Sesquiterpenes with inhibitory activity against CDC25 phosphatases from the soft coral Pseudopterogorgia rigida

Tetrahedron 72 (2016) 3262e3269

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Sesquiterpenes with inhibitory activity against CDC25 phosphatases from the soft coral Pseudopterogorgia rigida Panagiota Georgantea a, Efstathia Ioannou a, Emilie Evain-Bana b, Denyse Bagrel b, Nadine Martinet c, Constantinos Vagias a, y, Vassilios Roussis a, * a

Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece Laboratoire Structure et R eactivit e des Syst emes Mol eculaires Complexes, UMR CNRS 7565, Universit e de Lorraine, Campus Bridoux, Rue du G en eral Delestraint, 57070, Metz, France c Institut de Chimie, UMR CNRS 7272, UNSA, F-06108 Nice, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 November 2015 Received in revised form 1 April 2016 Accepted 21 April 2016 Available online 23 April 2016

Twenty one sesquiterpenes were isolated from the organic extract of the Caribbean gorgonian Pseudopterogogia rigida. Among them, seven bisabolanes (1, 2, 9e11, 13 and 14), six cadinanes (15e20) and one sesquiterpene featuring a new tricyclic carbon skeleton (21) are new natural products, while two (6 and 7) are reported for the first time from a natural source. The structures of the new compounds were established by detailed analyses of their NMR and MS data. The isolated metabolites were evaluated for their inhibitory activity against CDC25 phosphatases, which represent possible targets related to cancer therapy. Compounds 4 and 18 were the most active with IC50 values of 1.88 and 3.44 mM, respectively, against the CDC25A isoform. Ó 2016 Elsevier Ltd. All rights reserved.

Keywords: Pseudopterogorgia rigida Bisabolane Cadinane Eleutherane CDC25 phosphatases inhibitory activity

1. Introduction Gorgonian octocorals are found throughout the world’s oceans and are ecologically important members of coral reef communities. Gorgonians represent a seemingly endless source of structurally complex and biologically active compounds.1,2 In particular, soft corals of the genus Pseudopterogorgia are considered to be among the most prolific producers of bioactive metabolites, such as pseudopterosin A with anti-inflammatory activity, ileabethoxazole with anti-tuberculosis activity, curcuphenol with antimicrobial activity, bis(pseudopterane) amine with antitumor activity and aberrarone with antimalarial activity.3 Their interesting structural diversity, coupled with their biomedical potential, have rendered these compounds prime targets for natural product and synthetic chemists alike.4 In 1978 Fenical and co-workers reported the isolation and characterization of four bisabolane sesquiterpenes with antibacterial properties from the Caribbean sea plume Pseudopterogorgia

* Corresponding author. Tel./fax: þ30 210 7274592; e-mail address: roussis@ pharm.uoa.gr (V. Roussis). y Deceased. http://dx.doi.org/10.1016/j.tet.2016.04.059 0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

rigida Bielschowsky.5 For more than two decades that report remained the only in-depth study on the chemistry of this species. Recently, we initiated a thorough investigation of P. rigida, which has led to the isolation of two new bisabolane dimers and a variety of new bisabolane and chamigrane sesquiterpenes.6,7 Herein, we report the isolation and structure elucidation of seven new bisabolanes (1, 2, 9e11, 13 and 14), six new cadinanes (15e20) and eleutheradione (21) featuring a new carbocycle, along with seven previously described compounds (3e8 and 12). Among the known metabolites, 6 and 7 were previously reported as semisynthetic compounds and are isolated for the first time from a natural source. Compounds 1e6, 8, 10, 11, 13 and 15e19 were evaluated for their inhibitory activity against the Cell Division Cycle (CDC) 25 proteins. CDC25s are key phosphatases, which exist as three isoforms (AeC), regulating cell cycle transitions and thus stimulating proliferation by activating CDK/cyclin complexes. Over-expression of these enzymes is frequently observed in several types of tumors and is related to cancer aggressiveness, high-grade tumors and poor prognosis.8,9 Thus, within the context of searching for new targets related to cancer therapy, CDC25 phosphatases are interesting candidates, due to the fact that their inhibition can slow down tumor growth and eventually improve the cancer treatments currently in use.10

P. Georgantea et al. / Tetrahedron 72 (2016) 3262e3269

2. Results and discussion Specimens of the gorgonian P. rigida were freeze-dried and exhaustively extracted at room temperature with mixtures of CH2Cl2/ MeOH to afford a dark orange oily residue. The organic extract was subsequently subjected to a series of chromatographic separations that led to the isolation of compounds 1e21. Rigidamide (1), obtained as a yellowish oil, displayed an ion peak at m/z 304.1551 (HR-ESIMS), corresponding to C17H22NO4 and consistent with [MH]. The 13C NMR spectrum and DEPT experiments revealed the presence of eight quaternary, two methine, two methylene and five methyl carbon atoms, including three carbonyl groups at dC 162.3, 182.1 and 183.2 and six olefinic carbons resonating at dC 124.2, 124.5, 131.3, 134.6, 142.9 and 150.7 (Table 1). The 1H NMR spectrum of 1 exhibited four singlet methyls at dH 1.51, 1.62, 2.04 and 3.09, one doublet methyl at dH 1.19, one olefinic methine at dH 5.03 and one aldehyde proton at dH 7.96 (Table 2). Taking into consideration the three carbonyls and the three carboncarbon double bonds as six of the seven degrees of unsaturation,

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the molecule was assumed to be monocyclic. The COSY cross-peaks of H-7/H3-14, H-7/H2-8, H2-8/H2-9 and H2-9/H-10 defined the only spin system in the molecule, which in combination with the HMBC correlations of H3-12 and H3-13 with C-10 and C-11 identified the 6-methylhept-5-en-2-yl side chain. The HMBC correlations of H315 with C-2, C-3 and C-4, of OH-5 with C-4, C-5 and C-6 and of H-7 with C-1, C-5 and C-6 outlined a fully substituted p-quinone ring, as in the case of hydroxy-perezone (5), and verified the connection of the side chain at C-6. The HMBC correlations of H3-17 with C-2 and C-16 concluded the fully substituted p-quinone ring and placed the amide functionality at C-2. Riboperezone (2), isolated as a purple oil, displayed a pseudomolecular ion peak at m/z 365.1875 (HR-ESIMS), corresponding to C20H29O6 and consistent with [MþH]þ. The 13C NMR spectrum and DEPT experiments revealed the presence of seven quaternary, five methine, four methylene and four methyl carbon atoms. Among them, six olefinic carbons at dC 121.2, 126.5, 131.4, 141.2, 148.8 and 148.9, two carbonyl groups both resonating at dC 181.3 and four oxygenated carbons at dC 63.2, 72.5, 86.6 and 89.6 were evident

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5,9-Epoxy-curcuquinone (9) was isolated as a yellowish oil. Its molecular formula was postulated to be C15H18O3 by a combined analysis of the HR-FABMS and 13C NMR data. The IR spectrum showed an absorption band at 1718 cm1 supporting the presence of carbonyl functionalities, while the absorption band at 1095 cm1 indicated an ether moiety in the molecule. The 1H and 13C NMR spectra displayed resonances for an isopropylidene group (dH/C 5.30/122.9, 1.71/18.4, 1.76/25.8), a doublet methyl on a benzylic methine (dH/C 1.22/21.5, 2.90/23.3), a vinylic methyl (dH/C 1.99/15.3), an olefinic methine (dH/C 6.44/133.6), an oxygenated methine (dH/C 4.79/71.0) and an aliphatic methylene (dH/C 1.63, 1.72/33.9), as well as three olefinic quaternary carbons (dC 122.9, 142.6 and 153.5) and two carbonyls (dC 182.4 and 186.9) (Tables 1 and 2). The COSY crosspeaks of H-7/H3-14, H-7/H2-8, H2-8/H-9 and H-9/H-10 defined the only spin system in the molecule. The HMBC correlations of H-2 with C-4, C-6 and C-15, of H3-15 with C-2, C-3 and C-4 and of H-7 with C-1, C-5 and C-6 completed a trisubstituted quinone ring and verified the connection of the side chain at C-6. The downfield shift of the phenolic carbon C-5 at dC 153.5, in combination with the bicyclic structure of the molecule as indicated by the seven degrees of unsaturation, implied the formation of an ether bridge between C-5 and the oxygenated methine C-9. The observed correlations could be consistent with either an o-quinone or a p-quinone system. Proof that compound 9 possessed a p-quinone ring was provided when addition of o-phenylenediamine to 9 failed to yield the condensation phenazine product that would be expected for an oquinone system. The relative configurations at C-7 and C-9 were determined as 7R*,9S* on the basis of an NOE correlation observed between H-9 and H3-14. Oxazocurcuphenol (10), isolated as a yellowish oil, displayed a pseudomolecular ion peak at m/z 260.1652 (HR-ESIMS), corresponding to C16H22NO2 and consistent with [MþH]þ. The assigned molecular formula C16H21NO2 indicated seven degrees of unsaturation. The IR spectrum showed an absorption band at 3263 cm1 for hydroxy groups, as well as absorption bands at 1098 cm1 and 1040 cm1 indicative of the presence of CeN bonds in the molecule. Its 1H and 13C NMR spectra exhibited signals characteristic for aromatic bisabolane sesquiterpenes (Tables 1 and 2). In particular, the 1H NMR spectrum of 10 included signals for an aliphatic methyl (dH 1.37) on a benzylic methine, an aromatic methyl (dH

Table 1 13 C NMR (50 MHz) spectroscopic dataa of compounds 1, 2 and 8e11 Position 1

2

8

9

10

11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 10 20 30 40 50

181.3, C 148.8, C 141.2, C 181.3, C 148.9, C 121.2, C 30.8, CH 35.9, CH2 28.0, CH2 126.5, CH 131.4, C 25.9, CH3 17.8, CH3 19.2, CH3 11.3, CH3

187.4, C 133.0, CH 145.2, C 188.4, C 131.0, CH 153.8, C 31.3, CH 32.5, CH2 32.5, CH2 75.8, CH 147.1, C 17.4, CH3 111.4, CH2 19.5, CH3 15.4, CH3

186.9, C 133.6, CH 142.6, C 182.4, C 153.5, C 122.9, C 23.3, CH 33.9, CH2 71.0, CH 122.9, CH 138.4, C 25.8, CH3 18.4, CH3 21.5, CH3 15.3, CH3

149.0, C 133.2, C 127.9, C 114.3, CH 148.7, C 113.9, C 30.4, CH 35.8, CH2 26.5, CH2 124.5, CH 131.9, C 25.9, CH3 18.0, CH3 20.3, CH3 16.4, CH3 150.1, CH

111.4, CH 147.6, C 122.6, C 111.4, CH 153.1, C 130.9, C 42.0, CH 91.4, CH 33.9, CH2 119.7, CH 134.6, C 26.3, CH3 18.8, CH3 19.9, CH3 16.8, CH3

a

182.1, C 142.9, C 134.6, C 183.2, C 150.7, C 124.2, C 30.0, CH 33.4, CH2 26.9, CH2 124.5, CH 131.3, C 26.0, CH3 18.3, CH3 18.5, CH3 11.9, CH3 162.3, CH 33.4, CH3

86.6, 41.9, 72.5, 89.6, 63.2,

CH CH2 CH CH CH2

Recorded in CDCl3 for compounds 1 and 8e11 and in CD3OD for compound 2.

(Table 1). In the 1H NMR spectrum the presence of an isopropylidene group at dH 1.53, 1.61 and 5.08, an aromatic methyl at dH 1.85 and a doublet methyl at dH 1.18 was obvious. Additionally, three oxygenated methines at dH 4.02, 4.56 and 6.47 and an oxygenated methylene at dH 3.74 and 3.82 were observed (Table 2). The presence of three carbon-carbon double bonds and two carbonyls, in combination with the seven degrees of unsaturation suggested by the molecular formula C20H28O6, indicated that metabolite 2 was bicyclic. Analysis of the 2D spectra revealed that 2 possessed a bisabolane structure with a fully substituted p-quinone ring, as in the case of compound 1. The COSY cross-peaks of H-10 /H2-20 , H2-20 / H-30 , H-30 /H-40 and H-40 /H2-50 , in conjunction with the HMBC correlations of H-10 with C-2 and C-3 indicated that the p-quinone ring was substituted at C-2 with 2-deoxyribose.

Table 2 1 H NMR (400 MHz) spectroscopic dataa of compounds 1, 2 and 9e11 Position 1 2 4 7 8 9 10 12 13 14 15 16 17 10 20 30 40 50 2-OH 5-OH 30 -OH 50 -OH a

1

2

9

10

11 6.55 s

6.44 d (1.7) 3.06 1.76 1.92 5.03 1.62 1.51 1.19 2.04 7.96 3.09

m m, 1.60 m m, 1.83 m brs s s d (5.9) s s s

3.08 1.90 1.90 5.08 1.61 1.53 1.18 1.85

m m, 1.51 m m m s s brd (6.5) s

6.47 2.76 4.56 4.02 3.82

dd (6.6, 6.6) m, 2.49 m ddd (6.0, 3.3, 3.3) ddd (3.3, 3.3, 3.3) m, 3.74 m

2.90 1.72 4.79 5.30 1.76 1.71 1.22 1.99

m m, 1.63 m ddd (11.2, 8.7, 2.5) brd (8.7) s s d (7.0) d (1.7)

6.63 3.23 1.94 1.84 5.08 1.64 1.44 1.37 2.52 7.93

s m m, 1.74 m m m s s d (7.0) s s

6.52 3.06 4.19 2.44 5.22 1.71 1.62 1.24 2.17

s dq (6.9, 6.9) ddd (6.9, 6.9, 6.9) m m s s d (6.9) s

4.24 brs 7.14 brs

8.40 brs 8.38 brs 8.00 brs

Recorded in CDCl3 for compounds 1 and 9e11 and in CD3OD for compound 2.

4.77 brs

P. Georgantea et al. / Tetrahedron 72 (2016) 3262e3269

2.52), an isopropylidene group (dH 5.08, 1H; 1.64, 3H; 1.44, 3H) and an aromatic proton (dH 6.63). In addition, a quite deshielded methine at dH 7.93 was observed. The sequence of the carbons in the molecule was confirmed by the strong HMBC correlations of H4 with C-2, C-6 and C-15, of H3-15 with C-2, C-3 and C-4, of H-7 with C-5, of H3-14 with C-6, C-7 and C-8 and of H3-12 and H3-13 with C-10 and C-11. The key HMBC interactions of the olefinic methine at dH 7.93 with both C-1 (dC 149.0) and C-2 (dC 133.2), combined with the necessity of a second ring in the molecule to justify the seven degrees of unsaturation, dictated the presence of an oxazoline ring. Heliannuol N (11), obtained as yellowish oil, displayed in the HR-FABMS a molecular ion peak [M]þ at m/z 232.1453, corresponding to the molecular formula C15H20O2. Detailed comparison of the 1H and 13C NMR spectra of 11 with those of heliannuol H (12) indicated the dehydration of the isopropyl group and the simultaneous transposition of the double bond between C-10 and C-11 resulting to an isopropylidene group (Tables 1 and 2). This hypothesis was verified by the correlations observed in the 2D NMR spectra of 11. The NOE correlation between H-8 and H3-14 established the relative configurations at C-7 and C-8 as 7R*,8S*. Additionally, two metabolites identified as perezone sodium salt (13) and rigidamide sodium salt (14) were isolated from the polar fractions of the extract as purple powders. Interestingly, P. rigida is colored purple in its natural environment. Both compounds exhibited rather similar spectroscopic data to those of their respective parent metabolites. The structures were verified when small quantities of perezone (4) and rigidamide (1) were treated with NaOH 1 N to yield compounds with identical spectroscopic and physical characteristics with those of 13 and 14, respectively. Pseudorigidone A (15) was isolated as a brownish oil and displayed a molecular ion peak [M]þ at m/z 232.1444 in the HR-FABMS, corresponding to the molecular formula C15H20O2. The IR spectrum showed absorption bands at 1650 cm1 and 1125 cm1, characteristic for unsaturated ketone carbonyls and ether moieties, respectively. The 13C NMR spectrum and DEPT experiments revealed the presence of 15 carbons, which corresponded to five quaternary, four methine, two methylene and four methyl carbon atoms, including four olefinic carbons and one carbonyl (Table 3). The 1H NMR spectrum disclosed signals for a doublet methyl (dH 1.15), two deshielded singlet methyls (dH 1.49 and 1.62), a vinylic methyl (dH 1.89) and two olefinic methines (dH 6.06 and 7.07) (Table 4). The two carbon-carbon double bonds and the carbonyl accounted for three of the six degrees of unsaturation, thus indicating a tricyclic structure for metabolite 15. The correlations of the vinylic H3-15

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with the carbonyl C-2 (dC 186.5), the quaternary C-3 (dC 133.8) and the tertiary C-4 (dC 145.4), of H-4 with C-2 and the quaternary C-6 (dC 163.7) and of H-1 with the aliphatic C-7 (dC 29.1) and the oxygenated C-5 (dC 74.7) observed in the HMBC spectrum defined the first six-membered ring. The HMBC correlations of H-10 with C-4 and C-5 and of H3-14 with C-6 and C-7, in combination with the COSY cross-peaks of H-7/H3-14, H-7/H2-8, H2-8/H2-9 and H2-9/H10 completed the second six-membered ring of the cadinane skeleton. Furthermore, the HMBC correlations of H3-12 and H3-13 with the oxygenated C-11 (dC 82.1) and the aliphatic C-10 (dC 51.0), in conjunction with the necessity for an additional ring dictated the formation of an oxetane between C-5 and C-11. The relative configurations at C-5, C-7 and C-10 were determined as 5R*,7R*,10S* on the basis of the observed NOESY cross-peaks of H-4/H-10, H-4/H312 and H-7/H3-13. Pseudorigidones B (16) and C (17), obtained as pale green oil and yellowish oil, respectively, were assigned the molecular formula C16H22O3, consistent with six degrees of unsaturation. Their IR spectra showed absorption bands at approx. 1170 cm1 supporting the presence of ether moieties, while the absorption bands at approx. 1645 cm1 indicated the presence of unsaturated ketone carbonyls in the molecules. The 1H and 13C NMR spectra of 16 and 17 revealed a considerable degree of similarity to those of 15, implying that both molecules featured the cadinane skeleton (Tables 3 and 4). Characteristic differences in the case of 16 and 17 were the appearance of a methyl of a methoxy group (dH 2.98 for both compounds) and of an oxygenated methine (dH 4.36 for 16 and 4.29 for 17), whereas H3-12 and H3-13 resonances were shifted upfield (dH 1.23 and 1.04 for 16 and 1.03 and 0.95 for 17). The HMBC correlations of H-1 with C-3, C-5, and C-7, of H-4 with C-2, C-5 and C15, of H3-15 with C-2, C-3 and C-4, of H3-14 with C-6, C-7 and C-8, of H3-12 and H3-13 with C-10 and C-11, and, finally, of H3-16 with C11, C-12 and C-13, as well as the HMBC correlation of H-8 with C-5 supporting the formation of the oxygen bridge connecting C-5 and C-8, enabled the assembly of the planar structure of the two epimeric molecules. The NOE enhancements of H-1/H-7, H-7/H-8, H-7/ H-10 and H-4/H3-13, as well as the absence of COSY cross-peak between H-7 and H-8 implying a dihedral angle of 90 , determined the relative configurations at C-5, C-7, C-8 and C-10 of 16 as 5R*,7R*,8S*,10S*. In contrast, the relative configurations at C-5, C7, C-8 and C-10 of 17 were assigned as 5R*,7R*,8S*,10R* on the basis of the NOESY cross-peaks of H-1/H3-14, H-7/H-9a, H-9a/H3-12, H4/H-10 and H-10/H3-13 and the lack of COSY correlation between H-7 and H-8. Therefore, metabolites 16 and 17 were identified as epimers at C-10.

Table 3 13 C NMR (50 MHz) spectroscopic dataa of compounds 15e21 Position

15

16

17

18

19

20

21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

122.6, CH 186.5, C 133.8, C 145.4, CH 74.7, C 163.7, C 29.1, CH 32.4, CH2 18.7, CH2 51.0, CH 82.1, C 32.4, CH3 24.6, CH3 17.2, CH3 15.2, CH3

117.2, CH 187.1, C 135.4, C 138.6, CH 82.9, C 171.9, C 42.8, CH 82.4, CH 34.0, CH2 51.6, CH 76.7, C 22.8, CH3 23.9, CH3 18.6, CH3 16.5, CH3 48.4, CH3

118.9, CH 187.1, C 138.8, C 140.1, CH 82.7, C 171.7, C 42.9, CH 83.3, CH 33.0, CH2 57.1, CH 73.4, C 25.7, CH3 23.8, CH3 19.0, CH3 17.3, CH3 49.4, CH3

199.2, C 138.5, CH 149.6, C 198.8, C 46.9, CH 49.2, CH 27.6, CH 27.1, CH2 22.9, CH2 40.0, CH 143.1, C 24.0, CH3 113.1, CH2 13.1, CH3 16.6, CH3

146.6, C 115.4, CH 115.5, C 149.4, C 129.0, C 124.1, C 26.2, CH 31.2, CH2 18.4, CH2 49.8, CH 92.1, C 27.2, CH3 21.4, CH3 21.0, CH3 14.8, CH3

147.2, C 115.8, CH 116.8, C 149.1, C n.d.b, C 122.9, C 28.8, CH 34.2, CH2 24.4, CH2 49.9, CH 91.8, C 28.1, CH3 22.5, CH3 20.8, CH3 15.3, CH3

201.3, C 140.2, CH 152.4, C 202.1, C 45.4, CH 58.9, C 31.6, CH 24.6, CH2 25.1, CH2 45.7, CH 45.4, C 19.9, CH3 25.0, CH3 18.8, CH3 16.6, CH3

a b

Recorded in CDCl3 for compounds 15e21. n.d.: not detected.

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Table 4 1 H NMR (400 MHz) spectroscopic dataa of compounds 15e21 Position

15

16

17

1 2 4 5 6 7 8 9

6.06 s

5.92 s

5.95 s

7.07 d (1.4)

7.22 d (1.4)

6.77 d (1.5)

3.32 m 2.34 m, 1.36 m 1.76 m, 1.55 m

2.59 q (7.1) 4.36 t (2.9) 1.77 dd (7.7, 2.9)

10 12 13 14 15 16 1-OH

2.85 1.62 1.49 1.15 1.89

2.15 1.23 1.04 1.12 1.94 2.98

a

dd (7.2, 1.7) s s d (6.5) d (1.4)

t (7.7) s s d (7.1) d (1.4) s

2.67 q (7.1) 4.29 d (5.5) a 1.83 dd (11.6, 5.5), b 2.14 ddd (11.6, 11.6, 5.5) 2.32 dd (11.6, 5.5) 1.03 s 0.95 s 1.13 d (7.1) 1.91 d (1.5) 2.98 s

18

19

20

21

6.59 d (1.1)

6.29 s

6.27 s

6.51 d (1.5)

2.92 3.33 2.63 1.56 1.80

dd (11.3, 3.7) dd (11.3, 3.7) m m, 1.38 m m, 1.63 m

2.55 s 2.99 m 1.85 m 1.68 m, 1.47 m

2.85 m a 2.19 m, b 1.32 m 1.82 m, 1.29 m

2.63 m 1.94 m, 1.21 m 1.98 m, 1.85 m

3.13 1.76 4.96 0.95 1.98

m s s, 4.27 s d (7.0) d (1.1)

2.94 1.59 1.10 1.18 2.08

2.94 1.58 1.05 1.34 2.08

2.46 0.96 0.91 0.82 2.04

dd (12.0, 5.7) s s d (7.2) s

4.19 brs

m s s d (7.2) s

dd (4.1, 1.4) s s d (6.6) d (1.5)

4.20 s

Recorded in CDCl3 for compounds 15e21.

Pseudorigidone D (18) was obtained as a yellowish amorphous solid. The molecular ion at m/z 232.1473 (HR-FABMS) and 13C NMR data established the molecular formula C15H20O2, dictating six degrees of unsaturation. The 13C NMR spectrum and DEPT experiments revealed the presence of 15 carbons, which corresponded to four quaternary, five methine, three methylene and three methyl carbon atoms. Among them, four olefinic carbons at dC 113.1, 138.5, 143.1 and 149.6 and two carbonyl groups at dC 198.8 and 199.2 were evident (Table 3). The 1H NMR spectrum of 18 included signals for an aliphatic methyl on a tertiary carbon at dH 0.95, two vinylic methyls at dH 1.76 and 1.98, a terminal methylene at dH 4.27 and 4.96 and an olefinic methine at dH 6.59 (Table 4). Since the molecule contained two carbon-carbon double bonds and two carbonyls, the structure of 18 was assumed to be bicyclic. The cross-peaks of H-5/ H-6, H-6/H-7, H-7/H2-8, H-7/H3-14, H2-8/H2-9, H2-9/H-10 and H10/H-5 observed in the COSY spectrum concluded the cyclohexane ring of the molecule, while the HMBC correlations of H3-12 and H213 with C-10 placed the isopropenyl group at C-10. Moreover, the HMBC correlations of H3-15 with C-2, C-3 and C-4, of H-6 with C-1 and of H-5 with C-4 completed the second ring of the cadinane skeleton. The relative configurations of the asymmetric centers of 18 were proposed on the basis of the interactions observed in the NOESY spectrum. Specifically, the NOE enhancements of H-5/H-10, H-5/H3-14 and H-6/H-13b determined the relative configurations at C-5, C-6, C-7 and C-10 as 5R*,6R*,7R*,10R*. Pseudorigidol A (19) was obtained as a brownish oil. Its molecular formula was determined as C15H20O2 by a combined analysis of the HR-FABMS and 13C NMR data. The absorption bands at 3390 cm1 and 1086 cm1 in the IR spectrum indicated the presence of hydroxy and ether moieties, respectively. The 13C NMR spectrum revealed 15 signals, including an oxygenated carbon resonating at dC 92.1 and six sp2 carbon atoms resonating at dC 115.4, 115.5, 124.1, 129.0, 146.6 and 149.4 (Table 3). In the 1H NMR spectrum three singlet methyls at dH 1.10, 1.59 and 2.08, one doublet methyl at dH 1.18 and an aromatic proton at dH 6.29 were evident (Table 4). Since the three carbon-carbon double bonds accounted for three of the six degrees of unsaturation, the molecule was assumed to be tricyclic. The HMBC correlations of H-2 with C-4 (dC 149.4), C-6 (dC 124.1) and C-15 (dC 14.8), of H3-15 with C-2 (dC 115.4), C-3 (dC 115.5) and C-4 and of OH-1 with C-1 (dC 146.6) concluded the pentasubstituted aromatic ring. The COSY cross-peaks of H-7/ H3-14, H-7/H2-8, H2-8/H2-9 and H2-9/H-10 defined the only spin system in the molecule, which in conjunction with the HMBC correlations of H-10 with C-6 and of H3-14 with C-6, C-7 (dC 26.2) and C-8 (dC 31.2) completed the second six-membered ring of the cadinane carbocycle. Furthermore, the two singlet methyls

resonating at dH 1.10 and 1.59 (H3-13 and H3-12, respectively) were strongly correlated with C-10 (dC 49.8) and the oxygenated C-11 (dC 92.1). Given the tricyclic skeleton of 19, the presence of an ether bridge between the oxygenated C-11 and C-4 was dictated. The relative configurations of the chiral centers of 19 were assigned on the basis of 1D NOE experiments, which were measured using C6D6 as the solvent, since H2-8 were resolved and could be more readily distinguished. In particular, the strong NOE interactions between H-8a (dH 1.56) and both H-7 (dH 3.02) and H-10 (dH 2.76) established the relative configurations at C-7 and C-10 as 7R*,10S*. Pseudorigidol B (20) was isolated as a yellowish oil. The molecular ion peak [M]þ at m/z 232.1480 observed in the HR-FABMS suggested the same molecular formula as in 19. The structural elements displayed in the 1H and 13C NMR spectra of 20 exhibited a high degree of similarity with those of metabolite 19 (Tables 3 and 4). In agreement with the molecular formula, it was obvious that 20 was an isomer of the latter. Analyses of its 2D NMR spectra resulted in the establishment of the same planar structure, implying that 20 was an epimer of 19 at C-10. Indeed, the key NOE enhancements between H-7/H-8a and H-8b/H-10 observed in the NOESY spectrum and 1D NOE experiments established the relative configurations of the two chiral centres of the molecule as 7R*,10R*. Eleutheradione (21), isolated as a yellowish oil, had the molecular formula C15H20O2, as deduced from the HR-ESIMS and 13C NMR data. The 13C NMR spectrum and DEPT experiments revealed four methyl, two methylene, three methine and two quaternary carbon atoms in the aliphatic region and one methine and one quaternary carbon atom in the olefinic region, as well as two carbonyls (Table 3). Detailed comparison of the spectroscopic data of 21 with those of 18 showed that the 1H NMR spectrum of 21 lacked the signals of H-6, the vinylic methyl H3-12 and the terminal methylene H2-13. Instead, two singlet methyls at dH 0.96 and 0.91 corresponding to H3-12 and H3-13, respectively, appeared (Table 4). In the HMBC spectrum correlations of H3-12, H3-13 and H3-14 with C-6 were observed, indicating the formation of a single bond between C-6 and C-11. Furthermore, the correlations of H-5 with C-1, C-4, C-6, C7 and C-10 concluded the cyclohex-2-en-1,4-dione ring (Fig. 1a). The NOESY cross-peaks of H-5/H3-14 and H-7/H3-12, in conjunction with the absence of coupling between H-5 and H-10 in the COSY spectrum indicating a dihedral angle of approx. 90 , established the relative configurations at C-5, C-6, C-7 and C-10 as 5R*,6R*,7R*,10R* (Fig. 1b). Metabolite 21 possesses a new tricyclic carbon skeleton, designated as eleutherane after Eleuthera island where the gorgonian was collected, resulting from the additional linkage between C-6 and C-11 of the cadinane carbocycle.

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Fig. 1. (a) COSY and important HMBC correlations for compound 21. (b) Key NOESY interactions for compound 21.

Compounds 3e8 and 12 were identified by comparison of their spectroscopic and physical characteristics with those reported in the literature as curcuquinone (3) isolated from the soft coral P. rigida,5 perezone (4) isolated from the roots of Perezia alamani var. oolepis,11e15 hydroxy-perezone (5) isolated from the rhizome of the plant Perezia hebeclada,16 as well as glandulone E (8)17 and heliannuol H (12)18,19 isolated from the plant Helianthus annuus. Metabolites 620 and 721 were previously described as semisynthetic compounds and are reported here for the first time as natural products. The 13C NMR chemical shifts for compound 8 are presented in Table 1, supplementing the relevant literature, since only the 1H NMR resonances were available. On the basis of biosynthetic considerations the absolute configuration of compounds 1, 2, 9e11 and 13e20 at C-7 is expected to be R. In support of this, all known marine and terrestrial bisabolanes and cadinanes exhibit a 7R configuration with the exception of sponge-derived analogues which possess a 7S configuration.22 Compounds 1e6, 8, 10, 11, 13 and 15e19 were evaluated for their inhibitory activity against A, B and C recombinant human CDC25 phosphatases. According to the results (Table 5), metabolites 3, 4, 8 and 18 displayed significant levels of inhibitory activity against the three CDC25 isoforms. Since compounds 4 and 18 exhibited inhibitory activity higher than 85% against CDC25A, CDC25B and CDC25C at 100 mM, their IC50 was subsequently determined (Table 6). The IC50 values were lower than 10 mM, thus indicating a strong potential in CDC25 inhibition. It is worth-noting that even

Table 5 Inhibitory activity (expressed as percentage of inhibition) of compounds 1e6, 8, 10, 11, 13 and 15e19 evaluated at 100 mM against CDC25A, CDC25B and CDC25C phosphatases Compound

CDC25A

CDC25B

CDC25C

1 2 3 4 5 6 8 10 11 13 15 16 17 18 19 Naphthoquinone

60.772.50 79.111.31 92.800.92 93.372.76 46.013.42 78.703.94 93.933.29 57.513.15 74.392.50 31.672.89 64.863.29 65.981.31 77.851.18 93.682.76 72.501.58 98.681.58

47.230.79 64.723.94 79.661.71 87.453.15 41.630.66 51.711.18 85.682.63 41.201.84 50.902.89 28.863.15 31.142.89 35.883.68 52.372.10 86.161.58 40.892.23 99.661.71

65.353.68 77.323.02 83.623.94 90.901.71 49.972.63 67.302.63 78.033.02 53.971.05 67.832.89 28.991.97 55.000.66 50.521.45 65.852.37 90.090.92 61.281.58 99.972.63

though several of the tested compounds possessed a p-quinone ring, a structural feature that has been correlated in the past with CDC25 inhibitory activity,23,24 only perezone (4) displayed high activity with IC50 values below 5 mM. Further studies on the activity of 4 against cancer cell lines, including IC50 determination, cell cycle status evaluation, oxidative stress evaluation and correlation with proteic CDK/phospho-CDK status, would be of interest.

3. Experimental 3.1. General experimental procedures Optical rotations were measured on a PerkineElmer model 341 polarimeter with a 1 dm cell. UV spectra were acquired on a Shimadzu UV-160A spectrophotometer. IR spectra were obtained on a Paragon 500 PerkineElmer spectrometer. NMR spectra were recorded on Bruker AC 200 and Bruker DRX 400 spectrometers. Chemical shifts are given on a d (ppm) scale using TMS as internal standard. The 2D experiments (HSQC, HMBC, COSY, NOESY) were performed using standard Bruker pulse sequences. High resolution FAB mass spectra were recorded on a JEOL JMS AX505 HA mass spectrometer. High resolution ESI mass spectra were recorded on a Thermo Scientific LTQ Orbitrap Velos mass spectrometer. Low resolution EI mass spectra were measured on a Hewlett Packard 5973 mass spectrometer. Column chromatography separations were performed with Kieselgel 60 or C18-RP silica gel (Merck). HPLC separations were conducted on an Agilent 1100 liquid chromatography system equipped with refractive index detector, using the following columns: (i) Kromasil 100 C18 5 mm (MZ-Analysentechnik, 25 cm8 mm), (ii) Nucleosil SP250/10 100-7 C18 (Macherey-Nagel, 25 cm10 mm), (iii) Econosphere C18 5 mm (Alltech, 25 cm4.6 mm) and (iv) Chiralcel OD 10 mm (Daicel Chemical Industries Ltd., 25 cm10 mm). TLC were performed with Kieselgel 60 F254 (Merck aluminum support plates) and spots were detected after spraying with 15% H2SO4 in MeOH reagent and heating at 100  C for 1 min. The lyophilization was carried out in a Freezone 4.5 freeze dry system (Labconco).

Table 6 Inhibitory activity (expressed as IC50 values in mM) of compounds 4 and 18 against CDC25A, CDC25B and CDC25C phosphatases Compound

CDC25A

CDC25B

CDC25C

4 18

1.881.19 3.440.48

4.300.60 7.421.23

3.190.74 6.640.34

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3.2. Animal material Specimens of P. rigida were collected by SCUBA diving southeast of Lighthouse Point in Eleuthera island in the Bahamas, at a depth of 20e30 m. A voucher specimen has been deposited at the animal collection of the Department of Pharmacognosy and Chemistry of Natural Products, University of Athens (ATPH/MO/57). 3.3. Extraction and isolation Specimens of the freeze-dried gorgonian (280 g) were exhaustively extracted with mixtures of CH2Cl2/MeOH at room temperature. Evaporation of the solvents in vacuo afforded a dark orange oily residue (27.0 g) which was subjected to vacuum column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc, followed by EtOAc with increasing amounts of MeOH as the mobile phase, to yield 10 fractions (1e10). Fraction 2 (13.2 g) was divided into two parts (2A and 2B) and handled with two different ways. Part 2A (8.6 g) was fractionated by gravity column chromatography on silica gel, using cyclohexane/EtOAc (95:5) as the mobile phase, to yield 12 fractions (2A1e2A12). Part of fraction 2A8 (284.0 mg) was subjected to gravity column chromatography on silica gel, using cyclohexane/EtOAc (80:20) to afford 13 fractions (2A8Ae2A8M). Fraction 2A8B (12.4 mg) was purified by reversed phase HPLC, using MeOH/H2O (75:5) as eluent, to afford 4 (6.8 mg) and 13 (3.3 mg), whereas fraction 2A12 (90.0 mg) was purified using MeOH/H2O (80:20) as eluent, to afford 1 (6.5 mg) and 14 (1.5 mg). Part 2B (4.6 g) was fractionated by reversed phase vacuum column chromatography, using H2O with increasing amounts of MeOH as the mobile phase, to yield 15 fractions (2B1e2B15). Fraction 2B13 (390.0 mg) was subjected to reversed phase HPLC, using MeOH/H2O (80:20) as eluent, to yield 7 fractions (2B13Ae2B13G). Fractions 2B13C and 2B13E were metabolites 11 (1.6 mg) and 6 (4.4 mg), respectively. Fraction 2B13F (197.0 mg) was further fractionated by reversed phase HPLC, using MeOH/H2O (90:10) as eluent, to afford 7 fractions (2B13F1e2B13F7). Fraction 2B13F5 (8.2 mg) was purified by reversed phase HPLC, using MeOH/ H2O (70:30) as eluent, to afford 16 (1.0 mg) and 17 (1.0 mg). Fraction 2B13F7 (141.0 mg) was purified by chiral HPLC, using n-Hex/i-Prop (95:5) as eluent, to afford 3 (75.0 mg), 15 (1.0 mg), 18 (1.5 mg), 19 (2.2 mg), 20 (1.0 mg) and 21 (1.0 mg). Fraction 3 (1.0 g) was fractionated by gravity column chromatography on silica gel, using cyclohexane/EtOAc (95:5) as the mobile phase, to yield 12 fractions (3Ae3L). Fraction 3I (173 mg) was purified by reversed phase HPLC, using MeOH/H2O (90:10) as eluent, to afford 9 (2.2 mg). Fraction 4 (3.1 g) was fractionated by gravity column chromatography on silica gel, using cyclohexane/EtOAc (90:10) as the mobile phase, to yield 15 fractions (4Ae4O). Fraction 4G (835.0 mg) was purified by reversed phase HPLC, using MeOH/H2O (80:20) as eluent, to afford 10 (1.8 mg). Fraction 6 (408.0 mg) was purified by reversed phase HPLC, using MeOH/H2O (70:30) as eluent, to afford 12 (2.5 mg). Fraction 7 (487.0 mg) was fractionated by gravity column chromatography on silica gel, using cyclohexane/EtOAc (70:30) as the mobile phase, to yield 11 fractions (7Ae7K). Fraction 7F (50.0 mg) was purified by chiral HPLC, using n-Hex/i-Prop (85:15) as eluent, to afford 7 (1.2 mg) and 8 (2.5 mg). Fraction 10 (1.0 g) was fractionated by reversed phase vacuum column chromatography, using H2O with increasing amounts of MeOH as the mobile phase to afford 3 fractions (10Ae10C). Fraction 10A (70 mg) was purified by reversed phase HPLC, using MeCN/H2O (20:80) as eluent, to afford 2 (6.6 mg) and 5 (2.5 mg). 3.3.1. Rigidamide (1). Yellowish oil; [a]20 D þ5.4 (c 0.092, CHCl3); UV (CHCl3) lmax (log 3) 262 (3.70), 306 (3.47) nm; IR (thin film)

nmax 3400, 2926, 1680, 1655, 1373, 1267, 1082, 975, 755 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; EIMS 70 eV m/ z (rel int. %) 305 (27), 287 (5), 277 (6), 262 (5), 248 (10), 223 (59), 195 (100), 180 (79), 166 (29), 152 (12), 96 (12), 67 (22), 55 (26), 41 (45); HR-ESIMS m/z 304.1551 [MH] (calcd for C17H22NO4, 304.1554). 3.3.2. Riboperezone (2). Purple oil; [a]20 D þ12.0 (c 0.033, MeOH); UV (MeOH) lmax (log 3) 212 (3.6) nm; IR (thin film) nmax 3338, 2942, 2833, 1698, 1546, 1377, 1102, 1026 cm1; 1H NMR data, see Table 1; 13 C NMR data, see Table 2; HR-ESIMS m/z 365.1875 [MþH]þ (calcd for C20H29O6, 365.1959). 3.3.3. 5,9-Epoxy-curcuquinone (9). Yellowish oil; [a]20 D 11.0 (c 0.1, CHCl3); UV (CHCl3) lmax (log 3) 269 (3.59) nm; IR (thin film) nmax 1718, 1652, 1095, 736 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; EIMS 70 eV m/z (rel int. %) 246 (23), 231 (13), 218 (17), 204 (15), 191 (6), 176 (9), 136 (17), 108 (16), 96 (31), 82 (100), 67 (67), 53 (12); HR-FABMS m/z 246.1259 [M]þ (calcd for C15H18O3, 246.1256). 3.3.4. Oxazocurcuphenol (10). Yellowish oil; [a]20 D 4.0 (c 0.1, CHCl3); UV (CHCl3) lmax (log 3) 250 (3.23), 278 (3.00) nm; IR (thin film) nmax 3263, 2963, 1732, 1520, 1392, 1266, 1098, 1040 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; EIMS 70 eV m/z (rel int. %) 259 (33), 242 (1), 216 (1), 202 (5), 189 (23), 176 (100), 162 (16), 95 (13), 41 (9); HR-ESIMS m/z 260.1652 [MþH]þ (calcd for C16H22NO2, 260.1645). 3.3.5. Heliannuol N (11). Yellowish oil; [a]20 D 21.4 (c 0.019, CHCl3); UV (CHCl3) lmax (log 3) 265 (3.29), 301 (3.43) nm; IR (thin film) nmax 3418, 2911, 2357, 2225, 1650, 1169, 861 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; EIMS 70 eV m/z (rel int. %) 232 (95), 217 (2), 189 (3), 175 (60), 163 (100), 150 (15), 135 (60), 91 (19), 41 (16); HR-FABMS m/z 232.1453 [M]þ (calcd for C15H20O2, 232.1463). 3.3.6. Perezone sodium salt (13). Purple powder; 1H NMR (CD3OD) 6.20 (q, J¼1.6 Hz, H-2), 5.06 (m, H-10), 2.96 (m, H-7), 1.84 (m, H2-9), 1.83 (d, J¼1.6 Hz, H3-15), 1.58 (s, H3-12), 1.49 (s, H3-13), 1.48 (m, H8b), 1.45 (m, H-8a), 1.10 (d, J¼7.3 Hz, H3-14); 13C NMR (CD3OD) 189.4 (C-4), 187.4 (C-1), 170.6 (C-5), 140.3 (C-3), 139.0 (C-2), 130.8 (C-11), 126.9 (C-10), 121.1 (C-6), 35.9 (C-8), 30.5 (C-7), 28.2 (C-9), 25.9 (C12), 19.6 (C-14), 17.7 (C-13), 14.9 (C-15). 3.3.7. Rigidamide sodium salt (14). Purple powder; 1H NMR (CD3OD) 7.93 (s, H-16), 5.09 (m, H-10), 3.04 (m, H-7), 3.03 (s, N-Me), 1.89 (s, H3-15), 1.62 (s, H3-12), 1.53 (s, H3-13), 1.16 (d, J¼6.9 Hz, H3-14). 3.3.8. Pseudorigidone A (15). Brownish oil; [a]20 D 19.3 (c 0.05, CHCl3); UV (CHCl3) lmax (log 3) 251 (3.95) nm; IR (thin film) nmax 1650, 1125, 746 cm1; 1H NMR data, see Table 3; 13C NMR data, see Table 4; EIMS 70 eV m/z (rel int. %) 232 (92), 217 (23), 203 (12), 189 (100), 175 (54), 161 (72), 147 (37), 91 (35), 77 (29); HR-FABMS m/z 232.1444 [M]þ (calcd for C15H20O2, 232.1463). 3.3.9. Pseudorigidone B (16). Pale green oil; [a]20 D þ7.2 (c 0.042, CHCl3); UV (CHCl3) lmax (log 3) 253 (3.59) nm; IR (thin film) nmax 2347, 2235, 1645, 1173 cm1; 1H NMR data, see Table 3; 13C NMR data, see Table 4; EIMS 70 eV m/z (rel int. %) 262 (25), 230 (27), 215 (27), 201 (13), 188 (11), 174 (95), 162 (100), 147 (37), 91 (24), 73 (30); HR-ESIMS m/z 263.1639 [MþH]þ (calcd for C16H23O3, 263.1647). 3.3.10. Pseudorigidone C (17). Yellowish oil; [a]20 D 24.0 (c 0.042, CHCl3); UV (CHCl3) lmax (log 3) 252 (3.58) nm; IR (thin film) nmax

P. Georgantea et al. / Tetrahedron 72 (2016) 3262e3269

2349, 2234, 1644, 1171 cm1; 1H NMR data, see Table 3; 13C NMR data, see Table 4; EIMS 70 eV m/z (rel int. %) 262 (31), 230 (29), 215 (31), 201 (14), 187 (13), 174 (100), 162 (78), 147 (31), 91 (23), 73 (36); HR-ESIMS m/z 285.1458 [MþNa]þ (calcd for C16H22O3Na, 285.1467). 3.3.11. Pseudorigidone D (18). Yellowish amorphous solid; [a]20 D 9.6 (c 0.083, CHCl3); UV (CHCl3) lmax (log 3) 246 (3.56) nm; IR (thin film) nmax 2338, 2235, 1678, 1164 cm1; 1H NMR data, see Table 3; 13C NMR data, see Table 4; EIMS 70 eV m/z (rel int. %) 232 (100), 215 (57), 202 (42), 189 (58), 175 (60), 161 (20), 145 (18), 128 (16), 115 (19), 91 (24), 77 (17), 41 (11); HR-FABMS m/z 232.1473 [M]þ (calcd for C15H20O2, 232.1463). 3.3.12. Pseudorigidol A (19). Brownish oil; [a]20 D 30.0 (c 0.053, CHCl3); UV (CHCl3) lmax (log 3) 247 (3.17), 300 (3.33) nm; IR (thin film) nmax 3390 (broad), 2860, 1652, 1086 cm1; 1H NMR data, see Table 3; 13C NMR data, see Table 4; EIMS 70 eV m/z (rel int. %) 232 (100), 217 (33), 204 (6), 189 (43), 175 (45), 161 (11), 128 (8), 115 (8), 91 (8), 77 (6); HR-FABMS m/z 232.1468 [M]þ (calcd for C15H20O2, 232.1463). 3.3.13. Pseudorigidol B (20). Yellowish oil; [a]20 D 16.9 (c 0.083, CHCl3); UV (CHCl3) lmax (log 3) 246 (3.39), 302 (3.41) nm; IR (thin film) nmax 3390 (broad), 2860, 1652, 1086 cm1; 1H NMR data, see Table 3; 13C NMR data, see Table 4; EIMS 70 eV m/z (rel int. %) 232 (100), 217 (33), 215 (33), 202 (21), 189 (48), 175 (45), 161 (12), 128 (13), 115 (14), 91 (15), 77 (11); HR-FABMS m/z 232.1480 [M]þ (calcd for C15H20O2, 232.1463). 3.3.14. Eleutheradione (21). Yellowish oil; [a]20 D 51.6 (c 0.083, CHCl3); UV (CHCl3) lmax (log 3) 248 (3.84) nm; IR (thin film) nmax 2939, 2347, 2235, 1664, 1183 cm1; 1H NMR data, see Table 3; 13C NMR data, see Table 4; EIMS 70 eV m/z (rel int. %) 232 (23), 217 (17), 204 (3), 189 (37), 175 (18), 161 (20), 151 (100), 122 (61), 91 (43), 77 (36), 41 (66); HR-ESIMS m/z 232.1473 [M]þ (calcd for C15H20O2, 232.1463). 3.4. Evaluation of inhibitory activity against CDC25 phosphatases 3.4.1. Production and purification of recombinant human CDC25 phosphatases. Recombinant CDC25-GST proteins of the three isoforms were obtained as previously described.25 Briefly, Escherichia coli strain BL21-DE3 pLys S was transformed by a plasmidic vector lizy, France) containing the (pGEX 2T, Amersham, Ge-Healthcare, Ve sequences encoding full length CDC25A, B and C. Recombinant protein production was induced by addition of isopropyl-thio-bgalactoside (Sigma Aldrich, Saint Quentin Fallavier, France) in the medium. Cells were lysed and centrifuged to recover the supernatant which was purified on a GSH-agarose column (Sigma Aldrich) and recombinant GST-CDC25 proteins were eluted and collected in fractions. Activity, purity and protein concentration of the fractions were evaluated. 3.4.2. In vitro enzymatic assays. CDC25 phosphatase activity was quantified in vitro by a fluorimetric assay as previously described.26 Measurement of GST-CDC25 enzymatic activity was performed in 96-well plates in (50 mM TriseHCl, 50 mM NaCl, 1 mM EDTA and

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0.1% SAB, pH 8.1) buffer containing 500 mM 3-O-methylfluorescein phosphate (Sigma Aldrich) as substrate. The GSTeCDC25 proteins, diluted in assay buffer, were used at a final concentration of 1 mg/ well. After 2 h at 30  C, fluorescent emission of 3-O-methylfluorescein was measured with a CytoFluor system (Perspective Applied Biosystems, Life Technologies, Villebon sur Yvette, France) with excitation filter at 475 nm and emission filter at 510 nm. Tests were performed in triplicates, and the experiment was performed three times independently (nine values in total). DMSO was used as control and naphthoquinone as reference for complete inhibition. Results are expressed as percentage of inhibition of CDC25 phosphatase activity in the presence of the tested compounds. Acknowledgements The authors thank Prof. W. Fenical (SIO, UCSD, U.S.A.), coordinator of a National Science Foundation project, for allowing V.R. and C.V. to participate and collect specimens of P. rigida during a collection campaign in the Bahamas Islands aboard the R/V ‘Seward Johnson’. References and notes 1. Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2015, 32, 116e211 and earlier reviews in this series. 2. MarinLit. Royal Society of Chemistry, 2015 Available online at: http://pubs.rsc. org/marinlit/. 3. Rocha, J.; Peixem, L.; Gomes, N. C. M.; Calado, R. Mar. Drugs 2011, 9, 1860e1886. 4. Marrero, J.; Rodrıguez, A. D.; Baran, P.; Raptis, R. G. Eur. J. Org. Chem. 2004, 3909e3912. 5. McEnroe, F.; Fenical, W. Tetrahedron 1978, 34, 1661e1664. 6. Georgantea, P.; Ioannou, E.; Vagias, C.; Roussis, V. Tetrahedron Lett. 2013, 54, 6920e6922. 7. Georgantea, P.; Ioannou, E.; Vagias, C.; Roussis, V. Phytochem. Lett. 2014, 8, 86e91. 8. Boutros, R.; Dozier, C.; Ducommun, B. Curr. Opin. Cell Biol. 2006, 18, 185e191. 9. Boutros, R.; Lobjois, V.; Ducommun, B. Nat. Rev. Cancer 2007, 7, 495e507. 10. Contour-Galcera, M. O.; Sidhu, A.; Prevost, G.; Bigg, D.; Ducommun, B. Pharmacol. Ther. 2007, 115, 1e12. 11. Archer, D. A.; Thomson, R. H. Chem. Commun. 1965, 354e355. 12. Enriquez, R. G.; Fernandez, G. J. M.; Gnecco, D.; Penicaud, A.; Reynolds, W. F. J. Chem. Crystallogr. 1998, 28, 529e537. €gl, F.; Boer, A. G. Rec. Trav. Chim. 1935, 54, 779e794. 13. Ko 14. Soriano-Garcia, M.; Toscano, R. A.; Flores-Valverde, E.; Montoya-Vega, F.; LopezCelis, I. Acta Crystallogr., Sect. C 1986, 42, 327e329. 15. Wagner, E. R.; Moss, R. D.; Broker, R. M.; Heeschen, J. P.; Potts, W. J.; Dilling, M. L. Tetrahedron Lett. 1965, 47, 4233e4239. 16. Joseph-Nathan, P.; Gonzalez, P.; Rodriguez, V. Phytochemistry 1972, 11, 1803e1808. 17. Spring, O.; Pfannstiel, J.; Klaiber, I.; Conrad, J.; Beifuß, U.; Apel, L.; Aschenbrenner, A. K.; Zipper, R. Phytochemistry 2015, 119, 83e89. 18. Macias, F. A.; Varela, R. M.; Torres, A.; Molinillo, J. M. G. J. Nat. Prod. 1999, 62, 1636e1639. 19. Morimoto, S.; Shindo, M.; Yoshida, M.; Shishido, K. Tetrahedron Lett. 2006, 47, 7353e7356. 20. Joseph-Nathan, P.; Burgueno-Tapia, E.; Santillan, R. L. J. Nat. Prod. 1993, 56, 1758e1765. 21. Joseph-Nathan, P.; Hernandez-Medel, M. D. R.; Martinez, E.; Rojas, M.; Cerda, C. M. J. Nat. Prod. 1988, 51, 675e689. 22. Harrison, B.; Crews, P. J. Org. Chem. 1997, 62, 2646e2648. 23. Lavecchia, A.; Di Giovanni, C.; Novellino, E. Mini-Rev. Med. Chem. 2012, 12, 62e73. 24. Bana, E.; Sibille, E.; Valente, S.; Cerella, C.; Chaimbault, P.; Kirsch, G.; Dicato, M.; Diederich, M.; Bagrel, D. Mol. Carcinog. 2015, 54, 229e241. , M.; Banaszak, E.; El Maadidi, S.; Battaglia, E.; Bagrel, D.; 25. Brault, L.; Denance Samadi, M. Eur. J. Med. Chem. 2007, 42, 243e247. 26. Viry, E.; Anwar, A.; Kirsch, G.; Jacob, C.; Diederich, M.; Bagrel, D. Int. J. Oncol. 2011, 38, 1103e1111.