Cytotoxic and Protein Kinase Inhibiting Nakijiquinones and

Article pubs.acs.org/jnp

Cytotoxic and Protein Kinase Inhibiting Nakijiquinones and Nakijiquinols from the Sponge Dactylospongia metachromia Georgios Daletos,† Nicole J. de Voogd,‡ Werner E. G. Müller,§ Victor Wray,⊥ WenHan Lin,∥ Daniel Feger,▽ Michael Kubbutat,▽ Amal H. Aly,*,† and Peter Proksch*,† †

Institut für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany ‡ Netherlands Centre for Biodiversity Naturalis, P.O. Box 9517, 2300 RA Leiden, The Netherlands § Institut für Physiologische Chemie, Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Duesbergweg 6, 55128 Mainz, Germany ⊥ Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany ∥ National Research Laboratories of Natural and Biomimetic Drugs, Peking University, Health Science Center, 100083 Beijing, People’s Republic of China ▽ ProQinase GmbH, Breisacher Straße 117, 79106 Freiburg, Germany S Supporting Information *

ABSTRACT: Chemical investigation of the sponge Dactylospongia metachromia afforded five new sesquiterpene aminoquinones (1−5), two new sesquiterpene benzoxazoles (6 and 7), the known analogue 18-hydroxy-5-epi-hyrtiophenol (8), and a known glycerolipid. The structures of all compounds were unambiguously elucidated by one- and two-dimensional NMR and by MS analyses, as well as by comparison with the literature. Compounds 1−5 showed potent cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values ranging from 1.1 to 3.7 μM. When tested in vitro for their inhibitory potential against 16 different protein kinases, compounds 5, 6, and 8 exhibited the strongest inhibitory activity against ALK, FAK, IGF1-R, SRC, VEGF-R2, Aurora-B, MET wt, and NEK6 kinases (IC50 0.97−8.62 μM).

M

including antitumor,7 anti-inflammatory,8 and antiviral activities.9 A literature survey revealed that Dactylospongia metachromia has not been intensively investigated so far. It is important to mention, however, that this species was originally described as Hippospongia metachromia by De Laubenfels in 1954.10 The reassignment by Bergquist in 1965 led to the currently accepted name D. metachromia.11 Reports on D. metachromia12 describe the isolation of a sesterterpene lactone,13 sesterterpene sulfates,14 and sesquiterpene quinones/quinols.15−19 As part of our ongoing research on bioactive natural products from marine sponges,20,21 we investigated a specimen of D. metachromia collected at Ambon, Indonesia. The extract exhibited considerable in vitro cytotoxicity against mouse lymphoma L5178Y cells. Subsequent bioactivity-guided isolation yielded five new sesquiterpene aminoquinones (1−5), two new sesquiterpene benzoxazoles (6 and 7), and two known compounds. Herein, we report the isolation and structure elucidation of the new compounds, as well as bioassay results employing the

arine organisms represent a largely unexploited source of potential pharmaceuticals with a great diversity of fascinating structures.1 Such bioactive metabolites are believed to play a significant role in the protection, adaptation, and survival of marine organisms in the unique environmental conditions of the sea.2 Many marine invertebrates, including sponges, are sessile and soft-bodied organisms lacking a hard outer protective shell, which makes them vulnerable to marine predators. It is assumed that sponges like other marine invertebrates rely mainly on chemical rather than on physical defense to deter predators or compete with neighbors for resources or space.3,4 Sponges of the genus Dactylospongia are a rich source of bioactive secondary metabolites, the majority of which are sesquiterpene quinones/quinols.5,6 This class of compounds includes constituents of mixed biogenetic origin, which frequently consist of sesquiterpene moieties linked to quinones, quinols, or structural analogues. The sesquiterpene unit is of biosynthetic interest, as it usually features a drimane- or a 4,9friedodrimane-type skeleton comprising a trans- or a less common cis-fused ring junction. These compounds have attracted considerable interest due to their pronounced biological activities © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 1, 2013

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dx.doi.org/10.1021/np400633m | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

the structure of 1, thus revealing a nakijiquinone core structure. The sesquiterpenoid moiety comprised two continuous spin systems, CH(10)CH2(1)CH2(2)CH(3) and CH2(6)CH2(7)CH(8)CH3(13), as indicated by the COSY spectrum. The corresponding HMBC correlations (Table 1, Figure 1) confirmed the sesquiterpenoid substructure as identical to that found in 18-hydroxy-5-epi-hyrtiophenol (8).22 This was further corroborated by the prominent fragment ion peak at m/z 191, characteristic of the decalin moiety (C14H23), that was observed in the mass spectrum of 1. Further HMBC correlations of H2-15 to C-16, C-17, and C-21 and of H-19 to C-17 and C-21 and the downfield-shifted signal of C-17 (δC 157.2 ppm) established the 17-hydroxyquinone subunit and its connection to the sesquiterpenoid moiety at C-16. The chemical shift of C-20 (δC 150.3 ppm) suggested its attachment to an amino substituent, the HMBC correlations of the 20-NH to C-19, C-21, and C-22 confirmed the presence of the amino substituent on C-20, and the upfield chemical shift of H-19 (δH 5.38 ppm) was consistent with location ortho to an amino group. All remaining signals were assigned to a tyramine unit, which included the NHCH2(22)CH2(23) substructure and the AA′BB′ spin system observed in the COSY spectrum (Table 1). This was further corroborated by inspection of the respective HMBC correlations (Table 1, Figure 1). The relative configuration of the sesquiterpenoid unit in 1 was deduced from analysis of the ROESY spectrum. Key correlations were observed from H2-15 to both H-8 and H-10, as well as from H-10 to H3-12, indicating their cofacial orientation. According to the literature, the 13C signals of CH3-12 in transdecalin moieties of structural analogues resonate upfield from those in cis-decalins (Δ ca. 10 ppm).5,22 Thus, the deshielded resonance of C-12 (δC 32.5 ppm) in 1 offered additional evidence and confirmed a cis rather than trans junction of the decalin ring. Hence, the structure of 1 was assigned representing a new natural product named 5-epi-nakijiquinone S.23 Compound 2 was obtained as a red, amorphous solid. The molecular formula was established as C29H37NO3 from the prominent ion peak at m/z 448.2844 for the protonated molecule [M + H]+ in the HRESIMS spectrum, thus revealing a 16 amu decrease in the molecular weight compared to 1. The 1 H and 13C NMR data of 2 (Table 1) were similar to those of 1 except for the loss of the hydroxy group at C-27, which accounts for the molecular weight difference between both compounds. Furthermore, comparison of 2 to the data reported for the known nakijiquinone Q24 indicated that both compounds are epimers with different configurations at the stereogenic center C-5. This was corroborated by interpretation of ROESY spectra and the downfield chemical shift of C-12 (δC 32.5 ppm), establishing a cis junction of the decalin ring as in 1, in contrast to the trans junction as reported for nakijiquinone Q. Thus, the structure of 2 was identified, and the trivial name 5-epinakijiquinone Q is proposed. Compound 3 was obtained as a red, amorphous solid. The molecular formula C31H38N2O3 was established by HRESIMS, in accordance with the signal observed at m/z 487.2953 [M + H]+. The spectroscopic data of 3 had some features in common with those of 1 and 2, suggesting the same sesquiterpenoid aminoquinone core structure, but a different amine function connected to C-20. 1H NMR and COSY spectra (Table 2) revealed an aminoethyl residue, an additional NH proton resonating at δH 8.04 ppm, which is coupled to an aromatic proton at δH 7.04 ppm (H-2′), and an aromatic ABCD spin system (H-5′ to H-8′). Hence, the last two spin systems revealed

L5178Y mouse lymphoma cell line and a panel of protein kinases.

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RESULTS AND DISCUSSION The combined MeOH and MeOH/CH2Cl2 (1:1) extracts of D. metachromia were subjected to solvent−solvent partitioning to give n-hexane, CH2Cl2, EtOAc, and n-BuOH fractions. Column chromatography of the CH2Cl2 and n-hexane fractions followed by purification using semipreparative HPLC afforded nine compounds. Compound 1 was obtained as a red, amorphous solid. The HRESIMS spectrum exhibited a prominent peak at m/z 464.2792 [M + H]+ consistent with the molecular formula C29H37NO4. The UV spectrum, revealing absorbances at λmax 319 and 490 nm, suggested the presence of a quinone chromophore in the molecule.15 The 1H NMR spectrum (Table 1) indicated the presence of one NH proton resonating at δH 6.46 ppm, two olefinic protons at δH 5.30 and 5.38 ppm (H-3 and H-19, respectively), a para-substituted aromatic ring (H-25/29 and H-26/28), an olefinic methyl group at δH 1.62 ppm (H3-11), a secondary methyl group split into a doublet at δH 0.88 ppm (H3-13), and two tertiary methyl signals at δH 0.88 and 0.93 ppm (H3-14 and H3-12, respectively). The 13C NMR (Table 1) and HMQC spectra confirmed the corresponding carbon signals and revealed in addition eight sp2 quaternary carbons, including two carbonyl groups at δC 178.6 and 183.1 ppm (C-18 and C-21, respectively), two sp3 quaternary carbons, two sp3 methines, and seven sp3 methylene groups. Thorough inspection of the 2D NMR spectra disclosed the presence of a sesquiterpenoid and an aminoquinone moiety in B


a

19.3, CH2

24.7, CH2

124.1, 139.2, 37.5, 37.6,

29.3, CH2

38.9, CH 44.3, C 46.1, CH

20.0, 32.5, 18.4, 16.5, 33.1,

114.3, 157.2, 178.6, 92.0, 150.3, 183.1, 44.5,

33.6, 129.8, 130.0, 116.0, 154.8, 116.0, 130.0,

1

2

3 4 5 6

7

8 9 10

11 12 13 14 15

16 17 18 19 20 21 22

C

23 24 25 26 27 28 29 17-OH 20-NH 27-OH 24, 26, 27 23, 25, 27 19, 21, 22

26 25 29 28 22

6.77, 7.03, 8.27, 6.46, 4.86,

d (8.3) d (8.3) brs brt brs

23, 27, 29 24, 27, 28

22, 24, 25/29

7.03, d (8.3) 6.77, d (8.3)

2.86, t (7.0)

23, 20-NH 22

20, 23, 24

17, 21

1, 2, 4, 5, 6, 9, 12, 14, 15 3, 4, 5 4, 5, 6, 10 7, 8, 9 8, 9, 10, 15 8, 9, 10, 14, 16, 17, 21 8, 9, 10, 14, 16, 17, 21

5, 6, 9, 13 5, 6 6, 9, 10, 13, 14, 15

4, 5, 7, 8 7, 8, 10

1, 2, 5

2, 3, 9, 10 2, 3, 5, 9, 10

HMBC

3.37, td (7.0, 6.7)

5.38, s

15b

2.56, d (13.8)

8 15a

3

1.62, 0.93, 0.88, 0.88, 2.40,

d (1.1) s d (6.2) s d (13.8)

1

1.09, brd (6.3)

6a, 7 6b, 7

1a, 2, 10 1b, 2, 10 1, 2a, 3 1, 2b, 3 2, 11

COSY

6, 7a, 8 6, 7b 7b, 13

m m m m brs

1

0.91a 1.86, ddd (13.4, 3.9, 1.8) 1.01, m 1.22, m 1.19, m

1.94, 2.15, 2.01, 2.15, 5.30,

δH (J in Hz)

Overlapped with CH3-12, CH3-13, and CH3-14.

CH2 C CH CH C CH CH

C C C CH C C CH2

CH3 CH3 CH3 CH3 CH2

CH C C CH2

δC, type

position

CH C C CH2

34.5, 137.7, 128.8, 129.1, 127.3, 129.1, 128.8,

114.3, 157.2, 178.7, 92.1, 150.2, 183.1, 44.3,

20.0, 32.5, 18.4, 16.5, 33.1,

CH2 C CH CH CH CH CH

C C C CH C C CH2

CH3 CH3 CH3 CH3 CH2

38.9, CH 44.3, C 46.1, CH

29.3, CH2

124.1, 139.2, 37.5, 37.6,

24.7, CH2

19.3, CH2

δC, type m m m m brs

d (1.1) s d (6.2) s d (13.8)

7.17, 7.31, 7.24, 7.31, 7.17, 8.29, 6.47,

d (7.4) dd (7.4, 7.5) t (7.5) dd (7.4, 7.5) d (7.4) brs brt

2.94, t (7.0)

3.42, td (7.0, 6.7)

5.40, s

2.56, d (13.8)

1.62, 0.93, 0.88, 0.88, 2.41,

1.10, brd (6.3)

0.92a 1.86, brdd (13.3, 3.5) 1.01, m 1.22, m 1.19, m

1.94, 2.15, 2.00, 2.15, 5.30,

δH (J in Hz)

2

22

26 25, 27 26/28 27, 29 28

23, 20-NH 22

15b

15a

8

3

1

6, 7a, 8 6, 7b 7b, 13

6a, 7 6b, 7

1a, 2, 10 1b, 2, 10 1, 2a, 3 1, 2b, 3 2, 11

COSY

23, 27, 29 24, 28 25/29 24, 26 23, 25, 27

22, 24, 25/29

20, 23, 24

17, 21

1, 2, 4, 5, 6, 9, 12, 14, 15 3, 4, 5 4, 5, 6, 10 7, 8, 9 8, 9, 10, 15 8, 9, 10, 14, 16, 17, 21 8, 9, 10, 14, 16, 17, 21

5, 6, 13 5, 6 6, 9, 10, 13, 14, 15

4, 5, 7, 8 7, 8, 10

1, 2, 5

2, 3, 9, 10 2, 3, 5, 9, 10

HMBC

CH C C CH2

37.1, 26.2, 22.5, 22.5,

114.0, 157.5, 178.4, 91.7, 150.5, 183.2, 41.4,

20.0, 32.5, 18.4, 16.5, 33.1,

CH2 CH CH3 CH3

C C C CH C C CH2

CH3 CH3 CH3 CH3 CH2

38.8, CH 44.2, C 46.0, CH

29.4, CH2

124.1, 139.2, 37.5, 37.5,

24.7, CH2

19.3, CH2

δC, type

Table 1. 1H and 13C NMR, COSY, and HMBC Data of 1, 2, and 5 at 600 (1H) and 75 (13C) MHz (chloroform-d, δ in ppm)

m m m m brs

5

d (1.1) s d (6.2) s d (13.8)

td (8.0, 7.1) m d (6.5) d (6.5)

8.33, brs 6.39, brt

1.55, 1.66, 0.93, 0.93,

3.16, td (8.0, 6.2)

5.37, s

2.56, d (13.8)

1.62, 0.93, 0.90, 0.89, 2.41,

1.11, brd (6.2)

0.90a 1.86, ddd (13.4, 3.7, 2.1) 1.01, m 1.23, m 1.19, m

1.94, 2.17, 2.00, 2.15, 5.30,

δH (J in Hz) COSY

22

23, 20-NH 22, 24 23, 25/26 24 24

15b

15a

8

3

1

6, 7a, 8 6, 7b 7b, 13

6a, 7 6b, 7

1a, 2, 10 1b, 2, 10 1, 2a, 3 1, 2b, 3 2, 11

HMBC

22, 23, 23, 23,

25/26 25/26 24, 26 24, 25

20, 23, 24

17, 21

1, 2, 4, 5, 6, 9, 12, 14, 15 3, 4, 5 4, 5, 6, 10 7, 8, 9 8, 9, 10, 15 8, 9, 10, 14, 16, 17, 21 8, 9, 10, 14, 16, 17, 21

5, 6, 13 5, 6 6, 9, 10, 13, 14, 15

4, 5, 7, 8 7, 8, 10

1, 2, 5

2, 3, 9, 10 2, 3, 5, 9, 10

Journal of Natural Products Article



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Figure 1. COSY (─) and key HMBC (→) correlations of 1, 3, and 4.

Thus, 5 was characterized as a new natural product and was named 5-epi-nakijiquinone N. Compound 6 was obtained as a pale yellow, amorphous solid. The molecular formula was determined to be C24H33NO3 by HRESIMS (m/z 384.2532 [M + H]+), indicating nine elements of unsaturation. Thorough inspection of 1D and 2D NMR data disclosed the presence of the same sesquiterpenoid moiety as in 1−5. In addition, the 1H NMR spectrum (Table 3) revealed the presence of a hydroxy group resonating at δH 5.85 ppm, an aromatic proton at δH 6.98 ppm (H-19), a methoxy group at δH 3.91 ppm (18-OCH3), and an olefinic methyl group at δH 2.54 ppm (CH3-23). The 13C NMR (Table 3) and HMQC spectra confirmed the corresponding carbon signals and revealed in addition six sp2 quaternary carbons, four of which were oxygenated, as indicated by their downfield chemical shifts at δC 162.1, 146.8, 144.6, and 143.7 ppm (C-22, C-17, C-18, and C-21, respectively). HMBC correlations (Table 3) of H-19 to C-17, C-18, C-20, and C-21 established a pentasubstituted aromatic ring, which was connected to the sesquiterpenoid moiety at C-16 based on correlations observed for H2-15 to C-16, C-17, and C-21. The methoxy group 18-OCH3 was attached to C-18, as indicated by the respective HMBC correlation, as well as by its ROESY correlation to H-19. The downfield chemical shifts of C-20 (δC 132.5), C-21 (δC 143.7), and C-22 (δC 162.1), as well as the HMBC correlation observed for the methyl group H3-23 to C-22, were indicative of a 2-methyloxazole moiety and hence accounted for the remaining two elements of unsaturation in the structure of 6. This was further confirmed by the upfield chemical shift of C-23 at δC 14.6 ppm, which is characteristic for respective methyl substituents in structural analogues.25 Accordingly, 6 was identified as a new natural product and was given the name 5-epi-nakijinol C.23 Compound 7 was obtained as a pale yellow, amorphous solid. The molecular formula C25H32N2O2 was established by HRESIMS (m/z 393.2535 [M + H]+), thus corresponding to 11 elements of unsaturation. NMR data of 7 revealed the presence of the same cis-4,9-friedodrim-3-ene subunit as for 1−6. The remaining 1H NMR signals (Table 3) included an aromatic proton at δH 7.71 ppm (H-19) and two overlapping olefinic methyl groups at δH 2.61 ppm (CH3-23/25), both

an indole moiety. This was confirmed by HMBC cross-peaks detected from H-2′ to C-3′, C-4′, and C-9′, from H-5′ to C-3′, C-7′, and C-9′, and from H-8′ to C-4′ and C-6′ (Table 2). In addition, HMBC correlations of H2-23 to C-2′, C-3′, C-4′, and C-22, as well as of H2-22 to C-3′ and C-23, established the connection of the indole ring with the aminoethyl unit at C-3′ (Figure 1), thus indicating a tryptamine unit. Accordingly, the structure of 3 was elucidated and designated as 5-epinakijiquinone T.23 Compound 4 was obtained as a red, amorphous solid. HRESIMS showed an ion peak at m/z 432.2567 [M + H]+ indicating the molecular formula C25H37NO3S. NMR data denoted the same sesquiterpenoid aminoquinone basic structure as in 1−3. In addition, 1H NMR and COSY spectra (Table 2) showed an aminopropyl spin system including three methylene groups resonating at δH 3.30, 1.95, and 2.56 ppm (H2-22, H2-23, and H2-24, respectively) and one methyl group singlet at δH 2.09 (H3-25). The downfield chemical shifts of the methyl group and of CH2-24 at δH 2.09 (δC 15.8) and 2.56 (δC 31.7) ppm respectively, as well as the presence of one sulfur atom in the molecule, as indicated by HRESIMS, suggested the attachment of the methyl group to CH2-24 through sulfur. This was further confirmed by the observed HMBC correlation of the methyl group to C-24 (Figure 1). Further HMBC correlations were observed from H2-22 to C-20, C-23, and C-24, from H2-23 to C-22 and C-24, and from H2-24 to C-22, C-23, and C-25 (Table 2). Thus, 4 was identified as the new natural product 5-epi-nakijiquinone U.23 On the basis of ROESY and 13C NMR spectra interpretation, the relative configuration of the sesquiterpenoid unit in 3 and 4 was found to be identical to that observed for 1 and 2. Compound 5 was obtained as a red, amorphous solid. The molecular formula C26H39NO3 was established by HRESIMS analysis (m/z 414.3006 [M + H]+). Close similarity of the 1 H and 13C NMR data (Table 1) to those reported for nakijiquinone N24 indicated both compounds to be epimers differing in the configuration of C-5. In contrast to the transfused decalin ring reported for nakijiquinone N, ROESY and 13 C NMR data indicated a cis fusion in 5, by analogy with 1−4. D



Article

Table 2. 1H and 13C NMR, COSY, and HMBC Data of 3 and 4 (chloroform-d, δ in ppm) 3 position

δC,a type

1

19.3, CH2

2

24.7, CH2

3 4 5 6

CH C C CH2

7

29.3, CH2

8 9 10

38.8, CH 44.3, C 46.0, CH

11 12 13 14 15

20.0, 32.5, 18.4, 16.5, 33.1,

CH3 CH3 CH3 CH3 CH2

16 17 18 19 20 21 22

114.2, 157.3, 178.6, 91.9, 150.3, 183.1, 43.1,

C C C CH C C CH2

23 24 25 17-OH 20-NH NH-1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ a

124.1, 139.2, 37.5, 37.5,

24.4, CH2

122.4, 112.0, 127.1, 118.6, 120.0, 122.8, 111.6, 136.7,

CH C C CH CH CH CH C

4

δHb (J in Hz) 1.92, 2.15, 2.00, 2.14, 5.30,

m m m m brs

COSY

δC,c type

HMBC

1a, 2, 10 1b, 2, 10 1, 2a, 3 1, 2b, 3 2, 11

2, 3, 9, 10 2, 3, 5, 9, 10

19.3, CH2 24.7, CH2

1, 2, 5

124.1, 139.2, 37.5, 37.5,

0.89d 1.85, ddd (13.3, 4.0, 1.7) 1.00, m 1.24, m 1.18, m

6a, 7 6b, 7

4, 5, 7, 8 7, 8, 10

6, 7a, 8 6, 7b 7b, 13

5, 6, 9, 13 5, 6 6, 9, 10, 13, 14, 15

1.10, brd (6.3)

1

1.62, 0.93, 0.87, 0.88, 2.40, 2.55,

(1.1)

3

(6.2)

8

(13.8) (13.8)

15a 15b

1, 2, 4, 5, 6, 9, 12, 14, 15 3, 4, 5 4, 5, 6, 10 7, 8, 9 8, 9, 10, 15 8, 9, 10, 14, 16, 17, 21 8, 9, 10, 14, 16, 17, 21

d s d s d d

5.41, s

3.49, td (6.9, 6.3)

17, 21

3.12, t (6.9)

23, 20-NH 22

20, 23, 3′

8.29, 6.54, 8.04, 7.04,

brs brt brs d (1.7)

22 2′ NH-1′

3′, 4′, 9′

7.56, 7.13, 7.21, 7.37,

brd (7.8) dd (7.8, 7.4) dd (8.0, 7.4) brd (8.0)

6′ 5′, 7′ 6′, 8′ 7′

3′, 4′, 5′, 4′,

22, 2′, 3′, 4′

CH C C CH2

29.3, CH2 38.8, CH 44.3, C 46.1, CH 20.0, 32.5, 18.4, 16.5, 33.1,

CH3 CH3 CH3 CH3 CH2

114.2, 157.3, 178.6, 92.0, 150.4, 183.1, 41.8,

C C C CH C C CH2

27.3, CH2 31.7, CH2 15.8, CH3

δHb (J in Hz) 1.93, 2.17, 2.00, 2.15, 5.30,

m m m m brs

COSY

HMBC

1a, 2, 10 1b, 2, 10 1, 2a, 3 1, 2b, 3 2, 11

2, 3, 9, 10 2, 3, 5, 9, 10

0.89d 1.86, ddd (13.4, 4.5, 3.2) 1.01, m 1.24, m 1.20, m

6a, 7 6b, 7

4, 5, 7, 8 7, 8, 10

6, 7a, 8 6, 7b 7b, 13

5, 6, 9, 13 5, 6 6, 9, 10, 13, 14, 15

1.11, brd (5.9)

1

1.62, 0.93, 0.90, 0.89, 2.41, 2.56,

(1.1)

3

(6.2)

8

(13.8) (13.8)

15a 15b

1, 2, 4, 5, 6, 9, 12, 14, 15 3, 4, 5 4, 5, 6, 10 7, 8, 9 8, 9, 10, 15 8, 9, 10, 14, 16, 17, 21 8, 9, 10, 14, 16, 17, 21

d s d s d d

5.41, s

3.30, td (6.9, 6.5) 1.95, 2.56, 2.09, 8.27, 6.51,

q (6.9) t (6.9) s brs brt

1, 2, 5

17, 21

23, 20-NH 22, 24 23

20, 23, 24 22, 24 22, 23, 25 24

22

7′, 9′ 8′ 9′ 6′

100 MHz. b600 MHz. c75 MHz. dOverlapped with CH3-12, CH3-13, and CH3-14.

Moreover, the prominent fragment ion peaks detected at m/z 191 and 202, which are characteristic for the decalin (C14H23) and the 2,6,8-trimethylbisbenzoxazole (C11H10N2O2) subunits, respectively, offered further evidence. Thus, the structure of 7 was assigned, and it was designated 5-epi-nakijinol D.23 The known compounds were identified as 18-hydroxy-5-epihyrtiophenol (8)22 and a known glycerolipid (C18H38O3)26 by comparison of their spectroscopic data (UV, 1H and 13C NMR, MS) with values in the literature. Interpretation of 13C NMR and ROESY spectra and the observed [α]D value ([α]23D −70, c 0.1, MeOH) of 8 proved the same relative configuration of the sesquiterpenoid unit as determined for 1−7. Previous reports include the detection of the isolated glycerolipid in the Myxobacterium Myxococcus xanthus27 and its isolation from a

familiar features from the spectra of 6. In addition to the corresponding carbon signals, only four out of seven expected sp2 quaternary carbon signals were detected in the 13C NMR spectrum. Accordingly, six carbons appeared as three pairs of overlapped signals (C-17/21, C-18/20, and C-22/24), suggesting a plane of symmetry in the structural subunit. HMBC correlations (Table 3) from H-19 to C-17/21 and C-18/20 and from H3-23/25 to C-22/24, as well as comparison of 13C chemical shifts of C-18/20 (δC 138.5), C-17/21 (δC 149.2), and C-22/24 (δC 163.9) with those detected in 6, corroborated the presence of a 2,6-dimethylbisbenzoxazole moiety, rationalizing the remaining elements of unsaturation. The connection to the sesquiterpenoid moiety at C-16 was deduced from HMBC correlations of H2-15 to C-16 and C-17/21 by analogy with 6. E



Article

Table 3. 1H and 13C NMR, COSY, and HMBC Data of 6 and 7 at 600 (1H) and 150 (13C) MHz (chloroform-d, δ in ppm) 6 δH (J in Hz)

COSY

1

19.6, CH2

2.05, m 2.32, brdd (13.3, 9.1)

1a, 2, 10 1b, 2, 10

2

24.9, CH2

2.06, m 2.21, m 5.32, brs

1, 2a, 3 1, 2b, 3 2, 11

1, 2, 5

0.74, td (13.6, 2.8) 1.79, ddd (13.6, 3.5, 2.8) 1.03, qd (13.3, 2.5) 1.19, dq (13.4, 3.5) 1.35, m

6a, 7 6b, 7

4, 5, 7, 8 7, 8, 10

6, 7a, 8 6, 7b 7b, 13

5, 6, 13 5, 6, 9, 13 6, 7, 9, 13, 14, 15

1.32, brd (5.7)

1

1.62, 0.87, 0.99, 0.99, 2.82, 3.01,

(1.2)

3

(6.2)

8

(14.0) (14.0)

15a 15b

1, 2, 4, 5, 6, 9, 12, 14, 15 3, 4, 5 4, 5, 6, 10 7, 8, 9 8, 9, 10, 15 8, 9, 10, 14, 16, 17, 21 8, 9, 10, 14, 16, 17, 21

3 4 5 6

124.1, 139.4, 37.4, 37.3,

CH C C CH2

7

29.4, CH2

8 9 10

38.1, CH 44.2, C 45.3, CH

11 12 13 14 15

20.0, 32.4, 18.7, 16.7, 34.9,

CH3 CH3 CH3 CH3 CH2

109.7, 146.8, 144.6, 98.9, 132.5, 143.7, 162.1, 14.6,

C C C CH C C C CH3

16 17 18 19 20 21 22 23 24 25 18-OCH3 17-OH a

7

δC, type

position

56.7, CH3

d s d s d d

2, 9, 10 2, 3, 5, 9, 10

19.3, CH2

24.7, CH2

6.98, s

17, 18, 20, 21

2.54, s

22

3.91, s 5.85, s

18

123.9, 139.4, 37.3, 37.2,

CH C C CH2

29.1, CH2 38.0, CH 44.2, C 45.2, CH 19.9, 32.4, 18.1, 16.7, 35.2,

CH3 CH3 CH3 CH3 CH2

106.7, 149.2, 138.5, 106.7, 138.5, 149.2, 163.9, 14.9, 163.9, 14.9,

C C C CH C C C CH3 C CH3

δH (J in Hz)

COSY

HMBC

2.11, m 2.44,brdd (13.7, 9.8) 2.12, m 2.26, m 5.34, brs

1a, 2, 10 1b, 2, 10

2, 9, 10 2, 3, 5, 9, 10

1, 2a, 3 1, 2b, 3 2, 11

1, 2

0.64, td (13.8, 3.0) 1.78, brdt (13.8, 3.1) 1.08a 1.19, m 1.25, m

6a, 7 6b, 7

4, 5, 7, 8, 12 4, 7, 8, 10

6, 7a, 8 6, 7b 7b, 13

5, 6, 8 8, 13 9, 13, 14

1.22, m

1

1, 2, 15 3, 4, 4, 5, 7, 8, 8, 9, 8, 9, 8, 9,

1.63, 0.81, 1.08, 1.06, 3.04, 3.25,

d s d s d d

(1.3)

3

(6.5)

8

(14.2) (14.2)

15a 15b

4, 5, 6, 9, 12, 14, 5 6, 10 9 10, 15 10, 14, 16, 17/21 10, 14, 16, 17/21

7.71, s

17/21, 18/20

2.61, s

22

2.61, s

24

Overlapped with CH3-13 and CH3-14.

control (IC50 4.3 μM). Interestingly, the loss of the aminoquinone core structure, as in 6−8, resulted in a significant decrease of cytotoxic activity, indicating that the aminoquinone moiety plays an important role in mediating cytotoxicity. Compounds 1−8 were further tested against 16 protein kinases, which have been shown to be involved in the regulation of tumor growth and metastasis (Table 5),29−44 at a dose of 10 μM each. The protein kinase activity assay results (Table S1) revealed comparable activity for 1−5, particularly inhibiting ALK, FAK, IGF1-R, SRC, and VEGF-R2. The nakijinols (6 and 7) showed a slightly narrower spectrum of protein kinase inhibition. Both compounds inhibited ALK, FAK, and IGF1-R, and only 6 inhibited VEGF-R2 as well. 18-Hydroxy-5-epihyrtiophenol (8) exhibited a broader spectrum of activity, inhibiting ALK, Aurora-B, FAK, MET wt, NEK6, SRC, and VEGF-R2. IC50 values were determined for compounds 5, 6, and 8, which inhibited the activity of at least one of the 16 kinases by more than 75% (Table 6). The lack of cytotoxic activity for 6−8 in spite of their protein kinase inhibitory activity indicated that the pronounced cytotoxic activity of 1−5 is most likely due to another cellular mechanism that needs to be further studied.

Sarcotragus sp. (Dictyoceratida) sponge.26 The S configuration was established from the positive specific rotation value ([α]23D +10, c 0.06, MeOH) as established for long-chain 1-O-alkyl-sn-glycerols.28 Compounds 1−8 were subjected to a cellular cytotoxicity (MTT) assay against L5178Y mouse lymphoma cells. The sesquiterpene quinones (1−5) showed pronounced cytotoxicity against L5178Y cells with IC50 values ranging between 1.1 and 3.7 μM (Table 4) compared to kahalalide F as a positive Table 4. Cytotoxic Activities of 1−8

a

δC, type

HMBC

compound

IC50 (μM)a

1 2 3 4 5 6 7 8 kahalalide F

1.7 1.1 3.7 1.8 1.3 >10.0 >10.0 >10.0 4.3

Only IC50 values < 10.0 μM are reported. F



Article

Table 5. Relevance of the Tested Protein Kinases to the Regulation of Tumor Growth and Metastasis kinase AKT1 ALK

deregulation found in tumors

cancer-related signaling

relevant cancers

ARK5 Aurora-B AXL

deregulation of upstream effectors expression of fusionproteins, mutations, gene amplification overexpression overexpression overexpression

FAK IGF1-R MEK1 MET NEK2 NEK6 PIM1

overexpression, deregulation of upstream effectors overexpression of ligands mutation of upstream B-Raf gene amplification, mutations overexpression overexpression overexpression

unknown cell cycle checkpoint metastasis, angiogenesis, proliferation, survival, migration proliferation, survival, migration proliferation proliferation, migration, apoptosis metastasis, proliferation deregulation of centrosomes, proliferation anchorage-independent cell growth survival, proliferation, differentiation, apoptosis

PLK1 PRK1 SRC VEGF-R2

overexpression, mutations overexpression overexpression, deregulation of upstream effectors activated by tumor cells

cell cycle progression migration proliferation, adhesion, invasion, motility tumor angiogenesis

many human cancers ALCL, neuroblastoma

29 30

hepatocellular carcinoma many human cancers many human cancers

31 32 33

many human cancers breast cancer melanoma and all cancers many human cancers breast cancer most human cancers hematopoietic malignancies/prostate cancer many human cancers androgen-dependent prostate cancer many human cancers solid tumors

34 35 36 37 38 39 40 41 42 43 44

Sephadex LH-20 and Merck MN silica gel 60 M (0.04−0.063 mm). Solvents were distilled before use, and spectral grade solvents were used for spectroscopic measurements. TLC plates with silica gel F254 (Merck) were used to monitor fractions (CH2Cl2/MeOH mixtures as developing systems), and detection was by UV absorption at 254 and 366 nm. Animal Material. A specimen of Dactylospongia metachromia was collected at Ambon, Indonesia, in August 1996, and subsequently identified by one of the authors (N.d.V.). The sponge was preserved in a mixture of EtOH and H2O (70:30) and stored in a −20 °C freezer until extraction. A voucher specimen (reference number RMNHPOR8010) is deposited at the Zoological Museum, Amsterdam, The Netherlands. Extraction and Isolation. The thawed sponge material (wet weight 600 g) was cut into small pieces and exhaustively extracted with MeOH (2 L × 2) followed by MeOH/CH2Cl2 (1:1, 2 L × 2) at room temperature. The extracts were combined and concentrated under vacuum to yield 6.7 g. Liquid−liquid fractionation afforded n-hexane (2 g), CH2Cl2 (1.8 g), EtOAc (617 mg), and n-BuOH (1.2 g) fractions. The bioactive CH2Cl2 fraction was further subjected to vacuum liquid chromatography (VLC) on silica gel using a step gradient of n-hexane/ EtOAc, followed by CH2Cl2/MeOH, to yield 10 fractions (F1−F10). Fractions 3 (40% EtOAc in n-hexane, 110 mg), 4 (40% n-hexane in EtOAc, 60 mg), and 6 (100% EtOAc, 85 mg) were further purified by column chromatography on Sephadex LH-20, using CH2Cl2/MeOH (1:1) as a mobile phase, followed by semipreparative HPLC for final purification using an eluting gradient of MeOH/H2O or CH3CN/H2O to yield 8 (10 mg) from F3, 2 (8 mg) and 5 (5 mg) from F4, and 1 (4 mg), 3 (2.5 mg), and 4 (2 mg) from F6. Following the same procedure, the bioactive n-hexane fraction was subjected to VLC on silica gel to yield 10 fractions. Fractions 3 (40% EtOAc in n-hexane, 77 mg), 4 (40% n-hexane in EtOAc, 87 mg), and 5 (20% n-hexane in EtOAc, 87 mg) afforded the glycerolipid (3 mg), 6 (8 mg), and 7 (2.5 mg), respectively. 5-epi-Nakijiquinone S (1): red, amorphous solid; [α]23D −23 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203.1 (4.23), 318.6 (3.92), 490.1 (2.80) nm; 1H and 13C NMR data, see Table 1; EIMS m/z (relative intensity %) 463 (5), 369 (2), 273 (100), 257 (18), 238 (9), 191 (21), 179 (61), 166 (30), 152 (9), 135 (11), 107 (4) 95 (52); HRESIMS m/z 464.2792 [M + H]+ (calcd for C29H38NO4, 464.2795). 5-epi-Nakijiquinone Q (2): red, amorphous solid; [α]23D −18 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 207.1 (4.33), 319.4 (4.07), 490.1 (2.98) nm; 1H and 13C NMR data, see Table 1; EIMS m/z (relative intensity %) 447 (9), 257 (100), 241 (4), 209 (5), 191 (5), 166 (20), 152 (6), 105 (12), 95 (16); HRESIMS m/z 448.2844 [M + H]+ (calcd for C29H38NO3, 448.2846). 5-epi-Nakijiquinone T (3): red, amorphous solid; [α]23D −17 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 219.0 (4.12), 322.4 (3.73),

Literature surveys revealed that sesquiterpene quinones/ quinols structurally related to 1−8 are reported from sponges of the order Dictyoceratida. Compounds incorporating a nakijiquinone core structure, as in 1−5, have hitherto been isolated only from Spongia sp.,45 Dactylospongia elegans,5,6 Smenospongia sp.,46 and Hippospongia sp.47 These compounds have attracted considerable interest, as they display a wide range of biological activities, including cytotoxic,6,48 antimicrobial,49 inhibitory activity against the tyrosine kinase EGFR24 and protein kinase C,48 and differentiation-inducing activity of K562 cells into erythroblasts.50 The nakijiquinones belong to a relatively rare group of natural products that selectively inhibit the Her-2/Neu receptor tyrosine kinase48 known to be overexpressed in approximately 30% of primary breast and gastric carcinomas.51−53 Structurally related sesquiterpene benzoxazoles (6 and 7) are rarely encountered in nature. Nakijinols A and B, isolated from Spongia sp.54 and Dactylospongia elegans,6 respectively, are so far the only representatives of this class of compounds. Structural analogues with a trans-fused 4,9friedodrimane skeleton are more commonly described in the literature. In this context, our report highlights the potential of derivatives with cis-fused 4,9-friedodrimane subunits as promising cytotoxic and protein kinase inhibiting agents.

■

ref

survival, proliferation, growth proliferation, survival

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on a JASCO P-2000 polarimeter. UV spectra were measured using a Perkin-Elmer Lambda 25 UV/vis spectrometer. NMR spectra were recorded on Bruker DMX 600, Bruker ARX 400, and Bruker DPX 300 spectrometers. EIMS was conducted on a Thermo Finnigan TCQ 7000 mass spectrometer, and HRESIMS spectra were obtained on a LTQ Orbitrap Velos Pro (Thermo Scientific). HPLC analysis was performed using a Dionex Ultimate 3000 System coupled to a photodiode array detector (DAD300RS). The separation column (125 × 4 mm, L × i.d.) was prefilled with Eurospher-10 C18 (Knauer, Germany), and the following gradient was used (MeOH, 0.1% HCOOH in H2O): 0 min, 10% MeOH; 5 min, 10% MeOH; 35 min, 100% MeOH; 45 min, 100% MeOH. Routine detection was at 235, 254, 280, and 340 nm. Semipreparative purification was accomplished on a Merck Hitachi system consisting of an L-7400 UV detector and an L-7100 pump connected with a Kipp & Zonen flatbed recorder. The attached column was a Knauer VertexPlus C18 column (Eurospher 100-10, 300 × 8 mm, L × i.d.). Column chromatography included G


2.22 3.66 4.81 2.20 19.7 8.62

SRC PRK1

>100 >100 >100 100.0 >100 21.7

PLK1 PIM1

62.6 >100 20.3 90.8 >100 7.72

NEK6 NEK2

29.9 >100 14.7 40.3 >100 3.12

MET wt

1.94 3.31 11.8 3.03 7.78 4.56

MEK1 wt IGF1-R FAK

■

61.0 >100 3.89 0.97 3.38 2.19

>100 14.7 13.1

Aurora-B ARK5

>100 >100 29.4

ALK

AXL

490.5 (2.50) nm; 1H and 13C NMR data, see Table 2; EIMS m/z (relative intensity %) 486 (M+, 11), 359 (7), 296 (100), 273 (9), 191 (8), 166 (12), 144 (35), 130 (58), 107 (12), 95 (25); HRESIMS m/z 487.2953 [M + H]+ (calcd for C31H39N2O3, 487.2955). 5-epi-Nakijiquinone U (4): red, amorphous solid; [α]23D −54 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204.5 (4.17), 327.8 (3.93), 493.9 (2.33) nm; 1H and 13C NMR data, see Table 2; EIMS m/z (relative intensity %) 431 (M+, 7), 241 (100), 223 (7), 191 (5), 166 (7), 152 (16), 121 (9), 107 (11), 95 (26); HRESIMS m/z 432.2567 [M + H]+ (calcd for C25H38NO3S, 432.2567). 5-epi-Nakijiquinone N (5): red, amorphous solid; [α]23D −26 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 206.3 (4.19), 322.2 (3.97), 495.1 (2.99) nm; 1H and 13C NMR data, see Table 1; EIMS m/z (relative intensity %) 413 (M+, 4), 223 (100), 209 (21), 191 (7), 166 (11), 152 (12), 121 (8), 107 (10), 95 (24); HRESIMS m/z 414.3006 [M + H]+ (calcd for C26H40NO3, 414.3003). 5-epi-Nakijinol C (6): pale yellow, amorphous solid; [α]23D −33 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204.1 (3.97), 227.4 sh (3.58), 294.0 (3.46) nm; 1H and 13C NMR data, see Table 3; EIMS m/z (relative intensity %) 383 (M+, 6), 193 (100), 191 (21), 177 (4), 147 (3), 135 (6), 121 (12), 107 (15), 95 (41); HRESIMS m/z 384.2532 [M + H]+ (calcd for C24H34NO3, 384.2533). 5-epi-Nakijinol D (7): pale yellow, amorphous solid; [α]23D −41 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 219.0 (4.01), 248.9 (3.62), 284.1 (3.53), 288.9 (3.52), 295.0 (3.58) nm; 1H and 13C NMR data, see Table 3; EIMS m/z (%) 392 (M+, 2), 202 (100), 191 (21), 135 (4), 121 (9), 107 (11), 95 (29); HRESIMS m/z 393.2535 [M + H]+ (calcd for C25H33N2O2, 393.2537). Cell Proliferation Assay. Cytotoxicity was tested against L5178Y mouse lymphoma cells using a microculture tetrazolium (MTT) assay and compared to that of untreated controls as previously described.55 Experiments were repeated three times and carried out in triplicate. As negative controls, media with 0.1% EGMME-DMSO were included in the experiments. Protein Kinase Activity Assay. The inhibitory profiles of the compounds were determined using 16 protein kinases, namely, AKT1, ALK, ARK5, Aurora-B, AXL, FAK, IGF1-R, MEK1 wt, METwt, NEK2, NEK6, PIM1, PLK1, PRK1, SRC, and VEGF-R2. A radiometric protein kinase assay (33PanQinase activity assay) was used for measuring the kinase activity of the 16 protein kinases as previously described.56 Briefly, recombinant protein kinases were incubated with a mixture of [γ-33P]-labeled ATP, unlabeled ATP, and kinase substrate. After kinase reaction, incorporation of labeled ATP on the substrate was measured using 96-well FlashPlates from Perkin-Elmer/NEN.

ASSOCIATED CONTENT

S Supporting Information *

Residual activity values of tested protein kinases upon treatment with 1−8, 1H and 13C NMR and HMBC spectra of 1−8, and the ROESY spectrum of 1 are available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*Tel: +49 211 81-14173. Fax: +49 211 81-11923. E-mail: amal. [email protected] (A. H. Aly). *Tel: +49 211 81-14163. Fax: +49 211 81-11923. E-mail: [email protected] (P. Proksch).

73.2 >100 48.4

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Financial support by BMBF is gratefully acknowledged. The authors wish to acknowledge the assistance and collaboration of Dr. E. Ferdinandus from Ambon University, Indonesia, during the sponge collection.

5 6 8

AKT1

Notes

compound

IC50 [μM]

Table 6. IC50 Values of 5, 6, and 8 against 16 Different Protein Kinases

Article

>100 >100 >100

VEGF-R2


H



■

Article

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Cytotoxic and Protein Kinase Inhibiting Nakijiquinones and

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