Additional bioactive guanidine alkaloids from the Mediterranean sponge Crambe crambe

Additional bioactive guanidine alkaloids from the Mediterranean spongeCrambe crambe{

Stephanie Bondu,a Gregory Genta-Jouve,a Marta Leiros,b Carmen Vale,b Jean-Marie Guigonis,c Luis M.

Botanab and Olivier P. Thomas*a

Received 6th January 2012, Accepted 6th January 2012

DOI: 10.1039/c2ra00045h

The full chemical reinvestigation of the Mediterranean marine sponge Crambe crambe led to the

isolation and structural characterization of 11 crambescin derivatives, including 8 new compounds,

together with the known crambescidin 816. HRMS/MS studies allowed the complete assignment of

the alkyl chain lengths of these guanidine alkaloids while the absolute configurations of all

compounds were inferred from the comparison between experimental and theoretical circular

dichroism spectra. Crambescidin 816 was proven to be more cytotoxic against neuronal cell lines than

crambescin C1.

Introduction

Marine sponges of the order Poecilosclerida are known to

produce a large array of structurally diverse bioactive polycyclic

guanidine alkaloids.1 This family of sponge marine natural

products is today considered as a chemotaxonomic marker of the

Crambeidae family.2 Crambe crambe (Schmidt, 1862) is a red

encrusting marine sponge, widely distributed in the Western

Mediterranean Sea but also in the Macaronesian archipelagos.

The first chemical studies on this sponge date back to the early

90s where crambines A, B, C1 and C2 were first isolated by the

group of Braekman.3 At the same time, the polycyclic

crambescidins 800, 816, 830, 844, isocrambescidin 800 and

crambidine, which are oxidized analogues of the previously

described ptilomycalin A,4 were reported by the groups of

Braekman and Rinehart in the same sponge.5 We decided to

reinvestigate the entire secondary metabolome of this sponge

because several structural revisions have been added later for the

crambines, mainly on the basis of chemical synthetic studies.6

Our interest in these compounds also arose from the vast array

of biological activities mainly associated with the polycyclic

crambescidin derivatives.7

As proposed earlier by the group of Rinehart,6b we will change

the name crambines to crambescins because crambin has been

previously ascribed to a peptide.8 While crambescidins were

patented due to their highly interesting cytotoxic and antiviral

activities,9 there are only limited data on the pharmaceutical

potential of crambescins.6b We describe herein the isolation and

structural elucidation of 11 crambescin derivatives (1–11)

together with crambescidin 816 (12) from the sponge C. crambe,

including for the first time the determination of the absolute

configurations deduced from comparisons between experimental

and theoretical electronic circular dichroism (ECD) spectra.

Four of these compounds have been reported earlier but usually

in a mixture.3a A further biological evaluation of these alkaloids

was performed against cortical neurons.

According to the proposition of the group of Rinehart, we will

classify the crambescins into three groups according to the

structural features of the left propyl side chain at C-8 of the

guanidinium core.6b The structure of all crambescins A includes a

pyrrolidine ring, while crambescins B are characterized by a

spiroaminal, and crambescins C by a linear 3-hydroxypropyl side-

chain at C-8 (Fig. 1). Controversies on the structure of crambescins

came from the number of methylenes present in the upper alkyl

side-chain (n+2) and in the lower guanidinoalkyl side-chain (m+2)

but also from the relative configurations of the substituents around

the cyclic core. Using chemical synthesis the group of Snider

inverted the relative configuration of crambescin B at C-8 initially

proposed by the group of Braekman.3b,6c While crambescin B (first

named crambine B) was first described with n = 8 and m = 3, the

groups of Rinehart and Snider then demonstrated independently

using mass spectrometry and chemical synthesis respectively that

the chain lengths should be revised as n = 6 and m = 5.6a,6b In the

same manner, the structure of crambescin C1 (previously named

crambine C1) was revised and the initial values of n = 8 and m = 3

were here also replaced by n = 6 and m = 5.6a,6b

The crambescins C2 (previously named crambine C2) will

correspond to a shorter guanidino alkyl side chain with m = 2. In

aNice Institute of Chemistry UMR 7272 CNRS, University of Nice Sophia-Antipolis, Faculte des Sciences, Parc Valrose 06108 Nice, France.E-mail: [email protected]; Fax: +33 49207 6151;Tel: +33 49207 6134bDepartment of Pharmacology, Facultad Veterinaria, Universidad deSantiago de Compostela, 27002, Lugo, Spain.E-mail: [email protected]; Fax: +34 982822233cPlate forme Bernard ROSSI Proteome et Metabolome, University of NiceSophia-Antipolis, Faculte de Medecine, 06107, Nice, France. E-mail: [email protected]; Tel: +33 493377708{ Electronic supplementary information (ESI) available: NMR andHRMS spectra are given for compounds 1–11. See DOI: 10.1039/c2ra00045h

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the same manner, we propose to use crambescin A2 and

crambescin B2 for the analogues with m = 2 because their

structures strongly differ from the usual crambescins with m

higher than four. Thus, most batzelladines, a large family of

crambescin ‘dimers’, all incorporate a crambescin A2 unit.10 The

biological evaluations and full structural assignments (including

the absolute configurations of these compounds) were hampered

by the difficulties associated with the purification processes, as

crambescins were usually isolated as a complex mixture of

homologues with added methylene units. We then decided to

purify all compounds to give additional data on the pure

crambescins.

Results and discussion

After an organic extraction of the lyophilized material with

CH2Cl2/MeOH (1 : 1), the extract was first submitted to fractiona-

tion by reversed phase Vacuum Liquid Chromatography. The

methanolic fraction was further purified by two successive HPLC

on semi-preparative C18 and analytical C3Phenyl reversed phase to

yield 11 pure crambescin derivatives and crambescidin 816. The 1H

NMR and HRMS spectra were used to confirm the high purity of

each isolated compound (.95%).

Each compound was then assigned to a crambescin family on

the basis of characteristic signals on their 1H NMR spectra. The

non-equivalent methylenes resonating at dH 2.99 and 3.33 (H-9),

2.11 and 2.23 (H-10) and 3.67 and 3.82 (H-11) ppm were

reminiscent of a crambescin A skeleton for compounds 1–4.

Compounds 6–8 were members of the crambescin B group due

to the absence of the signal at 4.40 (dd, H-13) ppm and the

presence of a characteristic signal at 2.99 (d, H-7) ppm.

Compounds 9–11 were part of the crambescin C family because

of the characteristic signals of the hydroxypropyl side chain

methylenes at 2.80 and 2.83 (H2-9), 1.81 (H2-10) and 3.61 (H2-11)

ppm (Table 1). Compound 5 exhibited unusual signals in its 1H

NMR spectrum which required deeper NMR analyses.

Comparison of the 1H NMR spectra for compounds 1–4

revealed clear differences in the intensity of the broad signals

around dH 1.30 and 1.40 ppm, assigned to the aliphatic side

chains protons of both the alkyl side chain at C-13 (n+2) and the

guanidinoalkyl chain at C-6 (m+2). These data were inferred to

differences in the lengths of both alkyl side chains. This

explanation may also account for the differences in the chemical

shifts at dH 4.25 and 4.22 (H2-5) ppm for 2, while these value

were dH 4.21 and 4.17 ppm for compound 4.

However, it was not possible to give clear data on the chain

lengths by NMR spectroscopy, due to signal overlapping and

imprecision in the signal integrations. As previously reported by

the group of Rinehart, mass spectrometry proved to be the

method of choice to assess the side-chain lengths.6b To be more

confident with our results, we performed (+)-HRESIMS/MS

analyses of each pure compound. All four crambescidin A

analogues differ by one or two methylene units (differences of 14

or 28 Da) (Table 2). Fragmentation analyses on compound 1 at

Table 1 1H NMR data of crambescin derivatives (500 MHz, CD3OD)

Position

Crambescin

A2 2 A1 4 5 B1 7 C1 10

2 3.23 3.17 3.16 3.16 3.173 1.68 1.60 1.61 1.59 1.60

3A 1.42 1.41 1.38 1.383B 1.42 1.41 1.38 1.413C 1.42 1.41 1.38 1.43

4 1.75 1.69 1.80 1.67 1.715 4.25 4.21 4.36 4.17 4.20

4.22 4.17 4.13 4.177 2.999 3.33 3.31 3.57 2.12 2.83

2.99 2.95 2.07 2.8010 2.23 2.23 2.39 2.10 1.81

2.11 2.10 2.0511 3.82 2.82 4.26 4.02 3.61

3.67 3.66 3.9313 4.40 4.39 3.86 4.4214 1.56 1.60 3.07 1.59 1.6015 1.41 1.41 1.72 1.47 1.4116 1.30 1.30 1.41 1.30 1.30

17-(15+n) 1.25–1.35 1.25–1.35 1.25–1.35 1.25–1.35 1.25–1.3516+n 0.90 0.90 0.90 0.90 0.90

Fig. 1 Crambescins and crambescidin skeletons.

Table 2 Crambescins isolated from C. crambe

Compound Crambescin m/z [M+H]+ m n

1 A2 449.36017 2 82 A2 463.37552 2 93 A2 477.39197 2 104 A1 463.37531 5 65 (Didehydro) A1 461.35976 5 66 B1 467.37051 4 67 B1 481.38629 5 68 B1 495.40207 6 69 C1 467.37027 4 6

10 C1 481.38635 5 611 C1 495.40182 6 6

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m/z 449.36017 [M+H]+ yielded two major fragments at m/z

336.26456 and 241.16585 corresponding to the molecular

formulae C19H34N3O2 and C11H21N4O2, respectively and were

consistent with the fragmentation scheme shown in Fig. 2.

The fragment peak at m/z 336.26456 [M–C5H11N3]+ indicated

the loss of a guanidine alkyl chain with m = 2 (Fig. 2). The

fragment at m/z 241.16285 [M–C13H24N2]+ was consistent with

an upper alkyl chain length of n = 8. The occurrence of the

analogous fragments at m/z 350.28018 and m/z 364.39654 in the

(+)-HRESIMS/MS spectra of compounds 2 and 3, respectively,

implied that both compounds had the same guanidinoalkyl chain

length (m = 2). The additional methylene units were then located

on the upper alkyl side chain which was confirmed by the

presence of the same fragments for all three compounds at m/z

241. Consequently, compounds 1–3 are all members of the

crambescin A2 family. The (+)-HRESIMS/MS mass fragmenta-

tion pattern of the molecular ion corresponding to compound 4

at m/z 463.37531 was completely different from those observed

for compounds 1–3. The presence of an intense fragment ion at

m/z 283.21280 was only consistent with a longer guanidinoalkyl

chain (m = 5). The length of the upper alkyl chain was then

deduced to be n = 6. According to this study, compound 4 is the

first isolated member of the crambescin A1 family and was

proven to be an isomer of compound 2, a member of the

crambescin A2 family, which underlined the importance of the

HRMS/MS study. In the original paper of the group of

Braekman only crambescins A2 (crambine A) were described

in a mixture.3b

The molecular formula of compound 5 was determined to be

C25H44N6O2 by HRESIMS (D 20.2 ppm by internal calibra-

tion), indicating seven degrees of unsaturation, one more than

compounds 2 and 4. The characteristic signals of crambescin A

were absent in the 1H NMR spectrum of 5 and the signals of the

methylenes at C-5 (dH, 4.36, t), C-9 (dH, 3.57, t) and C-11 (dH,

4.26, t) became equivalent and deshielded comparing to the data

obtained for 2 and 4. Furthermore, the upper alkyl chain was

connected to a quaternary carbon at C-13 (dC 180 ppm) due to

the H-14/C-13 HMBC. In consequence the additional unsatura-

tion was located at C-13 which induced an aromatized planar

pyrimidine cycle. The length of the upper alkyl chain was

estimated as being n = 6 from the integration of signals at dH

1.31 ppm (10H) assigned to H2-17 to H2-21 (Table 1). The length

of the guanidinoalkyl chain was assessed by the integration of

the signal at 1.41 ppm (8H) which corresponds to H2-3A, H2-3B

and H2-3C and H2-16, thus suggesting m = 5. The lengths of

the alkyl chains at C-13 and C-6 were further confirmed by

(+)-HRESIMS/MS fragmentation pattern of the molecular peak

at m/z 231.18352 [M+2H]2+ which gave two majors fragments at

m/z 306.21762 and m/z 156.14943, in accordance with the

molecular formulae C17H26N2O3 and C8H18N3, respectively.

The fragment peak at m/z 306.21762 [M–C8H17N3]+ corre-

sponds to the loss of a molecular ion of 155 Da, a 7–guanidino-1-

heptene. This result is consistent with m = 5 and n = 6 for both

alkyl chains and we could conclude that 5 is an oxidized form of

compound 4, belonging to the crambescin A1 family. 5 is then a

didehydrocrambescin A1 derivative. A dehydrocrambine A has

already been reported from an unknown sponge of the genus

Monanchora in Palau.11 The described dehydrocrambine A is an

oxidized equivalent of crambescin A2 (named crambine A) they

also isolated from that sponge. Aromatization into a pyrimidine

is not rare in this family of compounds.12 Just like for the

crambescin A2 derivative found in Monanchora sp., compound

4 was also isolated together with its oxidized form 5 which

reinforced the idea that an oxidation could partially take place

during the purification process, especially in the acidic conditions

required for the HPLC purification.

The 1H NMR spectra of compounds 6–8 first indicated that

they belong to the crambescin B family, especially for the

occurrence of the small doublet at 2.99 (J = 4 Hz, H-7) ppm.

The chain lengths were assigned on the basis of HRMS/MS data.

The fragmentation pattern differed from crambescins A and

both guanidines remain intact during the fragmentation whereas

the presence of the pyrrolidine in crambescins A induced a

fragmentation of the cyclic guanidine (Fig. 2).

Fragments were then observed at m/z 270.18124, 284.19693

and 298.21896 for compounds 6, 7 and 8 respectively, which

Fig. 2 Fragmentation pattern of crambescins.


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indicated a change in the chain lengths for the guanidino alkyl

chain with m = 4, 5 and 6 respectively and a common upper alkyl

chain with n = 6 for all crambescin B1 derivatives. The relative

configurations for compounds 6–8 were assigned by interpreta-

tion of the coupling constant between H-7 and H-13 which was

reported as 4–4.2 Hz in the cis isomers and 11.5–12 Hz in the

trans isomer.6a All isolated crambescins B1 had JH7–H13 close to

4.2 Hz revealing a cis configuration between H-7 and H-13.

Comparison of the chemical shifts for the proton of the spiro

cycle with the previously described crambescins B1 (crambine B)

led us to propose the same relative configurations at C-8 than

those proposed by Snider.6a

The 1H NMR spectra of compounds 9–11 indicated that they

belong to the crambescin C family, especially for the occurrence

of the triplet at 3.61 (H2-11) ppm. The same fragmentation

patterns as those obtained for crambescins B were observed

which allowed us to assign the chain lengths of all three

crambescin C derivatives. Here also, differences between the

three compounds 9–11 were identified in the length of the lower

guanidine alkyl chain with m values of 4, 5 and 6 while the upper

alkyl chain at C-13 remain unchanged with the same n = 6. In

consequence these compounds are all crambescin C1 analogues.

At this point, it is interesting to note that most of the crambescin

A derivatives are from the crambescin A2 family while

crambescins B1 and C1 are only found in the sponge C. crambe.

In contrast to the second study of the group of Braekman,3a we

did not manage to identify a sufficient quantity of crambescin

C2. The presence of short guanidino alkyl chains with m = 2 in

crambescin A2 is remarkable and much more if we observe that

all batzelladine derivatives incorporate only this monomer.12

This observation raises the question of the biosynthetic path-

ways leading to these compounds, as studied first by the group of

Snider,6a and then by the group of Quinn for the mirabilins,

which are tricyclic monomers, analogues of the mono or bicyclic

crambescins.13 The addition of a guanidine to a unique polyketide

chain is questionable as no enzyme has been described to perform

such transformation. We rather propose the involvement of an

arginine or a homologue for the guanidines. Indeed, we previously

shown that the 2-aminoimidazole side chain of oroidin originates

from homoarginine,14 and, recently, the group of Molinski

proposed the same origin for a guanidinated derivative of the

polyacetylene family.15 We will address this issue for crambescins

by in vivo biosynthetic experiments.

With all pure crambescins in hand, we were able to assign the

absolute configurations of the three families A, B and C using

ECD and comparison with theoretical values as already reported

by us for other families of marine natural products.16 We

anticipated strong Cotton effects due to the presence of

chromophoric groups (nAp* transition of the enone) placed in

the vicinity of asymmetric centers as for the asymmetric center at

C-13 always substituted by the guanidine and an enone in the

cases of crambescins A and C. Experimental ECD spectra were

then compared to the calculated ECD spectra of both

enantiomers. As illustrated on Fig. 3, there is a good overlapping

between both experimental and theoretical ECD spectra for one

enantiomer of the three different kinds of crambescins. All our

results converge to suggest an absolute S configuration at C-13

which is in accordance with the only result obtained on the

biosynthetic congener crambidine using asymmetric chemical

synthesis.17 The only discrepancy we observed for these

11 compounds was for the ECD spectrum of one crambescin

C1 (9) where the signs of the Cotton effects opposite to the one

measured for 10 and 11. Nevertheless in this case the intensities

of the bands were very low and the other results obtained for

10 and 11 seemed more reliable.

While crambescidins were largely evaluated for their biological

properties, crambescins were much less studied because of the

difficulties to obtain pure compounds. For the first time, we

decided to test the activity of one representative of each family

isolated in sufficient amount on cortical neurons. In the MTT

assay, we observed that 24 h of exposure of the cortical neurons

to different concentrations (0.001 to 1 mM) of crambescidin

816 (12) caused a dose-dependent increase on the cytotoxic effect

and the almost complete cell death at 1 mM (86.3 ¡ 6.8%),

whereas crambescin C1 (10) only lowered cellular viability by

Fig. 3 Experimental and calculated CD spectra for crambescins A2 (1),

B1 (7) and C1 (11).


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22.6 ¡ 4.1% at 1 mM (Fig. 4). The effects after long term

treatment were similar, with an IC50 value of 432 nM for 12, and

a decrease in cell viability of 40.8 ¡ 12.6% for 10 at 1 mM.

Crambescin C1 (10) and crambescidin 816 (12), tested at

different concentrations (0.001 to 1 mM) caused reductions in

the number of cortical living cell at 1 mM compared to the cells

control. However no significant differences were observed for the

several concentrations of 10. There is not much literature about

crambescidin 816 (12) cytotoxicity. In a previous publication in

IC2 murine mast cell lines the crambescidin acid presents an IC50

value of 25.0 mg ml21,18 whereas our value in cortical neurons is

0.354 mg ml21. A full cytotoxic evaluation of crambescidin

analogues was also reported but crambescidin 816 (12) was not

evaluated.19 This observation suggests that cell type is a crucial

determinant for the potency of the compound. Therefore, the

toxic or therapeutic effect of 12 must the carefully studied in each

system, given the extremely high potency of the compound on

the nervous model.

Experimental

General experimental procedures

HPLC purifications were carried out on a Waters 600 system

equipped with a Waters 717 plus autosampler, a Waters 996

photodiode array detector, and a Sedex 55 evaporative light-

scattering detector (SEDERE, France). Detection wavelengths

were set at 214, 254 and 280 nm. 1H and 13C NMR spectra were

recorded on a 500 MHz Bruker Avance NMR spectrometer.

Chemical shifts (d) are recorded in ppm with CD3OD (dH 3.31 ppm

and dC 49.00 ppm) as internal reference with multiplicity (s singlet,

d doublet, t triplet, m multiplet, br broad). LCESIMS analyses

were carried out on a Waters 2695 system equipped with a Waters

2487 dual l absorbance detector and a Bruker Esquire 3000 Plus

spectrometer (Ion Trap). High-resolution mass spectra ESIMS

(HRESIMS/MS) were conducted on a LTQ Orbitrap mass

spectrometer (Thermo Fisher Scientific). The MSI system of the

LTQ-Orbitrap hybrid mass spectrometer was operated in the

positive mode at a voltage of 4.5 kV, with no sheath or auxiliary

gas and in maintaining the ion transfer tube at 275 uC. The

compounds were then infused at a flow rate of 0.5 mL min21. The

Orbitrap mass analyzer was calibrated according to the manufac-

turer’s directions using a mixture of caffeine, MRFA peptide, and

Ultramark for positive ionization mode. Selected ion monitoring

profile MS data (10 Da window and 40 scans) were acquired with

resolving power setting of 100 K. The injection time was from 50 to

350 ms with a target of 100 000 ions adjusted by automatic gain

control. To achieve the highest possible mass accuracy, the lock

mass function was enabled with the pollutant ion at an m/z of

391.28429 in the positive ionization mode used for real-time

internal recalibration. MS data acquisition and processing were

performed using Xcalibur software (version 2.0.7; Thermo Fisher

Scientific). Spectral accuracy was calculated by MassWorks using

sClips software (version 2.0; Cerno Bioscience). The mass tolerance

for the sClips searches was 2 ppm. Optical rotations were measured

on a Jasco P-1020 polarimeter. UV and CD spectra were measured

using a JASCO J-810 spectropolarimeter.

Collection

The sponge specimens of Crambe crambe were collected in

March 2011 by hand using SCUBA diving at Villefranche-Sur-

Mer (France), at depths ranging from 10 to 20 m, and kept

frozen until used.

Bioassays

1. Primary cultures of cortical neurons. Swiss mice were used

to obtain primary cultures of cortical neurons. All protocols

were approved by the University of Santiago de Compostela

Institutional animal care and use committee. Primary cortical

neurons were obtained from embryonic day 15–17 swiss mice as

previously described.20 Briefly, cerebral cortices were removed

and dissociated by mild trypsinisation, followed by mechanical

trituration in a DNAse solution (0.005% w/v) containing a

soybean trypsin inhibitor (0.05% w/v). The cells were suspended

in Neurobasal Medium supplemented with B-27 and penicillin-

streptomycin. The cell suspension was seeded in 96 multiwell

plates precoated with poly-D-lysine and in a humidified 5%

CO2/95% air atmosphere at 37 uC.

2. Determination of cellular viability. Cell viability was assessed

by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-

lium bromide) test, as previously described.21 This test, which

measures mitochondrial function, was used to assess cell viability

Fig. 4 Effects of crambescin C1 (10) and crambescidin 816 (12) on cell

viability in primary cultures of cortical neurons.


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as it has been shown that in neuronal cells there is a good

correlation between a drug-induced decrease in mitochondrial

activity and its cytotoxicity.22 The assay was performed in

cultures grown in 96 well plates and exposed to different

concentrations (0.001 mM, 0.01 mM, 0.1 mM and 1 mM) of 10 and

12 added to the culture medium. Cultures were maintained in the

presence of the toxin at 37 uC in humidified 5% CO2/95% air

atmosphere for 24 h or five days in vitro (3–7 div). Saponin was

used as cellular death control and its absorbance was subtracted

from the other data. After the exposure time, cells were rinsed

and incubated for 60 min with a solution of MTT (500 mg ml21)

dissolved in Locke’s buffer. After washing off excess MTT, the

cells were disaggregated with 5% sodium dodecyl sulfate and the

absorbance of the colored formazan salt was measured at

595 nM in a spectrophotometer plate reader.

Extraction and isolation

The samples of Crambe crambe were freeze dried and then ground

to obtain a dry powder (15 g) which was exhaustively extracted

with a mixture of MeOH/CH2Cl2 (1 : 1, v/v) to yield 2.4 g of crude

extract after concentration under reduced pressure. The crude

extract was fractionated by Reverse Phase Vacuum Liquid

Chromatography over RP-18 silica gel (200 g) with a step gradient

of H2O (F1 fraction, 581 mg), MeOH/H2O 2/1 v/v (F2 fraction

286 mg), MeOH/H2O 3/1 v/v (F3 fraction, 284 mg), MeOH (F4

fraction, 767 mg) and DCM (F5 fraction, 450 mg) (500 mL each).

The F2 (140 mg, 100 mg ml21) and F3 (160 mg, 100 mg ml21)

fractions were then subjected to semi-preparative HPLC purifica-

tion (Phenomenex Luna C6-Phenyl, 250 6 10 mm, 5 mm) eluted

with a gradient of H2O/ACN/TFA : isocratic step from 0 min to

5 min (90 : 10 : 0.1) and then gradient steps from 5 min to 6 min

(from 90 : 10 : 0.1 to 70 : 30 : 0.1) and from 6 to 36 min (from

70 : 30 : 0.1 to 55 : 45 : 0.1) (flow rate: 3.0 mL min21, injection

volume: 100 mL) to give 9 and 13 fractions respectively, named

from F2P1 to F2P9 and F3P1 to F3P13. The fractions F2P2 (tr

21.5 min), F2P3 (tr 23.0 min), F2P4 (tr 26.0 min), F2P5

(tr 28.0 min) and F2P9 (tr 36.0 min) were further subjected to

analytical HPLC purification (XSelect C6-Phenyl, 250 6 4.6 mm,

5 mm). Fractions were eluted with a gradient of H2O/ACN/TFA:

isocratic step from 0 min to 5 min (90 : 10 : 0.1) and then gradient

steps from 5 min to 6 min (from 90 : 10 : 0.1 to 68 : 32 : 0.1) and

isocratic step from 6 to 30 min (68 : 32 : 0.1) (flow rate: 0.80 mL

min21, injection volume: 30 mL). This step yielded the major

components of the F2P2, F2P3, F2P4 and F2P9 fractions and so

to yield compound 9 (1.2 mg), 10 (11.1 mg), 11 (2.4 mg), 12

(10.2 mg), respectively, and, in the case of F2P5 fraction, to afford

5 (tr 24.5 min, 0.8 mg), 6 (tr 25.2 min, 0.9 mg), 4 (tr 26.5 min,

0.9 mg) and 7 (tr 28.2 min, 1.1 mg). Similar analytical HPLC

analyses were performed on fractions F3PP7 (tr 29.0 min), F3P8

(tr 30.5 min), F3P9 (tr 32.0 min) and F3P12 (tr 37.0 min) and let to

afford 1 (2.0 mg), 8 (1.1 mg), 2 (2.0 mg), 3 (1.3 mg), respectively.

At the total, twelve guanidine alkaloids were purified. All of them

were identified by a combination of spectroscopic methods (1D

and 2D NMR, HRESIMS).

Norcrambescin A2 (1)

[a]20D +7.1 (c 0.2, MeOH); UV (MeOH) lmax (log e) 288 nm (2.7);

CD (MeOH, c 4.46 6 1024 M) De (lmax nm) +0.66 (209) –2.22

(248) –0.99 (291); ESIMS m/z 449.3 [M+H]+; HRESIMS m/z

449.36017 [M+H]+ (Calc for C24H45O2N6, 449.35985,

D 20.70 ppm). 1H NMR (500 MHz) and 13C NMR

(125 MHz, CD3OD) (lH, J, 13C) C-1 (158.6), H2C-2 (3.23 t,

6.8; 42.1), H2C-3 (1.68 m; 26.6), H2C-4 (1.75 m; 27.1), H2C-5

(4.22 m, and 4.23 m; 65.1), C-6 (166.1), C-7 (103.2), C-8 (152.7),

H2C-9 (2.99 ddd, 9.5/ 9.8/18.3 and 3.30; 31.9), H2C-10 (2.11 m

and 2.23 m; 22.9), H2C-11 (3.67 ddd, 7.2/9.7/9.93 and 3.82 ddd,

2.8/ 9.1/9.4; 49.2), C-12 (153.0), HC-13 (4.40 dd, 7.1; 51.3), H2C-

14 (1.56 m; 37.5), H2C-15 (1.41 m; 25.2), H2C-16 to H2C-21

(1.25–1.35 br s; 30.0–31.0), H2C-22 (1.25–1.35 br s; 33.1), H2C-23

(1.29 br s; 23.8), H3C-24 (0.90 t, 6.8; 14.3).

Crambescin A2 (2)

[a]20D +12.1 (c 0.18, MeOH); UV (MeOH) lmax 287 nm (log e 2.7);

CD (MeOH, c 4.33 6 1024 M) De (lmax nm) –1.86 (249) –0.81

(288); ESIMS m/z 463.3 [M+H]+; HRESIMS m/z 463.37552

[M+H]+ (Calc for C24H45O2N6, 463.37550, D 20.04 ppm).

Homocrambescin A2 (3)

(3) [a]20D +35.9 (c 0.1, MeOH); UV (MeOH) lmax 288 nm (log e 2.3);

CD (MeOH, c 4.20 6 1024 M) De (lmax nm) +0.58 (207) –1.93 (249)

–0.85 (288); ESIMS m/z 477.0 [M+H]+; HRESIMS m/z 477.39197

[M+H]+ (Calc for C26H49O2N6, 477.39115, D –1.71 ppm).

Crambescin A1 (4)

[a]20D +8.9 (c 0.06, MeOH); UV (MeOH) lmax (log e) 289 nm (1.5);

CD (MeOH, c 4.84 6 1024 M) De (lmax nm) 20.10 (218) 20.06

(288) +0.08 (330); ESIMS m/z 463.2 [M+H]+; HRESIMS m/z

463.37531 [M+H]+ (Calc for C25H47O2N6, 463.37550, D

–0.42 ppm). 1H NMR (500 MHz) and 13C NMR (125 MHz,

CD3OD) (lH, J, 13C) C-1 (158.6), H2C-2 (3.17 t, 6.9; 42.5), H2C-3

(1.60 m; 29.8), H2C-3A (1.42 br s; 27.6), H2C-3B (1.42 br s; 29.9),

H2C-3C (1.42 br s; 27.1), H2C-4 (1.69 m; 29.8), H2C-5 (4.17 m,

17.4 and 4.21 m, 17.4; 65.6), C-6 (166.9), C-7 (100.4), C-8 (155.1),

H2C-9 (2.95 ddd, 9.1/ 9.5/18.9 and 3.31; 31.9), H2C-10 (2.10 m

and 2.23 m; 22.9), H2C-11 (3.66 ddd, 7.5/9.5/10.3 and 3.82 ddd,

2.8/9.5/10.3; 49.5), C-12 (154.4), HC-13 (4.39 m; 51.4), H2C-14

(1.60 m; 37.6), H2C-15 (1.30 br m; 25.2), H2C-16 to H2C-19

(1.30 br m; 30.35, 30.44, 30.60, 30.64), H2C-20 (1.30 br m; 33.1),

H2C-21 (1.30 br m; 23.8), H3C-22 (0.90 t, 7.4; 14.5).

Didehydrocrambescin A1 (5)

ESIMS m/z 461.3 [M+H]+; HRESIMS m/z 461.35976 [M+H]+

(Calcd for C25H45N6O2 461.35985 found 461.35976, D –0.20 ppm).1H NMR (500 MHz) and 13C NMR (125 MHz, CD3OD) (lH, J,13C) C-1 (158.5), H2C-2 (3.16 t, 6.6; 42.5), H2C-3 (1.61 m; 30.1),

H2C-3A (1.41 br s; 28.9), H2C-3B (1.41 br s; 29.9), H2C-3C (1.41 br

s; 27.3), H2C-4 (1.80 m; 29.9), H2C-5 (4.36 t, 7.6; 67.0), C-6 (165.9),

C-7 (111.7), C-8 (170.6), H2C-9 (2.57 t, 8.6 and 3.30; 35.6), H2C-10

(2.05 m and 2.39 q; 20.8), H2C-11 (4.26 t, 9.1; 53.1), C-13 (180.2),

H2C-14 (3.07 t, 9.5; 38.2), H2C-15 (1.72 m; 30.0), H2C-16 (1.41 br

m; 27.3), H2C-17 to H2C-19 (1.31 br m; 30.0-31.0), H2C-20 (1.31 br

m; 32.8), H2C-21 (1.31 br m; 23.6), H3C-22 (0.90 t, 7.5; 14.5).

Norcrambescin B1 (6)

[a]20D 2114.0 (c 0.05, MeOH); UV (MeOH) lmax (log e) 285 nm

(1.8), 326 nm (1.9); CD (MeOH, c 4.03 6 1024M) De (lmax nm)


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20.53 (211) +0.75 (247) +0.55 (326); ESIMS m/z 467.3 [M+H]+;

HRESIMS m/z 467.37051 [M+H]+ (Calc for C24H47O3N6,

467.37042, D +0.21 ppm).

Crambescin B1 (7)


(1.9), 325 nm (1.9); CD (MeOH, c 3.58 6 1024 M) De (lmax nm)

–0.51 (211) +0.74 (247) +0.53 (328); ESIMS m/z 481.2 [M+H]+;


481.38607, D +0.47 ppm). 1H NMR (500 MHz) and 13C NMR

(125 MHz, CD3OD) (lH, J, 13C) C-1 (158.6), H2C-2 (3.16 t, 7.3;

42.6), H2C-3 (1.59 m; 29.7), H2C-3A (1.38 br s; 27.8), H2C-3B

(1.38 br s; 30.3), H2C-3C (1.38 br s; 27.1), H2C-4 (1.67 m; 29.9),

H2C-5 (4.13 m and 4.17 m; 66.3), C-6 (169.9), HC-7 (2.99 d, 4.3;

50.2), C-8 (89.9), H2C-9 (2.07 m and 2.12, m; 36.1), H2C-10

(2.10 m; 25.7), H2C-11 (3.93 m and 4.02 m; 68.9), C-12 (155.2),

HC-13 (3.86 ddd, 4.3/6.7/7.3; 49.7), H2C-14 (1.59 m; 33.1), H2C-

15 (1.47 m; 26.5), H2C-16 to H2C-19 (1.31 br m; 30.0–31.0), H2C-

20 (1.31 br m; 32.8), H2C-21 (1.31 br m; 23.7), H3C-22 (0.90 t,

7.1; 14.4).

Homocrambescin B1 (8)


(1.8), 326 nm (1.8); CD (MeOH, c 1.34 6 1024 M) De (lmax nm)

–0.90 (211) +1.42 (247) +0.90 (326); ESIMS m/z 495.3 [M+H]+;


495.40172, D 20.71 ppm).

Norcrambescin C1 (9)

[a]20D +76.3 (c 0.08, MeOH); UV (MeOH) lmax (log e) 279 nm

(2.3); CD (MeOH, c 5.15 6 1024 M) De (lmax nm) +0.08 (207) –

0.20 (247); ESIMS m/z 467.5 [M+H]+; HRESIMS m/z 467.37027

[M+H]+ (Calc for C24H47O3N6, 467.37042, D 20.31 ppm).

Crambescin C1 (10)


(2.8); CD (MeOH, c 1.39 6 1024 M) De (lmax nm) 20.48 (213)

+2.24 (247); ESIMS m/z 481.5 [M+H]+; HRESIMS m/z

481.38635 [M+H]+ (Calc for C25H49N6O3, 481.38607, D

0.59551). 1H NMR (500 MHz) and 13C NMR (125 MHz,

CD3OD) (lH, J, 13C) C-1 (159.1), H2C-2 (3.17 t, 7.0; 42.8), H2C-3

(1.60 m; 30.9), H2C-3A (1.41 br s; 27.9), H2C-3B (1.41 br s; 30.4),

H2C-3C (1.41 br s; 27.4), H2C-4 (1.71 m; 30.2), H2C-5 (4.17 m

and 4.20 m; 66.3), C-6 (166.5), C-7 (106.4), C-8 (149.4), H2C-9

(2.80 m and 2.83, m; 29.1), H2C-10 (1.81q; 32.5), H2C-11 (3.61 t,

6.5; 62.5), C-12 (153.9), HC-13 (4.42 dd, 7.5/5.0; 51.5), H2C-14

(1.60 m; 37.4), H2C-15 (1.41 m; 25.4), H2C-16 to H2C-19 (1.30 br

m; 30.0-32.0), H2C-20 (1.30 br m; 33.4), H2C-21 (1.30 br m;

24.7), H3C-22 (0.90 t, 6.5; 15.1).

Homocrambescin C1 (11)


(2.9); CD (MeOH, c 0.68 6 1024 M) De (lmax nm) –0.58 (214)

+3.03 (249); ESIMS m/z 495.0 [M+H]+; HRESIMS m/z

495.40182 [M+H]+ (Calc for C26H51O3N6, 495.40172, D

+0.22 ppm).

Crambescidin 816 (12)

As reported in ref. 8.

Theoretical calculations of the electronic dichroism spectra

Quantum chemical calculations have been performed for all

examined compounds 1–11. Conformational analysis was

performed using the conformer research algorithm implemented

in the Conflex-Barista software.23 Given the high degree of

conformational freedom of the side chains, the conformational

analysis led to a large number of structures (n . 500). The

Gaussian03W package24 has been used for the electronic circular

dichroism calculations on the most stable conformer of each

compound. Density functional theory (DFT) with B3LYP

functional25 and Pople’s 6.31++G(d,p)26 basis set was used on

the lowest energy conformers. TDDFT was employed to

calculate excitation energy (in eV) and rotatory strength R in

dipole velocity (Rvel) and dipole length (Rlen) forms. The

calculated rotatory strengths were simulated in ECD curve by

using a Gaussian function:

De Eð Þ~ 1

2:296|10{39ffiffiffiffiffiffiffiffiffi

2pDp

X

a

DE0aR0ae{E{DE0að Þ

2D

� �2

(1)

where D is half the width of the band at 1/e peak height expressed

in energy units. The parameters DE0a and R0a are the excitation

energies and the rotatory strengths for transition from the

ground state 0 to an excited state a, respectively, D = 0.1 eV and

Rvel were used.

Conclusions

The full chemical study of the Mediterranean sponge C. crambe

led to the isolation and structural characterization of 11

crambescins A1, A2, B1 and C1 in their pure forms. Eight of

the 12 isolated compounds are described for the first time and the

lengths of the alkyl chains were deduced from the careful analyses

of HRMS/MS data. The presence of the new didehydrocrambes-

cin A1 (5) derivative supports the assumption that aromatization

could occur in this family during the acidic purification process.

Our studies allowed the assignment of the absolute configurations

of all compounds and revealed a high cytotoxic activity of

crambescidin 816 (12) against cortical neurons.

Acknowledgements

This work was supported by the European Coordinative

Project FP7-KBBE-2010-4-BAMMBO (265896) and the

French ECIMAR project (ANR-06-BDIV-01). We are grateful

to Thierry Perez (Centre d’Oceanologie d’Endoume) for

taxonomic identification and to Marc Gaysinski (PFTC Nice)

for recording the NMR spectra. Assistance for submarine

collection was kindly given by David Luquet (Observatoire

Oceanologique de Villefranche sur Mer).

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Documents

Additional bioactive guanidine alkaloids from the Mediterranean sponge Crambe crambe