3448, 1712, 1642, 1384, 1259, 1055, 812, 752, 697 cm−1; 1H NMR and 13C NMR spectroscopic data (see Tables 1 and 3); HRESIMS m/z 397.1625 [M + Na]+ (calcd for C21H26NaO6, 397.1622). Cryptoconcatone C (3): yellowish gum; [α]25 D +25 (c 0.1, MeOH); UV λmax (log ε) 209 (4.19), 250 (4.05), 282 (3.19), 291 (2.91) nm; ECD (c 2.5 × 10−4 g mL−1, MeOH) λ (Δε) 200 (+10.93), 233 (+2.64), 253 (+5.69) nm; IR (KBr) νmax 3450, 1723, 1639, 1384, 1255, 1048, 813, 752, 697 cm−1; 1H NMR and 13C NMR spectroscopic data (see Tables 1 and 3); HRESIMS m/z 439.1713 [M + Na]+ (calcd for C23H28NaO7, 439.1727). Cryptoconcatone D (4): yellowish gum; [α]25 D +12 (c 0.1, MeOH); UV λmax (log ε) 210 (4.22), 250 (4.10), 282 (3.15), 291 (3.00) nm; ECD (c 2.5 × 10−4 g mL−1, MeOH) λ (Δε) 200 (+11.73), 235 (+1.84), 254 (+3.16) nm; IR (KBr) νmax 3449, 1720, 1642, 1384, 1258, 1050, 818, 752, 697 cm−1; 1H NMR and 13C NMR spectroscopic data (see Tables 1 and 3); HRESIMS m/z 355.1518 [M + Na]+ (calcd for C19H24NaO5, 355.1516). Cryptoconcatone E (5): yellowish gum; [α]25 D +18 (c 0.1, MeOH); UV λmax (log ε) 210 (4.11), 250 (4.09), 282 (3.08), 291 (2.94) nm; ECD (c 2.3 × 10−4 g mL−1, MeOH) λ (Δε) 200 (+5.47), 256 (+1.02) nm; IR (KBr) νmax 3443, 2919, 1705, 1638, 1384, 1269, 1070, 831, 752, 695 cm−1; 1H NMR and 13C NMR spectroscopic data (see Tables 2 and 3); HRESIMS m/z 371.1467 [M + Na]+ (calcd for C19H24NaO6, 371.1465). Cryptoconcatone F (6): yellowish gum; [α]25 D +24 (c 0.1, MeOH); UV λmax (log ε) 210 (4.01), 250 (4.08), 282 (3.05), 291 (2.89) nm; ECD (c 2.2 × 10−4 g mL−1, MeOH) λ (Δε) 200 (+6.07), 253 (+0.78) nm; IR (KBr) νmax 3444, 2921, 1716, 1638, 1384, 1264, 1053, 831, 752, 696 cm−1; 1H NMR and 13C NMR spectroscopic data (see Tables 2 and 3); HRESIMS m/z 413.1574 [M + Na]+ (calcd for C21H26NaO7, 413.1571). Cryptoconcatone G (7): yellowish gum; [α]25 D +32 (c 0.1, MeOH); UV λmax (log ε) 211 (4.14), 250 (4.05), 282 (3.08), 291 (2.91) nm; ECD (c 2.2 × 10−4 g mL−1, MeOH) λ (Δε) 200 (+3.58), 254 (+1.17) nm; IR (KBr) νmax 3444, 2918, 1712, 1639, 1384, 1264, 1055, 831, 752, 696 cm−1; 1H NMR and 13C NMR spectroscopic data (see Tables 2 and 3); HRESIMS m/z 413.1572 [M + Na]+ (calcd for C21H26NaO7, 413.1571). Cryptoconcatone H (8): yellowish gum; [α]25 D −24 (c 0.1, MeOH); UV λmax (log ε) 209 (3.82), 253 (3.58), 284 (3.58), 292 (3.57) nm; ECD (c 2.3 × 10−4 g mL−1, MeOH) λ (Δε) 200 (+5.29), 235 (+1.10), 258 (+1.17) nm; IR (KBr) νmax 3453, 1708, 1641, 1384, 1262, 1111, 817, 701 cm−1; 1H NMR and 13C NMR spectroscopic data (see Tables 2 and 3); HRESIMS m/z 337.1412 [M + Na]+ (calcd for C19H22NaO4, 337.141). Cryptoconcatone I (9): yellowish gum; [α]25 D −5 (c 0.1, MeOH); UV λmax (log ε) 208 (4.21), 224 (3.68), 253 (4.08) nm; ECD (c 2.0 × 10−4 g mL−1, MeOH) λ (Δε) 200 (+1.23), 230 (+0.41), 260 (+0.86) nm; IR (KBr) νmax 3465, 1724, 1641, 1385, 1257, 1031, 814, 754, 699 cm−1; 1H NMR and 13C NMR spectroscopic data (see Tables 1 and 3); HRESIMS m/z 395.1464 [M + Na]+ (calcd for C21H24NaO6, 395.1465). Cryptoconcatone J (10): yellowish gum; [α]25 D −25 (c 0.1, MeOH); UV λmax (log ε) 207 (4.17), 225 (3.75), 253 (4.08) nm; ECD (c 2.1 × 10−4 g mL−1, MeOH) λ (Δε) 200 (+1.44), 231 (+0.58), 260 (+1.10) nm; IR (KBr) νmax 3455, 1722, 1640, 1495, 1383, 1257, 1030, 814, 752, 698 cm−1; 1H NMR and 13C NMR spectroscopic data (see Tables 1 and 3); HRESIMS m/z 395.1467 [M + Na]+ (calcd for C21H24NaO6, 395.1465).

activity of this type of compound. In addition, compounds with an α,β-unsaturated-δ-lactone ring but no acetoxy group attached to the alkenyl chain (4, 5, and 8) were more potent than those that did possess such an acetoxy group (1−3 and 6). The present study showed that compounds 4 and 8−10 exhibited a significant NO inhibitory effect, indicating that arylalkenyl α,β-unsaturated δ/γ-lactones merit further investigation as potential anti-inflammatory agents.

■

EXPERIMENTAL SECTION

General Experimental Procedures. A JASCO P-1020 polarimeter was used for optical rotations. UV spectra were acquired on a Shimadzu UV-2450 spectropolarimeter. A JASCO 810 spectropolarimeter was used to collect ECD data, and a Bruker Tensor 27 spectrometer was used to obtain IR data. NMR data were measured on a Bruker AV III-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C). Tetramethylsilane was used as the internal standard. HRESIMS data were acquired on an Agilent UPLC-Q-TOF (6520B) instrument. Column chromatography (CC) was done on Sephadex LH-20 (Pharmacia, Uppsala, Sweden), D101 macroporous resin (The Chemical Plant of Nankai University), silica gel (200−300 and 100−200 mesh, Qingdao Marine Chemical Co., Ltd.), and ODS (40− 63 μm, Fuji). Semipreparative HPLC was performed on a Shimadzu LC-6A instrument using an SPD-20A detector and a Shim-pack PRCODS column (20 × 250 mm, i.d.). An Agilent 1200 Series instrument coupled to a DAD detector using a Shim-pack VP-ODS column (4.6 × 250 mm, i.d.) was used for HPLC analysis. Plant Material. The leaves and twigs of C. concinna were collected in Guangdong Province, People’s Republic of China, in April 2014 and authenticated by Prof. Zhong-Liang Huang, South China Botanical Garden, Chinese Academy of Sciences. A voucher specimen (No. CCH-20140422) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. The air-dried leaves and twigs of C. concinna (2.5 kg) were extracted with 95% EtOH (3 × 10 L) under reflux. The EtOH extract was concentrated under reduced pressure, yielding a viscous residue (145.0 g), which was dissolved in 40% EtOH (4 L) and filtered to remove chlorophyll. Subsequently, the filtrate was subjected to chromatography on a D101 macroporous resin column using EtOH−H2O (0:100−90:10, v/v) as eluent to obtain four fractions, A−D. Fraction B (10.7 g) was subjected to silica gel column chromatography using CH2Cl2−MeOH in a gradient (20:1−5:1, v/v) to yield five subfractions (Fr. B1−B5). Fr. B5 was further chromatographed via preparative HPLC using MeOH−H2O (45:55, v/v) to yield 5 (28.5 mg, tR = 46.2 min). Fraction C (8.0 g) was subjected to ODS column chromatography with MeOH−H2O (30:70−100:0, v/v) to yield four subfractions (Fr. C1−C4). Fr. C1 was further separated via MPLC successively to produce five subfractions (C1.1−C1.5). Fr. C1.2 was subjected to preparative HPLC with MeCN−H2O (30:70, v/v) to yield 4 (2.7 mg, tR = 58.9 min), 7 (70.5 mg, tR = 72.1 min), and Fr. C1.2.1 (81.4 mg). Fr. C1.2.1 was separated by recycling-preparative HPLC using MeOH−H2O (50:50, v/v) to yield 6 (6.9 mg, tR = 16.5 min). Fr. C2 was chromatographed over an ODS column with MeOH−H2O (35:65, v/ v) to obtain Fr. C2.1−C2.4. Fr. C2.2 and Fr. C2.3 were separated using Sephadex LH-20 with MeOH to obtain Fr. C2.2.1, Fr. C2.3.1, and Fr. C2.3.2, respectively. Fr. C2.2.1 was purified by preparative HPLC with MeOH−H2O (50:50, v/v) to afford 8 (7.0 mg, tR = 26.3 min). Fr. C2.3.1 was subjected to preparative HPLC with MeOH− H2O (60:40, v/v) to obtain 1 (30.5 mg, tR = 12.8 min) and 2 (28.2 mg, tR = 25.3 min). Fr. C2.3.2 was further purified via preparative HPLC with MeOH−H2O (55:45, v/v) to obtain 3 (7.2 mg, tR = 18.7 min). Fr. C3 was isolated by an MCI column to obtain Fr. C3.1−C3.6. Fr. C3.2 was chromatographed via preparative HPLC using MeCN− H2O (35:65, v/v) to yield Fr. C3.2.1 (8.7 mg), which was subsequently purified with recycling-preparative HPLC using MeOH−H2O (60:40) to yield 9 (2.6 mg, tR = 16.1 min) and 10 (4.0 mg, tR = 34.8 min). F

DOI: 10.1021/acs.jnatprod.5b00839 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

tion); HRESIMS m/z 476.2282 [M + NH4]+ (calcd for C25H34NO8, 476.2279). O-Acetyl derivatives of 9 and 10 (9c/10c): 1H NMR (500 MHz, CDCl3) δH 7.36 (2H, d, J = 7.5 Hz, H-2″/H-6″), 7.33 (1H, d, J = 5.4 Hz, H-4), 7.31 (2H, overlap, H-3″/H-5″), 7.29 (1H, overlap, H-4″), 6.62 (1H, d, J = 15.9 Hz, H-8′), 6.19 (1H, d, J = 5.4 Hz, H-3), 6.10 (1H, dd, J = 15.9, 7.3 Hz, H-7′), 5.42 (1H, m, H-6′), 5.25 (1H, t, J = 7.9 Hz, H-6), 5.13 (1H, m, H-4′), 5.03 (1H, m, H-2′), 2.66 (1H, m, H1′), 2.06 (3H, s, 6′-COCH3), 2.01 (3H, s, 4′-COCH3), 1.99 (3H, s, 2′COCH3); CD spectra (Figure S78, Supporting Information); HRESIMS m/z 474.2118 [M + NH4]+ (calcd for C25H32NO8, 474.2122). Cell Culture and Nitrite Determination. The NO inhibitory assay was achieved as previously reported.26 All experiments were carried out in triplicate, with N-monomethyl-L-arginine (L-NMMA) as a positive control.

Preparation of the Bis(S)- and Bis(R)-MTPA Esters of Compounds 1, 2, and 10. (R)-(−)-α-Methoxy-α-trifluoromethylphenylacetyl chloride (5 μL) and dimethylaminopyridine (1.5 mg) were dissolved in a solution of 1 (2 or 10) (1.0 mg) in dried pyridine (50 μL). MeOH (150 μL) was used to quench the reaction after the mixture stood overnight at room temperature. Preparative HPLC was used to purifiy the product with MeOH−H2O (85:15 or 80:20, v/v) to obtain pure bis(S)-MTPA esters of 1 (2 or 10) (0.7 mg). The bis(R)MTPA ester of 1 (2 or 10) was obtained with the method described above, but using the (S)-(+)-MTPA-Cl. Bis(S)-MTPA Eester of 1 (1a): 1H NMR (500 MHz, CDCl3) δH 6.83 (1H, m, H-4), 6.57 (1H, d, J = 15.9 Hz, H-8′), 6.08 (1H, dd, J = 15.9, 8.2 Hz, H-7′), 6.00 (1H, d, J = 9.5 Hz, H-3), 5.44 (1H, m, H-6′), 5.15 (1H, m, H-4′), 4.97 (1H, m, H-2′), 4.42 (1H, m, H-6), 3.60 (3H, s, OCH3), 3.55 (3H, s, OCH3), 2.25 (2H, m, H-5), 2.12 (1H, m, H-5′a), 1.97 (3H, s, COCH3), 2.02 (1H, m, H-5′b). Bis(R)-MTPA ester of 1 (1b): 1H NMR (500 MHz, CDCl3) δH 6.82 (1H, m, H-4), 6.60 (1H, d, J = 15.9 Hz, H-8′), 5.98 (1H, dd, J = 15.9, 8.2 Hz, H-7′), 5.49 (1H, m, H-6′), 5.28 (1H, m, H-4′), 4.86 (1H, m, H-2′), 4.35 (1H, m, H-6), 3.58 (3H, s, OCH3), 3.56 (3H, s, OCH3), 2.24 (2H, m, H-5), 2.16 (1H, m, H-5′a), 2.07 (1H, m, H-5′b), 1.93 (3H, s, COCH3). Bis(S)-MTPA ester of 2 (2a): 1H NMR (500 MHz, CDCl3) δH 6.76 (1H, d, J = 15.9 Hz, H-8′), 6.72 (1H, m, H-4), 6.19 (1H, dd, J = 15.9, 8.2 Hz, H-7′), 5.94 (1H, d, J = 9.5 Hz, H-3), 5.71 (1H, m, H-6′), 5.29 (1H, m, H-2′), 4.82 (1H, m, H-4′), 3.98 (1H, m, H-6), 3.52 (3H, s, OCH3), 3.50 (3H, s, OCH3), 2.04 (3H, s, COCH3), 1.83 (1H, m, H1′a), 1.77 (1H, m, H-1′b). Bis(R)-MTPA ester of 2 (2b): 1H NMR (500 MHz, CDCl3) δH 6.81 (1H, m, H-4), 6.65 (1H, d, J = 15.9 Hz, H-8′), 6.00 (1H, d, J = 15.9 Hz, H-7′), 6.03 (1H, d, J = 9.5 Hz, H-3), 5.61 (1H, m, H-6′), 5.32 (1H, m, H-2′), 4.98 (1H, m, H-4′), 4.32 (1H, m, H-6), 3.53 (3H, s, OCH3), 3.41 (3H, s, OCH3), 2.07 (1H, m, H-1′a), 2.05 (1H, m, H1′b), 2.03 (3H, s, COCH3). Bis(S)-MTPA ester of 10 (10a): 1H NMR (500 MHz, CDCl3) δH 6.73 (1H, dd, J = 15.9 Hz, H-8′), 6.16 (1H, d, J = 5.1 Hz, H-3), 6.12 (1H, dd, J = 15.9, 7.8 Hz, H-7′), 5.67 (1H, m, H-6′), 5.19 (1H, m, H2′), 4.19 (1H, t, J = 7.8 Hz, H-6), 4.91 (1H, m, H-4′), 3.50 (3H, s, OCH3), 3.47 (3H, s, OCH3), 2.64 (2H, m, H-1′), 2.01 (3H, s, COCH3). Bis(R)-MTPA ester of 10 (10b): 1H NMR (500 MHz, CDCl3) δH 6.62 (1H, dd, J = 15.9 Hz, H-8′), 6.19 (1H, d, J = 5.1 Hz, H-3), 5.94 (1H, dd, J = 15.9, 7.8 Hz, H-7′), 5.57 (1H, m, H-6′), 5.30 (1H, m, H2′), 5.16 (1H, t, J = 7.8 Hz, H-6), 4.96 (1H, m, H-4′), 3.52 (3H, s, OCH3), 3.43 (3H, s, OCH3), 2.78 (2H, m, H-1′), 2.02 (3H, s, COCH3). Preparation of the (R)- and (S)-MTPA Ester Derivatives of Compound 8. The (R)- and (S)-MTPA esters of 8 were prepared in the same way as the esters of compound 1. (S)-MTPA ester of 8 (8a): 1H NMR (500 MHz, CDCl3) δH 6.65 (1H, dd, J = 15.9 Hz, H-8′), 6.21 (1H, dd, J = 15.9, 4.0 Hz, H-7′), 4.66 (1H, m, H-6′), 3.55 (3H, s, OCH3), 2.25 (1H, m, H-5′a), 2.07 (1H, m, H-3′a), 1.86 (2H, m, H-1′), 1.71 (1H, m, H-5′b). (R)-MTPA ester of 8 (8b): 1H NMR (500 MHz, CDCl3) δH 6.69 (1H, dd, J = 15.9 Hz, H-8′), 6.24 (1H, dd, J = 15.9, 4.0 Hz, H-7′), 4.76 (1H, m, H-6′), 3.56 (3H, s, OCH3), 2.35 (1H, m, H-5′a), 2.00 (1H, m, H-3′a), 1.95 (1H, m, H-5′b), 1.81 (2H, m, H-1′). Acetylation of 1, 2, 3, 4, 9, and 10. Each compound (1.0 mg) was treated with Ac2O (100 mL) and dried pyridine (50 μL). The reaction mixture was purified using preparative HPLC with MeOH− H2O (65:35 or 70:30 v/v) after standing overnight at room temperature. O-Acetyl derivatives of 1, 2, 3, and 4 (1c/2c/3c/4c): 1H NMR (500 MHz, CDCl3) δH 7.42 (2H, d, J = 7.5 Hz, H-2″/H-6″), 7.36 (2H, t, J = 7.5 Hz, H-3″/H-5″), 7.29 (1H, m, H-4″), 6.90 (1H, m, H-4), 6.67 (1H, d, J = 15.9 Hz, H-8′), 6.16 (1H, dd, J = 15.9, 7.3 Hz, H-7′), 5.48 (1H, m, H-6′), 5.16 (2H, m, H-4′/H-2′), 4.54 (1H, m, H-6), 2.38 (2H, m, H-5), 2.11 (3H, s, 6′-COCH3), 2.05 (3H, s, 4′-COCH3), 2.05 (3H, s, 2′-COCH3); CD spectra (Figure S77, Supporting Informa-

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00839. 1D NMR, 2D NMR, and the HRESIMS of 1−10; 1H NMR of 1c−4c, 9c, 10c, 1a, 1b, 2a, 2b, 8a, 8b, 10a, and 10b; HRESIMS of 1c and 9c; and ECD spectra of 1−8, 1c−4c, 9c, and 10c (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86-25-8327-1405. E-mail: [email protected] (L.-Y. Kong). *Tel/Fax: +86-25-8327-1402. E-mail: [email protected] (J.-G. Luo). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research work was supported by New Century Excellent Talents in University (NCET-12-09-77) and was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We also thank Prof. Z.-L. Huang for collecting and identifying the plants.

■

REFERENCES

(1) Liu, Y.; Rakotondraibe, L. H.; Brodie, P. J.; Wiley, J. D.; Cassera, M. B.; Miller, J. S.; Ratovoson, F.; Rakotobe, E.; Rasamison, V. E.; Kingston, D. G. I. J. Nat. Prod. 2015, 78, 1330−1338. (2) Davis, R. A.; Demirkiran, O.; Sykes, M. L.; Avery, V. M.; Suraweera, L.; Fechner, G. A.; Quinn, R. J. Bioorg. Med. Chem. Lett. 2010, 20, 4057−4059. (3) Feng, R.; Wang, T.; Wei, W.; Tan, R. X.; Ge, H. M. Phytochemistry 2013, 90, 147−153. (4) Chou, T. H.; Chen, J. J.; Lee, S. J.; Chiang, M. Y.; Yang, C. W.; Chen, I. S. J. Nat. Prod. 2010, 73, 1470−1475. (5) Awang, K.; Hadi, A. H. A.; Saidi, N.; Mukhtar, M. R.; Morita, H.; Litaudon, M. Fitoterapia 2008, 79, 308−310. (6) Wu, T. S.; Lin, F. W. J. Nat. Prod. 2001, 64, 1404−1407. (7) Wang, Z. F.; Zhu, P.; Yan, W. H. Conserv. Genet. 2009, 10, 1841− 1843. (8) Huang, W.; Zhang, W. J.; Cheng, Y. Q.; Jiang, R.; Wei, W.; Chen, C. J.; Wang, G.; Jiao, R. H.; Tan, R. X.; Ge, H. M. Planta Med. 2014, 80, 925−930. (9) Lin, C. T.; Chu, F. H.; Tseng, Y. H.; Tsai, J. B.; Chang, S. T.; Wang, S. Y. Pharm. Biol. 2007, 45, 638−644.

G

DOI: 10.1021/acs.jnatprod.5b00839 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(10) Shen, C. P.; Luo, J. G.; Yang, M. H.; Kong, L. Y. J. Nat. Prod. 2015, 78, 1322−1329. (11) Yang, M. H.; Wang, J. S.; Luo, J. G.; Wang, X. B.; Kong, L. Y. Bioorg. Med. Chem. 2011, 19, 1409−1417. (12) Yin, H.; Luo, J. G.; Shan, S. M.; Wang, X. B.; Luo, J.; Yang, M. H.; Kong, L. Y. Org. Lett. 2013, 15, 1572−1575. (13) Cavalheiro, A. J.; Yoshida, M. Phytochemistry 2000, 53, 811− 819. (14) Snatzke, G. Angew. Chem., Int. Ed. Engl. 1968, 7, 14−25. (15) Novaes, L. F. T.; Drekener, R. L.; Avila, C. M.; Pilli, R. A. Tetrahedron 2014, 70, 6467−6473. (16) Grkovic, T.; Blees, J. S.; Colburn, N. H.; Schmid, T.; Thomas, C. L.; Henrich, C. J.; McMahon, J. B.; Gustafson, K. R. J. Nat. Prod. 2011, 74, 1015−1020. (17) Seco, J. M.; Quiñoá, E.; Riguera, R. Chem. Rev. 2012, 112, 4603−4641. (18) Guo, J. P.; Zhu, C. Y.; Zhang, C. P.; Chu, Y. S.; Wang, Y. L.; Zhang, J. X.; Wu, D. K.; Zhang, K. Q.; Niu, X. M. J. Am. Chem. Soc. 2012, 134, 20306−20309. (19) Freire, F.; Seco, J. M.; Quiñoá, E.; Riguera, R. J. Org. Chem. 2005, 70, 3778−3790. (20) Seco, J. M.; Quiñoá, E.; Riguera, R. Chem. Rev. 2004, 104, 17− 117. (21) Kobayashi, Y.; Tan, C. H.; Kishi, Y. Helv. Chim. Acta 2000, 83, 2562−2571. (22) Shao, C. L.; Linington, R. G.; Balunas, M. J.; Centeno, A.; Boudreau, P.; Zhang, C.; Engene, N.; Spadafora, C.; Mutka, T. S.; Kyle, D. E.; Gerwick, L.; Wang, C. Y.; Gerwick, W. H. J. Org. Chem. 2015, 80, 7849−7855. (23) Burke, C. P.; Swingle, M. R.; Honkanen, R. E.; Boger, D. L. J. Org. Chem. 2010, 75, 7505−7513. (24) Das, B.; Nagendra, S.; Reddy, C. R. Tetrahedron: Asymmetry 2011, 22, 1249−1254. (25) Seco, J. M.; Quiñoá, E.; Riguera, R. Tetrahedron: Asymmetry 2001, 12, 2915−2925. (26) Li, Q. M.; Luo, J. G.; Wang, X. B.; Yang, M. H.; Kong, L. Y. Fitoterapia 2013, 86, 29−34.

H

DOI: 10.1021/acs.jnatprod.5b00839 J. Nat. Prod. XXXX, XXX, XXX−XXX