Acantholactam and Pre-neo-kauluamine, Manzamine

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Acantholactam and Pre-neo-kauluamine, Manzamine-Related Alkaloids from the Indonesian Marine Sponge Acanthostrongylophora ingens Ahmed H. El-Desoky,†,‡ Hikaru Kato,†,‡ Keisuke Eguchi,† Tetsuro Kawabata,† Yukio Fujiwara,§ Fitje Losung,⊥ Remy E. P. Mangindaan,⊥ Nicole J. de Voogd,∥ Motohiro Takeya,§ Hideyoshi Yokosawa,∇ and Sachiko Tsukamoto*,† †

Graduate School of Pharmaceutical Sciences, Kumamoto University, Oe-honmachi 5-1, Kumamoto 862-0973, Japan Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan ⊥ Faculty of Fisheries and Marine Science, Sam Ratulangi University, Kampus Bahu, Manado 95115, Indonesia ∥ Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands ∇ School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan §

S Supporting Information *

ABSTRACT: Two new manzamine alkaloids, acantholactam (3) and pre-neo-kauluamine (4), were isolated from the marine sponge Acanthostrongylophora ingens along with manzamine A (1) and neo-kauluamine (2). Acantholactam contains a γlactam ring N-substituted with a (Z)-2-hexenoic acid moiety and is proposed to be biosynthetically derived from manzamine A by oxidative cleavage of the eight-membered ring. Compound 4 was converted to the dimer 2 during storage, suggesting nonenzymatic dimer formation. Among the four isolated compounds, 1, 2, and 4 showed proteasome inhibitory activity.

M

Acantholactam (3) was obtained as a pale yellow, amorphous solid, and HRESITOFMS established its molecular formula as C36H42N4O4. 2D NMR spectra (DMSO-d6) including COSY, HOHAHA, HSQC, and HMBC (Figure 1 and Table 1) indicated the presence of a fused six-, six-, and 13-membered ring system connected to a β-carboline, which exists in 1. The residual structure composed of C8H11NO3 was further analyzed

anzamines are complex polycyclic alkaloids composed of a fused and bridged tetra- or pentacyclic ring system that is connected to a β-carboline. More than 80 manzamine derivatives have been isolated since the first isolation of manzamine A (1),1 including the dimers kauluamine2 and neokauluamine (2).3 Manzamines have been shown to exhibit various biological activities, including cytotoxic,1 antimicrobial,4 antimalarial,5 antiviral,6 antineuroinflammatory,6 antiatherosclerotic,7 and insecticidal8 effects. During our search for biologically active natural products, we found that the extracts of two specimens of the marine sponge Acanthostrongylophora ingens, collected at two locations, Ti Toi and Bajotalawaan, North Sulawesi, Indonesia, showed cytotoxicity and an inhibitory effect on the chymotrypsin-like activity of the proteasome. We here reported the isolation, structure determination, and biological activities of two new manzamine derivatives, acantholactam (3) and pre-neo-kauluamine (4), together with the known alkaloids 1 and 2. Sponges collected at Ti Toi and Bajotalawaan were immediately soaked in EtOH and extracted with EtOH. The extracts were partitioned between EtOAc and H2O. The EtOAc-soluble fractions of the extracts were subjected to column chromatography followed by HPLC to afford 1 and 3 from the sponge collected at Ti Toi and 1, 2, and 4 from the sponge collected at Bajotalawaan. © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. COSY and key HMBC correlations of 3. Received: April 1, 2014

A

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Table 1. 1H and 13C NMR Dataa for 3 (DMSO-d6) position 1 3 4 4a 4b 5 6 7 8 8a 9a 10 11 12 13

by NMR spectra. COSY and HMBC correlations [δH 5.75 (d, J = 11.5 Hz, H-32) and 6.24 (dt, J = 11.5, 7.6 Hz, H-31) to δC 167.0 (C-33)] showed the presence of a 2-hexenoic acid moiety, which was supported by the presence of an Dexchangeable hydrogen at δH 12.10 (33-OH). The Z-geometry of Δ31,32 was determined by the coupling constant (11.5 Hz). HMBC correlations from hydrogens [δH 1.81 and 2.35 (H235)] to C-24 (δC 39.2), C-25 (δC 40.3), C-26 (δC 64.8), C-34 (δC 172.7), and C-36 (δC 65.5) and from H-26 (δH 4.18) to C34 and C-36 showed the presence of a γ-lactam ring. Methylene hydrogens (δH 3.24 and 3.80) at C-28 (δC 41.9) exhibited HMBC correlations to C-26 and C-34, which indicated that the (Z)-2-hexenoic acid moiety was attached to the nitrogen atom of the γ-lactam ring. To date, enantiomers of 8-hydroxymanzamine A,9 manzamine F,10 and 12,34-oxamanzamine E11 have been isolated from various marine sponges. The absolute configuration of 3 was deduced from the similar CD spectrum to 1 (Figures S9 and S16, respectively, Supporting Information). There is the possibility that 3 may have been derived from 1 by oxidative cleavage of the C33−C34 bond. Pre-neo-kauluamine (4) was obtained as a pale yellow, amorphous solid, and HRESITOFMS showed its molecular formula as C36H44N4O3, two oxygens more than 1. The 1H and 13 C NMR spectra readily revealed that 4 was a congener of 1 (Table 2). The 1H NMR spectrum of 4 showed the presence of two coupled low-field hydrogens at δH 4.11 (δC 65.6, C-31) and 4.20 (δC 69.2, C-30) together with the absence of olefin signals corresponding to H-32 and H-33 in 1. The COSY spectrum showed the connection C28−C29−C30−C31−C32−C33 (Figure 2). HMBC correlations from H2-28 (δH 4.01 and 4.11), H-33 (δH 1.85), and H2-35 (δH 1.93 and 1.99) to C-34 (δC 91.7) and from H-26 (δH 4.24) to C-28 (δC 47.0) indicated that 4 had the same pentacyclic ring system as that in 1 and the three carbons

δC, type 143.3, 137.8, 113.7, 128.5, 121.0, 121.6, 119.5, 128.2, 111.9, 140.6, 132.6, 138.6, 137.9, 72.2, 41.6,

C CH CH C C CH CH CH CH C C C CH C CH2

14

21.1, CH2

15 16 17

128.5, CH 131.8, CH 25.8, CH2

18 19 20

26.5, CH2 24.5, CH2 52.9, CH2

22

49.5, CH2

23

32.7, CH2

24 25 26 28

39.2, 40.3, 64.8, 41.9,

29

25.9, CH2

30

25.9, CH2

31 32 33 34 35 36 9-NH 12-OH 33-OH a1

148.1, 121.0, 167.0, 172.7, 41.7,

CH C CH CH2

CH CH C C CH2

65.5, CH2

δH (J in Hz)

HMBC

8.33, d (5.1) 8.02, d (5.1)

1, 4, 4a 9a

8.23, 7.25, 7.56, 7.64,

4b, 7, 8a 4b, 7, 8 5, 6, 8a 4b, 6

d (7.4) t (7.4) t (7.4) d (7.4)

6.40, s 1.57, 1.94, 2.07, 2.31, 5.58, 5.47, 1.86, 2.43, 1.38, 1.47, 2.44, 2.54, 1.97, 2.80, 1.51, 2.34, 2.88,

m m m m m m m m m m m m m m m m dd (12.0, 6.2)

4.18, 3.24, 3.80, 1.61, 1.70, 2.56, 2.58, 6.24, 5.75,

s m m m m m m dt (11.5, 7.6) d (11.5)

1.81, d (16.5) 2.35, d (16.5) 2.20, d (11.9) 2.60, d (11.9) 11.10, s 5.17, s 12.10, s

1, 24, 26

14, 17 14, 17

1, 10, 23, 25, 26, 36 11, 26, 26, 31 31 31, 31, 30, 30,

13, 25, 34, 36 29, 34 29, 34

32 32 33 33

25, 26, 34, 36 24, 25, 34, 36 26 24, 26 4a, 4b, 8a, 9a 11, 12, 13

H NMR spectrum was recorded at 500 MHz and spectrum at 125 MHz.

13

C NMR

at C-30, C-31, and C-34 in 4 were oxygenated based on their chemical shifts. The molecular formula of 4 implied that two additional oxygen atoms existed as a hydroxy group and an ether linkage in the eight-membered ring. The HMBC correlation between H-30 and C-34 unambiguously determined the position of the ether linkage C30−O−C34. Therefore, the carbon at C-31 possessed a hydroxy group. Thus, the structure B

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Table 2. 1H and 13C NMR Dataa for 4 (CDCl3) position

δC, type

1 3 4 4a 4b 5 6 7 8 8a 9a 10 11 12 13

143.0, 137.7, 114.0, 129.6, 121.2, 121.0, 119.5, 128.2, 112.9, 141.5, 133.4, 142.4, 135.3, 72.0, 38.0,

C CH CH C C CH CH CH CH C C C CH C CH2

14 15 16 17

20.8, 127.0, 133.1, 24.8,

CH2 CH CH CH2

18 19

26.9, CH2 24.4, CH2

20

54.0, CH2

22

50.2, CH2

23

34.0, CH2

24 25 26 28

40.9, 44.2, 75.9, 47.0,

CH C CH CH2

29 30 31 32 33

16.1, 69.2, 65.6, 27.3, 28.6,

CH2 CH CH CH2 CH2

34 35

91.7, C 52.3, CH2

36

70.4, CH2

9-NH 12-OH 27-NH

δH (J in Hz)

HMBC

8.32, d (5.1) 7.82, d (5.1)

1, 4, 4a 9a

8.05, 7.20, 7.49, 7.78,

4a, 7, 8a 4b, 7, 8 5, 8a 4b, 6

d (7.8) t (7.8) t (7.8) d (7.8)

6.59, s 1.64, 2.33, 2.27, 5.58, 5.58, 1.68, 2.53, 1.53, 1.43, 1.78, 2.34, 2.67, 1.87, 2.95, 1.81, 2.94, 2.61,

m m m m m m m m m m m m m m m m m

4.24, 4.01, 4.11, 2.31, 4.20, 4.11, 2.09, 1.85, 2.93,

d (7.7) dd (14.4, 6.8) m m brt (5.6) m m m m

1.93, m 1.99, m 2.22, d (12.7) 3.51, d (12.7) 11.63, s 5.70, s 10.41, s

Figure 2. COSY and key HMBC correlations of 4.

10, 24, 26

C-30′ (unit B) to iminium carbons C-34′ (unit B) and C-34 (unit A), respectively, to furnish 2 (Scheme 1). The structure of 2 was originally reported by El-Sayed et al.,3 but the relative configuration of C-30′, C-31′, and C-34′ has yet to be determined. Our result that 4 dimerized to produce 2 clearly indicated that the hemispheres A and B of 2 were derived from the same molecule 4. Therefore, the relative configuration of C30′, C-31′, and C-34′ in 2 was the same as that of C-30, C-31, and C-34, which has been determined by NOE experiments in the literature,3 and the relative configuration of C-30, C-31, and C-34 in 4 was also the same. The CD spectrum of 2 converted from 4 during storage was identical to that of 2 isolated from the sponge extract (Figures S18 and S17, respectively, Supporting Information) and also similar to that of 1 (Figure S16, Supporting Information), which indicated that 1, 2, and 4 had the same absolute configuration in the fused six-, six-, 13-, and five-membered ring system. It should be noted that 4 spontaneously changed to the dimer nonenzymatically after isolation. The existence of the dimer in the sponge remains to be determined. The ubiquitin−proteasome system plays a critical role in selective protein degradation and regulates various cellular events.12 After the approval of the synthetic proteasome inhibitor bortezomib13 for the treatment of relapsed multiple myeloma, drugs targeting the proteasome, as well as those targeting the ubiquitin system, have been expected to be excellent anticancer agents. Therefore, we have been searching for inhibitors of the ubiquitin−proteasome system from natural sources.14 We recently reported that manzamine A (1) inhibited the accumulation of cholesterol esters in macrophages and suppressed hyperlipidemia and atherosclerosis in mice.7 In this study, we examined the biological activities of 1−4, such as cytotoxic activity, inhibitory activity against the proteasome, and inhibitory activity against the accumulation of cholesterol esters in macrophages (Table 3). Cytotoxicity was tested against HeLa cells, and only 2 was active, with an IC50 value of 5.4 μM. Proteasome inhibitory activity was measured against the chymotrypsin-like activity of the proteasome.14e Compounds 2 and 4 inhibited the proteasome with IC50 values of 0.13 and 0.34 μM, respectively, while 1 was less active (IC50 = 2.0 μM) and 3 was a very weak inhibitor (IC50 = 33 μM). Inhibiting the accumulation of cholesterol esters was evaluated using human monocyte-derived macrophages7 at a concentration of 20 μM. Compounds 1, 2, and 4 exhibited 80−92% inhibition, whereas 3 showed no inhibition. Thus, we found that these three activities were markedly lower in 3 than in 1, 2, and 4. These results clearly indicated that the presence of the

14, 17 14, 17

25 1, 10, 11, 23, 25, 26, 35 11, 29, 29, 31 32, 29 30,

13, 25, 28, 36 30, 34 30, 34 34 31, 33

32, 34 24, 25, 34, 36 24, 25, 34, 36 20, 22, 24, 25, 26 20, 22, 24, 25, 26 4a, 4b, 8a, 9a 11, 12, 13

a1 H NMR spectrum was recorded at 500 MHz and spectrum at 125 MHz.

13

C NMR

of 4 was determined except for the relative configuration at C30, C-31, and C-34. Incidentally, during storage at −20 °C for 2 months, 4 was found to have been converted to the dimer neokauluamine (2), as the NMR spectra were identical to those of 2 reported in the literature.3 The dimerization of 4 may have occurred due to the nucleophilic attacks in two molecules that were equilibrated into a ring-opened diol form with a strained iminium carbon, i.e. from oxygen atoms at C-31 (unit A) and C

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Scheme 1. Possible Mechanism of the Dimerization of 4 to 2

fraction (11.3 mg) that eluted with MeOH was further purified by HPLC (Asahipack GS-310P column, Asahi Chemical Industry Co., Ltd., 21.5 × 500 mm) with CHCl3 to yield acantholactam (3) (3.8 mg). The sponge (RMNH POR 3991, wet weight 600 g) was extracted with EtOH, and the extract was partitioned between EtOAc and H2O. The EtOAc fraction (3.1 g) was subjected to SiO2 column chromatography with a stepwise gradient elution using hexane/EtOAc, CHCl3/MeOH (9:1), MeOH, and n-BuOH/AcOH/H2O (4:4:1). The fraction (1.3 g) that eluted with MeOH was further purified by SiO2 column chromatography with hexane/EtOAc (2:1, 1:1, and 1:2) and EtOAc. The fraction that eluted with hexane/EtOAc (2:1) afforded 1 (380 mg). The fraction that eluted with EtOAc was repeatedly purified by HPLC (Asahipack GS-310, CHCl3/MeOH (1:1); Inertsil NH2 column, GL Sciences Inc., 20 × 250 mm, CHCl3 (0−40 min), 0−1% MeOH−CHCl3 (40−100 min), and 1−2% MeOH−CHCl3 (100−102 min), 5 mL/min) to yield pre-neokauluamine (4) (1.7 mg) and neo-kauluamine (2) (2.6 mg). Manzamine A (1): colorless, amorphous solid; [α]20D +44 (c 0.19, CHCl3); lit. [α]20D +50 (c 0.28, CHCl3).1 neo-Kauluamine (2): colorless, amorphous solid; [α]20D +62 (c 0.41, CH2Cl2); lit. [α]25D +94.6 (c 0.1, CHCl3).3 Acantholactam (3): colorless, amorphous solid; [α]21D +48 (c 2.3, MeOH); UV (MeOH) λmax (log ε) 347 (4.14), 281 (4.45), and 236 (4.69) nm; CD (200 μM, MeOH) λmax (Δε), 275 (+22.19), 248 (+8.24), 230 (+61.72), 218 (+14.72), and 208 (+73.08) (Figure S9); IR (film) νmax 3222, 2929, 2854, 1708, 1650, 1452, 1232, 746, and 669 cm−1; 1H and 13C NMR data, see Table 1; HRESITOFMS m/z 595.3296 [M + H]+ (calcd for C36H43N4O4, 595.3284). Pre-neo-kauluamine (4): colorless, amorphous solid; 1H and 13C NMR data, see Table 2; HRESITOFMS m/z 581.3497 [M + H]+ (calcd for C36H45N4O3, 581.3492). The specific rotation and UV, IR, and CD spectra of 4 could not be measured, as the compound converted to 2 before these measurements could be obtained. Biological Assays. Cytotoxicity,14e inhibition of the proteasome,14e and inhibition of the accumulation of cholesterol esters in macrophages7 were tested using procedures reported previously.

Table 3. Biological Activities of 1−4 compd

cytotoxicity IC50 (μM)a

inhibition of the proteasome IC50 (μM)

inhibition of the accumulation of the cholesterol ester (%)b

1 2 3 4

13 5.4 >50 16

2.0 0.13 33 0.34

80 92 0 91

a

Tested against HeLa cells. bTested at a concentration of 20 μM.

eight-membered ring in the manzamines was essential for them to show their biological activities. Yousaf et al. also demonstrated in their molecular modeling study that the antimicrobial and anti-HIV activities of manzamines were closely related to the conformation of the eight-membered ring.6 Taken together, these findings suggest that the eightmembered ring in the manzamines plays a critical role in their various biological activities.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO DIP-1000 polarimeter in MeOH, CHCl3, or CH2Cl2. UV absorption was measured on a JASCO V-550 spectrophotometer in MeOH. CD spectra were measured on a JASCO J-820 spectropolarimeter in MeOH. The IR spectrum was recorded on a JEOL JIR-6500W spectrophotometer. 1H and 13C NMR spectra were recorded on a Bruker Avance 500 NMR or JEOL ECX400 spectrometer in CDCl3, CD3OD, or DMSO-d6. Chemical shifts were referenced to the residual solvent peaks (δH 7.24 and δC 77.0 for CDCl3, δH 3.30 and δC 49.0 for CD3OD, or δH 2.49 and δC 39.5 for DMSO-d6). ESIMS spectra were measured on a Bruker Bio-TOF or Bruker micrOTOF II mass spectrometer. Animal Material, Extraction, and Isolation. The Acanthostrongylophora ingens sponge specimens were collected by scuba at a depth of 10 m in Ti Toi and Bajotalawaan, North Sulawesi, Indonesia, in December 2006 and soaked in EtOH immediately. Voucher specimens (RMNH POR 8527 and RMNH POR 3991, respectively) of the sponges have been deposited in the Naturalis Biodiversity Center. The sponge (RMNH POR 8527, wet weight 250 g) was extracted with EtOH. After evaporation, the residual aqueous solution was extracted with EtOAc. The EtOAc fraction (2.7 g) was subjected to SiO2 column chromatography with a stepwise gradient elution using hexane/EtOAc, EtOAc, and MeOH. The fraction that eluted with hexane/EtOAc (4:6) (280 mg) was purified by Sephadex LH-20 with CHCl3/MeOH (1:1) to give manzamine A (1) (34.2 mg). The

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ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra of 3 and 4 and CD spectra of 1−3. These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-96-371-4380. Fax: +81-96-371-4380. E-mail: [email protected]. D

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Author Contributions ‡

A. H. El-Desoky and H. Kato contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. H. Kobayashi of the University of Tokyo and Dr. H. Rotinsulu of Universitas Pembangunan for collecting the sponges. This work was supported by Grants-in-Aid for Scientific Research (Nos. 18406002 and 25293025 to S.T. and 2586501 to Y.F.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and also by a grant from the Uehara Memorial Foundation.

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REFERENCES

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