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Polycyclic Hybrid Isoprenoids from a Reed Rhizosphere Soil Derived Streptomyces sp. CHQ-64 Qian Che,† Tianjiao Zhu,† Robert A. Keyzers,‡ Xiaofang Liu,† Jing Li,§ Qianqun Gu,† and Dehai Li*,† †
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington 6140, New Zealand § College of Marine Life Sciences, Ocean University of China, Qingdao 266003, People’s Republic of China S Supporting Information *
ABSTRACT: Two new hybrid isoprenoids, named indotertine B (2) and drimentine H (3), along with the known analogue drimentine C (4), were isolated from the reed rhizosphere soil derived actinomycete Streptomyces sp. CHQ-64. The structures of these compounds, including absolute configurations, were elucidated by extensive NMR, MS, and CD analyses. Indotertine B (2) exists as a pair of rotamers about the N−C(O) bond with a 2:1 ratio and displays activities against HCT-8 and A549 tumor cell lines with IC50 values of 6.96 and 4.88 μM, respectively.
T
Preliminary HPLC-UV profiles of the broth indicated the presence of various undiscovered HI alkaloids with UV absorptions similar to indotertine A.11 All the compounds were isolated using a combination of chromatographic techniques including column chromatography and culminating with semipreparative HPLC, and their structures were elucidated by spectroscopic methods (IR, UV, NMR, and MS). Indotertine B (2) exists as a pair of rotamers inseparable by HPLC because of fast interconversion. However, a set of separated signals with different intensities in the 1H NMR spectrum suggested that 2a and 2b exist in two different forms, in a ratio of 2:1. The EIMS spectrum of the mixture revealed a parent ion peak at m/z 531 [M]+, while the HRESIMS established the molecular formula to be C33H45N3O3. The 1H and 13C NMR data of the major compound 2a displayed resonances that were assigned to eight quaternary carbons, 12 methines including a formamide group, seven methylenes, and six methyls (including one N-methyl) (Table 1). The physicochemical properties and NMR data obtained for 2a showed a striking resemblance to those of indotertine A (1).11 In fact, the only major difference was the appearance of a formyl signal at δH 8.95 and δC 157.5, which implied that it was attached to either nitrogen or oxygen. The HMBC correlation from the formyl proton to C-2 (δC 63.5) placed the formyl group on N-1. The relative configuration of 2a was deduced by analyzing its NOESY spectrum. A correlation between H-2 and H2-22a (δH 2.32) suggested they have a cis relationship. The correlations
erpenoids are among the most diverse secondary metabolites observed in nature.1 In addition, terpenoid moieties can also be attached to molecules produced via nonterpenoid biosynthetic routes, forming products known as hybrid isoprenoids (HIs).2 Although common as secondary metabolites of eukaryotes, terpenoids and especially HIs are surprisingly rare in prokaryotes. 3 For instance, many indolosesquiterpenes such as suaveolindole,4 greenwayodendrine,5 polyveoline,6 and 3-farnesylindole derivatives7 have been reported from plants, while only seven structurally related indolosesquiterpenes have been reported from bacteria, the first being in 2010.8−10 We have previously reported indotertine A (1) from cultures of the reed rhizosphere soil derived actinomycete Streptomyces sp. CHQ-64.11 Indotertine A comprises a novel scaffold, characterized by a condensed ring system containing a tryptophan-derived indole moiety and a sesquiterpene unit, and represents a new class of HIs merging amino acid and mevalonate pathways.11 In an effort to obtain more insight into the biosynthesis and possible structure− activity relationships in this family of metabolites, investigations guided by characteristic indole-based UV absorptions from a new batch of this strain led to the discovery of two new hybrid isoprenoids containing a formyl-N amide (2, 3), with a known hybrid isoprenoid, drimentine C (4).12 The new indotertine analogue (2) comprises a pair of rotamers about the N−C (O) bond, which exist as stable conformers 2a and 2b with a ratio of 2:1. Herein, we report the isolation, structural determination, and cytotoxicities of these compounds against a panel of five human tumor cell lines (HCT-8, Bel-7402, BGC-823, A-549, and A-2780). The strain Streptomyces sp. CHQ-64 (GenBank No: JQ405211) was fermented (100 L) as previously described.11 © 2013 American Chemical Society and American Society of Pharmacognosy
Received: December 19, 2012 Published: April 2, 2013 759
dx.doi.org/10.1021/np3008864 | J. Nat. Prod. 2013, 76, 759−763
Journal of Natural Products
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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for Compounds 2a, 2b, and 3 2a position 1 2 3 4 5 6 7 8 9 10 11 12
δC 63.5, 45.2, 135.1, 123.1, 124.8, 129.2, 111.7, 140.0, 117.4, 142.8, 34.4,
CH C C CH CH CH CH C CH C CH2
13
20.9, CH2
14 15
53.8, CH 33.3, C
16
41.8, CH2
17 18
18.9, CH2 39.9, CH2
19 20
37.6, C 47.2, CH
21
26.4, CH2
22
49.0, CH2
23 24
52.0, CH 166.8, C
26
67.7, CH
27 28 29 30 31 32 33 34 35 36
2b
δH, mult (J in Hz) 4.75, d (2.2)
7.15, 7.16, 7.29, 7.22,
d (7.7) m m d (7.7)
5.33, d (2.2) 2.22, 1.82, 1.49, 1.27, 0.95,
m m m m m
1.41, br d (13.2) 1.15, m 1.49 m 1.90, m 1.05, m 1.65, m a: 2.41, m b: 1.59, m a: 2.32, dd (14.3, 2.2) b: 1.80, m 4.00, m
3.60, d (4.9)
165.5, C
15.0, CH3 33.7, 22.1, 31.7, 18.5, 19.4, 34.5, 157.5,
CH3 CH3 CH CH3 CH3 CH3 CH
δC 64.4, 45.0, 135.1, 122.0, 125.0, 129.2, 119.2, 140.0, 120.0, 143.7, 34.4,
CH C C CH CH CH CH C CH C CH2
20.9, CH2 53.8, CH 33.3, C 41.8, CH2 18.9, CH2 39.9 CH2 37.6, C 47.2, CH 24.9, CH2 48.9, CH2 52.0, CH 166.8, C 67.7, CH
3
δH, mult (J in Hz) 4.44, d (1.9)
7.12, 7.11, 7.30, 8.08,
d (8.3) m m d (8.3)
5.18, d (1.9) 2.22, 1.82, 1.49, 1.27, 0.95,
m m m m m
0.82, 0.85, 2.11, 0.90, 1.05, 2.96, 8.95,
s s m d (6.6) d (7.1) s s
15.0, CH3 33.8, 22.1, 31.7, 18.5, 19.4, 34.5, 159.5,
CH3 CH3 CH CH3 CH3 CH3 CH
δC
1 2 3 4 5 5a 6 6a 7 8 9 10
141.1, 116.4, 129.2, 125.4, 123.9,
10a
134.2, C
δH, mult (J in Hz)
165.0, C 67.6, CH 163.9, C 77.7, CH C CH CH CH CH
3.90, br s
6.26, s
8.03, 7.29, 7.17, 7.22,
d (7.7) dd (7.7,7.1) dd (7.7, 7.1) d (7.1)
10b 11
55.2, C 42.9, CH2
11a
57.8, CH
2.63, dd (12.1, 5.0) 2.11, dd (12.6, 12.1) 3.91, dd (12.6, 5.0)
12 13
31.1, CH2 52.5, CH
1.97, d (6.1) 1.32, m
14 15
149.7, C 38.3, CH2
16
24.4, CH2
16a
55.7, CH
17 18
33.6, C 41.8, CH2
19
19.2, CH2
20
38.9, CH2
5.09, d (2.2) 0.81, s
20a 21
40.2, C 107.5, CH2
0.85, 0.83, 2.11, 0.90, 1.05, 2.96, 8.57,
22 23 24 25 26 27 28- NMe 29
33.8, 21.7, 14.5, 31.3, 19.6, 16.7, 33.6, 162.1,
1.41, 1.15, 1.49, 1.90, 1.05,
br d (13.2) m m m m
1.71, m a: 2.40, m b: 1.59, m a: 2.32, dd (14.3, 2.2) b: 1.80, m 4.03, m
3.62, d (4.4)
165.4, C 4.83, d (2.8) 0.81, s
position
between H-34 (δH 1.05) and H-22b (δH 1.80) indicated the syn orientation of H-23 (δH 4.00) and H-26 (δH 3.60). Diagnostic NOE correlations of H-22a/H-21b (δH 1.59) and H-21b/Me29 (δH 0.81) positioned them on the same side and established the cis relationships of these protons. On the other hand, NOE correlations between H-21a (δH 2.41)/H-23, H-21a/H-20 (δH 1.65), and H-20/H-14 (δH 0.95) indicated that these protons are on the other face of the molecule. Complete analysis of the 1D and 2D NMR spectroscopic data of the minor rotamer 2b confirmed it had the same planar structure as and similar relative configuration to 2a. The 1D NMR spectra of 2b differed only for resonances CH-2, CH-8, C-10, and CH3-36, suggesting that the rotamers varied only in
s s m d (6.6) d (7.1) s s
CH3 CH3 CH3 CH CH3 CH3 CH3 CH
2.42, 1.81, 1.70, 1.28, 0.85,
m dt (12.6, 4.4) m m dd (12.7, 2.2)
1.32, 1.07, 1.50, 1.40, 1.61, 0.58,
m m m m m m
4.97, 4.72, 0.81, 0.74, 0.60, 2.35, 1.23, 0.94, 2.93, 8.97
s s s s s m d (7.1) d (7.1) s
the N-1−C-36 geometry. The NOESY spectrum of the major rotamer 2a showed a significant NOE between H-36 (δH 8.95) and H-8 (δH 7.22) with no NOE detected between H-36 and H-2 (δH 4.75), while rotamer 2b exhibited a strong NOE from H-36 (δH 8.57) to H-2 (δH 4.44) and H-10 (δH 5.18) but no NOE between H-36 and H-8 (δH 8.08) (Figure 2). These results implied that the formyl−N-1 amide bond was S-trans in 2a and S-cis in 2b. The absolute configuration of 2 was determined to be (2R,3S,14S,19S,20S,23S,26S) from analysis of its CD spectrum, which showed one negative Cotton effect at 200 nm and two positive Cotton effects at 220 and 240 nm, similar to indotertine A (1). 760
dx.doi.org/10.1021/np3008864 | J. Nat. Prod. 2013, 76, 759−763
Journal of Natural Products
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162.1) in 3, which was connected to the nitrogen of the dihydroindole system based on HMBC correlations from the formyl proton to C-6a (δC 141.1) (Figure 2). The NOESY spectrum of 3 showed a significant NOE between H-29 and H5a (δH 6.26) with no NOE correlation between H-29 and H-7 (δH 8.03), which implied the formyl−N-6 amide bond was Strans in 3. The obvious lack of stable rotameric forms for 3 can be rationalized by hydrogen bond formation between formyl proton H-29 with adjacent N-5 and the carbonyl oxygen attached to C-4. This would stabilize one single conformation in a position where H-29 is in close spatial proximity to H-5a, as evidenced by the NOE correlations. The experimental ECD spectra of 3 and drimentine F11,13 were very similar. The Cotton effects observed at 230 nm (Δε −0.2) and 245 nm (Δε −1.7) for 3 and 220 nm (Δε −0.9) and 241 nm (Δε −5.3) for drimentine F indicated the absolute configuration of 3 was the same as that of drimentine F and therefore was determined as (3S,5aS,10bS,11aS,13S,16aS,20aS). Compounds 2 and 3 constitute a rare heterocyclic ring system co-occurring in a bacterium. On the basis of these findings, it is plausible to conclude that they share a common prenylated indole-diketopiperazine precursor. Subsequently, nucleophilic addition to the α position of the indole could take place from either the exocyclic olefin or amide nitrogen and then with further methylation and oxidation would afford the pentacyclic indotertine B (2) or drimentine H (3) skeletons.10,14,15 Interestingly, this possible biochemical pathway involves the acid-promoted rearrangement of a substituted 3H-indole to an uncommon hydrogenated naphtho[2,1-b]carbazole ring system. Compounds 2−4 were evaluated in vitro for their cytotoxicities against five human tumor cell lines (HCT-8, Bel-7402, BGC-823, A549, and A2780 cells) using the MTT method previously described,16 using paclitaxel as a positive control. The rotamer mixture (2) showed the best cytotoxic activities against the HCT-8 and A549 cell lines, with IC50 values of 6.96 and 4.88 μM, respectively, while the other compounds showed no significant cytotoxicity against the tested cell lines (IC50 > 10 μM) (Table 2). The possible molecular targets of these compounds were also investigated in various models including topoisomerase I, NF-κB, and PKCα with no inhibitory effect observed. In conclusion, three hybrid amino acid−terpenoid metabolites were isolated from a marine-derived Streptomyces sp. CHQ-64. Together with the previously reported indotertine A, indotertine B (2) possesses the same scaffold with an indolesesquiterpene connected to a diketopiperazine unit via a methylene bridge. In contrast to the known derivative, indotertine B has a formyl group, which improved the observed cytotoxicities against two human tumor cell lines (HCT-8 and A-549). For drimentines F−H, the exchangeable NH-proton in the diketopiperazine seems essential to the cytotoxic activity (noted for drimentine G),11 as when the nitrogen was
Figure 1. Structures of indotertine A (1), indotertine B (2a and 2b), drimentine H (3), and drimentine C (4).
Figure 2. Selected 2D NMR correlations for indotertine B (2) and drimentine H (3).
Drimentine H (3) was obtained as an optically active, colorless, and amorphous powder. Its molecular formula was determined as C33H45N3O3 on the basis of HRESIMS, requiring 13 degrees of unsaturation. Comparison of the 1H and 13C NMR data between 3 and drimentine F11 suggested that they shared similar structural features. The only difference was the presence of one additional formyl group (δH 8.97, δC
Table 2. Cytotoxicities of Compounds 2−4 for Five Human Tumor Cell Lines IC50 (μM) compound
HCT-8
Bel-7402
BGC-823
A549
A2780
2 3 4 paclitexal
6.96 ± 0.04 >10 >10 0.051
>10 >10 >10 0.006
>10 >10 >10 10 >10 0.016
>10 >10 >10
