Isolation of a Cyclic Depsipetide, Aspergillicin F, and Synthesis of

Aug 14, 2015 - Innate immunity is the front line of self-defense against microbial infection. After searching for natural compounds that regulate inna...
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Isolation of a Cyclic Depsipetide, Aspergillicin F, and Synthesis of Aspergillicins with Innate Immune-Modulating Activity Haruhisa Kikuchi,*,† Tsuyoshi Hoshikawa,† Shimpei Fujimura,† Noriaki Sakata,‡ Shoichiro Kurata,† Yasuhiro Katou,† and Yoshiteru Oshima† †

Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai 980-8578, Japan Bioresource Laboratories, MicroBiopharm Japan Co., Ltd. (MBJ), 1808 Nakaizumi, Iwata, Shizuoka 438-0078, Japan

‡

S Supporting Information *

ABSTRACT: Innate immunity is the front line of self-defense against microbial infection. After searching for natural compounds that regulate innate immunity using an ex vivo Drosophila culture system, we identified a new cyclic depsipeptide, aspergillicin F, from the fungus Aspergillus sp., as an innate immune suppressor. The total synthesis and biological evaluation of the aspergillicin family, including aspergillicin F, were performed, revealing that slight structural differences in the side chains of amino acid residues alter innate immunity-regulating activity.

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nnate immunity is the front line of self-defense against microbial infection.1,2 The basic mechanisms of this process, including pathogen recognition and immune response activation, have been evolutionarily conserved.3 In mammals, innate immunity interacts with adaptive immunity and plays a key role in regulating immune response.4 Innate immunity is a good pharmaceutical target for the development of immune regulators to suppress unwanted immune responses, such as septic shock,5 inflammatory diseases,6 and autoimmune diseases.7 The innate immune system also provides targets for the development of agents that stimulate protective immune responses toward some diseases, such as infections with pathogenic organisms and cancer.8 To screen for compounds that target innate immunity, we established an ex vivo culture system based on the Drosophila IMD (IMmunoDeficiency) signaling pathway.9,10 Drosophila is a model organism for genetic and molecular studies of innate immunity because of the striking conservation between the mechanisms that regulate insect immunity and mammalian innate immunity.2,3 Specifically, the TNF-α signaling pathway in humans plays a critical role in the inflammatory response by producing co-stimulatory molecules, cytokines, chemokines, and adhesion molecules through the activation of NF-kB,11 which shares some similarity with the IMD pathway in Drosophila innate immunity. This system has been used to search for natural substances that regulate innate immunity of both humans and Drosophila.12−14 In this paper, we describe the isolation and the elucidation of the structure of a cyclic depsipeptide, aspergillicin F (6) (Figure 1), from the fungus Aspergillus sp. and the synthesis of aspergillicin A−F (1−6), which show innate immunity-modulating activity. © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Structures of aspergillicin A−F (1−6).

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RESULTS AND DISCUSSION A culture broth (2 L) of Aspergillus sp. f19703, isolated by MicroBiopharm Japan Co., Ltd., was extracted with butanol to yield an extract (20.5 g). In the ex vivo Drosophila culture system,10 100 μg/mL of the extract suppressed the innate immune response by 32%. Activity-guided fractionation of the extract yielded aspergillicin F (6) (1.3 mg), which suppressed the innate immune response in a concentration-dependent manner, despite only weak cytotoxicity against Drosophila S2 cells (Figure 2). HRFABMS (m/z 755.4344 [M + H]+) and 1H and 13C NMR spectra (Table 1) indicated that the molecular formula of 6 was C39H58O9N6. The seven carbonyl carbon signals (δ 173.2, 171.2, 171.1, 170.3, 170.1, 169.7, 168.4) in the 13C NMR spectrum and the three amide proton signals (δ 8.13 (1H, d, J = Received: April 2, 2015

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Table 1. 1H and 13C NMR Spetcra of Aspergillicin F (6)a 13

1

C

H

N,O-dimethyl-L-Tyr

Figure 2. Innate immune-suppressive activity of aspergillicin F (6). Effects of aspergillicin F (6) on the DAP-type peptidoglycan-mediated activation of Drosophila Dpt-lacZ. The DAP-type peptidoglycanmediated activation of Dpt-lacZ (open triangles) and the Drosophila S2 cell viability (black circles) are presented as the percentage relative to the control (DMSO). The bars indicate the standard errors of three independent measurements. *p < 0.05 vs control (DMSO).

8.8 Hz), 7.03 (1H, d, J = 9.7 Hz), 6.57 (1H, d, J = 9.7 Hz)) in the 1H NMR spectrum suggested that aspergillicin F (6) is a peptide. 1H−1H COSY and HMBC spectra revealed the presence of six amino acid residues, including N,Odimethyltyrosine (diMeTyr), N-acetylthreonine (AcThr), two isoleucines (Ile1, Ile2), and two prolines (Pro1, Pro2) (Figure 3). Moreover, the HMBC correlations for H3-11−C-12, NH23−C-28, H-30−C-34, and NH-35−C-1 revealed a depsipeptide chain, Pro1−diMeTyr−Ile2−AcThr−Ile1. Finally, taking into account the molecular formula, C39H 58O9N 6, the remaining proline residue (Pro2) may be linked to another proline residue (Pro1) and an isoleucine residue (Ile1) to give the planar structure of aspergillicin F (6) as a cyclic hexadepsipeptide. The planar structure of 6 is the same as that of aspergillicin E (5), which has been isolated from Aspergillus carneus.15 Although the signal assignments for all protons in the 1H NMR data of 5 have not been reported, almost all the 1H NMR signals observed for aspergillicin F (6) are identical to those of 5. However, the chemical shift (δ 4.52) of H-35 in the 1H NMR of 6 appears to be different from that of 5 (δ 4.68). Because H35 is the α-proton of L-allo-isoleucine in aspergillicin E (5), aspergillicin F (6) may contain its stereoisomer, e.g., Lisoleucine, D-isoleucine, and D-allo-isoleucine, as Ile2. If L-alloisoleucine is changed to the D-form, it is expected that a conformational change of the macrocycle occurs, and the 1H NMR spectrum of 6 is completely different from that of 5. Thus, Ile2 of aspergillicin F (6) is likely to be an L-isoleucine residue. To confirm the structure of 6, we synthesized both 5 and 6. These compounds can be synthesized from the linear depsipeptide precursors 7e and 7f through macrolactamization between Pro1 and diMeTyr to avoid racemization.16 The precursor 7e/7f was synthesized through a combination of solid- and liquid-phase peptide synthesis (Scheme 1). To start with, the introduction of N-Fmoc-L-proline into 2chlorotrityl chloride resin provided polymer-supported 8. Successive condensation with Fmoc-L-proline, Fmoc-D-alloisoleucine, and Fmoc-L-threonine was carried out in the usual way for solid-phase peptide synthesis to give tetrapeptide 11. Removal of the Fmoc group in 11, followed by N-acetylation, provided 12. After cleavage from the resin under acidic

1 2 3

169.7 62.5 33.5

4 5, 9 6, 8 7 10 11

129.7 130.5 114.4 158.7 55.4 29.4

12 13 14

173.2 55.2 28.5

15

25.4

16

47.4

17 18 19

170.1 58.3 27.9

20

24.8b

21

47.6

22 23 23-NH 24 25 26 27 28 29 29-NH 30 31 32 33

171.2 54.7 37.7 26.1 11.7 14.5 168.4 55.8 71.6 16.7 171.1 23.1

4.97 (1H, dd, J = 11.1, 3.0 Hz) 3.15 (1H, dd, J = 14.9, 3.0 Hz) 2.95 (1H, dd, J = 14.9, 11.1 Hz) 7.02 (1H, d, J = 8.5 Hz) 6.82 (1H, d, J = 8.5 Hz) 3.76 (3H, s) 2.81 (3H, s) L-Pro 4.30 (1H, dd, J = 8.1, 5.1 Hz) 1.01−1.08 (1H, m) 0.84−0.90 (1H, m)d 2.05−2.12 (1H, m) 1.64−1.71 (1H, m) 3.57−3.63 (1H, m) 3.44−3.49 (1H, m) L-Pro 4.48 (1H, dd, J = 8.5, 5.1 Hz) 2.15−2.22 (1H, m) 1.81−1.86 (1H, m) 1.94−2.00 (1H, m) 1.88−1.96 (1H, m)c 3.64−3.71 (1H, m) 3.53−3.60 (1H, m) D-allo-Ile 4.61 (1H, dd, J = 9.7, 8.0 Hz) 6.57 (1H, d, J = 9.7 Hz) 1.57−1.63 (1H, m)g 1.33−1.40 (1H, m)e 1.07−1.13 (1H, m)f 0.90 (3H, t, J = 7.2 Hz) 0.87 (3H, d, J = 8.3 Hz) N-Ac-L-Thr 4.84 7.03 5.56 1.26

(1H, (1H, (1H, (3H,

dd, J = 9.7, 2.4 Hz) d, J = 9.7 Hz) qd, J = 6.5, 2.4 Hz) d, J = 6.5 Hz)

2.13 (3H, s) L-Ile

34 35 35-NH 36 37 38 39

170.3 57.6 36.9 24.8b 11.1 15.7

4.52 (1H, dd, J = 8.8, 5.6 Hz) 8.13 (1H, d, J = 8.8 Hz) 1.88−1.96 (1H, m)c 1.33−1.40 (1H, m)e 1.07−1.13 (1H, m)f 0.81 (3H, t, J = 7.3 Hz) 0.88 (3H, d, J = 8.4 Hz)

a

600 MHz for 1H and 150 MHz for 13C in CDCl3. b−fThese signals are overlapped. gThis chemical shift was estimated from a cross-peak of the HMQC spectrum, as it was obscured by a signal from water. B

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was performed under high dilution conditions (1 mM), to give aspergillicins E (5) and F (6), respectively. 1 H NMR and 13C NMR spectra of synthetic aspergillicin F (6) were identical with those of the natural compound, whereas aspergillicin E (5) showed different spectra from natural 6. Specifically, the chemical shifts and splitting patterns of all the signals, including H-35 (α-proton of L-isoleucine), were perfectly matched in the α-proton region (δ 4.2−5.2) of the 1 H NMR spectra of natural and synthetic 6 (Figure 4), allowing confirmation of the structure of aspergillicin F (6). In addition to aspergillicin E (5), aspergillicins A−D (1−4) were also isolated from A. carneus.15 We thus synthesized compounds 1−4 to reveal the structural characteristics of the aspergillicin family required for innate immune-modulating activity. For the synthesis of aspergillicins A (1), C (3), and D (4) (Scheme 2), tetrapeptide 13 can be used as an intermediate, in common with the synthesis of 5 and 6. OAcylation with Boc-L-valine, followed by N-acylation with CbzN,O-dimethyl-L-tyrosine, Cbz-N-methyl-L-phenylalanine, or Cbz-L-phenylalanine, was carried out to give hexapeptides 7a/ 7c/7d. After removal of the benzyl and benzyloxycarbonyl

Figure 3. Structural elucidation of aspergillicin F (6).

conditions, treatment with benzyl bromide afforded benzyl ester 13. O-Acylation with Boc-L-allo-isoleucine/L-isoleucine followed by N-acylation with Cbz-N,O-dimethyl-L-tyrosine was carried out to give the hexapeptide 7e/7f. After the removal of the benzyl and benzyloxycarbonyl groups, macrolactamization Scheme 1. Synthesis of Aspergillicins E (5) and F (6)a

a Reagents and conditions: (a) N-Fmoc-L-prolin, N,N-diisopropylethylamine (DIPEA), CH2Cl2, rt; (b) 20% piperidine/N,N-dimethylformamide (DMF), rt; (c) N-Fmoc-L-Prolin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 1-hydroxybenzotriazole (HOBt), DIPEA, DMF, rt; (d) 20% piperidine/DMF, rt; (e) N-Fmoc-D-allo-isoleucine, EDCI, HOBt, DIPEA, DMF, rt; (f) 20% piperidine/DMF, rt; (g) N-Fmoc-L-threonine, EDCI, HOBt, DIPEA, DMF, rt; (h) 20% piperidine/DMF, rt; (i) Ac2O, Et3N, CH2Cl2, rt; (j) 50% trifluoroacetic acid (TFA)/CH2Cl2, rt; (k) BnBr, K2CO3, DMF, 0 °C; (l) N-Boc-L-allo-isoleucine or N-Boc-L-isoleucine, MNBA, DMAP, DIPEA, CH2Cl2, rt (68% (for 14e) and 55% (for 14f)); (m) 10% TFA/CH2Cl2, 0 °C; (n) N-Cbz-N,O-dimethyl-L-tyrosine, EDCI, HOBt, DIPEA, CH2Cl2, rt (63% (for 7e) and 44% (for 7f), 2 steps); (o) H2 (balloon), Pd/C, MeOH, rt; (p) HATU, DIPEA, CH2Cl2 (1 mM), rt (38% (for 5) and 34% (for 6), 2 steps).

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aspergillicin B (2) was synthesized from tetrapeptide 15. Compound 15 was synthesized in the same manner as 13, but with Fmoc-D-norvaline instead of Fmoc-D-allo-isoleucine (Scheme 3). O-Acylation was carried out with Boc-L-valine, following N-acylation with Cbz-N,O-dimethyl-L-tyrosine to form hexapeptide 7b. Finally, removal of the benzyl groups and macrolactamization resulted in aspergillicin B (2). The innate immunity-regulating activities of aspergillicins A− F (1−6) were evaluated using the ex vivo Drosophila culture system (Figure 5).10 As described earlier, 10 μg/mL of aspergillicin F (6) suppressed the innate immune response by 37%. This suppression was similar to that of 1 μg/mL of zeaenol, a TAK1 (TGF-β-activated kinase-1) inhibitor in the Drosophila IMD pathway and manmalian TNF-α pathway.6,17 On the other hand, aspergillicins A−E (1−5) showed no suppressive activity at a concentration of 10 μg/mL. In particular, while aspergillicin E (5) is a 36-epimer of aspergillicin F (6), it showed no innate immunity-regulating activity. In addition, while compounds 2 and 3 had the tendency to moderately promote innate immune responses, this was of no statistical significance (p > 0.05 vs DMSO). These results indicate that slight structural differences in the side chains of amino acid residues alter innate immunityregulating activity. In a similar case, a cyclic depsipetide, destruxin E, showed V-ATPase inhibitory activity at submicromolar levels, but its 1γ-epimer was not active until 10 μM.18 Thus, synthesis of more side-chain-modified aspergillicin derivatives would lead to specifying the structural requirements of 6 for the activity and to the discovery of new potent innate immune-suppressive agents.

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EXPERIMENTAL SECTION

General Experimental Procedures. Starting materials were either commercially available or prepared as reported previously in the literature. Analytical thin-layer chromatography was performed on silica gel 60 F254 (Merck). Column chromatography was carried out on silica gel 60 (70−230 mesh, Merck). Nuclear magnetic resonance spectra were recorded on a JEOL JNM ECA-600 and an AL-400. Mass spectra were measured on a JEOL JMS AX-500 and an AX-700. Optical rotations were measured on a JASCO P-1030 polarimeter.

Figure 4. α-Proton region (δ4.2−5.2) in the 1H NMR spectra of synthesized aspergillicin E (5) (A), aspergillicin F (6) (B), and natural 6 (C).

groups, macrolactamization was performed to give aspergillicins A (1), C (3), and D (4), respectively. On the other hand, Scheme 2. Synthesis of Aspergillicins A (1), C (3), and D (4)a

Reagents and conditions: (a) N-Boc-L-valine, MNBA, DMAP, DIPEA, CH2Cl2, rt (57%); (b) 10% TFA/CH2Cl2, 0 °C; (c) N-Cbz-N,O-dimethyl-Ltyrosine or N-Cbz-N-methyl-L-phenylalanine or N-Cbz-L-phenylalanine, EDCI, HOBt, DIPEA, CH2Cl2, rt (50% (for 7a), 66% (for 7c), and 70% (for 7d), 2 steps); (d) H2 (balloon), Pd/C, MeOH, rt; (p) HATU, DIPEA, CH2Cl2 (1 mM), rt (21% (for 1), 29% (for 3), and 20% (for 4), 2 steps). a

D

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Scheme 3. Synthesis of Aspergillicin B (2)a

a

Reagents and conditions: (a−k) The same reagents and conditions described in Scheme 1 were used, but N-Fmoc-D-norvaline was used instead of N-Fmoc-D-allo-isoleucine; (l) N-Boc-L-valine, MNBA, DMAP, DIPEA, CH2Cl2, rt (40%); (b) 10% TFA/CH2Cl2, 0 °C; (c) N-Cbz-N,O-dimethyl-Ltyrosine, EDCI, HOBt, DIPEA, CH2Cl2, rt (53%); (d) H2 (balloon), Pd/C, MeOH, rt; (p) HATU, DIPEA, CH2Cl2 (1 mM), rt (25%, 2 steps). Industry Co., Ltd.); solvent, chloroform−methanol (4:1)] to give aspergillicin F (6) (1.3 mg). Data for 6: colorless, amorphous solid; [α]D −73.0 (c 0.212, CHCl3); 1H NMR and 13C NMR data are shown in Table 1; HRFABMS m/z 755.4328 [M + H] + (755.4344 calcd for C39H59O9N6). Immobilization of N-Fmoc-L-proline onto 2-Chlorotrityl Chloride Resin. To a suspension of 2-chlorotrityl chloride resin (1.6 mmol/g resin) (1.72 g) in dichloromethane (10 mL) were added N-Fmoc-L-proline (1.30 g, 3.85 mmol) and DIPEA (0.96 mL, 5.51 mmol) at room temperature. After being stirred for 10 h, the suspension was filtered and then washed with dichloromethane (50 mL). The washed resin was dried in vacuo to afford polymer-supported N-Fmoc-L-proline 8 (2.58 g). Polymer-Supported Dipeptide 9. The polymer-supported NFmoc-L-proline 8 (2.58 g) was treated with a solution of 20% piperidine (10 mL) in DMF at room temperature. After being stirred for 3 h, the suspension was filtered and then washed with DMF (30 mL). To a supension of the washed resin in DMF (10 mL) were added N-Fmoc-L-proline (1.39 g, 4.13 mmol), DIPEA (1.44 mL, 8.26 mmol), HOBt (632 mg, 4.13 mmol), and EDCI·HCl (791 mg, 4.13 mmol) at room temperature. After being stirred for 10 h, the suspension was filtered and then washed with dichloromethane (50 mL). The washed resin was dried in vacuo to afford polymer-supported N-Fmoc-L-Pro-L-Pro 9 (2.42 g). Polymer-Supported Tripeptide 10. A similar procedure to the synthesis of 9 was used. After deprotection of the polymer-supported N-Fmoc-L-Pro-L-Pro 9 (2.42 g), condensation with N-Fmoc-D-alloisoleucine (1.42 g, 4.01 mmol) in the presence of DIPEA (1.40 mL, 8.02 mmol), HOBt (614 mg, 4.01 mmol), and EDCI·HCl (768 mg, 4.01 mmol) afforded polymer-supported N-Fmoc-D-allo-Ile-L-Pro-LPro 10 (2.61 g). Polymer-Supported Tetrapeptide 11. A similar procedure to that used for compound 9 was employed. After deprotection of the polymer-supported N-Fmoc-D-allo-Ile-L-Pro-L-Pro 10 (2.05 g), condensation with N-Fmoc-L-threonine (1.10 g, 3.22 mmol) in the presence of DIPEA (1.12 mL, 6.45 mmol), HOBt (494 mg, 3.22 mmol), and EDCI·HCl (618 mg, 3.22 mmol) afforded polymersupported N-Fmoc-L-Thr-D-allo-Ile-L-Pro-L-Pro 11 (2.07 g). Polymer-Supported Tetrapeptide 12. The polymer-supported N-Fmoc-L-Thr-D-allo-Ile-L-Pro-L-Pro 11 (2.07 g) was treated with a solution of 20% piperidine (10 mL) in DMF at room temperature. After being stirred for 3 h, the suspension was filtered and then washed with DMF (30 mL). To a supension of the washed resin in dichloromethane (10 mL) were added DIPEA (680 μL, 3.91 mmol)

Figure 5. Effects of aspergillicins A−F (1−6) on the innate immune response. Effects of 10 μg/mL aspergillicin on the DAP-type peptidoglycan-mediated activation of Drosophila Dpt-lacZ. The DAPtype peptidoglycan-mediated activation of Dpt-lacZ is presented as the percent activity relative to DMSO. Zeaenol (1 μg/mL) was used as a positive control. The bars indicate the standard errors of three independent measurements. *p < 0.05 vs control (DMSO). Organism and Culture Conditions. Aspergillus sp. f19703 was isolated from a soil sample collected in Kitadaito Island, Okinawa Prefecture, Japan. This strain was cultured on a rotary shaker (220 rpm) at 25 °C for 3 days in a 250 mL Erlenmeyer flasks containing 20 mL of a medium consisting of 2% soluble starch (Junsei Chemical, Tokyo, Japan), 1% glucose (Junsei Chemical, Tokyo, Japan), 2% soybean meal (SoyPro, J-Oil Mills, Tokyo, Japan), 1% KH2PO4 (Wako Pure Chemical Industries), and 0.5% MgSO4·7H2O (Wako Pure Chemical Industries); 0.5 mL of this culture was transferred to 500 mL Erlenmeyer flasks containing 50 mL of the same medium. The cultures were incubated on a rotary shaker (220 rpm) at 25 °C for 4 days. Isolation of Aspergillicin F (6). The culture broth (2 L) was extracted with n-butanol at room temperature to yield the extract (20.5 g). This extract was chromatographed over silica gel, and the column eluted with hexane−ethyl acetate and ethyl acetate−methanol solutions with increasing polarity to afford an ethyl acetate−methanol (9:1) eluent (948 mg). This elutant was chromatographed over silica gel, and the column eluted with chloroform−methanol solutions with increasing polarity to afford a chloroform−methanol (49:1) eluent (13.0 mg). This elutant was subjected to recycle preparative HPLC [column, JAIGEL-GS310 (⦶ 20 mm × 500 mm, Japan Analytical E

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and acetic anhydride (370 μL, 3.91 mmol) at 0 °C. After being stirred for 2 h, the suspension was filtered and then washed with dichloromethane (50 mL). The washed resin was dried in vacuo to afford polymer-supported N-Ac-L-Thr-D-allo-Ile-L-Pro-L-Pro 12 (1.68 g). Benzyl Ester 13. The polymer-supported N-Ac-L-Thr-D-allo-Ile-LPro-L-Pro 12 (1.42 g) was treated with a mixture of TFA− dichloromethane−water−anisole (50:49:1:1) (10 mL) at room temperature. After being stirred for 2 h, the suspension was filtered and then washed with dichloromethane (50 mL). The combined filtrate was concentrated in vacuo. The residue was dissolved in DMF (4 mL), and benzyl bromide (160 μL, 1.33 mmol) and potassium carbonate (184 mg, 1.33 mmol) were added to the solution. After being stirred for 3 h at room temperature, the mixture was poured into 0.5 M hydrochloric acid and extracted with ethyl acetate three times. The combined organic layer was washed with saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was chromatographed over silica gel eluted by chloroform−methanol (19:1) to afford the benzyl ester N-Ac-L-Thr-D-allo-Ile-L-Pro-L-Pro-OBn 13 (158 mg, 0.283 mmol). Data for 13: colorless oil; 1H NMR (400 MHz, CDCl3) δ 7.29− 7.38 (5H, m), 6.70−6.77 (2H, m), 5.18 (1H, d, J = 12.0 Hz), 5.08 (1H, d, J = 12.0 Hz), 4.68 (1H, dd, J = 9.2, 7.2 Hz), 4.59 (1H, dd, J = 8.0, 3.2 Hz), 4.54 (1H, dd, J = 8.8, 4.4 Hz), 4.45 (1H, dd, J = 6.4, 1.6 Hz), 4.36 (1H, dd, J = 8.0, 1.2 Hz), 4.25−4.32 (1H, m), 3.98−4.05 (1H, m), 3.73−3.81 (1H, m), 3.52−3.63 (3H, m), 2.04 (3H, s), 1.88− 2.27 (8H, m), 1.75−1.84 (1H, m), 1.37−1.45 (1H, m), 1.14 (3H, d, J = 6.4 Hz), 0.93 (3H, t, J = 7.2 Hz), 0.91 (3H, d, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 171.7, 170.9, 170.8, 170.7, 170.5, 135.4, 128.4 (2C), 128.2, 128.0 (2C), 66.8, 66.5, 58.8, 58.5, 57.9, 54.5, 47.5, 46.6, 36.6, 28.6, 28.0, 26.3, 24.7, 24.4, 22.9, 19.5, 14.3, 11.4; HRFABMS m/z 559.3115 [M + H]+ (559.3132 calcd for C29H43O7N4). Pentadepsipeptide 14e. To a solution of 13 (62.8 mg, 0.113 mmol) in dichloromethane (2 mL) were added N-Boc-L-alloisoleucine (27.0 mg, 0.116 mmol), triethylamine (100 μL, 0.725 mmol), DMAP (3.0 mg, 0.025 mmol), and 2-methyl-6-nitrobenzoic anhydride (80.5 mg, 0.234 mmol) at room temperature. After being stirred for 8 h, the mixture was poured into 0.5 M hydrochloric acid and extracted with ethyl acetate three times. The combined organic layer was washed with saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was chromatographed over silica gel eluted by chloroform−methanol (49:1) to afford the pentadepsipeptide NBoc-L-allo-Ile-N-Ac-L-Thr-D-allo-Ile-L-Pro-L-Pro-OBn 14e (59.0 mg, 0.076 mmol, 68%). Data for 14e: colorless oil; 1H NMR (400 MHz, CDCl3) showed a mixture of rotamers; HRFABMS m/z 772.4510 [M + H]+ (772.4497 calcd for C40H62O10N5). Hexadepsipeptide 7e. Compound 14e (57.7 mg, 0.075 mmol) was treated with a mixture of TFA−dichloromethane (1:10) (3 mL) at room temperature. After being stirred for 1 h, the solution was concentrated in vacuo. The residue was dissolved in dichloromethane (1 mL), and N-Cbz-N,O-dimethyl-L-tyrosine (31.6 mg, 0.092 mmol), DIPEA (50 μL, 0.287 mmol), HOBt (19.3 mg, 0.126 mmol), and EDCI·HCl (24.9 mg, 0.130 mmol) were added to the solution. After being stirred for 5 h at room temperature, the mixture was poured into 0.5 M hydrochloric acid and extracted with ethyl acetate three times. The combined organic layer was washed with saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was chromatographed over silica gel eluted by chloroform−methanol (49:1) to afford the hexadepsipeptide N-Cbz-N,O-diMe-L-Tyr-L-allo-Ile-N-Ac-L-Thr-D-alloIle-L-Pro-L-Pro-OBn 7e (47.5 mg, 0.048 mmol, 63%, 2 steps). Data for 7e: colorless oil; 1H NMR (400 MHz, CDCl3) showed a mixture of rotamers; HRFABMS m/z 997.5292 [M + H]+ (997.5286 calcd for C54H73O12N6). Synthesis of Aspergillicin E (5). Compound 7e (40.7 mg, 0.042 mmol) and 5% palladium on carbon (5.0 mg) in methanol (1.5 mL) was stirred at room temperature for 3 h under a hydrogen atmosphere.

After filtration through a Celite pad, the filtrate was concentrated in vacuo. The residue was dissolved in dichloromethane (36 mL), and DIPEA (20 μL, 0.115 mmol) and HATU (20.9 mg, 0.055 mmol) were added to the solution. After being stirred for 25 h at room temperature, the mixture was poured into 0.5 M hydrochloric acid and extracted with ethyl acetate three times. The combined organic layer was washed with saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was chromatographed over silica gel eluted by chloroform−methanol (79:1) to afford aspergillicin E (5) (12.0 mg, 0.016 mmol, 38%, 2 steps). Data for 5: colorless, amorphous solid; [α]D −68.7 (c 0.571, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.15 (1H, d, J = 9.2 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.84 (2H, d, J = 8.4 Hz), 6.80 (1H, d, J = 10.4 Hz), 6.61 (1H, d, J = 9.6 Hz), 5.60 (1H, qd, J = 6.6, 2.4 Hz), 4.97 (1H, dd, J = 11.6, 4.0 Hz), 4.86 (1H, dd, J = 10.0, 2.8 Hz), 4.68 (1H, dd, J = 8.8, 4.8 Hz), 4.61 (1H, dd, J = 9.2, 7.6 Hz), 4.50 (1H, dd, J = 8.4, 5.2 Hz), 4.30 (1H, dd, J = 8.4, 5.2 Hz), 3.78 (3H, s), 3.59−3.72 (2H, m), 3.47−3.58 (2H, m), 3.17 (1H, dd, J = 14.4, 3.6 Hz), 2.98 (1H, dd, J = 14.4, 12.0 Hz), 2.82 (3H, s), 2.15 (3H, s), 1.80−2.27 (9H, m), 1.57− 1.80 (2H, m), 1.30−1.41 (1H, m), 1.28 (3H, d, J = 6.4 Hz), 1.05−1.28 (3H, m), 0.92 (3H, t, J = 7.5 Hz), 0.89 (3H, d, J = 6.7 Hz), 0.88 (3H, t, J = 7.4 Hz), 0.85 (3H, d, J = 7.0 Hz); 13C NMR (100 MHz, CDCl3) δ 173.2, 171.0 (2C), 170.9, 169.9, 169.8, 168.3, 158.7, 130.4 (2C), 129.6, 114.3 (2C), 71.7, 62.5, 58.2, 56.3, 55.8, 55.4, 55.1, 54.7, 47.5, 47.3, 37.9, 36.9, 33.6, 29.3, 28.5, 27.9, 26.5, 26.1, 25.4, 24.7, 23.1, 16.6, 15.0, 14.5, 11.7, 11.6; HRFABMS m/z 755.4321 [M + H]+ (755.4344 calcd for C39H59O9N6). Pentadepsipeptide 14f. A similar procedure to that employed to prepare compound 14e was used. From compound 13 (115.4 mg, 0.207 mmol) and N-Boc-L-isoleucine (57.4 mg, 0.248 mmol), the pentadepsipeptide N-Boc-L-Ile-N-Ac-L-Thr-D-allo-Ile-L-Pro-L-Pro-OBn 14f (87.6 mg, 0.114 mmol, 55%) was obtained. Data for 14f: colorless oil; 1H NMR (400 MHz, CDCl3) showed a mixture of rotamers; HRFABMS m/z 772.4515 [M + H]+ (772.4497 calcd for C40H62O10N5). Hexadepsipeptide 7f. A similar procedure to the synthesis of 7e was used. From compound 14f (83.4 mg, 0.108 mmol) and N-CbzN,O-dimethyl-L-tyrosine (46.1 mg, 0.134 mmol), the hexadepsipeptide N-Cbz-N,O-diMe-L-Tyr-L-Ile-N-Ac-L-Thr-D-allo-Ile-L-Pro-L-Pro-OBn 7f (47.5 mg, 0.048 mmol, 44%) was obtained. Data for 7f: colorless oil; 1H NMR (400 MHz, CDCl3) showed a mixture of rotamers; HRFABMS m/z 997.5291 [M + H]+ (997.5286 calcd for C54H73O12N6). Synthesis of Aspergillicin F (6). A similar procedure to the synthesis of aspergillicin E (5) was used. From compound 7f (41.5 mg, 0.042 mmol), aspergillicin F (6) (10.7 mg, 0.014 mmol, 34%) was obtained. All spectral data of the synthetic product were identical with those of the natural product. Pentadepsipeptide 14a. A similar procedure to the synthesis of 14e was used. From compound 13 (172.5 mg, 0.309 mmol) and NBoc-L-valine (80.1 mg, 0.369 mmol), the pentadepsipeptide N-Boc-LVal-N-Ac-L-Thr-D-allo-Ile-L-Pro-L-Pro-OBn 14a (132.8 mg, 0.175 mmol, 57%) was obtained. Data for 14a: colorless oil; 1H NMR (400 MHz, CDCl3) showed a mixture of rotamers; HRFABMS m/z 758.4353 [M + H]+ (758.4340 calcd for C39H60O10N5). Hexadepsipeptide 7a. A similar procedure to the synthesis of 7e was used. From compound 14a (53.0 mg, 0.070 mmol) and N-CbzN,O-dimethyl-L-tyrosine (28.4 mg, 0.083 mmol), the hexadepsipeptide N-Cbz-N,O-diMe-L-Tyr-L-Val-N-Ac-L-Thr-D-allo-Ile-L-Pro-L-Pro-OBn 7a (34.0 mg, 0.035 mmol, 50%) was obtained. Data for 7a: colorless oil; 1H NMR (400 MHz, CDCl3) showed a mixture of rotamers; HRFABMS m/z 983.5139 [M + H]+ (983.5130 calcd for C53H71O12N6). Synthesis of Aspergillicin A (1). A similar procedure to the synthesis of aspergillicin E (5) was used. From compound 7a (33.0 mg, 0.034 mmol), aspergillicin A (1) (5.3 mg, 0.007 mmol, 21%) was obtained. F

DOI: 10.1021/acs.jnatprod.5b00286 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Data for 1: colorless, amorphous solid; [α]D −90.9 (c 0.368, CHCl3) (lit.15 [α]D −51.9 (c 0.03, CHCl3)); 1H NMR (400 MHz, CDCl3) δ 8.22 (1H, d, J = 8.6 Hz), 7.00−7.10 (1H, m), 7.04 (2H, d, J = 8.4 Hz), 6.84 (2H, d, J = 8.4 Hz), 6.34 (1H, d, J = 10.0 Hz), 5.58 (1H, qd, J = 6.8, 2.8 Hz), 4.99 (1H, dd, J = 12.0, 3.8 Hz), 4.87 (1H, dd, J = 10.0, 2.5 Hz), 4.64 (1H, dd, J = 9.7, 7.2 Hz), 4.51 (1H, dd, J = 9.2, 5.2 Hz), 4.40 (1H, dd, J = 8.4, 6.1 Hz), 4.34 (1H, dd, J = 8.0, 5.2 Hz), 3.78 (3H, s), 3.57−3.71 (2H, m), 3.46−3.58 (2H, m), 3.17 (1H, dd, J = 14.4, 3.6 Hz), 2.97 (1H, dd, J = 14.4, 11.8 Hz), 2.83 (3H, s), 2.16 (3H, s), 2.07−2.25 (3H, m), 1.81−2.00 (4H, m), 1.57−1.80 (2H, m), 1.35−1.44 (2H, m), 1.28 (3H, d, J = 6.4 Hz), 1.02−1.14 (2H, m), 0.83−0.96 (12H, m); 13C NMR (100 MHz, CDCl3) δ 173.2, 171.4, 171.1, 170.5, 169.93, 169.91, 168.3, 158.7, 130.5 (2C), 129.8, 114.4 (2C), 72.0, 62.6, 59.0, 58.4, 55.9, 55.5, 55.3, 54.6, 47.6, 47.4, 38.0, 33.5, 30.5, 29.4, 28.6, 27.8, 26.1, 25.5, 24.7, 23.1, 19.6, 18.3, 16.5, 14.5, 11.8; HRFABMS m/z 741.4189 [M + H] + (741.4187 calcd for C38H57O9N6). Hexadepsipeptide 7c. A similar procedure to the synthesis of 7e was used. From compound 14a (33.3 mg, 0.044 mmol) and N-Cbz-Nmethyl-L-tyrosine (16.3 mg, 0.052 mmol), the hexadepsipeptide NCbz-N-Me-L-Tyr-L-Val-N-Ac-L-Thr-D-allo-Ile-L-Pro-L-Pro-OBn 7c (27.6 mg, 0.029 mmol, 66%) was obtained. Data for 7c: colorless oil; 1H NMR (400 MHz, CDCl3) showed a mixture of rotamers; HRFABMS m/z 983.5038 [M + H]+ (953.5024 calcd for C52H69O11N6). Synthesis of Aspergillicin C (3). A similar procedure to the synthesis of aspergillicin E (5) was used. From compound 7c (27.6 mg, 0.029 mmol), aspergillicin C (3) (6.1 mg, 0.009 mmol, 30%) was obtained. Data for 3: colorless, amorphous solid; [α]D −104.5 (c 0.321, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.24 (1H, d, J = 8.4 Hz), 7.20−7.34 (3H, m), 7.11−7.18 (3H, m), 6.65 (1H, d, J = 9.7 Hz), 5.59 (1H, qd, J = 6.8, 2.8 Hz), 5.04 (1H, dd, J = 11.8, 3.6 Hz), 4.88 (1H, dd, J = 10.0, 2.4 Hz), 4.64 (1H, dd, J = 9.6, 7.5 Hz), 4.50 (1H, dd, J = 9.2, 5.2 Hz), 4.40 (1H, dd, J = 8.4, 6.2 Hz), 4.29 (1H, dd, J = 7.6, 5.0 Hz), 3.47−3.71 (4H, m), 3.23 (1H, dd, J = 14.4, 3.2 Hz), 3.03 (1H, dd, J = 14.4, 11.5 Hz), 2.84 (3H, s), 2.17 (3H, s), 2.17−2.25 (2H, m), 1.81− 2.11 (4H, m), 1.59−1.73 (2H, m), 1.33−1.43 (1H, m), 1.29 (3H, d, J = 6.8 Hz), 1.08−1.16 (1H, m), 0.96−1.04 (1H, m), 0.83−0.96 (12H, m), 0.68−0.75 (1H, m); 13C NMR (100 MHz, CDCl3) δ 173.2, 171.3, 171.1, 170.5, 169.9, 169.8, 168.3, 137.9, 129.5 (2C), 129.0 (2C), 127.0, 72.0, 62.5, 59.0, 58.3, 55.9, 55.3, 54.6, 47.6, 47.4, 38.0, 34.4, 30.4, 29.4, 28.5, 27.8, 26.1, 25.4, 24.7, 23.1, 19.6, 18.3, 16.6, 14.5, 11.8; HRFABMS m/z 711.4069 [M + H] + (711.4081 calcd for C37H55O8N6). Hexadepsipeptide 7d. A similar procedure to the synthesis of 7e was used. From compound 14a (33.6 mg, 0.044 mmol) and N-Cbz-Ltyrosine (15.5 mg, 0.052 mmol), the hexadepsipeptide N-Cbz-L-Tyr-LVal-N-Ac-L-Thr-D-allo-Ile-L-Pro-L-Pro-OBn 7d (28.8 mg, 0.031 mmol, 70%) was obtained. Data for 7d: colorless oil; 1H NMR (400 MHz, CDCl3) showed a mixture of rotamers; HRFABMS m/z 939.4872 [M + H]+ (939.4868 calcd for C51H67O11N6). Synthesis of Aspergillicin D (4). A similar procedure to the synthesis of aspergillicin E (5) was used. From compound 7d (28.8 mg, 0.031 mmol), aspergillicin D (4) (4.3 mg, 0.006 mmol, 20%) was obtained. Data for 4: colorless, amorphous solid; [α]D −103.3 (c 0.316, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.22−7.32 (3H, m), 7.14− 7.20 (2H, m), 6.95−7.03 (2H, m), 6.01 (1H, d, J = 10.4 Hz), 5.60 (1H, d, J = 9.8 Hz), 5.47 (1H, q, J = 6.4 Hz), 4.80 (1H, d, J = 11.4 Hz), 4.53−4.63 (2H, m), 4.42−4.53 (2H, m), 4.07−4.13 (1H, m), 3.77−3.86 (1H, m), 3.65−3.71 (1H, m), 3.53−3.62 (2H, m), 3.42− 3.51 (1H, m), 3.17 (1H, dd, J = 14.9, 8.0 Hz), 2.14 (3H, s), 2.03−2.27 (3H, m), 1.87−1.95 (2H, m), 1.60−1.83 (4H, m), 1.28−1.39 (2H, m), 1.23 (3H, d, J = 6.3 Hz), 1.01−1.10 (1H, m), 0.99 (3H, d, J = 6.0 Hz), 0.89 (3H, t, J = 6.8 Hz), 0.87 (3H, d, J = 6.8 Hz), 0.83 (3H, d, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 172.8, 172.1, 171.5, 170.6, 169.7, 169.3, 168.7, 137.1, 129.6 (2C), 128.7 (2C), 127.2, 70.4, 62.3, 59.0, 58.3, 56.5, 54.9, 54.3, 47.7, 47.4, 36.0, 35.8, 32.1, 29.5, 27.4, 26.1, 25.4,

25.3, 23.0, 19.7, 19.2, 16.9, 14.8, 11.5; HRFABMS m/z 697.3925 [M + H]+ (697.3925 calcd for C36H53O8N6). Benzyl Ester 15. A similar procedure to the synthesis of 13 was used. From the polymer-supported N-Ac-L-Thr-D-norVal-L-Pro-L-Pro, the benzyl ester N-Ac-L-Thr-D-norVal-L-Pro-L-Pro-OBn 14 (49.3 mg, 0.091 mmol) was obtained. Data for 15: colorless oil; 1H NMR (400 MHz, CDCl3) δ 7.29− 7.38 (5H, m), 6.87 (1H, d, J = 9.2 Hz), 6.71 (1H, d, J = 8.2 Hz), 5.18 (1H, d, J = 12.2 Hz), 5.08 (1H, d, J = 12.2 Hz), 4.78−4.87 (1H, m), 4.54−4.63 (2H, m), 4.41−4.47 (1H, m), 4.38 (1H, dd, J = 8.0, 1.2 Hz), 4.05 (1H, brs), 3.93−4.01 (1H, m), 3.73−3.81 (1H, m), 3.52− 3.62 (3H, m), 2.06 (3H, s), 2.17−2.28 (2H, m), 1.88−2.15 (6H, m), 1.67−1.76 (1H, m), 1.56−1.63 (1H, m), 1.28−1.36 (2H, m), 1.13 (3H, d, J = 6.4 Hz), 0.92 (3H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) δ 171.7, 171.0, 170.58, 170.56, 170.5, 135.5, 128.5 (2C), 128.3, 128.1 (2C), 66.8, 66.7, 58.9, 58.1, 58.0, 50.2, 47.5, 46.7, 33.9, 28.7, 28.1, 24.8, 24.5, 23.1, 19.3, 18.7, 13.8; HRFABMS m/z 545.2965 [M + H]+ (545.2975 calcd for C28H41O7N4). Pentadepsipeptide 14b. A similar procedure to the synthesis of 14e was used. From compound 15 (40.0 mg, 0.073 mmol) and N-BocL-valine (18.9 mg, 0.087 mmol), the pentadepsipeptide N-Boc-L-ValN-Ac-L-Thr-D-norVal-L-Pro-L-Pro-OBn 14b (21.4 mg, 0.029 mmol, 40%) was obtained. Data for 14b. colorless oil; 1H NMR (400 MHz, CDCl3) showed a mixture of rotamers; HRFABMS m/z 744.4211 [M + H]+ (744.4184 calcd for C38H58O10N5). Hexadepsipeptide 7b. A similar procedure to the synthesis of 7e was used. From compound 14b (18.4 mg, 0.025 mmol) and N-CbzN,O-dimethyl-L-tyrosine (10.1 mg, 0.029 mmol), the hexadepsipeptide N-Cbz-N,O-diMe-L-Tyr-L-Val-N-Ac-L-Thr-D-norVal-L-Pro-L-Pro-OBn 7b (12.7 mg, 0.013 mmol, 53%) was obtained. Data for 7b: colorless oil; 1H NMR (400 MHz, CDCl3) showed a mixture of rotamers; HRFABMS m/z 969.4997 [M + H]+ (969.4973 calcd for C52H69O12N6). Synthesis of Aspergillicin B (2). A similar procedure to the synthesis of aspergillicin E (5) was used. From compound 7b (12.0 mg, 0.012 mmol), aspergillicin B (2) (2.2 mg, 0.003 mmol, 25%) was obtained. Data for 2: colorless, amorphous solid; [α]D −85.3 (c 0.188, CHCl3) (lit.15 [α]D −46.6 (c 0.025, CHCl3)); 1H NMR (600 MHz, CDCl3) δ 8.25 (1H, d, J = 8.4 Hz), 7.02 (2H, d, J = 8.4 Hz), 7.00−7.05 (1H, m), 6.83 (2H, d, J = 8.4 Hz), 6.67 (1H, d, J = 9.2 Hz), 5.53 (1H, qd, J = 6.6, 2.7 Hz), 4.97 (1H, dd, J = 11.6, 3.5 Hz), 4.85 (1H, dd, J = 10.0, 2.6 Hz), 4.77 (1H, q, J = 8.1 Hz), 4.47 (1H, dd, J = 8.8, 5.2 Hz), 4.41 (1H, dd, J = 8.5, 5.9 Hz), 4.30 (1H, dd, J = 8.0, 5.5 Hz), 3.77 (3H, s), 3.62−3.68 (1H, m), 3.55−3.62 (1H, m), 3.46−3.54 (2H, m), 3.15 (1H, dd, J = 14.8, 3.3 Hz), 2.96 (1H, dd, J = 14.8, 11.7 Hz), 2.82 (3H, s), 2.15−2.23 (2H, m), 2.11 (3H, s), 2.04−2.12 (1H, m), 1.96−2.02 (1H, m), 1.88−1.93 (1H, m), 1.82−1.88 (1H, m), 1.66−1.73 (1H, m), 1.53−1.66 (3H, m), 1.25−1.44 (3H, m), 1.26 (3H, d, J = 6.5 Hz), 1.04−1.12 (1H, m), 0.90 (3H, d, J = 6.9 Hz), 0.89 (3H, t, J = 7.5 Hz), 0.85 (3H, d, J = 6.9 Hz); 13C NMR (150 MHz, CDCl3) δ 173.2, 171.4, 171.1, 170.6, 169.9, 169.6, 168.0, 158.7, 130.5 (2C), 129.7, 114.4 (2C), 72.3, 62.5, 58.9, 58.4, 55.9, 55.4, 55.3, 50.0, 47.5, 47.3, 34.9, 33.5, 30.5, 29.4, 28.6, 27.8, 25.5, 24.7, 23.2, 19.6, 18.7, 18.1, 16.5, 13.8; HRFABMS m/z 727.4037 [M + H] + (727.4031 calcd for C37H55O9N6). Ex Vivo Drosophila Culture Assay. The experimental procedure has been described previously.10 Briefly, the abdominal cavity of thirdinstar larva was opened using fine pincettes. Individual whole larval tissues were cultured in Schneider’s Drosophila medium (Gibco-BRL, Invitrogen, Carlsbad, CA, USA) containing 20% fetal bovine serum (Valley Biomedical, Winchester, VA, USA) and 1% antibiotics/ antimycotics (Gibco-BRL) in each well of a 96-well plate at 25 °C. For each condition, six females were cultured to produce six replicates. The test compounds were dissolved in DMSO and added to the culture medium. To determine the effects of the test compounds on the innate immune response, Dpt-lacZ larvae were cultured in the presence of 100 ng/mL peptidoglycans from Escherichia coli (InvivoGen, San Diego, CA, USA) and the compound at 25 °C for 12 h. The cultured G

DOI: 10.1021/acs.jnatprod.5b00286 J. Nat. Prod. XXXX, XXX, XXX−XXX

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individual larvae were sonicated with 200 μL of reaction buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, and 1 mM MgCl2) using an Ultrasonic Processor (Misonix, New York, NY, USA). After centrifugation (10000g) at 4 °C for 10 min, supernatant was harvested, and β-galactosidase activity and total protein were determined as previously described.9 β-Galactosidase activity was normalized to total protein amount. Measurement of Cytotoxicity. Drosophila S2 cells were cultured in Schneider’s medium (Gibco-BRL) supplemented with 10% FBS and 1% antibiotics/antimycotics at 25 °C. Cytotoxicity was measured using the colorimetric thiazolyl blue coversion assay using WST-8 solution (Nacalai Tesque) as described previously.10

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(14) Kikuchi, H.; Isobe, M.; Sekiya, M.; Abe, Y.; Hoshikawa, T.; Ueda, K.; Kurata, S.; Katou, Y.; Oshima, Y. Org. Lett. 2011, 13, 4624− 4627. (15) Capon, R. J.; Skene, C.; Stewart, M.; Ford, J.; O’Hair, R. A. J.; Williams, L.; Lacey, E.; Gill, J. H.; Heiland, K.; Friedel, T. Org. Biomol. Chem. 2003, 1, 1856−1862. (16) Kopple, K. D. J. Pharm. Sci. 1972, 61, 1345−1356. (17) Rawlins, P.; Mander, T.; Sadeghi, R.; Hill, S.; Gammon, G.; Foxwell, B.; Wrigley, S.; Moore, M. Int. J. Immunopharmacol. 1999, 21, 799−814. (18) Yoshida, M.; Takeuchi, H.; Ishida, Y.; Yashiroda, Y.; Yoshida, M.; Takagi, M.; Shin-ya, K.; Doi, T. Org. Lett. 2010, 12, 3792−3795.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00286. NMR spectra of the new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81-22-7956824. Fax: +81-22-795-6821. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Scientific Research (No. 23710247 and No. 25350959) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN); the Strategic International Cooperative program from Japan Science and Technology Agency; the SUNBOR GRANT from the Suntory Institute for Bioorganic Research; and the Astellas Foundation for Research on Metabolic Disorders.

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DOI: 10.1021/acs.jnatprod.5b00286 J. Nat. Prod. XXXX, XXX, XXX−XXX