Two novel pentacyclic guanidine alkaloids, celeromycalin 3 and fromiamycalin 4, have been isolated from the New Caledonian starfishes Celerina heffernani and Fromia monilis, respectively. The new compounds exhibited an high cytotoxic activity like the previous crambescidins. These complex pentacyclic guanidines are typical sponges metabolites and their occurence in starfishes is noteworthy. Fromia monilis also contained the less active component 5, made up from an hydroxyspermidine residue linked to a long chain omega-hydroxyacid

Carré, Jean-Baptiste

De Marino, S.

Debitus, Cécile

Iorizzi, M.

Lucarain, L.

Minale, L.

Palagiano, E.

Provost, J.

Riccio, R.

Zollo, F.

Name not available

Ptilomycalin A, crambescidin 800 and related new highly cytotoxic guanidine alkaloids from the starfishes Fromia monilis and Celerina heffernani

Horizon / Pleins textes

Pergamon 
I '  
I 
't 
Tefruhedron Vol. 51, No. 12, pp. 3675-3682, 1995 
Copyright O 1995 Elsevier Science Ltd 
Printed in Great Britain. All rights reserved 
0040-4020/95 $9.50+0.00 
0040-4020(95)00082-8 
Ptilomycalin A, Crambescidin SOO and Related 
New Highly Cytotoxic Guanidine Alkaloids from the 
Starfishes Fromia rnonìlis and Celerina iteffernani. 
Elio Palagiano, Simona De Marino, Luigi Minale*, Raffaele Riccio 
and Franco Zoll0 
Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli "Federico U", 
Via Domenico Montesano, 49,80131 Napoli, ITALY. 
Maria Iorizzi 
Universita degli Studi del Molise, Via Tiberio 21M, 86100 Campobasso. 
Jean Baptiste Carré and Cécile Debitus 
Centre ORSTOM, B.  P. A5, Noumea, Nouvelle Calaonie 
Laetitia Lucarain and Jean Provost 
Rhône-Poulenc Rorer Centre de Recherche de VITRY-ALFORTVLLJ3 
13 quai Jules Guesde, BP14 94403 VITRYKEINE Cedex FRANCE 
Abstract: Two novel pentacyclic guanidine alkaloids, celeromycalin 3 and fromiamycalin 4, have been 
isolated from the New Caledonian starfishes Celerina heffernani and Fromia monilis, respectively. 
Also found in Celerina heflemani are the known ptilomycalii A (1) and crambescidin 800 (2), which 
latter has been also isolated from Fromia nwnilis. The new compounds exhibited an high cytotoxic 
activity like the previous crambescidins. These complex pentacyclic guanidines are typical sponges 
metabolites and their occurence in starfishes is noteworthy. Frotnia nzunilis also contained the less 
active component 5, made up from an hydroxyspermidine residue linked to a long chain a- 
hydroxyacid. 
Ptilomycalin A (1) from the sponges Ptilocaulis spiculver and Hemimycale SP.',~ and the related 
crambescidins (e. g. crambescid9 800,2) from the sponge Crambe crambe 3,4 are an unique f d y  of complex 
pentacyclic guanidines linked by a linear o-hydroxy fatty acid to a spermidine or hydroxyspermidine unit. These 
compounds, which differ from one another in the chain length of the long-chain hydroxy-acid and in the 
presence or absence of a hydroxyl group at C-13 in the guanidine-containing portion andor in the spermidine 
moiety, were found to be very antiviral, cytotoxic and antifungal agents. In connection with our systematic 
investigation of the chemical constituents of echinoderms 6 we have examined the polar extracts of the 
starfishes Fromia Nlonilis and Celerina heffentani, collected off New Caledonia, which both showed marked 
activity in antiviral assays vs. Herpes sinplex virus HSV-1. In a previous paper we described the steroidal 
- _ - - _ _  - -  - -  - 
y 3676 E. PALAGIANO et al. 
glycoside constituents from Fromia monilis 7, which were modestly cytotoxic, and in this paper we report the 
discovery of crambescidin 800 (2) in both Fromia monilis and Celerina heffernani, which latter also contained 
ptilomycalin A (l), and of two new related guanidine alkaloids, named celeromycalin 3, from Celerina 
heffemani, and fromiamycalin 4, from Fromia manilis, as the highly cytotoxic agents. Fromia monilis also 
contained the modestly active compound 5, made up from the hydroxyspermidine residue linked to long chain 
w-hydroxy'acid, the so called "anchor part" of this unique family of compounds, the pentacyclic guanidine 
moiety having been indicated the "vessel part" z. 
R=R,=H Ptilomycalin A (1) 
R=H, 'R,=OH Crambescidin 800 (2) 
R=OH, R,=H Celeromycalin (3) 
Fromiamycalin (4) 
These compounds were submitted to anti-HIV assays on cells CEM 4 infected by HIV-1 by the ANTI- 
AIDS UNIT of Rhône Poulenc Rorer and compounds 1-4 were found to be highly cytotoxic to the target cells 
with CC-50 of 0.11 yg/ml compounds (1, 2 and 4) and 0.32 pg/ml (compound 3) without cytoprotective 
effects at a dose of < O. 1 yglml. Interestingly compound 5 exhibited a weaker cytotoxicity with CC-50 of 2.7 
yglml. These results related a structure activity relationship among these compounds, showing that the activity 
is mostly due to the pentacyclic guanidine portions of the molecules. 
Ptilomycalin A and crambescidin SOO, 
RESULTS AND DISCUSSION 
3677 
Compounds 1-5 were isolated from the methanol soluble portion of the acetone extracts of the fresh 
organisms by DCCC separations, the solvents used being CHC1,-MeOH-H,O 7: 1323 (ascending) and n-BuOH- 
Me$O-H,O 3:1:5 (descending), followed by reverse phase HPLC (see Experimental). The known compounds 
1 and 2 were identified by FABMS and NMR spectra and comparison with the literature data ,s3. 
Celeromycalin 3, [aID=-4.5', isolated from C. heffemani, exhibited in the FAB mass spectrum a 
pseudomolecular ion at m/z 801 [M + HI+, already observed in the spectrum of crambescidin SOO (2). 
Comparison of the lH and 13C NMR data of crambescidin 800 (2) and ptilomycalin A (1) with those for 3 
(Table 1) argues the presence of the same ring system, and probably, the same relative stereochemistry, which 
was then confirmed by NOE difference spectra (CD,OD). NOE's between H-10 and H-13, between CH,-1 and 
both H-10, H-13, and between H-13 and H-14, established the relative stereochemistry at C-8, C-10, C-13 and 
C-14. Similarly, NOE between the CH,-1 and H-19 assigned the stereochemistry at C-15 and C-19, in 
agreement with the stereochemistry reported for crambescidins and ptilomycalin A (l), and eliminated that 
reported for isocrambescidin 800, which requires inversion of the configuration of C-13, C-14 and C-15 with 
respect to 1 and 2 4. Further analysis of the 1H and 13C NMR data for 3 established the presence of the 
spermidine residue, like in ptilomycalin A (I). The position of the hydroxyl group in the polymethylene chain of 
3 was assigned by HMBC NMR,experiment (Fig. l), which indicated that the proton at 6 2.62 ppm (H-37) 
correlated with both the carbonyl carbon at 6 175.3 ppm (C-38) and the hydroxymethine carbon at 6 70.1 ppm 
(C-36). The carbon signal at 175.3 ppm, in turn, correlated with the protons on C-39 and C-42 of the 
spermidine residue, thus confirming unambiguously the assignement of signal at 6 175.3 ppm to the amide 
carbonyl-38, and accordingly located the hydroxyl group at C-36. The configuration 36R was assigned based 
on the results of the application of the Mosher's method 8 ~ 9 9  10. In the 1H NMR spectrum of the 36-(S)-a- 
methoxy-a-(trifluoromethyl)phenyl acetate (MTPA ester), the C-37 methylene proton signal were observed 
upfield shifted relative to the corresponding ones in the lH NMR spectrum of the 36-(R)-MTPA ester [A6 (&- 
aR); for H-37,=-0.03; and H-37@=-0.06 1, in agreement with the 36R configuration. The proton signals at C-35 
overlap with those of the polymethylene chain and were not observed as separate peaks. The absolute 
configuration of the pentacyclic guanidine moiety of one member of the crambescidin family was recently 
determined 4. Thus, assuming the same absolute configuration, formula 3 can be regarded as the absolute 
stereostructure of the novel celeromycalin. 
OH - 
i" 
Fig. 1 J1 Some HMBC correlations of Celeromycalin (3) and Fromiamycalin, (4) 
3678 E. PALAGIANO et al. 
Table 1. 1H 13C NMR data for Celeromycalin 3 and Fromiamycalin 4 (CD30D). 
Position 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
22 
23 
24 
25 
26-34 
35 
36 
37 
38 
39 
40 
41 
42 
43 
44 
45 
3 
0.89 t (7.3) 
1.48 m, 1.58 m 
4.45 brd (10.3) 
5.55 brd (10.5) 
5.75 brt (9.5) 
2.22 m, 2.45 m 
2.04 dd (13.4,5.7), 
2.41 m 
2.68 dd, 1.45 t 
4.08 m 
2.32 m, 1.60 
2.38, 1.85 m 
4.38 m 
3.12 d (5.0) 
1.75 m, 1.55 m 
1.87 m, 1.95 m 
1.32 m, 1.72 m 
3.89 m 
1.13 d (6.3) 
4.17 t (6.6) 
1.63 m 
1.32 m 
s 1.32m 
1.43 m, 1.55 m 
4.04 m 
2.62 dd (14.5,9.3), 
2.42 dd 
3.51 m, 3.62 m 
2.1 m 
2.94 m 
3.40 dd (14.0,3.5), 
3.54 dd (14.0,lO.O) 
1.98 m, 2.05 m 
1.68 m, 1.74m 
3.00 m 
6, 
10.7 
30.7 
72.3 
134.3 
131.3 
24.4 
38.1 
85.1 
38.4 
55.5 
31.5 
27.6 
54.0 
50.9 
82.1 
32.7 
19.4 
33.0 
68.4 
21.7 
150.1 
170.0 
66.5 
30.2 
30.1-30.6" 
30.1-30.6" 
38.5 
70.1 
41.3 
175.3 
43.3 
29.6 
37.9 
48.6 
26.8 
26.9 
40.5 
4 
0.89 t (7.3) 
1.48 m, 1.58 m 
4.45 brd (10.3) 
5.55 brd (10.5) 
5.75 brt (9.5) 
2.22 m, 2.45 m 
2.04 dd (13.4,5.7), 
2.41 m 
2.68 dd, 1.45 t 
4.08 m 
2.32 m, 1.60 
2.38, 1.85 m 
4.38 m 
3.12 d (5.0) 
1.75 m, 1.55 m 
1.87 m, 1.95 m 
1.32 m, 1.72 m 
3.89 m 
1.13 d (6.3) - 
4.17 t (6.6) 
1.63 m 
1.32 m 
1.32 m 
1.32 m 
2.1 m 
2.80 m, 2.52 m 
3.54 m, 3.61 m 
2.1 m 
3.42 m, 3.64 m 
3.42 dd (14.0,3.5), 
3.64 dd (14.0,lO.O) 
4.08 m 
1.88 m, 1.78 m 
3.16 m 
6, 
80.8 
30.6 
72.3 
134.3 
131.3 
24.4 
38.1 
85.1 
37.8 
55.5 
31.5 
27.6 
54.0 
50.7 
82.1 
32.6 
19.5 
33.0 
68.4 
21.8 
l'SO. 1 
170.2 
66.5 
29.6 
27.0 
30.1-30.6" 
27.7 
30.1-30.6" 
4 32.8 
166.2 
47.8 
19.8 
39.9 
58.5 
67.1 
32.6 
38.4 
a : several signals between 30.12 and 30.62. The coupling constants are given in Hz and are enclosed in parentheses. 
The assigments were aided by 2D-COSY, 2D-HOHAHA, HhIQC, HMBC experiments, and comparison with the spectra described 
for the known 1 and 2. 
Ptilomycalin A and crambescidin SOO 3679 
The FABMS spectrum of fromiamycalin 4, [a],=-12', isolated from F. modis, exhibited a 
pseudomolecular ion at m/z 783 [M + HI+, eighteen mass units shifted relative to 2 and 3 (mlz 801). The 
presence of a 3-hydroxyspermidine residue was at f is t  indicated by a fragment peak in the FAT3 mass spectrum 
at m/z 696 [M +H - 871, corresponding to the loss of a C,H,NO (aminohydroxybutyl residue) from [M + W3s4. 
The 1H and 13C NMR chemical shifts in the C-42K-45 residue in 4 were nearly identical to those in 
crambescidin SOO (Z), and related crambescidins 3-4, suggesting that the stereochemistry of the secondary 
alcohol in a l l  compounds is the same 5. The presence of the pentacyclic guanidine moiety was derived from 
NMR measurements (Table 1) and as in the other crambescidins a polymethylene chain from C-37 to C-23 was 
also indicated by NMR. In the 13C NMR spectrum of 4 the carbonyl signal observed in the spectra of all 
crambescidins at ca. 175.0 ppm was found upfield shifted to 166.2 ppm. Thus, the structure of fromiamycalin 
was, consequently, concluded to be 4. The HMBC spectrum (Fig. 1) gave further support to this conclusion by 
showing that the H,-42 proton signals exhibited crosq peaks to C-39, to the hydroxymethine carbon-43 at 67.1 
ppm, and to the amidine carbon-38 at 166.2 ppm, which latter is also correlated with H,-37 (6 2.5 and 2.8 
Compound 5, [a], =+3.5", isolated from F. "lis, exhibited in the FABMS a pseudomolecular ion at 
m/z 416 [M+H]+, accompained by a fragment at d z  329 [M + H -87]+, corresponding to the loss of an 
aminohydroxybutyl residue, which is characteristic of the hydroxyspermidine in the previous crambescidins 3,4. 
The structure of the hydroxyspermidine and the long chain o-hydroxyacid units were then confirmed by NMR 
data (Table 2). 1H and 13C chemical shifts in the hydroxyspermidine unit in 5 were nearly identical to those in 
crambescidins 3~4,5 and fromiamycalin 4 suggesting that the stereochemistry of the secondary alcohol is the 
PPd .  
same. 
Table 2.1H and I3C NMR data of Compound 5 (CD30D) 
Position 6, s, 
1 
2 
3- 13 
14 
15 
16 
17 
18 
20 
21 
22 
23 
19 . 
63.0 
33.6 
26.5 
34.1 
177.5 
43.7 
27.0 
38.0 
54.7 
68.6 
32.9 
38.6 
30.5-30.8" 
3.57 t (7) 
1.55 m 
1.32 m 
1.32 m 
2.52 m 
3.70 m 
1.98 m 
2.91 m 
3.50 dd (13,5), 3.41 (10) 
3.98 m 
1.72 m, 1.85 m 
3.14 m 
a : several signals between 30.5 and 30.8. The coupling constants are given in Hz and are enclosed in parentheses 
3680 E. PALAGIANO et al. 
EXPERIMENTAL 
General Methods. 
NMR measurements were performed on a Bruker AMX-500 spectrometer equipped with a Bruker X-32 
computer, using the UXNMR software package. Some of the HNLBC experiments, using field gradients, were 
performed on a Bruker DRX-500 spectrometer at Bruker, Karlsruhe (Germany), and on a Varian Unity 500 at 
Varian, Darmstadt (Germany). 
Two-dimensional homonuclear proton chemical shift correlation (COSY) experiments were measured by 
employing the conventional sequence. The COSY spectra were obtained using a data set (tl x tJ of 1024 X 
512 points for a spectral data width of 2994.05 Hz (relaxation delay 1 sec.). The data matrix was processed 
using an unshifted sine bell window function, following transformation to give a magnitude spectrum with 
symmetrization (digital resolution in both F2 and F1 dimensions was 2.994 Hdpt). 
The 2D-HOHAHA experiment was performed in the phase-sensitive mode (TPPI) using an MLEV-17 
sequence for mixing ll.The spectral width (tJ was 2.994 Hz; 512 experiments of 160 scans each (relaxation 
delay 1.5 sec., mixing time 100 ms) acquired in 1K data points. For processing, an unshifted sine bell window 
function was applied in both dimensions before transformation. Resulting digital resolution in F2 was 2.9 Wpt .  
The {lH, 13C) shift correlation experiments, at 300 K, utilized a 5-mm probe with reverse geometry and the 
sample was not spun. 
'H-detected heteronuclear multiple quantum coherence (HMQC) experiments were performed according 
to Bax and Subramanian 12, using an initial BIRD pulse to suppress 1H resonances not coupled to 13C and a 
GARP sequence for 13C decoupling during data acquisition. The spectral width in 1H dimension was 2994.05 
Hz; 256 experiments of 240 scans each (relaxation delay 1.5 s, delay after BIRD pulse 0.4 s, fixed delay t, 3.3 
ms) were acquired in 1K points. A sine square function was applied in t, dimension and a trapezoidal window 
in t, dimension (TM, 0.03 Hz, TM, 0.6 Hz) before Fourier transformation (digital resolution in F2 dimension 
2.994 Hdpt). 
1H detected heteronwlear multiple bond correlation (HMBC) spectroscopy was performed according to 
Bax and co-workers l 3 8 l 4 .  
Optical rotation were measured on a Perkin-Elmer 141 polarimeter using a sodium lamp operating at 589 
nm. Fast atom bombardment mass spectra (FABMS) were recorded in a glycerol-thioglycerol ma& (with 10 
1-11 of a solution of NaCl O. 1 N) in the positive ion mode on a VG ZAB instrument (Argon atoms of energy of 
2-6 kV). 
Animal Collection and Preliminary Experiments. 
Fromia monilis (family Ophidiasteridae) Penrier and Celerìna heffemani (family Ophidiasteridae) Livingstone 
were collected in the South of New Caledonia at depth of 5-40 m, in January. Taxonomic identifkation was 
performed by Jean Lotis Menou of the ORSTOM Centre de Nouméa where reference specimens are on fila 
(reference EA 13 and EA 17, respectively). Preliminary assays for antiviral activity where done on Herpes 
simplex, grown on Viro cells (monkey kidney epithelium cells) and showed marked actidly ["um viral 
inhibition concentration 3 pg/ml(lOO %)I of both the acetone extracts of both starfishes. 
Ptilomycalin A and crambescidin 800 3681 
Extraction and Isolation. 
The fresh organisms (C. heffernani and F. mnilis), were extracted with acetone to give, after evaporation 
under reduced pressure, 4.57 g and 5.54 g residues, respectively, which were then partioned between n-hexane 
and MeOH. The MeOH layer of C. heffernani (1.8 g) was chromatographed by DCCC using CHCI,-MeOH- 
H,O (7:13:8) in the ascending mode (the lower phase was stationary phase). Fractions were collected and 
combined into three main ones: 8-16 contained a mixture of guanidine alkaloids (160 mg),whereas fractions 17- 
30 (15 mg) and 31-70 (35 mg) were a complex mixture of sulphated steroidal glycosides. Fractionation of 8-16 
(160 mg) was continued by DCCC using BuOH-Me,CO-H,O (3:2:5) in the descending mode (the upper phase 
was used as stationary phase ) to give three main fractions. All fractions were separated by HPLC on a Waters 
C-18 p-Bondapak column (30 cm x 3.9 mm i.d.; flow rate 1.5 ml m-1) with MeOH-H,O 4357 as eluent to give 
ptilomycalin A (1,6.0 mg), crambescidin 800 (2,45 mg) and celeromycalin (3, 13 mg). 
The MeOH layer of F. nwnilis (2.06 g) was chromatographed by DCCC in the same conditions as above 
and each fraction was purified straight by HPLC ((218-p Bondapak column; MeOH:H,O 43:57) to give 
crambescidin 800 (2, 3.9 mg), celeromycalin (3, 3.8 mg), fromiamycalin (4, 3.5 mg) and compound (5, 3.2 
ad. ' ' 
i 
I 
I 
mg>. 
ACKNOWLEDGEMENTS 
This contribution is part of the project SMIB "Substances Marines d Intérêt Biologique" ORSTOM- 
CNRS, Nouméa, New Caledonia. The chemical work was supported by "P. F. Chimica Fina 2" CNR and 
Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST), Rome. 
Mass spectra were provided by Servizio Spettrometria di Massa del C.N.R. e dell' Università di Napoli. The 
assistance of the staff is gratefully acknowledged. The NMR spectra were in part performed at "Centro 
Interdipartimentale di Analisi Strumentale" and in part at Bruker, Karlshruhe and at Varian, at Darmstadt, 
REFERENCES 
1. Kashman, Y.; Hirsh, S., Mc Connell, O. J.; Ohtani, I.; Kusumi, T.; Kakisawa, H. J. Am Chem. Sac. 
1989,111,8925. 
Ohtani, I. ;.Kusumi, T; Kakisawa, H.; Kashman, Y.; Hirst, S., J. A m  Chem Soc. 1992,114,8472. 
Jares-Eriyman, E. A.;. Sakai, R.; Rinehart, K. L.; J. Org. Chenz 1991,56,5712. 
Jares-Eriyman, E. A. ; .Ingkm, A. L.; Carney, J. R Rinehart, K. L.; Sakai; R. J. Org. Chem 1993, 58, 
4805. 
2. 
3. 
4. 
\ 
L ~ 
3682 
5. 
6. 
7. 
8. 
9. 
10. 
11. 
12. 
13. 
14. 
E. PALACIANO et al. 
Berlinck, R. G. S.; Braekman, J. C.; Daloze, D.; Bruno, I.; Riccio, R.; Ferri, S.; Spampinato, S.; Speroni, 
E. J. Nat. Prod. 1993,56, 1007. 
Minale, L. ; Riccio, R.; Zollo, F. in "Progress in the Chemistry of Organic Natural Products". Ed. by 
Herz, W.; Kirby, H.G.W.; Moore, R. E.; Steglich, W.; T a "  Ch., Springer-Verlag, Wien 1993, Vol. 62, 
Casapullo, A.; Finamore, E.; Minale, L.; Zollo, F.; Carré, 5. B.; Dbbitus, C.; Laurent, D.; Folgsre, A.; 
Dale, J. A.; Mosher, J. A. J. Am. Chem Soc. 1973,95,512. 
Sullivan, G. R.;. Dale, J. A, Mosher, H. S. J. Org. Chem. 1973,38,2143. 
Ohtani, I.; Kusumi, T.; Kakisawa, H. J. Am. Chem. Soc. 1991,113,4092. 
Bax, A.; Davis, D. G. J. Magn. Res. 1956,49,67. 
Bax, A.; Subramanian, S. J. Mugn. Res. 1956,67,565. 
Bax, A.; Summers, F. J. Magn. Res. 1986, 108,2093. 
Bax A.; Aszalos A.; Dinya, Z.; Sudo, K. J. Am, Chem. Soc. 1956,108,8056. 
pp. 75-308. 
6 
Galdiero, F. J. Nat. Prod. 1993,56, 105. 4 
(Received in UK 13 December 1994; revised 24 January 1995; accepted 27 January 1995) 


Iorizzi, Maria

Università degli Studi del Molise: IRIS

Ptilomycalin A, Crambescidin 800 and related new highly cytotoxic guanidine alkaloids from the starfishes Fromia monilis and Celerina heffernani.

E. Palagiano

L. Minale

R. Riccio

M. Iorizzi

J. B. Carrè

J. Provost

DE MARINO, SIMONA

ZOLLO, FRANCO

Archivio della ricerca - Università degli studi di Napoli Federico II

Ptilomycalin A, crambescidin 800 and related new highly cytotoxic guanidine alkaloids from starfishes Fromia monilis and Celerina heffernani

Elio Palagiano

Simona De Marino

Luigi Minale

Franco Zollo

Maria Iorizzi

J. B. Carré

Cecile Debitus

L. Lucarain

RICCIO, Raffaele

Archivio della Ricerca - Università di Salerno

Ptilomycalin A, Crambescidin 800 and related new highly cytotoxic guanidine alkaloids from the starfishes Fromia monilis and Celerina heffernani

Ptilomycalin A, crambescidin 800 and related new highly cytotoxic guanidine alkaloids from the starfishes Fromia monilis and Celerina heffernani

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