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Okaramines S–U, three new indole diketopiperazine alkaloids from Aspergillus taichungensis ZHN-7-07
Tetrahedron xxx (2014) 1e5
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Okaramines SeU, three new indole diketopiperazine alkaloids from Aspergillus taichungensis ZHN-7-07 Shengxin Cai y, Shiwei Sun y, Jixing Peng, Xianglan Kong, Huinan Zhou, Tianjiao Zhu, Qianqun Gu, Dehai Li * Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao, Shandong 266003, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2014 Received in revised form 3 September 2014 Accepted 8 September 2014 Available online xxx
Three new indole diketopiperazine alkaloids, okaramines SeU (1e3), with two (1 and 2) of them prenylated, were obtained from the Aspergillus taichungensis ZHN-7-07. Their structures including the absolute configurations were determined by combination of NMR, MS, TDDFT ECD calculation, and Marfey’s analysis. Okaramine S (1) showed the best cytotoxic activity against HL-60 cell with IC50 value of 0.78 mM. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Indole diketopiperazine alkaloids Okaramine Mangrove plant Aspergillus taichungensis Cytotoxicity
1. Introduction Prenylated indole diketopiperazine (DKP) alkaloids are hybrid natural products derived from coupling a prenylated tryptophan with the second amino acid such as proline, tryptophan, histidine or alanine, and widely distributed in filamentous fungi, especially in the genera Penicillium and Aspergillus of Ascomycota.1 Among them, the family composed by two tryptophan units is an important group with high structure diversity and various biological activities, attracting much attention of the synthetic and biosynthetic chemists.1e5 In our previous research, a series of tryptophan-proline composed indole DKPs, including a dimer, aspergilazine A with a rare N1 to C-6 linkage,6 and three reversely prenylated indole alkaloids with a rare anti bicyclo-[2.2.2]dizaoctane core ring,7 were discovered from the fungus Aspergillus taichungensis ZHN-7-07 isolated from the rhizosphere soil of the mangrove plant Acrostichum aureum. Encouraged by the novelty and bioactivities of them, further studies of this fungus led to the isolation of three pyrrolo[2,3-b] indole contained DKPs composed by two tryptophan moieties (Fig. 1), okaramines SeU (1e3), with two of them prenylated (1 and
* Corresponding author. Tel.: þ86 532 82031619; fax: þ86 532 82033054; e-mail addresses: [email protected], [email protected] (D. Li). y These authors contributed equally to this work.
2), together with a known C-7 prenylated cyclo-L-Trp-L-Trp.8 Cytotoxic and antibiotic evaluations revealed that only compound 1 exhibited cytotoxic activity against HL-60 and K-562 cell lines, and
Fig. 1. The structures of compounds 1e3 and okaramine J.
http://dx.doi.org/10.1016/j.tet.2014.09.019 0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.
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S. Cai et al. / Tetrahedron xxx (2014) 1e5
none of them displayed antibiotic activity against Enterobacter aerogenes, Staphylococcus aureus, Bacillus proteus, Micrococcus tetragenus, Bacillus subtilis, and Candida albicans strains. In this paper, we report the isolation, structure elucidation, and bioactivities of the new indole DKP alkaloids (1e3). 2. Results and discussion The fungal isolate A. taichungensis was grown in a liquid medium under static condition for 4 weeks. The culture broth was extracted with EtOAc, and the organic extract was separated using silica gel, Sephadex LH-20 and C18 HPLC to yield the new compounds (1e3). Okaramine S (1) was obtained as a pale yellow oil, and its molecular formula was established as C32H36N4O3 by the HRESIMS peak at m/z 525.2862 [MþH]þ (calcd 525.2866), indicating seventeen degrees of unsaturation. The IR data displayed the absorptions for amides (3289 and 1674 cm1). The 1D NMR data of compound 1 displayed signals for two carbonyls, 10 quarternary carbons, 12 methines, four methylenes, and four methyls (Table 1). The UV, IR, and 1D NMR data of metabolite 1 were similar to those of okaramine J,9 suggested that they share the same skeleton. The difference between them was that the C-8a0 reverse prenyl of okaramine J was replaced by a C-7 prenyl. This change and the planar structure of 1 were supported by the proton chemical shifts and coupling patterns of H-40 (dH 7.44, d, J¼7.8 Hz), H-50 (6.92, dd, J¼7.8, 6.9 Hz), Table 1 1 H (600 MHz) and No.
13
H-60 (6.84, d, J¼6.9 Hz) and H-8a0 (dH 7.26, d, J¼2.3 Hz), and the COSY correlations of H-40 /H-50 /H-60 and 80 -NH/H-8a0 , as well as the HMBC correlations from H-100 to C-60 and C-7a0 (Fig. 2). The relative configuration of 1 was established by the NOESY and NOE experiments. The NOESY correlation of H-2 and H-20 indicated that they were on the same face of the diketopiperazine ring. The cis configuration of H-8a and 3a-OH was also deduced from the NOESY correlation between them, which was further suggested to be cofacial to H-2 based on the enhancement of H-8a when irradiated H-2 (Fig. 3). The molecular formulae of compounds 2 and 3 were determined by HRESIMS to be C27H28N4O3 and C22H20N4O3, respectively. The 1H and 13C NMR data of both metabolites revealed they possessed the same diketopiperazine core as compound 1 (Table 1), with the difference in the absence of one and two prenyls in 2 and 3, respectively. The COSY correlations of H-4/H-5/H-6/H-7 suggested the disappearance of C-7 prenyl in compound 2, while the remained prenyl was assigned to C-70 based on the HMBC correlations from H-100 to C-7a0 (Fig. 2). For okaramine U (3), the COSY correlations of H-4/H-5/H-6/H-7 and H-40 /H-50 /H-60 /H-70 indicated the appearance of two 1,2-disubstituted benzenes and confirmed the absence of the two prenyls (Fig. 2). The relative configurations of compounds 2 and 3 were elucidated the same as 1 based on the NOESY and NOE experiments (Fig. 3). The absolute configuration of compounds 3 were determined by combination of Marfey’s analysis and experimental/calculated ECD
C (150 MHz) NMR data for compounds 1e3 (DMSO-d6) 1
2
3
dC, mult.
dH, (mult., J in Hz)
dC, mult.
dH, (mult., J in Hz)
dC, mult.
dH, (mult., J in Hz)
2 3
59.1 d 41.8 t
4.65 (dd, 11.0, 6.5) 2.42 (dd, 13.3, 6.5) 1.84 (dd, 13.3, 11.0)
59.2 d 41.8 t
4.66 (dd, 11.0, 6.5) 2.42 (dd, 13.0, 6.5) 1.83 (dd, 13.0, 11.0)
59.2 d 41.8 t
4.66 (dd, 11.5, 6.5) 2.43 (dd, 13.0, 6.5) 1.83 (dd, 13.0, 11.5)
3a 3b 4 5 6 7 7a 8 8a 9 3a-OH 10
86.5 s 131.7 s 120.7 d 119.2 d 128.7 d 123.4 s 146.7 s
11 12 13 14 10 20 30
122.3 d 132.8 s 18.2 q 26.1 q
3a0 3b0 40 50 60 70 7a0 80 8a0 90 100 110 120 130 140
110.5 124.8 116.8 119.2 120.6 127.9 135.4
84.5 d 170.3 s 29.0 t
55.6 d 25.3 t s s d d d s s
124.6 d 168.5 s 29.6 t 122.8 d 132.5 s 18.2 q 26.1 q
7.05 (d, 7.3) 6.67 (dd, 7.8, 7.3) 6.87 (d, 7.8)
6.04 (d, 4.3) 5.35 (d, 4.3) 6.01 3.21 3.09 5.25
(br s) (dd, 16.3, 7.7) (dd, 16.3, 6.0) (m)
1.66 1.71 7.68 4.45 3.37 3.05
(s) (s) (s) (dd, 5.5, 5.0) (dd, 15.1, 4.6) (dd, 15.1, 6.4)
7.44 (d, 7.8) 6.92 (dd, 7.8, 6.9) 6.84 (d, 6.9)
10.78 (br s) 7.26 (d, 2.3) 3.50 (d, 7.3) 5.41 (m) 1.71 (s) 1.72 (s)
86.4 s 131.7 s 123.0 d 118.3 d 129.5 d 110.4 d 149.0 s 84.5 d 170.3 s
7.18 6.67 7.05 6.54
(d, 7.3) (dd, 7.3, 7.3) (dd, 7.8, 7.3) (d, 7.8)
6.68 (d, 4.1) 5.33 (d, 4.1)
86.4 s 131.7 s 123.0 d 118.3 d 129.5 d 110.3 d 148.9 s 84.5 d 170.3 s
6.03 (br s)
55.6 d 25.3 t 110.6 124.8 116.7 119.2 120.6 127.9 135.4
s s d d d s s
124.4 d 168.3 s 29.6 t 122.8 d 132.5 s 18.3 q 26.1 q
7.71 4.45 3.37 3.04
(s) (dd, 5.5, 5.0) (m) (dd, 15.6, 6.9)
7.42 (d, 8.2) 6.92 (dd, 7.3, 7.3) 6.85 (d, 6.9)
10.81 (br s) 7.25 (br s)
7.18 6.66 7.05 6.54
(d, 7.3) (dd, 7.3, 7.3) (dd, 8.0, 7.3) (d, 8.0)
6.69 (d, 4.1) 5.33 (d, 4.1) 6.04 (br s)
55.6 d 25.3 t 110.1 128.0 119.0 118.9 121.5 111.8 136.6
s s d d d d s
124.7 d 168.3 s
3.50 (d, 6.9) 5.42 (m) 1.71 (s) 1.71 (s)
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7.73 4.46 3.36 3.06
(s) (dd, 5.5, 5.0) (m) (dd, 15.6, 6.9)
7.60 6.99 7.07 7.33
(d, 7.8) (dd, 7.8, 7.3) (dd, 8.0, 7.3) (d, 8.0)
10.87 (br s) 7.25 (br s)
S. Cai et al. / Tetrahedron xxx (2014) 1e5
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Fig. 5. The CD spectra comparison of compounds 1e3.
Fig. 2. The Key 1He1H COSY and HMBC correlations of 1e3.
Up to now, around 30 nature occurred prenylated indole diketopiperazine alkaloids consisting of two L-tryptophan units were reported mainly covering three structure types represented as fellutanine, amauromine, and okaramine.1,9e18 Among them, okaramine is a major group containing about 20 analogs with some of them showed insecticidal activity, mainly discovered by Hideo Hayashi and co-workers.9e18 In our bioactivity evaluation for cytotoxic and antibiotic activities, only okaramine S (1) exhibited cytotoxic activity against HL-60 and K-562 cell lines with IC50 values 0.78 and 22.4 mM, respectively, while none of them showed antibiotic activities against microbial strains, Enterobacter aerogenes, Staphylococcus aureus, Bacillus proteus, Micrococcus tetragenus, Bacillus subtilis, and Candida albicans. 3. Experimental 3.1. General experimental procedures
Fig. 3. The NOESY and NOE correlations of compounds 1e3.
comparison. The tryptophan residue in 3 was determined as L-Trp using Marfey’s methodology indicating the 2S,3aS,8aR,20 S absolute configuration of compound 3, which was further confirmed by the comparison of experimental ECD curve with the calculated one (Fig. 4). The stereochemistry of compounds 1 and 2 were determined to be the same as 3 based on the biogenetic relationship and the similar CD curves (Fig. 5), and further confirmed by the comparison of the calculated and experimental ECD data (Figs. 30S and 32S, Supplementary data).
Specific rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded on Beckman DU 640 spectrophotometer. CD spectra were measured on JASCO J-715 spectropolarimeter. 1H, 13C NMR, DEPT and 2D NMR spectra were recorded on a JEOL Eclips-600 spectrometer. ESI-MS was measured on a Micromass Q-TOF ULTIMA GLOBAL GAAo76 LC Mass spectrometer. Semipreparative HPLC was performed using an ODS column (YMC-Pack ODS-A, 10250 mm, 5 mm). The seawater was collected from Jiaozhou Bay, Qingdao, China. 3.2. Fermentation and extraction A. taichungensis19 was cultured under static condition at room temperature in 1000-mL Erlenmeyer flasks containing 300 mL fermentation media (mannitol 20 g, maltose 20 g, glucose 10 g, monosodium glutamate 10 g, KH2PO4 0.5 g, MgSO4$7H2O 0.3 g, yeast extract 3 g, and corn steep liquor 1 g, dissolved in 1 L sea water, pH 6.5). After 4 weeks of cultivation, 30 L of whole broth was filtered through cheesecloth to separate the broth supernatant and mycelia. The former was extracted with ethyl acetate, while the latter was extracted with acetone. The acetone extract was evaporated under reduced pressure to afford an aqueous solution, and then extracted with ethyl acetate. The two ethyl acetate extracts were combined and concentrated under reduced pressure to give an extract (40.0 g). 3.3. Compound purification
Fig. 4. Calculated and experimental ECD data for compound 3.
The extract (40.0 g) was subjected to a silica gel (300e400 mesh) column and was separated into five fractions
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(Fr.1-Fr.5) using a step gradient elution of petroleum ether/acetone. Fr.4, which was eluted with 8:2 petroleum ether/acetone, was further fractionated on a C-18 ODS column using a step gradient elution of MeOH/H2O, resulting in 10 subfractions (Fr.4.1-Fr.4.10). The Fr.4.7 was applied on Sephadex LH-20 using CHCl3/CH3OH (50:50) and further purified by semi-preparative HPLC (70:30 CH3OH/H2O, 4 mL/min) to yield 2 (25.0 mg, tR 12.7 min). The Fr.4.8 was applied on semi-preparative HPLC (80% CH3OH/H2O, 4 mL/ min) to afford compound 1 (15.0 mg, tR 11.9 min). The Fr.5, eluted with 7:3 petroleum ether/acetone, was subjected to a C-18 ODS column using a step gradient elution of MeOH/H2O, giving 10 subfractions (Fr.5.1-Fr.5.10). The Fr.5.1 was applied on Sephadex LH20 using CHCl3/CH3OH (50:50) and finally purified by semipreparative HPLC (50:50 CH3OH/H2O, 4 mL/min) to yield 3 (16.5 mg, tR 11.2 min). 3.3.1. Okaramine S (1). Pale yellow oil; [a]25 D 3.0 (c 0.25, MeOH); UV (MeOH) lmax (log ε): 211 (4.53), 283 (3.68) nm; CD (MeOH) lmax (Dε) 210.5 (þ4.39), 223.0 (þ1.42), 241.5 (þ6.26), 271.0 (þ1.98), 287.5 (þ2.53); IR (KBr) nmax 3290, 2967, 2917, 1674, 1607, 1435, 1339, 1303, 1194, 1104, 1025, 750 cm1; 1H NMR and 13C NMR, see Table 1; HRESIMS [MþH]þ m/z 525.2862 (calcd for C32H37N4O3, 525.2866). 3.3.2. Okaramine T (2). Pale yellow oil; [a]25 D þ16.7 (c 0.25, MeOH); UV (MeOH) lmax (log ε): 209 (4.43), 282 (3.78) nm; CD (MeOH) lmax (Dε) 206.5 (1.76), 220.5 (5.15), 240.5 (þ8.25), 284.5 (þ0.99), 305.5 (þ0.52); IR (KBr) nmax 3288, 2914, 1671, 1613, 1433, 1340, 1305, 1190, 1104, 1025, 750 cm1; 1H NMR and 13C NMR, see Table 1; HRESIMS [MþH]þ m/z 457.2245 (calcd for C27H29N4O3, 457.2240). 3.3.3. Okaramine U (3). Pale yellow oil; [a]25 D þ16.6 (c 0.25, MeOH); UV (MeOH) lmax (log ε): 209 (4.39), 282 (3.62) nm; CD (MeOH) lmax (Dε) 204.5 (1.63), 217.5 (5.60), 240 (þ5.56), 285 (þ0.94), 302.5 (þ0.58); IR (KBr) nmax 3319, 1671, 1614, 1420, 1339, 1305, 1104, 1025, 748 cm1; 1H NMR and 13C NMR, see Table 1; HRESIMS [MþH]þ m/z 389.1604 (calcd for C22H21N4O3, 389.1614). 3.4. Acid hydrolysis of okaramine U (3) Okaramine U (3) (1 mg) was hydrolyzed in 1 mL of 6 N HCl at 90 C for 12 h in a 3 mL reaction vial. The cooled reaction mixture was evaporated to dryness, and traces of HCl were removed from the residual hydrolysate by repeated evaporation from H2O (31 mL) using N2 gas.
following retention times: 37.5 min for FDAA, 45.1 min for L-Trp, and 47.3 min for D-Trp. 3.6. Biological assay Cytotoxic activities of 1e3 were assayed using the MTT20,21 method with HL-60 and K-562 cell lines. The detailed methodology for biological testing has already been described by the authors in their previous reports.20 The antimicrobial activities were evaluated by an agar dilution method.22 3.7. Computation section Conformational searches were run employing the ‘systematic’ procedure implemented in Spartan’14,23 using MMFF (Merck molecular force field). All MMFF minima were reoptimized with DFT calculations at the B3LYP/6-31þg(d) level using the Gaussian09 program.24 The geometry was optimized starting from various initial conformations, with vibrational frequency calculations confirming the presence of minima. Time-dependent DFT calculations were performed on lowest-energy conformations (>5% population) for each configuration using 30 excited states, and using a polarizable continuum model (PCM) for MeOH. ECD spectra were generated using the program SpecDis25 by applying a Gaussian band shape with 0.18e0.36 eV width, from dipole-length rotational strengths. The dipole velocity forms yielded negligible differences. The spectra of the conformers were combined using Boltzmann weighting, with the lowest-energy conformations accounting for about 99% of the weights. The calculated spectrum was red-shifted by 5e18 nm to facilitate comparison to the experimental data. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 21372208 and 41176120), the NSFC-Shandong Joint Fund (No. U1406402), the National High Technology Research and Development Program of China (No. 2013AA092901), the Program for New Century Excellent Talents in University (No. NCET-12-0499) and the State Key Laboratory of Bioorganic and Natural Products Chemistry (SKLBNPC12331). Supplementary data 1
3.5. Absolute configuration of amino acid To a 1.5 mL vial containing 0.25 mmol of pure amino acid standards in 50 mL of H2O was added 0.25 mmol of N-R-(2,4-dinitro-5fluorophenyl)-L-alanine amide (L-FDAA) in 100 mL of acetone followed by 25 mL of 1 N NaHCO3. After 1 h at 40 C, the mixture was cooled to room temperature (rt), followed by adding 25 mL of 2 N HCl and filtering through a small 4.5 mm filter. A quarter of the peptide hydrolysate mixture was dissolved in 50 mL of H2O, and added 0.25 mmol of L-FDAA in 100 mL of acetone and 25 mL of 1 N NaHCO3. The derivatization was carried out and worked up as described above. An 8 mL aliquot of the resulting mixture of L-FDAA derivative was analyzed by reversed-phase HPLC (5 mm250 mm YMC C18 column, 5 mm; flow rate, 1 mL/min). The gradient was 5%e 55% CH3CN in 0.05% TFA aqueous over 50 min. UV wavelength was set to 340 nm to detect the L-FDAA derivatives. Each chromatographic peak was identified by comparing its retention time with the L-FDAA derivative of the pure D- and L-amino acid standards. The FDAA derivative of tryptophan liberated from compound 3 displayed a sharp peak at 45.1 min. The standards gave the
H, 13C NMR, DEPT, HMQC, 1He1H COSY, HMBC, NOESY, NOE, and HRESIMS spectroscopic data for compounds 1e3, the HPLC profiles of the L-FDAA derivatives, and the computional details of compounds 1e3. Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.tet.2014.09.019. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
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Okaramines SeU, three new indole diketopiperazine alkaloids from Aspergillus taichungensis ZHN-7-07 Shengxin Cai y, Shiwei Sun y, Jixing Peng, Xianglan Kong, Huinan Zhou, Tianjiao Zhu, Qianqun Gu, Dehai Li * Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao, Shandong 266003, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2014 Received in revised form 3 September 2014 Accepted 8 September 2014 Available online xxx
Three new indole diketopiperazine alkaloids, okaramines SeU (1e3), with two (1 and 2) of them prenylated, were obtained from the Aspergillus taichungensis ZHN-7-07. Their structures including the absolute configurations were determined by combination of NMR, MS, TDDFT ECD calculation, and Marfey’s analysis. Okaramine S (1) showed the best cytotoxic activity against HL-60 cell with IC50 value of 0.78 mM. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Indole diketopiperazine alkaloids Okaramine Mangrove plant Aspergillus taichungensis Cytotoxicity
1. Introduction Prenylated indole diketopiperazine (DKP) alkaloids are hybrid natural products derived from coupling a prenylated tryptophan with the second amino acid such as proline, tryptophan, histidine or alanine, and widely distributed in filamentous fungi, especially in the genera Penicillium and Aspergillus of Ascomycota.1 Among them, the family composed by two tryptophan units is an important group with high structure diversity and various biological activities, attracting much attention of the synthetic and biosynthetic chemists.1e5 In our previous research, a series of tryptophan-proline composed indole DKPs, including a dimer, aspergilazine A with a rare N1 to C-6 linkage,6 and three reversely prenylated indole alkaloids with a rare anti bicyclo-[2.2.2]dizaoctane core ring,7 were discovered from the fungus Aspergillus taichungensis ZHN-7-07 isolated from the rhizosphere soil of the mangrove plant Acrostichum aureum. Encouraged by the novelty and bioactivities of them, further studies of this fungus led to the isolation of three pyrrolo[2,3-b] indole contained DKPs composed by two tryptophan moieties (Fig. 1), okaramines SeU (1e3), with two of them prenylated (1 and
* Corresponding author. Tel.: þ86 532 82031619; fax: þ86 532 82033054; e-mail addresses: [email protected], [email protected] (D. Li). y These authors contributed equally to this work.
2), together with a known C-7 prenylated cyclo-L-Trp-L-Trp.8 Cytotoxic and antibiotic evaluations revealed that only compound 1 exhibited cytotoxic activity against HL-60 and K-562 cell lines, and
Fig. 1. The structures of compounds 1e3 and okaramine J.
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none of them displayed antibiotic activity against Enterobacter aerogenes, Staphylococcus aureus, Bacillus proteus, Micrococcus tetragenus, Bacillus subtilis, and Candida albicans strains. In this paper, we report the isolation, structure elucidation, and bioactivities of the new indole DKP alkaloids (1e3). 2. Results and discussion The fungal isolate A. taichungensis was grown in a liquid medium under static condition for 4 weeks. The culture broth was extracted with EtOAc, and the organic extract was separated using silica gel, Sephadex LH-20 and C18 HPLC to yield the new compounds (1e3). Okaramine S (1) was obtained as a pale yellow oil, and its molecular formula was established as C32H36N4O3 by the HRESIMS peak at m/z 525.2862 [MþH]þ (calcd 525.2866), indicating seventeen degrees of unsaturation. The IR data displayed the absorptions for amides (3289 and 1674 cm1). The 1D NMR data of compound 1 displayed signals for two carbonyls, 10 quarternary carbons, 12 methines, four methylenes, and four methyls (Table 1). The UV, IR, and 1D NMR data of metabolite 1 were similar to those of okaramine J,9 suggested that they share the same skeleton. The difference between them was that the C-8a0 reverse prenyl of okaramine J was replaced by a C-7 prenyl. This change and the planar structure of 1 were supported by the proton chemical shifts and coupling patterns of H-40 (dH 7.44, d, J¼7.8 Hz), H-50 (6.92, dd, J¼7.8, 6.9 Hz), Table 1 1 H (600 MHz) and No.
13
H-60 (6.84, d, J¼6.9 Hz) and H-8a0 (dH 7.26, d, J¼2.3 Hz), and the COSY correlations of H-40 /H-50 /H-60 and 80 -NH/H-8a0 , as well as the HMBC correlations from H-100 to C-60 and C-7a0 (Fig. 2). The relative configuration of 1 was established by the NOESY and NOE experiments. The NOESY correlation of H-2 and H-20 indicated that they were on the same face of the diketopiperazine ring. The cis configuration of H-8a and 3a-OH was also deduced from the NOESY correlation between them, which was further suggested to be cofacial to H-2 based on the enhancement of H-8a when irradiated H-2 (Fig. 3). The molecular formulae of compounds 2 and 3 were determined by HRESIMS to be C27H28N4O3 and C22H20N4O3, respectively. The 1H and 13C NMR data of both metabolites revealed they possessed the same diketopiperazine core as compound 1 (Table 1), with the difference in the absence of one and two prenyls in 2 and 3, respectively. The COSY correlations of H-4/H-5/H-6/H-7 suggested the disappearance of C-7 prenyl in compound 2, while the remained prenyl was assigned to C-70 based on the HMBC correlations from H-100 to C-7a0 (Fig. 2). For okaramine U (3), the COSY correlations of H-4/H-5/H-6/H-7 and H-40 /H-50 /H-60 /H-70 indicated the appearance of two 1,2-disubstituted benzenes and confirmed the absence of the two prenyls (Fig. 2). The relative configurations of compounds 2 and 3 were elucidated the same as 1 based on the NOESY and NOE experiments (Fig. 3). The absolute configuration of compounds 3 were determined by combination of Marfey’s analysis and experimental/calculated ECD
C (150 MHz) NMR data for compounds 1e3 (DMSO-d6) 1
2
3
dC, mult.
dH, (mult., J in Hz)
dC, mult.
dH, (mult., J in Hz)
dC, mult.
dH, (mult., J in Hz)
2 3
59.1 d 41.8 t
4.65 (dd, 11.0, 6.5) 2.42 (dd, 13.3, 6.5) 1.84 (dd, 13.3, 11.0)
59.2 d 41.8 t
4.66 (dd, 11.0, 6.5) 2.42 (dd, 13.0, 6.5) 1.83 (dd, 13.0, 11.0)
59.2 d 41.8 t
4.66 (dd, 11.5, 6.5) 2.43 (dd, 13.0, 6.5) 1.83 (dd, 13.0, 11.5)
3a 3b 4 5 6 7 7a 8 8a 9 3a-OH 10
86.5 s 131.7 s 120.7 d 119.2 d 128.7 d 123.4 s 146.7 s
11 12 13 14 10 20 30
122.3 d 132.8 s 18.2 q 26.1 q
3a0 3b0 40 50 60 70 7a0 80 8a0 90 100 110 120 130 140
110.5 124.8 116.8 119.2 120.6 127.9 135.4
84.5 d 170.3 s 29.0 t
55.6 d 25.3 t s s d d d s s
124.6 d 168.5 s 29.6 t 122.8 d 132.5 s 18.2 q 26.1 q
7.05 (d, 7.3) 6.67 (dd, 7.8, 7.3) 6.87 (d, 7.8)
6.04 (d, 4.3) 5.35 (d, 4.3) 6.01 3.21 3.09 5.25
(br s) (dd, 16.3, 7.7) (dd, 16.3, 6.0) (m)
1.66 1.71 7.68 4.45 3.37 3.05
(s) (s) (s) (dd, 5.5, 5.0) (dd, 15.1, 4.6) (dd, 15.1, 6.4)
7.44 (d, 7.8) 6.92 (dd, 7.8, 6.9) 6.84 (d, 6.9)
10.78 (br s) 7.26 (d, 2.3) 3.50 (d, 7.3) 5.41 (m) 1.71 (s) 1.72 (s)
86.4 s 131.7 s 123.0 d 118.3 d 129.5 d 110.4 d 149.0 s 84.5 d 170.3 s
7.18 6.67 7.05 6.54
(d, 7.3) (dd, 7.3, 7.3) (dd, 7.8, 7.3) (d, 7.8)
6.68 (d, 4.1) 5.33 (d, 4.1)
86.4 s 131.7 s 123.0 d 118.3 d 129.5 d 110.3 d 148.9 s 84.5 d 170.3 s
6.03 (br s)
55.6 d 25.3 t 110.6 124.8 116.7 119.2 120.6 127.9 135.4
s s d d d s s
124.4 d 168.3 s 29.6 t 122.8 d 132.5 s 18.3 q 26.1 q
7.71 4.45 3.37 3.04
(s) (dd, 5.5, 5.0) (m) (dd, 15.6, 6.9)
7.42 (d, 8.2) 6.92 (dd, 7.3, 7.3) 6.85 (d, 6.9)
10.81 (br s) 7.25 (br s)
7.18 6.66 7.05 6.54
(d, 7.3) (dd, 7.3, 7.3) (dd, 8.0, 7.3) (d, 8.0)
6.69 (d, 4.1) 5.33 (d, 4.1) 6.04 (br s)
55.6 d 25.3 t 110.1 128.0 119.0 118.9 121.5 111.8 136.6
s s d d d d s
124.7 d 168.3 s
3.50 (d, 6.9) 5.42 (m) 1.71 (s) 1.71 (s)
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7.73 4.46 3.36 3.06
(s) (dd, 5.5, 5.0) (m) (dd, 15.6, 6.9)
7.60 6.99 7.07 7.33
(d, 7.8) (dd, 7.8, 7.3) (dd, 8.0, 7.3) (d, 8.0)
10.87 (br s) 7.25 (br s)
S. Cai et al. / Tetrahedron xxx (2014) 1e5
3
Fig. 5. The CD spectra comparison of compounds 1e3.
Fig. 2. The Key 1He1H COSY and HMBC correlations of 1e3.
Up to now, around 30 nature occurred prenylated indole diketopiperazine alkaloids consisting of two L-tryptophan units were reported mainly covering three structure types represented as fellutanine, amauromine, and okaramine.1,9e18 Among them, okaramine is a major group containing about 20 analogs with some of them showed insecticidal activity, mainly discovered by Hideo Hayashi and co-workers.9e18 In our bioactivity evaluation for cytotoxic and antibiotic activities, only okaramine S (1) exhibited cytotoxic activity against HL-60 and K-562 cell lines with IC50 values 0.78 and 22.4 mM, respectively, while none of them showed antibiotic activities against microbial strains, Enterobacter aerogenes, Staphylococcus aureus, Bacillus proteus, Micrococcus tetragenus, Bacillus subtilis, and Candida albicans. 3. Experimental 3.1. General experimental procedures
Fig. 3. The NOESY and NOE correlations of compounds 1e3.
comparison. The tryptophan residue in 3 was determined as L-Trp using Marfey’s methodology indicating the 2S,3aS,8aR,20 S absolute configuration of compound 3, which was further confirmed by the comparison of experimental ECD curve with the calculated one (Fig. 4). The stereochemistry of compounds 1 and 2 were determined to be the same as 3 based on the biogenetic relationship and the similar CD curves (Fig. 5), and further confirmed by the comparison of the calculated and experimental ECD data (Figs. 30S and 32S, Supplementary data).
Specific rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded on Beckman DU 640 spectrophotometer. CD spectra were measured on JASCO J-715 spectropolarimeter. 1H, 13C NMR, DEPT and 2D NMR spectra were recorded on a JEOL Eclips-600 spectrometer. ESI-MS was measured on a Micromass Q-TOF ULTIMA GLOBAL GAAo76 LC Mass spectrometer. Semipreparative HPLC was performed using an ODS column (YMC-Pack ODS-A, 10250 mm, 5 mm). The seawater was collected from Jiaozhou Bay, Qingdao, China. 3.2. Fermentation and extraction A. taichungensis19 was cultured under static condition at room temperature in 1000-mL Erlenmeyer flasks containing 300 mL fermentation media (mannitol 20 g, maltose 20 g, glucose 10 g, monosodium glutamate 10 g, KH2PO4 0.5 g, MgSO4$7H2O 0.3 g, yeast extract 3 g, and corn steep liquor 1 g, dissolved in 1 L sea water, pH 6.5). After 4 weeks of cultivation, 30 L of whole broth was filtered through cheesecloth to separate the broth supernatant and mycelia. The former was extracted with ethyl acetate, while the latter was extracted with acetone. The acetone extract was evaporated under reduced pressure to afford an aqueous solution, and then extracted with ethyl acetate. The two ethyl acetate extracts were combined and concentrated under reduced pressure to give an extract (40.0 g). 3.3. Compound purification
Fig. 4. Calculated and experimental ECD data for compound 3.
The extract (40.0 g) was subjected to a silica gel (300e400 mesh) column and was separated into five fractions
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S. Cai et al. / Tetrahedron xxx (2014) 1e5
(Fr.1-Fr.5) using a step gradient elution of petroleum ether/acetone. Fr.4, which was eluted with 8:2 petroleum ether/acetone, was further fractionated on a C-18 ODS column using a step gradient elution of MeOH/H2O, resulting in 10 subfractions (Fr.4.1-Fr.4.10). The Fr.4.7 was applied on Sephadex LH-20 using CHCl3/CH3OH (50:50) and further purified by semi-preparative HPLC (70:30 CH3OH/H2O, 4 mL/min) to yield 2 (25.0 mg, tR 12.7 min). The Fr.4.8 was applied on semi-preparative HPLC (80% CH3OH/H2O, 4 mL/ min) to afford compound 1 (15.0 mg, tR 11.9 min). The Fr.5, eluted with 7:3 petroleum ether/acetone, was subjected to a C-18 ODS column using a step gradient elution of MeOH/H2O, giving 10 subfractions (Fr.5.1-Fr.5.10). The Fr.5.1 was applied on Sephadex LH20 using CHCl3/CH3OH (50:50) and finally purified by semipreparative HPLC (50:50 CH3OH/H2O, 4 mL/min) to yield 3 (16.5 mg, tR 11.2 min). 3.3.1. Okaramine S (1). Pale yellow oil; [a]25 D 3.0 (c 0.25, MeOH); UV (MeOH) lmax (log ε): 211 (4.53), 283 (3.68) nm; CD (MeOH) lmax (Dε) 210.5 (þ4.39), 223.0 (þ1.42), 241.5 (þ6.26), 271.0 (þ1.98), 287.5 (þ2.53); IR (KBr) nmax 3290, 2967, 2917, 1674, 1607, 1435, 1339, 1303, 1194, 1104, 1025, 750 cm1; 1H NMR and 13C NMR, see Table 1; HRESIMS [MþH]þ m/z 525.2862 (calcd for C32H37N4O3, 525.2866). 3.3.2. Okaramine T (2). Pale yellow oil; [a]25 D þ16.7 (c 0.25, MeOH); UV (MeOH) lmax (log ε): 209 (4.43), 282 (3.78) nm; CD (MeOH) lmax (Dε) 206.5 (1.76), 220.5 (5.15), 240.5 (þ8.25), 284.5 (þ0.99), 305.5 (þ0.52); IR (KBr) nmax 3288, 2914, 1671, 1613, 1433, 1340, 1305, 1190, 1104, 1025, 750 cm1; 1H NMR and 13C NMR, see Table 1; HRESIMS [MþH]þ m/z 457.2245 (calcd for C27H29N4O3, 457.2240). 3.3.3. Okaramine U (3). Pale yellow oil; [a]25 D þ16.6 (c 0.25, MeOH); UV (MeOH) lmax (log ε): 209 (4.39), 282 (3.62) nm; CD (MeOH) lmax (Dε) 204.5 (1.63), 217.5 (5.60), 240 (þ5.56), 285 (þ0.94), 302.5 (þ0.58); IR (KBr) nmax 3319, 1671, 1614, 1420, 1339, 1305, 1104, 1025, 748 cm1; 1H NMR and 13C NMR, see Table 1; HRESIMS [MþH]þ m/z 389.1604 (calcd for C22H21N4O3, 389.1614). 3.4. Acid hydrolysis of okaramine U (3) Okaramine U (3) (1 mg) was hydrolyzed in 1 mL of 6 N HCl at 90 C for 12 h in a 3 mL reaction vial. The cooled reaction mixture was evaporated to dryness, and traces of HCl were removed from the residual hydrolysate by repeated evaporation from H2O (31 mL) using N2 gas.
following retention times: 37.5 min for FDAA, 45.1 min for L-Trp, and 47.3 min for D-Trp. 3.6. Biological assay Cytotoxic activities of 1e3 were assayed using the MTT20,21 method with HL-60 and K-562 cell lines. The detailed methodology for biological testing has already been described by the authors in their previous reports.20 The antimicrobial activities were evaluated by an agar dilution method.22 3.7. Computation section Conformational searches were run employing the ‘systematic’ procedure implemented in Spartan’14,23 using MMFF (Merck molecular force field). All MMFF minima were reoptimized with DFT calculations at the B3LYP/6-31þg(d) level using the Gaussian09 program.24 The geometry was optimized starting from various initial conformations, with vibrational frequency calculations confirming the presence of minima. Time-dependent DFT calculations were performed on lowest-energy conformations (>5% population) for each configuration using 30 excited states, and using a polarizable continuum model (PCM) for MeOH. ECD spectra were generated using the program SpecDis25 by applying a Gaussian band shape with 0.18e0.36 eV width, from dipole-length rotational strengths. The dipole velocity forms yielded negligible differences. The spectra of the conformers were combined using Boltzmann weighting, with the lowest-energy conformations accounting for about 99% of the weights. The calculated spectrum was red-shifted by 5e18 nm to facilitate comparison to the experimental data. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 21372208 and 41176120), the NSFC-Shandong Joint Fund (No. U1406402), the National High Technology Research and Development Program of China (No. 2013AA092901), the Program for New Century Excellent Talents in University (No. NCET-12-0499) and the State Key Laboratory of Bioorganic and Natural Products Chemistry (SKLBNPC12331). Supplementary data 1
3.5. Absolute configuration of amino acid To a 1.5 mL vial containing 0.25 mmol of pure amino acid standards in 50 mL of H2O was added 0.25 mmol of N-R-(2,4-dinitro-5fluorophenyl)-L-alanine amide (L-FDAA) in 100 mL of acetone followed by 25 mL of 1 N NaHCO3. After 1 h at 40 C, the mixture was cooled to room temperature (rt), followed by adding 25 mL of 2 N HCl and filtering through a small 4.5 mm filter. A quarter of the peptide hydrolysate mixture was dissolved in 50 mL of H2O, and added 0.25 mmol of L-FDAA in 100 mL of acetone and 25 mL of 1 N NaHCO3. The derivatization was carried out and worked up as described above. An 8 mL aliquot of the resulting mixture of L-FDAA derivative was analyzed by reversed-phase HPLC (5 mm250 mm YMC C18 column, 5 mm; flow rate, 1 mL/min). The gradient was 5%e 55% CH3CN in 0.05% TFA aqueous over 50 min. UV wavelength was set to 340 nm to detect the L-FDAA derivatives. Each chromatographic peak was identified by comparing its retention time with the L-FDAA derivative of the pure D- and L-amino acid standards. The FDAA derivative of tryptophan liberated from compound 3 displayed a sharp peak at 45.1 min. The standards gave the
H, 13C NMR, DEPT, HMQC, 1He1H COSY, HMBC, NOESY, NOE, and HRESIMS spectroscopic data for compounds 1e3, the HPLC profiles of the L-FDAA derivatives, and the computional details of compounds 1e3. Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.tet.2014.09.019. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
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