Khayseneganins A–H, Limonoids from Khaya senegalensis

Article pubs.acs.org/jnp

Khayseneganins A−H, Limonoids from Khaya senegalensis Chun-Mao Yuan,†,‡ Yu Zhang,† Gui-Hua Tang,† Ying-Tong Di,† Ming-Ming Cao,† Xiao-Ying Wang,† Guo-Ying Zuo,§ Shun-Lin Li,† Hui-Ming Hua,*,‡ Hong-Ping He,*,† and Xiao-Jiang Hao*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China § Research Center of Natural Medicine, Clinical School of Kunming General Hospital of Chengdu Military Command, Kunming, 650032, People’s Republic of China S Supporting Information *

ABSTRACT: Eight new limonoids, khayseneganins A−H (1−8), and 31 known limonoids were isolated from the leaves and twigs of Khaya senegalensis. The structures of the new compounds were elucidated by 2DNMR spectroscopy and mass spectrometry, and the absolute configuration of 1 was determined by the CD exciton chirality method. Compounds 9, 10, 12, and 15 showed antimicrobial activities against Pseudomonas aeruginosa, MRSA 92#, and MRSA 98#, all with a MIC value of 12.5 μg/mL.

imonoids have attracted much attention in the fields of natural products and synthetic chemistry because of their marked insect antifeedant, growth-regulating, cytotoxic, and antiviral activities.1 The genus Khaya (about eight species), belonging to the Meliaceae family, is distributed extensively in tropical Africa and Madagascar, and the bark of these species has been used in traditional medicine for the treatment of fever and malaria in Africa.2 Crude extracts of the bark of Khaya species showed cytotoxic, antifungal, anti-inflammatory, and antimalarial activities.3 Previous studies on the genus Khaya have led to the isolation of a variety of ring B,D-seco limonoids.4 In the course of a search for tetranortriterpenoids with new carbon skeletons and significant biological activities,5 eight new limonoids, khaysenagnins A−H (1−8), along with 31 known compounds were isolated from the leaves and twigs of Khaya senegalensis (Desr.) A. Juss. In the current paper, the isolation, structure elucidation, and antimicrobial activities of these compounds are presented.

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RESULTS AND DISCUSSION Khayseneganin A (1) was obtained as a yellow, amorphous solid, and its molecular formula, C27H30O9, was deduced from the positive-ion HRESIMS at m/z 521.1790 (calcd for C27H30O9 Na, 521.1787), with 13 indices of hydrogen deficiency. From the 1H and 13C NMR data (Tables 1 and 3), the presence of a typical β-furan ring, a C-29-methylene (δH 2.32, d, J = 12.3 Hz and 2.59, d, J = 12.3 Hz; δC 39.8), and the fragment MeO2CCH(OH)− implied that 1 is a phragmalintype limonoid.4c Extensive analysis of the 1D- and 2D-NMR data of 1 suggested a close similarity between 1 and khayanolide C.6 The differences were the presence of a Δ8(9) double bond, a hydroxy group, and a ketone carbonyl in 1. The © 2012 American Chemical Society and American Society of Pharmacognosy

proton signal at δH 6.00 (s, H-15) along with carbon signals at δC 130.1 (C-8), 157.7 (C-9), 157.2 (C-14), 112.9 (C-15), and 166.3 (C-16) indicated the presence of an α,β,γ,δ-unsaturated Special Issue: Special Issue in Honor of Lester A. Mitscher Received: October 4, 2012 Published: December 4, 2012 327

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Table 1. 1H NMR Spectroscopic Data for Compounds 1−4

position 1 2a 2b 5 6a

1a,d

2b,c

3a,c

4a,c

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

4.91, d (8.0) 2.10, m

3.2, d (3.2)

4.49, d (1.9)

4.42, d (3.2)

9 11α 11β 12α 12β 15a 15b 17 18 19 21 22

4.70, d (8.3)

2.10, m (2H)

2.10, dd (12.1, 8.3) 1.60, m

1.72, 1.29, 1.92, 6.09,

5.10, 0.95, 1.05, 7.47, 6.43,

5.20, 1.25, 1.16, 7.60, 6.52,

5.04, s 1.17, s 1.16, s 7.47, s 6.39, d (1.2) 7.40, t (1.2) 1.41, s 2.10, d (12.3) 2.75, d (12.3) 3.28, d (8.3)

s s s s s

s s s s s

7.55, t (1.5)

28 29a

1.34, s 2.32, d (12.3) 2.59, d (12.3) 3.42, d (8.0)

1.35, s 1.99, m

30b OMe-7 OAc-1

1.58, m

1.77, m 1.27, m 2.0, m 6.12, s

7.42, s

30a

3.08, d (2.2) 4.45, d (2.2) 2.04, m

1.49, m 1.32, m 6.00, s

23

29b

4.87, d (8.3)

3.85, s

2.21, d (11.9) 2.92, d (8.3)

3.75, s

m m m s

3.80, s 2.05, s

Table 2. 1H NMR Spectroscopic Data for Compounds 5−8 5b,d

3.38, dd (5.6, 2.2) 2.84, dd (15.2, 5.6) 3.25, dd (15.2, 2.2) 3.18, s

7.38, t (1.5) 1.38, s 1.79, s

δH (J in Hz)

δH (J in Hz)

3.29, s 2.89, d (8.3)

6a

4.17, d (8.3)

3.72, d (10.3) 3.36, dd (11.4, 3.0) 2.59, dd (16.0, 11.4) 2.50, dd (16.0, 3.0) 2.79, d (9.3) 1.75, dd (15.1, 2.3) 2.01, m 1.15, dd (14.0, 2.3) 2.31, dt (14.0, 4.4) 4.09, d (18.4) 3.76, d (18.4) 6.16, s 1.33, s 1.24, s 7.59, brs 6.55, brs 7.62, brs 1.10, s 1.85, d (12.0)

9 11α

2.11, d (8.8) 1.78, m

11β 12α

1.90, m 0.90, m

12β

1.92, m

15a 15b 17 18 19 21 22 23 28 29a

3.74, s 3.70, s 5.78, s 1.06, s 1.22, s 7.54, s 6.48, s 7.49, s 1.01, s 1.53, d (12.0) 1.91, m

30 OMe-7 OAc-1 OMe-2

5.20, s 3.85, s

δH (J in Hz)

δH (J in Hz)

29b

4.92, s

8a,d

3 5

2.32, d (3.0) 2.25, ddd (15.1, 4.6, 3.0) 1.54, tt (15.1, 3.5) 1.04, dd (13.5, 3.5) 1.91, dt (13.5, 4.6) 2.56, d (18.2) 2.93, d (18.2) 5.67, s 0.88, s 1.29, s 7.46, s 6.40, d (1.5)

7b,d

position

6b

4.25, s

6c,d

a

2.64, s 3.73, s

2.07, d (12.0) 3.47, brs 3.67, s OH-3 4.16, d (10.3) 3.62, s

2.90, d (8.3)

2.57, d (2.9)

4.22, d (8.3)

4.38, d (2.9)

2.29, d (8.6) 1.99, m

2.31, d (9.4) 1.90, m

1.82, m 0.95, m

1.30, m 0.96, d (12.0)

1.75, m

1.58, dt (12.0, 3.5) 3.23, d (18.5) 3.13, d (18.5) 5.54, s 1.05, s 1.38, s 7.42, s 6.37, s 7.40, s 1.32, s 2.12, d (12.8)

3.20, s (2H) 5.57, s 1.08, s 1.33, s 7.47, brs 6.47, s 7.50, s 1.02, s 1.86, d (12.7) 2.11, d (12.7) 2.87, s 3.72, s

3.50, s

Recorded in CDCl3. bRecorded in methanol-d4. pyridine-d5. dRecorded at 500 MHz.

Recorded in CDCl3. bRecorded in methanol-d4. cRecorded at 500 MHz. dRecorded at 600 MHz.

2.93, d (12.8) 3.48, s 3.75, s 2.08, s 3.54, s c

Recorded in

a

The molecular formula of khayseneganin B (2) was determined to be C27H32O10 from the pseudomolecular ion peak [M + Na]+ at m/z 539.1897 (calcd C27H32O10Na, 539.1893) in the HRESIMS, 18 mass units more than 1. The 1 H and 13C NMR data resembled closely those of 1 (Tables 1 and 3), except for the absence of signal for a Δ8(9) double bond and the presence of signal for a hydroxy group at C-8, which were supported by the HMBC correlations from Me-19 to C-9 (δC 48.2) and from H-15 and H-2 to C-8 (δC 82.9). Comparison of the 13C NMR data of C-8 (δC 82.9) in 2 with those of C-8 (δC 87.1) in khayanolide C6 indicated that OH-8 is β-oriented. However, it was not possible to determine the configuration of OH-8 due to the absence of any ROESY correlation between the OH-8 signal and the other proton resonances, when measured in methanol-d4. Therefore, the NMR spectrum performed in DMSO-d6 showed the crucial ROESY correlations of OH-8 (δH 3.95) with H-5 (δH 2.93) and H-12β (δ H 1.83), revealing that OH-8 is β-oriented (Supporting Information). Accordingly, the structure of 2 was established as shown. Khayseneganin C (3) gave a molecular formula of C29H34O11 from the HRESIMS, 42 mass units more than 2, in accordance with the presence of an additional acetyl group. The 1D-NMR data of 3 were similar to those of 2, with the only difference being the signals for an additional acetyl that could be located

lactone, which was supported by the HMBC correlations of Me-19/C-9, H-15/C-8, Me-18/C-14, and H-17/C-16 as well as the UV absorption band at λmax 293 nm.7 Furthermore, the hydroxy group located at C-2 and a ketone carbonyl assigned at C-3 were readily confirmed by the 1H−1H COSY correlation of H-30/H-2 and the HMBC correlations of H-5/C-3 and Me28/C-3, respectively (Figure 1). Thus, the gross structure of 1 was elucidated as depicted. The relative stereochemistry of 1 was assigned from the ROESY spectrum (Figure 1), in which correlations of H-5/H11β and H-12β/H-17 indicated that such groups are cofacial, and these were assigned arbitrarily as β-oriented. The ROESY cross-peaks of Me-19/H-11α, Me-18/H-12α, H-2/H2-29, and H-2/H-30 showed the α-orientation of those groups. Therefore, the relative stereochemistry of compound 1 was established as shown. The absolute configuration of 1 was determined by the CD exciton chirality method.8 The CD spectrum of 1 showed a positive Cotton effect at λmax 293 nm (Δε +26.30) and a negative Cotton effect at λmax 257 nm (Δε −7.30) due to the transition interaction between two different chromophores of the α,β,γ,δ-unsaturated lactone and the furan ring,7,8 indicating a positive chrality for 1 (Figure 2). The absolute configuration of 1 was thus assigned as shown. 328

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Table 3. 13C NMR Spectroscopic Data for Compounds 1−8 1a,e

2b,d

3a,d

4a,d

5b,d

6c,d

7b,d

8a,d

position

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 OMe-7 OAc-1

86.8, 74.9, 217.8, 50.5, 51.0, 70.4, 175.3, 130.1, 157.7, 61.7, 19.6, 29.9, 37.7, 157.2, 112.9, 166.3, 80.4, 16.1, 14.3, 120.3, 141.1, 110.1, 143.0, 17.2, 39.8, 52.5, 53.1,

88.0, 72.5, 218.4, 50.5, 49.3, 72.7, 177.0, 82.9, 48.2, 57.3, 14.6, 28.0, 40.3, 172.8, 113.9, 168.4, 84.7, 20.6, 18.1, 121.9, 142.9, 111.2, 144.4, 17.5, 41.1, 56.5, 52.7,

91.8, s 71.4, d 216.0, s 49.7, s 46.3, d 71.3, d 175.6, s 81.5, s 47.3, d 57.4, s 15.0, t 26.4, t 38.9, s 169.0, s 113.7, d 165.4, s 82.9 d 20.0, q 17.8, q 120.0, s 141.2, d 109.9, d 142.8, d 17.0, q 36.3, t 51.0, d 52.8, q 170.8, s 21.7, q

74.3, d 38.4, t 171.0, s 84.2, s 49.2, d 71.0, d 176.3, s 146.2, s 52.6, d 48.6 s 24.5, t 28.6, t 42.1, s 81.4, s 34.4, t 170.1, s 79.6, d 14.2, q 24.1, q 121.1, s 141.4, d 110.4, d 143.1, d 31.9, q 27.0, q 112.5, t 54.0, q

85.8, s 104.1, s 88.6, d 42.9, s 41.1, d 72.4, d 177.0, s 88.5, s 57.0, d 61.3, s 17.3, t 28.1, t 39.0, s 83.9, s 37.5, t 174.9, s 82.4, d 16.0, q 18.4, q 122.5, s 142.5, d 111.3, d 144.1 d 19.9, q 45.6, t 73.3, d 52.6, q

84.5, 106.7, 83.9, 44.0, 35.2, 34.5, 175.6, 87.5, 55.8, 58.5, 16.7, 27.3, 38.5, 84.2, 37.3, 171.6, 81.0, 15.6, 17.9, 122.1, 141.7, 111.0, 143.3, 19.5, 45.0, 74.5, 51.9,

84.9, 101.9, 204.8, 50.2, 44.1, 71.5, 175.5, 88.1, 56.8, 61.0, 17.2, 28.1, 38.7, 86.4, 37.5, 173.9, 82.5, 15.7, 18.7, 122.1, 142.7, 111.2, 144.2, 15.7, 43.9, 73.8, 52.6,

90.6, 99.8, 202.7, 51.6, 42.5, 71.4, 174.8, 87.1, 57.0, 62.0, 16.4, 25.9, 37.2, 84.7, 35.9, 170.5, 80.1, 14.6, 20.2, 120.4, 141.1, 110.0, 142.9, 15.8, 39.0, 68.4, 52.6, 170.2, 21.9, 52.8,

s d s s d d s s s s t t s s d s d q q s d d d q t d q

s d s s d d s s d s t t s s d s d q q s d d d q t d q

OMe-2 a

s s d s d t s s d s t t s s t s d q q s d d d q t d q

50.7, q

s s s s d d s s d s t t s s t s d q q s d d d q t d q

53.1, q

s s s s d d s s d s t t s s t s d q q s d d d q t d q s q q

Recorded in CDCl3. bRecorded in methanol-d4. cRecorded in pyridine-d5. dRecorded at 100 MHz. eRecorded at 125 MHz.

Figure 1. 1H−1H COSY (bold), key HMBC (a), and key ROESY (b) correlations of 1.

characteristic β-furan ring, three carbonyl groups, and a pair of olefinic carbons. Therefore, compound 4 was assigned as a tetracyclic ring system. Comparison of the NMR spectroscopic data of 4 with those of methyl ivorensate9 indicated that 4 is a ring A,B,D-seco limonoid bearing the same A, C, D, E, and 1,14epoxy rings as in methyl ivorensate. A difference between the two compounds was the presence of a hydroxy group signal at C-6 in 4, which was confirmed by the HMBC correlations of H6 (δH 4.25) to C-5 (δC 49.2), C-7 (δC 176.3), and C-10 (δC 48.6) (Figure 3). The relative configuration of 4 was determined from the ROESY spectrum, in which correlations of Me-19/Me-29, Me-19/H-1, and H-17/H-12β indicated that these groups were on the same side of the molecule, and they

at C-1, which was supported by the HMBC correlations observed from Me-19 and H-30 to C-1, along with the downfield shifted carbon signal of C-1 (from δC 88.0 to δC 91.8). Comparison of 13C NMR data of C-8 (δC 81.5) in 3 with those of C-8 (δC 82.9) in 2 indicated that OH-8 in the former is also β-oriented. The relative configurations of 2 and 3 were assigned as the same as that of 1 by analysis of their ROESY spectra. Khayseneganin D (4) was found to possess the molecular formula C27H34O9 on the basis of its positive-ion [M + Na]+ at m/z 525.2095 in the HRESIMS, with 11 indices of hydrogen deficiency. From the 1H and 13C NMR spectra, it was evident that seven degrees of unsaturation were represented by a 329

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Figure 2. CD and UV spectra of 1 (in MeOH).

Figure 3. 1H−1H COSY (bold), key HMBC (a), and key ROESY (b) correlations of 4.

were arbitrarily assigned as β-oriented. The singlet of H-6 suggested that the dihedral angle between H-6 and H-5 is approximately 90° in its most favorable orientation, which was consistent with the ROESY correlations from H-6 to H-5, H-9, and Me-19 (Figure 3).10 The ROESY correlations of Me-28/H5 and Me-18/H-12α implied that Me-28, Me-18, and H-5 are α-oriented. Thus, the structure of 4 was identified as 6βhydroxylmethyl ivorensate, and it was named khayseneganin D. The molecular formula of khayseneganin E (5) was determined as C27H34O11 from a [M + Na]+ peak at m/z 557.2014 in the HRESIMS. Further analysis of the 1H and 13C NMR spectra indicated that 5 is similar to 1-O-deacetyl-2αhydroxykhayanolide E.11 The chemical shift differences resulted

from the absence of a ketone carbonyl signal and the presence of a hydroxy group resonance (δH 3.29, s; δC 88.6) in the former compound. The HMBC correlations from H-5, H-28, and H-30 to C-3 (δC 88.6) suggested that a hydroxy group is located at C-3 (Figure 4). The relative configuration of 5 was determined by analysis of the ROESY spectrum, in which correlations of H-5/H-6, H-5/H-11β, H-5/H-12β, H-12β/H17, and H-6/Me-28 suggested that these groups are all βoriented (Figure 4). In addition, Me-19, H-9, and H-3 were assigned as α-oriented on the basis of the ROESY correlations between Me-19/H-9 and between H2-29/H-3, suggesting that OH-3 is β-oriented. Therefore, the structure of 5 was 330

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Figure 4. 1H−1H COSY (bold), key HMBC (a), and key ROESY (b) correlations of 5.

hydroxykhayalactone (32),25 3-acetylkhayalactone (33),11 khayanolide A (34),26 methyl angolensate (35),17 17-epimethyl-6-hydroxyangolensate (36),27 methyl 6-acetoxyangolensate (37),6 proceranolide (38),17 and khayalactol (39),28 were identified by comparison of their spectroscopic data with those reported in the literature. In addition, the 13C NMR data of 3deacetylkhivorin (9) were reported for the first time, which were assigned by analysis of its 2D-NMR spectra. Selected compounds were screened for their antimicrobial activities against four microorganisms, Pseudomonas aeruginosa, Staphylococcus aureus, MRSA (methicillin-resistant Staphylococcus aureus) 92#, and MRSA 98#. The minimum inhibitory concentrations (MICs) of these compounds were determined by the 2-fold dilution method.29 The results revealed that compounds 9, 10, 12, and 15 showed antimicrobial activities against P. aeruginosa, MRSA 92#, and MRSA 98#, all with a MIC value of 12.5 μg/mL (Table 4).

established as 1-O-deacetyl-2α,3β-dihydroxylkhayanolide E (khayseneganin E). Khayseneganin F (6) was determined to have a molecular formula of C28H36O10 from its positive-ion HRESIMS. Compound 6 exhibited similar NMR spectra to those of 5, except for the absence of the hydroxy group signal and the presence of an O-methyl signal. In the former, the absence of the hydroxy group at C-6 was confirmed by the HMBC correlation between H-5 and C-6 (δC 34.5) as well as the 1 H−1H COSY correlation between H-5/H2-6 (δH 2.50, dd, J = 16.0, 3.0 Hz and 2.59, dd, J = 16.0, 11.4 Hz). The O-methyl group was located at C-2 on the basis of HMBC correlation of the O-methyl (δH 3.62) with C-2 (δC 106.7). Therefore, the structure of compound 6 was determined as shown. Khayseneganin G (7) gave a quasimolecular ion peak [M + Na]+ at m/z 569.1997 in the HRESIMS, corresponding to the molecular formula C28H34O11, with 12 indices of hydrogen deficiency. The 1D-NMR data of 7 resembled those of 1-Odeacetyl-2α-hydroxykhayanolide E,11 except for the occurrence of an O-methyl signal at C-2. This was supported by the HMBC correlation of the O-methyl (δH 3.50) with C-2 (δC 101.9). Therefore, the structure of compound 7 was established as 1-O-deacetyl-2α-methoxylkhayanolide E (khayseneganin G). Khayseneganin H (8) was found to possess the molecular formula C30H36O12 by means of HRESIMS, 42 mass units more than 7. The NMR data of 8 showed striking similarities to those of 7, with the only difference being the presence of an acetyl group signal at C-1 on the basis of HMBC correlations from Me-19 and H-30 to C-1, along with the downfield shifted carbon signal of C-1 (from δC 84.9 to δC 90.6). The relative configurations of 5−8 were almost the same on the basis of the ROESY correlations of these compounds. Accordingly, the structure of compound 8 was established as 2α-methoxylkhayanolide E, and it was named khayseneganin H. Thirty-one known compounds, 3-deacetylkhivorin (9),12 1deacetylkhivorin (10),13 7-oxokhivorin (11),14 swietmanin B (12),15 khayalenoid E (13),16 11α-acetoxy-2α-hydroxy-6deoxydestigloylswietenine acetate (14),11 Δ8(30)-3β-acetoxy-1oxo-methylmeliacate (15),14 3-O-acetylswietenolide (16),17 khayanone (17),18 3α,7α-dideacetylkhivorin (18),19 swietmanin C (19),15 seneganolide (20),18 3-acetoxy-8,14-dien-8,30seco-khayalactone (21),20 methyl ivorensate (22),9 khayanolide B (23),21 1α-acetoxy-3β,6,8α-trihydroxy-2α-methoxy-2β,14βepoxy-[4.2.1.10,301.1,4]-tricyclomeliac-7-oate (24),22 6-dehydroxyl-1-O-acetylkhayanolide B (25),23 1-O-acetylkhayanolide B (26),21 1-O-deacetyl-2α-hydroxylkhayanolide E (27),11 1-Odeacetylkhayanolide E (28),21 khayanolide E (29),21 6dehydroxylkhayanolide E (30),21 khayalactone (31),24 6S-

Table 4. Antimicrobial Activities of 3, 4, and 8−22a antimicrobial activities (MICs in μg/mL) compound 4 9 10 11 12 13 14 15 16 17 vancomycin hydrochloridec

S. aureusb

P. aeruginosab

MRSA 92#b

MRSA 98#b

50 25 25 50 25 25 25 25 25 25 0.78

25 12.5 25 25 12.5 25 25 12.5 25 >50 25

25 12.5 25 50 50 25 25 12.5 25 25 0.78

50 12.5 12.5 25 12.5 25 25 12.5 25 25 0.78

a Compounds 3, 8, and 18−22 were inactive against all the tested strains (MIC > 50 μg/mL). bS. aureus (Staphylococcus aureus), P. aeruginosa (Pseudomonas aeruginosa), MRSA (methicillin-resistant Staphylococcus aureus) 92#, and MRSA 98#. cPositive control.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 polarimeter. UV spectra were recorded on a Shimadzu UV-2401A. CD spectra was recorded with an Applied Photophysics Chirascan spectrometer. IR spectra were determined on a Bruker Tensor-27 infrared spectrophotometer with KBr disks. 1H and 13C NMR and 2D-NMR spectra were recorded on Bruker AM-400, Bruker DRX-500, and Bruker Avance III 600 spectrometers using TMS as an internal standard. ESIMS and

331

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see Tables 1 and 3; positive ESIMS m/z 521 [M + Na]+; HRESIMS m/z 521.1790 [M + Na]+ (calcd for C27H30O9Na, 521.1787). Khayseneganin B (2): white, amorphous power; [α]25D +88.6 (c 0.16, CH3OH); UV (MeOH) λmax (log ε) nm 208 (3.41); IR (KBr) νmax 3453, 1737, 1682, 1289, 1213, 1038 cm−1; 1H NMR and 13C NMR data in methanol-d4, see Tables 1 and 3; 1H NMR (600 MHz, DMSO-d6) δ 7.72 (1H, s, H-21), 7.67 (1H, s, H-23), 6.51 (1H, s, H22), 5.99 (1H, s, OH-2), 5.91 (1H, s, H-15), 5.43 (1H, s, OH-6), 5.15 (1H, s, H-17), 5.08 (1H, s, OH-1), 4.60 (1H, d, J = 8.3 Hz, H-2), 4.22 (1H, d, J = 3.3 Hz, H-6), 3.95 (1H, s, OH-8), 3.60 (1H, s, CH3O-7), 2.93 (1H, d, J = 3.3 Hz, H-5), 2.71 (1H, d, J = 8.3 Hz, H-6), 2.50 (1H, d, J = 11.9 Hz, H-29b), 1.92 (1H, d, J = 11.9 Hz, H-29a), 1.90 (1H, m, H-5), 1.83 (1H, m, H-12β), 1.54 (1H, m, H-11β), 1.42 (1H, m, H12α), 1.16 (1H, s, CH3-28), 1.10 (1H, s, CH3-18), 1.02 (1H, m, H11α), 1.00 (1H, s, CH3-19); 13C NMR (150 MHz, DMSO-d6) δ 215.4 (C, C-3), 174.9 (C, C-7), 170.3 (C, C-14), 165.1 (C, C-16), 143.4 (CH, C-23), 141.7 (CH, C-21), 120.4 (C, C-20), 112.3 (CH, C-15), 110.4 (CH, C-22), 86.0 (C, C-1), 81.9 (CH, C-17), 81.0 (C, C-8), 71.0 (CH, C-2), 70.7 (CH, C-6), 55.4 (C, C-10), 55.1 (CH, C-30), 51.6 (CH3, CH3O-7), 48.6 (C, C-4), 47.1 (CH, C-5), 46.1 (CH, C-9), 40.0 (CH2, C-29), 38.3 (C, C-13), 26.4 (CH2, C-12), 20.1 (CH3, C18), 17.4 (CH3, C-19), 17.0 (CH3, C-28), 15.1 (CH2, C-11); positive ESIMS m/z 539 [M + Na]+; HRESIMS m/z 539.1897 [M + Na]+ (calcd for C27H32O10Na, 539.1893). Khayseneganin C (3): white, amorphous power; [α]25D −123.5 (c 0.35, CH3OH); UV (MeOH) λmax nm (log ε) 209 (3.51); IR (KBr) νmax 3444, 1728, 1633, 1247, 1038, 875 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 3; positive ESIMS m/z 581 [M + Na]+; HRESIMS m/z 581.1994 [M + Na]+ (calcd for C29H34O11Na, 581.1998). Khayseneganin D (4): white, amorphous power; [α]25D −56.8 (c 0.16, CH3OH); UV (MeOH) λmax nm (log ε) 204 (3.27); IR (KBr) νmax 3441, 1738, 1716, 1631, 1250, 1127, 1021 cm−1; 1H NMR and 13 C NMR data, see Tables 1 and 3; positive ESIMS m/z 525 [M + Na]+; HRESIMS m/z 525.2095 [M + Na]+ (calcd for C27H34O9Na, 525.2100). Khayseneganin E (5): white, amorphous power; [α]25D +35.2 (c 0.14, CH3OH); UV (MeOH) λmax nm (log ε) 209 (3.20); IR (KBr) νmax 3432, 1722, 1638, 1025 cm−1; 1H NMR and 13C NMR data, see Tables 2 and 3; positive ESIMS m/z 557 [M + Na]+; HRESIMS m/z 557.2014 [M + Na]+ (calcd for C27H34O11Na, 557.1998). Khayseneganin F (6): white, amorphous power; [α]25D +34.7 (c 0.1, CH3OH); UV (MeOH) λmax nm (log ε) 209 (3.10); IR (KBr) νmax 3439, 1720, 1629, 1038, 1026 cm−1; 1H NMR and 13C NMR data, see Tables 2 and 3; positive ESIMS m/z 555 [M + Na]+; HRESIMS m/z 555.2199 [M + Na]+ (calcd for C28H36O11Na, 555.2206). Khayseneganin G (7): white, amorphous power; [α]25D +31.0 (c 0.1, CH3OH); UV (MeOH) λmax nm (log ε) 206 (3.31); IR (KBr) νmax 3441, 1729, 1631, 1461, 1246, 1032 cm−1; 1H NMR and 13C NMR data, see Tables 2 and 3; positive ESIMS m/z 569 [M + Na]+; HRESIMS m/z 569.1997 [M + Na]+ (calcd for C28H34O11Na, 569.1998). Khayseneganin H (8): white, amorphous power; [α]25D +28.4 (c 0.35, CH3OH); UV (MeOH) λmax nm (log ε) 207 (3.13); IR (KBr) νmax 3442, 1736, 1630, 1245, 1034 cm−1; 1H NMR and 13C NMR data, see Tables 2 and 3; positive ESIMS m/z 611 [M + Na]+; HRESIMS m/z 611.2096 [M + Na]+ (calcd for C30H36O12Na, 611.2104). 3-Deacetylkhivorin (9): 13C NMR (100 MHz, CDCl3) δ 170.0 (C, COCH3-7), 169.6 (C, COCH3-1), 169.3 (C, C-16), 143.0 (CH, C-23), 141.0 (C, C-21), 120.4 (C, C-20), 109.8 (CH, C-22), 78.4 (CH, C17), 75.7 (CH, C-3), 74.3 (CH, C-1), 73.8 (CH, C-7), 69.6 (C, C-14), 56.2 (CH, C-15), 42.3 (C, C-8), 40.8 (C, C-10), 38.7 (C, C-13), 37.2 (C, C-4), 36.6 (CH, C-9), 36.2 (CH, C-5), 28.2 (CH3, C-28), 28.0 (CH2, C-2), 26.2 (CH2, C-12), 22.6 (CH2, C-6), 21.5 (CH3, COCH31), 21.8 (CH3, C-29), 21.2 (CH3, COCH3-7), 18.2 (CH3, C-30), 17.5 (CH3, C-18), 16.5 (CH3, C-19), 14.4 (CH2, C-11). Antimicrobial Assays. The minimum inhibitory concentrations of selected compounds against Staphylococcus aureus (ATCC25923), Pseudomonas aeruginosa (ATCC27853) (National Institutes for Food and Drug Control (NIFDC), China), MRSA 92#, and MRSA 98#

HRESIMS were measured with a Finnigan MAT 90 instrument. Column chromatography was performed on silica gel (90−150 μm, Qingdao Marine Chemical Company, Qingdao People's Republic of China), Sephadex LH-20 (40−70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), and Lichroprep RP-C18 gel (40−63 μm, Merck, Darmstadt, Germany). TLC spots were visualized under UV light and by dipping into 5% H2SO4 in EtOH followed by heating. Plant Material. The dried leaves and twigs of K. senegalensis were collected in Xishuangbanna in Yunnan Province of China in August 2010 and were identified by Mr. Yu Chen (Kunming Institute of Botany, Chinese Academy of Sciences). A voucher specimen (H20100808) was deposited at the Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. Extraction and Isolation. The air-dried powdered plant material (8.0 kg) was extracted with MeOH (3 × 20 L) under reflux three times (4, 3, and 3 h), respectively. The combined MeOH extracts were concentrated under vacuum to give a crude residue (630 g), which was suspended in water and then partitioned successively with petroleum ether, EtOAc, and n-BuOH. The EtOAc portion (200.6 g) was chromatographed on a silica gel column, eluted with petroleum ether− acetone (from 1:0 to 0:1), to yield six fractions (1−6). Fr. 3 (8.1 g) was then separated over a MCI-gel column (MeOH−H2O from 2:8 to 10:0) to obtain four fractions (3A−3D). Fr. 3A (672 mg) was chromatographed on Sephadex LH-20 (MeOH) to give 10 (50 mg) and Fr. 3A1 (400 mg), which was further purified by a silica gel column (CHCl3−Me2CO, 100:1) to obtain 14 (10 mg), 16 (8 mg), and 38 (15 mg). Fr. 3C (1.89 g) was chromatographed over a silica gel column to yield three fractions (3C1−3C3). Fr. 3C1 (400 mg) was purified by Sephadex LH-20 (MeOH) and then chromatographed on a silica gel column (CHCl3−Me2CO, 100:2) to obtain 13 (17 mg) and 12 (10 mg). Compounds 9 (17 mg), 11 (15 mg), and 20 (22 mg) were isolated from Fr. 3C3 (400.0 mg) by repeated silica gel columns, eluted with CHCl3−Me2CO (100:4). Fr. 3D (1.61 g) was purified by Sephadex LH-20 (MeOH) to yield 19 (20 mg) and a major component, which was separated over preparative HPLC (MeOH− H2O, 60:40) to give 18 (10 mg) and 15 (10 mg). Fr. 4 (15.0 g) was separated over a MCI gel column (MeOH−H2O from 2:8 to 8:2) to obtain five fractions (4A−4E). Fr. 4A (900 mg) was applied to a silica gel column, eluted with CHCl3−MeOH (100:1), to afford 26 (25.7 mg), 39 (17.5 mg), and subfractions 4A1 (60 mg), 4A2 (30 mg), and 4A3 (26 mg). Subfractions 4A1, 4A2, and 4A3 were further purified by semipreparative HPLC (MeOH−H2O, 4:6) to afford 5 (15.7 mg), 7 (11.6 mg), and 1 (3.7 mg). Compounds 37 (11.7 mg), 21 (4.5 mg), and 31 (6.5 mg) were obtained from Fr. 4B (2 g) by a silica gel column eluted with CHCl3−Me2CO (from 100:1 to 100:5). Fraction 4C (1.4 g) was chromatographed on a C18 silica gel column, eluted with a gradient of MeOH−H2O (40:60, 45:55, and 50:50), to afford five subfractions (4C1−4C5), and each fraction was purified by Sephadex LH-20 (MeOH) to yield 8 (20 mg), 23 (3.0 mg), 24 (3.0 mg), 29 (7.6 mg), and 32 (3.3 mg). Fraction 4D (1.2 g) was applied to Sephadex LH-20 (MeOH) to afford 3 (20.8 mg), 4 (9 mg), and subfractions 4D1 (350 mg) and 4D2 (60 mg). Fr. 4D1 was chromatographed over a silica gel column, eluted with CHCl3− MeOH (from 100:1 to 100:5), to afford 31 (6.7 mg), 33 (61.3 mg), 34 (34 mg), and 35 (8.1 mg). Fraction 4E (950 mg) was chromatographed on a silica gel column, eluted with CHCl3−Me2CO (from 100:2 to 100:8), to afford 22 (20 mg), 25 (2.1 mg), 30 (9 mg), and 36 (15.3 mg). Fr. 5 (15.0 g) was separated over a C18 silica gel column, eluted with a gradient of MeOH−H2O (from 3:7 to 7:3), to obtain five fractions (5A−5E). Fr. 5A was further chromatographed over a C18 silica gel column, eluted with a gradient of MeOH−H2O (30:70 and 35:65), to give three subfractions (5A1−5A3). Fr. 5A1 (2.9 g) was subjected to a silica gel column, eluted with CHCl3−Me2CO (from 9:1 to 7:3), to yield 28 (10 mg), 2 (20 mg), 17 (6 mg), 27 (10.6 mg), and 6 (7 mg). Khayseneganin A (1): yellow, amorphous power; [α]25D +134.6 (c 0.14, CH3OH); UV (MeOH) λmax (log ε) nm 293 (3.43), 205 (3.24); CD (0.0009 M) λmax (Δ ε) 293 (+26.30), 257 (−7.30) nm; IR (KBr) νmax 3441, 1630, 1462, 1052, 874 cm−1; 1H NMR and 13C NMR data, 332

dx.doi.org/10.1021/np3006919 | J. Nat. Prod. 2013, 76, 327−333

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(9) Adesogan, E. K.; Taylor, D. A. H. J. Chem. Soc. (D) 1969, 15, 889−889. (10) Liu, J.; Yang, S. P.; Su, Z. S.; Lin, B. D.; Wu, Y.; Yue, J. M. Phytochemistry 2011, 72, 2189−2196. (11) Zhang, B.; Yang, S. P.; Yin, S.; Zhang, C. R.; Wu, Y.; Yue, J. M. Phytochemistry 2009, 70, 1305−1308. (12) Adesogan, E. K.; Powell, J. W.; Taylor, D. A. H. J. Chem. Soc. (C) 1967, 7, 554−556. (13) (a) Taylor, D. A. H.; Jibodu, K. O.; Ohochuku, N. S. J. Chem. Soc. (C) 1970, 17, 2396−2401. (b) Bickii, J.; Njifutie, N.; Ayafor, F. J.; Basco, L. K.; Ringwald, P. J. Ethnopharmacol. 2000, 69, 27−33. (14) Narender, T.; Khaliq, T.; Shweta, T. Nat. Prod. Res. 2008, 22, 763−800. (15) Lin, B. D.; Yuan, T.; Zhang, C. R.; Dong, L.; Zhang, B.; Wu, Y.; Yue, J. M. J. Nat. Prod. 2009, 72, 2084−2090. (16) Yuan, T.; Zhang, C. R.; Yang, S. P.; Yue, J. M. J. Nat. Prod. 2010, 73, 669−674. (17) Kadota, S.; Marpaung, L.; Kikuchi, T.; Ekimoto, H. Chem. Pharm. Bull. 1990, 38, 639−651. (18) Nakatani, M.; Abdelgaleil, S. A. M.; Kurawaki, J.; Okamura, H.; Iwagawa, T.; Doe, M. J. Nat. Prod. 2001, 64, 1261−1265. (19) Zhang, H.; Wang, X.; Chen, F.; Androulakis, X. M.; Wargovich, M. J. Phytother. Res. 2007, 21, 731−734. (20) Ferreira, I. C. P.; Cortez, D. A. G.; Da Silva, M. F. d. G. F.; Fo, E. R.; Vieira, P. C.; Fernandes, J. B. J. Nat. Prod. 2005, 68, 413−416. (21) Zhang, H.; Tan, J.; VanDerveer, D.; Wang, X.; Wargovich, M. J.; Chen, F. Phytochemistry 2009, 70, 294−299. (22) Olmo, L. R. V.; Da Silva, M. F. D. G. F.; Fo, E. R.; Vieira, P. C.; Fernandes, J. B.; Pinheiro, A. L.; Vilela, E. F. Phytochemistry 1997, 44, 1157−1161. (23) Nakatani, M.; Abdelgaleil, S. A. M.; Okamura, H.; Iwagawa, T.; Doe, M.; Hirotsu, K. Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 2000, 42, 37−42. (24) Tchuendem, M. H. K.; Ayafor, J. F.; Connolly, J. D.; Sterner, O. Tetrahedron Lett. 1998, 39, 719−722. (25) Zhang, H.; Odeku, O. A.; Wang, X. N.; Yue, J. M. Phytochemistry 2008, 69, 271−275. (26) Nakatani, M.; Abdelgaleil, S. A. M.; Okamura, H.; Iwagawa, T.; Sato, A.; Doe, M. Tetrahedron Lett. 2000, 41, 6473−6477. (27) Attaur, R.; Zareen, S.; Choudhary, M. I.; Akhtar, M. N.; Khan, S. N. J. Nat. Prod. 2008, 71, 910−913. (28) Abdelgaleil, S. A. M.; Okamura, H.; Iwagawa, T.; Doe, M.; Nakatani, M. Heterocycles 2000, 53, 2233−2240. (29) Xu, S. Y.; Bian, R. L.; Chen, X. Pharmacological Experiment Methodology, 3rd ed.; People’s Medical Publishing House: Beijing, 2002; pp 1647−1719. (30) Tang, G. H.; Zhang, Y.; Gu, Y. C.; Li, S. F.; Di, Y. T.; Wang, Y. H.; Yang, C. X.; Zuo, G. Y.; Li, S. L.; He, H. P.; Hao, X. J. J. Nat. Prod. 2012, 75, 996−1000.

(clinically isolated strains, from Kunming General Hospital of Chengdu Military Command) were determined by the 2-fold dilution method.29 The strains used in antimicrobial tests were obtained from the Research Center of Natural Medicine, Clinical School of Kunming General Hospital of Chengdu Military Command. Antimicrobial tests were performed according to the previously described method.30

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ASSOCIATED CONTENT

* Supporting Information S

This material (1D- and 2D-NMR, ESIMS, HRESIMS, UV, and IR spectra of khayseneganins A−H (1−8) and 3-deacetylkhivorin (9) and the CD spectrum of khayseneganin A) is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-871-5223070. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was financially supported by the National Natural Science Foundation of China (No. 30830114), the Ministry of Science and Technology (2009CB522300 and 2009CB940900), and the Young Academic and Technical Leader Raising Foundation of Yunnan Province (2010CI047).

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DEDICATION Dedicated to Dr. Lester A. Mitscher, of the University of Kansas, for his pioneering work on the discovery of bioactive natural products and their derivatives.

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REFERENCES

(1) (a) Fang, X.; Di, Y. T.; Hao, X. J. Curr. Org. Chem. 2011, 15, 1363−1391. (b) Tan, Q. G.; Luo, X. D. Chem. Rev. 2012, 112, 2591− 2591. (2) (a) Dalziel, J. M. The Useful Plants of West Tropical Africa; The Crown Agents for the Colonies: London, 1937; p 325. (b) Peng, H.; David, J. M. Flora of China; Science Press: Beijing, 2008; Vol. 11, pp 116−117. (3) (a) Agbedahunsi, J. M.; Fakoya, F. A.; Adesanya, S. A. Phytomedicine 2004, 11, 504−508. (b) Abdelgaleil, S. A. M.; Hashinaga, F.; Nakatani, M. Pest Manage. Sci. 2005, 61, 186−190. (4) (a) Olmo, L. R. V.; Da Silva, M. F. D. G. F.; Fo, E. R.; Vieira, P. C.; Fernandes, J. B.; Marsaioli, A. J.; Pinheiro, A. L.; Vilela, E. F. Phytochemistry 1996, 42, 831−837. (b) Zhang, H.; Van Derveer, D.; Wang, X.; Chen, F.; Androulakis, X. M.; Wargovich, M. J. J. Chem. Crystallogr. 2007, 37, 463−467. (c) Yuan, C. M; Zhang, Y.; Tang, G. H.; Li, S. L.; Di, Y. T.; Hou, L.; Cai, J. Y.; Hua, H. M.; He, H. P.; Hao, X. J. Chem. Asian J. 2012, 7, 2024−2027. (5) (a) Di, Y. T.; He, H. P.; Liu, H. Y.; Yi, P.; Zhang, Z.; Ren, Y. L.; Wang, J. S.; Sun, Q. Y.; Yang, F. M.; Fang, X.; Li, S. L.; Zhu, H. J.; Hao, X. J. J. Nat. Prod. 2007, 70, 1352−1355. (b) Fang, X.; Di, Y. T.; He, H. P.; Liu, H. Y.; Zhang, Z.; Ren, Y. L.; Gao, Z. L.; Gao, S.; Hao, X. J. Org. Lett. 2008, 10, 1905−1908. (c) Yin, J. L.; Di, Y. T.; Fang, X.; Liu, E. D.; Liu, H. Y.; He, H. P.; Li, S. L.; Li, S. F.; Hao, X. J. Tetrahedron Lett. 2011, 52, 3083−3085. (d) Cai, J. Y.; Zhang, Y.; Luo, S. H.; Chen, D. Z.; Tang, G. H.; Yuan, C. M.; Di, Y. T.; Li, S. L.; Hao, X. J.; He, H. P. Org. Lett. 2012, 14, 2524−2529. (6) Abdelgaleil, S. A. M.; Okamura, H.; Iwagawa, T.; Sato, A.; Miyahara, I.; Doe, M.; Nakatani, M. Tetrahedron 2001, 57, 119−126. (7) Yuan, T.; Yang, S. P.; Zhang, C. R.; Zhang, S.; Yue, J. M. Org. Lett. 2009, 11, 617−620. (8) Berova, N.; Nakanishi, K. Circular Dichroism: Principles and Applications, 2nd ed.; Wiley-VCH: New York, 2000; pp 337−382. 333

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