Article pubs.acs.org/jnp
Cite This: J. Nat. Prod. 2018, 81, 394−399
Cyclizidine-Type Alkaloids from Streptomyces sp. HNA39 Yong-Jun Jiang, Jia-Qi Li, Hao-Jian Zhang, Wan-Jing Ding, and Zhong-Jun Ma* Institute of Marine Biology, Ocean College, Zhejiang University, Zhoushan 316021, People’s Republic of China S Supporting Information *
ABSTRACT: Eight new cyclizidine-type alkaloids (1−8) and one known alkaloid (9) were identified from the chemical investigations of a marine-derived actinomycete, Streptomyces sp. HNA39. Among these alkaloids, compounds 3, 7, and 8 contain a chlorine atom, and the known alkaloid, (+)-ent-cyclizidine (9), is now first reported as a natural product. Their structures were elucidated by extensive NMR-spectroscopic analysis and HRESIMS data. The absolute configurations of all of the compounds were established by ECD calculations. Cytotoxicity evaluations of all of the compounds showed that compound 2 exhibited significant activity against the PC3 and HCT116 humancancer-cell lines with IC50 values of 0.52 ± 0.03 and 8.3 ± 0.1 μM, respectively. Interestingly, compounds 2, 5, 7, and 8 exhibited moderate inhibition against the ROCK2 protein kinase with IC50 values from 7.0 ± 0.8 to 42 ± 3 μM.
C
Carefully analysis of the 1D-NMR-spectroscopic data (Tables 1 and 2) combined with HSQC correlations of 1 revealed a typical monosubstituted cyclopropyl ring [δH 0.45 (2H, m), 0.79 (2H, m), 1.49 (1H, m)]; two methyl groups [δH 1.29 (3H, s), 1.87 (3H, s)]; three olefinic protons [δH 5.42 (1H, dd, J = 15.6, 9.6 Hz), 5.44 (1H, d, J = 8.5 Hz), 6.25 (1H, d, J = 15.6 Hz)]; four methylenes [δC 22.6, 22.6, 23.8, 52.1]; three methines, including one oxygenated carbon [δH 3.07 (1H, br s), 3.67 (1H, d, J = 3.5 Hz), 3.91 (1H, dd, J = 10.5, 3.5 Hz); and two nonprotonated carbons (δC 78.6, 143.5). Inspired by the special characteristic NMR signals of the coisolated (+)-ent-cyclizidine (9),2,5 we determined that compound 1 should be a cyclizidine analogue (Tables 1 and 2). The major differences between the two were the absence of an epoxide bond in (+)-ent-cyclizidine (9) and the presence of two saturated methylenes in 1 at C-7 and C-8. These deductions were further confirmed by the key correlation system of H2-6/H2-7/H2-8/H-8a in the COSY spectrum of 1 (Figure 1). The relative configuration of 1 was assigned on the basis of the analysis of the proton-coupling constants and NOESY correlations (Figure 2). A large coupling constant of 15.6 Hz indicated the 12E configuration, and a 10E configuration was deduced from significant NOESY correlations (H-10/H12, H17/H-3, and H-17/H-13). The NOESY correlations of H-3/H8a and H-3/H-9 suggested the β-orientation of H-8a and H-9. Furthermore, the observation of NOESY cross-peaks between H-2 and H-10 confirmed the α-orientation of H-2. The absolute configuration of 1 was determined by using the solution TDDFT ECD calculation. First, conformational analyses were carried out via random searching in Sybyl-X 2.0 using the MMFF94S force field with an energy cutoff of 2.5 kcal/ mol.11 The results showed the two lowest-energy conformers for (1S, 2S, 3S, 8aR)-1. Subsequently, the conformers were reoptimized using DFT at the b3lyp/6-31+g(d) level in gas
yclizidine alkaloids, belonging to the indolizidine alkaloids, have a fused 6/5 bicyclic ring system with a tertiary amine and a unique monosubstituted cyclopropyl trans-dienic subunit at C-3.1 The group of cyclizidine-type alkaloids has grown very slowly since the first isolation of a cyclizidine from Streptomyces NCIB 11649 in 1982.2−4 However, the unique structures and various biological activities of cyclizidine-type alkaloids have attracted considerable attention from the chemical and biological communities.5−7 As a part of our research work in discovering structurally novel and bioactive structures from marine-derived microorganisms,8−10 an actinomycete classified as Streptomyces sp. HNA39 was chemically investigated. As a result, eight new cyclizidinetype alkaloids and one known derivative were isolated and identified. The known compound was identified as (+)-entcyclizidine (9), a new natural product that was known by synthesis, by comparison of its NMR, HRESIMS, and specificrotation data with those published.2,5 All of the compounds were tested for cytotoxicity against the PC3 and HCT116 cancer-cell lines and for ROCK2-protein-kinase-inhibitory activities. We herein described the details of the isolation, structure elucidation, and bioactivities of these isolated compounds.
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RESULTS AND DISCUSSION Spores of the titled strain growing on a Gause’s agar plate were inoculated into 500 mL Erlenmeyer flasks containing 250 mL of Gause’s liquid medium and then incubated at 28 °C for 8 days on a rotary shaker (180 rpm). EtOAc extracts from the cultures (25 L total) were separated by silica-gel, Sephadex LH-20 column chromatography and purified further by HPLC to afford nine compounds. Compound 1 possessed a molecular formula of C17H27NO2 on the basis of the NMR data and the HRESIMS ion peak at m/z 278.2118 [M + H]+, indicating five indices of hydrogen deficiency. The IR spectrum showed the presence of doublebond (1683 cm−1) and hydroxy (3421 cm−1) functionalities. © 2018 American Chemical Society and American Society of Pharmacognosy
Received: December 18, 2017 Published: February 1, 2018 394
DOI: 10.1021/acs.jnatprod.7b01055 J. Nat. Prod. 2018, 81, 394−399
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Table 1. 1H-NMR (600 MHz) Data of Compounds 1−8 (δH, J in Hz) no. 2 3
8a
3.67, d (3.5) 3.91, dd (10.5, 3.5) 3.38, m 2.96, m 1.91, overlap 1.70, m 1.98, overlap 1.57, m 1.94, overlap 1.74, m 3.07, br s
9 10
12 13
5 6 7 8
14 15 16 17 a
1a
2b
3a
4a
5a
3.34, overlap 2.65, dd (6.8, 2.5) 2.61, m 1.69, m 1.64, overlap 1.27, m 3.15, m
3.53, d (6.5) 2.90, dd (9.5, 6.5) 2.85, m 1.92, overlap 2.13, m 1.85, m 3.71, m
3.28, m
3.68, m
1.64, overlap
1.93, overlap
1.29, s 5.44, d (8.5)
1.13, s 5.21, d (8.8)
1.34, s 5.28, d (9.5)
1.60, s 5.51, overlap
1.68, s 5.51, dd (9.0, 6.4)
6.25, d (15.6) 5.42, dd (15.6, 9.6) 1.49, m 0.79, m 0.45, m 1.87, s
6.14, d (15.6) 5.13, dd (15.6, 9.0) 1.42, m 0.70, m 0.37, m 1.66, m
6.19, d (15.6) 5.21, dd (15.6, 8.8) 1.43, m 0.74, m 0.38, m 1.77, s
6.37, d (15.6) 5.58, dd (15.6, 9.2) 1.53, m 0.82, m 0.50, m 2.04, s
6.35, d (15.6) 5.54, dd (15.6, 9.2) 1.53, m 0.83, m 0.48, m 2.0, s
6a
7a
8a
4.35, d (7.6) 5.51, overlap
4.25, d (7.6) 5.40, br t
3.65, d (2.5) 3.20, m
3.45, d (4.8) 2.26, m
3.48, d (4.8) 2.27, m
8.54, d (6.5)
7.79, d (6.4)
8.0, t (6.5)
7.73, t (6.4)
8.63, t (6.5)
7.91, d (6.4)
3.67, overlap 2.99, m 1.91, overlap 1.80, overlap 1.98, overlap 1.57, overlap 1.93, overlap 1.76, overlap 3.07, dd, (10.0, 1.9) 1.26, s 2.71, dd, (13.5, 4.8) 2.44, dd (13.5, 10.6) 6.04, d (10.6) 6.37, dd (15.0, 10.6) 5.70, m 2.34, m 3.58, t (6.6) 1.84, s
3.11, m 2.03, m 2.13, m 1.89, m 3.71, m
3.15, m 2.03, m 2.15, m 1.90, overlap 3.72, m
3.66, t (9.0)
3.69, t (9.0)
1.97, d (9.0)
1.95, d (9.0)
1.32, s 2.42, dd (13.2, 4.8) 2.21, m
1.32, s 2.52, dd (13.5, 5.8) 2.44, dd (13.5, 5.8) 5.91, d (10.8) 6.47, dd (15.6, 10.8) 5.59, m 2.33, m 3.59, t (6.6) 1.84, s
8.15, d (6.5)
5.93, d (10.8) 6.34, dd (15.6, 10.8) 5.58, m 2.31, m 3.56, t (6.6) 1.79, s
Recorded in methanol-d4. bRecorded in DMSO-d6.
phase by the GAUSSIAN 09 program.12 The energies, oscillator strengths, and rotational strengths (velocity) of the first 60 electronic excitations were calculated using the TDDFT methodology at the b3lyp/6-311++g(d,p) level in a vacuum. The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at the 1/e peak height, σ = 0.35).13 To get the final spectra, the simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energies (ΔG). The theoretical ECD spectra of the corresponding enantiomers were obtained by directly inverting the ECD spectra of the calculated model molecules. By comparing the experimental spectrum to the calculated ECD spectra, it was determined that the calculated ECD curve of (1S, 2S, 3S, 8aR)-1 revealed a good agreement with the measured spectrum (Figure 3). Thus, the absolute structure of 1, named cyclizidine B, was determined as shown. Compound 2 had an [M + H]+ ion peak at m/z 310.2015 in the HRESIMS data, consistent with the molecular formula
C17H27NO4. Its 1H- and 13C-NMR spectra were similar to those of 1, with the exception that two methylenes (δC 22.6, 22.6) in 1 were replaced by two oxygenated methines (δC 73.7, 72.1) in 2. This suggested that two additional hydroxy groups were attached at C-7 and C-8 in 2, as further deduced from the HMBC correlations from H-7 (δH 3.15, m) to C-8 and H-8 (δH 3.28, m) to C-8a and from the COSY correlation system of H-5/H-6/H7/H-8/H-8a (Figure 2). The NOESY spectrum showed crosspeaks of H-3 with H-7, H-8a, and H3-9; H-7 with H-8a; and H-8 with H-5α and H-6α. This information supported the relative configuration of 2 as H-2α, H-3β, H-7β, H-8α, H-8aβ, and CH39β (Figure 3). The configurations of the double bonds were determined to be E by the NOEs (H-10/H12, H-17/H-3, H-17/ H-13). The absolute configuration of 2 was also established, like 1, by computational methods. Finally, the calculated ECD curve of (1S, 2S, 3S, 7S, 8S, 8aR)-2 showed good agreement with the experimental curve (Figure 4). Compound 2 was determined as shown and named cyclizidine C. 395
DOI: 10.1021/acs.jnatprod.7b01055 J. Nat. Prod. 2018, 81, 394−399
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Table 2. 13C-NMR (150 MHz) Data of Compounds 1−8 (δC, Type)
a
no.
1a
2b
3a
4a
5a
6a
7a
8a
1 2 3 5 6 7 8 8a 9 10 11 12 13 14 15 16 17
78.6, C 83.7, CH 73.9, CH 52.1, CH2 23.8, CH2 22.6, CH2 22.6, CH2 72.0, CH 18.2, CH3 120.4, CH 143.5, C 131.5, CH 138.8, CH 15.2, CH 7.8, CH2 7.8, CH2 13.2, CH3
77.9, C 86.0, CH 68.7, CH 48.0, CH2 32.4, CH2 73.7, CH 72.1, CH 74.6, CH 23.1, CH3 130.3, CH 135.8, C 132.0, CH 132.5, CH 14.0, CH 7.0, CH2 6.9, CH2 12.9, CH3
79.7, C 87.5, CH 70.4, CH 50.3, CH2 35.9, CH2 65.9, CH 73.9, CH 76.5, CH 22.6, CH3 129.2, CH 139.2, C 133.1, CH 134.7, CH 15.0, CH 7.5, CH2 7.5, CH2 13.3, CH3
80.0, C 84.1, CH 71.4, CH 140.9, CH 128.4, CH 147.9, CH 124.6, CH 163.0, C 20.8, CH3 120.8, CH 146.9, C 131.2, CH 139.8, CH 15.3, CH 7.9, CH2 7.9, CH2 13.4, CH3
80.8, C 83.9, CH 71.6, CH 132.9, CH 128.8, CH 130.0, CH 157.5, C 148.6, C 18.5, CH3 121.4, CH 146.1, C 131.3, CH 139.6, CH 15.3, CH 7.9, CH2 7.9, CH2 13.4, CH3
78.4, C 80.8, CH 74.1, CH 53.2, CH2 23.8, CH2 22.7, CH2 22.5, CH2 72.7, CH 17.9, CH3 41.3, CH2 131.4, C 130.2, CH 129.1, CH 131.9, CH 37.1, CH2 62.6, CH2 16.3, CH3
79.5, C 85.4, CH 70.4, CH 50.6, CH2 36.0, CH2 66.0, CH 73.8, CH 75.8, CH 21.7, CH3 44.2, CH2 135.2, C 128.3, CH 129.7, CH 129.7, CH 37.2, CH2 62.8, CH2 17.0, CH3
79.5, C 85.6, CH 70.5, CH 50.6, CH2 36.1, CH2 66.1, CH 73.9, CH 75.9, CH 21.7, CH3 36.3, CH2 135.4, C 128.7, CH 129.6, CH 129.8, CH 37.2, CH2 62.8, CH2 24.5, CH3
Recorded in methanol-d4. bRecorded in DMSO-d6.
Figure 1. Key COSY (bold lines) and HMBC (arrows) correlations of compounds 1−8.
Figure 2. Key NOESY correlations (dashed arrows) of compounds 1−8.
isotopic peak at m/z 330.1648 [M + H]+ (ratio = 3:1), indicating the presence of a chlorine atom. Its HRESIMS data combined
The HRESIMS data of compound 3 gave a protonatedmolecule peak at m/z 328.1673 [M + H]+, accompanied by an 396
DOI: 10.1021/acs.jnatprod.7b01055 J. Nat. Prod. 2018, 81, 394−399
Journal of Natural Products
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Figure 5. Experimental ECD spectra for 4 and 5 and calculated ECD spectra of 4. The calculated ECD spectra for 5 were similar to those for 4 (Supporting Information).
Figure 3. Experimental ECD spectrum and calculated ECD spectra of 1.
The NMR spectra of 5 were similar to those of 4 except for the presence of an oxygenated aromatic carbon at δC 157.5 in 5 and the absence of the aromatic proton (H-8) in 4. One hydroxy was presumed to be attached to C-8 because of the HMBC correlations from H-6 (δH 7.73, t, J = 6.5 Hz) to C-8. The absolute configuration of 5 was also determined by NOESYspectra information (H-2/H-10, and H-3/H3-9) and ECD calculations (Figures 2 and 5). Thus, compound 5 was named cyclizidine F. Compound 6 was assigned the molecular formula C17H29NO3 deduced from its HRESIMS data ([M + H]+ m/z 296.2227), which implied four indices of hydrogen deficiency. Its 1H- and 13 C-NMR-spectroscopic data (Tables 1 and 2) were similar to those of 1. The main differences included the absence of a Δ10 double bond and a cyclopropyl ring in 6 and the presence of a Δ14 double bond and an oxygenated methylene group in 1. The structure was supported by HMBC correlations from H-10 to C2, C-3, C-11, and C-17 and COSY correlations of H-2/H-3/H-10 and H-12/H-13/H-14/H-15/H-16 (Figure 1). The NOESY cross-peaks of H-3/H-8a, Hβ-8/H-9, and H-2/H-10 established the β-orientations of H-3, H-8a, and Me-9, and H-2 was deduced to be α-oriented (Figure 2). The configurations of the double bonds were determined to be 11E and 13E by NOEs (H-10/ H12, H-12/H-14, and H-13/H-15). ECD calculations were performed to establish the absolute configuration of 6 as 1S, 2S, 3S, and 8aR (Figure S57 in the Supporting Information). On the basis of all of the above evidence, the structure of 6 was established, and it was named cyclizidine G. Compound 7 was given the molecular formula C17H28ClNO4 by HRESIMS ([M + H]+ m/z 346.1781, isotopic peak at 348.1756). Its 1H- and 13C-NMR spectra were almost identical to the corresponding spectra of 6 except for the absence of two saturated methylenes in 7. The COSY, HSQC, and HMBC correlations revealed the replacement of a chlorine atom and a hydroxy group at C-7 and C-8, respectively. The configurations of the double bonds were all determined to be E by NOESY correlations of H-10/H-12, H-12/H-14, and H-13/H-15. The absolute configuration of 7 was established on the basis of a NOESY experiment (Figure 2) and ECD calculations (Figure S76 in the Supporting Information). Accordingly, the structure of 7 was determined to be as shown, and it was named cyclizidine
Figure 4. Experimental ECD spectra for 2 and 3 and calculated ECD spectra of 2. The calculated ECD spectra for 3 were similar to those for 2 (Supporting Information).
with NMR data further revealed a molecular formula of C17H26ClNO3. The 1H- and 13C-NMR data of 3 (Tables 1 and 2) were almost same as those of 2 except for the H-7 (3: δH 3.71, 2: δH 3.15) and C-7 (3: δC 65.9, 2: δC 73.7) chemical shifts, indicating that 2 and 3 have the same skeleton but differ in the substituents at C-7. This can be further verified from the HMBC correlations from H-8 (δH 3.68, m) to C-7 and C-8a together with the COSY spin system of H-5/H-6/H-7/H-8/H-8a (Figure 1). The absolute configuration of 3, consistent with that of 2, was established on the basis of the NOESY experiment and ECD calculations (Figures 2 and 4). Accordingly, the structure of 3 was named cyclizidine D. The HRESIMS data of compound 4 gave an M+ ion at m/z 272.1649, indicating the molecular formula was C17H22NO2. The 1 H- and 13C-NMR spectra of compound 4 showed a number of similarities to those of 1 except for the absence of a piperidine ring and the presence of a pyridinium ring in 4. These differences indicated that the six-membered piperidine ring in 1 had been oxidized to a pyridinium ring. The proposed structure was further rigorously determined by HMBC correlations (Figure 1), and the absolute configurations of its C-1, C-2, and C-3 were established as S, S, and S by NOESY cross-peaks (H-2/H-10, and H-3/H3-9) and ECD calculations (Figures 2 and 5). Compound 4 was named cyclizidine E. Compound 5 had the molecular formula C17H22NO3, determined on the basis of HRESIMS (M+ m/z 288.1592). 397
DOI: 10.1021/acs.jnatprod.7b01055 J. Nat. Prod. 2018, 81, 394−399
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H. The 1H- and 13C-NMR spectra of 8 were almost imitated those of 7 except for the chemical shifts of C-10 (8: δC 36.3, 7: δC 44.2) and C-17 (8: δC 24.5, 7: δC 17.0) (Table 2). This difference suggested that the Δ11 double bond in 8 had a different configuration than that in 7, which was further supported by the NOESY cross-peaks from H-10 to H-13 and from H-12 to H-14 in 8. The similar ECD spectra between 8 and 7 indicated that the absolute configuration of 8 was 1S, 2S, 3S, 7S, 8S, 8aR (Figure S76 in the Supporting Information). Thus, the structure of 8 was established as a C-11 Z isomer of 7. Compound 8 was named cyclizidine I. All of the isolates were evaluated for cytotoxicity in the PC-3 and HCT-116 cancer-cell lines and for ROCK2-protein-kinaseinhibitory activities (Table 3). Among the test results, compound
province, China, N 19° 95′ E 110° 58′) by using a standard dilution-plating method. The strain was maintained in Gause’s solid medium consisting of (per liter) 20 g of soluble starch, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, 20 g of agar, and 25 g of artificial sea salt (pH = 7.2−7.4) and subcultured monthly. Strain HNA39 was identified with a 16S rDNA sequence analysis performed by TaKaRa (Dalian, China), and its DNA sequence was compared via BLAST (nucleotide sequence comparison) to the GenBank database. The 16S rDNA sequence of strain HNA39 has been deposited in GenBank (accession number CTI0507HNA39). A voucher strain (Streptomyces sp. HNA39) is preserved at the Laboratory of the Institute of Marine Biology, Ocean College, Zhejiang University, China. Large Cultures of Streptomyces sp. HNA39. Colonies of strain HNA39 growing on Gause’s agar plate were inoculated into 500 mL Erlenmeyer flasks containing 250 mL of Gause’s liquid medium and then incubated at 28 °C for 8 days on a rotary shaker (180 rpm). A total of 100 Erlenmeyer-flask cultures were prepared for this study. Extraction and Isolation. The whole broths were extracted with ethyl acetate three times. The resulting EtOAc fraction was vacuum dried and yielded an organic extract (3 g), which was subjected to silica-gel CC (60 g) and eluted with CH2Cl2/MeOH (50:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1; 500 mL each) to give seven fractions (A−G). Fraction A (300 mg) was separated by a Sephadex LH-20 (MeOH) and further purified by semipreparative HPLC (MeCN/H2O, 10−100%, 40 min, flow rate of 10 mL/min) to give 2 (2.1 mg, tR = 27 min) and 9 (6.3 mg, tR = 35 min). Fraction C (150 mg) was chromatographed over a Sephadex LH-20, with MeOH as the eluent, and purified by semipreparative HPLC (MeCN/H2O, 10−60%, 40 min, flow rate of 10 mL/min) to provide compound 7 (3.3 mg, tR = 21 min), compound 8 (1.8 mg, tR = 32 min), and compound 3 (2.6 mg, tR = 36 min). Fraction D (100 mg) was also fractionated by a Sephadex LH-20, with MeOH as the eluent, to give three subfractions, D1−D3. Subfraction D1 was directly purified by semipreparative HPLC (25% MeCN and 0.05% TFA in H2O, flow rate of 10 mL/min) to give 5 (2.3 mg, tR = 12 min). Subfraction D2 was subjected to semipreparative HPLC (10− 50% MeCN/H2O, 40 min, flow rate of 10 mL/min) to yield 6 (3.4 mg, tR = 16 min) and 1 (1.8 mg, tR = 17 min). Fraction F (42 mg) was purified by semipreparative HPLC (10−100% MeCN and 0.05% TFA in H2O, 40 min, flow rate of 10 mL/min) to give compound 4 (8.6 mg, tR = 25 min). Cyclizidine B (1). Colorless oil; [α]20D +22 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 247 (2.96) nm; ECD (c 1.1 × 10−3 M, MeOH), λmax (Δε) 200 (−0.41), 251 (0.56) nm; IR νmax: 3421, 1683, 1200, 1141 cm−1; 1H- and 13C-NMR data, Tables 1 and 2; HRESIMS m/z 278.2118 [M + H]+ (calcd for C17H28NO2, 278.2120). Cyclizidine C (2). Amorphous powder; [α]20D +20 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 249 (2.98) nm; ECD (c 1.3 × 10−3 M, MeOH), λmax (Δε) 233 (−1.2), 256 (1.58) nm; IR νmax: 3396, 1680, 1198, 1138, 1062 cm−1; 1H- and 13C-NMR data, Tables 1 and 2; HRESIMS m/z 310.2015 [M + H]+ (calcd for C17H28NO4, 310.2018). Cyclizidine D (3). Amorphous powder; [α]20D +19 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 243 (4.01) nm; ECD (c 1.5 × 10−3 M, MeOH), λmax (Δε) 228 (−0.95), 256 (2.13) nm; IR νmax: 3426, 1684, 1204, 1140 cm−1; 1H- and 13C-NMR data, Tables 1 and 2; HRESIMS m/z 328.1673, 330.1648 [M + H]+ (calcd for C17H27ClNO3, 328.1679, 330.1650).
Table 3. Cytotoxicity and ROCK2-Protein-Kinase-Inhibitory Activities of 1−9 IC50 (μM) compounds
PC-3
HCT-116
ROCK2
1 2 3 4 5 6 7 8 9 staurosporine
>40 0.52 ± 0.03 33 ± 1 >40 >40 >40 17 ± 1 >40 23 ± 1 0.017 ± 0.004
>40 8.3 ± 0.1 40 ± 1 >40 >40 >40 >40 >40 28 ± 1 0.055 ± 0.001
>100 7.0 ± 0.8 >100 >100 27 ± 1 >100 42 ± 3 39 ± 1 >100 0.009 ± 0.001
2 exhibited significant cytotoxicity against PC3 and HCT116 cells with IC50 values of 0.52 ± 0.03 and 8.3 ± 0.1 μM, respectively. In addition, the ROCK2-protein-kinase-inhibitory assay showed that compounds 2, 5, 7, and 8 exhibited moderate inhibitory activities on ROCK2 with IC50 values of 7.0 ± 0.8, 27 ± 1, 42 ± 3, 39 ± 1 μM, respectively.
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EXPERIMENTAL SECTION General Experimental Procedures. Optical rotations and UV spectra were obtained by using a JASCO DIP-370 digital polarimeter (Easton, MD) and a SHIMADZU UV-1800 spectrophotometer (Kyoto, Japan), respectively. ECD spectra were collected on a JASCO J-1500-150ST. IR spectra were recorded on a Bruker Vector 22 spectrophotometer (Billerica, MA). 1D- and 2D-NMR spectra were measured on a Bruker AV III instrument using TMS as an internal standard. Highresolution mass data were recorded on an Agilent 1260 HPLC6230 TOF tandem spectrometer (Santa Clara, CA). The extracts were separated by silica-gel column chromatography (CC, 100− 200 or 300−400 mesh, Qingdao Haiyang Chemical Company, Qingdao, China) and Sephadex LH-20 (Amersham Pharmacia Biotech, Little Chalfont, U.K.) column chromatography and further purified with preparative HPLC in a Beijing Chuangxintongheng LC3000 system (Beijing, China) equipped with an Agilent Pursuit C-18 column (10 μm, 21.2 × 250 mm). Fractions were monitored by TLC under UV light, and spots were visualized by heating silica-gel plates sprayed with 8−10% H2SO4 in EtOH. The artificial sea salt was a commercial product (Zhejiang Province Salt Industry Group Company, Ltd., Zhejiang province, China). Strain Isolation and Identification. Strain HNA39 was isolated from marine sediments of Hainan Island (Hainan 398
DOI: 10.1021/acs.jnatprod.7b01055 J. Nat. Prod. 2018, 81, 394−399
Journal of Natural Products
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Cyclizidine E (4). Yellowish oil; [α]20D +20 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 252 (3.91) nm; ECD (c 7.3 × 10−4 M, MeOH), λmax (Δε) 259 (17.1) nm; IR νmax: 3295, 1682, 1197, 1136, 962, 721 cm−1; 1H- and 13C-NMR data, Tables 1 and 2; HRESIMS m/z 272.1649 [M]+ (calcd for C 17 H 22 NO2 , 272.1645). Cyclizidine F (5). Yellowish oil; [α]20D +20 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 252 (3.41) nm; ECD (c 1.7 × 10−3 M, MeOH), λmax (Δε) 262 (15.6) nm; IR νmax: 3425, 1680, 1204, 1143 cm−1; 1H- and 13C-NMR data, Tables 1 and 2; HRESIMS m/z 288.1592 [M]+ (calcd for C17H22NO3, 288.1594). Cyclizidine G (6). Colorless oil; [α]20D +22 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 237 (3.86) nm; ECD (c 1.4 × 10−3 M, MeOH), λmax (Δε) 204 (−3.28), 231 (1.22) nm; IR νmax: 3424, 1680, 1204, 1143 cm−1; 1H- and 13C-NMR data, Tables 1 and 2; HRESIMS m/z 296.2227 [M + H]+ (calcd for C17H30NO3 296.2226). Cyclizidine H (7). Colorless oil; [α]20D +21 (c 0.7, MeOH); UV (MeOH) λmax (log ε) 240 (3.93) nm; ECD (c 5.8 × 10−4 M, MeOH), λmax (Δε) 200 (−3.85), 230 (1.76) nm; IR νmax: 3441, 1683, 1201, 1140, 1054 cm−1; 1H- and 13C-NMR data, Tables 1 and 2; HRESIMS m/z 346.1781, 348.1756 [M + H]+ (calcd for C17H29ClNO4, 346.1785, 348.1756). Cyclizidine I (8). Colorless oil; [α]20D +23 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 243 (4.02) nm; ECD (c 8.7 × 10−4 M, MeOH), λmax (Δε) 200 (−5.7), 229 (1.92) nm; IR νmax: 3248, 1683, 1201, 1139, 1050 cm−1; 1H- and 13C-NMR data, Tables 1 and 2; HRESIMS m/z 368.1596, 370.1569 [M + Na]+ (calcd for C17H28ClNNaO4, 368.1605, 370.1575). (+)-ent-Cyclizidine (9). Colorless needles (EtOAc); [α]20D +60 (c 1.0, MeOH), lit. value:5 [α]D +36 (c 0.5, MeOH); ECD (c 1.7 × 10−3 M, MeOH), λmax (Δε) 229 (−1.55), 254 (1.55) nm; HRESIMS m/z 292.1912 [M + H]+ (calcd for C17H25NO3, 292.1913). Cytotoxicity Assay. The SRB assay, performed as previously described,14 was used for assessing the cytotoxicity of compounds 1−9 against PC3 and HCT-116 human cancer cells. Staurosporine was simultaneously tested as a positive control. ROCK2 Assay. The ROCK2-protein-kinase-inhibitory activities of the compounds were monitored using the HTRF KinEASE Kit protocol. Briefly, a 10 μL reaction mixture consisting of 4 μL of one of the compounds or buffer, 2 μL of the substrate, 2 μL of the enzyme, and 2 μL of ATP was incubated for 30 min at 37 °C, which was followed by the addition of 5 μL of SA-XL665 and 5 μL of STK-antibody−cryptate, and the mixture was incubated again at room temperature for 60 min. The HTRF signal was measured by a microplate reader, SPARK 10 M (Tecan, Männedorf Switzerland). Staurosporine was used as a positive control. All of the reactions were performed in triplicate.
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Article
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Zhong-Jun Ma: 0000-0002-5825-5095 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the Zhejiang University Cross Researching Fund under grant JCZZ-2013021. REFERENCES
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01055. 1D- and 2D-NMR, HRESIMS, IR, and UV spectra of the new compounds and ECD spectra for compounds 3 and 5−9 (PDF) 399
DOI: 10.1021/acs.jnatprod.7b01055 J. Nat. Prod. 2018, 81, 394−399
