Cytotoxic Guanidine Alkaloids from a French Polynesian Monanchora

Jul 15, 2016 - Four bicyclic and three pentacyclic guanidine alkaloids (1–7) were isolated from a French Polynesian Monanchora n. sp. sponge, along ...
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Cytotoxic Guanidine Alkaloids from a French Polynesian Monanchora n. sp. Sponge Amr El-Demerdash,†,‡ Céline Moriou,† Marie-Thérèse Martin,† Alice de Souza Rodrigues-Stien,† Sylvain Petek,§ Marina Demoy-Schneider,⊥ Kathryn Hall,∥ John N. A. Hooper,∥,# Cécile Debitus,§ and Ali Al-Mourabit*,† †

Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Univ. Paris-Sud, Université Paris-Saclay, 1, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France ‡ Organic Chemistry Division, Chemistry Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt § Institut de Recherche pour le Développement (IRD), UMR-241 EIO, BP529, 98713, Papeete, Tahiti, French Polynesia ⊥ Université de la Polynésie Française, UMR-241 EIO, BP 6570, 98702 Faa’a Aéroport,Tahiti, French Polynesia ∥ Queensland Museum, PO Box 3300, South Brisbane BC, Queensland 4101, Australia # Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland 4111, Australia S Supporting Information *

ABSTRACT: Four bicyclic and three pentacyclic guanidine alkaloids (1−7) were isolated from a French Polynesian Monanchora n. sp. sponge, along with the known alkaloids monalidine A (8), enantiomers 9−11 of known natural product crambescins, and the known crambescidins 12−15. Structures were assigned by spectroscopic data interpretation. The relative and absolute configurations of the alkaloids were established by analysis of 1H NMR and NOESY spectra and by circular dichroism analysis. The new norcrambescidic acid (7) corresponds to interesting biosynthetic variation within the pentacyclic core. All compounds exhibited antiproliferative and cytotoxic efficacy against KB, HCT116, HL60, MRC5, and B16F10 cancer cells, with IC50 values ranging from 4 nM to 10 μM.

M

continued chemical studies of new sponges. The genus Monanchora (family Crambeidae, order Poecilosclerida) was reported to contain several polycyclic guanidine alkaloids including crambescidin 800, crambescidin 826, fromiamycalin, dehydrocrambine A,22 merobatzelladines A and B,23 ptilomycalin D,24 and other derivatives.14,25 In the present paper, we report on the isolation, structure elucidation, and evaluation of cytotoxic activity of seven new guanidine structures (1−7), along with eight known compounds (8−15) from a French Polynesian Monanchora n. sp. marine sponge.

arine sponges of the orders Poecilosclerida and Axinellida such as the genera Batzella, Crambe, and Ptilocaulis have provided structurally diverse compounds including complex polycyclic guanidine alkaloids with various bioactivities.1 Since the discovery of the cytotoxic tricyclic guanidine alkaloids ptilocaulin and isoptilocaulin,2 the pentacyclic guanidine alkaloid ptilomycalin A from Ptilocaulis spiculifer and Hemimycale sp.,3 and the crambescins from Crambe crambe,4 a large number of other derivatives have been reported. Their diverse guanidine architecture and potent biological activities have made them interesting target molecules for bioactivity and synthetic purposes.5−10 These classes of metabolites have bicyclic, tricyclic, and pentacyclic guanidine cores coupled to polyketide chains and spermidine moieties.11−14 Some acyclic members have been reported.15 Several metabolites from this family showed potent cytotoxicity against cancer cell lines and antiviral activities.16−19 Studies regarding the synaptic transmission in the central nervous system have been reported recently.20 Although the permeability of the cell membranes for these polar guanidine compounds limits their use as biological tools,21 they remain interesting as rich sources of new natural derivatives through © XXXX American Chemical Society and American Society of Pharmacognosy



RESULTS AND DISCUSSION The dried CH2Cl2/MeOH extract of Monanchora n. sp. 4696 (14 g) was partitioned between n-BuOH and H2O. The resulting organic residue (9.6 g) was subjected to silica-gel flash chromatography eluting with a gradient of CH2Cl2/MeOH. Further repetitive chromatographic purification of the obtained fractions using semipreparative reversed-phase C18-HPLC and Received: February 25, 2016

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DOI: 10.1021/acs.jnatprod.6b00168 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

Table 1. 1H and 13C NMR Data for the Salts 1·HCO2H (CD3OD) and 3·HCO2H (DMF-d7) monanchoradin A (1) δC, type

position 1 2 3 4

172.4, 107.3, 145.7, 30.7,

monanchoradin C (3)

δH mult, (J in Hz)

a

a

C C C CH2

COSY

HMBC

a: 3.35, m b: 2.86, m

5

2, 3, 5, 6

172.1, C 108.3 C 146.3, C 30.9, CH2

5

23.3, CH2

a: 2.12, m b: 2.05, m

4, 6

4

23.3, CH2

6

48.4, CH2

a: 3.71, ddd (3.5, 2.5, 3.0) b: 3.59, m

5

3, 4, 5

48.0, CH2

4.41, m

9

2, 3, 7, 10

7 8

153.1, C 53.0, CH

153.9, C 51.8, CH

9

37.7, CH2

1.57, m

8, 10

37.8, CH2

10

25.5, CH2

1.42, m

9, 11

25.5, CH2

11−14 15 11−16

23.8−30.5−30.9−33.1, CH2 14.6, CH3

1.30−1.36, br s 0.89, t (7)

10, 15 14

H 500 MHz, 13C 125 MHz.

δH mult, (J in Hz)a

COSY

HMBC

a: 3.36, m b: 2.88, t (8) (2.89, 3.38, m)b a: 2.12, m b: 1.99, m (2.06, 2.16, m)b a: 3.79, m b: 3.66, dddd, (2, 2.5, 3, 2.5) (3.60, 3.73, m)b

5

2, 3, 5, 6

4, 6

4, 6

5

3, 4, 5

4.41, br d (4.5) (4.40, m)b 1.52, m (1.57, m)b 1.62, m (1.41, m)b

9

2, 7, 9, 10

8, 10

8, 10

9, 11

8, 9

1.29−1.35, m (1.29−1.35, m)b 0.88, t (6) (0.90, t (7))b

10, 17

17

16

16

15 14 23.6−32.9,c CH2

17 a1

δC, type

a

14.8, CH3 b1

H NMR were recorded in CD3OD for comparison. cOverlapped.

nonprotonated carbons (δC 172.4 for a conjugated carboxylic acid, δC 153.1 for a cyclic guanidine group, one olefin bond at δC 145.7 and δC 110.3), 10 methylenes including carbons bearing distinguishable diastereotopic protons, and a methine C-8 (δC 52.9). The methine proton H-8 (δH 4.41) is similar to that of the characteristic H-13 in the known crambescins A.26,27 So the compound must contain two rings to satisfy five degrees of unsaturation. Comparison of the 1H NMR data between 1 and known crambescins showed the presence of signals with

solvents containing 0.1% formic acid resulted in the isolation of the formic acid salts of 1−7, along with the known members 8−15. The molecular formula C15H26N3O2 of the protonated compound 1 was established by HRESIMS at m/z 280.2012 [M + H]+, indicating five degrees of unsaturation. Direct examination of the 13C NMR spectrum (Table 1) in combination with HSQC correlations revealed the presence of 15 signals, corresponding to the carbon resonances of four B

DOI: 10.1021/acs.jnatprod.6b00168 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. COSY and HMBC correlations for monanchoradins A (1) and C (3) and dihydrocrambescin A2 418 (4).

of the COSY and HMBC spectra. The HMBC correlations found from H2-2 (δH 3.23/δC 42.2) to C-1 (δC 159.1) and from H2-5 (δH 4.39/66.5) to C-6 (δC 165.0) confirmed the terminal guanidine group and the connection of the side chain to the ester group. The fragment corresponding to the saturated aliphatic chain showed expected chemical shifts assigned from the methylene group H2-14 (δH 3.10) to the terminal methyl group H3-22 (δH 0.89) (Table 2). COSY correlations from H2-

chemical shifts similar to those of the aliphatic chains, particularly the characteristic H-8 (δH 4.41), which displayed HMBC correlations with C-2 (δC 110.3), C-3 (δC 145.7), C-7 (δC 153.1), and C-10 (δC 25.5) (Figure 1). Hydrogen H-10 was further correlated to the methylene protons H-9 and H-11 by COSY NMR, and the remaining methylenes were attributed to the aliphatic side chain C-9 to C-15. The HMBC correlations from the terminal methyl group H-15 (δH 0.89, C-15 δC 14.6) to the methylene carbon C-14 (δC 23.9) and from H-8 (δH 4.41) to C-2 (δC 110.3) were important for the connection of the chain to the bicyclic core. Further analysis of both COSY and HMBC spectra clearly indicated the fused guanidine moiety in the bicyclic part (Figure 1 and Table 1). Finally, the IR spectrum showed a strong and wide band at 3000−3500 cm−1 for the −OH group and the α,β-unsaturated carbonyl group at 1694 cm−1, corresponding to a free carboxylic acid group. These data were consistent with the proposed structure of the new monanchoradin A (1). The 1H NMR spectrum showed a signal at δH 8.52 ppm, corresponding to the C−H of the formate counterion. The 1H NMR spectrum for compounds 2−15 also exhibited this same signal, indicating a formate counterion for all the alkaloids. The molecular formulas of compounds 2 and 3 were established by HRESIMS at m/z 294.2213 [M + H] + (C16H28N3O2) and 308.2146 [M + H]+ (C17H30N3O2) respectively. The determined degrees of unsaturation were the same as for monanchoradin A (1). Comparison of the 1H NMR spectra of 1, 2, and 3 indicated the same carbon skeleton for 2 and 3. The difference was only the number of methylene groups within the alkyl side chains, indicating octyl and nonyl chains for monanchoradin B (2) and monanchoradin C (3), respectively (Table 1 and Supporting Information). This is the first report of crambescin analogues devoid of the 4guanidinobutyl ester chain. Additionally, we isolated monalidine A (8) from this sponge, a compound that was recently reported by Berlinck and co-workers from a Brazilian specimen of Monanchora arbuscula.28 High-resolution ESIMS of 4 gave a protonated molecule at m/z 419.3137 [M + H]+, indicating a molecular formula of C22H39N6O2 that requires seven degrees of unsaturation. The IR spectrum showed infrared absorption at 2855 cm−1 of methyl and methylene groups and 2926 cm−1 of CH methine groups and a strong vibration at 1609 cm−1 indicating an α,βunsaturated carbonyl function. The UV spectrum showed maxima at 295, 248, and 217 nm. The number of nitrogen atoms and a literature survey of the sponges of the genus Monanchora, together with cursory examination of the 1H and 13C NMR spectra, suggested that compound 4 was close to the known dehydrocrambine A222,29 and the dehydrocrambescine A1.24 Three characteristic fragments were found by 2D NMR analysis. The guanidinobutyl ester fragment C-1−C-6 (Figure 1) was determined by analysis

Table 2. NMR Data for Compound 4 in CD3OHa dehydrocrambescin A2 418 (4) δC, type

position 1 2 3 4 5 6 7 8 9

159.1, 42.2, 26.7, 26.9, 66.5, 165.0, 113.1, 168.5, 35.2,

C CH2 CH2 CH2 CH2 C C C CH2

10 11 12 13 14 15 16 17−20 21 22

21.3, 53.9, 155.5, 180.9, 38.4, 29.1, 30.6, 30.5, 23.8, 14.6,

CH2 CH2 C C CH2 CH2 CH2 30.6, 30.7, 33.0, CH2 CH2 CH3

δH mult, (J in Hz)

COSY

HMBC

3.23, 1.73, 1.84, 4.39,

3 2, 4 3, 5 4

1, 2, 2, 3,

3.58, t (8)

10

2.40, m 4.28, t (7)

9, 11 10

7, 8, 10, 11 8, 9, 11 8, 9, 10

3.10, t (7.5) 1.71, m 1.39, m 1.29, br s 1.28, br s 0.89, t (7)

15 14, 16 15, 17

7, 13, 15 16

22 21

22 21

t (7) m m t (7)

3, 4, 3, 4,

4 5 5 6

a

NMR spectra for monanchoradin 4 were recorded in CD3OH in order to avoid H2-9 and H2-14 deuterium exchange.

9 (δH 3.58) to H2-11 (δH 4.28), along with the presence of sp2 carbon C-12 (δC 155.5), enabled us to identify the bicyclic guanidine fragment. The 13C NMR spectrum showed three sp2 carbons, C-7 (δC 113.0), C-8 (δC 168.5), and C-13 (δC 180.9). The key HMBC correlations from H2-9 (δH 3.58) and H2-14 to C-7, from H2-9 (δH 3.58), H2-10 (δH 2.40), and H2-11 (δH 4.28) to C-8, and from H2-14 (δH 3.10) to C-13 indicated a similar structure to that for dehydrocrambine A.22 Dehydrocrambescin A2 418 (4) was identified as a new member of the dehydrocrambine A family of sponge guanidine alkaloids.14,22 As shown in Scheme 1, upon standing in CD3OD, it was noticed that the H2-9 and H2-14 proton signals for compound 4 disappeared in the 1H NMR spectrum. Hydrogen/deuterium exchange within compound 4 in CD3OD was faster at H2-9 (5 days) than at H2-14 (10 days; Figure S24). C

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Scheme 1. Hydrogen/Deuterium Exchange of H2-9 and H2-14 in 4 Observed in MeOH-d4a

a

For clarity, the HCO2− counterion is not indicated with the structures.

C46H83N6O6, and 801.6205 [M + H]+ for C45H81N6O6, respectively, indicating that these three molecules had in common nine degrees of unsaturation. The 1H and 13C NMR spectra of 5 and 6 are almost superimposable with the spectrum of 12 (Figure S39) and other closely related pentacyclic crambescidin members such as crambescidin 826 isolated from a Monanchora sp.22 Analysis of the 13C NMR spectra of the three compounds showed identical chemical shifts for the characteristic olefinic carbons C-4 (δC 134.6) and C-5 (δC 131.3), the carbon of the guanidine C-21 (δC 150.6), the ester carbon C-22 (δC 170.3), the amidic carbon C-38 (δC 177.6), two methyl groups C-1 and C-20 (δH 0.87, 1.09; δC 11.1, 22.0), two oxymethines C-3 and C-19 (δH 4.43, 3.85; δC 72.4, 68.7), two N-substituted CH groups C-10 and C-13 (δH 4.04, 4.34; δC 55.6, 54.2.0), one oxymethylene C-23 (δH 4.13, δC 66.6), and two nonprotonated carbons of aminocarbinols C-8 and C-15 (δC 85.3, 82.2). Further HMBC analysis (Figure 3) completed the arguments for the pentacyclic part. Similar reasoning using comparison of NMR and MS data of 5 with those of 6 suggested a homologous structure. Comparative analysis of the COSY, HSQC, and HMBC data for 5 and the close derivative 6 established that their “vessel” part was identical. The 28-unit mass difference between 5 and 6 was then attributed to chain length, although this was not evident by NMR alone. The polymethylene side chain between the ester and the amide functions (n = 12 for 5 instead of n = 14 for 6) were attributed to the new crambescidins at 786 and 814, respectively. The relative configurations of compounds 5 and 6, which were established by NOESY and ROESY correlations, were identical to the known compound crambescidin 800 (12). On the basis of the literature, the chemical shift of the H-14α in crambescidin 800 (12)22 (δH 3.07, d, J = 5 Hz) is characteristic of a syn relationship between H-10 and H-13, while in isocrambescidin 800,12,26 the H-14 presented a higher chemical shift (δH 3.80, d, J = 3.4 Hz). Our compounds 5 and 6 displayed H-14α values of a syn relationship, δH 3.05, d, J = 5 Hz, and δH 3.04, d, J = 5 Hz, respectively.

As close derivatives of dehydrocrambescin A2 418 (4), we isolated enantiomers of two known derivatives, (−)-crambescin A2 392 (9) and (−)-crambescin A2 406 (10), along with (−)-crambescin A2 420 (12).30 The isolation of (+)-crambescin A2 392 (9) and (+)-crambescin A2 406 (10) by Molinski and co-workers from a Pseudaxinella reticulate sponge suggests that there is a dependence of the chirality on the sponge species. As pointed out by Molinski,31 it is also important to note that in some cases the value of the recorded [α]D of chiral ammonium salts may be influenced by the nature of the counterion. Indeed, the recent disconcerting example of sceptrin32 may allow some uncertainty regarding [α]D values of chiral ammonium salts. In order to control whether the sign and magnitude of the optical rotation change with the salt form, compounds 9−11 were mixed with TFA in MeOH and dried. The [α]D and electronic circular dichroism (ECD) spectra of resulting TFA salts were recorded and compared with those reported by Molinski and indicated indeed the opposite forms.

Figure 2. ECD spectra of the TFA salts of (−)-crambescins A2 392 (9), 406 (10), and 420 (11).

HRESIMS analysis of compounds 5 and 6 and the known crambescidin 800 (12) showed protonated molecules at m/z 787.6102 [M + H]+ for C44H79N6O6, 815.6379 [M + H]+ for

Figure 3. Important COSY and HMBC correlations for compounds 5, 6, and 7 and HRMS-MS fragmentation for 7. D

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Figure 4. ECD spectra for (−)-crambescidins (6, 12, 14, 15) and the (−)-norcrambescidin (7).

the crambescidins 5, 6, and 12 (Table 4). It is important to note that the activity varies with the length of the side chain of the compounds. The highest activity was observed for crambescidin 800 (12, n = 13), while the shorter homologue crambescidin 786 (5, n = 12) and the longer one crambescidin 814 (6, n = 14) were less active. This might be indicative of how the chain can play an important role as a spacer between two sites of interaction. Indeed, compounds possessing the pentacyclic core (vessel) linked by a linear ω-hydroxy fatty acid to a hydroxyspermidine moiety (anchor) were more active than compounds possessing only the vessel. Bicyclic compound 1, bearing only a carboxylic acid group, was found to be less potent against cancer cell lines but still in the nanomolar range. Furthermore, crambescidic acid 14 and norcrambescidic acid 7 terminated with the carboxylic acid functionality were found to have significant activity; however, they were less active than the pentacyclic molecules possessing the spermidine terminus. Activities of the crambescidins were also evaluated against B16F10 murine melanoma cells: crambescidins 800 (12), 814 (6), and 826 (13) displayed moderate activity against this cell line with an IC50 of 0.2 μM for 12 and 6 and 0.85 μM for compound 13. Among the polycyclic guanidine metabolites described here, some possess uncommon structural features including the new variant 20-norcrambescidic acid (7). It should be noted that the replacement of the methyl group by an ethyl group in monanchomycalin A was previously described.34 Although, the biosynthetic pathway is not known, these interesting variations of the unique pentacyclic guanidinium core can give some insight into its mode of cyclization and raise interesting questions. All evaluated compounds exhibited strong antiproliferative activities on several cancer cell lines.

The molecular formula of compound 7 was determined to be C37H62N3O6, with nine degrees of unsaturation, by HRESIMS at m/z 644.4555 [M + H]+. Comparison of the 1H NMR spectrum of 7 with the spectrum for the known and co-isolated pentacyclic crambescidic acid (14) (Figure S63) indicated the presence of the guanidine core with the notable absence of the characteristic methyl H3-20 of the series. Further analysis of the COSY, HSQC, and HMBC data established that the typical 2methylyltetrahydro-2H-pyran in crambescidins like 14 was replaced with the simple tetrahydro-2H-pyran in 7. Evidence for the absence of the methyl group included the two multiplets at δH 3.78 and 3.70, corresponding to the methylene H2-19. The HRESIMS mass spectrum showed fragment ions at m/z 390.2398 (Figure 3, fragment F1), 372.2292 (fragment F1− H2O), and 346.2494 (fragment F1−CO2) corresponding to pentacyclic fragments of the norcrambescidin (Figure 3). The length of the linear fatty acid chain as well as its attachment through the ester function was confirmed by mass fragmentation and HMBC correlation from H-22 to C-21. On the basis of the above analyses, 20-norcrambescidic acid (7) was assigned as a new guanidine alkaloid. Starting from the absolute configuration of the isolated known crambescidin 800 (12), which was established by enantioselective synthesis,33 we could take advantage of the almost superimposable NMR spectra and ECD curves to propose the absolute configurations of our compounds. The [α]25D −3.5 (c 0.72, CHCl3 as HCl salt) of our crambescidin 800 (13) was close to the reported [α]25D −4.4 (c 0.7, CHCl3 as HCl salt).33 We next recorded ECD spectra for compounds 6, 7, 12, 14, and 15 and found similar curves, indicating the same configuration. Therefore, the 3S, 8S, 10S, 13S, 14S, 15S, 19R absolute configuration was assigned for 6, 12, 14, and 15. The absolute configuration of the C-43 hydroxyspermidine side chain of crambescidin 800 (13) was not determined.13 Coffey and co-workers demonstrated that C-43 S and R epimers were indistinguishable by Mosher’s derivatization.33 Finally, the configuration of the polycyclic part of the new 20norcrambescidic acid (7) was assigned as 3S, 8S, 10S, 13S, 14S, 15S. Most of the compounds were assayed for their cytotoxic activities. The CH2Cl2/MeOH and the n-BuOH extracts were primarily subjected to cytotoxicity bioassay against KB tumor cell lines and showed 91 ± 1% and 91 ± 3% at 1 μg/mL concentration. Consequently, several pure isolated compounds showed strong cytotoxicity in the nanomolar range, particularly



EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations [α]D were measured using an Anton Paar MCP-300 polarimeter. UV spectra were recorded on a PerkinElmer Lambda 5 spectrophotometer. ECD experiments were performed on a Jasco J-810 spectropolarimeter. IR spectra were recorded on a PerkinElmer BX FT-IR spectrometer. 1D and 2D NMR spectra were recorded on a Bruker Avance 500 MHz or a Bruker Avance 600 MHz (TXI 1.7 mm probe) (CNRS-ICSN). The chemical shifts are relative to the residual signal solvent (CD3OD: δH 3.31; δC 49.20; DMF-d7: δH 8.03, 2.92, 2.75; δC 163.2, 34.9, 29.8). High-resolution mass spectra were obtained on a Waters LCT Premier XE spectrometer in electrospray ionization mode by direct infusion of the purified compounds. Preparative HPLC was performed using an E

DOI: 10.1021/acs.jnatprod.6b00168 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. General 1H and 13C NMR Data for Crambescidins 786 (5) and 814 (6) and 20-Norcrambescidic Acid (7) in CD3OD crambescidin 786 (5) position 1 2 3 4 5 6

δC, type 11.1, CH3 30.5, CH2 72.4, 134.6, 131.5, 24.6,

CH CH CH CH2

7

38.2, CH2

8 9

85.3, C 38.0, CH2

10 11

55.6, CH 30.9, CH2

12

27.7, CH2

13 14 15 16 17 18

54.2, 51.4, 82.2, 32.9, 19.4, 32.9,

19

68.7, CH

20 21 22 23 *24−34 35 36 37 38

CH CH C CH2 CH2 CH2

22.0, CH3 150.6, C 170.3, C 66.6, CH2 28.2−32.0, CH2 28.2−31.0, CH2 34.3, CH2 177.6, C 44.1, CH2

39 40

26.8, CH2 38.6, CH2

41 42 43

55.5, CH2 68.4, CH 33.1, CH2

44 45

38.6, CH

46

δH mult, (J in Hz) 0.87 a: 1.55, m b: 1.50, m 4.43, m 5.50, br d (11) 5.70, br t (8) a: 2.45, m b: 2.14, m a: 2.44, m b: 1.95, m a: 2.61, m b:1.41, m 4.04, m a: 2.30, m b: 1.62, m a: 2.34, m b: 1.82, m 4.34, m 3.05, d (5) 1.69, m 1.81, m a: 1.70, m b: 1.60, m 3.85, m 1.09, d (6)

4.13, m 1.26−1.34, m 1.26−1.34, m 2.44, m a: 3.65, m b: 3.55, m 1.85, m 2.96, m 3.42, m 3.94, m a: 1.80, m b: 1.70, m 2.88, m

crambescidin 814 (6) δC, type 11.1, CH3 30.5, CH2 72.5, 134.6, 131.5, 24.8,

0.85, t (7) a: 1.55, m b: 1.48, m 4.42, br d (9.5) 5.50, br d (11) 5.70, br t (8) a: 2.45, m b: 2.17, m a: 2.45, m b: 1.98, m

CH CH CH CH2

38.3, CH2 85.3, C 38.1, CH2

27.7, CH2 CH CH C CH2 CH2 CH2

22.1, CH3 150.6, C 170.5, C 66.7, CH2 28.2−32.0, 28.2−31.0, 28.2−31.0, 28.2−31.0, 34.4, CH2

CH2 CH2 CH2 CH2

177.7, C 44.2, CH2

72.3, 134.6, 131.4, 24.8,

CH CH CH CH2

38.1, CH2

27.9, CH2 54.2, 50.1, 82.0, 33.6, 19.5, 33.6,

CH CH C CH2 CH2 CH2

62.7, CH2

1.08, d (6.5)

4.14, m 1.24−1.35, 1.24−1.35, 1.24−1.35, 1.24−1.35, 2.43, m

11.1, CH3 30.4, CH2

55.4, CH 31.6, CH2

1.70, m 1.81, m a: 1.70, m b: 1.0, m 3.85, m

68.8, CH

δC, type

85.3, C 38.0, CH2

a: 2.62, dd (13, 4.5) b: 1.40, m 4.04, m a: 2.30, m b: 1.60, m a: 2.34, m b: 1.82, m 4.34, m 3.04, d (5)

55.7, CH 30.9, CH2

54.2, 51.4, 82.3, 32.8, 19.7, 32.8,

norcrambescidic acid (7)

δH mult, (J in Hz)

m m m m

150.7, C 170.4, C 66.6, CH2 26.0, CH2 29.8−31.0, CH2 26.0, CH2 37.3, CH2 181.0, C

δH mult, (J in Hz) 0.85, t (5.5) a: 1.56, m b: 1.46, m 4.44, m 5.50, br d (9) 5.70, m a: 2.42, m b: 2.16, m a: 2.40, m b: 1.98, m a: 2.61, dd (11, 4) b: 1.43, d (13) 4.03, m a: 2.29, m b: 1.60, m a: 2.34, m b: 1.80, m 4.26, m 3.16, d (4) 1.69, m 1.80, m a: 1.70, m b: 1.60, m a: 3.79, ddd (3, 2, 3) b: 3.70, br d (10)

4.13, m 1.64, m 1.28−1.35, m 1.64, m −2.22, t (6)

a: 3.65, m b: 3.56, m 1.89, m 2.96, m 3.42, m

26.8, CH2 38.4, CH2 55.1, CH2 68.5, CH 33.2, CH2

3.95, m a: 1.75, m b: 1.68, m 3.11, m

38.5, CH2

exhaustively in a 1:1 mixture of CH2Cl2/MeOH (2 L). The colored extract was dried under reduced pressure to afford a reddish-brown residue (40.5 g). Fourteen grams of this residue was partitioned between n-BuOH and H2O to give 3.65 and 9.6 g of material. The resulting organic n-BuOH reddish-brown residue was subjected to normal-phase silica-gel flash chromatography (35−70 μm), using the gradient from 1:0 to 0:1 mixtures of CH2Cl2/MeOH (12 L, gradient of MeOH increased by 5%) to afford 25 fractions. All fractions were monitored by TLC using different eluent systems such as heptane/ CH2Cl2 and CH2Cl2/MeOH mixtures and assembled according to

Auto Prep system (Waters 600 controller and Waters 600 pump, equipped with a Waters 996 photodiode array detector). Animal Material. The sponge was collected off the coast of Hiva Oa (9°45.421′ S; 139°08.275′ W) at 20 m depth using scuba on July 9, 2009.35 It was identified as Monanchora n. sp. 4696, and a reference specimen is deposited at the Queensland Museum (Brisbane, Australia) under the accession number QM G331116. The complete description is given in the Supporting Information. Extraction and Isolation. The freeze-dried sponge samples of Monanchora n. sp. 4696 (240 g) were extracted at room temperature F

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Monanchoradin B (2): colorless oil (0.5 mg); UV (MeOH) λmax (log ε) 211 (0.38), 291 (0.26) nm; 1H and 13C NMR data Table 1; HRESIMS m/z 294.2213 [M + H]+ (calcd for C16H27N3O2, 294.2182). Monanchoradin C (3): pale yellow oil (0.7 mg); UV (MeOH) λmax (log ε) 210 (1.00), 291 (0.75) nm; IR (neat) νmax 2942, 2854, 1693, 1385, 1256 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 308.2146 [M + H]+ (calcd for C17H29N3O2, 308.2338). Dehydrocrambescin A2 418 (4): pale yellow oil (7 mg); UV (MeOH) λmax (log ε) 218 (1.60), 248 (2.60), 295 (1.00) nm; IR (neat) νmax 3166, 2926, 2855, 1609, 1465, 1374, 1347, 1261, 1119, 762 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 419.3137 [M + H]+ (calcd for C22H39N6O2, 419.3134). Crambescidin 786 (5): pale yellow oil (5 mg); UV (MeOH) λmax (log ε) 213 (0.65) nm; IR (neat) νmax 3377, 2926, 2344, 1731, 1618, 1394, 1057 cm−1; 1H NMR and 13C NMR data, Table 3; HRESIMS m/z 787.6102 [M + H]+ (calcd for C44H79N6O6, 787.6101). (−)-Crambescidin 814 (6): yellow oil (6.6 mg); [α]25D −2.5 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 201 (0.55) nm; ECD (0.02 mg/ mL, MeOH), λmax (Δε) 204 (11.14) nm; IR (neat) νmax 2925, 2852, 1732, 1622, 1349, 1165, 1052, 718 cm−1; 1H NMR and 13C NMR data, Table 3; HRESIMS m/z 815.6379 [M + H]+ (calcd for C46H83N6O6, 815.6374). 20-Norcrambescidic acid (7): pale yellow powder (1 mg); UV (MeOH) λmax (log ε) 202 (0.15) nm; ECD (0.03 mg/mL, MeOH), λmax (Δε) 205 (2.58) nm; IR (neat) νmax 3394 (broad band, COOH), 292, 1710, 1626, 1400, 1054 cm−1; 1H NMR and 13C NMR data, Table 3; HRESIMS m/z 644.4555 [M + H]+ (calcd for C37H62N3O6, 644.4639). (−)-Crambescin A2 392 (9): pale yellow oil (10.8 mg); [α]25D −31 (c 0.54, MeOH as formate salt); [α]25D −20 (c 0.25, MeOH as TFA salt);30 UV (MeOH) λmax (log ε) 222 (1.80), 293 (2.00) nm; ECD (0.02 mg/mL, MeOH), λmax (Δε) 200 (0.96), 247 (−3.00), 288 (−1.27) nm; IR (neat) νmax 3173, 2929, 2857, 1677, 1552, 1459, 1349, 1271, 1201, 1134, 837, 801, 721 cm−1; 1H NMR and 13C NMR data, Supporting Information; HRESIMS m/z 393.2982 [M + H]+ (calcd for C20H37N6O2, 393.2978). (−)-Crambescin A2 406 (10): pale yellow oil (5.5 mg); [α]25D −31 (c 0.35, MeOH as formate salt), [α]25D −12 (c 0.18, MeOH as TFA salt); UV (MeOH) λmax (log ε) 222 (1.80), 293 (2.00) nm; ECD (0.02 mg/mL, MeOH), λmax (Δε) 204 (0.39), 248 (−1.82), 282 (−0.79) nm; IR (neat) νmax 3173, 2943, 1680, 1349, 1271, 1202, 1134, 1066, 837, 801, 721 cm−1; 1H NMR and 13C NMR data, Supporting Information; HRESIMS m/z 407.3123 [M + H]+ (calcd for C21H39N6O2, 407.3134). (−)-Crambescin A2 420 (11): pale yellow oil (32.6 mg); [α]25D −31 (c 0.53, MeOH as formate salt); [α]25D −30 (c 0.25, MeOH as TFA salt); UV (MeOH) λmax (log ε) 232 (2.30), 281 (2.40) nm; ECD (0.02 mg/mL, MeOH), λmax (Δε) 205 (0.42), 248 (−1.54), 288 (−0.73) nm; IR (neat) νmax 3173, 2927, 2855, 1683, 1584, 1462, 1376, 1347, 1270, 1198, 1094, 764 cm−1; 1H NMR and 13C NMR data, Supporting

Table 4. Cytotoxic Activities in Vitro (KB Cell Line) for Pure Isolated Natural Products concentration extract/compound

10 μM

CH2Cl2/MeOH extract n-butanol extract monanchoradin A (1) monanchoradin B (2) monanchoradin C (3) dehydrocrambescin A2 418 (4) crambescidin 786 (5) crambescidin 814 (6) norcrambescidic acid (7) monalidine A (8) (−)-crambescin 392 (9) (−)-crambescin 406 (10) (−)-crambescin 420 (11) crambescidin 800 (12) crambescidin 826 (13) crambescidic acid (14) crambescidin 359 (15)

100 ± 1 100 ± 1 96 ± 1 n.d.c 72 ± 3 98 ± 1 100 ± 1 100 ± 1 95 ± 1 99 ± 1 100 ± 1 100 ± 1 100 ± 1 99 ± 1 100 ± 1 97 ± 2 84 ± 1

a

1 μMb

IC50, μMb

91 ± 1 91 ± 3 61 ± 4 n.d. 0±1 89 ± 1 89 ± 1 96 ± 1 80 ± 3 82 ± 1 80 ± 1 86 ± 1 97 ± 1 100 ± 1 100 ± 1 85 ± 1 0±9

0.3/0.3 0.2/0.4 7.7/6.7 n.d. n.d. 0.1/0.1 0.3/0.3 0.005/0.02 0.5/0.6 0.2/0.4 0.1/0.2 0.3/0.3 0.03/0.03 0.005/0.005 0.07/0.05 0.5/0.6 n.d.

a

Cell proliferation was measured with Celltiter 96 Aqueous One solution reagent (Promega), and results are expressed as the percentage of inhibition of cellular proliferation of KB cells treated for 72 h with compounds compared to cells treated with DMSO only (mean ± SE of triplicate). bFor IC50 values results were expressed as individual values in experiments performed in duplicate. IC50 values were determined only for those compounds exhibiting 100% inhibition at both preliminary concentrations. cn.d. = not determined.

their polarities in 15 fractions, A−O (Supporting Information). The promising fractions E, F, H, J, and K (Supporting Informaion) were selected based on the LC/MS analytical profiles, and each fraction was submitted for further purification by preparative reversed-phase HPLC (column: Waters Sunfire C18 19 × 150 mm, 5 μm, H2O + 0.1% formic acid/CH3CN + 0.1% formic acid). Further repetitive ultrapurifications were developed using semipreparative or analytical columns (Waters Sunfire C18, 10 × 150 mm, 5 μm, or Waters Sunfire C18, 4.5 × 150 mm, 5 μm, respectively) with the eluent system H2O + 0.1% formic acid/CH3CN + 0.1% formic acid. The detailed isolation scheme indicating the relative polarities of the isolated compounds and their quantities is given in the Supporting Information. Monanchoradin A (1): colorless oil (4.5 mg); UV (MeOH) λmax (log ε) 220 (1.30), 293 (1.20) nm; IR (neat) νmax 3620, 2948, 2854, 1693, 1384, 1256, 1059 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 280.2012 [M + H]+ (calcd for C15H26N3O2, 280.2025).

Table 5. Cytotoxic Activities of Selected Compounds against HTC-116, MDA-435, HL-60, and MRC-5 Cell Lines IC50, μMa compound monanchoradin A (1) dehydrocrambescin A2 418 (4) crambescidin 786 (5) crambescidin 814 (6) monalidine A (8) (−)-crambescin 406 (10) crambescidin 800 (12) crambescidin 826 (13)

HCT-116 9.9/9.9 3.4/3.5 3.1/3.4 0.02/0.05 0.84/0.74 3.4/4.2 0.007 n.d.

MDA-435 11/9.3 n.d. n.d. 0.04/0.07 0.32/0.86 n.d. 0.009/0.015 n.d.

HL-60 3.8/7.1 3.6/5.4 5.0/5.4 0.01/0.03 1.3/1.3 8.0/9.1 0.004/0.006 n.d.

MRC-5 b

n.d. 3.4/3.9 3.2/3.4 n.d. 0.55/0.60 4.1/3.6 n.d. n.d.

B16-F10 n.d. n.d. n.d. 0.20 n.d. n.d. 0.2 0.8

a For IC50 values results were expressed as individual values in experiments performed in duplicate. IC50 values were determined only for those compounds exhibiting 100% inhibition at both preliminary concentrations. bn.d. = not determined.

G

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Information; HRESIMS m/z 421.3286 [M + H]+ (calcd for C22H41N6O2, 421.3291). Cell Culture and Cell Proliferation Assay. The human cell line KB originated from the NCI and was grown in D-MEM medium supplemented with 10% fetal calf serum, in the presence of penicillin, streptomycin, and fungizone in a 75 mL flask under 5% CO2. A total of 600 cells were plated in 96-well tissue culture plates in 200 μL of medium and treated 24 h later with 2 μL of stock solution of compounds dissolved in DMSO using a Biomek 3000 (BeckmanCoulter). Controls received the same volume of DMSO (1% final volume). After 72 h of exposure, cell titer 96 Aqueous One solution (Promega) was added and incubated for 3 h at 37 °C; the absorbance was monitored at 490 nm, and results were expressed as the inhibition of cell proliferation calculated as the ratio [1 − (OD490 treated/OD490 control)] × 100 in triplicate experiments. For IC50 determination [50% inhibition of cell proliferation], cells were incubated for 72 h following the same protocol with compound concentrations ranging from 5 nM to 100 μM in separate duplicate experiments. Murine melanoma cells B16F10 were purchased from the American Type Culture Collection (ATCC, CRL-6475). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with sodium pyruvate and without glutamine (D6546, Sigma), which was supplemented with 10% fetal bovine serum (N4637, Sigma), 2% glutamine (G7513, Sigma), and 1% penicillin−streptomycin (P4333 Sigma) in 75 mL flasks, at 37 °C in 5% CO2. A total of 20 000 cells per well were plated in 96-well tissue culture plates in 200 μL of medium and treated 24 h later with 2 μL of stock solution of compounds dissolved in DMSO. Controls received the same volume of DMSO (1% final volume). After 72 h of exposure, cells were rinsed with 200 μL of PBS and the test medium was replaced by 100 μL of medium containing 40 μg/mL of Neutral Red according to Borenfreund and Puerner.15 After incubation for 3 h at 37 °C, the cells were rinsed with PBS again and then destained with 100 μL/well stop solution consisting of glacial acetic acid, 96% EtOH, and H2O at 1:50:49 (by volume). After agitation for 30 min at room temperature, absorbance of the solution for each well was measured at 540 nm. Results were expressed as the inhibition of cell proliferation calculated as the ratio [1 − (OD540 treated/OD540 control)] × 100 in triplicate experiments. For IC50 determination [50% inhibition of cell proliferation], cells were incubated for 72 h following the same protocol with compound concentrations ranging from 10 ng/mL to 10 μg/mL in separate duplicate experiments.



edged. We thank F. Pelissier and O. Thoison for HPLC assistance. Special thanks go to J.-F. Gallard and K. Hammad for technical assistance and NMR measurements. We thank J. Bignon for cytotoxicity evaluations.



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00168. Spectroscopic data and NMR spectra (1H NMR, 13C NMR) for compounds 1−15 (PDF)



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AUTHOR INFORMATION

Corresponding Author

*Tel: (+33) 1-6982-4585. Fax: (+33) 1-6907-7247. E-mail: ali. [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank the French Polynesian authorities as well as the communities for allowing us to collect in Marquesas Islands. Financial support from CNRS-ICSN, IRD for the collecting trip aboard R/V Alis, the French and French Polynesian governments for the Marquesas project, and ANR (POMARE project, 2011-EBIM-006-01) are gratefully acknowledged. A.E.-D.’s Ph.D. was granted and financed by the Egyptian Government (Ministry of Higher Education), which is gratefully acknowlH

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