Linear diterpene with antimitotic activity from the brown alga Bifurcaria bifurcata

Phytochemrstry, Vol. 34, No. 6 pp. 1585.-1588, 1993 Printedin Great Britain.

LINEAR DITERPENE

0031-9422:93 %6.00+0.00 Q 1993 PergamonPress Ltd

WITH ANTIMITOTIC ACTIVITY ALGA BIFURCARIA BIFURCATA

ROBERT VALLS, BERNARD BANAIGS,*

LOUIS PIOVEIX,?

FROM THE BROWN

ALBERT ARCHAVLIS~

and

JACQUES ARTAUD

Laboratoire d’Analyse et

de Valorisation des Biomolbcules, I.U.T. Saint J&&me, Universitt d’Aix-Marseille III, BP 157, F-13388 Marseille Cedex 13, France; *Groupe d’Etude des MCtabolites Marins d’Int6rit Biologique, UniversitC de Perpignan, F-66860 Perpignan Cedex, France; TLaboratoire de Recherches de Chimie Marine des Organomktalliques (RCMO), Universitk de Toulon et du Var, BP 132, F-83957 La Garde Cedex, France; JENSSPICAM, Universiti d’Aix-Marseille III, Avenue Escadrille Normandie Niemen, F-13397 Marseille Cedex, France (Received in revised form 14 May 1993)

Key Word Index-Bifurcaria bifurcata; Cystoseiraceae; antimitotic; seasonal and geographical variations.

brown algae; diterpene;

geranylgeraniol;

Abstract-A new linear diterpene, isolated from the brown alga Bijiicaria bijiucata collected on the Atlantic coasts from Morocco, has been characterized by spectral methods and chemical reactions. This compound revealed a potent cytotoxicity to fertilized sea urchin eggs which was compared to that of previously described acyclic diterpenes. The seasonal and geographical variations in the diterpenoid composition of this species are also studied and the results discussed.

INTRODUCTION

Phaeophycean, family Cystoseiraceae, contains a major genus, Cystoseira, which abounds in species (more than SO) in Mediterranean and contiguous Atlantic marine environments and has had many chemical studies [e.g. l-33. In contrast Bijiircaria is a minor genus with two species. B. galapagensis occurring in equatorial environments and B. b@rcata found on the Atlantic coasts from Morocco (southern limit) to Great Britain (northern limit). Bfircaria bifitrcata, collected initially from French coasts (Brittany) [4, 51 and later from Moroccan coasts [6, 71, contains a rich array of linear diterpenes. A new one is described in this paper. It showed potent cytotoxicity to fertilized sea urchin eggs. This activity is compared to that of acyclic diterpenes previously described [6, 71. The various collections of B. bifurcata revealed qualitative and quantitative differences in their secondary metabolite composition, although the same types of compounds were present in all cases. The seasonal and geographical variations in these compounds was examined.

RESULTS AND DISCUSSION

The ether extracts of the freeze-dried B. bificata, collected near Rabat, Morocco, were fractionated by liquid chromatography using silica gel. The fraction that eluted with hexane-ether (2:3) was further purified by HPLC on rormal phase silica. From this separation, we

obtained the previously reported diterpenes 1 and 5 as well as a small amount of a new compound (6). Diterpene 6, &H,,O, (high resolution mass spectrometry of the TMSi ether and EIMS, m/z 288 [M -HzO]+), was optically inactive and showed hydroxyl absorption (3400 cm- ‘) in its IR spectrum. The metabolite exhibited similar spectral data (‘H, 13C) to S except for the signals corresponding to the last isoprenoid unit. The 13C NMR spectrum revealed five methyl groups, six methylenes (among them one oxygenated), five olefinic methine carbons, one oxygenated quaternary carbon and three olefmic quaternary carbons. Acetylation of 6 gave a monoacetate derivative which showed a weak hydroxyl absorption in its IR (vOH= 3400 cm- ‘). Thus 6 is a linear, tetraunsaturated diterpene with two hydroxyl substituents. Comparison of the ‘H and 13CNMR data of 6 with those of l-5 suggested that the same first three isoprenoid units were present in 6. Examination of the ‘HNMR spectrum and single-frequency-decoupled spectra revealed the complete structure to be that of 6. The two olefinic protons (C-13, C-14) appeared as an AB system at 65.57 and 5.63, with a trans coupling (J = 15.6 Hz), one of which was coupled (5.6 Hz) to methylene protons (62.67) which were in turn long range coupled to the methyl protons positioned at C-18 (61.57). The tertiary alcohol was located at C-15 as evidenced by two methyl groups, C-16 and C-17, having the same chemical shifts (61.32) in the ‘HNMR spectrum. The assignments of ‘H and 13CNMR spectra are summarized in Table 1. To further confirm the C-15 location of the tertiary alcohol function, the monoacetate of 6 was dehydrated

1585

1586

R.

VALLS

OH

et al.

with POCl, in pyridine to yield the monoacetate compound 5 (NMR data given in Table 1).

of

1 Eleganediol OH

2 Eleganolone

: 3 12-(S)-hydroxygeranylgeraniol 9H OH

4 12-(S)-hydroxygeranylgeranic

acid

Cytotoxic

activity

Sea urchin egg development is frequently used as a pharmacological screen for compounds that inhibit cell division. The details of the experiment procedure which is used primarily as a general cytotoxicity screen to assess overall biological activity of the metabolites has been reported elsewhere [a]. The activity of compound 6 was compared to that of compounds 1, 3 and 4 and ether extracts of the alga from the three locations. Table 2 shows the ED,, for inhibition of development of fertilized eggs of the common sea urchin Paracentrotus lividus. Compound 6 was active at the lowest concentration (4~gml-1=10-6M).

OH

Seasonal and geographical

17

18

19

variations in B. bifurcata

Terpenoid metabolites have been useful as chemotaxonomic markers in terrestrial environments because of their structural complexity and resulting uniqueness. As a consequence of the availability of numerous collections (more than 150) of B. bifurcata from different years (1984-1986), seasons and areas (Moroccan Atlantic

20

6

Table 1. NMR spectral data of compounds 5 and 6 (400 MHz ‘H NMR, CDCI,; 13C NMR, CDCI,; TMS as int. standard)* 6 C AC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

6 acetate

‘H

l’C

4.15 d (6.8) 5.42 t (6.8)

59.4 123.4 139.7 39.6 26.3 123.9 135.3 39.6 26.7 125.2 133.7 42.4 125.4

2.00 m 2.07 m 5.11 t (6.8) 2.00 m 2.07 m 5.13 t (6.8) 2.67 d (5.6) 5.57 dt (15.6, 5.6) 5.63 d (15.6) 1.32 1.32 1.57 1.60 1.68

s s s s s

139.2 70.7 29.8 29.8 16.0 16.0 16.3

gt

100.5 MHz

5 acetate.?

‘H

‘H

‘“C

2.04 s 4.55 d (6.8) 5.33 t (6.8)

4.10 d (6.8) 5.37 t (6.8)

2.00 m 2.07 m 5.03 t (6.8)

2.0 m 207 m 5.09 t (6.8)

2.00 m 2.07 m 5.12 t (6.8)

2.0 m 2.07 m 5.13 t (6.8)

2.65 d (5.6) 5.57 dt (15.6, 5.6) 5.63 d (15.6)

2.72 d (7.2) 5.58 dt (15.6, 7.2) 6.10 d (15.6)

59.0 123.3 139.1 39.4 26.5 123.8 135.0 39.4 26.2 125.1 133.5 42.7 128.7

1.30 s 1.30 s 1.56 s 1.58 s 1.69 s

1.81 s 4.84 s 1.57 s 1.58 s 1.65 s

133.7 141.9 18.5 114.3 15.9 15.8 16.1

‘HNMR 2.05 s 4.57 d (6.8) 5.33 t (6.8) 2.0 m 2.07 m 5.06 t (6.8) 2.0 m 2.07 m 5.12 t (6.8) 2.74 d (7.2) 5.58 dt (15.6, 7.2) 6.12 d (15.6) 1.84 s 4.86 s 1.58 s 1.59 s 1.69 s

*Coupling constants (J in parentheses) are given in Hz; assignments were confirmed decoupling, 2D NMR (COSY ‘H-IH, ‘H-r3C correlation) and DEPT experiments. tNMR data of ref. [7] added for comparison.

by

Diterpenes from brown alga

1587

Table 2. Effects on cell division in fertilized sea urchin eggs

Et,0 extracts

ED,, 019 ml-‘)

Compounds

Rabat

El Jadida

Oualidia

35

50

38

1

3

4

6

36

18

60

4

Table 3. Seasonal and geographical variations in the diterpenoid composition of B. bijiircata

Compounds (mg g-l algal dry wt) Rabat* Month@

1

J F M A M J J A S 0 N D

El Jadidat

2

OualidiaS

5

6

1

6

3

4

3.63

0.28

0.45

1.06

3.15

0.11

3.27

0.70

3.70 3.82 3.70 3.71 3.62 3.73 3.61 3.71 3.59 3.68 3.68

0.30 0.25 0.31 0.33 0.32 0.26 0.28 0.29 0.26 0.29 0.25

0.48 0.44 0.38 0.42 0.52 0.50 0.47 0.52 0.52 0.52 0.50

1.12 1.18 1.24 1.09 1.01 0.95 1.02 1.03

3.08 3.06 3.01 3.11 3.06 292 2.91 3.05

0.14 0.12 0.12 0.13 0.14 0.13 0.12 0.14

3.34 3.41 3.35 3.33 3.30 3.26 3.30 3.36

0.79 0.82 0.79 0.88 0.96 0.85 0.82 0.80

*Compounds 2,3 and 4 not detected. tCompounds 3,4 and 5 not detected. $Compounds 1, 2,s and 6 not detected. $Month of collection. -Not collected.

coasts from Rabat in the north to Essaouira in the south), we observed a number of morphological and chemical variations (terpenoid composition). Btjimaria bifircata was collected monthly between January and December 1985, at three separate locations: Rabat, El Jadida and Oualidia. Certain collections of this species were also carried out during 1984 and 1986. The different collections were treated and extracted in an identical fashion. Preliminary examination of the ether extract from each collection by TLC revealed that all samples possessed the usual mixtures of lipids, pigments, fatty acids and sterols. In addition to these compounds, extracts of B. bijkrcata contained several geranylgeraniol homologues (Rf 0.39-0.65 in ethylacetate-isooctane, 2:3). The ether extracts were linally analysed by HPLC (ethylacetate-isooctane, 3: 2) for the presence of compounds 1 to 6. Concentrations of metabolites were calculated as mg per g of algal dry weight. Table 3 lists the values obtained. The results (Table 3) show: secondary metabolite production, in terms of total diterpene concentration, was relatively constant from month to month and place to place, no, or very minor, seasonal fluctuation occurred in

the types of diterpenoid metabolites within each location, the expected population variability was observed in populations of B. bifurcata collected from various habitats. Thus the Rabat collection contained a high proportion of 1 (eleganediol = 13S-hydroxygeranylgeraniol) with compounds 5 and 6 as minor constituents. The El Jadida collection possessed 2 (eleganolone = 13-ketohydroxygeranylgeraniol) and 1 as the major metabolites with 6 as a minor one. In the Oualidia collection 3 and 4 were observed. The Rabat and El Jadida collections contained compounds directly or indirectly derived from hydroxylation of geranylgeraniol at C-13 (1 could give rise to 6 by a homoallylic transposition and 5 is the dehydrated derivative of 6). By contrast, the Oualidia collection contained only compounds biosynthetically derived from geranylgeraniol by C-12 hydroxylation. Factors influencing the variation in secondary metabolite production are reasonably well understood in terrestrial plants but little is known about such variation in marine algae. The observed geographical variations could be due to chemical adaptations of B. bifurcata to biological or physical environmental conditions.

R.

1588

VALLS

EXPEffIMENTAL

General MS: direct inlet, 70 eV, ‘H NMR: 400 MHz; 13CNMR: 100.5 MHz. Chemical shifts are quoted in ppm (6) relative to TMS and coupling constants are in Hz. Final purification of all metabolites was achieved by HPLC on silica gel (Intersphere a-60, 5 pm), with RI monitoring. Plant material. Bifurcaria bifurcata Ross was collected between January and December in 1984-1986 (Atlantic coast of Morocco). A voucher specimen of this species is deposited in the Herbarium of Dr Pdlegrini, Laboratoire de Biologie Fondamentale et Appliquee, University of Marseille II, France. Extraction and purification. The freeze-dried material (100 g) was ground and extracted with Et,0 at room temp. After filtration and evaporation, the extract was partitioned between H,O and Et,O. The Et,O-soluble material was dried over MgSO, and the filtrate was evaporated to yield 1.65 g of a crude extract which was subjected to CC on silica gel eluted with a solvent gradient (hexane to EtOAc). The new compound 6 was eluted with hexane-EtOAc (1: 1) and subsequently purified by HPLC (EtOAc-isooctane, 3: 2) to give 45 mg of pure product. B~rcanol(6). Oil; [E-J;’ 0”; IR vzi: cm-‘: 34OQ 1685, 1625, 1015; UV Jb:::‘3 nm (E): 285 (8500), 245 (12200); HRMS of the TMSi ether: [M-TMSiOH-j+ 360.2855 (talc. for C,,H,,SiO, 360.2848): EIMS (70 eV) m/z (rel. int.): 288 (1) [M-H,O]‘, 257 (l), 147 (ll), 135 (31), 109 (36), 93 (57), 81 (lOtI), 43 (77); ‘H and 13C NMR: Table 1. ‘H-13C shift correlution. The applied pulse sequence ‘T)-(t,,,)-rt-(n/2, ‘H; $2, was ($2, ‘Hf-(t&-(x, r3C)-r2-(BB, ‘H; FID, tz) with r, =3.3 msec and zZ = 1.67 msec. Spectra width in F, was B’, = + 500 Hz and in F2, W, = 6024 Hz. Acetytation of compounds 5 and 6. Excess Ac,O and dry pyridine were added to 25 mg of compounds 5 and 6 and the resultant soln was stirred at room temp. overnight. The soln was then diluted with H,O and extracted with

et d.

EtzO. Evaporation of the solvent left a residue which was subjected to semi-prep. HPLC (EtOAc-isooctane, 1:4). Dehydration of compound 6 acetate to give 5 acetate.

POCl, (1 ml) was added to a soln of 6 acetate (10 mg) in pyridine (2.5 ml) and the mixture was stirred at 0” for 30 min. The soln was then diluted with H,O (5 ml), extracted with Et,0 and evapd. The residue was purified by semi-prep. HPLC (EtOAc-isooctane, 1:4). HPLC analysis of compounds l-6. The method previously described for the determination of sterols and diterpenoids from Cystoseiraceae [9] was used. Ac~nowZedgements-.~e

Pellegrini (University classification of plant S.A.M.M. (Chatenay spectra of compounds

authors wish to thank Dr M. of Marseille II, France) for the material and Dr Becue of the Malabry, France) for the mass l-6.

REFERENCES

1. Amico, V., Piattelli, M., Cunsolo, F,, Neri, P. and Ruberto, G. (1989) J. Nat. Prod. 52, 962. 2. Amico, V., Piattelli, M., Cunsolo, F., Recupero, M. and Ruberto, G. (1990) Guzz. Chim. ital. 120, 9. 3. Valls, R., Piovetti, L., Banaigs, B. and Praud, A. (1993) Phytochemistry 32, 961.

4. Biard, J. F., Verbist, J. F., Floch, R. and Letourneux, Y. (1980) Tetrahedron Letters 21, 1849. 5. Combaut, G. and Piovetti, L. (1983) Phytochemistry 22, 1787.

6. Valls, R., Banaigs, B., Francisco, C., Codomier, L. and Cave, A. (1986) Phytochemistry 25, 751. 7. Valls, R., Banaigs, B., Francisco, C., Codomier, L. and Cave, A. (1988) Phytochemistry 27, 2347. 8. Jacobs, R. S., White, S. and Wilson, L. (1981) Fred. Proc. Am. Sot. Exp. Biol. 40, 26. 9. Piovetti, L., Deffo, P., Vails, R. and Peiffer, G. (1991) J. Chromatogr. S&99.