Article pubs.acs.org/jnp
Xanthoquinodins from the Endolichenic Fungal Strain Chaetomium elatum Guo-Dong Chen,†,⊥,∥ Ying Chen,†,∥ Hao Gao,*,† Li-Qing Shen,† Yang Wu,† Xiao-Xia Li,† Yan Li,‡ Liang-Dong Guo,§ Ying-Zhou Cen,⊥ and Xin-Sheng Yao*,† †
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China ‡ State Key Laboratory of Photochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China § State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ⊥ Department of Chemistry, Jinan University, Guangzhou 510632, People’s Republic of China S Supporting Information *
ABSTRACT: Five new xanthoquinodins, A4−A6 (1−3), B4 (4), and B5 (5), were isolated from the crude extract of the endolichenic fungal strain Chaetomium elatum (No. 63-10-31), along with three known xanthoquinodins, A1−A3 (6−8). Their structures were determined by detailed spectroscopic analysis and comparison of the NMR data with those of the closely related compounds previously reported. The absolute configuration of 1 was established by X-ray crystallographic analysis and ECD calculation. The cytotoxic activity of all compounds was tested against HL-60, SMMC-7721, A-549, MCF-7, and SW480 human cancer cell lines.
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RESULTS AND DISCUSSION Compound 1 was obtained as yellow needles. The quasimolecular ion at m/z 575.1547 [M + H]+ by HRESIMS indicated the molecular formula of 1 was C31H26O11 (19 degrees of unsaturation). The 13C NMR spectrum showed 31 carbon signals, which was consistent with the deduction of the HRESIMS. Combined with the DEPT experiment, these carbons can be categorized into 15 sp2 quaternary carbons [including three α,β-conjugated keto carbonyls (δC 193.1, 187.1, and 185.5) and two ester carbonyls (δC 175.5 and 168.7)], five sp2 methine carbons (δC 133.4, 131.4, 122.0, 118.8, and 110.7, respectively), two sp3 quaternary carbons [including an oxygenated carbon (δC 83.9)], three sp3 methine carbons [including two oxygenated (δC 80.8 and 73.1)], four sp3 methylene carbons (δC 39.2, 35.4, 27.4, and 21.8, respectively), one methoxyl carbon (δC 53.6), and one methyl carbon (δC 22.0). The proton resonances were assigned to relevant carbon atoms through the HSQC experiment. The analysis of the 1H−1H COSY experiment and the coupling values of protons revealed the presence of the two isolated spin systems, shown in bold in Figure 1a. Combined with the analysis of the HMBC spectrum and the 1H−1H COSY experiment, the partial structure of 1 (Figure 1a) was revealed.
anthone−anthraquinone heterodimers are a series of fungal metabolites with an unusual skeleton and antibacterial, antifungal, anticoccidial, antiplasmodial, and cytotoxic activities.1−5 On the basis of Omura’s work,6 these compounds originate from two identical C16 polyketide chains, one of which forms an anthraquinone-like monomer (A-unit) via decarboxylation of the tail, and the second forms a xanthone-like monomer (B-unit) by decarboxylation and oxidization, which fuses the anthraquinone-like monomer (Aunit) in “tail to body” mode (C-1, C-15 of the B-unit binding to C-6, C-9 of the A-unit) or in “tail to tail” mode (C-1, C-15 of the B-unit binding to C-4, C-15 of the A-unit). According to the binding modes of the two monomers, this family can be divided into xanthoquinodin-type heterodimers (such as xanthoquinodins,1,2 acremoxanthones,3,4 and acremonidins4) and beticolin-type heterodimers (such as beticolins5). In our search for bioactive secondary metabolites from endolichenic fungi,7−9 a chemical investigation of the fungal strain Chaetomium elatum (No. 63-10-3-1) was carried out, which led to the isolation of five new xanthoquinodins, A4-A6 (1−3), B4 (4), and B5 (5), along with three known xanthoquinodins, A1−A3 (6−8).6 Details of the isolation, structure characterization, and cytotoxicity of 1−8 are reported herein. © XXXX American Chemical Society and American Society of Pharmacognosy
Received: January 16, 2013
A
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from the existence of a β-keto−enol tautomeric system (C-10− C-12 moiety), which is similar to that found in beticolin-1.11,12 Due to the fast exchange between tautomers a and b (Figure 3) in the room-temperature NMR experiments, C-10 and C-12 appeared similar at intermediate chemical shifts between those corresponding to typical α,β-conjugated keto and enol carbons. Nevertheless, the exchange of tautomers slows at low temperature, so that low-temperature X-ray data could show the presence of one of the β-keto−enol tautomeric forms.13,14 Furthermore, the predicted ECD curves of 1 and its relevant enantiomer were calculated by a quantum chemical method at the [B3P86/6-311++G(2d,p)] level, and the predicted ECD curve of 1 was similar to the experimental one (Figure 4a), consistent with the deduction from the X-ray crystallography analysis. On the basis of these data, the structure of 1 was proposed as the reduzate of xanthoquinodin A3 at the 5-ketone and named xanthoquinodin A4. Compound 2 was obtained as a yellow, amorphous powder. The quasi-molecular ion at m/z [M + H]+ by HRESIMS indicated that the molecular formula of 2 was C32H30O12 (18 degrees of unsaturation). The 13C NMR spectrum showed 32 carbon signals, consistent with the deduction from the HRESIMS. Combined with the DEPT experiment, these carbons can be categorized into 15 sp2 quaternary carbons [including three α,β-conjugated keto carbonyls (δC 195.2, 187.4, and 185.4) and two ester carbonyls (δC 173.8 and 170.0)], five sp2 methine carbons (δC 133.3, 131.6, 121.9, 119.0, and 110.5, respectively), two sp3 quaternary carbons [including one oxygenated (δC 86.4)], three sp3 methine carbons [including two oxygenated (δC 73.8 and 73.3)], four sp3 methylene carbons (δC 38.4, 35.5, 30.3, and 25.6, respectively), two methoxyl carbons (δC 53.3 and 51.8), and one methyl carbon (δC 22.0). Except for the signal assignment of C-6′ to C-10′, most of the NMR data of 2 were similar to those of 1, indicating that 2 also possesses a xanthoquinodin skeleton. Compared to 1, the degrees of unsaturation of 2 and the significantly different chemical shifts of C-6′ to C-10′ in 2 indicated the structural change in the γ-lactone ring (C-7′−C10′). The key 1H−1H COSY correlations of Hb-8′ (δH 1.67, m) with H-7′ [δH 3.96, d (10.0)] and H-9′ (δH 2.51, m) and the HMBC cross-peak between the additional methoxyl protons at δH 3.66 and C-10′ (δC 173.8) confirmed that the γ-lactone ring (C-7′−C-10′) was cleaved to give the butyric acid methyl ester unit. During the structure elucidation, it was found that 2 can fully convert into 1 in CDCl3 in two weeks (Figure 5), while 1 was partial converted to 2 in MeOH after two weeks (Figure 6), suggesting that 1 and 2 share the same absolute configurations at C-5, C-6, C-9, C-6′, and C-7′ and the same double-bond configuration (C-7 to C-8). Therefore, 2 is the
On the basis of the above analysis, the degrees of unsaturation, and the molecular formula, the planar structure of 1 was elucidated as shown in Figure 1b, and the assignments of all proton and carbon resonances are provided in Table 1. The geometrical configuration of the double-bond moiety (C-7 to C-8) was deduced as Z on the basis of the coupling constant of the olefinic protons (J7−8 = 8.4 Hz). In the ROESY experiment, the ROESY correlations of H-5 with Ha-1′ and Hb-1′ indicated that 5-hydroxyl should have the same orientation as the doublebond moiety (C-7 to C-8). The single-crystal X-ray crystallography10 (Figure 2) of 1 confirmed the above deduction and assigned the absolute configuration of 1 as 5S, 6R, 9S, 6′R, and 7′S. Interestingly, the C-12 displayed the keto carbonyl property and the C-10 possessed the enol carbon property (the bond lengths of O(3)−C(12) and O(2)−C(10) were 1.275 and 1.335 Ǻ , respectively) in the X-ray crystallography (Figure 2). However, C-10 and C-12 showed the α,β-conjugated keto carbonyl property (δC 187.1 and 185.5) in the NMR experiments. The difference in properties of C-10 in the X-ray analysis and the NMR experiments results
Figure 1. 1H−1H COSY and key HMBC of 1. B
dx.doi.org/10.1021/np400041y | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. NMR Data for 1−5 (in CDCl3) 1 no.
δC, mult
1 2 3 4 5 6 7
22.0, CH3 147.9, C 122.0,CH 140.8, C 73.1, CH 42.7, C 133.4, CH
8
131.4, CH
9
37.4, CH
10 11 12 13 14 15 1′
2′ 3′ 4′ 5′ 6′ 7′
187.1, 104.8, 185.5, 111.4, 161.8, 118.8, 35.4,
149.5, 110.7, 157.2, 168.7, 83.9, 80.8,
C C C C C CH CH2
C CH C C C CH
2 δH (J in Hz) 2.38, s 6.77, s 4.51, s 6.44, dd (8.4, 1.9) 6.43, dd (8.4, 5.8) 4.73, dd (5.8, 1.9)
6.72, s 2.74, d (18.0), Ha 2.54, d (18.0), Hb 6.10, s
4.78, t (6.3)
δC, mult 22.0, CH3 147.9, C 121.9,CH 140.8, C 73.3, CH 42.7, C 133.3, CH 131.6, CH 37.5, CH 187.4, 104.6, 185.4, 111.4, 161.9, 119.0, 35.5,
148.8, 110.5, 157.2, 170.0, 86.4, 73.8,
C C C C C CH CH2
C CH C C C CH
8′
21.8, CH2
2.33, m
25.6, CH2
9′
27.4, CH2
2.52, m
30.3, CH2
10′ 11′
175.5, C 39.2, CH2
12′ 13′ 14′ 15′ 5′OCH3 10′OCH3 12-OH 14-OH 4′-OH 10′-OH 14′-OH
193.1, 105.3, 158.2, 117.3, 53.6,
a
C C C C CH3
3.04, d (17.2), Ha 2.87, d (17.2), Hb
3.71, s
173.8, C 38.4, CH2
195.2, 105.4, 158.3, 117.2, 53.3,
2.38, s
δC, mult
6.45a
22.1, 147.8, 121.9, 140.8, 73.4, 42.8, 133.1,
6.45a
131.9, CH
6.78, s 4.53, s
4.76, br s
6.78, s 2.72, d (17.3), Ha 2.54, m, Hb
6.05, s
3.96, br d (10.0) 1.81, m, Ha 1.67, m, Hb 2.51, m
3.08, s
C C C C CH3
51.8, CH3
11.90, s
3 δH (J in Hz)
4 δH (J in Hz)
CH3 C CH C CH C CH
37.4, CH
2.40, s
6.45a
22.0, 147.9, 122.1, 140.6, 73.4, 42.8, 132.9,
6.45a
131.8, CH
6.79, s 4.53, s
4.77, br d (5.7)
187.2, C 104.8,b, C 185.4, C 111.6, C 162.0, C 119.0, CH 35.3, CH2
δC, mult
6.81, s 2.71, m, Ha
CH3 C CH C CH C CH
39.1, CH 188.7, 105.0, 183.4, 111.0, 161.5, 119.0, 35.3,
C C C C C CH CH2
2.60, m, Hb 148.2, 110.8, 156.8, 169.5, 84.3, 71.7,
C CH C C C CH
6.07, s
23.7, CH2 27.5, CH2
4.24, dd (11.7, 3.9) 2.15, m, Ha 2.06, m, Hb 2.65, m
148.0, 114.0, 159.9, 170.0, 85.2, 71.6,
C CH C C C CH
23.8, CH2 27.7, CH2
5 δH (J in Hz) 2.36, s
δC, mult
6.46a
22.1, 148.1, 121.9, 140.8, 73.4, 42.7, 133.4,
6.46a
131.7, CH
6.78, s 4.52, s
4.83, br s
6.75, s 2.71, d (17.8), Ha 2.56, d (17.8), Hb 6.07, s
4.36, br d (10.0) 2.20, m, Ha 2.08, m, Hb 2.65, m
CH3 C CH C CH C CH
38.1, CH 187.0, C 105.2,c, C 186.0, C 111.5, C 162.1, C 119.1, CH 35.3, CH2
147.7, 114.7, 160.1, 170.9, 84.5, 66.9,
C CH C C C CH
22.9, CH2 24.4, CH2
178.6, C 101.6, C
180.2, C 100.2, C
186.5, C 104.9,b C 158.3, C 117.4, C 53.2, CH3
186.7, 105.3, 154.9, 115.4, 53.2,
186.6, C 105.3,c C 153.7, C 115.4, C 53.5, CH3
3.68, s
3.72, s
6.79, s 4.56, s 6.47a 6.47a 4.77, m
6.79, s 2.72, d (17.6), Ha 2.61, d (17.6), Hb
178.0, C 101.2, C
C C C C CH3
δH (J in Hz) 2.39, s
6.13, s
4.48, br s 2.15, m, Ha 1.98, m, Hb 2.83, ddd (18.8, 11.7, 7.3), Ha 2.42, m, Hb
3.73, s
3.66, s 14.22, s 11.54, s
14.24, br s 11.58, s
12.08, s
13.84, s 11.75, s
:Overlapped signals are reported without designating multiplicity.
b,c
14.27, 11.56, 11.04, 13.95,
br s s s s
14.36, 11.56, 11.23, 14.09,
br s s s s
The assignment may be exchanged in each column.
quaternary carbons [including three α,β-conjugated keto carbonyls (δC 187.2, 186.5, and 185.4) and one ester carbonyl (δC 169.5)], five sp2 methine carbons (δC 133.1, 131.9, 121.9, 119.0, and 110.8, respectively), two sp3 quaternary carbons [including one oxygenated (δC 84.3)], three sp3 methine carbons [including two oxygenated (δC 73.4 and 71.7)], three sp3 methylene carbons (δC 35.3, 27.5, and 23.7, respectively), one methoxyl carbon (δC 53.2), and one methyl carbon (δC 22.1). The NMR spectroscopic data of 3 (Table 1) were similar
methanolysis derivative of xanthoquinodin A4 and named xanthoquinodin A5. Compound 3 was obtained as a yellow, amorphous powder. The molecular formula was established as C31H26O12 (19 degrees of unsaturation) by the quasi-molecular ion at m/z 575.1571 [M + H]+ in the HRESIMS. The 13C NMR spectrum showed 31 carbon signals, which was consistent with the deduction of the HRESIMS. Combined with the DEPT experiment, these carbons can be categorized into 15 sp2 C
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(δC 121.9), C-11 (δC 104.8), and C-13 (δC 111.6) confirmed the above deduction. Analysis of the 1H−1H COSY and HMBC data led to the assignment of proton and carbon resonances (Table 1). On the basis of the NMR data of the B-unit carbons (C-5′ to C-11′) in 3 being similar to those in xanthoquinodin A2 (7), the configurations of C-6′ and C-7′ were deduced to be the same as those of xanthoquinodin A2 (7). On the other hand, the configurations of 5-hydroxyl and the double-bond moiety (C-7 to C-8) were assigned as the same as those of 1, because the NMR data of C-1′ and the A-unit carbons (C-3 to C-13) in 3 were similar to 1. Considering the co-occurrence of 1 and 3 in C. elatum (No. 63-10-3-1), it was deduced that these compounds should possess the same absolute configurations at C-5, C-6, C-9, and C-7′. Furthermore, the predicted ECD curves of 3 and its relevant enantiomer were calculated by a quantum chemical method at the [B3P86/6-311++G (2d,p)] level, and the predicted ECD curve of 3 was similar to the experimental one (Figure 4b). Therefore, the absolute configuration of 3 was assigned as 5S, 6R, 9S, 6′S, and 7′S. On the basis of the above data, 3 was deduced as the reduzate of xanthoquinodin A2 at the 5-ketone and named xanthoquinodin A6. Compound 4 was isolated as a yellow, amorphous powder. Its molecular formula was also determined by its HRESIMS. The 13C NMR spectrum showed 31 carbon signals, which was consistent with the deduction of the HRESIMS. Combined with the DEPT experiment, these carbons can be categorized into 15 sp2 quaternary carbons [including two α,β-conjugated keto carbonyls (δC 188.7, 186.7 and 183.4) and one ester carbonyl (δC 170.0)], five sp2 methine carbons (δC 132.9,
Figure 2. ORTEP drawing of the asymmetric unit of xanthoquinodin A4 (1).
Figure 3. Exchange between tautomers a and b.
to those of xanthoquinodin A2 (7), expect for an oxymethine carbon at δC 73.4 (s) and the loss of a ketone carbonyl at δC 195.7 (s), indicating the 5-ketone in xanthoquinodin A2 (7) had been reduced to the 5-hydroxyl. The key HMBC correlations from the oxymethine proton at δH 4.53 to C-3
Figure 4. ECD spectra of 1−5 (in MeOH) (4a: experimental spectra of 1 and 2 and calculated ECD spectrum of 1 (5S, 6R, 9S, 6′R, 7′S) and its enantiomer (5R, 6S, 9R, 6′S, 7′R); 4b: experimental spectrum of 3 and calculated ECD spectrum of 3 (5S, 6R, 9S, 6′S, 7′S) and its enantiomer (5R, 6S, 9R, 6′R, 7′R); 4c: experimental spectrum of 4 and calculated ECD spectrum of 4 (5S, 6R, 9S, 6′S, 7′S) and its enantiomer (5R, 6S, 9R, 6′R, 7′R); 4d: experimental spectrum of 5 and calculated ECD spectrum of 5 (5S, 6R, 9S, 6′R, 7′S) and its enantiomer (5R, 6S, 9R, 6′S, 7′R)). D
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Figure 5. Conversion of 2 to 1 in CDCl3 (the conversion of 2 was detected by 1H NMR after 0, 1, and 2 weeks).
Figure 6. Comparison of 1, 1 (kept in MeOH after 2 weeks), and 2 carried out by HPLC.
131.8, 122.1, 119.0, and 114.0, respectively), two sp3 quaternary carbons [including one oxygenated (δC 85.2)], three sp3 methine carbons [including two oxygenated (δC 73.4 and 71.6)], three sp3 methylene carbons (δC 35.3, 27.7, and 23.8, respectively), one methoxyl carbon (δC 53.2), and one methyl carbon (δC 22.0). The NMR spectroscopic data of 4 (Table 1) resembled those of xanthoquinodin B2,6 expect for the lack of a 5-ketone carbonyl and the appearance of an oxymethine carbon at δC 73.4 (s), indicating the 5-ketone had been reduced. The key HMBC correlations from the additional oxymethine proton at δH 4.52 to C-3 (δC 122.1), C-11 (δC 105.0), and C-13 (δC 111.0) confirmed the above deduction. Analysis of the 1H−1H COSY and HMBC data led to the assignment of proton and carbon resonances (Table 1). On the basis of the NMR data of the B-unit carbons (C-5′ to C-11′) in 4 being similar to those of xanthoquinodin B2,6 4 and xanthoquinodin B2 should share the same configurations at C-6′ and C-7′. Likewise, the
configurations of the 5-hyhroxyl and the double-bond moiety (C-7 to C-8) were assigned as the same as those of 1 through comparing the NMR data of C-1′ and the A-unit carbons (C-3 to C-13) in 4 with those of 1. Considering the co-occurrence of 1 and 4 in C. elatum (No. 63-10-3-1), it can be concluded that the absolute configurations at C-5, C-6, C-9, and C-7′ of 4 should be the same as those of 1. Furthermore, the predicted ECD curves of 4 and its relevant enantiomer were calculated by a quantum chemical method at the [B3P86/6-311++G (2d,p)] level, and the predicted ECD curve of 4 was similar to the experimental one (Figure 4c). Therefore, the absolute configuration of 4 was assigned as 5S, 6R, 9S, 6′S, and 7′S. On the basis of the above data, 4 was deduced as the reduzate of xanthoquinodin B2 at the 5-ketone and named xanthoquinodin B4. Compound 5, obtained as a yellow, amorphous powder, was an isomer of 4 and was assigned as C31H26O12 (19 degrees of E
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unsaturation) based on the quasi-molecular ion at m/z 575.1568 [M + H]+ in its HRESIMS. The 13C NMR spectrum showed 31 carbon signals. Combined with the DEPT experiment, these carbons can be categorized into 15 sp2 quaternary carbons [including two α,β-conjugated keto carbonyls (δC 187.0 186.6 and 186.0) and one ester carbonyl (δC 170.9)], five sp2 methine carbons (δC 133.4, 131.7, 121.9, 119.1, and 114.7, respectively), two sp3 quaternary carbons [including one oxygenated (δC 84.5)], three sp3 methine carbons [including two oxygenated (δC 73.4 and 66.9)], three sp3 methylene carbons (δC 35.3, 24.4, and 22.9, respectively), one methoxyl carbon (δC 53.5), and one methyl carbon (δC 22.1). The NMR spectroscopic data of 5 (Table 1) resembled those of 4, with the main chemical shift difference located at the positions 6′−11′ in the B-unit, which indicated epimerization at C-6′ or C-7′. On the basis of the NMR data of the B-unit carbons (C-5′ to C-13′) in 5 being similar to xanthoquinodin B1,6 the configurations of C-6′ and C-7′ in 5 should be the same as those of xanthoquinodin B1. Likewise, the configurations of the 5-hydroxyl and the double-bond moiety (C-7 to C-8) were assigned as the same as 1 based on the NMR data of C-1′ and the A-unit carbons (C-3 to C-13) in 1 and 5. Considering the co-occurrence of 1 and 5 in C. elatum (No. 6310-3-1), the absolute configurations at C-5, C-6, C-9, and C-7′ of 5 were proposed to be the same as those of 1. Furthermore, the predicted ECD curves of 5 and its relevant enantiomer were calculated by a quantum chemical method at the [B3P86/6311++G (2d,p)] level, and the predicted ECD curve of 5 was similar to the experimental one (Figure 4d). Therefore, the absolute configuration of 5 was assigned as 5S, 6R, 9S, 6′R, and 7′S. On the basis of the above data, 5 was deduced to be the reduzate of xanthoquinodin B1 at the 5-ketone and named xanthoquinodin B5. The absolute configuration of 1 was assigned as 5S, 6R, 9S, 6′R, and 7′S by single-crystal X-ray crystallography, which is the first report of the absolute configurations of xanthoquinodintype heterodimers. The ECD experiments of 6−8 also had been carried out, and the data are shown in (Figure 7).
Table 2. Cytotoxicity of 1−8 against Human Tumor Cell Lines (IC50: μM) no.
HL-60
SMMC-7721
A-549
MCF-7
SW480
1 2 3 4 5 6 7 8 cisplatin paclitaxel
>40 26.62 3.75 3.01 4.74 6.22 5.50 4.18 1.86
