Fitoterapia 96 (2014) 115–122
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Stereochemical determination of new cytochalasans from the plant endophytic fungus Trichoderma gamsii Lin Chen a, Yue-Tao Liu a, Bo Song a, Hong-Wu Zhang a, Gang Ding a,b,⁎, Xing-Zhong Liu c, Yu-Cheng Gu d, Zhong-Mei Zou a,⁎⁎ a
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China c Institute of Microbiology. Chinese Academy of Sciences, Beijing, PR China d Syngenta Jealott’s Hill International Research Center, Bracknell, Berkshire RG42 6EY, United Kingdom b
a r t i c l e
i n f o
Article history: Received 2 March 2014 Accepted in revised form 11 April 2014 Available online 18 April 2014 Keywords: Endophytes Trichoderma gamsii Cytochalasans Stereochemistry
a b s t r a c t Three new cytochalasans, trichalasins E (1), F (2) and H (7), together with four known analogues, trichalasin C (3), aspochalasin K (4), trichalasin G (5) and aspergillin PZ (8), were isolated from one endophytic fungus Trichoderma gamsii inhabiting in the traditional medicinal plant Panax notoginseng (BurK.) F.H. Chen. Trichalasins E (1) contains a unique hydroperoxyl group, which is the first report in all known analogues, whereas trichalasin H (7) possesses the rare 6/5/6/6/5 pentacyclic skeleton with 12-oxatricyclo [6.3.1.02,7] moiety as that of aspergillin PZ (8). The relative configurations of the new compounds were characterized by analysis of coupling constants and ROESY correlations, and the absolute configurations of trichalasins E (1), H (7) and aspergillin PZ (8) were determined by modified Mosher’s reaction. In addition, compounds 1–5, 7 and 8 were tested cytotoxic activities against several cancer cell lines. © 2014 Published by Elsevier B.V.
1. Introduction Cytochalasans is a big member of importantly bio-functional molecules, which displayed a wide range of biological activities, such as anticancer, antimicrobial and antiparasitic activities, and was also found to possess phytotoxic activities. In addition, cytochalasans might play a major ecological role to its producer by deterring predators or competitors and enhance fitness to access nutrition, food and living space [1]. Cytochalasans (fungal toxin) are produced by different fungal strains such as Chaetomium, Penicillium, Zygosporium, Aspergillus, Phomopsis, Rosellinia and Metarrhizium [2, 3], and recently also found in ⁎ Correspondence to: G. Ding, No.151, Malianwa North Road, Haidian District, Beijing 100193, PR China. Tel./fax: + 86 10 57833290. ⁎⁎ Correspondence to: Z.-M. Zou, No.151, Malianwa North Road, Haidian District, Beijing 100193, PR China. Tel./fax: + 86 10 57833290. E-mail addresses: [email protected] (L. Chen), [email protected] (G. Ding), [email protected] (Z.-M. Zou).
http://dx.doi.org/10.1016/j.fitote.2014.04.009 0367-326X/© 2014 Published by Elsevier B.V.
Trichoderma [4–7]. The bio-genetic investigation revealed that this group of toxin is originated from polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) hybrid biosynthetic pathways, which form one isoindole unit fused with one macrocyclic ring [2, 3]. Up to date, more than 100 cytochalasan analogues are reported. The wide structural diversity of cytochalasans is mainly due to the variation of amino acid moiety including Leu, Phe, Ala, Trp and Val [8–11] and the different oxidation regions in the macrocyclic ring. Several cytochalasans including trichalasins A–D, polycyclic trichoderones A and B and the first spiro-cytochalasans trichodermone have been obtained from the plant endophytic fungus Trichoderma gamsii [4–7]. In our ongoing chemical investigation of this plant endophytic fungus for new analogues or possibly unique intermediates, further seven compounds including three new ones named trichalasins E (1), F (2) and H (7) were isolated, and other three known ones were trichalasin C (3), aspochalasin K (4) and aspergillin PZ (8) [12]. The fifth
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Fig. 1. Structures of compounds 1–8.
one (5) had a CAS Registry Number 1222847-89-3 in Chemical Abstract (CA) but without any reference; thus, we named it trichalasin G (5) (Fig. 1). Trichalasin E (1) contains a unique
hydroperoxyl group representing the first analogue in this member of mycotoxin. Trichalasin H (7) is the stereoisomer of aspergillin PZ (8) possessing the rare 6/5/6/6/5 pentacyclic
Table 1 NMR data of compounds 1, 2 and 5 (in CD3OD). Pos
Trichalasin E (1) a
1 3 4 5 6 7 8 9 10a 10b 11 12 13 14 15a 15b 16a 16b 17a 17b 18 19 20a 20b 21 22 23 24 25 19-OCH3 a b
Trichalasin F (2) δC , mult.
δH (J in Hz)
δC , mult.
δHa (J in Hz)
δCb, mult.
– 3.44, 3.80, – – 4.08, 3.81, – 1.47, 1.58, 1.79, 1.75, 6.02, – 2.08, 2.39, 1.35, 2.09, 3.72,
175.1, s 57.2, d 53.0, d 129.7, d 133.7, s 85.1, d 41.0, d 87.0, s 47.3, t
174.6, s 54.1, d 53.9, d 36.5, d 142.4, s 124.0, d 43.5, d 88.7, s 48.0, t
28.9, t
1.64, m
24.4, t
79.5, d
1.40, 1.73, 4.36, 3.70, 2.50, 2.53, – 1.64 0.86, 0.87, 1.60 –
34.0, t
– 2.44, 2.54, 3.18, – 5.29, 3.29, – 1.07, 1.14, 1.18, 1.48, 6.10, – 1.91, 2.36, 1.21, 1.79, 4.16,
178.0, s 52.4, d 55.8, d 34.5, d 140.8, s 126.9, d 44.7, d 69.9, s 50.4, t
14.6, q 18.3, q 122.9, d 142.4, s 41.5, t
– 2.97, 2.33, 2.71, – 5.28, 3.38, – 1.11, 1.84, 1.17, 1.74, 5.98, – 2.20,
d (10.0) d (10.0) m m s s d (11.0) m dd (13.0, 10.0) m m dd (5.5, 1.5)
4.48, s 7.08, dd (15.5, 2.0) 5.91, dd (15.5, 2.5)
74.1, d 154.1, d 120.4, d
– 1.69, 0.93, 0.95, 1.43, –
168.6, s 26.4, d 22.6, q 23.5, q 15.7, q –
m d (7.0) d (7.0) s
Recorded at 500 MHz. Recorded at 125 MHz.
a
Trichalasin G (5)
δH (J in Hz) dd (7.5, 7.0) d (6.0)
b
dt (11.0, 4.0) t (4.0) m s m m m d (7.0) s d (10.5) m
m m m m dd (14.0, 4.0) dd (14.0, 4.0)
d (6.5) d (6.5)
b
14.7, q 20.2, q 124.8, d 139.5, s 40.2, t
69.7, d 71.5, d 40.5, t 171.3, s 26.1, d 21.5, q 21.8, q 15.7, q –
3.18, 3.61, 2.10, 3.56, – 1.56, 0.86, 0.85, 1.70, –
m m m s s m dd (9.0, 5.0) d (2.0) s d (11.0) m dd (6.0, 2.0) m m dd (8.5, 2.0) m dd (8.0, 4.5) d (13.0) dd (13.0, 8.0) m d (1.5) d (1.5) s
13.8, q 19.8, q 126.8, d 138.3, s 36.7, t 29.6, t 75.0, d 72.8, d 79.8, d 43.4, t 215.1, s 25.8, d 22.1, q 23.9, q 15.6, q 57.4, q
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Table 2 NMR spectroscopic data of 7 and 8. No.
Aspergillin PZ (8)
Trichalasin H (7) a
1 2-NH3 4 5 6 7 8 9 10a 10b 11 12 13 14 15a 15b 16a 16b 17 18 19 20 21 22 23 24 25 a b
b
δH (J in Hz) (CDCl3)
δH (J in Hz) (CD3OD)
− 5.93, s 3.10, br.d (12.0) 2.67, br.s 2.37, br.s − 5.43, br, s 2.46, br, d (12.0) − 1.30, m 1.74, m 1.16, d (7.5) 1.78, br, s 2.99, m (overlapped) − 1.45, dd (10.0, 5.0) 1.91, dt (10.0, 5.0) 1.78, m 2.05, m 3.59, br.s 3.72, br, s 2.99, m (overlapped) 2.59, dd (13.0, 5.0) 2.53, dd (13.0, 11.5) − 1.59, m 0.93, d (6.5) 0.95, d (6.5) 1.23, s
− − 3.16, m, (overplapped) 2.54, dd (4.8, 3.6) 2.47, m (overlapped) − 5.47, br, s 2.57, br, d (12.0) − 1.30, m 1.65, m 1.18, d (7.2) 1.80, br, s 2.95, dd (12.6, 9.0) − 1.41, m 1.96, m 1.65, m 2.06, m 3.55, br, s 3.67, br, s 2.85, ddd (13.8, 9.0, 5.4) 2.62, dd (15.6, 13.8) 2.47, dd (15.6,4.8) − 1.66, m 0.93, d (6.6) 0.95, d (6.6) 1.21, s
δC, mult.
δH (J in Hz) (CDCl3)
δH (J in Hz) (CD3OD)
δC, mult.
174.4, s − 52.0, d 51.8, d 35.2, d 140.0, s 127.3, d 36.4, d − 47.7, t
210.8, s 25.4, d 21.3, q 23.8, q 23.3, q
− 1.55, m 0.93, d (6.5) 0.95, d (6.5) 1.21, s
− − 3.16, m, (overplapped) 2.54, dd (4.8, 3.6) 2.48, m − 5.47, br, s 2.57, br, d (12.0) − 1.33, m 1.68, m 1.18, d (7.2) 1.80, br, s 2.91, dd (12.6, 9.0) − 1.33, m 1.69, m 1.68, m 1.91, m 3.73, m 3.59, d (3.6) 3.17, m (overlapped) 2.40, dd (15.6, 5.4) 2.63, dd (15.6, 13.8) − 1.68, m 0.93, d (6.6) 0.95, d (6.6) 1.20, s
173.5 − 52.1, d 51.7, d 35.1, d 139.7, s 127.2, d 36.6, d − 47.4, t
66.8, d 84.5, d 38.9, d 42.7, t
− 5.84, s 3.10, dt (10.5, 3.5) 2.65,dd (5.0, 3.5) 2.36, br.s − 5.42, br, s 2.44, br, d (12.0) − 1.30, m 1.71, m 1.15, d (7.5) 1.77, br, s 2.89, dd (12.0, 9.0) − 1.59, m 1.71, m 1.74, m 1.99, m 3.82, br.s 3.66, d (3.5) 3.26, dt (9.0, 9.0) 2.56, d (9.0)
13.7, d 20.2 42.7 82.7 35.1, t 24.8, t
13.6, 20.2, 44.3, 82.0, 38.5,
d d d s t
27.0, t 68.1, 83.3, 36.4, 42.8,
d d d t
210.9, s 25.3, d 21.2, q 23.7, q 22.5, q
Recorded at 500 MHz. Recorded at 125 MHz.
skeleton with 12-oxatricyclo [6.3.1.02,7] moiety [12]. In this paper, we reported the isolation, structure elucidation and their cytotoxic activities of these compounds. 2. Experimental
G2. Purification was performed by semiprep-HPLC with a Lumtech apparatus equipped with UV detector under ODS column (250 × 10 mm; YMC Co., Ltd.). TLC was performed on silica gel GF254 (10–40 μm; Qingdao Marine Chemical, Inc.). Column chromatography was performed on silica gel (100–200 or 200–300 mesh; Qingdao Marine Chemical, Inc.).
2.1. General 2.2. Fungal material Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on Beckman Coulter DU 800 spectrometer. IR data were recorded using a Shimadzu FTIR-8400S spectrophotometer. NMR spectra were measured on a Bruker AM 500 NMR spectrometer with tetramethylsilane as the internal reference and chemical shifts are expressed in ppm. TOF-ESI-MS spectra were measured on a Waters Synapt
The culture of T. gamsii was isolated from traditional Chinese medicinal plant Panax notoginseng (BurK.) F.H. Chen. The isolate was identified based on sequence (GenBank accession no. JF964996) analysis of the ITS region of the rDNA and assigned the accession no. SQP 79-1 in X. L’s culture collection at the Institute of Microbiology, Chinese Academy of Sciences, Beijing.
Fig. 2. 1H–1H COSY, key HMBC and ROESY of 1. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
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Fig. 3. Δδ values (in ppm) = δS − δR obtained for (R)- and (S)-MTPA esters 1a, and 1b.
The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C for 10 days. The agar plugs were used to inoculate 250 mL Erlenmeyer flasks, each containing 40 mL of media (0.4% glucose, 1% malt extract and 0.4% yeast extract), and the final pH of the media was adjusted to 6.5 before sterilization. Flask cultures were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days. Fermentation was carried out in Fernbach flasks (500 mL) each containing 80 g of rice. Spore inoculum was prepared by suspension in sterile, distilled H2O to give a final spore/cell suspension of 1 × 106/mL. Distilled H2O (100 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 lb/in−2 for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of seed culture and cultivated at 25 °C for 40 days. 2.3. Extraction and isolation The fermented material was extracted with ethyl acetate (5 L for 4 times). The solution was concentrated to dryness under vacuum to afford a crude extract (50.0 g), which was fractionated by silica gel column chromatography (CC) (10 × 100 cm) using CH2Cl2–MeOH gradient elution. The fraction (1.2 g) eluted with 100:4 CH2Cl2–MeOH was separated by column chromatography to afford 5 fractions (A1–A5). Fraction A2 (40 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 80% MeOH in H2O for 40 min) to afford trichalasin E (1; 5.4 mg, tR 36.2 min). Fraction A3 (70 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 75% MeOH in H2O for 40 min) afforded trichalasin G (5; 15.0 mg, tR 34.2 min)
and trichalasin F (2; 10.3 mg, tR 13.5 min). Fraction A4 (55 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 75% MeOH in H2O for 35 min) afforded aspochalasin K (4; 7.5 mg, tR 30.5 min) and trichalasin C (3; 6.0 mg, tR 15.8 min). The other fraction (900 mg) eluted with 100:4 CH2Cl2–MeOH was separated by column chromatography to afford 5 fractions (B1–B5). Fraction B3 (35 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 75% MeOH in H2O for 40 min) to afford trichalasin H (7; 20 mg, tR 25.2 min) and aspergillin PZ (8; 8 mg, tR 19.0 min). Trichalasin E (1): white powder; UV (CH3OH)λmax(log ε) 208 (2.738), 277 (0.007) nm; IR (Neat) νmax 3372, 2953 and 1726 cm−1; HRESIMS obsd m/z 472.2316 [M + Na]+ (calcd for C24H35O7Na, 472.2311); 1H and 13C NMR data, see Table 1. Trichalasin F (2): white powder; UV (CH3OH)λmax(log ε) 209 (2.809), 269 (0.021) nm; IR (Neat) νmax 3432, 2932 and 1721 cm−1; HRESIMS obsd m/z 420.2706 [M + H]+ (calcd for C24H38NO5, 420.2750); 1H and 13C NMR data, see Table 1. Trichalasin H (7): white powder, [α]22 D = + 45.5 (c 0.11, CH3OH); IR (neat): νmax = 3421, 3264, 2952, 1685, 1654, 1386 and 1225 cm−1; 1H and 13C NMR data, see Table 2; TOF-ESI-MS calcd for C24H36NO4, [M + H]+ 402.2644, found 402.2640. 2.3.1. Preparation of (S)-MTPA Ester (7a/8a) and (R)-MTPA Ester (7b/8b) [13] The powder of 7/8 (1.0 mg) was transferred to two small clean bottles, respectively, and then pyridine-d5 (0.5 mL) and (R)-MTPACl/(S)-MTPACl (8.0 μL) were quickly added into the clean bottles, and all contents were mixed thoroughly by shaking
Fig. 4. 1H–1H COSY, key HMBC and ROESY of 2. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
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Fig. 5. Key ROESY correlations of compounds 7 and 8. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
the clean bottles carefully. The reaction was performed at room temperature, and the solution was allowed to stand for 24 h. The S-MTPA and the R-MTPA ester (7a/7b) were obtained by HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 75% MeOH in H2O for 30 min) to obtain 7a and 7b (tR 21.8 and 19.9 min), respectively. The S-MTPA and the R-MTPA esters (8a/8b) were obtained by HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 75% MeOH in H2O for 30 min) to obtain 8a and 8b (tR 28.2 and 26.5 min), respectively. 2.3.2. Cytotoxic activity assay All compounds evaluated for their growth inhibition against A549, MDA-MB-231 and PANC-1 human cancer cell lines by the
MTT method as shown in the reference [14]. The positive control was 5-fluorouracil, and the negative control is 1% DMSO. 3. Results and discussion Trichalasin E (1) was obtained as a white amorphous powder. TOF-ESI-MS gave a molecular ion peak at m/z 472.2316 [M + Na]+, which was consistent with the molecular formula of C24H35NO7 with eight degrees of unsaturation. The IR spectrum displayed the absorption bands at 3372 and 1726 cm− 1 revealing the presence of hydroxyl and carbonyl units, respectively. The molecular weight and formula suggested that compound 1 had one more oxygen atom than that of trichalasin C (3) [5]. Comparison of the 1H and 13C NMR data
Fig. 6. Δδ values (in ppm) = δS − δR obtained for (R)- and (S)-MTPA esters 8a, and 8b.
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Fig. 7. Chem3Ddraw pictures of 7 and 8. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
(Table 1) revealed the similar structural fragment as found in trichalasin C (3) except for the different chemical shift value at C-7. The much lower chemical shift value of C-7 (δC = 85.1) in 1 compared with δC (71.2) in trichalasin C together with the difference of molecular formula implied that a hydroperoxyl group was attached at C-7 in 1 instead of the hydroxyl group in trichalasin C. This hypothesis was further supported by 1H–1H COSY and HMBC data. Thus, the structure of 1 was assigned (Fig. 2). The relative configuration of 1 was determined to be same as that of trichalasin C (3) by analysis of its coupling constants and ROESY correlations (Fig. 2). The small coupling constant between H-17 and H-18 (J ≈ 0 Hz) suggested the cis-configuration of these two protons. The correlations from CH2-10 to H-4, H-4 to H-8 and from H-8 to CH3-25 displayed that these protons were in closeness in space. The distinct cross peaks of H-13, H-17 and H-18 with H-19 implied their proximity. Thus, the relative configuration was established. From the biosynthetic pathway, compounds 1 might possess the same stereochemistry as its analogues of compound 3. To support this hypothesis, modified
Mosher’s reaction was used to determine the absolute configuration for compound 1. Treatment of 1 with (R)-MTPACl and (S)-MTPACl afforded the S-MTPA ester (1a) and R-MTPA ester (1b), respectively. The 1H NMR data revealed one methoxyl group present in the target product, which implied that 17-OH or 18-OH reacted with (R) and (S) MTPACl. Analyzing the 1H–1H COSY data of products 1a and 1b revealed that hydroxyl group at C-17 was reacted with MTPACl. The difference in chemical shift values (Δδ) δS − δR) for the diastereomeric esters 1a and 1b was calculated in order to assign the absolute configuration at C-17, implying that the absolute configuration at C-17 in 1 was to be R (Fig. 3). Considering the relative configuration established by analysis of coupling constant and ROESY correlations, the stereochemistry of compound 1 was determined. Trichalasin F (2) was isolated as a white amorphous powder. Its molecular formula was determined as C24H37NO5 (seven degrees of unsaturation) by TOF-ESI-MS spectrum data, which showed a pseudomolecular ion at m/z 420.2706 [M + H]+. The IR spectrum showed the absorption bands at 3432 and 1721 cm−1, indicating the existence of hydroxyl and
Fig. 8. Postulated biosynthesis of compounds 1–8. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
L. Chen et al. / Fitoterapia 96 (2014) 115–122 Table 3 Growth inhibition of 1–5, 7 and 8 against three cancer cell lines. Compounds
1 2 3 4 5 7 8 5-Fluorouracil
Cytotoxicity (IC50 in μM) A549
MDA-MB-231
PANC-1
N100 N100 N100 N100 N100 N100 N100 0.47
N100 N100 N100 N100 60.60 N100 N100 0.12
N100 N100 N100 N100 N100 N100 N100 0.67
carbonyl groups, respectively. The 1H, 13C NMR and HMQC NMR spectra data of 2 (Table 1) displayed five methyl groups, five methylene units, seven methines (two oxygen-substituted), one oxygenated quaternary carbon, four olefinic carbons (two of which were protonated) and two carbonyl groups. 1H–1H COSY spectrum revealed three isolated proton spin-systems corresponding to C-15–C-20, C-11–C-5–C-4–C-3–C-10–C-22-C-23/C-24 and C-7–C-8–C-13 fragments (Fig. 4), and the remaining connectivity was obtained by HMBC correlations (Fig. 4). The isoindol-1-one unit was established by HMBC correlations from H-3 to C-1, H-4 to C-1, C-8 and C-9, 11-CH3 to C-4, C-5 and C-6 as well as the cross peaks from 12-CH3 to C-5, C-6 and C-7. HMBC correlations from CH3-25 to C-13, C-14 and C-15 confirmed the location of the methyl group as those of other analogues. Accounting for the chemical shift value for C-9 at δC 88.7 and degree of unsaturation, and also comparison with other similar analogues, one ester bond must be formed between C-9 and C-21. Thus, the planar structure for 1 was established. The relative stereochemistry of 2 was determined on the basis of analysis of 1H NMR coupling constants and ROESY spectrum. The cis configuration for diol groups at C-18 and C-19 was supported by a small coupling constant (J = 4.0) between H-18 and H-19. The ROESY correlations of H-13 with H-18 and of Me-25 with H-8 implied that these protons were close to each other in space. The correlation of H-4 with H2-10 put these protons on the same orientation of the corresponding pyrrolidin-2-one ring. The cross peaks from H-8 to H-4 and H-5 implied these protons on the same orientation on the cyclohexene ring system. Thus, the relative configuration of 2 was confirmed. The absolute configuration of 2 was suggested the same as that 1 on the basis of similar biosynthetic origin. Trichalasin G (5) was isolated as a white amorphous powder. The molecular formula of 5 was established as C25H39NO5 (seven degrees of unsaturation) on the basis of TOF-ESI-MS (m/z 433.2797 [M + Na]+). The structure of 5 was confirmed by analysis of its 1H, 13C (Table 1), 1H–1H COSY, HMQC and HMBC NMR data. The structure of 5 had a CAS registry number 1222847-89-3 in Chemical Abstract (CA) but without any reference, and we named it as trichalasin G (5). The compound 8 was determined to be aspergillin PZ compared with its NMR with those of reports [12, 15]. Trichalasin H (7) was isolated as a white amorphous powder. Its molecular formula was determined as C24H36NO4(8 degrees of unsaturation) by TOF-ESI-MS spectrum data (m/z 402.2640 [M + H]+), which had the same molecular formula as that of aspergillin PZ (8). The resolution (in deuterated chloroform, CDCl3) for proton signals H-13, H-17, H-18, H-19 and H-20 were
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not good, and this problem was solved in deuterated methanol (CD3OD) (Table 2). The 1H, 13C NMR, HMQC especially 2D NMR spectra data (including 1H–1H COSY and HMBC correlations) of 7 revealed the same planar structure as that aspergillin PZ (8), whereas the two compounds (7,8) had difference retention time in HPLC, which supported that these two compounds possessed different stereochemistry. Comparison of the NMR of 7 and 8, especially analysis of the coupling constant between these two compounds implied the possible difference of C-17, C-18 and C-19. The relative configuration of 7 was determined by detailed analysis of NEOSY correlations (Fig. 5). Most correlations such as ones from H-8 to H-4 and H-5, from H-4 to H2-10 and from CH3-11 to H-3 and the correlations of H-13 with H-19 and CH3-25, of H-18 with H-17, H-19 and H2-20 were same in the two compounds, whereas the correlations between H-17 with H-19 was present in 8 and absent in 7, which implied C-17 possessed different configuration in these two compounds. The X-ray diffraction method obtained the relative configuration of aspergillin PZ (8), and the absolute stereochemistry of this compound was determined by total synthesis [15]. Modified Mosher’s reaction was first introduced to determine the absolute configuration of C-17 for compounds 7 and 8 in this report. The difference in chemical shift values (Δδ) δS − δR) for the diastereomeric esters 8a and 8b led to solve the absolute configuration of C-17 (R), which was same as that of total synthetic result. Interestingly, compound 7 did not react with Mosher’s reagent (Fig. 6). Analysis of the configuration of 7 and 8 implied the fact that neighboring protons surrounding the 17-OH in 7 precluded the reaction to happen, whereas the Mosher’s reagent reacted with 8 easily due to no effect of steric hindrance (see Fig. 7). Thus, the absolute configurations of 7 and 8 were determined. (See Fig. 6.) Isotope labeling together with genetic investigation confirmed that cytochalasans were biosynthesized from a polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid pathway [2, 16, 17]. In this report, we postulated the possible biosynthesis of compounds 1–8 (Fig. 8). First the Claisen condensation connected the fragments produced by PKS and NRPS hybrid pathway, and then the Diels–Alder reaction shaped the cyclohexene ring. Oxygenation at double bond C-17/18 formed two stereoisomers: compounds 6 and its 17-epimer 6′. The Baeyer–Villiger oxygenation at C-9 and C-20 produced 2 by 6, from which post-modification obtained compounds 3–5. One intramolecular 1,4-Michael addition (IMA) at C-13 and C-19 in 6 and 6′ together with one molecular of H2O loss happened to finally shape the products 7 and 8, respectively. All compounds were evaluated inhibitor activities against the A549, MDA-MB-231 and PANC-1 cell lines by the MTT method (Table 3). Trichalasin G (5) showed modest activity against MDA-MB-231 cells, with IC50 values of 60.6 μM. The previous reports revealed that aspochalasins displayed different cytotoxicities, while the bioactive results of this member of mycotoxins were weak, which was similar with our results (Prof. L. E. Overman also told us that their synthetic aspergillin PZ (8) did not display strong cytotoxicities against different cancer cell lines, by private communication). Acknowledgements We thank Syngenta for the fellowship to L Chen. We gratefully acknowledge financial support from Program for
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Innovative Research Team in IMPLAD (PIRTI), the Chinese National S&T Special Project on Major New Drug Innovation (2011ZX09307-002-01), the Open Funding Project of the State Key Laboratory of Bioactive Substance and Function of Natural Medicines, PUMC Youth Fund and the Fundamental Research Funds for the Central Universities. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.04.009. References [1] Scherlach K, Boettqer D, Remme N, Hertweck C. Nat Prod Rep 2010;27: 869–86. [2] Schümann J, Hertweck C. J Am Chem Soc 2007;129:9564–5. [3] Qiao K, Chooi Y, Tang Y. Metab Eng 2001;13:723–32. [4] Ding G, Wang H, Chen L, Chen A, Lan J, Chen X, et al. J Antibiot 2012;65: 143–5.
[5] Ding G, Chen L, Chen A, Tian X, Zhang H, Chen H, et al. Fitoterapia 2012; 83:541–4. [6] Ding G, Wang H, Li L, Chen A, Chen L, Chen H, et al. Eur J Org Chem 2012;13:2516–9. [7] Ding G, Wang H, Li L, Song B, Chen H, Zhang H, et al. J Nat Prod 2014;77: 164–7. [8] Espada A, Rivera-Sagredo A, de la Fuente J, Hueso-Rodriguez J, Elson S. Tetrahedron 1997;53:6485–92. [9] Hyuncheol O, Swenson D, Gloer J, Wicklow D, Dowd P. Tetrahedron Lett 1998;39:7633–6. [10] Lin Z, Zhu T, Wei H, Zhang G, Wang H, Gu Q, et al. Eur J Org Chem 2009: 3045–51. [11] Zhou G, Kithsiri Wijeratne E, Bigelow D, Pierson L, Vanetten H, Gunatilaka A. J Nat Prod 2004;67:3282–332. [12] Zhang Y, Wang T, Pei Y, Hua H, Feng B. J Antibiot 2002;55:693–5. [13] Ohtani I, Kusumi T, Kashaman Y, Kakisawa H. J Am Chem Soc 1991;113: 4092–6. [14] Zheng Y, Zhang W, Ben K, Wang J. Immunopharmacol Immunotoxicol 1995;17:692-79. [15] Canham S, Overman L, Tanis P. Tetrahedron 2011;67:9837–43. [16] Binder M, Kiechel JR, Tamn C Helv. Chim Acta 1970;53:1797–812. [17] Qiao K, Chooi Y, Tang Y. Metab Eng 2011;13:723–32.
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Stereochemical determination of new cytochalasans from the plant endophytic fungus Trichoderma gamsii Lin Chen a, Yue-Tao Liu a, Bo Song a, Hong-Wu Zhang a, Gang Ding a,b,⁎, Xing-Zhong Liu c, Yu-Cheng Gu d, Zhong-Mei Zou a,⁎⁎ a
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China c Institute of Microbiology. Chinese Academy of Sciences, Beijing, PR China d Syngenta Jealott’s Hill International Research Center, Bracknell, Berkshire RG42 6EY, United Kingdom b
a r t i c l e
i n f o
Article history: Received 2 March 2014 Accepted in revised form 11 April 2014 Available online 18 April 2014 Keywords: Endophytes Trichoderma gamsii Cytochalasans Stereochemistry
a b s t r a c t Three new cytochalasans, trichalasins E (1), F (2) and H (7), together with four known analogues, trichalasin C (3), aspochalasin K (4), trichalasin G (5) and aspergillin PZ (8), were isolated from one endophytic fungus Trichoderma gamsii inhabiting in the traditional medicinal plant Panax notoginseng (BurK.) F.H. Chen. Trichalasins E (1) contains a unique hydroperoxyl group, which is the first report in all known analogues, whereas trichalasin H (7) possesses the rare 6/5/6/6/5 pentacyclic skeleton with 12-oxatricyclo [6.3.1.02,7] moiety as that of aspergillin PZ (8). The relative configurations of the new compounds were characterized by analysis of coupling constants and ROESY correlations, and the absolute configurations of trichalasins E (1), H (7) and aspergillin PZ (8) were determined by modified Mosher’s reaction. In addition, compounds 1–5, 7 and 8 were tested cytotoxic activities against several cancer cell lines. © 2014 Published by Elsevier B.V.
1. Introduction Cytochalasans is a big member of importantly bio-functional molecules, which displayed a wide range of biological activities, such as anticancer, antimicrobial and antiparasitic activities, and was also found to possess phytotoxic activities. In addition, cytochalasans might play a major ecological role to its producer by deterring predators or competitors and enhance fitness to access nutrition, food and living space [1]. Cytochalasans (fungal toxin) are produced by different fungal strains such as Chaetomium, Penicillium, Zygosporium, Aspergillus, Phomopsis, Rosellinia and Metarrhizium [2, 3], and recently also found in ⁎ Correspondence to: G. Ding, No.151, Malianwa North Road, Haidian District, Beijing 100193, PR China. Tel./fax: + 86 10 57833290. ⁎⁎ Correspondence to: Z.-M. Zou, No.151, Malianwa North Road, Haidian District, Beijing 100193, PR China. Tel./fax: + 86 10 57833290. E-mail addresses: [email protected] (L. Chen), [email protected] (G. Ding), [email protected] (Z.-M. Zou).
http://dx.doi.org/10.1016/j.fitote.2014.04.009 0367-326X/© 2014 Published by Elsevier B.V.
Trichoderma [4–7]. The bio-genetic investigation revealed that this group of toxin is originated from polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) hybrid biosynthetic pathways, which form one isoindole unit fused with one macrocyclic ring [2, 3]. Up to date, more than 100 cytochalasan analogues are reported. The wide structural diversity of cytochalasans is mainly due to the variation of amino acid moiety including Leu, Phe, Ala, Trp and Val [8–11] and the different oxidation regions in the macrocyclic ring. Several cytochalasans including trichalasins A–D, polycyclic trichoderones A and B and the first spiro-cytochalasans trichodermone have been obtained from the plant endophytic fungus Trichoderma gamsii [4–7]. In our ongoing chemical investigation of this plant endophytic fungus for new analogues or possibly unique intermediates, further seven compounds including three new ones named trichalasins E (1), F (2) and H (7) were isolated, and other three known ones were trichalasin C (3), aspochalasin K (4) and aspergillin PZ (8) [12]. The fifth
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Fig. 1. Structures of compounds 1–8.
one (5) had a CAS Registry Number 1222847-89-3 in Chemical Abstract (CA) but without any reference; thus, we named it trichalasin G (5) (Fig. 1). Trichalasin E (1) contains a unique
hydroperoxyl group representing the first analogue in this member of mycotoxin. Trichalasin H (7) is the stereoisomer of aspergillin PZ (8) possessing the rare 6/5/6/6/5 pentacyclic
Table 1 NMR data of compounds 1, 2 and 5 (in CD3OD). Pos
Trichalasin E (1) a
1 3 4 5 6 7 8 9 10a 10b 11 12 13 14 15a 15b 16a 16b 17a 17b 18 19 20a 20b 21 22 23 24 25 19-OCH3 a b
Trichalasin F (2) δC , mult.
δH (J in Hz)
δC , mult.
δHa (J in Hz)
δCb, mult.
– 3.44, 3.80, – – 4.08, 3.81, – 1.47, 1.58, 1.79, 1.75, 6.02, – 2.08, 2.39, 1.35, 2.09, 3.72,
175.1, s 57.2, d 53.0, d 129.7, d 133.7, s 85.1, d 41.0, d 87.0, s 47.3, t
174.6, s 54.1, d 53.9, d 36.5, d 142.4, s 124.0, d 43.5, d 88.7, s 48.0, t
28.9, t
1.64, m
24.4, t
79.5, d
1.40, 1.73, 4.36, 3.70, 2.50, 2.53, – 1.64 0.86, 0.87, 1.60 –
34.0, t
– 2.44, 2.54, 3.18, – 5.29, 3.29, – 1.07, 1.14, 1.18, 1.48, 6.10, – 1.91, 2.36, 1.21, 1.79, 4.16,
178.0, s 52.4, d 55.8, d 34.5, d 140.8, s 126.9, d 44.7, d 69.9, s 50.4, t
14.6, q 18.3, q 122.9, d 142.4, s 41.5, t
– 2.97, 2.33, 2.71, – 5.28, 3.38, – 1.11, 1.84, 1.17, 1.74, 5.98, – 2.20,
d (10.0) d (10.0) m m s s d (11.0) m dd (13.0, 10.0) m m dd (5.5, 1.5)
4.48, s 7.08, dd (15.5, 2.0) 5.91, dd (15.5, 2.5)
74.1, d 154.1, d 120.4, d
– 1.69, 0.93, 0.95, 1.43, –
168.6, s 26.4, d 22.6, q 23.5, q 15.7, q –
m d (7.0) d (7.0) s
Recorded at 500 MHz. Recorded at 125 MHz.
a
Trichalasin G (5)
δH (J in Hz) dd (7.5, 7.0) d (6.0)
b
dt (11.0, 4.0) t (4.0) m s m m m d (7.0) s d (10.5) m
m m m m dd (14.0, 4.0) dd (14.0, 4.0)
d (6.5) d (6.5)
b
14.7, q 20.2, q 124.8, d 139.5, s 40.2, t
69.7, d 71.5, d 40.5, t 171.3, s 26.1, d 21.5, q 21.8, q 15.7, q –
3.18, 3.61, 2.10, 3.56, – 1.56, 0.86, 0.85, 1.70, –
m m m s s m dd (9.0, 5.0) d (2.0) s d (11.0) m dd (6.0, 2.0) m m dd (8.5, 2.0) m dd (8.0, 4.5) d (13.0) dd (13.0, 8.0) m d (1.5) d (1.5) s
13.8, q 19.8, q 126.8, d 138.3, s 36.7, t 29.6, t 75.0, d 72.8, d 79.8, d 43.4, t 215.1, s 25.8, d 22.1, q 23.9, q 15.6, q 57.4, q
L. Chen et al. / Fitoterapia 96 (2014) 115–122
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Table 2 NMR spectroscopic data of 7 and 8. No.
Aspergillin PZ (8)
Trichalasin H (7) a
1 2-NH3 4 5 6 7 8 9 10a 10b 11 12 13 14 15a 15b 16a 16b 17 18 19 20 21 22 23 24 25 a b
b
δH (J in Hz) (CDCl3)
δH (J in Hz) (CD3OD)
− 5.93, s 3.10, br.d (12.0) 2.67, br.s 2.37, br.s − 5.43, br, s 2.46, br, d (12.0) − 1.30, m 1.74, m 1.16, d (7.5) 1.78, br, s 2.99, m (overlapped) − 1.45, dd (10.0, 5.0) 1.91, dt (10.0, 5.0) 1.78, m 2.05, m 3.59, br.s 3.72, br, s 2.99, m (overlapped) 2.59, dd (13.0, 5.0) 2.53, dd (13.0, 11.5) − 1.59, m 0.93, d (6.5) 0.95, d (6.5) 1.23, s
− − 3.16, m, (overplapped) 2.54, dd (4.8, 3.6) 2.47, m (overlapped) − 5.47, br, s 2.57, br, d (12.0) − 1.30, m 1.65, m 1.18, d (7.2) 1.80, br, s 2.95, dd (12.6, 9.0) − 1.41, m 1.96, m 1.65, m 2.06, m 3.55, br, s 3.67, br, s 2.85, ddd (13.8, 9.0, 5.4) 2.62, dd (15.6, 13.8) 2.47, dd (15.6,4.8) − 1.66, m 0.93, d (6.6) 0.95, d (6.6) 1.21, s
δC, mult.
δH (J in Hz) (CDCl3)
δH (J in Hz) (CD3OD)
δC, mult.
174.4, s − 52.0, d 51.8, d 35.2, d 140.0, s 127.3, d 36.4, d − 47.7, t
210.8, s 25.4, d 21.3, q 23.8, q 23.3, q
− 1.55, m 0.93, d (6.5) 0.95, d (6.5) 1.21, s
− − 3.16, m, (overplapped) 2.54, dd (4.8, 3.6) 2.48, m − 5.47, br, s 2.57, br, d (12.0) − 1.33, m 1.68, m 1.18, d (7.2) 1.80, br, s 2.91, dd (12.6, 9.0) − 1.33, m 1.69, m 1.68, m 1.91, m 3.73, m 3.59, d (3.6) 3.17, m (overlapped) 2.40, dd (15.6, 5.4) 2.63, dd (15.6, 13.8) − 1.68, m 0.93, d (6.6) 0.95, d (6.6) 1.20, s
173.5 − 52.1, d 51.7, d 35.1, d 139.7, s 127.2, d 36.6, d − 47.4, t
66.8, d 84.5, d 38.9, d 42.7, t
− 5.84, s 3.10, dt (10.5, 3.5) 2.65,dd (5.0, 3.5) 2.36, br.s − 5.42, br, s 2.44, br, d (12.0) − 1.30, m 1.71, m 1.15, d (7.5) 1.77, br, s 2.89, dd (12.0, 9.0) − 1.59, m 1.71, m 1.74, m 1.99, m 3.82, br.s 3.66, d (3.5) 3.26, dt (9.0, 9.0) 2.56, d (9.0)
13.7, d 20.2 42.7 82.7 35.1, t 24.8, t
13.6, 20.2, 44.3, 82.0, 38.5,
d d d s t
27.0, t 68.1, 83.3, 36.4, 42.8,
d d d t
210.9, s 25.3, d 21.2, q 23.7, q 22.5, q
Recorded at 500 MHz. Recorded at 125 MHz.
skeleton with 12-oxatricyclo [6.3.1.02,7] moiety [12]. In this paper, we reported the isolation, structure elucidation and their cytotoxic activities of these compounds. 2. Experimental
G2. Purification was performed by semiprep-HPLC with a Lumtech apparatus equipped with UV detector under ODS column (250 × 10 mm; YMC Co., Ltd.). TLC was performed on silica gel GF254 (10–40 μm; Qingdao Marine Chemical, Inc.). Column chromatography was performed on silica gel (100–200 or 200–300 mesh; Qingdao Marine Chemical, Inc.).
2.1. General 2.2. Fungal material Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on Beckman Coulter DU 800 spectrometer. IR data were recorded using a Shimadzu FTIR-8400S spectrophotometer. NMR spectra were measured on a Bruker AM 500 NMR spectrometer with tetramethylsilane as the internal reference and chemical shifts are expressed in ppm. TOF-ESI-MS spectra were measured on a Waters Synapt
The culture of T. gamsii was isolated from traditional Chinese medicinal plant Panax notoginseng (BurK.) F.H. Chen. The isolate was identified based on sequence (GenBank accession no. JF964996) analysis of the ITS region of the rDNA and assigned the accession no. SQP 79-1 in X. L’s culture collection at the Institute of Microbiology, Chinese Academy of Sciences, Beijing.
Fig. 2. 1H–1H COSY, key HMBC and ROESY of 1. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
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Fig. 3. Δδ values (in ppm) = δS − δR obtained for (R)- and (S)-MTPA esters 1a, and 1b.
The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C for 10 days. The agar plugs were used to inoculate 250 mL Erlenmeyer flasks, each containing 40 mL of media (0.4% glucose, 1% malt extract and 0.4% yeast extract), and the final pH of the media was adjusted to 6.5 before sterilization. Flask cultures were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days. Fermentation was carried out in Fernbach flasks (500 mL) each containing 80 g of rice. Spore inoculum was prepared by suspension in sterile, distilled H2O to give a final spore/cell suspension of 1 × 106/mL. Distilled H2O (100 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 lb/in−2 for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of seed culture and cultivated at 25 °C for 40 days. 2.3. Extraction and isolation The fermented material was extracted with ethyl acetate (5 L for 4 times). The solution was concentrated to dryness under vacuum to afford a crude extract (50.0 g), which was fractionated by silica gel column chromatography (CC) (10 × 100 cm) using CH2Cl2–MeOH gradient elution. The fraction (1.2 g) eluted with 100:4 CH2Cl2–MeOH was separated by column chromatography to afford 5 fractions (A1–A5). Fraction A2 (40 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 80% MeOH in H2O for 40 min) to afford trichalasin E (1; 5.4 mg, tR 36.2 min). Fraction A3 (70 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 75% MeOH in H2O for 40 min) afforded trichalasin G (5; 15.0 mg, tR 34.2 min)
and trichalasin F (2; 10.3 mg, tR 13.5 min). Fraction A4 (55 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 75% MeOH in H2O for 35 min) afforded aspochalasin K (4; 7.5 mg, tR 30.5 min) and trichalasin C (3; 6.0 mg, tR 15.8 min). The other fraction (900 mg) eluted with 100:4 CH2Cl2–MeOH was separated by column chromatography to afford 5 fractions (B1–B5). Fraction B3 (35 mg) was purified by RP-HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 75% MeOH in H2O for 40 min) to afford trichalasin H (7; 20 mg, tR 25.2 min) and aspergillin PZ (8; 8 mg, tR 19.0 min). Trichalasin E (1): white powder; UV (CH3OH)λmax(log ε) 208 (2.738), 277 (0.007) nm; IR (Neat) νmax 3372, 2953 and 1726 cm−1; HRESIMS obsd m/z 472.2316 [M + Na]+ (calcd for C24H35O7Na, 472.2311); 1H and 13C NMR data, see Table 1. Trichalasin F (2): white powder; UV (CH3OH)λmax(log ε) 209 (2.809), 269 (0.021) nm; IR (Neat) νmax 3432, 2932 and 1721 cm−1; HRESIMS obsd m/z 420.2706 [M + H]+ (calcd for C24H38NO5, 420.2750); 1H and 13C NMR data, see Table 1. Trichalasin H (7): white powder, [α]22 D = + 45.5 (c 0.11, CH3OH); IR (neat): νmax = 3421, 3264, 2952, 1685, 1654, 1386 and 1225 cm−1; 1H and 13C NMR data, see Table 2; TOF-ESI-MS calcd for C24H36NO4, [M + H]+ 402.2644, found 402.2640. 2.3.1. Preparation of (S)-MTPA Ester (7a/8a) and (R)-MTPA Ester (7b/8b) [13] The powder of 7/8 (1.0 mg) was transferred to two small clean bottles, respectively, and then pyridine-d5 (0.5 mL) and (R)-MTPACl/(S)-MTPACl (8.0 μL) were quickly added into the clean bottles, and all contents were mixed thoroughly by shaking
Fig. 4. 1H–1H COSY, key HMBC and ROESY of 2. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
L. Chen et al. / Fitoterapia 96 (2014) 115–122
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Fig. 5. Key ROESY correlations of compounds 7 and 8. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
the clean bottles carefully. The reaction was performed at room temperature, and the solution was allowed to stand for 24 h. The S-MTPA and the R-MTPA ester (7a/7b) were obtained by HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 75% MeOH in H2O for 30 min) to obtain 7a and 7b (tR 21.8 and 19.9 min), respectively. The S-MTPA and the R-MTPA esters (8a/8b) were obtained by HPLC (Lumtech; YMC-Pack ODS-A column; 10 μm; 250 × 10 mm; 2 mL/min, 75% MeOH in H2O for 30 min) to obtain 8a and 8b (tR 28.2 and 26.5 min), respectively. 2.3.2. Cytotoxic activity assay All compounds evaluated for their growth inhibition against A549, MDA-MB-231 and PANC-1 human cancer cell lines by the
MTT method as shown in the reference [14]. The positive control was 5-fluorouracil, and the negative control is 1% DMSO. 3. Results and discussion Trichalasin E (1) was obtained as a white amorphous powder. TOF-ESI-MS gave a molecular ion peak at m/z 472.2316 [M + Na]+, which was consistent with the molecular formula of C24H35NO7 with eight degrees of unsaturation. The IR spectrum displayed the absorption bands at 3372 and 1726 cm− 1 revealing the presence of hydroxyl and carbonyl units, respectively. The molecular weight and formula suggested that compound 1 had one more oxygen atom than that of trichalasin C (3) [5]. Comparison of the 1H and 13C NMR data
Fig. 6. Δδ values (in ppm) = δS − δR obtained for (R)- and (S)-MTPA esters 8a, and 8b.
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Fig. 7. Chem3Ddraw pictures of 7 and 8. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
(Table 1) revealed the similar structural fragment as found in trichalasin C (3) except for the different chemical shift value at C-7. The much lower chemical shift value of C-7 (δC = 85.1) in 1 compared with δC (71.2) in trichalasin C together with the difference of molecular formula implied that a hydroperoxyl group was attached at C-7 in 1 instead of the hydroxyl group in trichalasin C. This hypothesis was further supported by 1H–1H COSY and HMBC data. Thus, the structure of 1 was assigned (Fig. 2). The relative configuration of 1 was determined to be same as that of trichalasin C (3) by analysis of its coupling constants and ROESY correlations (Fig. 2). The small coupling constant between H-17 and H-18 (J ≈ 0 Hz) suggested the cis-configuration of these two protons. The correlations from CH2-10 to H-4, H-4 to H-8 and from H-8 to CH3-25 displayed that these protons were in closeness in space. The distinct cross peaks of H-13, H-17 and H-18 with H-19 implied their proximity. Thus, the relative configuration was established. From the biosynthetic pathway, compounds 1 might possess the same stereochemistry as its analogues of compound 3. To support this hypothesis, modified
Mosher’s reaction was used to determine the absolute configuration for compound 1. Treatment of 1 with (R)-MTPACl and (S)-MTPACl afforded the S-MTPA ester (1a) and R-MTPA ester (1b), respectively. The 1H NMR data revealed one methoxyl group present in the target product, which implied that 17-OH or 18-OH reacted with (R) and (S) MTPACl. Analyzing the 1H–1H COSY data of products 1a and 1b revealed that hydroxyl group at C-17 was reacted with MTPACl. The difference in chemical shift values (Δδ) δS − δR) for the diastereomeric esters 1a and 1b was calculated in order to assign the absolute configuration at C-17, implying that the absolute configuration at C-17 in 1 was to be R (Fig. 3). Considering the relative configuration established by analysis of coupling constant and ROESY correlations, the stereochemistry of compound 1 was determined. Trichalasin F (2) was isolated as a white amorphous powder. Its molecular formula was determined as C24H37NO5 (seven degrees of unsaturation) by TOF-ESI-MS spectrum data, which showed a pseudomolecular ion at m/z 420.2706 [M + H]+. The IR spectrum showed the absorption bands at 3432 and 1721 cm−1, indicating the existence of hydroxyl and
Fig. 8. Postulated biosynthesis of compounds 1–8. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
L. Chen et al. / Fitoterapia 96 (2014) 115–122 Table 3 Growth inhibition of 1–5, 7 and 8 against three cancer cell lines. Compounds
1 2 3 4 5 7 8 5-Fluorouracil
Cytotoxicity (IC50 in μM) A549
MDA-MB-231
PANC-1
N100 N100 N100 N100 N100 N100 N100 0.47
N100 N100 N100 N100 60.60 N100 N100 0.12
N100 N100 N100 N100 N100 N100 N100 0.67
carbonyl groups, respectively. The 1H, 13C NMR and HMQC NMR spectra data of 2 (Table 1) displayed five methyl groups, five methylene units, seven methines (two oxygen-substituted), one oxygenated quaternary carbon, four olefinic carbons (two of which were protonated) and two carbonyl groups. 1H–1H COSY spectrum revealed three isolated proton spin-systems corresponding to C-15–C-20, C-11–C-5–C-4–C-3–C-10–C-22-C-23/C-24 and C-7–C-8–C-13 fragments (Fig. 4), and the remaining connectivity was obtained by HMBC correlations (Fig. 4). The isoindol-1-one unit was established by HMBC correlations from H-3 to C-1, H-4 to C-1, C-8 and C-9, 11-CH3 to C-4, C-5 and C-6 as well as the cross peaks from 12-CH3 to C-5, C-6 and C-7. HMBC correlations from CH3-25 to C-13, C-14 and C-15 confirmed the location of the methyl group as those of other analogues. Accounting for the chemical shift value for C-9 at δC 88.7 and degree of unsaturation, and also comparison with other similar analogues, one ester bond must be formed between C-9 and C-21. Thus, the planar structure for 1 was established. The relative stereochemistry of 2 was determined on the basis of analysis of 1H NMR coupling constants and ROESY spectrum. The cis configuration for diol groups at C-18 and C-19 was supported by a small coupling constant (J = 4.0) between H-18 and H-19. The ROESY correlations of H-13 with H-18 and of Me-25 with H-8 implied that these protons were close to each other in space. The correlation of H-4 with H2-10 put these protons on the same orientation of the corresponding pyrrolidin-2-one ring. The cross peaks from H-8 to H-4 and H-5 implied these protons on the same orientation on the cyclohexene ring system. Thus, the relative configuration of 2 was confirmed. The absolute configuration of 2 was suggested the same as that 1 on the basis of similar biosynthetic origin. Trichalasin G (5) was isolated as a white amorphous powder. The molecular formula of 5 was established as C25H39NO5 (seven degrees of unsaturation) on the basis of TOF-ESI-MS (m/z 433.2797 [M + Na]+). The structure of 5 was confirmed by analysis of its 1H, 13C (Table 1), 1H–1H COSY, HMQC and HMBC NMR data. The structure of 5 had a CAS registry number 1222847-89-3 in Chemical Abstract (CA) but without any reference, and we named it as trichalasin G (5). The compound 8 was determined to be aspergillin PZ compared with its NMR with those of reports [12, 15]. Trichalasin H (7) was isolated as a white amorphous powder. Its molecular formula was determined as C24H36NO4(8 degrees of unsaturation) by TOF-ESI-MS spectrum data (m/z 402.2640 [M + H]+), which had the same molecular formula as that of aspergillin PZ (8). The resolution (in deuterated chloroform, CDCl3) for proton signals H-13, H-17, H-18, H-19 and H-20 were
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not good, and this problem was solved in deuterated methanol (CD3OD) (Table 2). The 1H, 13C NMR, HMQC especially 2D NMR spectra data (including 1H–1H COSY and HMBC correlations) of 7 revealed the same planar structure as that aspergillin PZ (8), whereas the two compounds (7,8) had difference retention time in HPLC, which supported that these two compounds possessed different stereochemistry. Comparison of the NMR of 7 and 8, especially analysis of the coupling constant between these two compounds implied the possible difference of C-17, C-18 and C-19. The relative configuration of 7 was determined by detailed analysis of NEOSY correlations (Fig. 5). Most correlations such as ones from H-8 to H-4 and H-5, from H-4 to H2-10 and from CH3-11 to H-3 and the correlations of H-13 with H-19 and CH3-25, of H-18 with H-17, H-19 and H2-20 were same in the two compounds, whereas the correlations between H-17 with H-19 was present in 8 and absent in 7, which implied C-17 possessed different configuration in these two compounds. The X-ray diffraction method obtained the relative configuration of aspergillin PZ (8), and the absolute stereochemistry of this compound was determined by total synthesis [15]. Modified Mosher’s reaction was first introduced to determine the absolute configuration of C-17 for compounds 7 and 8 in this report. The difference in chemical shift values (Δδ) δS − δR) for the diastereomeric esters 8a and 8b led to solve the absolute configuration of C-17 (R), which was same as that of total synthetic result. Interestingly, compound 7 did not react with Mosher’s reagent (Fig. 6). Analysis of the configuration of 7 and 8 implied the fact that neighboring protons surrounding the 17-OH in 7 precluded the reaction to happen, whereas the Mosher’s reagent reacted with 8 easily due to no effect of steric hindrance (see Fig. 7). Thus, the absolute configurations of 7 and 8 were determined. (See Fig. 6.) Isotope labeling together with genetic investigation confirmed that cytochalasans were biosynthesized from a polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid pathway [2, 16, 17]. In this report, we postulated the possible biosynthesis of compounds 1–8 (Fig. 8). First the Claisen condensation connected the fragments produced by PKS and NRPS hybrid pathway, and then the Diels–Alder reaction shaped the cyclohexene ring. Oxygenation at double bond C-17/18 formed two stereoisomers: compounds 6 and its 17-epimer 6′. The Baeyer–Villiger oxygenation at C-9 and C-20 produced 2 by 6, from which post-modification obtained compounds 3–5. One intramolecular 1,4-Michael addition (IMA) at C-13 and C-19 in 6 and 6′ together with one molecular of H2O loss happened to finally shape the products 7 and 8, respectively. All compounds were evaluated inhibitor activities against the A549, MDA-MB-231 and PANC-1 cell lines by the MTT method (Table 3). Trichalasin G (5) showed modest activity against MDA-MB-231 cells, with IC50 values of 60.6 μM. The previous reports revealed that aspochalasins displayed different cytotoxicities, while the bioactive results of this member of mycotoxins were weak, which was similar with our results (Prof. L. E. Overman also told us that their synthetic aspergillin PZ (8) did not display strong cytotoxicities against different cancer cell lines, by private communication). Acknowledgements We thank Syngenta for the fellowship to L Chen. We gratefully acknowledge financial support from Program for
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Innovative Research Team in IMPLAD (PIRTI), the Chinese National S&T Special Project on Major New Drug Innovation (2011ZX09307-002-01), the Open Funding Project of the State Key Laboratory of Bioactive Substance and Function of Natural Medicines, PUMC Youth Fund and the Fundamental Research Funds for the Central Universities. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.04.009. References [1] Scherlach K, Boettqer D, Remme N, Hertweck C. Nat Prod Rep 2010;27: 869–86. [2] Schümann J, Hertweck C. J Am Chem Soc 2007;129:9564–5. [3] Qiao K, Chooi Y, Tang Y. Metab Eng 2001;13:723–32. [4] Ding G, Wang H, Chen L, Chen A, Lan J, Chen X, et al. J Antibiot 2012;65: 143–5.
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