Anti-inflammatory Coumarin and Benzocoumarin Derivatives from

Jan 26, 2015 - activities.7 Murraya alata Drake (Rutaceae) is a shrub that is distributed in ... Murraya species,9 the 95% aqueous ethanol extract of ...
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Anti-inflammatory Coumarin and Benzocoumarin Derivatives from Murraya alata Hai-Ning Lv,† Shu Wang,‡ Ke-Wu Zeng,† Jun Li,§ Xiao-Yu Guo,† Daneel Ferreira,⊥ Jordan K. Zjawiony,⊥ Peng-Fei Tu,† and Yong Jiang*,† †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China ‡ Department of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People’s Armed Police Forces, Tianjin 300162, People’s Republic of China § Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ⊥ Department of BioMolecular Sciences, Division of Pharmacognosy, and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States S Supporting Information *

ABSTRACT: Two new rare 8-methylbenzo[h]coumarins, muralatins A and B (1, 2), nine new C-8-substituted coumarins, muralatins C−K (3−11), and 22 known analogues (12−33) were isolated from the leaves of Murraya alata. The absolute configurations of compounds 5, 11, 23, 24, 27, 30, and 33 were assigned via comparison of their specific rotations, by Mosher’s method, and by single-crystal X-ray diffraction and electronic circular dichroism (ECD) data of the in situ formed transition metal complexes. A putative biosynthesis pathway to 1 and 2 is proposed, and the chemical synthesis of 1 was accomplished through electrocyclization of 5,7-dimethoxy-8-[(Z)-3-methylbut1,3-dienyl)]coumarin (12). Compounds 1, 2, 8, 12, and 31 showed inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages with IC50 values of 6.0− 14.5 μM.

C

new compounds and the inhibitory effects of the isolates on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages are reported.

oumarins, one of the groups of bioactive constituents of the genus Murraya, particularly of M. sect. Murraya, have been demonstrated to possess anti-inflammatory,1,2 antiplatelet aggregation,3,4 antidote,5 antitumor,6 and AChE inhibitory activities.7 Murraya alata Drake (Rutaceae) is a shrub that is distributed in the thickets of sandy areas or near sea level in the Guangdong, Guangxi, and Hainan Provinces of mainland China and in Vietnam. There has been no report on the chemical constituents of this plant except for its essential oil.8 As a continuation of a search for bioactive natural products from Murraya species,9 the 95% aqueous ethanol extract of the leaves of M. alata was investigated and afforded 33 coumarin derivatives including two new 8-methylbenzo[h]coumarins, named muralatins A and B (1, 2), nine new C-8-substituted coumarins, named muralatins C−K (3−11), and 22 known analogues (12−33) (Figure S2, Supporting Information). A putative biosynthesis pathway to the new 8-methylbenzo[h]coumarins (1, 2) is proposed, and the chemical synthesis of 1 has been accomplished through electrocyclization of 5,7dimethoxy-8-[(Z)-3-methylbut-1,3-dienyl)]coumarin (12). Herein, the isolation and structural characterization of the © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The 95% aqueous EtOH extract of the leaves of M. alata was suspended in H2O and partitioned with petroleum ether and CHCl3, successively. The petroleum ether- and CHCl3-soluble portions were subjected repeatedly to silica gel and Sephadex LH-20 column chromatography (CC) followed by preparative TLC to afford two new 8-methylbenzo[h]coumarins (1, 2) and nine new (3−11) and 22 known (12−33) C-8-substituted coumarins. Muralatin A (1) was obtained as colorless needles via crystallization from MeOH. Its molecular formula was assigned as C15H12O3 based on the 13C NMR spectroscopic data and the observed pseudomolecular ion at m/z 241.0872 [M + H]+ (calcd for C15H13O3, 241.0865) in the positive-ion HRESIMS. Received: November 2, 2014

A

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data provided support that 1 is a benzocoumarin.10−12 The additional aromatic ring was located at the h-face of the coumarin backbone on the basis of the HMBC correlations of H-6 and C-6a/C-7/C-10a; H-7 and C-6a/C-6/C-10a; and H10 and C-6a/C-10a/C-10b (Figure S1, Supporting Information). The HMBC correlations between the methoxy protons and C-5 and between the methyl protons and C-7/C-8/C-9 indicated that the locations of the methoxy and methyl groups are at C-5 and C-8, respectively. The NOE correlations between the methoxy protons and H-4/H-6 supported the above assignment and confirmed the structure of muralatin A (1) as 5-methoxy-8-methylbenzo[h]coumarin. Muralatin B (2) was obtained as colorless needles after crystallization from MeOH. Its molecular formula was established as C20H20O5 from the 13C NMR and positive-ion HRESIMS data (m/z 341.1386 [M + H]+), indicating 11 indices of hydrogen deficiency. The UV spectrum showed characteristic absorptions of a benzocoumarin (λmax 226, 270, 278, 321 nm) similar to 1. The 1H and 13C NMR data obtained for 2 were similar to those of 1 (Table 1), suggesting that they share the same benzocoumarin skeleton. The main difference is that the C-8 methyl group in 1 [δH 2.53 (3H, s); δC 21.9] is replaced by a hydroxymethylene moiety [δH 5.27 (2H, s); δC 65.7] in 2. In addition, resonances ascribed to a 3methylbutanoyl group [δH 0.98 (6H, d, J = 6.6 Hz), 2.16 (1H, m), 2.30 (2H, d, J = 7.2 Hz); δC 22.4 (×2), 25.7, 43.3, 172.9] were observed in the NMR spectra of 2. The deshielded hydroxymethylene resonance (δH 5.27, 2H, s) indicated that its hydroxy group is acylated, which was supported by the HMBC correlation between the methylene protons (δH 5.27) and the carbonyl carbon (δC 172.9) of the 3-methylbutanoyl group. Therefore, the structure of muralatin B (2) was defined as 5methoxy-8-(3-methylbutanoyloxy)methylbenzo[h]coumarin. Muralatin C (3) was obtained as a light yellow oil. Its molecular formula was deduced as C17H20O5 on the basis of its 13 C NMR and HRESIMS data (m/z 305.1390 [M + H]+, calcd for C17H21O5, 305.1389), indicating eight indices of hydrogen deficiency. The UV spectrum showed characteristic absorptions at 213 and 304 nm for a coumarin core.6,7 The 1H NMR spectrum revealed the presence of H-3 and H-4 of the coumarin moiety at δH 6.29 and 7.95 (each 1H, d, J = 9.6 Hz), three methoxy resonances at δH 3.89 (3H, s, OCH3-6), 3.95 (3H, s, OCH3-7), and 3.99 (3H, s, OCH3-5), and the protons of an isopentenyl group at δH 1.69 (3H, s, H-4′), 1.85 (3H, s, H-5′), 3.50 (2H, d, J = 7.2 Hz, H-1′), and 5.21 (1H, t, J = 7.2 Hz, H-2′). The ESIMS data also supported the presence of an isopentenyl group via the generation of a fragment ion at m/z 249.0766 [M + H − C4H8]+ due to cleavage of the C-1′−C-2′ bond. The 1H and 13C NMR data of 3 (Tables 2 and 3) were nearly superimposable on those of coumurrayin (16),13 except for the presence of an additional methoxy group [δH 3.89 (3H, s); δC 61.0], which was deduced to be located at C-6 by the HMBC correlations of H-1′ and C-7/C-9, H-4 and C-5/C-9/ C-10, OCH3-5/C-5, OCH3-6/C-6, and OCH3-7/C-7. Thus, the structure of muralatin C (3) was defined as 6methoxycoumurrayin. Muralatin D (4) gave a molecular formula of C17H20O6 determined from its 13C NMR and HRESIMS data (m/z 343.1158 [M + Na]+, calcd for C17H20O6Na, 343.1158), indicating eight indices of hydrogen deficiency. Comparison of the NMR spectroscopic data of 4 with those of 3 suggested that the isopentenyl group in 3 is replaced by a (1E)-3-hydroxy-3methyl-1-butenyl group [δH 1.50 (6H, s), 6.78, 6.87 (each 1H,

The UV spectrum showed characteristic absorptions for a benzocoumarin (λmax 227, 271, 281, 324 nm),10,11 and the IR spectrum showed absorption bands for carbonyl (1735 cm−1) and aromatic ring (1637 and 1462 cm−1) functionalities. The 1 H NMR spectrum exhibited a pair of characteristic doublets for H-3 and H-4 of a coumarin skeleton [δH 6.44 and 8.19 (each 1H, d, J = 9.7 Hz)], as well as resonances for the protons of a 1,2,4-trisubstituted phenyl group at δH 7.30, 8.32 (each 1H, d, J = 8.5 Hz), 7.51 (1H, brs), an aromatic singlet at δH 6.84 (1H, s), a methoxy singlet at δH 4.01 (3H, s), and a methyl group at δH 2.53 (3H, s). The 13C NMR data (Table 1) showed 15 carbon resonances comprising 12 olefinic carbons, one carbonyl carbon, one methyl, and one methoxy group. These Table 1. NMR Data of 1 and 2 in CDCl3 (δ in ppm, J in Hz, 500 MHz for 1H NMR, 125 MHz for 13C NMR) 1 position 2 3 4 4a 5 6 6a 7 8 9 10 10a 10b 1′ 2′ 3′ 4′ 5′ CH2-8 CH3-8 OCH3-5 OCH3-6

δH 6.44, d (9.7) 8.19, d (9.7)

6.84, s 7.51, brs 7.30, d (8.5) 8.32, d (8.5)

2 δC, type 161.0, 114.2, 139.3, 108.1, 152.6, 100.3, 135.7, 126.0, 139.5, 126.8, 122.3, 117.1, 152.4,

C CH CH C C CH C CH C CH CH C C

δH 6.46, d (9.7) 8.18, d (9.7)

6.90, s 7.70, brs 7.42, d (8.5) 8.39, d (8.5)

2.30, 2.16, 0.98, 0.98, 5.27, 2.53, s 4.01, s

21.9, CH3 55.8, CH3

d (7.2) m d (6.6) d (6.6) s

4.02, s

δC, type 160.7, 114.8, 139.1, 108.8, 152.8, 100.8, 135.3, 125.7, 137.2, 124.4, 122.8, 118.4, 152.1, 172.9, 43.3, 25.7, 22.4, 22.4, 65.7,

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

56.2, CH3

B

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Table 2. 1H NMR (500 MHz) Data of Compounds 3−11 in CDCl3 (δH in ppm, J in Hz) position

3

4

3 4 5 6 1′ 2′ 3′ 4′ 5′ 1″ 2″ OCH3-5 OCH3-6 OCH3-7

6.29, d (9.6) 7.95, d (9.6)

6.32, d (9.7) 7.99, d (9.7)

6.25, d (9.6) 7.93, d (9.6)

6.18, d (9.6) 8.01, d (9.6)

6.15, d (9.6) 8.00, d (9.6)

6.16, d (9.7) 8.00, d (9.7)

3.50, d (7.2) 5.21, t (7.2)

6.87, d (16.7) 6.78, d (16.7)

2.93, m 3.73, dd (9.3, 3.4)

6.35, s 3.81, d (7.4) 6.50, td (7.4, 1.1)

6.35, s 3.46, s

6.36, s 3.87, d (1.1) 9.71, brs

1.69, s 1.85, s

1.50, s 1.50, s

3.99, s 3.89, s 3.95, s

4.04, s 3.90, s 3.94, s

1.27, 1.30, 3.48, 1.17, 3.98, 3.87, 3.98,

5

6

s s m t (7.0) s s s

7

8

4.51, brs, 4.69, brs 1.80, s

9.36, s 1.94, s

9 6.23, 7.62, 7.54, 6.88,

d d d d

10 (9.6) (9.6) (8.7) (8.7)

3.47, m 1.26, d (7.0) 1.26, d (7.0)

3.96, s

3.95, s

3.96, s

3.95, s

3.92, s

3.91, s

6.18, d (9.8) 7.95, d (9.8) 6.31, s

3.42, m 1.30, d (7.0) 1.30, d (7.0)

11 6.26, 7.63, 7.40, 6.90, 5.30, 4.76,

d d d d d d

(9.5) (9.5) (8.6) (8.6) (6.0) (6.0)

4.83, 1.87, 3.43, 1.17,

4.92, s s m t (7.0)

4.00, s 3.86, s

3.91, s

3.94, s

Table 3. 13C NMR (125 MHz) Data of Compounds 3−11 in CDCl3 (δC in ppm) 3

4

5

6

7

8

9

10

11

position

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 1″ 2″ OCH3-5 OCH3-6 OCH3-7

161.1, C 113.8, CH 138.6, CH 147.7, C 142.3, C 155.3, C 119.1, C 148.6, C 109.9, C 22.4, CH2 121.7, CH 132.4, CH 17.9, CH3 25.7, CH3

160.8, C 113.9, CH 138.8, CH 142.2, C 140.7, C 155.6, C 114.8, C 148.6, C 109.9, C 139.9, CH 118.0, CH 83.0, C 24.4, CH3 24.4, CH3

161.1, C 111.0, CH 138.7, CH 156.1, C 90.3, CH 161.0, C 106.3, C 153.7, C 103.9, C 22.3, CH2 151.6, CH 139.4, C 195.5, CH 9.2, CH3

161.6, C 110.8, CH 138.7, CH 155.5, C 90.3, CH 161.3, C 108.4, C 154.0, C 103.7, C 29.9, CH2 143.7, C 110.0, CH2 22.9, CH3

161.0, C 111.0, CH 138.8, CH 156.6, C 90.2, CH 161.4, C 101.3, C 154.0, C 103.8, C 37.4, CH2 198.7, CH

159.2, C 114.0, CH 142.9, CH 132.0, CH 107.9, CH 161.0, C 106.3, C 153.4, C 112.9, C 190.3, C 202.5, C 34.7, CH 17.6, CH3 17.6, CH3

159.3, C 112.1, CH 137.9, CH 160.1, C 90.3, CH 163.4, C 105.3, C 155.5, C 103.7, C 190.4, C 203.7, C 35.2, CH 17.7, CH3 17.7, CH3

160.5, C 113.4, CH 143.6, CH 128.6, CH 108.2, CH 161.5, C 114.4, C 154.2, C 113.0, C 75.3, CH 76.5, CH 144.8, C 112.4, CH2 18.9, CH3 64.9, CH2 15.2, CH3

61.9, CH3 61.0, CH3 61.3, CH3

61.9, CH3 61.1, CH3 61.2, CH3

160.8, C 113.6, CH 138.7, CH 148.0, C 142.1, C 155.9, C 117.0, C 148.9, C 109.7, C 25.4, CH2 77.0, CH 76.3, C 20.5, CH3 21.5, CH3 56.6, CH2 16.0, CH3 61.8, CH3 60.8, CH3 61.2, CH3

56.0, CH3

56.1, CH3

56.1, CH3

56.0, CH3

55.9, CH3

56.0, CH3

d, J = 16.7 Hz); δC 24.4 (×2), 83.0, 118.0, 139.9], similar to the side chain of murraol (32).14 In combination with HMBC correlations (Figure S1, Supporting Information), the structure of muralatin D (4) was deduced as 5,6-dimethoxymurraol. Muralatin E (5) was obtained as a light yellow oil with [α]24D −75 (c 0.2, MeOH). Its molecular formula, C19H26O7, was deduced from the HRESIMS (m/z 389.1578 [M + Na]+, calcd for C19H26O7Na, 389.1576) and 13C NMR spectroscopic data, indicating seven indices of hydrogen deficiency. The NMR spectroscopic data of 5 were similar to those of 8-(3-ethoxy-2hydroxy-3-methylbutyl)-5,7-dimethoxycoumarin (33),15 except for the presence of an additional methoxy group [δH 3.87 (3H, s); δC 60.8], which was deduced to be located at C-6 by the HMBC correlations (Figure S1, Supporting Information). The sign of the specific rotation of 5 is the same as that of 33, for which the (2′S) configuration was defined by X-ray diffraction analysis in this study (Figure S43, Supporting Information). Therefore, the structure of muralatin E (5) was assigned as 8[(2S)-3-ethoxy-2-hydroxy-3-methylbutyl)]-5,6,7-trimethoxycoumarin.

56.3, CH3 56.5, CH3

56.5, CH3

56.5, CH3

Muralatin F (6) gave a molecular formula of C16H16O5 on the basis of its 13C NMR and HRESIMS data (m/z 289.1078 [M + H]+, calcd for C16H17O5, 289.1076), indicating nine indices of hydrogen deficiency. Analysis of the 1D and 2D NMR data suggested that the structure of 6 is similar to that of coumurrayin (16),13 except that a methyl group in 16 is replaced by a formyl group [δH 9.36 (1H, s); δC 195.5] in 6. This was supported further by the HMBC correlations of H-4′ and C-2′/C-3′/C-5′. Furthermore, in the selective 1D NOE experiment, irradiation of H-2′ resulted in a 32% enhancement of the signal intensity of H-4′, suggesting an E configuration of the double bond. Thus, muralatin F (6) was assigned as 5,7dimethoxy-8-[(2E)-3-methylbut-2-enal]coumarin. Muralatin G (7) was obtained as a light yellow, amorphous powder. Its positive-ion HRESIMS data showed a pseudomolecular ion at m/z 261.1127 [M + H]+, which, in conjunction with the 13C NMR data, established a molecular formula of C 15 H 16 O 4 , with eight indices of hydrogen deficiency. Comparison of NMR spectroscopic data of 7 with those of 6 indicated that the (2E)-3-methylbut-2-enal moiety in 6 is replaced by a 2-methyl-2-propenyl group [δH 1.80 (3H, s), 3.46 C

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(2H, s), 4.51 and 4.69 (each 1H, brs); δC 22.9, 29.9, 110.0, 143.7]. In combination with HMBC correlation analysis, the structure of muralatin G (7) was thus elucidated as 5,7dimethoxy-8-(2-methyl-2-propenyl)coumarin. Muralatin H (8) was obtained as a white, amorphous powder. Its molecular formula was established as C13H12O5 on the basis of 13C NMR and HRESIMS data (m/z 249.0766 [M + H]+, calcd for C13H13O5, 249.0763). The NMR spectra of 8 displayed resonances reminiscent of a 5,7-dimethoxycoumarin skeleton carrying a formylated methyl group [δH 3.87 (2H, d, J = 1.1 Hz, H-1′), 9.71 (1H, brs, H-2′); δC 37.4, 198.7]. The formylmethyl group was connected to C-8 of the 5,7dimethoxycoumarin skeleton via the HMBC correlations of H-1′ and C-8 as well as H-2′ and C-8. Hence, the structure of muralatin H (8) was assigned as 2-(5,7-dimethoxy-2-oxo-2Hchromen-8-yl)acetaldehyde. Muralatin I (9) was obtained as a light yellow, amorphous powder with a molecular formula of C15H14O5 deduced from the 13C NMR and HRESIMS data (m/z 275.0917 [M + H]+, calcd for C15H15O5, 275.0919). Comparison of the NMR spectroscopic data of 9 with those of murranganon (30)16 indicated that the hydroxymethine group in 30 is replaced by a carbonyl group in 9, which was supported by the HMBC correlations between H-3′ and C-1′/C-2′ and of H-4′ and H-5′ with C-2′. Thus, the structure of muralatin I (9) was defined as 8-(1,2-dioxo-3-methylbutyl)-7-methoxycoumarin. Muralatin J (10) was obtained as a white, amorphous powder. It exhibited an [M + H]+ quasimolecular ion at m/z 305.1019 in the positive HRESIMS, which, in conjunction with the 13C NMR data, suggested a molecular formula of C16H16O6 (calcd for C16H17O6, 305.1025), indicating nine indices of hydrogen deficiency. The 1H NMR data of 10 were found to be similar to those of 9, except for the presence of an additional methoxy group at δH 4.00 (s, 3H) in 10 and the replacement of two aromatic doublets at δH 6.88 and 7.54 (each 1H, d, J = 8.7 Hz) in 9 by an aromatic singlet at δH 6.31 (s, 1H) in 10. The additional methoxy group could be located at C-5 on the basis of the HMBC correlations of H-4 [δH 7.95 (d, J = 9.8 Hz)] and C-5 (δC 160.1) and the methoxy protons (δH 4.00) and C-5 (δC 160.1). Thus, the structure of muralatin J (10) was defined as 5,7-dimethoxy-8-(1,2-dioxo-3-methylbutyl)coumarin. Muralatin K (11), [α]24D −27 (c 0.1, MeOH), was obtained as a white, amorphous powder with a molecular formula of C17H20O5 as deduced from the 13C NMR and positive-ion HRESIMS data (m/z 327.1196 [M + Na]+, calcd for C17H20O5Na, 327.1208). Slight differences in the 1H and 13C NMR spectroscopic data (Tables 2 and 3) of 11 in comparison with those of murpanicin (27)17 suggested that they could be a pair of stereoisomeric compounds. The coupling constant between H-1′ and H-2′ (J = 6.0 Hz) indicated that 11 is the erythro isomer. The (2′S) absolute configuration was defined via the ECD data [a positive E band at 375 nm (Figure S40, Supporting Information)] of the in situ generated [Rh2(OCOCF3)4] complex.18 Thus, the structure of muralatin K (11) was defined as 8-[(1R,2S)-1-ethoxy-2-hydroxy-3methyl-3-butenyl)]-7-methoxycoumarin. The known compounds were identified as 5,7-dimethoxy-8[(Z)-3-methylbut-1,3-dienyl]coumarin (12),19 5-methoxyseselin (13),20 6-(3,3-dimethylallyl)seselin (14),21 osthol (15),22 coumurrayin (16),13 gleinene (17),23 ayapanin (18),24 gleinadiene (19),23 5,7-dimethoxy-8-(3-methyl-2-oxobutyl)coumarin (20),17 muraculatin (21),25 seselinal (22),26 murragleinin (23),27 mexoticin (24),16 murrangatin (25),28

peroxyauraptenol (26),29 murpanicin (27),17 omphamurin (28),30 murralongin (29),31 murranganon (30),16 toddalenone (31),23 murraol (32),14 and 8-(3-ethoxy-2-hydroxy-3-methylbutyl)-5,7-dimethoxycoumarin (33)15 by comparison of observed and reported physical data. In addition, the configurations of compounds 23, 27, 30, and 33 were determined for the first time by induced ECD spectroscopy, Mosher’s method, and/or single-crystal X-ray diffraction analysis. Compounds 27 and 30 were assigned (1′S, 2′S) and (1′S) configurations, respectively, based on the positive signs of the E bands at 345 and 354 nm in their Rh2(OCOCF3)4induced ECD spectra (Figures S41 and S42, Supporting Information). The absolute configuration of 30 was confirmed by Mosher’s method (Figure S3, Supporting Information).32 By the same method, a (2′S) configuration was established for both 23 and 24 (Figure S3, Supporting Information), in accord with a previous assignment based on Horeau’s empirical rule for 24.26 The (2′S) absolute configuration of 33 was unambiguously defined by single-crystal X-ray diffraction analysis (Figure S43, Supporting Information). The 33 coumarins described here are dominated by C-8substituted analogues. A putative biogenetic relationship could thus be proposed for the structurally related coumarins (Figure S4, Supporting Information). Among the isolated coumarins, muralatins A (1) and B (2) possess a new 8-methylbenzo[h]coumarin skeleton, reported here for the first time from a natural source. The isolation of structurally related compound 12 from the same plant provided a biosynthetic clue for the synthesis of 1 and 2, which was inspired by the synthesis of diphenyls from stilbene or diphenylbutadiene derivatives.33,34 A putative biosynthesis pathway to 1 and 2 from 12 was proposed as illustrated in Figure 1, and the chemical synthesis of 1 was

Figure 1. Putative biosynthesis pathway to 1 and 2.

accomplished through electrocyclization reactions. Compound 12 was cyclized to 1 in a yield of 17% when refluxed in diphenyl ether at 260 °C for 1 h, and higher yields (73 and 59%) were obtained when 12 was irradiated with 365 and 254 nm ultraviolet light in tetrahydrofuran solution, respectively (Figure 2). These provide insight into the possible biosynthesis of benzo[h]coumarins 1 and 2 via the electrocyclization of 12 by photon or enzyme catalysis. These results may also provide insight into an alternative chemical synthesis of benzocoumarin derivatives or other related natural products.35 In order to assess whether the benzo[h]coumarins were produced during the isolation process, a rapid extraction was performed in the dark and the extract was swiftly examined by LC-MS, which D

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compounds were due to their cytotoxicities, the effects of the compounds on cell proliferation were measured using the MTT method. None showed obvious cytotoxicity at a dosage of 80 μM.

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showed unambiguously the presence of compounds 1 and 2 in M. alata (see Supporting Information). Inflammation consists of a complex mechanism developed as the first response of the immune system to viral, bacterial, or parasitic infections. NO is a signaling molecule that plays a key role in the pathogenesis of inflammation. It has long been considered as one of the pro-inflammatory mediators or regulators that induce inflammation due to overproduction in abnormal situations. NO reacts with soluble guanylate cyclase to generate cyclic guanosine monophosphate (cGMP), which mediates many properties of NO. NO can also interact with molecular oxygen and superoxide anion to form reactive nitrogen species that can modify various cellular functions.36−38 Therefore, inhibition of NO release represents an important therapeutic target in the management of inflammatory diseases. Thus, the isolates from M. alata were tested for their inhibitory effects on LPS-induced NO production in RAW 264.7 macrophages. As shown in Table 4, compounds 2 and 31 Table 4. Inhibitory Effects of the Isolates on LPS-Activated NO Production in RAW 264.7 Cellsa compound

IC50 (μM)

1 2 5 6 8 9 12 16 17 20 27 31 33 indomethacinb

12.4 9.1 34.2 28.3 13.8 30.5 14.5 33.1 20.5 20.6 43.3 6.0 31.9 6.3

± ± ± ± ± ± ± ± ± ± ± ± ± ±

EXPERIMENTAL SECTION

General Experimental Procedures. IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer. NMR spectra were recorded on a Varian INOVA-500 NMR spectrometer, using CDCl3 as solvent, and the chemical shifts were referenced to the solvent residual peak. HRMS data were acquired on a Waters Xevo G2 Q-TOF spectrometer fitted with an ESI source. X-ray data were collected using an Agilent Gemini E X-ray single-crystal diffractometer with Cu Kα radiation (Agilent Technologies, Yarnton, Oxfordshire, UK). CC was performed on silica gel (100−200 mesh or 200−300 mesh, Qingdao Marine Chemical Inc., People’s Republic of China). TLC analysis and preparative TLC were carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., People’s Republic of China). Spots were visualized under UV light (254 and 365 nm) or by heating after spraying with 2% vanillin−H2SO4 solution. All the solvents were of analytical grade. Plant Material. The leaves of Murraya alata were collected in Sanya, Hainan Province, People’s Republic of China, in August 2011. The plant material was identified by Professor P.-F. Tu. A voucher specimen (No. YY201108) was deposited at the Herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine. Extraction and Isolation. The leaves of M. alata (8.5 kg) were extracted three times with 95% aqueous EtOH (80 L × 2 h). The extract was evaporated under reduced pressure, and the residue (1.8 kg) was suspended in H2O and partitioned successively with petroleum ether and CHCl3. The petroleum ether extract (275 g) was fractionated by silica gel CC (2.6 kg, 100−200 mesh) using petroleum ether−acetone (19:1, 9:1, 8:2, and 5:5, v/v) as eluents to produce eight fractions (F1−F8) on the basis of TLC analysis. F4 (120 g) was subjected to silica gel CC, eluting with petroleum ether−EtOAc (100:0, 97:3, 92:8, and 85:15, v/v), to obtain four subfractions, F4a−F4d. F4c (12 g) was separated by silica gel CC eluting with n-hexane−CHCl3 (2:8, v/v) to afford five fractions, F4c1−F4c5. F4c2 was chromatographed by silica gel CC (petroleum ether−acetone, 4:1, v/v) to obtain two subfractions, F4c2a and F4c2b; F4c2a was further purified by Sephadex LH-20 CC (CHCl3−MeOH, 1:1, v/v) and preparative TLC (petroleum ether−EtOAc, 8:2, v/v) to yield 1 (13 mg). F4c4 was separated by silica gel CC eluting with petroleum ether−acetone (8:2, v/v) and then purified by silica gel CC (petroleum ether−CHCl3, 1:4, v/v) to obtain 3 (2 mg). F5 (25 g) was fractioned on silica gel CC eluting with petroleum ether−acetone (9:1, 8:2, and 7:3, v/v) to afford five fractions, F5a−F5e. F5b (9 g) was separated by CC on silica gel eluting with petroleum ether−EtOAc (8:2 and 7.5:2.5, v/v) to afford fractions F5b1−F5b6. F5b1 was subjected to silica gel CC eluting with CH2Cl2 to obtain 2 (15 mg). F6 (32 g) was fractioned on a silica gel column eluting with petroleum ether−acetone (8:2, 7:3, and 5:5, v/v) to afford fractions F6a−F6f. F6c (6 g) was further chromatographed on silica gel CC eluting with CH2Cl2 to give F6c1 and F6c2. F6c2 was purified further by preparative TLC (CHCl3) to yield 6 (6 mg) and 8 (8 mg). F6d (8 g) was subjected to silica gel CC eluting with petroleum ether−EtOAc (6:4, v/v) to obtain 5 (83 mg). F7 (24 g) was fractioned by silica gel CC eluting with petroleum ether−EtOAc (7.5:2.5, v/v) to give eight fractions, F7a−F7e. F7a (1 g) was separated by preparative TLC (CHCl3−MeOH, 98:2, v/v) to yield 7 (2 mg). The CHCl3 extract (500 g) was subjected to silica gel CC and eluted with a stepwise gradient of petroleum ether−acetone (9:1, 8:2, 7:3, 6:4, and 5:5, v/v) to afford 10 fractions (F1−F10). F4 (3 g) was separated on silica gel CC eluting with CH2Cl2 to afford fractions F4a−F4d. F4b (1 g) was further chromatographed over silica gel CC eluting with CHCl3−MeOH (95:5, v/v) to yield 4 (1 mg). F7 (36 g) was fractioned on silica gel CC, with petroleum ether−EtOAc (5:5, v/ v) as eluent, to afford four subfractions, F7a−F7d. F7d (3 g) was

Figure 2. Synthesis of muralatin A (1) from 5,7-dimethoxy-8-[(Z)-3methylbut-1,3-dienyl]coumarin (12).

0.2 0.4 4.4 2.2 0.3 1.5 1.0 3.9 1.3 0.3 6.6 0.7 2.3 0.4

a Only compounds with observable inhibitory effects (IC50 < 50 μM) are listed. bPositive control.

showed inhibitory effects against NO production with IC50 values of 9.1 and 6.0 μM, respectively, comparable to that of the positive control, indomethacin (6.3 μM). The remaining compounds exhibited weaker activities, with IC50 values in the range 12.4−43.3 μM. The preliminary structure−activity relationship analysis indicated that the α,β-unsaturated carbonyl group in the side chain (31) contributed much to the NO inhibitory activity followed by the naphthalene moiety (1 and 2) and the conjugated diene unit (12). On the contrary, the carboxylic or hydroxy group in the side chain (21, 23−25) might have a slight influence on the activity. When the hydroxy group was alkylated (5, 31), the activity was increased, which could be related to the increase of lipophilicity. In order to investigate whether the inhibitory activities of these active E

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chromatographed on silica gel CC (CHCl3−MeOH, 95:5, v/v) and recrystallized from MeOH to give 9 (7 mg). F8f (18 g) was chromatographed on silica gel CC, eluting with CHCl3−MeOH (95:5, v/v), and purified by preparative TLC (CH2Cl2−MeOH, 95:5, v/v) to give 10 (2 mg) and 11 (1 mg). Muralatin A (1): colorless needles (MeOH); mp 171−173 °C; UV (MeOH) λmax (log ε) 227 (4.96), 271 (4.72), 281 (4.77), 324 (4.52) nm; IR (KBr) νmax 2956, 2933, 2852, 1735, 1637, 1462, 1109, 825 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 241.0872 [M + H]+ (calcd for C15H13O3, 241.0865). Muralatin B (2): colorless needles (MeOH); mp 97−98 °C; UV (MeOH) λmax (log ε) 226 (4.96), 270 (4.67), 278 (4.69), 321 (4.51) nm; IR (KBr) νmax 2958, 2922, 2850, 1737, 1639, 1462, 1110, 828 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 341.1386 [M + H]+ (calcd for C20H21O5, 341.1389). Muralatin C (3): light yellow oil; UV (MeOH) λmax (log ε) 213 (4.24), 304 (3.84) nm; IR (KBr) νmax 2956, 2917, 2851, 1738, 1462, 1377 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 305.1390 [M + H]+ (calcd for C17H21O5, 305.1389), 249.0766 [M + H − C4H8]+. Muralatin D (4): light yellow oil; UV (MeOH) λmax (log ε) 210 (3.90), 303 (3.37) nm; IR (KBr) νmax 2956, 2925, 2852, 1736, 1463, 1378, 1048 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 343.1158 [M + Na]+ (calcd for C17H20O6Na, 343.1158), 303.1234 [M + H − H2O]+. Muralatin E (5): light yellow oil; [α]24D −75 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 210 (4.33), 309 (3.90) nm; IR (KBr) νmax 2953, 2928, 2852, 1737, 1599, 1463, 1380, 1114, 1058 cm−1; ECD (MeOH) λmax (Δε) 204 (+2.26), 215 (−4.08), 306 (−4.52) nm; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 389.1578 [M + Na]+ (calcd for C19H26O7Na, 389.1576). Muralatin F (6): light yellow, amorphous powder; UV (MeOH) λmax (log ε) 207 (4.14), 260 (3.57), 321 (3.69) nm; IR (KBr) νmax 2954, 2922, 2850, 1714, 1679, 1605, 1459, 1328, 1249, 1114, 1101, 819 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 289.1078 [M + H]+ (calcd for C16H17O5, 289.1076). Muralatin G (7): light yellow, amorphous powder; UV (MeOH) λmax (log ε) 216 (4.13), 261 (3.82), 324 (3.90) nm; IR (KBr) νmax 2918, 1719, 1603, 1456, 1332, 1251, 1119, 1101, 816 cm−1; 1H and 13 C NMR data, see Tables 2 and 3; HRESIMS m/z 261.1127 [M + H]+ (calcd for C15H17O4, 261.1127). Muralatin H (8): white, amorphous powder; UV (MeOH) λmax (log ε) 215 (4.20), 260 (3.88), 322 (4.07) nm; IR (KBr) νmax 2955, 2922, 2850, 1724, 1603, 1464, 1334, 1249, 1135, 1110, 821 cm−1; 1H and 13 C NMR data, see Tables 2 and 3; HRESIMS m/z 249.0766 [M + H]+ (calcd for C13H13O5, 249.0763). Muralatin I (9): light yellow, amorphous powder; UV (MeOH) λmax (log ε) 212 (4.44), 320 (4.33) nm; IR (KBr) νmax 2955, 2924, 2852, 1734, 1605, 1461, 1252, 1092, 457 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 275.0917 [M + H]+ (calcd for C15H15O5, 275.0919). Muralatin J (10): white, amorphous powder; UV (MeOH) λmax (log ε) 212 (4.35), 260 (3.94), 319 (4.06) nm; IR (KBr) νmax 2918, 2850, 1733, 1597, 1467, 1339, 1115, 1018, 817, 468 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 305.1019 [M + H]+ (calcd for C16H17O6, 305.1025). Muralatin K (11): white, amorphous powder; [α]24D −27 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (4.20), 260 (3.44), 322 (3.78) nm; IR (KBr) νmax 3711, 2961, 2907, 2867, 1730, 1604, 1460, 1376, 1249, 1161, 1093, 457 cm−1; ECD (MeOH) λmax (Δε) 301 (−2.19) nm; Rh2(OCOCF3)4-induced ECD (CH2Cl2) λmax (Δε) 375 (+0.02) nm; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 327.1196 [M + Na]+ (calcd for C17H20O5Na, 327.1208). Murragleinin (23): light yellow oil; [α]24D −82 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 211 (−8.54), 303 (−5.65) nm. Mexoticin (24): light yellow oil; [α]24D −60 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 235 (−2.64), 286 (−2.70) nm. Murpanicin (27): white, amorphous powder; [α]24D −31 (c 0.1, MeOH); Rh2(OCOCF3)4-induced ECD (CH2Cl2) λmax (Δε) 345 (+0.06) nm.

Murranganon (30). light yellow oil; [α]24D +9 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 203 (+8.08), 210 (−9.39), 254 (−5.10), 286 (+7.32) nm; Rh2(OCOCF3)4-induced ECD (CH2Cl2) λmax (Δε) 354 (+0.17) nm. 8-(3′-Ethoxy-2′-hydroxy-3′-methylbutyl)-5,7-dimethoxy-2H-chromen-2-one. (33): colorless plates (MeOH); mp 131−132 °C; [α]24D −47 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 204 (+1.96), 217 (−3.67), 290 (−2.94) nm. X-ray Crystallography of Compound 33. The crystal structure and absolute configuration of 33 were determined using data collected at T = 174.7 K with Cu Kα radiation on an Agilent Gemini E X-ray single-crystal diffractometer, equipped with an Oxford Cryostream cooler. Structures were solved by direct methods using SHELXS-97 and refined anisotropically by full-matrix least-squares on F2 using SHELXL-97. The H atoms were placed in calculated positions and refined using a riding model. The absolute configuration was determined by refinement of the Flack parameter based on resonant scattering of the light atoms. Crystal data: 5,7-dimethoxy-8-(2′hydroxy-3′-ethoxy-3′-methylbutyl)coumarin (33), C18H24O6, M = 336.37, size 0.60 × 0.40 × 0.15 mm3, orthorhombic, a = 6.8035(11) Å, b = 9.6710(10) Å, c = 26.398(3) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1736.9(4) Å3, T = 174.7 K, space group P212121 (no. 19), Z = 4, μ(Cu Kα) = 0.796 mm−1; 6344 reflections measured, 3248 unique (Rint = 0.0307), which were used in all calculations. The final wR(F2) was 0.1327 (all data), and the Flack parameter −0.1(2). Crystallographic data for compound 33 have been filed with the Cambridge Crystallographic Data Centre (CCDC, deposition number: CCDC 1012370). These data can be obtained free of charge from the CCDC via http://www.ccdc.cam.ac.uk/Community/ Requestastructure/Pages/DataRequest.aspx. Culture, Viability Assay, and Measurement of NO Production. Murine monocytic RAW 264.7 macrophages (Peking Union Medical College Cell Bank, Beijing, People’s Republic of China) were used for the anti-inflammatory assay. The cells were cultured in DMEM (Hyclone, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Hyclone), penicillin (Macgene Biotech., Beijing, People’s Republic of China, 100 U/mL), and streptomycin (Macgene Biotech., 100 μg/mL) in a humidified incubator containing 95% air and 5% CO2 at 37 °C. Accumulation of NO in the culture medium was determined using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, People’s Republic of China), according to the manufacturer’s instructions. Briefly, RAW 264.7 cells were seeded into 48-well plates at a density of 1 × 105 cells/well and stimulated with 1.0 μg/mL LPS (Escherichia coli 0111:B4, Sigma, St. Louis, MO, USA) in the presence or absence of test compounds at 37 °C for 24 h. Supernatants (160 μL) were allowed to react with 80 μL of the Griess reagent (1% sulfanilamide/ 0.1% naphthylethylene diaminedihydrochloride/2% phosphoric acid) for 10 min at room temperature in the dark. The optical density was measured at 540 nm using a microplate reader (Tecan Trading AG, Switzerland). Sodium nitrite was used to prepare a standard curve in the assay. The experiments were performed in parallel three times, and the results are presented as the mean ± SD (n = 3). Indomethacin was used as a positive control. Cell viability was evaluated by MTT assay.

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ASSOCIATED CONTENT

S Supporting Information *

HRESIMS and 1H and 13C NMR spectra of 1−11. Isolation procedures and structures of the known compounds 12−32. Δδ (δR − δS) values (in ppm) for the MPA esters of 23, 24, and 30. Rh2(OCOCF3)4-induced ECD spectra of 11, 27, and 30. These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +86-10-82802719. F

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Notes

(29) Ito, C.; Furukawa, H. Heterocycles 1987, 26, 1731−1734. (30) Wu, T. S. Phytochemistry 1981, 20, 178−179. (31) Talapatra, S. K.; Dutta, L. N.; Talapatra, B. Tetrahedron Lett. 1973, 50, 5005−5008. (32) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512−519. (33) Anjaneyulu, A. S. R.; Raghu, P.; Ramakrishna Rao, K. V.; Row, L. R. Indian J. Chem. 1979, 18B, 391−394. (34) Zaki, M. A.; Balachandran, P.; Khan, S.; Wang, M.; Mohammed, R.; Hetta, M. H.; Pasco, D. S.; Muhammad, I. J. Nat. Prod. 2013, 76, 679−684. (35) Lv, H. N.; Tu, P. F.; Jiang, Y. Mini-rev. Med. Chem. 2014, 14, 603−622. (36) Sharma, J. N.; Al-Omran, A.; Parvathy, S. S. Inflammopharmacology 2007, 15, 252−259. (37) Blantz, R. C.; Munger, K. Nephron 2002, 90, 373−378. (38) Korhonen, R.; Lahti, A.; Kankaanranta, H.; Moilanen, E. Curr. Drug Targets Inflamm. Allergy 2005, 4, 471−479.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Sciences Foundation of China (NSFC; Nos. 81222051 and 81473106) and National Key Technology R&D Program “New Drug Innovation” of China (Nos. 2012ZX09301002-002-002 and 2012ZX09304-005).

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