Nitrogen Oxide Inhibitory Trimeric and Dimeric ... - ACS Publications

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Nitrogen Oxide Inhibitory Trimeric and Dimeric Carbazole Alkaloids from Murraya tetramera Hai-Ning Lv,† Ran Wen,† Ying Zhou,‡ Ke-Wu Zeng,† Jun Li,§ Xiao-Yu Guo,† Peng-Fei Tu,† and Yong Jiang*,†

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 1, 2015 | http://pubs.acs.org Publication Date (Web): September 1, 2015 | doi: 10.1021/acs.jnatprod.5b00527

†

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China ‡ Zhejiang Institute for Food and Drug Control, Hangzhou 310004, People’s Republic of China § Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China S Supporting Information *

ABSTRACT: Two new structurally unique trimeric carbazole alkaloids, murratrines A and B (1, 2), and 11 new carbazole dimers, murradines A−K (3−13), and four known analogues (14−17) were isolated from the leaves and stems of Murraya tetramera. The structures and relative configurations of 1−13 were elucidated on the basis of comprehensive 1D and 2D NMR spectroscopy, high-resolution mass spectrometry, and electronic circular dichroism (ECD) data analysis. Murratrines A and B (1, 2) both contain an unprecedented carbazole trimeric skeleton, and murradines A and D (3, 6) are the first natural C-1−C-3′-methyl-linked and C-6−C-3′-methyl-linked dimeric carbazole alkaloids, respectively. Compounds 4, 10, 14, 15, and 17 exhibited inhibition of nitric oxide production stimulated by lipopolysaccharide in BV-2 microglial cells with IC50 values in the range of 11.2−19.3 μM.

C

C6−C3′-methyl linkages, respectively. Murradines I and K (11 and 13) are the first examples of naturally occurring unsymmetrical carbazole dimers having a C-1−C-1′ linkage and an ether linkage, respectively.1−3 Herein, we described the isolation and structural characterization of compounds 1−13, and the inhibitory effects of these isolates on lipopolysaccharide (LPS)induced nitric oxide (NO) production in BV-2 microglial cells.

arbazole alkaloids are a group of natural products possessing a broad range of intriguing pharmacological activities, such as antioxidant, cytotoxic, analgesic, anti-HIV, antimicrobial, antidiarrheal, and anti-inflammatory effects.1−3 Many of the biologically active carbazole alkaloids have been isolated from taxonomically related higher plants of the genera Murraya, Clausena, and Glycosmis from the family Rutaceae, among which, the genus Murraya is the most common plant source of this type of compounds.1−3 Murraya tetramera C. C. Huang, a small tree, is distributed widely in Guangxi and Yunnan Provinces of mainland China. Several parts of this plant have been used as folk medicines by local people, and for example, the leaves and stems have been used for the treatment of cough, bronchitis, asthma, eczema, and acute conjunctivitis, and the roots administrated for rheumatism, dropsy, traumatic injury, and insect and snake bites.4,5 Previous chemical investigations of M. tetramera have led to reports of flavonoids, anthraquinones, coumarins, sesquiterpenes, and long chain alkanes from this species.6−8 In a search for new bioactive compounds from this plant, the 95% aqueous ethanol extract of the leaves and stems was investigated to afford 13 new carbazole alkaloids, named murratrines A and B (1 and 2), and murradines A−K (3−13), along with four known analogues (14−17). Murratrines A and B (1 and 2) possess an unprecedented trimeric skeleton, which represents a new class of carbazole scaffolds. Murradines A−K (3−13) are new carbazole dimers, among which, murradines A and D (3 and 6) contain unprecedented C-1−C-3′-methyl and © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The 95% aqueous EtOH extract of the leaves and stems of M. tetramera was suspended in H2O and partitioned with CHCl3. The CHCl3-soluble portion was subjected to silica gel and Sephadex LH-20 column chromatography (CC) followed by semipreparative RP-HPLC to afford two new carbazole trimers (1, 2), and 11 new (3−13) and four known (14−17) carbazole dimers. Murratrine A (1) was obtained as a brown amorphous powder. Its molecular formula, C42H35N3O4, was deduced from the positive-ion HRMALDIMS (m/z 645.2623 [M]+, calcd for C42H35N3O4, 645.2622) and 13C NMR spectroscopic data, indicating 27 indices of hydrogen deficiency. The UV spectrum showed absorptions at 237, 265, 294, and 335 nm, suggesting the presence of a carbazole skeleton in the molecule.9−11 Analysis of the 1H and 13C NMR data (Table 1) revealed the presence of two Received: June 14, 2015

A

DOI: 10.1021/acs.jnatprod.5b00527 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. NMR Spectroscopic Data of 1 and 2 in CDCl3 (δ in ppm, J in Hz, 500 MHz for 1H NMR, 125 MHz for 13C NMR)


1

ortho-disubstituted phenyl moieties [δH 7.99 (1H, d, J = 7.8 Hz, H-5), 7.24 (1H, t, J = 7.8 Hz, H-6), 7.52 (1H, t, J = 7.8 Hz, H-7), and 7.50 (1H, d, J = 7.8 Hz, H-8); δH 8.03 (1H, d, J = 7.3 Hz, H-5″), 7.21 (1H, t, J = 7.3 Hz, H-6″), 7.49 (1H, t, J = 7.3 Hz, H-7″), and 7.50 (1H, d, J = 7.3 Hz, H-8″)], four 1,2,4-trisubstituted phenyl unities [δH 7.45, 7.50 (each 1H, d, J = 8.3 Hz, H-1 and H-2), 8.02 (1H, s, H-4); δH 7.45, 7.50 (each 1H, d, J = 8.3 Hz, H-1′ and H-2′), 8.06 (1H, s, H-4′); δH 7.38, 7.50 (each 1H, d, J = 8.2 Hz, H-7′ and H-8′), 7.90 (1H, s, H-5′); δH 7.50, 7.38 (each 1H, d, J = 8.2 Hz, H-1″ and H-2″), 7.91 (1H, s, H-4″)], three methylene groups [δH 4.74, 4.74, and 4.33 (each 2H, s)], and three N-methoxy signals [δH 4.12, 4.11, 4.11 (each 3H, s)].9 Thus, 1 was assigned as a carbazole trimer. The HMBC correlation of the CH2-3 protons and CH2-3′, and their reverse correlation implied the presence of a bismethylene ether linkage between two of the carbazole units, the same as for 3,3′[oxybis(methylene)]bis(9-methoxy-9H-carbazole) (16).9 The third carbazole unit was deduced to be connected to the middle carbazole unit via a C-6′−C-3″-methyl-linked mode, on the basis of HMBC correlations from the CH2-3″ protons to C-5′/C-6′/ C-7′/C-2″/C-3″/C-4″, from H-5′/H-7′ to CH2-3″, and from H-2″/H-4″ to CH2-3″ (Figure 1). The ROESY correlations of the protons of CH2-3″ and H-5′/H-7′/H-2″/H-4″ also supported the above deduction. Thus, the structure of murratrine A (1), the first trimeric carbazole alkaloid, was proposed as shown. Murratrine B (2) gave a molecular formula of C41H33N3O3 on the basis of its 13C NMR and HRMALDIMS data [m/z 615.2516 [M]+ (calcd for C41H33N3O3, 615.2516)]. Analysis of the NMR data (Table 1) indicated that 2 is also a trimeric carbazole alkaloid. The main differences found were in the disappearance of one of the N-methoxy proton resonances of 1 in 2. A deshielded methylene resonance at δH 5.71 (2H, s, CH2-3″)

2

position

δH

δC, type

δH

δC, type

1 2 3 4 4a 4b 5 6 7 8 8a 9a 1′ 2′ 3′ 4′ 4a′ 4b′ 5′ 6′ 7′ 8′ 8a′ 9a′ 1″ 2″ 3″ 4″ 4a″ 4b″ 5″ 6″ 7″ 8″ 8a″ 9a″ CH2−3 CH2−3′ CH2−3″ OCH3-9 OCH3-9′ OCH3-9″

7.45, d (8.3) 7.50, d (8.3)

108.4, CH 126.6, CH 133.8, C 120.5, CH 120.5, C 120.0, C 120.6, CH 120.1, CH 126.0, CH 108.4, CH 137.9, C 138.1, C 108.2, CH 126.7, CH 134.0, C 120.6, CH 120.3, C 120.3, C 120.5, CH 130.1, C 127.4, CH 108,4, CH 136.5, C 137.3, C 108.5, CH 127.4, CH 130.2, C 120.5, CH 120.5, C 120.0, C 120.5, CH 119.9, CH 126.1, CH 108.4, CH 137.8, C 136.8, C 72.4, CH2 72.4, CH2 42.0, CH2 63.4, CH3 63.4, CH3 63.4, CH3

7.52 d (8.3) 7.50 d (8.3)

4.78, s 4.79, s 5.71, s 4.13, s

108.3, CH 126.7, CH 130.3, C 120.5, CH 120.2, C 120.0, C 120.5, CH 120.1, CH 126.1, CH 108.4, CH 138.0, C 137.3, C 108.7, CH 126.5, CH 129.1, C 120.5, CH 123.1, C 123.1, C 120.6, CH 120.1, CH 125.9, CH 109.0, CH 141.1, C 140.5, C 109.1, CH 124.7, CH 129.0, C 118.5, CH 119.6, C 120.0, C 120.6, CH 119.2, CH 126.3, CH 108.3, CH 137.9, C 137.1, C 72.4, CH2 72.6, CH2 47.1, CH2 63.5, CH3

4.08, s

63.5, CH3

8.02, s

7.99, d (7.8) 7.21, t (7.8) 7.52, t (7.8) 7.50, d (7.8)

7.45, d (8.3) 7.50, d (8.3) 8.06, s

7.90, s 7.38, d (8.2) 7.50, d (8.2)

7.50, d (8.2) 7.38, d (8.2) 7.91, s

8.03, d (7.3) 7.24, t (7.3) 7.49, t (7.3) 7.50, d (7.3)

4.74, s 4.74, s 4.33, s 4.11, s 4.11, s 4.12, s

8.08, s

8.04, d (7.9) 7.25, t (7.9) 7.48, t (7.9) 7.53, d (7.9)

7.46 d (8.3) 7.49 d (8.3) 8.18, s

8.15, d (7.5) 7.27, t (7.5) 7.45, t (7.5) 7.45, d (7.5)

7.40, d (8.5) 7.29, d (8.5) 7.88, s

7.93, d (7.6) 7.20, t (7.6) 7.45, t (7.6) 7.51, d (7.6)

implied the presence of a N−C-3″-methyl linkage in 2,12−14 which was supported by HMBC correlations between the protons of CH2-3″ and C-8a′/C-9a′/C-2″/C-3″/C-4″. Therefore, the structure of murratrine B (2), the second naturally occurring trimeric carbazole alkaloid obtained, was established as shown. Murradine A (3) was obtained as a brown amorphous powder, and its molecular formula was determined as C27H22N2O2 from the 13C NMR spectroscopic data and the HRMALDIMS ion at m/z 406.1674 [M]+ (calcd 406.1676). The UV spectrum showed absorptions at 239, 292, and 302 nm, which are typical for a carbazole nucleus.9−11 Two sets of ortho-disubstituted phenyl protons [δH 7.98 (1H, d, J = 7.5 Hz, H-5), 7.17 (1H, t, J = 7.5 Hz, H-6), 7.40 (1H, t, J = 7.5 Hz, H-7), and 7.28 (1H, d, J = 7.5 Hz, H-8); δH 7.96 (1H, d, J = 7.7 Hz, H-5′), 7.19 (1H, t, J = 7.7 Hz, B




Article

methoxy group at C-1. Accordingly, the structure of murradine C (5) was established as 1-methoxy-4-[(9H-carbazol-3-yl)methyl]-3-methyl-9H-carbazole, a new C-4−C-3′-methyl-linked dimeric carbazole alkaloid. Murradine D (6) was obtained as a brown amorphous powder. Its molecular formula, C28H24N2O3, was deduced from the HRMALDIMS (m/z 436.1781 [M]+, calcd for C28H24N2O3, 436.1781) and 13C NMR spectroscopic data. The 1H NMR data (Table 2) displayed the resonances of a group of orthodisubstituted aromatic protons [δH 8.02 (1H, d, J = 7.8 Hz, H-5′), 7.20 (1H, t, J = 7.8 Hz, H-6′), 7.39 (1H, t, J = 7.8 Hz, H-7′), and 7.44 (1H, d J = 7.8 Hz, H-8′)], a set of ortho-coupled aromatic doublets at δH 7.25 and 7.33 (each 1H, d, J = 8.3 Hz, H-7 and H-8), four aromatic singlets at δH 7.81, 7.57, 7.52, and 6.79 (each 1H, s), a methylene singlet at δH 4.31 (2H, s), a methyl singlet at δH 2.39 (3H, s), and two methoxy groups at δH 4.02 and 3.95 (each 3H, s). The above information indicated a dimeric carbazole skeleton for 6.9−11 The HMBC spectrum showed correlations of the methylene protons with C-5/C-6/C-7/C-2′/ C-3′/C-4′, revealing a C-6−C-3′-methyl-linked pattern between the two carbazole units (Figure 1). The locations of the two methoxy groups at C-1 and C-1′ were inferred from the HMBC correlations between the methoxy protons and C-1/C-1′, respectively. HMBC correlations from H-4 to C-2 (δC 145.1) and from the methyl protons to C-2/C-3/C-4 were used to place the hydroxy and methyl groups at C-2 and C-3, respectively. Therefore, the structure of murradine D (6), the first example of a C-6−C-3′-methyl-linked dimeric carbazole alkaloid, was defined as 2-hydroxy-1-methoxy-6-[(1-methoxy-9H-carbazol-3-yl) methyl]-3-methyl-9H-carbazole. Murradine E (7) was obtained as a brown amorphous powder. Its molecular formula was established as C28H24N2O3 on the basis of the 13C NMR and HRMALDIMS data (m/z 436.1780 [M]+, calcd for C28H24N2O3, 436.1781). The NMR spectrum of 7 (Tables 2 and 3) displayed resonances consistent with a N−C3′-methyl-linked biscarbazole skeleton.12−14 Comparison of the 1 H and 13C NMR data of 7 (Tables 2 and 3) with those of bismurrayafoline A12 indicated that 7 is the hydroxylated product of bismurrayafoline A. The hydroxylated position was deduced to be located at C-2 (δC 146.4) from the HMBC correlations of the methyl protons and H-4 with C-2. Hence, the structure of murradine E (7) was assigned as 2-hydroxy-1-methoxy-9-[(1methoxy-9H-carbazol-3-yl)methyl]-3-methyl-9H-carbazole, a new N−C-3′-methyl-linked dimeric carbazole alkaloid. Murradine F (8) was isolated as an amorphous powder. A [M]+ ion peak at m/z 406.1675 in the HRMALDIMS, together with its 13C NMR data, suggested a molecular formula of C27H22N2O2 (calcd 406.1676). Its 1D and 2D NMR data showed many similarities to those of murradine E (7), except for the disappearance of a methoxy singlet, and the replacement of an aromatic singlet in 7 by a pair of ortho-coupled doublets in 8. This suggested that 8 is a demethoxy derivative of 7. Therefore, when supported with confirmatory HMBC correlations, the structure of murradine F (8) was deduced as 2-hydroxy-1-methoxy-9[(9H-carbazol-3-yl)methyl]-3-methyl-9H-carbazole. Murradine G (9) was shown to have the same molecular formula as 8, according to its 13C NMR data and the [M]+ ion at m/z 406.1675 in the HRMALDIMS (calcd for C27H22N2O2, 406.1676). It was found to be a demethoxy derivative of 7 by comparing their 1H and 13C NMR data (Tables 2 and 3). A newly appearing aromatic singlet at δH 6.84 exhibited HMBC correlations with C-3 and C-4a, suggesting it to be the H-1 proton. Accordingly, the signal of OCH3-1 of 7 was absent.

Figure 1. Key HMBC correlations of compounds 1, 3, and 6.

H-6′), 7.27 (1H, t, J = 7.7 Hz, H-7′), and 7.44 (1H, d, J = 7.7 Hz, H-8′)], three aromatic protons at δH 7.77, 6.81, and 7.60 (each 1H, s, H-4, H-2′, and H-4′), a methylene singlet at δH 4.48 (2H, s), a methoxy singlet at δH 3.90 (3H, s), and a methyl singlet at δH 2.46 (3H, s) were observed in the 1H NMR spectrum (Table 2). The 13C NMR data (Table 3) showed 27 carbon resonances, comprising 24 olefinic carbons, a methyl group, a methylene group, and a methoxy group. The above information coupled with biogenetic considerations and literature information1−3,9−11 indicated the dimeric carbazole skeleton of 3. The HMBC correlations of the methyl protons with C-2/C-3/C-4, the protons of CH2-3′ and H-4 with C-2 (δC 151.1), and the methoxy protons with C-1′ were used to locate methyl, hydroxy, and methoxy groups at C-3, C-2, and C-1′, respectively. Additionally, the HMBC correlations from the CH2-3′ protons to C-1/C-2/C-9a/C-2′/C-3′/C-4′, and H-2′/H-4′ to CH2-3′ revealed a C-1−C-3′-methyl linkage between the two carbazole moieties (Figure 1). Thus, the structure of murradine A (3) could be defined as 2-hydroxy-1-[(1-methoxy-9H-carbazol-3yl)methyl]-3-methyl-9H-carbazole. This is the first report of C-1−C-3′-methyl-linked dimeric carbazole alkaloid from a plant source. Murradine B (4) gave a molecular formula of C28H24N2O2, as established by 13C NMR data and a [M]+ ion at m/z 420.1831 (calcd 420.1832) in the HRMALDIMS. Analysis of 1H and 13C NMR data of 4 (Tables 2 and 3) showed a close structural resemblance to murrafoline F,10 a dimeric carbazole isolated from M. euchrestifolia with the same molecular formula. The difference between these two compounds is that one of the methoxy groups in murrafoline F is shifted from the nitrogen atom to the C-1′ position, as deduced for 4 from the HMBC correlations of the methoxy group protons [δH 3.92 (3H, s)] and C-1′ (δC 145.5), and H-2′ and C-1′/C-9a′/C-3′/C-4′/ CH2-3′. Hence, the structure of murradine B (4) was assigned as 1-methoxy-2-[(1-methoxy-9H-carbazol-3-yl)methyl]-3-methyl9H-carbazole, a new C-2−C-3′-methyl-linked dimeric carbazole alkaloid. Murradine C (5) was obtained as a brown amorphous powder with a molecular formula of C27H22N2O, as deduced from the 13C NMR and HRMALDIMS data (m/z 390.1726 [M]+, calcd for C27H22N2O, 390.1727). The UV and NMR data (Tables 2 and 3) of 5 were closely comparable to those of chrestifoline A (14),11 except for the disappearance of one methoxy group in 5, and the replacement of an aromatic singlet in 14 by two aromatic doublets. This suggested that 5 is the demethoxy derivative of 14. Further 2D NMR analysis was used to place the remaining C


1 2 4 5 6 7 8 1′ 2′ 4′ 5′ 6′ 7′ 8′ CH3-3 CH3-3′ CH2−3 CH2−3′ OCH3-1 OCH3-7 OCH3-9 OCH3-1′ OCH3-7′ CHO-3 CHO-8 CHO-8′

position

4.43, s 3.90, s

4.48, s

3.92, s

6.77, s 7.33, s 7.92, d (7.9) 7.14, t (7.9) 7.35, t (7.9) 7.41, d (7.9) 2.42, s

6.81, s 7.60, s 7.96, d (7.7) 7.19, t (7.7) 7.27, t (7.7) 7.44, d (7.7) 2.46, s

3.90, s

7.72, s 8.05, d (7.6) 7.24, t (7.6) 7.42, t (7.6) 7.48, d (7.6)

4

7.77, s 7.98, d (7.5) 7.17, t (7.5) 7.40, t (7.5) 7.28, d (7.5)

3

4.79, s 4.05, s

8.05, d (7.7) 7.15, t (7.7) 7.37, t (7.7) 7.45, d (7.7) 7.34, d (8.5) 7.25, d (8.5) 7.86, s 7.93, d (7.8) 7.05, t (7.8) 7.33, t (7.8) 7.34, d (7.8) 2.47, s

6.84, s

5

3.95, s

4.31, s 4.02, s

6.79, s 7.57, s 8.02, d (7.8) 7.20, t (7.8) 7.39, t (7.8) 7.44, d (7.8) 2.39, s

7.25, d (8.3) 7.33, d (8.3)

7.52, s 7.81, s

6

3.85, s

5.92, s 3.74, s

6.75, s 7.46, s 7.90, d (7.6) 7.16, t (7.6) 7.33, t (7.6) 7.41, d (7.6) 2.47, s

7.68, s 8.00, d (7.5) 7.21, t (7.5) 7.37, t (7.5) 7.33, d (7.5)

7

5.93, s 3.72, s

7.67, s 7.99, d (7.5) 7.19, t (7.5) 7.39, t (7.5) 7.31, d (7.5) 7.29, d (8.2) 7.18, d (8.2) 7.88, s 7.95, d (7.8) 7.19, t (7.8) 7.38, t (7.8) 7.30, d (7.8) 2.45, s

8

Table 2. 1H NMR (500 MHz) Spectroscopic Data of Compounds 3−13 in CDCl3 (δH in ppm, J in Hz)

3.84

5.57, s

6.71, s 7.51, s 7.94, d (7.9) 7.18, t (7.9) 7.35, t (7.9) 7.43, d (7.9) 2.42, s

7.86, s 8.02, d (7.7) 7.21, t (7.7) 7.39, t (7.7) 7.39, d (7.7)

6.84, s

9

D

10.12, s

3.84, s

5.73, s

6.70, s 7.51, s 7.93, d (7.8) 7.19, t (7.8) 7.40, t (7.8) 7.44, d (7.8)

7.58, d (8.3) 8.00, d (8.3) 8.69, s 8.22, d (7.5) 7.36, t (7.5) 7.51, t (7.5) 7.54, d (7.5)

10


10.46, s

4.00, s

7.97, s 8.03, d (7.4) 7.22, t (7.4) 7.29, t (7.4) 7.24, d (7.4) 2.50, s 2.52, s

7.87, s 8.12, d (8.5) 6.81, d (8.5)

11

10.46, s 10.46, s

4.00, s

4.00, s

2.52, s 2.52, s

7.91, s 8.15, d (8.5) 6.81, d (8.5)

7.91, s 8.15, d (8.5) 6.81, d (8.5)

12

4.13, s

4.78, s 4.78, s

7.52, brs 7.52, brs 8.08, s 8.04, d (7.4) 7.26, t (7.4) 7.47, t (7.4) 7.52, d (7.4) 7.48, d (8.4) 7.44, d (8.4) 8.11, s 8.09, d (7.4) 7.24, t (7.4) 7.44, t (7.4) 7.50, d (7.4)

13

Journal of Natural Products Article


4

δC, type

143.7, C 110.0, C 129.9, C 117.3, CH 123.7, C 123.9, C 120.5, CH 119.1, CH 125.6, CH 110.8, CH 139.7, C 131.6, C 145.5, C 106.9, CH 132.7, C 111.7, CH 123.6, C 124.2, C 120.2, CH 119.5, CH 125.4, CH 110.8, CH 139.4, C 128.2, C 20.3, CH3

32.6, CH2 61.3, CH3

55.5, CH3

3

δC, type


32.0, CH2

55.6, CH3

position

1 2 3 4 4a 4b 5 6 7 8 8a 9a 1′ 2′ 3′ 4′ 4a′ 4b′ 5′ 6′ 7′ 8′ 8a′ 9a′ CH3-3 CH3-3′ CH2-3 CH2-3′ OCH3-1 OCH3-7 OCH3-9 OCH3-1′ OCH3-7′ CHO-3 CHO-8 CHO-8′

E

35.3, CH2 55.6, CH3

143.8, C 109.0, CH 125.6, C 127.9, C 124.1, C 123.9, C 122.7, CH 119.1, CH 125.0, CH 110.5, CH 139.9, C 128.7, C 110.4, CH 126.3, CH 131.0, C 119.5, CH 123.5, C 123.4, C 120.4, CH 119.3, CH 125.6, CH 110.7, CH 140.0, C 138.0, C 19.6, CH3

δC, type

5

55.5, CH3

42.5, CH2 60.7, CH3

131.3, C 145.1, C 117.6, C 117.0, CH 117.9, C 124.2, C 119.6, CH 133.4, C 125.8, CH 110.4, CH 138.2, C 131.2, C 145.6, C 107.5, CH 133.9, C 112.8, CH 124.3, C 123.6, C 120.6, CH 119.2, CH 125.5, CH 110.9, CH 139.4, C 128.4, C 16.0, CH3

δC, type

6

55.5, CH3

48.6, CH2 62.2, CH3


δC, type

7

48.2, CH2 62.2, CH3

132.4, C 146.4, C 117.6, C 117.3, CH 118.2, C 123.8, C 119.3, CH 119.4, CH 124.6, CH 109.5, CH 141.4, C 131.7, C 110.6, CH 124.1, CH 129.5, C 117.9, CH 123.0, C 123.6, C 120.4, CH 119.2, CH 126.0, CH 110.8, CH 139.8, C 138.7, C 16.0, CH3

δC, type

8

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

55.5, CH3

47.7, CH2

95.3, CH 153.2, C 116.0, C 121.9, CH 117.0, C 123.2, C 119.4, CH 119.0, CH 124.4, CH 108.7, CH 140.9, C 141.0, C 146.0, C 104.6, CH 129.2, C 110.8, CH 124.4, C 123.3, C 120.6, CH 119.3, CH 125.8, CH 111.0, CH 139.4, C 129.1, C 16.1, CH3

δC, type

9

191.7, CH

55.5, CH3

47.9, CH2

109.4, CH 127.5, CH 128.9, C 123.8, CH 123.4, C 123.4, C 120.8, CH 120.6, CH 126.9, CH 109.9, CH 141.7, C 144.6, C 146.1, C 104.4, CH 128.0, C 110.9, CH 124.3, C 123.2, C 120.6, CH 119.5, CH 126.0, CH 111.0, CH 139.4, C 129.4, C

δC, type

10


190.5, CH

56.3, CH3

100.3, C 151.0, C 118.9, C 122.3, CH 117.3, C 118.7, C 127.2, CH 102.8, CH 161.0, C 108.9, C 139.8, C 138.3, C 98.5, C 151.2, C 117.8, C 123.4, CH 115.9, C 123.8, C 119.7, CH 119.5, CH 124.7, CH 110.6, CH 139.4, C 137.7, C 16.7, CH3 16.8, CH3

δC, type

11

190.6, CH 190.6, CH

56.3, CH3

56.3, CH3

99.7, C 150.9, C 118.9, C 122.7, CH 116.1, C 108.9, C 127.4, CH 102.8, CH 161.0, C 118.8, C 139.9, C 138.3, C 99.7, C 150.9, C 118.9, C 122.7, CH 116.1, C 108.9, C 127.4, CH 102.8, CH 161.0, C 118.8, C 139.9, C 138.3, C 16.7, CH3 16.7, CH3

δC, type

12

63.5, CH3

72.4, CH2 72.6, CH2

108.3, CH 126.7, CH 130.3, C 130.4, CH 120.0, C 120.1, C 120.6, CH 120.2, CH 126.1, CH 108.4, CH 138.0, C 137.4, C 108.4, CH 126.5, CH 129.5, C 120.5, CH 118.5, C 119.5, C 120.5, CH 119.5, CH 126.0, CH 108.4, CH 139.8, C 139.2, C

δC, type

13

Journal of Natural Products Article




Article

By comparing their spectroscopic data with literature values, the four known compounds obtained in this investigation were identified as chrestifoline A (14),11 bismurrayafolinol (15),13 chrestifoline D (16),14 and 3,3′-[oxybis(methylene)]bis(9methoxy-9H-carbazole) (17).9 All the isolated compounds (1−17) were evaluated for their inhibitory effects on NO production in LPS-stimulated BV-2 microglial cells using the Griess assay, consistent with the traditional anti-inflammation utilization of the plant, M. tetramera.4,5,19,20 As shown in Table 4, compounds 4, 10,

Thus, the structure of murradine G (9) was established as 2-hydroxy-9-[(1-methoxy-9H-carbazol-3-yl)methyl]-3-methyl9H-carbazole. Murradine H (10) was obtained as an off-white amorphous powder. It exhibited a [M + H]+ quasimolecular ion at m/z 405.1597 in the positive HRMALDIMS, which, in conjunction with the 13C NMR data, suggested a molecular formula of C27H20N2O2 (calcd for C27H21N2O2, 405.1598). The 1H NMR data of 10 (Table 2) were found to be similar to those of chrestifoline D (16).14 The apparent differences were the replacement of an aromatic singlet and a methoxy singlet in 16 by a pair of aromatic doublets in 10, suggesting that 10 is a demethoxy derivative of 16. Therefore, after the performance of confirmatory HMBC correlations, the structure of murradine H (10) was assigned as 9-[(1-methoxy-9H-carbazol-3-yl)methyl]-9H-carbazole-3-carboxaldehyde. Murradine I (11) was obtained as a brown amorphous powder, in optically active form ([α]22D + 93, c 0.1, MeOH). The HRMALDIMS gave a pseudomolecular ion peak at m/z 473.1474 [M + Na]+ (calcd for C28H22N2O4Na, 473.1472), corresponding to a molecular formula of C28H22N2O4. Analysis of the 1D and 2D NMR data suggested that the structure of 11 is similar to that of bis-2-hydroxy-3-methylcarbazole,15 except for the presence of an additional methoxy group [δH 4.00 (3H, s); δC 56.3] and a formyl group [δH 10.46 (1H, s); δC 190.5], which were deduced to be located at C-7 and C-8, respectively, via the HMBC correlations of the methoxy protons and C-7, and the formyl proton and C-7/C-8/C-8a. The electronic circular dichorism (ECD) spectrum of 11 showed positive Cotton effects at 218 and 240 nm, indicating a aR-biphenyl configuration for 11.16,17 Accordingly, the structure of murradine I (11) was defined as (aR)-2,2′-dihydroxy-7-methoxy-3,3′-dimethyl-(1,1′bi-9H-carbazole)-8-carboxaldehyde. This is the first naturally occurring C-1−C-1′-linked unsymmetrical dimeric carbazole alkaloid to have been found. Murradine J (12) was isolated as an off-white amorphous powder, [α]22D − 72. Its molecular formula was determined as C30H24N2O6 via its 13C NMR and HRMALDIMS data (m/z 531.1529 [M + Na]+). The UV spectrum showed a close similarity to that of 11, suggesting that these two compounds bear the same carbazole skeleton. The 13C NMR data of 12 exhibited only 15 carbon signals, suggesting that it is a symmetrical carbazole dimer. The NMR data of the monomeric unit of 12 resembled those of murrayaline B,18 except for the absence of the H-1 proton and a shift of the C-1 signal downfield to δC 99.7, indicating that the two units are linked through C-1− C-1′. In the ECD spectrum, 12 showed a negative Cotton effect at 226 nm, indicating a aS-biphenyl configuration.16,17 Therefore, the structure of 12 was established as (aS)-2,2′-dihydroxy-7,7′dimethoxy-3,3′-dimethyl-[1,1m-bis(9H-carbazole-8-carboxaldehyde)]. Murradine K (13) gave a molecular formula of C27H22N2O2, as determined from its 13C NMR and HRMALDIMS data (m/z 406.1675 [M]+, calcd for C27H22N2O2, 406.1676), indicating 18 indices of hydrogen deficiency. The UV and NMR data (Tables 2 and 3) of 13 resembled those of 3,3′-[oxybis(methylene)]bis(9-methoxy-9H-carbazole) (17),9 except for the absence of a N-OCH3 singlet. In combination with relevant HMBC correlations, the structure of murradine K (13), the first naturally occurring ether-linked unsymmetrical dimeric carbazole alkaloid, was deduced as 9-methoxy-3,3′-[oxybis(methylene)]bis(9H-carbazole).

Table 4. Inhibitory Effects of the Isolates on LPS-Activated NO Production in BV-2 Microglial Cells compounda

IC50 (μM)

1 4 5 10 13 14 15 16 17 quercetinb

27.2 ± 1.8 11.4 ± 3.2 26.7 ± 3.1 17.9 ± 2.0 21.9 ± 3.9 18.6 ± 2.7 19.3 ± 4.1 38.5 ± 2.5 11.2 ± 3.2 17.4 ± 0.8

Compound 2 was inactive (

Nitrogen Oxide Inhibitory Trimeric and Dimeric ... - ACS Publications

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