Tetrahedron Letters 58 (2017) 3223–3225
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Actinomadurone, a polycyclic tetrahydroxanthone from Actinomadura sp. BCC 35430 Taridaporn Bunyapaiboonsri ⇑, Seangaroon Yoiprommarat, Chanwit Suriyachadkun, Sumalee Supothina, Rungtiwa Chanthaket, Chanikul Chutrakul, Vanicha Vichai National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand
a r t i c l e
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Article history: Received 26 April 2017 Revised 14 June 2017 Accepted 1 July 2017 Available online 4 July 2017
a b s t r a c t A polycyclic tetrahydroxanthone, actinomadurone, was isolated from the actinomycete Actinomadura sp. BCC 35430. Its structure was elucidated by extensive spectroscopic analyses. Actinomadurone showed antifungal activities against Curvularia lunata, Alternaria brassicicola, Colletotrichum capsici, and Colletotrichum gloeosporioides; and displayed cytotoxic activity against Vero cell lines. Ó 2017 Elsevier Ltd. All rights reserved.
Keywords: Antifungal activity Actinomadura sp. Polycyclic tetrahydroxanthone
Polycyclic xanthones, a family of polyketides possessing highly oxygenated, angular hexacyclic aromatic framework, have been produced from several actinomycete genera, including Actinomadura, Actinoplanes, Amycolatopsis, Kibdelosporangium, and Streptomyces.1 Compounds in this class contain typical cyclic amide moiety, and can be classified based on oxidation states of xanthone rings into fully aromatic-, tetrahydro-, and hexahydro-xanthone derivatives.1 In addition to the hexacyclic core skeleton, formation of methylenedioxy bridge is observed in several members of this family, such as simaomicin a from Actinomadura madurae,2 actinoplanones from Actinoplanes sp.,3 kigamicins from Amycolatopsis sp.,4 and xantholipin from Streptomyces flavogriseus.5 Apart from the structural aspects, polycyclic xanthones have also been shown to possess a wide range of potent biological activities, including anticoccidial, antimalarial, antimicrobial, and anticancer properties.1–3,5–9 In our ongoing research on novel bioactive compounds from Thai actinomycete, five polycyclic tetrahydroxanthones (chrestoxanthones A-C, albofungin and chloroalbofungin) have been reported from Streptomyces chrestomyceticus BCC 24770.10 In this study, the strain Actinomadura sp. BCC 35430, isolated from soil sample collected in northeastern Thailand, was investigated as its EtOAc extract from small scale culture showed antifungal activity against Curvularia lunata, Alternaria brassicicola, Colletotrichum capsici, and Colletotrichum gloeosporioides with MIC values of 6.25, 12.5, 12.5, and 12.5 lg/mL, respectively. Bioassay-guided fraction⇑ Corresponding author. E-mail address:
[email protected] (T. Bunyapaiboonsri). http://dx.doi.org/10.1016/j.tetlet.2017.07.008 0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.
ation of the extract from scale-up fermentation of this actinomycete led to the isolation and structure elucidation of a new polycyclic tetrahydroxanthone, actinomadurone (1) (Fig. 1). Actinomadurone (1)11 was obtained as a yellow-brown solid from the crude EtOAc extract of Actinomadura sp. BCC 35430 after purification by Sephadex LH-20 column chromatography and reversed-phase HPLC. Its molecular formula, C34H31NO10, was deduced based on the quasimolecular ion peak at m/z 614.2032 [M + H]+, indicating twenty degrees of unsaturation. The 13C NMR spectrum of 1 included resonances of a ketone carbonyl carbon (dC 200.2), an amide and an ester carbonyl carbons (dC 167.4 and 166.1), thirteen quarternary sp2 carbons/oxycarbons (dC 152.2– 107.5), seven olefinic/aromatic methines (dC 146.6–100.9), a dioxymethylene (dC 90.9), three oxymethines (dC 77.9, 73.1, and 66.3), a methine (dC 39.6), four methylenes (dC 35.1, 30.1, 25.4, and 22.1), and two methyl groups (dC 19.5 and 14.2), representing thirteen sites of unsaturation, thus suggesting the presence of seven rings. Analysis of its 2D NMR data revealed the presence of isoquinolin1(2H)-one and tetrahydroxanthone subunits. A chelated hydroxy proton (dH 13.25, 3-OH), an amide proton (dH 11.74, NH), and a sp2 methine (dH 6.66, H-24) have HMBC correlations with C-2. Additional HMBC correlations from the chelated hydroxy proton (3-OH) to C-3 and C-4; from the amide proton to C-24 and C-26; from the methine H-24 to C-22, C-23, C-25, and C-26; and from 22-OH to C-21, C-22, and C-23 (as shown in Fig. 2 and Table 1) also supported the isoquinolin-1(2H)-one moiety. The tetrahydroxanthone subunit was deduced based on a spin system of cyclohexene ring and HMBC correlations from a chelated hydroxy proton (dH 12.03, 6-OH) to C-5, C-6, and C-7; and from H-9 to C-8. The
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T. Bunyapaiboonsri et al. / Tetrahedron Letters 58 (2017) 3223–3225 11
O HO O HO 1
HN H3C
25
23
24
26
3
2
4
21
OH
6
H
10 9
8
13
14
7
16 17
5 18 19 20
H
O
12
H
O
O
3'
5'
7'
1'
O 27
O
Fig. 1. Structure of actinomadurone (1).
11 10
O O H
HO HO 3
1
6
5
N
8
9
13
23
26
O
14
7 16
1'
3'
5'
7'
O
17
O
18
21
25
O
12
19
O
27
OH Fig. 2. COSY (in bold) and key HMBC correlations for 1.
Table 1 H (400 MHz) and
1
position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 17 18 19 20
13
remaining of the two rings together with a spin system of -CH2CH- and HMBC correlations observed from H2-20 to C-4 and C18; and from dioxymethylene protons H2-27 to C-17 and C-19 supported the connectivity between the isoquinolin-1(2H)-one and the tetrahydroxanthone subunits. The linear side chain was assigned based on a spin system of ACH@CHACH@CHACH2ACH2ACH3 and HMBC correlations from trans olefinic protons [dH 5.90 (d, J = 15.4 Hz), H-20 ; and dH 7.33 (dd, J = 15.4, 10.0 Hz), H-30 ] to an ester carbonyl carbon C-10 (dC 166.1). Due to the overlapping 1 H NMR signals of H-40 and H-50 , the E-configuration between C40 and C-50 could not be determined by using the 3J coupling constant, but suggested based on unobserved NOESY correlation between H-30 and H2-60 . A downfield shift of oxymethine signal H-13 [dH 5.57 (dd, J = 5.6, 3.5 Hz)] proposed an attachment of the linear side chain to C-13. The stereochemistry of 1 was deduced on the basis of 3JH-H coupling constants, NOESY correlations, and circular dichroism (CD) spectral data. The cyclohexene ring adopted pseudochair conformation as depicted (Fig. 3) by structural comparison with known polycyclic xanthones including kigamicins,12 kibdelones,13 actinoplanones,3 chrestoxanthones and albofungin.10 The large coupling constant of 14.1 Hz suggested pseudo-trans-diaxial relationship between H-9 and H-14, which was supported by NOESY correlation between H-14 and H-10b. Based on the small coupling constant,
C NMR (100 MHz) data for actinomadurone (1) in acetone-d6. dH, mult. (J in Hz)
dC
type
167.4 109.7 152.2 113.6 110.8b 152.0 107.5b 200.2 39.6 25.4
C C C C C C C C CH CH2
a 2.27, dd (13.9, 13.0)
133.1 122.9 66.3 77.9 147.8 131.5 133.6 73.1 30.1
CH CH CH CH C C C CH CH2
3.57, m
a 2.74, ddd (19.1, 5.6, 4.8) b 2.32, m 6.08, ddd (9.6, 4.8, 2.3) 5.86, m 5.57, dd (5.6, 3.5) 4.87, dd (14.1, 3.5)
4.74, dd (13.0, 4.7)
a b
C-14 C-9, C-11, C-12, C-14
H-27b, H-20a, H-20b
C-2, C-22, C-23, C-25, C-26
H3-26, 22-OH
C-24, C-25
H-24, NH H-27b H-19, H-27a
6.66, s 2.26, s
138.1 19.5
C CH3
27
a 5.46, d (6.0)
90.9
CH2
166.1 119.4 146.0 129.2 146.6 35.1 22.1 14.2
C CH CH CH CH CH2 CH2 CH3
b 5.24, d (6.0)
Overlapping signals. Interchangeable signals.
H-10a, H-10b, H-14 H-9, H-11 H-9, H-11, H-14 H-10a, H-10b, H-12 H-11, H-13 H-12, H-14 H-9, H-10b, H-13
C C C CH
25 26
5.90, d (15.4) 7.33, dd (15.4, 10.0) 6.28, ma 6.28, ma 2.11, dt (5.8, 7.3) 1.39, sext (7.3) 0.86, t (7.3) 11.74, s 13.25, s 12.03, s 8.66, s
C-8, C-10, C-11, C-14 C-9, C-11, C-12 C-9, C-11, C-12, C-14
H-19, 22-OH
127.4 139.1 130.1 100.9
10 20 30 40 50 60 70 80 NH 3-OH 6-OH 22-OH
NOESY
C-18, C-20 C-4, C-18, C-19, C-21, C-22 C-4, C-18, C-19, C-21, C-22
b 3.55, dd (13.9, 4.7)
21 22 23 24
HMBC
C-17, C-19 C-19 C-10 , C-40 C-10 , C-50 C-20 , C-50 , C-60 C-40 , C-60 C-40 , C-50 , C-70 , C-80 C-50 , C-60 , C-80 C-60 , C-70 C-2, C-24, C-26 C-2, C-3, C-4 C-5, C-6, C-7 C-21, C-22, C-23
H-19, 22-OH
H-30 , H-40 /H-50 H-20 , H-40 /H-50 H-30 , H2-60 H2-60 , H-30 H-40 /H-50 , H2-70 H2-60 , H3-80 H2-60 , H2-70 H3-26
H-24, H-20a, H-20b
T. Bunyapaiboonsri et al. / Tetrahedron Letters 58 (2017) 3223–3225
were closely similar, therefore the absolute configuration of 1 at C-19 was assigned as R-configuration. Actinomadurone (1) displayed antifungal activities against Colletotrichum capsici and Colletotrichum gloeosporioides (the causal agents of chilli anthracnose) with the same MIC90 value of 0.64 lM. It also exhibited antifungal activities against Alternaria brassicicola (a causal agent of black spot disease on most Brassica spp.) and Curvularia lunata (a causal agent of rice dirty panicle disease) with respective MIC70 = 0.64 lM and MIC90 = 5.10 lM. Cytotoxic activity against African green monkey kidney fibroblast (Vero) cells of 1 was observed with an IC50 value of 0.008 lM.
26 25
1
23
3
21 20
5
19
6
18 27
17
7 8 16
9
10
14
1'
11 13
Acknowledgments
12
Financial support from National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA) (Grant No. P-12-00041) is gratefully acknowledged. A. Supplementary data
Fig. 3. NOESY correlations for 1.
Supplementary data (detailed description of the experimental procedures; and 1H, 13C and 2D NMR spectra for 1) associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.tetlet.2017.07.008.
50 40 30
References
20
De
1. 2. 3. 4.
10 0 200 -10
250
300
350
-20
5. 6. 7. 8.
-30 -40
3225
l (nm) Fig. 4. CD spectrum of actinomadurone (1) in methanol.
JH-13,H-14 of 3.5 Hz, a pseudo-equatorial position of H-13 was proposed. The CD spectrum of 1 exhibited positive cotton effect at 219–248 nm region, and negative cotton effects at 208–219, and 248–350 nm regions as shown in Fig. 4. Since the CD spectral patterns of 1 and albofungol14 (a degradation product of albofungin)
9. 10. 11.
12. 13. 14.
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