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Acylphloroglucinols from Callistemon lanceolatus DC.
Tetrahedron 69 (2013) 6070e6075
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Acylphloroglucinols from Callistemon lanceolatus DC. Suthida Rattanaburi a, Wilawan Mahabusarakam a, b, *, Souwalak Phongpaichit b, c, Anthony R. Carroll d, e a
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Natural Products Research Center of Excellence, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand d Griffith School of Environment (Gold Coast Campus), Griffith University, Qld 4222, Australia e Environmental Futures Centre, Griffith University, Gold Coast, Qld 4222, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2013 Received in revised form 10 May 2013 Accepted 20 May 2013 Available online 24 May 2013
Five acylphloroglucinols, named callistenones AeE together with six known acylphloroglucinols, triterpenoids, and C-methylflavonoids were isolated from the leaves of Callistemon lanceolatus. Their structures were characterized by spectroscopic methods. Some of the compounds showed very strong antibacterial activity. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Callistemon lanceolatus Myrtaceae Acylphloroglucinols b-Triketones Antibacterial activity
1. Introduction
2. Result and discussion
Infectious diseases are a major problem worldwide because of the emergence of resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). S. aureus can cause a wide range of diseases from skin infections, hospital-acquired bacteremia to lifethreatening infections such as necrotizing fasciitis and pneumonia, septic arthritis, osteomyelitis, and endocarditis. MRSA now has emerged as a widespread cause of community infection. There is a concern because MRSA is becoming resistant to multiple antibiotics.1 Therefore, there is a need to find new antimicrobial agents to combat this pathogen. Natural products isolated from plants are an important source for the discovery of new antimicrobial substances. We have screened extracts from some Myrtaceae plants collected in Thailand for antibacterial activity and found that a crude extract from the leaves of Callistemon lanceolatus DC. (Myrtaceae), which is known as crimson bottlebrush, was very effective at inhibiting the growth of S. aureus. This prompted us to identify the active constituents from the leaves of C. lanceolatus.
The CH2Cl2 extract of the leaves of C. lanceolatus was tested for antibacterial and antifungal activities. The results showed that the extract was growth inhibitory against S. aureus ATTC25923, and MRSA SK1 with an MIC of 4 mg/mL for both strains, but showed no effect against Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922, Callistemon neoformans ATCC90113, Callistemon albicans NCPF3153, and Microsporum gypseum at a concentration of 200 mg/mL. Investigation of the active compounds present in the CH2Cl2 extract has resulted in the isolation of 11 compounds. On the basis of 1H, 13C NMR, COSY, HMQC, and HMBC spectra as well as by comparison with previous reports, callistenones AeE (1e5) were identified as new acylphloroglucinol derivatives while the remaining six compounds were found to be the known compounds, flavesone (6),2 leptospermone (7),2 rhodomyrtosone D (8),3 endoperoxide G3 (9),4 betulinaldehyde (10),5 and 5-hydroxy-7,40 dimethoxy-6,8-dimethylflavone (11).6 This is the first time that compounds 6e9 have been isolated from C. lanceolatus (Fig. 1). Callistenone A (1) was obtained as a yellowish gum. A molecular ion peak [MþH]þ at m/z 429.2270 in an HRFABMS represents C25H33O6 corresponded to a molecular formula of C25H32O6. The EIMS spectrum of 1 exhibited a base peak at m/z 385.0 [MCH(CH3)2], which suggested facile loss of an isopropyl group from the molecule. The IR spectrum exhibited stretching bands for
* Corresponding author. Tel.: þ66 7428 8432; fax: þ66 7428 8432/55 8841; e-mail address: [email protected] (W. Mahabusarakam). 0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2013.05.085
S. Rattanaburi et al. / Tetrahedron 69 (2013) 6070e6075 4' 12
H
13
O
5
4a O 4b
12
OH
3
11
9
1
7
8a 1''
O
10
OH
4a O 4b
11 10
5'
1''
O 3''
11'''
O 11 10
7''' 6'''
4a O 5 4 4b A B C 9
O 2''
1''
1'
OH
O
12
O
5'
4
9
O
8
15'''
1''
O
O
O
O
OH O
OH
O
5'
OH
4'
OH
O
O
3''
7
6
OCH 3
CH3
O O
5 D O 6 8 1'
5
O O
14'''
6'''
OH 2''
O H
O E 4'''
C
B
1
10
4'
4a O 4b
A
11
7'''
13
4
O
13'''
1'''
3''
O
12'''
O
10'''
14''' O OH
8
OH 2"
3
13'''
4'''
OH 12
1" 3"
2''
11'''
O 1'''
9'''
1
O
14
8
7
1
OH
12'''
10''' O 13
15
7
10
5
2
1
12
3
9
1
O OH OH 1'
3'
17
O
OH
5 8a
O
5'
4' 16
1'
3
3''
2''
5'
O
13
O
4'
1'
6071
CHO
O
O
H3 C
HO
9
H3 CO
OH 10
O 11
Fig. 1. Structures of compounds 1e11 from C. lanceolatus.
hydroxyl, saturated carbonyl, and unsaturated carbonyl groups at 3265, 1716, and 1624 cm1, respectively. The presence of carbonyl groups was also deduced from the carbon resonances at dC 207.6 (C10 ), 197.9 (C-1), and 212.6 (C-3) in the 13C NMR spectrum (Table 1). In addition the 13C NMR spectrum contained signals for 8 sp2 hybridized carbons between dC 94.2 and 168.5 and 14 aliphatic carbons upfield of dC 56.6. The 1H NMR spectrum (Table 1) showed resonances of four tertiary methyl groups at dH 1.37 (H3-10), 1.31 (H3-11), 1.57 (H3-12), and 1.43 (H3-13). In the HMBC spectrum, H310 and H3-11 showed HMBC correlations to carbonyl carbons C-1 (dC 197.9) and C-3 (dC 212.6) whereas H3-12 and H3-13 showed correlations to the carbonyl carbon C-3 (dC 212.6) and an oxygenated vinylic carbon C-4a (dC 168.5). These data indicated the presence of a b-triketone moiety.3 The presence of a di-C-substituted phloroglucinol moiety was deduced from HMBC correlations observed from an aromatic proton (dH 6.34, s, H-5) to two oxygenated aromatic carbons at dC 158.0 (C-4b) and dC 164.0 (C-6) and two upfield non-protonated sp2 carbons at dC 108.5 (C-7) and dC 104.8 (C-8a), and from a phenolic hydroxyl proton (dH 14.22, 8-OH) also to the two upfield non-protonated sp2 carbons C-7 and C-8a in addition to an oxygenated aromatic carbon at dC 160.9 (C-8). COSY correlations between two equivalent secondary methyl protons H340 and H3-50 (dH 0.96, d, J¼6.6 Hz) and a methine proton H-30 (dH 2.22e2.30, m) and further correlations from H-30 to methylene protons H2-20 (dH 3.99 dd, J¼13.6, 6.6 Hz and dH 3.07, dd, J¼13.6, 6.6 Hz) in addition to HMBC correlations from H2-20 and H-30 to the carbonyl carbon at dC 207.6 indicated that an isovaleryl group was present in the molecule. Since the phenolic proton (8-OH) resonated significantly downfield it must be strongly intramolecular hydrogen bonded suggesting that the isovaleryl group was attached to the carbon ortho to it.7 COSY correlations between the
resonances at dH 4.26 (d, J¼3.6 Hz, H-9), 1.92e2.01 (m, H-100 ), 0.77 (d, J¼6.6 Hz, H3-200 ), and 0.75 (d, J¼6.6 Hz, H3-300 ) were in agreement with the presence of an isobutyl group. This group was attached to both the b-triketone and phloroglucinol moieties since HMBC correlations were observed from H-9 to C-1 (dC 197.9), C-8 (dC 160.9), C-8a (dC 104.8), C-9a (dC 112.6), C-4a (dC 168.5), and C-4b (dC 158.0). Finally, the molecular formula for 1 dictated that an ether linkage was present between C-4a and C-4b. Thus callistenone A was assigned as 6,8-dihydroxy-9-isopropyl-2,2,4,4tetramethyl-7-(3-methylbutyryl)-4,9-dihydroxanthene-1,3-dione. The absolute configuration at C-9 remains unassigned in 1. Callistenone B (2) was a yellowish gum. Compound 2 had molecular formula C25H32O6, as derived from the ion peak at m/z 429.2285 [MþH]þ by HRFABMS and other spectroscopic data (UV, IR, MS, 1H NMR, and 13C NMR) were very similar to those of 1 with the only significant difference being the non-equivalent methylene signals of the isovaleryl group (dH 3.27, H-20 a and dH 3.01, H-20 b for 2; dH 3.07, H-20 a and dH 2.99, H-20 b for 1). Callistenone B was therefore proposed to be a regioisomer of 1. HMBC correlations (Table 1) from H-7 to C-5 (dC 104.7), C-6 (dC 164.2), C-8 (dC 161.0), C8a (dC 103.9), and that of 6-OH to C-5 (dC 104.7), C-6 (dC 164.2), C-8a (dC 103.9) were consistent with the aromatic proton being bonded to C-7 and the isovaleryl group attached at C-5. Therefore 5-(3methylbutanoyl)-6,8-dihydroxy-9-isopropyl-2,2,4,4-tetramethyl2H-xanthene-1,3(4H,9H)-dione was assigned for callistenone B (2). Callistenone C (3) was a yellowish gum. Its molecular ion [M]þ in the HREIMS at m/z 446.2305 corresponded to the molecular formula of C25H34O7. The IR spectrum showed absorption bands for a conjugated carbonyl group at 1652 cm1, a non-conjugated carbonyl group at 1715 cm1, and a hydroxyl group at 3163 cm1. Its 1H and 13C NMR spectra in CDCl3 (Table 2) showed doubling of all
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S. Rattanaburi et al. / Tetrahedron 69 (2013) 6070e6075
Table 1 13 C, 1H NMR, and HMBC spectroscopic data (300 MHz, acetone-d6) for 1 and 2 No.
1
2
dC
dH (J in Hz)
1 2 3 4 4a 4b 5 6 7 8 8a 9
197.9, C 56.6, C 212.6, C 47.9, C 168.5, C 158.0, C 94.2, CH 164.0, C 108.5, C 160.9, C 104.8, C 32.8, CH
9a 10 11 12 13 10 20
112.6, C 25.8, CH3 24.0, CH3 24.7, CH3 24.0, CH3 207.6, C 53.6, CH2
30 40 50 100 200 300 6-OH 8-OH
25.6, 23.0, 23.0, 34.7, 19.8, 18.0,
CH CH3 CH3 CH CH3 CH3
6.34, s
4b, 6, 7, 8a
4.26, d (3.6)
1, 4a, 4b, 8, 8a, 100 , 200 , 300
1.37, 1.31, 1.57, 1.43,
1, 1, 3, 3,
s s s s
2, 2, 4, 4,
3, 11 3, 10 4a, 13 4a, 12
10 , 30 , 40 , 50
3.07, dd (13.6, 6.6), 2.99, dd (13.6, 6.6) 2.22e2.30, m (6.6) 0.96, d (6.6) 0.96, d (6.6) 1.92e2.01, m 0.77, d (6.6) 0.75, d (6.6) 10.68, s 14.22, s
10 , 2 0 , 4 0 , 5 0 20 , 30 , 50 20 , 30 , 40 9, 100 , 300 9, 100 , 200 7, 8, 8a
Table 2 13 C, 1H NMR and HMBC spectroscopic data (300 MHz, CDCl3) for 3 No.
dC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 10 20 30 40 50 100 200 300 5-OH 9-OH 11-OH 13-OH
202.2, C 54.9, C 212.6, C 48.7, C 178.1, C 114.1, C 39.0, CH 108.9, C 163.2, C 103.7, C 163.6, C 97.0, CH 158.7, C 24.0, CH3 24.7, CH3 25.5, CH3 25.9, CH3 206.2, C 52.1, CH2 25.9, CH 22.8, CH3 22.8, CH3 26.0, CH 21.9, CH3 21.7, CH3
dC
HMBC
dH (J in Hz)
HMBC
3.76, d (10.5)
1, 5, 6, 8, 9, 13
5.91, s
8, 10, 11, 13
1.43, 1.35, 1.50, 1.00,
1, 1, 3, 3,
2, 2, 4, 4,
10 , 10 , 20 , 20 ,
30 , 20 , 30 , 30 ,
7, 7, 4, 8,
100 , 300 100 , 300 5, 6 9, 10
s s s s
2.97, d (6.6) 2.24e2.33, m (6.6) 0.99, d (6.6) 0.99, d (6.6) 1.91, obscured 0.82, d (6.6) 0.79, d (6.6) 11.20, s 16.78, s 6.44, br s 10.52, s
3, 3, 5, 5,
15 14 17 16
40 , 50 40 , 50 50 40
8, 12, 13
signals in a ratio of 2:1 due to the existence of two tautomers or rotamers.8,9 The signals for the major isomer revealed that 3 contained a b-triketone, an isobutyl group, and a phloroglucinol with an attached isovaleryl groups similar to that found in 1 and 2, in addition to two hydroxyl groups at dH 16.78 (9-OH) and dH 11.20 (5OH). The downfield phenolic hydroxyl group (dH 16.78) due to strong intramolecular hydrogen bonding was placed ortho to the
196.9, C 55.6, C 211.3, C 47.1, C 167.3, C 153.7, C 104.7, C 164.2, C 99.4, CH 161.0, C 103.9, C 31.3, CH 111.6, C 24.3, CH3 23.3, CH3 24.7, CH3 24.6, CH3 203.9, C 52.9, CH2 24.5, 22.1, 21.9, 34.6, 18.0, 18.6,
CH CH3 CH3 CH CH3 CH3
dH (J in Hz)
HMBC
6.31, s
5, 6, 8, 8a
4.29, d (3.6)
1, 4a, 4b, 8, 8a, 9a, 100 , 200 , 300
1.39, 1.33, 1.68, 1.50,
1, 1, 3, 3,
s s s s
3.27, dd (13.8, 6.6), 3.01, dd (13.8, 6.6) 2.30e2.38, m (6.6) 1.02, d (6.6) 0.99, d (6.6) 1.89e1.99, m 0.82, d (6.9) 0.79, d (6.9) 13.34, s 10.10, br s
2, 2, 4, 4,
3, 11 3, 10 4a, 13 4a, 12
10 , 30 , 40 , 50 10 , 20 , 40 , 50 20 , 30 , 50 20 , 30 , 40 9, 300 9, 200 5, 6, 7
isovaleryl group. While the upfield hydroxyl signal at dH 11.20 was assigned to 5-OH because HMBC correlations were observed to C-4 (dC 48.7), C-5 (dC 178.1), and C-6 (dC 114.1). The HMBC correlations from H-7 to C-1 (dC 202.2), C-5 (dC 178.1), C-9 (dC 163.2), and C-13 (dC 158.7) indicated that the isobutyl group was attached to the btriketone at C-6 and the phloroglucinol at C-8. Furthermore H-7, 9OH, and 13-OH all showed correlations to the same carbon (C-8) suggesting that 9-OH and 13-OH were ortho to this carbon substituted by the isobutyl group. The significant upfield shift of the only aromatic methine carbon (C-12, d 97.0) and its attached proton (d 5.91, H-12) was indicative of it being ortho to two hydroxyl groups. It was also meta to the carbon substituted by the isobutyl group because it showed HMBC correlations to C-8 (d 108.9), C-10 (d 103.7), C-11 (d 163.6), C-13 (d 158.7). Accordingly, callistenone C (3) was assigned as 5-hydroxy-2,2,6,6tetramethyl-4-(2-methyl-1-(2,4,6-trihydroxy-3-(3methylbutanoyl)phenyl)propyl)cyclohex-4-ene-1,3-dione. Callistenone D (4) was obtained as a yellowish gum. A molecular ion peak at m/z 665.3682 [MþH]þ in its HRFABMS spectrum allowed a molecular formula of C39H52O9 to be assigned to 4. The 1H and 13C NMR spectroscopic data (Table 1), the results obtained from HMBC correlations (Table 3) as well as comparison of NMR spectroscopic data for 1 indicated that the structure of 1 without an aromatic proton at H-5 was present in 4. In addition the 1H NMR spectrum of 4 showed signals for methyl groups (dH 1.32, H3-11000 ; 1.43, H3-12000 ; 1.47, H3-13000 ; 1.62, H3-14000 ) of a second b-triketone moiety, an isobutyl group (dH 4.17, dd, J¼6.6 and 3.9 Hz, H-7000 ; 2.36e2.43, m, H-8000 ; 0.92, d, J¼6.9 Hz, H-9000 ; 0.68, d, J¼6.9 Hz, H10000 ), and a methine proton (d 3.68, d, J¼3.6 Hz, H-6000 ). The 13C NMR spectrum also contained two additional ketone carbon signals at dC 205.0 and 213.5, two quaternary aliphatic carbons at dC 57.1 and 56.3, a deoxygenated quaternary carbon at dC 100.0, and nine aliphatic carbons upfield of dC 46.3. HMBC correlations from H3-11000 and H3-12000 to dC 57.1, 205.0 and 213.5 and from H3-13000 and H3-14000 to dC 56.3, 100.0 and 213.5 were consistent with 4 containing
S. Rattanaburi et al. / Tetrahedron 69 (2013) 6070e6075 Table 3 13 C, 1H NMR and HMBC spectroscopic data (500 MHz, CDCl3) for 4 No.
dC
1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 10 11 12 13 10 20
197.4, C 56.0, C 212.0, C 47.0, C 167.4, C 153.1, C 106.7, C 151.2, C 108.3, C 160.9, C 109.8, C 32.1, CH 111.4, C 24.2, CH3 25.2, CH3 25.4, CH3 25.0, CH3 205.5, C 54.3, CH2
30 40 50 100 200 300 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000
24.5, C 22.1, CH3 22.1, CH3 34.0, CH 18.8, CH3 19.0, CH3 205.0, C 57.1, C 213.5, C 56.3, C 100.0, C 46.3, CH 28.8, CH 32.3, CH 20.1, CH3 16.0, CH3 22.1, CH3 26.7, CH3 19.4, CH3 24.4, CH3
dH (J in Hz)
Table 4 13 C, 1H NMR and HMBC spectroscopic data (500 MHz, CDCl3) for 5 HMBC
4.39, d (3.3)
4a, 4b
1.38, 1.44, 1.60, 1.64,
1, 1, 3, 3,
s s s s
6073
2, 2, 4, 4,
3 3 4a 4a
3.14, dd (16.8, 6.9) 2.89, dd (16.8, 6.9) 2.31e2.39, m 0.99, d (6.9) 0.98, d (6.9) 2.00e2.12, m 0.74, d (3.9) 0.77, d (3.9)
1 0 , 30 , 40 , 50
3.68, d (6.3) 4.17, dd (6.3, 3.9) 2.36e2.43, m 0.92, d (6.9) 0.68, d (6.9) 1.32, s 1.43, s 1.47, s 1.62, s
1000 , 5000 , 7000 , 8000
2 0 , 30 , 50 2 0 , 30 , 40 9, 100 , 300 9, 100 , 200
7000 , 7000 , 1000 , 1000 , 3000 , 3000 ,
8000 , 8000 , 2000 , 2000 , 4000 , 4000 ,
10000 9000 3000 3000 5000 5000
a second b-triketone in which one of the ketone groups was present as a ketal or hemiketal. H-6000 (dH 3.68) showed a COSY correlation to H-7000 (dH 4.17) and HMBC correlations to C-7000 (dC 26.8), C-1000 (dC 205.0), and C-8000 (dC 32.2) suggesting it was adjacent to H-7 and the carbonyl carbon C-1000. H-6000 further correlated to C-5 (dC 106.7) allowing the second b-triketone to be linked to the phloroglucinol via a bond from C-7000 of the isobutyl group. Finally, an additional ring between C-6 and C-5000 via an oxygen bridge to the hemiketal carbon at C-5000 (dC 100.0)9 was required to fulfill the structure. The assigned structure was compared to the structure of myrtucommulone D9 with the isovaleryl group at C-7 instead of isobutyl ketone. The NOESY experiment was used to identify the relative configurations at C-9, C-5000 , C-6000 , and C-7000. Cross peak of H-6000 to H-12000 , H-14000 H-8000 and that of H-8000 and H-100 to H-12 indicated that methyl groups H-12000 , H-14000 , H-12; methine proton H-6000 and the isopropyl groups were arranged on the b-face of molecule. The methyl proton H-14000 further correlated to methylene H-20 suggesting that 5000 -OH was on the a-face. The relative configuration of 9R*,5000 S*,6000 R*,7000 S*10 was therefore assigned for callistenone D (4). Since the optical rotation determined for 4 [a]29 D þ39.6 (MeOH) was similar in magnitude and sign to that of myrtucommulone D ([a]17 D þ13.9 (MeOH))10 this suggested that both molecules possessed the same absolute configuration, however this configuration could not be determined. Callistenone E (5) was obtained as a yellowish gum. It molecular formula C40H52O8 was determined from analysis of HREIMS [m/z 660.3674]. The NMR spectroscopic data and HMBC correlations (Table 4) together with COSY correlations observed for 5 mostly
No
dC
1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 10 11 12 13 10 20
197.5, C 56.3, C 212.5, C 47.3, C 167.5, C 153.2, C 105.3, C 150.1, C 107.7, C 160.7, C 110.2, C 32.1, CH 111.9, C 24.2, CH3 24.3, CH3 24.9, CH3 25.0, CH3 204.5, C 53.9, CH2
30 40 50 100 200 300 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000 15000 8-OH
24.5, CH 22.3, CH3 22.5, CH3 34.4, CH 19.5, CH3 18.4, CH3 197.3, C 56.1, C 212.4, C 47.2, C 166.1, C 113.2, C 31.9, CH 46.7, CH2 25.3, CH2 23.8, CH3 23.3, CH3 24.1, CH3 24.9, CH3 24.8, CH3 25.0, CH3
dH (J in Hz)
HMBC
4.40, d (3.3)
1, 4a, 4b, 8a, 100 , 200 , 300
1.36, 1.43, 1.48, 1.65,
1, 1, 3, 3,
s s s s
2, 2, 4, 4,
3, 11 3, 10 4a, 13 4a, 12
3.21, dd (17.4, 7.2) 3.02, dd (17.4, 7.2) 2.34e2.46, m 1.05, d (6.0) 1.04, d (6.0) 2.02e2.12, m 0.79, d (6.9) 0.82, d (6.9)
1 0 , 30 , 40 , 50
4.37, t (6.0) 1.51, obscured 1.51, obscured 0.84, d (6.0) 0.93, d (6.0) 1.39, s 1.44, s 1.50, s 1.66, s 13.51, s
1000 , 5000 , 6000 , 8000 , 9000 , 4b, 5, 6
2 0 , 30 , 50 2 0 , 30 , 40 9, 100 , 300 9, 100 , 200
8000 , 9000 , 11000 8000 , 9000 , 10000 1000 , 2000 , 3000 , 13000 1000 , 2000 , 3000 , 12000 3000 , 4000 , 5000 , 15000 3000 , 4000 , 5000 , 14000 7, 8, 8a
exhibited the same results as for callistenone D (4) except for the replacement of the isobutyl resonances by isopentyl resonances and the absence of a methine proton H-6000 . The protons of the isopentyl group resonated at dC 4.37 (t, J¼3.6 Hz, H-7000 ), 1.51 (obscured, H-8000 and H-9000 ), 0.84 (d, J¼3.6 Hz, H-10), and 0.95 (d, J¼3.6 Hz, H-11000 ). HMBC correlations from H-7000 to C-4b (dC 153.2), C-6 (dC 150.1), C-1000 (dC 197.3), C-5000 (dC 166.1), and C-9000 (dC 25.3) indicated that the isopentyl group was attached to C-5 of the phloroglucinol and C-6000 of the second b-triketone moiety. The NOESY cross peaks from H-9 to H-10 and H-12 and H-7000 to H-13, H12000 , and H-14000 indicated that methyl groups H-10, H-12, H-12000 , and H-14000 are on the a-face whereas the isobutyl and isopropyl groups were on the b-face of molecule. The indicated that 5 possessed a relative configuration of 9R*,7000 S*. Structure 5 was thus assigned for callistenone E. Compounds 1, 2, 3, 6, and 10 inhibited the growth of S. aureus ATTC25923 with MIC values of 0.5, 8, 8, 8, and 200 mg/mL and MRSA SK1 with MIC values of 1, 8, 8, 16, and 200 mg/mL, respectively, while 7 and 11 showed no activity at a concentration up to 200 mg/ mL. Compounds 4, 5, 8, and 9 were not tested due to insufficient material. Compound 1, which is the most active compound, provided strong antibacterial activity comparable to vancomycin. It is interesting to note that 1 shows 16 and 8 more potent antibacterial activity against ATTC25923 and SK1 strains of S. aureus, respectively, compared to its isomer (2) and this is mostly likely associated with the point of attachment of the isovaleryl side chain (Table 5).
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S. Rattanaburi et al. / Tetrahedron 69 (2013) 6070e6075
Table 5 Antibacterial activity of the dichloromethane extract and some of the pure compounds from C. lanceolatus Compound
Antibacterial activity (MIC, mg/mL) S. aureus ATCC25923
MRSA SK1
CH2Cl2 extract 1 2 3 6 7 10 11 Vancomycin
4 0.5 8 8 8 NA 200 NA 0.5
4 1 8 8 16 NA 200 NA 1
NA¼no activity at 200 mg/mL.
In conclusion, five new phloroglucinols and six known compounds have been isolated from the leaves of C. lanceolatus. All of phloroglucinol compounds, which were tested showed good antibacterial activity. Interestingly, callistenone A (1) exhibited comparable antibacterial activity to vancomycin suggesting that it could be a good candidate for further antibacterial drug development. This compound is now undergoing further bioactivity studies in our laboratory. 3. Experimental
yielding 1 (5.0 mg). Fraction A5 (14.3 g) was separated by CC using Me2CO/hexane (1:49) to Me2CO/hexane (1:19) to provide 13 fractions (A5AeA5M). Fraction A5C (845.7 mg) was further subjected to CC and eluted with Me2CO/hexane (1:49) to give 2 (6.2 mg). Fraction A5L (40.4 mg) was rechromatographed on PTLC using Me2CO/ CH2Cl2/hexane (1:1:18) as an eluant to produce 8 (2.1 mg) and 9 (0.7 mg). Fraction A5M (752.1 mg) was further purified by CC using the same solvent system to produce 3 (2.8 mg). Fractions A7 and A9 were each purified by CC using Me2CO/hexane (1:19) as an eluant to provide 4 (1.8 mg), 10 (2.7 mg) and 11 (3.7 mg), 5 (2.0 mg), respectively. 3.3.1. Callistenone A (1). Yield 5.0 mg. A yellowish gum. [a]29 D þ2.5 (c 2.82, MeOH). UV (MeOH) lmax nm (log 3 ): 207 (4.33), 223 (4.44), 260 (4.19), 299 (4.34), 350 (3.70). IR (neat) n (cm1): 3265, 1716, 1624. 1H NMR data (300 MHz, acetone-d6) and 13C NMR data (75 MHz, acetone-d6): see Table 1. FABMS m/z (% rel int): 429 ([Mþ1]þ, (4)), 385 (1), 369 (5), 277 (14), 185 (100). HRFABMS m/z 429.2270 [Mþ1]þ for C25H33O6 (calcd 429.2272). 3.3.2. Callistenone B (2). Yield 6.2 mg. A yellowish gum. [a]29 D þ2.8 (c 4.55, CHCl3). UV (MeOH) lmax nm (log 3 ): 205 (4.04), 227 (3.76), 292 (3.48). IR (neat)n (cm1): 3316, 1716, 1650. 1H NMR (300 MHz, acetone-d6) and 13C NMR (75 MHz, acetone-d6): see Table 1. FABMS m/z (% rel int): 429 ([Mþ1]þ, 100), 385 (55), 371 (34), 357 (13), 315 (17), 185 (12), 133 (14). HRFABMS m/z 429.2285 [Mþ1]þ for C25H33O6 (calcd 429.2277).
3.1. General experimental procedures IR spectra were measured on an FTS 165 FT-IR PerkineElmer spectrophotometer. UV spectra were recorded by a SPECORD S100 spectrophotometer. 1H and 13C NMR spectra were recorded in CDCl3 or acetone-d6 using a Bruker Avance FT-NMR 300 MHz and 500 MHz spectrometers. Optical rotations were determined in CHCl3 or MeOH solution at the sodium D line (589 nm) on a JASCO P-1020 polarimeter. The EIMS, HREIMS, FABMS, and HRFABMS mass spectra were obtained using a MAT 95 XL mass spectrometer (Thermo Finnigan). Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60H (Merck) and silica gel 100 (Merck), respectively. Precoated plates of silica gel 60 GF254 were used for TLC analysis. 3.2. Plant material The leaves of C. lanceolatus (Myrtaceae) were collected from Amphur Mueang Nakhon Si Thammarat, Nakhon Si Thammarat Province on October 2007. The plant was identified by Mr. Ponlawat Pattarakulpisutti and the herbarium specimen (S. Rattanaburi 1) has been deposited in the Herbarium of the Department of Biology, Faculty of Science, Prince of Songkla University, Thailand.
3.3.3. Callistenone C (3). Yield 2.8 mg. A yellowish gum. [a]29 D þ4.5 (c 3.60, MeOH). UV (MeOH) lmax nm (log 3 ): 205 (4.08), 230 (3.78), 291 (3.61). IR (neat) n (cm1): 3163, 1715, 1652. 1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3): see Table 2. EIMS m/z (% rel int): 446 ([Mþ], (10)), 385 (53), 236 (20), 210 (16), 193 (18), 166 (22), 153 (100), 123 (30). HREIMS m/z 446.2305 for C25H34O7 (calcd 446.2305). 3.3.4. Callistenone D (4). Yield 1.8 mg. A yellowish gum. [a]29 D þ39.6 (c 0.10, MeOH). UV (MeOH) lmax nm (log 3 ): 208 (4.76), 214 (4.77), 221 (4.78), 267 (4.48). IR (neat) n (cm1): 3389, 2953, 2930, 1716, 1653, 1537, 1162, 1015. 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3): see Table 3. FABMS m/z (% rel int): 665 ([Mþ1]þ, (4)), 621 (3), 429 (8), 277 (10), 185 (89). HRFABMS m/z 665.3682 for C39H53O9 (calcd 665.3684). 3.3.5. Callistenone E (5). Yield 2.0 mg. A yellowish gum. [a]29 D þ6.5 (c 0.23, MeOH). UV (MeOH) lmax nm (log 3 ): 220 (4.10), 261 (3.93), 292 (3.94), 355 (3.32). IR (neat) n (cm1): 3420, 2957, 2933, 1716, 1652, 1455, 1384, 1182, 1154. 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3): see Table 4. EIMS m/z (% rel int): 660 ([M]þ), 659 (61), 615 (74), 184 (79), HREIMS m/z 660.3674 for C40H52O8 (calcd 660.3657).
3.3. Extraction and isolation 3.4. Antibacterial activity Ground-dried leaves (4.5 kg) of C. lanceolatus were extracted with CH2Cl2 at room temperature for 3 days. The viscous extract (349.5 g) after removal of solvent was sequentially dissolved in hexane to give hexane soluble (107.6 g) and insoluble fractions. The hexane soluble fraction was purified by QCC using Me2CO/hexane (1:49) as an eluant. Based on TLC analysis, fractions that contained similar components were combined to give 10 fractions (A1eA10). Fraction A2 (23.4 g) was further subjected to CC and eluted with Me2CO/hexane (1:49) to give 6 (7.4 mg). Fraction A4 (2.7 g) was purified by CC and eluted with Me2CO/hexane (1:49) to obtain 10 fractions (A4AeA4J). Fraction A4F (27.2 mg) was further purified by PTLC using Me2CO/hexane (1:99) gave 7 (2.3 mg). Fraction A4G (145.6 mg) was further purified by CC using Me2CO/hexane (1:49)
The crude extract and purified compounds were screened for antibacterial activity at a concentration of 200 mg/mL by a broth microdilution method against four bacteria: S. aureus ATCC25923, a clinical isolate of methicillin-resistant S. aureus SK1, E. coli ATCC25922, P. aeruginosa ATCC27853. Crude extracts and purified compounds having antibacterial activity were further determined for their minimum inhibitory concentrations (MICs) by broth microdilution methods according to a modification of the Clinical and Laboratory Standards Institute. Resazurin was added as viability indicator according to Drummonds and Waigh (2000). Colorimetric MIC end-points were interpreted as the lowest concentration that remained blue (indicating no growth) or the
S. Rattanaburi et al. / Tetrahedron 69 (2013) 6070e6075
first dilution that changed from blue to slightly purple (equivalent to prominent growth inhibition). Acknowledgements We are grateful to the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program for a scholarship given to S.R. (grant no. PHD/0284/2550) and the National Research University Project of Thailand’s Office of the Higher Education Commission for financial support. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2013.05.085.
6075
References and notes 1. Chambers, H. F.; DeLeo, F. R. Nat. Rev. Microbiol. 2009, 7, 629e641. 2. Klink, J. W. V.; Brophy, J. J.; Perry, N. B.; Weavers, R. T. J. Nat. Prod. 1999, 62, 487e489. 3. Hiranrat, A.; Mahabusarakam, W. Tetrahedron 2008, 64, 11193e11197. 4. Crow, W. D.; Nicholls, W.; Sterns, M. Tetrahedron Lett. 1971, 18, 1353e1356. 5. Macias, F. A.; Simonet, A. M.; Esteban, M. D. Phytochemistry 1994, 36, 1369e1379. 6. Huq, F.; Misra, L. N. Planta Med. 1997, 63, 369e370. 7. Salni, D.; Sargent, M. V.; Skelton, B. W.; Soediro, I.; Sutisna, M.; White, A. H.; Yulinah, E. Aust. J. Chem. 2002, 55, 229e232. 8. Appendino, G.; Bianchi, F.; Minassi, A.; Sterner, O.; Ballero, M.; Gibbons, S. J. Nat. Prod. 2002, 65, 334e338. 9. Carroll, A. R.; Urban, S.; Lamb, J.; Moni, R.; Guymer, G. P.; Forster, P. I.; Quinn, R. J. J. Nat. Prod. 2008, 71, 881e883. 10. Carroll, A. R.; Lamb, J.; Moni, R.; Guymer, G. P.; Forster, P. I.; Quinn, R. J. J. Nat. Prod. 2008, 71, 1564e1568.
Contents lists available at SciVerse ScienceDirect
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Acylphloroglucinols from Callistemon lanceolatus DC. Suthida Rattanaburi a, Wilawan Mahabusarakam a, b, *, Souwalak Phongpaichit b, c, Anthony R. Carroll d, e a
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Natural Products Research Center of Excellence, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand d Griffith School of Environment (Gold Coast Campus), Griffith University, Qld 4222, Australia e Environmental Futures Centre, Griffith University, Gold Coast, Qld 4222, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2013 Received in revised form 10 May 2013 Accepted 20 May 2013 Available online 24 May 2013
Five acylphloroglucinols, named callistenones AeE together with six known acylphloroglucinols, triterpenoids, and C-methylflavonoids were isolated from the leaves of Callistemon lanceolatus. Their structures were characterized by spectroscopic methods. Some of the compounds showed very strong antibacterial activity. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Callistemon lanceolatus Myrtaceae Acylphloroglucinols b-Triketones Antibacterial activity
1. Introduction
2. Result and discussion
Infectious diseases are a major problem worldwide because of the emergence of resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). S. aureus can cause a wide range of diseases from skin infections, hospital-acquired bacteremia to lifethreatening infections such as necrotizing fasciitis and pneumonia, septic arthritis, osteomyelitis, and endocarditis. MRSA now has emerged as a widespread cause of community infection. There is a concern because MRSA is becoming resistant to multiple antibiotics.1 Therefore, there is a need to find new antimicrobial agents to combat this pathogen. Natural products isolated from plants are an important source for the discovery of new antimicrobial substances. We have screened extracts from some Myrtaceae plants collected in Thailand for antibacterial activity and found that a crude extract from the leaves of Callistemon lanceolatus DC. (Myrtaceae), which is known as crimson bottlebrush, was very effective at inhibiting the growth of S. aureus. This prompted us to identify the active constituents from the leaves of C. lanceolatus.
The CH2Cl2 extract of the leaves of C. lanceolatus was tested for antibacterial and antifungal activities. The results showed that the extract was growth inhibitory against S. aureus ATTC25923, and MRSA SK1 with an MIC of 4 mg/mL for both strains, but showed no effect against Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922, Callistemon neoformans ATCC90113, Callistemon albicans NCPF3153, and Microsporum gypseum at a concentration of 200 mg/mL. Investigation of the active compounds present in the CH2Cl2 extract has resulted in the isolation of 11 compounds. On the basis of 1H, 13C NMR, COSY, HMQC, and HMBC spectra as well as by comparison with previous reports, callistenones AeE (1e5) were identified as new acylphloroglucinol derivatives while the remaining six compounds were found to be the known compounds, flavesone (6),2 leptospermone (7),2 rhodomyrtosone D (8),3 endoperoxide G3 (9),4 betulinaldehyde (10),5 and 5-hydroxy-7,40 dimethoxy-6,8-dimethylflavone (11).6 This is the first time that compounds 6e9 have been isolated from C. lanceolatus (Fig. 1). Callistenone A (1) was obtained as a yellowish gum. A molecular ion peak [MþH]þ at m/z 429.2270 in an HRFABMS represents C25H33O6 corresponded to a molecular formula of C25H32O6. The EIMS spectrum of 1 exhibited a base peak at m/z 385.0 [MCH(CH3)2], which suggested facile loss of an isopropyl group from the molecule. The IR spectrum exhibited stretching bands for
* Corresponding author. Tel.: þ66 7428 8432; fax: þ66 7428 8432/55 8841; e-mail address: [email protected] (W. Mahabusarakam). 0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2013.05.085
S. Rattanaburi et al. / Tetrahedron 69 (2013) 6070e6075 4' 12
H
13
O
5
4a O 4b
12
OH
3
11
9
1
7
8a 1''
O
10
OH
4a O 4b
11 10
5'
1''
O 3''
11'''
O 11 10
7''' 6'''
4a O 5 4 4b A B C 9
O 2''
1''
1'
OH
O
12
O
5'
4
9
O
8
15'''
1''
O
O
O
O
OH O
OH
O
5'
OH
4'
OH
O
O
3''
7
6
OCH 3
CH3
O O
5 D O 6 8 1'
5
O O
14'''
6'''
OH 2''
O H
O E 4'''
C
B
1
10
4'
4a O 4b
A
11
7'''
13
4
O
13'''
1'''
3''
O
12'''
O
10'''
14''' O OH
8
OH 2"
3
13'''
4'''
OH 12
1" 3"
2''
11'''
O 1'''
9'''
1
O
14
8
7
1
OH
12'''
10''' O 13
15
7
10
5
2
1
12
3
9
1
O OH OH 1'
3'
17
O
OH
5 8a
O
5'
4' 16
1'
3
3''
2''
5'
O
13
O
4'
1'
6071
CHO
O
O
H3 C
HO
9
H3 CO
OH 10
O 11
Fig. 1. Structures of compounds 1e11 from C. lanceolatus.
hydroxyl, saturated carbonyl, and unsaturated carbonyl groups at 3265, 1716, and 1624 cm1, respectively. The presence of carbonyl groups was also deduced from the carbon resonances at dC 207.6 (C10 ), 197.9 (C-1), and 212.6 (C-3) in the 13C NMR spectrum (Table 1). In addition the 13C NMR spectrum contained signals for 8 sp2 hybridized carbons between dC 94.2 and 168.5 and 14 aliphatic carbons upfield of dC 56.6. The 1H NMR spectrum (Table 1) showed resonances of four tertiary methyl groups at dH 1.37 (H3-10), 1.31 (H3-11), 1.57 (H3-12), and 1.43 (H3-13). In the HMBC spectrum, H310 and H3-11 showed HMBC correlations to carbonyl carbons C-1 (dC 197.9) and C-3 (dC 212.6) whereas H3-12 and H3-13 showed correlations to the carbonyl carbon C-3 (dC 212.6) and an oxygenated vinylic carbon C-4a (dC 168.5). These data indicated the presence of a b-triketone moiety.3 The presence of a di-C-substituted phloroglucinol moiety was deduced from HMBC correlations observed from an aromatic proton (dH 6.34, s, H-5) to two oxygenated aromatic carbons at dC 158.0 (C-4b) and dC 164.0 (C-6) and two upfield non-protonated sp2 carbons at dC 108.5 (C-7) and dC 104.8 (C-8a), and from a phenolic hydroxyl proton (dH 14.22, 8-OH) also to the two upfield non-protonated sp2 carbons C-7 and C-8a in addition to an oxygenated aromatic carbon at dC 160.9 (C-8). COSY correlations between two equivalent secondary methyl protons H340 and H3-50 (dH 0.96, d, J¼6.6 Hz) and a methine proton H-30 (dH 2.22e2.30, m) and further correlations from H-30 to methylene protons H2-20 (dH 3.99 dd, J¼13.6, 6.6 Hz and dH 3.07, dd, J¼13.6, 6.6 Hz) in addition to HMBC correlations from H2-20 and H-30 to the carbonyl carbon at dC 207.6 indicated that an isovaleryl group was present in the molecule. Since the phenolic proton (8-OH) resonated significantly downfield it must be strongly intramolecular hydrogen bonded suggesting that the isovaleryl group was attached to the carbon ortho to it.7 COSY correlations between the
resonances at dH 4.26 (d, J¼3.6 Hz, H-9), 1.92e2.01 (m, H-100 ), 0.77 (d, J¼6.6 Hz, H3-200 ), and 0.75 (d, J¼6.6 Hz, H3-300 ) were in agreement with the presence of an isobutyl group. This group was attached to both the b-triketone and phloroglucinol moieties since HMBC correlations were observed from H-9 to C-1 (dC 197.9), C-8 (dC 160.9), C-8a (dC 104.8), C-9a (dC 112.6), C-4a (dC 168.5), and C-4b (dC 158.0). Finally, the molecular formula for 1 dictated that an ether linkage was present between C-4a and C-4b. Thus callistenone A was assigned as 6,8-dihydroxy-9-isopropyl-2,2,4,4tetramethyl-7-(3-methylbutyryl)-4,9-dihydroxanthene-1,3-dione. The absolute configuration at C-9 remains unassigned in 1. Callistenone B (2) was a yellowish gum. Compound 2 had molecular formula C25H32O6, as derived from the ion peak at m/z 429.2285 [MþH]þ by HRFABMS and other spectroscopic data (UV, IR, MS, 1H NMR, and 13C NMR) were very similar to those of 1 with the only significant difference being the non-equivalent methylene signals of the isovaleryl group (dH 3.27, H-20 a and dH 3.01, H-20 b for 2; dH 3.07, H-20 a and dH 2.99, H-20 b for 1). Callistenone B was therefore proposed to be a regioisomer of 1. HMBC correlations (Table 1) from H-7 to C-5 (dC 104.7), C-6 (dC 164.2), C-8 (dC 161.0), C8a (dC 103.9), and that of 6-OH to C-5 (dC 104.7), C-6 (dC 164.2), C-8a (dC 103.9) were consistent with the aromatic proton being bonded to C-7 and the isovaleryl group attached at C-5. Therefore 5-(3methylbutanoyl)-6,8-dihydroxy-9-isopropyl-2,2,4,4-tetramethyl2H-xanthene-1,3(4H,9H)-dione was assigned for callistenone B (2). Callistenone C (3) was a yellowish gum. Its molecular ion [M]þ in the HREIMS at m/z 446.2305 corresponded to the molecular formula of C25H34O7. The IR spectrum showed absorption bands for a conjugated carbonyl group at 1652 cm1, a non-conjugated carbonyl group at 1715 cm1, and a hydroxyl group at 3163 cm1. Its 1H and 13C NMR spectra in CDCl3 (Table 2) showed doubling of all
6072
S. Rattanaburi et al. / Tetrahedron 69 (2013) 6070e6075
Table 1 13 C, 1H NMR, and HMBC spectroscopic data (300 MHz, acetone-d6) for 1 and 2 No.
1
2
dC
dH (J in Hz)
1 2 3 4 4a 4b 5 6 7 8 8a 9
197.9, C 56.6, C 212.6, C 47.9, C 168.5, C 158.0, C 94.2, CH 164.0, C 108.5, C 160.9, C 104.8, C 32.8, CH
9a 10 11 12 13 10 20
112.6, C 25.8, CH3 24.0, CH3 24.7, CH3 24.0, CH3 207.6, C 53.6, CH2
30 40 50 100 200 300 6-OH 8-OH
25.6, 23.0, 23.0, 34.7, 19.8, 18.0,
CH CH3 CH3 CH CH3 CH3
6.34, s
4b, 6, 7, 8a
4.26, d (3.6)
1, 4a, 4b, 8, 8a, 100 , 200 , 300
1.37, 1.31, 1.57, 1.43,
1, 1, 3, 3,
s s s s
2, 2, 4, 4,
3, 11 3, 10 4a, 13 4a, 12
10 , 30 , 40 , 50
3.07, dd (13.6, 6.6), 2.99, dd (13.6, 6.6) 2.22e2.30, m (6.6) 0.96, d (6.6) 0.96, d (6.6) 1.92e2.01, m 0.77, d (6.6) 0.75, d (6.6) 10.68, s 14.22, s
10 , 2 0 , 4 0 , 5 0 20 , 30 , 50 20 , 30 , 40 9, 100 , 300 9, 100 , 200 7, 8, 8a
Table 2 13 C, 1H NMR and HMBC spectroscopic data (300 MHz, CDCl3) for 3 No.
dC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 10 20 30 40 50 100 200 300 5-OH 9-OH 11-OH 13-OH
202.2, C 54.9, C 212.6, C 48.7, C 178.1, C 114.1, C 39.0, CH 108.9, C 163.2, C 103.7, C 163.6, C 97.0, CH 158.7, C 24.0, CH3 24.7, CH3 25.5, CH3 25.9, CH3 206.2, C 52.1, CH2 25.9, CH 22.8, CH3 22.8, CH3 26.0, CH 21.9, CH3 21.7, CH3
dC
HMBC
dH (J in Hz)
HMBC
3.76, d (10.5)
1, 5, 6, 8, 9, 13
5.91, s
8, 10, 11, 13
1.43, 1.35, 1.50, 1.00,
1, 1, 3, 3,
2, 2, 4, 4,
10 , 10 , 20 , 20 ,
30 , 20 , 30 , 30 ,
7, 7, 4, 8,
100 , 300 100 , 300 5, 6 9, 10
s s s s
2.97, d (6.6) 2.24e2.33, m (6.6) 0.99, d (6.6) 0.99, d (6.6) 1.91, obscured 0.82, d (6.6) 0.79, d (6.6) 11.20, s 16.78, s 6.44, br s 10.52, s
3, 3, 5, 5,
15 14 17 16
40 , 50 40 , 50 50 40
8, 12, 13
signals in a ratio of 2:1 due to the existence of two tautomers or rotamers.8,9 The signals for the major isomer revealed that 3 contained a b-triketone, an isobutyl group, and a phloroglucinol with an attached isovaleryl groups similar to that found in 1 and 2, in addition to two hydroxyl groups at dH 16.78 (9-OH) and dH 11.20 (5OH). The downfield phenolic hydroxyl group (dH 16.78) due to strong intramolecular hydrogen bonding was placed ortho to the
196.9, C 55.6, C 211.3, C 47.1, C 167.3, C 153.7, C 104.7, C 164.2, C 99.4, CH 161.0, C 103.9, C 31.3, CH 111.6, C 24.3, CH3 23.3, CH3 24.7, CH3 24.6, CH3 203.9, C 52.9, CH2 24.5, 22.1, 21.9, 34.6, 18.0, 18.6,
CH CH3 CH3 CH CH3 CH3
dH (J in Hz)
HMBC
6.31, s
5, 6, 8, 8a
4.29, d (3.6)
1, 4a, 4b, 8, 8a, 9a, 100 , 200 , 300
1.39, 1.33, 1.68, 1.50,
1, 1, 3, 3,
s s s s
3.27, dd (13.8, 6.6), 3.01, dd (13.8, 6.6) 2.30e2.38, m (6.6) 1.02, d (6.6) 0.99, d (6.6) 1.89e1.99, m 0.82, d (6.9) 0.79, d (6.9) 13.34, s 10.10, br s
2, 2, 4, 4,
3, 11 3, 10 4a, 13 4a, 12
10 , 30 , 40 , 50 10 , 20 , 40 , 50 20 , 30 , 50 20 , 30 , 40 9, 300 9, 200 5, 6, 7
isovaleryl group. While the upfield hydroxyl signal at dH 11.20 was assigned to 5-OH because HMBC correlations were observed to C-4 (dC 48.7), C-5 (dC 178.1), and C-6 (dC 114.1). The HMBC correlations from H-7 to C-1 (dC 202.2), C-5 (dC 178.1), C-9 (dC 163.2), and C-13 (dC 158.7) indicated that the isobutyl group was attached to the btriketone at C-6 and the phloroglucinol at C-8. Furthermore H-7, 9OH, and 13-OH all showed correlations to the same carbon (C-8) suggesting that 9-OH and 13-OH were ortho to this carbon substituted by the isobutyl group. The significant upfield shift of the only aromatic methine carbon (C-12, d 97.0) and its attached proton (d 5.91, H-12) was indicative of it being ortho to two hydroxyl groups. It was also meta to the carbon substituted by the isobutyl group because it showed HMBC correlations to C-8 (d 108.9), C-10 (d 103.7), C-11 (d 163.6), C-13 (d 158.7). Accordingly, callistenone C (3) was assigned as 5-hydroxy-2,2,6,6tetramethyl-4-(2-methyl-1-(2,4,6-trihydroxy-3-(3methylbutanoyl)phenyl)propyl)cyclohex-4-ene-1,3-dione. Callistenone D (4) was obtained as a yellowish gum. A molecular ion peak at m/z 665.3682 [MþH]þ in its HRFABMS spectrum allowed a molecular formula of C39H52O9 to be assigned to 4. The 1H and 13C NMR spectroscopic data (Table 1), the results obtained from HMBC correlations (Table 3) as well as comparison of NMR spectroscopic data for 1 indicated that the structure of 1 without an aromatic proton at H-5 was present in 4. In addition the 1H NMR spectrum of 4 showed signals for methyl groups (dH 1.32, H3-11000 ; 1.43, H3-12000 ; 1.47, H3-13000 ; 1.62, H3-14000 ) of a second b-triketone moiety, an isobutyl group (dH 4.17, dd, J¼6.6 and 3.9 Hz, H-7000 ; 2.36e2.43, m, H-8000 ; 0.92, d, J¼6.9 Hz, H-9000 ; 0.68, d, J¼6.9 Hz, H10000 ), and a methine proton (d 3.68, d, J¼3.6 Hz, H-6000 ). The 13C NMR spectrum also contained two additional ketone carbon signals at dC 205.0 and 213.5, two quaternary aliphatic carbons at dC 57.1 and 56.3, a deoxygenated quaternary carbon at dC 100.0, and nine aliphatic carbons upfield of dC 46.3. HMBC correlations from H3-11000 and H3-12000 to dC 57.1, 205.0 and 213.5 and from H3-13000 and H3-14000 to dC 56.3, 100.0 and 213.5 were consistent with 4 containing
S. Rattanaburi et al. / Tetrahedron 69 (2013) 6070e6075 Table 3 13 C, 1H NMR and HMBC spectroscopic data (500 MHz, CDCl3) for 4 No.
dC
1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 10 11 12 13 10 20
197.4, C 56.0, C 212.0, C 47.0, C 167.4, C 153.1, C 106.7, C 151.2, C 108.3, C 160.9, C 109.8, C 32.1, CH 111.4, C 24.2, CH3 25.2, CH3 25.4, CH3 25.0, CH3 205.5, C 54.3, CH2
30 40 50 100 200 300 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000
24.5, C 22.1, CH3 22.1, CH3 34.0, CH 18.8, CH3 19.0, CH3 205.0, C 57.1, C 213.5, C 56.3, C 100.0, C 46.3, CH 28.8, CH 32.3, CH 20.1, CH3 16.0, CH3 22.1, CH3 26.7, CH3 19.4, CH3 24.4, CH3
dH (J in Hz)
Table 4 13 C, 1H NMR and HMBC spectroscopic data (500 MHz, CDCl3) for 5 HMBC
4.39, d (3.3)
4a, 4b
1.38, 1.44, 1.60, 1.64,
1, 1, 3, 3,
s s s s
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2, 2, 4, 4,
3 3 4a 4a
3.14, dd (16.8, 6.9) 2.89, dd (16.8, 6.9) 2.31e2.39, m 0.99, d (6.9) 0.98, d (6.9) 2.00e2.12, m 0.74, d (3.9) 0.77, d (3.9)
1 0 , 30 , 40 , 50
3.68, d (6.3) 4.17, dd (6.3, 3.9) 2.36e2.43, m 0.92, d (6.9) 0.68, d (6.9) 1.32, s 1.43, s 1.47, s 1.62, s
1000 , 5000 , 7000 , 8000
2 0 , 30 , 50 2 0 , 30 , 40 9, 100 , 300 9, 100 , 200
7000 , 7000 , 1000 , 1000 , 3000 , 3000 ,
8000 , 8000 , 2000 , 2000 , 4000 , 4000 ,
10000 9000 3000 3000 5000 5000
a second b-triketone in which one of the ketone groups was present as a ketal or hemiketal. H-6000 (dH 3.68) showed a COSY correlation to H-7000 (dH 4.17) and HMBC correlations to C-7000 (dC 26.8), C-1000 (dC 205.0), and C-8000 (dC 32.2) suggesting it was adjacent to H-7 and the carbonyl carbon C-1000. H-6000 further correlated to C-5 (dC 106.7) allowing the second b-triketone to be linked to the phloroglucinol via a bond from C-7000 of the isobutyl group. Finally, an additional ring between C-6 and C-5000 via an oxygen bridge to the hemiketal carbon at C-5000 (dC 100.0)9 was required to fulfill the structure. The assigned structure was compared to the structure of myrtucommulone D9 with the isovaleryl group at C-7 instead of isobutyl ketone. The NOESY experiment was used to identify the relative configurations at C-9, C-5000 , C-6000 , and C-7000. Cross peak of H-6000 to H-12000 , H-14000 H-8000 and that of H-8000 and H-100 to H-12 indicated that methyl groups H-12000 , H-14000 , H-12; methine proton H-6000 and the isopropyl groups were arranged on the b-face of molecule. The methyl proton H-14000 further correlated to methylene H-20 suggesting that 5000 -OH was on the a-face. The relative configuration of 9R*,5000 S*,6000 R*,7000 S*10 was therefore assigned for callistenone D (4). Since the optical rotation determined for 4 [a]29 D þ39.6 (MeOH) was similar in magnitude and sign to that of myrtucommulone D ([a]17 D þ13.9 (MeOH))10 this suggested that both molecules possessed the same absolute configuration, however this configuration could not be determined. Callistenone E (5) was obtained as a yellowish gum. It molecular formula C40H52O8 was determined from analysis of HREIMS [m/z 660.3674]. The NMR spectroscopic data and HMBC correlations (Table 4) together with COSY correlations observed for 5 mostly
No
dC
1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 10 11 12 13 10 20
197.5, C 56.3, C 212.5, C 47.3, C 167.5, C 153.2, C 105.3, C 150.1, C 107.7, C 160.7, C 110.2, C 32.1, CH 111.9, C 24.2, CH3 24.3, CH3 24.9, CH3 25.0, CH3 204.5, C 53.9, CH2
30 40 50 100 200 300 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000 15000 8-OH
24.5, CH 22.3, CH3 22.5, CH3 34.4, CH 19.5, CH3 18.4, CH3 197.3, C 56.1, C 212.4, C 47.2, C 166.1, C 113.2, C 31.9, CH 46.7, CH2 25.3, CH2 23.8, CH3 23.3, CH3 24.1, CH3 24.9, CH3 24.8, CH3 25.0, CH3
dH (J in Hz)
HMBC
4.40, d (3.3)
1, 4a, 4b, 8a, 100 , 200 , 300
1.36, 1.43, 1.48, 1.65,
1, 1, 3, 3,
s s s s
2, 2, 4, 4,
3, 11 3, 10 4a, 13 4a, 12
3.21, dd (17.4, 7.2) 3.02, dd (17.4, 7.2) 2.34e2.46, m 1.05, d (6.0) 1.04, d (6.0) 2.02e2.12, m 0.79, d (6.9) 0.82, d (6.9)
1 0 , 30 , 40 , 50
4.37, t (6.0) 1.51, obscured 1.51, obscured 0.84, d (6.0) 0.93, d (6.0) 1.39, s 1.44, s 1.50, s 1.66, s 13.51, s
1000 , 5000 , 6000 , 8000 , 9000 , 4b, 5, 6
2 0 , 30 , 50 2 0 , 30 , 40 9, 100 , 300 9, 100 , 200
8000 , 9000 , 11000 8000 , 9000 , 10000 1000 , 2000 , 3000 , 13000 1000 , 2000 , 3000 , 12000 3000 , 4000 , 5000 , 15000 3000 , 4000 , 5000 , 14000 7, 8, 8a
exhibited the same results as for callistenone D (4) except for the replacement of the isobutyl resonances by isopentyl resonances and the absence of a methine proton H-6000 . The protons of the isopentyl group resonated at dC 4.37 (t, J¼3.6 Hz, H-7000 ), 1.51 (obscured, H-8000 and H-9000 ), 0.84 (d, J¼3.6 Hz, H-10), and 0.95 (d, J¼3.6 Hz, H-11000 ). HMBC correlations from H-7000 to C-4b (dC 153.2), C-6 (dC 150.1), C-1000 (dC 197.3), C-5000 (dC 166.1), and C-9000 (dC 25.3) indicated that the isopentyl group was attached to C-5 of the phloroglucinol and C-6000 of the second b-triketone moiety. The NOESY cross peaks from H-9 to H-10 and H-12 and H-7000 to H-13, H12000 , and H-14000 indicated that methyl groups H-10, H-12, H-12000 , and H-14000 are on the a-face whereas the isobutyl and isopropyl groups were on the b-face of molecule. The indicated that 5 possessed a relative configuration of 9R*,7000 S*. Structure 5 was thus assigned for callistenone E. Compounds 1, 2, 3, 6, and 10 inhibited the growth of S. aureus ATTC25923 with MIC values of 0.5, 8, 8, 8, and 200 mg/mL and MRSA SK1 with MIC values of 1, 8, 8, 16, and 200 mg/mL, respectively, while 7 and 11 showed no activity at a concentration up to 200 mg/ mL. Compounds 4, 5, 8, and 9 were not tested due to insufficient material. Compound 1, which is the most active compound, provided strong antibacterial activity comparable to vancomycin. It is interesting to note that 1 shows 16 and 8 more potent antibacterial activity against ATTC25923 and SK1 strains of S. aureus, respectively, compared to its isomer (2) and this is mostly likely associated with the point of attachment of the isovaleryl side chain (Table 5).
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Table 5 Antibacterial activity of the dichloromethane extract and some of the pure compounds from C. lanceolatus Compound
Antibacterial activity (MIC, mg/mL) S. aureus ATCC25923
MRSA SK1
CH2Cl2 extract 1 2 3 6 7 10 11 Vancomycin
4 0.5 8 8 8 NA 200 NA 0.5
4 1 8 8 16 NA 200 NA 1
NA¼no activity at 200 mg/mL.
In conclusion, five new phloroglucinols and six known compounds have been isolated from the leaves of C. lanceolatus. All of phloroglucinol compounds, which were tested showed good antibacterial activity. Interestingly, callistenone A (1) exhibited comparable antibacterial activity to vancomycin suggesting that it could be a good candidate for further antibacterial drug development. This compound is now undergoing further bioactivity studies in our laboratory. 3. Experimental
yielding 1 (5.0 mg). Fraction A5 (14.3 g) was separated by CC using Me2CO/hexane (1:49) to Me2CO/hexane (1:19) to provide 13 fractions (A5AeA5M). Fraction A5C (845.7 mg) was further subjected to CC and eluted with Me2CO/hexane (1:49) to give 2 (6.2 mg). Fraction A5L (40.4 mg) was rechromatographed on PTLC using Me2CO/ CH2Cl2/hexane (1:1:18) as an eluant to produce 8 (2.1 mg) and 9 (0.7 mg). Fraction A5M (752.1 mg) was further purified by CC using the same solvent system to produce 3 (2.8 mg). Fractions A7 and A9 were each purified by CC using Me2CO/hexane (1:19) as an eluant to provide 4 (1.8 mg), 10 (2.7 mg) and 11 (3.7 mg), 5 (2.0 mg), respectively. 3.3.1. Callistenone A (1). Yield 5.0 mg. A yellowish gum. [a]29 D þ2.5 (c 2.82, MeOH). UV (MeOH) lmax nm (log 3 ): 207 (4.33), 223 (4.44), 260 (4.19), 299 (4.34), 350 (3.70). IR (neat) n (cm1): 3265, 1716, 1624. 1H NMR data (300 MHz, acetone-d6) and 13C NMR data (75 MHz, acetone-d6): see Table 1. FABMS m/z (% rel int): 429 ([Mþ1]þ, (4)), 385 (1), 369 (5), 277 (14), 185 (100). HRFABMS m/z 429.2270 [Mþ1]þ for C25H33O6 (calcd 429.2272). 3.3.2. Callistenone B (2). Yield 6.2 mg. A yellowish gum. [a]29 D þ2.8 (c 4.55, CHCl3). UV (MeOH) lmax nm (log 3 ): 205 (4.04), 227 (3.76), 292 (3.48). IR (neat)n (cm1): 3316, 1716, 1650. 1H NMR (300 MHz, acetone-d6) and 13C NMR (75 MHz, acetone-d6): see Table 1. FABMS m/z (% rel int): 429 ([Mþ1]þ, 100), 385 (55), 371 (34), 357 (13), 315 (17), 185 (12), 133 (14). HRFABMS m/z 429.2285 [Mþ1]þ for C25H33O6 (calcd 429.2277).
3.1. General experimental procedures IR spectra were measured on an FTS 165 FT-IR PerkineElmer spectrophotometer. UV spectra were recorded by a SPECORD S100 spectrophotometer. 1H and 13C NMR spectra were recorded in CDCl3 or acetone-d6 using a Bruker Avance FT-NMR 300 MHz and 500 MHz spectrometers. Optical rotations were determined in CHCl3 or MeOH solution at the sodium D line (589 nm) on a JASCO P-1020 polarimeter. The EIMS, HREIMS, FABMS, and HRFABMS mass spectra were obtained using a MAT 95 XL mass spectrometer (Thermo Finnigan). Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60H (Merck) and silica gel 100 (Merck), respectively. Precoated plates of silica gel 60 GF254 were used for TLC analysis. 3.2. Plant material The leaves of C. lanceolatus (Myrtaceae) were collected from Amphur Mueang Nakhon Si Thammarat, Nakhon Si Thammarat Province on October 2007. The plant was identified by Mr. Ponlawat Pattarakulpisutti and the herbarium specimen (S. Rattanaburi 1) has been deposited in the Herbarium of the Department of Biology, Faculty of Science, Prince of Songkla University, Thailand.
3.3.3. Callistenone C (3). Yield 2.8 mg. A yellowish gum. [a]29 D þ4.5 (c 3.60, MeOH). UV (MeOH) lmax nm (log 3 ): 205 (4.08), 230 (3.78), 291 (3.61). IR (neat) n (cm1): 3163, 1715, 1652. 1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3): see Table 2. EIMS m/z (% rel int): 446 ([Mþ], (10)), 385 (53), 236 (20), 210 (16), 193 (18), 166 (22), 153 (100), 123 (30). HREIMS m/z 446.2305 for C25H34O7 (calcd 446.2305). 3.3.4. Callistenone D (4). Yield 1.8 mg. A yellowish gum. [a]29 D þ39.6 (c 0.10, MeOH). UV (MeOH) lmax nm (log 3 ): 208 (4.76), 214 (4.77), 221 (4.78), 267 (4.48). IR (neat) n (cm1): 3389, 2953, 2930, 1716, 1653, 1537, 1162, 1015. 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3): see Table 3. FABMS m/z (% rel int): 665 ([Mþ1]þ, (4)), 621 (3), 429 (8), 277 (10), 185 (89). HRFABMS m/z 665.3682 for C39H53O9 (calcd 665.3684). 3.3.5. Callistenone E (5). Yield 2.0 mg. A yellowish gum. [a]29 D þ6.5 (c 0.23, MeOH). UV (MeOH) lmax nm (log 3 ): 220 (4.10), 261 (3.93), 292 (3.94), 355 (3.32). IR (neat) n (cm1): 3420, 2957, 2933, 1716, 1652, 1455, 1384, 1182, 1154. 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3): see Table 4. EIMS m/z (% rel int): 660 ([M]þ), 659 (61), 615 (74), 184 (79), HREIMS m/z 660.3674 for C40H52O8 (calcd 660.3657).
3.3. Extraction and isolation 3.4. Antibacterial activity Ground-dried leaves (4.5 kg) of C. lanceolatus were extracted with CH2Cl2 at room temperature for 3 days. The viscous extract (349.5 g) after removal of solvent was sequentially dissolved in hexane to give hexane soluble (107.6 g) and insoluble fractions. The hexane soluble fraction was purified by QCC using Me2CO/hexane (1:49) as an eluant. Based on TLC analysis, fractions that contained similar components were combined to give 10 fractions (A1eA10). Fraction A2 (23.4 g) was further subjected to CC and eluted with Me2CO/hexane (1:49) to give 6 (7.4 mg). Fraction A4 (2.7 g) was purified by CC and eluted with Me2CO/hexane (1:49) to obtain 10 fractions (A4AeA4J). Fraction A4F (27.2 mg) was further purified by PTLC using Me2CO/hexane (1:99) gave 7 (2.3 mg). Fraction A4G (145.6 mg) was further purified by CC using Me2CO/hexane (1:49)
The crude extract and purified compounds were screened for antibacterial activity at a concentration of 200 mg/mL by a broth microdilution method against four bacteria: S. aureus ATCC25923, a clinical isolate of methicillin-resistant S. aureus SK1, E. coli ATCC25922, P. aeruginosa ATCC27853. Crude extracts and purified compounds having antibacterial activity were further determined for their minimum inhibitory concentrations (MICs) by broth microdilution methods according to a modification of the Clinical and Laboratory Standards Institute. Resazurin was added as viability indicator according to Drummonds and Waigh (2000). Colorimetric MIC end-points were interpreted as the lowest concentration that remained blue (indicating no growth) or the
S. Rattanaburi et al. / Tetrahedron 69 (2013) 6070e6075
first dilution that changed from blue to slightly purple (equivalent to prominent growth inhibition). Acknowledgements We are grateful to the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program for a scholarship given to S.R. (grant no. PHD/0284/2550) and the National Research University Project of Thailand’s Office of the Higher Education Commission for financial support. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2013.05.085.
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References and notes 1. Chambers, H. F.; DeLeo, F. R. Nat. Rev. Microbiol. 2009, 7, 629e641. 2. Klink, J. W. V.; Brophy, J. J.; Perry, N. B.; Weavers, R. T. J. Nat. Prod. 1999, 62, 487e489. 3. Hiranrat, A.; Mahabusarakam, W. Tetrahedron 2008, 64, 11193e11197. 4. Crow, W. D.; Nicholls, W.; Sterns, M. Tetrahedron Lett. 1971, 18, 1353e1356. 5. Macias, F. A.; Simonet, A. M.; Esteban, M. D. Phytochemistry 1994, 36, 1369e1379. 6. Huq, F.; Misra, L. N. Planta Med. 1997, 63, 369e370. 7. Salni, D.; Sargent, M. V.; Skelton, B. W.; Soediro, I.; Sutisna, M.; White, A. H.; Yulinah, E. Aust. J. Chem. 2002, 55, 229e232. 8. Appendino, G.; Bianchi, F.; Minassi, A.; Sterner, O.; Ballero, M.; Gibbons, S. J. Nat. Prod. 2002, 65, 334e338. 9. Carroll, A. R.; Urban, S.; Lamb, J.; Moni, R.; Guymer, G. P.; Forster, P. I.; Quinn, R. J. J. Nat. Prod. 2008, 71, 881e883. 10. Carroll, A. R.; Lamb, J.; Moni, R.; Guymer, G. P.; Forster, P. I.; Quinn, R. J. J. Nat. Prod. 2008, 71, 1564e1568.