Imidazole Alkaloids and Their Zinc Complexes from the Calcareous

Five new imidazole derivatives (1–5), together with eight related known alkaloids, were isolated from a calcareous marine sponge, Leucetta chagosens...
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Imidazole Alkaloids and Their Zinc Complexes from the Calcareous Marine Sponge Leucetta chagosensis Wei-Zhuo Tang,†,‡ Zhong-Zhen Yang,† Wei Wu,† Jie Tang,† Fan Sun,† Shu-Ping Wang,† Chun-Wei Cheng,† Fan Yang,*,† and Hou-Wen Lin*,† †

Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, People’s Republic of China ‡ College of Biological and Environmental Engineering, Changsha University, Changsha 410022, People’s Republic of China S Supporting Information *

ABSTRACT: Five new imidazole derivatives (1−5), together with eight related known alkaloids, were isolated from a calcareous marine sponge, Leucetta chagosensis, collected from the South China Sea. Their structures were fully characterized by spectroscopic methods. Structurally, 1 possesses an unusual skeleton featuring imidazole and oxazolone rings linked via a nitrogen atom, whereas 2 bears an intriguing guanylurea-substituted imidazole ring. Compounds 4 and 5 were identified as zinc complexes; they represent the metal complex analogues of naamidine J (6) and pyronaamidine (7), respectively. Among the isolated compounds, 2 and 5 showed significant inhibitory activities toward the LPS-induced production of IL-6 in the human acute monocytic leukemia cell line THP-1, and 7 displayed cytotoxicity against MCF-7, PC9, A549, and breast cancer stem cells (MCF-7-Oct4-GFP) with IC50 values of 5.2, 5.6, 7.8, and 10 μM, respectively.

C

alcareous sponges are prolific sources of 2-aminoimidazole alkaloids with a broad range of biological activities.1 To date, more than 60 naturally occurring 2-aminoimidazole alkaloids have been isolated from Calcarea sponges belonging to the genera Leucetta and Clathrina.2 Leucetta chagosensis has been thoroughly investigated and has yielded a number of imidazole alkaloids, including naamines,3−6 naamidines,4,7,8 isonaamine,9 and kealiinines.10 These alkaloids are characterized by a modified imidazole central core in conjugation with one or two functionalized benzyl moieties. Often these alkaloids also contain an amino- or imino-substituted hydantoin ring.11 Organometallic dimers of these alkaloids, including zinc complexes of clathridine,12 naamidines,13 and isonaamidines,14 along with spiro-linked imidazole derivatives, have also been isolated from several Leucetta sponges.15−18 The biological activities reported for alkaloids from Calcarea sponges include antifungal,14 antimicrobial,18 leukotriene B4 receptor antagonistic,19 and nitric oxide synthase inhibition activity6 as well as cancer cell toxicity.5,9,20 The discovery of these 2-aminoimidazole derivatives has received considerable attention from both biologists and chemists.21 Our chemical investigation of the sponge L. chagosensis collected from the South China Sea led to the isolation of five new imidazole alkaloids (1−5) and eight known imidazole derivatives. Herein, the isolation, structure elucidation, and evaluation of their biological activities are reported. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Leuchagodine A (1) was obtained as a white, amorphous solid and had a molecular formula of C16H15N5O4 based on its HRESIMS data, implying 12 degrees of unsaturation. Its IR spectrum displayed characteristic absorption bands of NH (3361 cm−1) and carbonyl (1690 cm−1) groups. The 1H NMR spectrum of 1 (Table 1) exhibited a set of coupled aromatic protons for a 1,3,4trisubstituted benzene (ring B) at δH 6.89 (1H, d, J = 1.0 Hz), Received: November 30, 2017

A

DOI: 10.1021/acs.jnatprod.7b01006 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data of 1−3 in DMSO-d6 1 position 2 2′-NH 4 5 6 7 8 9 10 11 12 13 14 16 18 20 22 15-NH 21-NH 15-OMe 16-OMe 17-OMe 1′ 2′ 3′ 4′ 5′ 6′

δC, type

2 δH (J in Hz)

δC, type

3 δH (J in Hz)

δC, type

δH (J in Hz)

149.4, C

140.9, C

156.9, C

124.4, C 119.4, CH 30.3, CH2 131.6, C 109.3, CH 147.3, C 145.8, C 108.2, CH 121.9, CH 100.8, CH2 31.6, CH3 151.8, C 162.6, C 161.2, C 26.2, CH3

136.7, C 115.6, CH 33.9, CH2 134.4, C 109.1, CH 147.0, C 145.2, C 107.9, CH 121.3, CH 100.6, CH2 32.1, CH3 166.7, C 158.1, C 27.4, CH3

122.6, C 129.6, C 109.5, C 137.6, C 154.0, C 102.8, CH 125.9, CH 152.2, C 122.1, C 183.2, C 25.4, CH3

8.14, s 7.26, s 4.34, s 6.89, d (1.0)

6.85, d (7.9) 6.77, dd (7.9, 1.0) 5.97, s 3.66, s

6.61, s 3.62, s 6.78, bs

6.79, d (8.0) 6.69, dd (8.0,1.2) 5.94, s 3.34, s

6.40, d (8.4) 6.70, d (8.4)

2.57, s

2.67, brs

2.75, d (4.8) 8.43, s 8.70, q (4.8) 54.9, CH3 59.8, CH3 55.5, CH3 127.5, C 130.8, CH 113.0, CH 157.9, C 113.0, CH 130.8, CH

3.68, s 3.65, s 3.71, s 6.94, d (8.0) 6.75, d (8.0) 6.75, d (8.0) 6.94, d (8.0)

signal at δC 161.2 and the strong IR band at 1690 cm−1. The mass spectrometric fragments at m/z 283 and 255 indicated the possibility of a CN at the C-16 position and the CO at C-20 (Figure 2). The location of the aminomethyl unit was verified by the COSY correlation between NH-21 and H3-22 (δH 2.75) as well as the HMBC cross-peaks of NH-21 to C-18 and H3-22 to C-18. Structurally, 1 possesses an unusual skeleton featuring imidazole and oxazolone cores connected via a nitrogen atom. This structural feature has not been previously observed for imidazole alkaloids; therefore, 1 was named leuchagodine A. Leuchagodine B (2) was isolated as a yellowish-brown, amorphous solid. Its molecular formula was determined to be C15H18N6O3 with 10 degrees of unsaturation by the HRESIMS ion at m/z 331.1522 [M + H]+. The IR spectrum showed absorption bands at 3345, 3273, 3188, and 1626 cm−1, which can be attributed to amine and carbonyl functionalities. The signals observed in its 1H and 13C NMR spectra (Table 1) for the CH2-6 to CH2-13 fragment were very similar to those of compound 1. An olefinic proton at δH 6.61 (1H, s) and two methyl groups at δH 3.34 (3H, s) and 2.67 (3H, brs) were also detected. The 13C NMR spectrum contained resonances for a carbonyl group at δC 166.7 and an imine group at δC 158.1. Characteristic 13C NMR signals of a 2-aminoimidazole moiety were observed at δC 140.9 (C-2), 136.7 (C-4), and 115.6 (C-5), and a characteristic 1H NMR proton was observed at δH 6.61 (s, H-5).9 HMBC correlations from H3-14 to C-2 and C-5 suggested that the N-CH3 unit was located at N-1 (Figure 1). Considering the 13C chemical shifts for this moiety in 1 and 2, the substituent attached to NH-15 in 2 was structurally distinct from that found in 1. The remaining C3H7N4O and two degrees of unsaturation were

6.85 (1H, d, J = 7.9 Hz), and 6.77 (1H, dd, J = 7.9, 1.0 Hz), an isolated olefinic proton at δH 7.26 (1H, s), two methylene singlets at δH 5.97 (2H, s) and 4.34 (2H, s), and two methyl groups at δH 3.66 (3H, s) and 2.75 (3H, d, J = 4.8 Hz). Additionally, one exchangeable proton signal was observed at δH 8.70 and was assigned as an NH proton. Analysis of 13C, DEPT, and HSQC NMR spectra of 1 confirmed 16 carbons, which were attributed to one carbonyl carbon (δC 161.2), seven olefinic nonprotonated carbons (δC 162.6, 151.8, 149.4, 124.4, 131.6, 147.3, and 145.8), four olefinic methines (δC 119.4, 109.3, 108.2, and 121.9), one dioxygenated methylene (δC 100.8), one methylene (δC 30.3), and two methyl groups (δC 31.6 and 26.2). The aforementioned spectroscopic data accounted for eight out of the 12 degrees of unsaturation, suggesting that 1 was tetracyclic. The presence of a 2-aminoimidazole core (ring A) was evident from the chemical shifts of C-2, C-4, and C-5 at δC 149.4, 124.4, and 119.4, respectively.5 The HMBC correlations from H3-14 to C-2 and C-5 positioned the CH3-14 methyl group at N-1 (Figure 1). The connectivity of rings A and B through the C-6 methylene group was assigned based on the HMBC cross-peaks of H2-6 to C-4, C-7 (δC 131.6), C-8 (δC 109.3), and C-12 (δC 121.9). Meanwhile, the HMBC correlations from dioxygenated methylene protons H2-13 to C-9 (δC 147.3) and C-10 (δC 145.8) constructed subunit C. Based on the molecular formula, the remaining four degrees of unsaturation combined with the three sp2 carbons still unaccounted for (δC 162.6, 161.2, and 151.8) could be attributed to an oxazolone moiety (ring D).22 The mass spectrometric fragmentation pattern observed by tandem mass spectrometry further supported this substructure (Figures 2 and S1). The presence of a carbonyl functionality was confirmed by the carbon B

DOI: 10.1021/acs.jnatprod.7b01006 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Key COSY, HMBC, and NOESY correlations of 1−5.

Figure 2. ESIMS/MS fragment ions (m/z) of 1 and 2.

three methoxy carbons, and one methyl group (Table 1). The 1H and 13C NMR spectra of 3 were similar to those recorded for kealiinine A,10 except for the presence of a para-dimethoxy tetrasubstituted aromatic ring and an additional carbonyl group. The para-dimethoxy aromatic ring (ring C) in 3 was readily indicated by HMBC correlations of H-9/C-7 and C-11, H3-16/ C-7 and C-8, and H3-17/C-11. The NOESY correlations of H-14/H-2′ and H3-15/H-3′and H-5′ along with HMBC correlations of H-3′/C-1′ and H-6′/C-4′ confirmed that the 1,4-disubstituted aromatic ring (D) is bound at C-6 (Figure 1). Considering the molecular formula and the remaining three degrees of unsaturation, the two unassigned nonprotonated carbons at δC 183.2 and 109.5 and one unassigned oxygen indicated the presence of another ring (ring B) with a carbonyl group incorporated at C-13. The shift of the carbonyl (δC 183.2) in ring B is consistent with that of a reported alkaloid analogue, 2-deoxy2-aminokealiiquinone (δC 182.0).5 Accordingly, the structure of 3 was determined as shown. Bis(naamidine J)zinc (4) was obtained as a yellowish, amorphous solid. Its ESIMS spectrum showed a molecular ion cluster [M + H]+ at m/z 1017, 1019, and 1021 (relative intensities 100, 66, and 33), which is clearly indicative of the presence of one zinc atom in the molecule.24 This deduction was confirmed by the HRESIMS spectrum obtained for 4, indicating the molecular formula of C50H52N10O10Zn. The 1H NMR spectrum of 4 (Table 2) exhibited aromatic proton doublets at δH 6.66 (2H, d, J = 8.6 Hz) and 6.59 (2H, d, J = 8.6 Hz) that correspond to a paradisubstituted aromatic ring (ring C) as well as three aromatic

accounted for by connecting a methyl guanylurea moiety at C-2 in one of two possible arrangements, A and B (Figure 2). These two substructures differ only in the connection positions of the guanidine and urea groups. The observed mass spectrometric fragment at m/z 258 (Figure 2) is more consistent with substructure A, in which the guanidine functionality is attached at C-16. Furthermore, the shift of the carbon at δC 158.1 also supported the presence of a terminal guanidine group in 2.23 Therefore, C-16 and C-18 can be assigned to the signals at δC 166.7 and 158.1, respectively. Moreover, the HMBC correlation from H3-20 (δH 2.67) to C-18 indicated that the isolated methyl group was attached to N-19 (Figure 1). Consequently, the structure of leuchagodine B (2) was elucidated as shown. The third compound characterized, kealiinine D (3), was isolated as a yellowish-brown, amorphous solid and was found to have a molecular formula of C21H19N3O4 based on its HRESIMS ion peak at m/z 378.1457 [M + H]+, which indicates 14 degrees of unsaturation. Its IR absorption band at 1659 cm−1 revealed the presence of a carbonyl group. The 1H NMR spectrum of 3 (Table 1) displayed six doublet aromatic signals at δH 6.40 (1H, d, J = 8.4 Hz), 6.70 (1H, d, J = 8.4 Hz), 6.94 (2H, d, J = 8.0 Hz), and 6.75 (2H, d, J = 8.0 Hz), three oxygenated singlets at δH 3.68 (3H), 3.65 (3H), and 3.71 (3H), and one aminomethyl singlet at δH 2.57 (3H). Moreover, an exchangeable proton indicative of an NH group was observed at δH 8.14. All 21 carbon resonances were fully resolved in the 13C NMR spectrum and were classified by DEPT and HSQC spectra as one carbonyl carbon (δC 183.2), 10 olefinic nonprotonated carbons, six olefinic methine carbons, C

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Table 2. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data of 4 and 5a 4 position

δC, type

2 4 5 6

148.2, C 132.7, C 127.2, C 29.9, CH2

7

31.1, CH2

9 11 13 14 15 16-OMe 17-OMe 18-OMe 1′ 2′ 3′ 4′ 5′ 6′ 2′-OH 1″ 2″ 3″ 4″ 5″ 6″

154.4, C 161.3, C 164.8, C 30.5, CH3 24.5, CH3 56.1, CH3 56.0, CH3 55.2, CH3 129.2, C 111.3, CH 148.2, C 149.5, C 111.6, CH 120.1, CH 130.0, C 128.8, CH 113.6, CH 158.2, C 113.6, CH 128.8, CH

spin coupling systems in ring B (Table 2). Compound 5 possesses a 1,2,3,4-tetrasubstituted benzene ring based on the two coupled doublets observed at δH 6.40 (1H, d, J = 8.6 Hz) and 6.51 (1H, d, J = 8.6 Hz) and the HMBC correlations from H-5′ to C-1′, C-3′, C-4′, and C-6′ (Figure 1). Moreover, 5 contains 32 more mass units than 4, indicating the presence of two additional oxygen atoms in 5. Thus, the molecular ligand of this zinc complex was determined to be pyronaamidine (7),26 which was coisolated from this sponge. The organometallic backbones of 4 and 5 were eventually established by analogy to the X-ray crystal study of Zn-clathridine, and assignments were made based on the assumption that Zn binds to both the N-3 and N-10 positions in the above structures (Figure S2). From the above data, the structure of compound 5 was determined as shown. Interestingly, a number of 2-aminoimidazoles have been isolated as Zn2+ coordination complexes from calcareous sponges and spongivorous nudibranchs over the past decades.12−14 Zinc is an extremely critical constituent of metalloenzymes, but the role of the Zn trapped in these imidazole alkaloids is unknown.13 Generally, structures with macrocyclic cavities or polar functional groups are ideal candidates for metal interactions.27 Zinc also prefers a tetrahedral arrangement of nitrogen-binding sites when coordinating to nitrogen-containing natural products.28 Because numerous similar imidazole alkaloids have been isolated from various calcareous sponges, some of them may also be coordinated to zinc, and their potential complexes await further exploration. In addition to new compounds 1−5, the known imidazole analogues naamidine J (6),25 pyronaamidine (7),26 Zn-clathridine,12 clathridine,12 kealiinine B,10 naamine C,5 leucettamine B,19 and leucettamine C29 were also isolated and elucidated by a comparison of their MS and NMR data to those reported in the literature. The inhibitory activities of all new compounds obtained in this study were evaluated against the production of five cytokines in the serum of the human acute monocytic leukemia cell line THP-1 by using the human inflammation cytometric bead array (CBA) assay (Table 3). Of these cytokines, IL-6, TNF-α, and IL-12p70

5 δH (J in Hz)

3.93, d (16.6) 3.72, d (16.6) 3.86, d (17.0) 3.41, d (17.0)

3.62, s 2.95, s 3.85, s 3.73, s 3,72, s 6.54, d (1.8)

6.77, d (8.2) 6.61, dd (8.2,1.8)

6.66, d (8.6) 6.59, d (8.6) 6.59, d (8.6) 6.66, d (8.6)

δC, type 148.1, C 132.3, C 127.9, C 22.7, CH2 30.3, CH2 153.1, C 160.5, C 163.9, C 29.9, CH3 24.1, CH3 60.2, CH3 55.6, CH3 54.7, CH3 117.5,C 146.7, C 136.3, C 151.7, C 103.2, CH 123.0, CH 130.0, C 128.4, CH 112.9, CH 157.3, C 112.9, CH 128.4, CH

δH (J in Hz)

3.90, d (17.0) 3.87, d (17.0) 3.73, d (16.7) 3.21, d (16.7)

3.62, s 2.78, s 3.63, s 3.71, s 3.61, s

6.40, d (8.6) 6.51, d (8.6) 9.09, s 6.44, d (8.9) 6.46, d (8.9) 6.46, d (8.9) 6.44, d (8.9)

a

The NMR data of 4 were measured in CDCl3, while the 1H and 13C NMR data of 5 were measured in DMSO-d6.

doublets at δH 6.54 (1H, d, J = 1.8 Hz), 6.77 (1H, d, J = 8.2 Hz), and 6.61 (1H, dd, J = 8.2, 1.8 Hz) that are characteristic of a 1,3,4trisubstituted benzene ring (ring B). A detailed comparison of the NMR data of 4 to those of naamidine J (6)25 intimated their similar structures. However, the benzylic regions of the 1H NMR spectra of 4 and 6 were significantly different, which provided an essential clue as to where the imidazole might be coordinated to a metal.12 The benzylic protons in 6 are magnetically equivalent within each pair, and they appear as broad singlets at δH 3.90 and 3.89 (Table S1). In contrast, the benzylic protons in 4 are diastereotopic due to the chelation of Zn2+ and thus appear as two sets of AB doublets at δH 3.93 (1H, d, J = 16.6 Hz), 3.72 (1H, d, J = 16.6 Hz), 3.86 (1H, d, J = 17.0 Hz), and 3.41 (1H, d, J = 17.0 Hz). Furthermore, compared to those of 6 (Table S1), the chemical shifts of C-2, C-9, C-11, and C-13 of 4 were deshielded to δC 148.2 (Δδ +1.7), 154.4 (Δδ +9.8), 161.3 (Δδ +5.8), and 164.8 (Δδ +2.5), respectively, which is also consistent with the presence of a tetrahedral Zn ion.13 Our structural assignment of 4 was further supported by the corresponding HMBC and NOESY spectra (Figure 1). Therefore, the structure of 4 was established as a zinc complex of naamidine J (6). Bis(pyronaamidine)zinc (5) was isolated as a yellowish, amorphous solid and was found to be another Zn complex based on the cluster of ESIMS ions [M + H]+ at m/z 1049, 1051, and 1053. Its molecular formula was determined to be C50H52N10O12Zn based on the HRESIMS data. The 1H and 13C chemical shifts of 5 and 4 were quite similar, but there were minor differences in the

Table 3. Effects of Compounds 1−5 on Cytokine Levels (pg/mL, mean ± SD) in the Serum of THP-1 Cells

a

group

IL-6

TNF-α

control LPS 1+LPS 2+LPS 3+LPS 4+LPS 5+LPS pomalidomidea corylifol Aa

3.7 ± 3.0 6100 ± 200b 7600 ± 500 5000 ± 400c 5400 ± 400 6200 ± 400 4300 ± 300d

610 ± 43 9900 ± 300b 9900 ± 500 7800 ± 400c 9700 ± 400 9900 ± 400 9600 ± 400 7500 ± 500

2700 ± 300

b

Positive control. P < 0.01, compared to that of the control group. P < 0.05, compared to that of the LPS-treated group. dP < 0.01, compared to that of the LPS-treated group. All the cells were treated with 10 μM of test compounds. The compounds had no effect on IL-10, MCP-1, and IL-12p70 levels. c

are classified as proinflammatory cytokines, while IL-10 is categorized as a Th2 cytokine, and MCP-1 is a chemotactic cytokine. Notably, lipopolysaccharide (LPS)-treated samples showed a significant increase in the secretion of TNF-α and IL-6 (P < 0.01). THP-1 cells pretreated with 2 and 5 at 10 μM showed decreases in the LPS-induced production of IL-6 (P < 0.05 for 2 and P < 0.01 for 5). D

DOI: 10.1021/acs.jnatprod.7b01006 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. Apoptosis increased after treatment with 7 in MCF-7 cells. Cells in the Q2 and Q4 quadrants were considered to be the late apoptotic and early apoptotic stages, respectively. Cells in the Q2 + Q4 quadrants were considered to be apoptotic. The data are presented as the mean ± SEM, n = 3. **P < 0.01, ***P < 0.001. Animal Material. Specimens of Leucetta chagosensis were collected off the Yongxing Islands in the South China Sea in June 2015. The sponge was identified by Prof. Jin-He Li (Institute of Oceanology, CAS) and was frozen shortly after collection and transported frozen to the laboratory. A voucher sample (No. XD150618) was deposited in the Laboratory of Marine Drugs, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, China. Extraction and Isolation. The freeze-dried sponges (3.2 kg, wet weight) were cut into pieces and exhaustively extracted with 95% EtOH (3 × 10 L) at room temperature. After extraction, the solvent was removed and the resulting extract was suspended in H2O and partitioned sequentially with EtOAc and n-BuOH to afford the EtOAc- and n-BuOH-soluble extracts. The EtOAc-soluble extract (25.2 g) was partitioned between 90% aqueous MeOH and n-hexane, and the n-hexane layer was collected and dried in vacuo to yield the n-hexanesoluble extract (13.4 g). The 90% aqueous MeOH layer was then diluted to 60% aqueous MeOH and extracted with CH2Cl2 to afford the CH2Cl2-soluble extract (10.2 g). The CH2Cl2-soluble extract was subjected to vacuum liquid chromatography on silica gel using a stepwise gradient of CH2Cl2/ MeOH (100:0−0:1) to afford fractions A−J. Fractions H and I were of interest due to their characteristic UV absorption and because they have molecular formulas different by HRESIMS from those of previously reported imidazoles. Fraction H (3.4 g) was separated by CC on ODS (50 μm), eluting with MeOH/H2O (1:9−1:0) to give subfractions H1−H13. Among these subfractions, subfraction H7 was further purified by reversed-phase HPLC eluting with 40% MeCN and monitoring at detection wavelengths of 230 and 360 nm to afford leucettamine B (2.0 mL/min, tR = 14.3 min, 1.6 mg) and leucettamine C (2.0 mL/min, tR = 18.5 min, 2.1 mg). H8 was purified by reversed-phase HPLC eluting with 60% MeCN and detecting at 230 and 360 nm to yield 7 (2.0 mL/min, tR = 16.5 min, 60.2 mg) and 6 (2.0 mL/min, tR = 19.5 min, 43.1 mg). Subfraction H10 was further separated by reversed-phase HPLC to provide clathridine (2.0 mL/min, tR = 16.5 min, 2.3 mg) using 45% MeCN as the eluent. Semipreparative HPLC separation of H11 eluting with 47% MeCN and detecting at 230 and 360 nm resulted in the isolation of Zn-clathridine (3.0 mL/min, tR = 17.5 min, 15.0 mg), 4 (3.0 mL/min, tR = 31.0 min, 2.0 mg), and 5 (3.0 mL/min, tR = 33.5 min, 3.0 mg). Compound 2 (2.0 mL/min, tR = 36.0 min, 1.8 mg) was purified from subfraction H13 by reversed-phase HPLC using 25% MeCN as the eluent. Fraction I (1.9 g) was further separated on an ODS (50 μm) column using a stepwise gradient elution with MeOH/H2O (1:9−1:0) to yield fractions I1−I9. Fraction I6 was separated by CC over silica gel with gradient elution using n-hexane/EtOAc (5:1 to 1:1, v/v) to provide 3 (2.3 mg). Fraction I9 was further isolated by reversed-phase HPLC eluting with 30% MeCN and detecting at 230 and 360 nm to afford 1 (2.0 mL/min, tR = 19.0 min, 2.1 mg), naamine C (2.0 mL/min, tR = 21.5 min, 1.8 mg), and kealiinine B (2.0 mL/min, tR = 42.0 min, 3.4 mg). Leuchagodine A (1): white, amorphous solid; UV (MeOH) λmax (log ε) 322 (3.74), 291 (3.71), 229 (4.04) nm; IR (KBr) νmax 3361, 3092, 2958, 2924, 2854, 1690, 1611, 1582, 1512, 1467, 1413, 1378, 1317, 1245, 1188, 1099, 1035, 924, 862, 791, 744, 716, 689, 578 cm−1;

Of the tested compounds, 5 displayed the highest inhibitory activity, causing a 29% decrease in the IL-6 level compared to that of the LPS-treated group, while 2 caused a decrease in the LPS-induced production of TNF-α (P < 0.05). The cytotoxicities of all compounds were tested against three human cancer cell lines, namely, the A549, PC9, and MCF-7 cell lines. Unfortunately, only the known compound pyronaamidine exhibited notable inhibitory effects on the growth of the A549, PC9, and MCF-7 cell lines with IC50 values of 7.8, 5.6, and 5.2 μM, respectively. All other compounds were inactive (IC50 > 20 μM). Pyronaamidine was previously identified as being cytotoxic to KB cells.26 In this work, pyronaamidine displayed additional inhibitory effects on the growth of several cancer cells, suggesting it possessed broad spectrum cytotoxicity. Moreover, 7 showed significant cytotoxicity against the test cells, while 6 showed no obvious cytotoxicity at the tested concentration levels, indicating that the 2′-hydroxy substituent may significantly impact cytotoxicity. To further determine the effect of 7 on the apoptosis of MCF-7 cells, annexin V/propidium iodide (PI) staining was performed to quantitatively determine the apoptotic cell percentage by flow cytometry. As shown in Figure 3, after treatment with 0−10 μM 7 for 24 h, the percentages of both early and late apoptotic cells increased relative to those of the untreated control in a dose-dependent manner. Cancer stem cells (CSCs) have been proposed as the cause of tumor metastasis and drug resistance, and therapeutic agents with CSC-specific toxicity are of great interest.30,31 All isolates were also evaluated for their in vitro CSC-specific activities, and 7 exhibited modest inhibitory activity against breast cancer stem cells (MCF-7-Oct4-GFP), with an IC50 value of 10 μM. An investigation of the possible targeting mechanism of 7 using MCF-7-Oct4-GFP cells is ongoing.

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EXPERIMENTAL SECTION

General Experimental Procedures. The UV spectra were obtained on a Hitachi U-3010 spectrophotometer, and the IR (KBr) spectra were recorded with a Jasco FTIR-400 spectrophotometer. NMR data were collected at room temperature on a Bruker Avance NMR spectrometer at 600 MHz for 1H NMR and 150 MHz for 13C NMR with tetramethylsilane as the internal reference. High-resolution mass spectra were acquired with a Waters Xevo G2-XS Q-TOF mass apparatus. Semipreparative HPLC separations were performed on a system composed of a Waters 1525 binary pump equipped with a Waters 2998 photodiode array detector and Waters XBridge C18 reversed-phase columns (250 × 10 mm, 5 μm). Column chromatography (CC) was carried out using silica gel 60 (200−300 mesh; Yantai), Sephadex LH-20 (18−110 μm, Pharmacia Co.), and ODS (50 μm, YMC Co.). Thin-layer chromatography was performed using HSGF 254 plates and visualized by spraying with an anisaldehyde−H2SO4 reagent. E

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1

H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), Table 1; ESIMS m/z 342.1 [M + H]+; HRESIMS m/z 342.1200 [M + H]+ (calcd for C16H16N5O4, 342.1202). Leuchagodine B (2): yellowish-brown, amorphous solid; UV (MeOH) λmax (log ε) 369 (3.02), 276 (4.02), 219 (3.98) nm; IR (KBr) νmax 3345, 3273, 3188, 2924, 1626, 1575, 1501, 1489, 1444, 1403, 1347, 1296, 1246, 1038 cm−1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), Table 1; ESIMS m/z 331.1 [M + H]+; HRESIMS m/z 331.1522 [M + H]+ (calcd for C15H19N6O3, 331.1519). Kealiinine D (3): yellowish-brown, amorphous solid; UV (MeOH) λmax (log ε) 276 (3.13), 230 (3.87) nm; IR (KBr) νmax 3358, 3182, 2921, 2851, 1699, 1659, 1634, 1610, 1558, 1510, 1467, 1419, 1377, 1360, 1304, 1249, 1205, 1180, 1140, 1105, 1028, 668 cm−1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), Table 1; ESIMS m/z 400.3 [M + Na]+; HRESIMS m/z 378.1457 [M + H]+ (calcd for C21H20N3O4, 378.1454). Bis(naamidine J)zinc (4): yellowish, amorphous solid; UV (MeOH) λmax (log ε) 391 (4.45), 277 (3.93), 233 (4.62) nm; IR (KBr) νmax 3387, 2927, 2852, 1769, 1720, 1619, 1507, 1461, 1390, 1354, 1307, 1283, 1246, 1161, 1097, 1034, 978, 897, 816, 769, 727, 649, 612, 566, 522 cm−1; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3), Table 2; ESIMS m/z 1019.3 [M + H]+; HRESIMS m/z 1019.3224 [M + H]+ (calcd for C50H53N10O10Zn, 1019.3206). Bis(pyronaamidine)zinc (5): yellowish, amorphous solid; UV (MeOH) λmax (log ε) 390 (4.48), 276 (4.01), 234 (4.65) nm; IR (KBr) νmax 3363, 2998, 2925, 2850, 1769, 1720, 1663, 1617, 1536, 1507, 1461, 1448, 1390, 1354, 1283, 1245, 1219, 1168, 1097, 1034, 979, 897, 817, 792, 769, 727, 647, 613, 566, 523 cm−1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), Table 2; ESIMS m/z 1051.3 [M + H]+; HRESIMS m/z 1051.3119 [M + H]+ (calcd for C50H53N10O12Zn, 1051.3105). Analysis of Cytokine Levels. The effects of compounds 1−5 on five cytokines in the serum of THP-1 cells were evaluated according to the established protocol.32,33 The cells were inoculated into 96-well plates. After 48 h, the cells were incubated with 10 μM of each tested compound for 2 h and then treated with LPS (0.1 μg/mL) for a further 22 h. The levels of five cytokines in the culture supernatant were measured on a FACSCalibur flow cytometer (BD Biosciences Pharmingen), and the concentrations were determined using FCAP Array software. Cytotoxicity Assay. The A549, PC9, and MCF-7 cell lines were cultured at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 10% nondialyzed fetal bovine serum and 2 mM L-glutamine. The overexpression of Oct4 induces the MCF-7 breast cancer cells to transform into cancer stem-like cells. Oct4-GFP shRNA and nontarget control lentiviral particles were purchased from Shanghai GenePharma Co., Ltd. The RNA interference procedure was performed according to the manufacturer’s instructions. Cell viability was analyzed using cell counting kit-8 (CCK-8) (Dojindo) according to the manufacturer’s instructions. The test compounds were solubilized in DMSO at six different concentrations. Cells were seeded into 96-well microplates at a density of 3000 cells/well and allowed to grow undisturbed for 24 h before treatments with various concentrations of the test compounds. After 72 h of incubation with the test substances, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37 °C for an additional 2 h. Cell viability was assayed by reading the absorbance of each well at 450 nm using a multifunction microplate reader (Scientific Vario, Thermo Scientific). The IC50 values for the positive control paclitaxel against the A549, PC9, MCF-7, and MCF-7-Oct4-GFP cell lines were 3.2 × 10−3, 2.7 × 10−3, 2.4 × 10−3, and 6.5 × 10−3 μM, respectively. Flow Cytometry Analysis. To investigate the apoptotic effect of 7 at concentrations ranging from 0 to 10 μM, MCF-7 cells were seeded in a six-well plate at a density of 1 × 105 cells/well. After 24 h, the cells were collected and analyzed for apoptosis using an Annexin V-FITC apoptosis detection kit (BD Pharmingen) according to the manufacturer’s instructions. Annexin V-FITC positive/PI negative cells were regarded as early apoptotic, whereas annexin V-FITC positive/PI positive cells were regarded as late apoptotic.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01006. Copies of HRESIMS, 1D NMR, 2D NMR, IR, and UV spectra of compounds 1−5 and structures for known compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86-21-68383346. Fax: +86-21-58732594. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Hou-Wen Lin: 0000-0002-7097-0876 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Natural Science Fund of China (Nos. 41506149, 81502936, 41106127, 81072573, 81602981), the Fund of the Science and Technology Commission of Shanghai Municipality (No. 15431900900), and the Beijing Medical Award Foundation (No. YJHYXKYJJ-125). We are also grateful for the financial support of the second-class General Financial Grant from the China Postdoctoral Science Foundation (2015M571573).

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