Fucophlorethols from the brown alga Carpophyllum maschalocarpum

0031 9422/91%3.00-t-0.00 cc; 1991Pergamon Pressplc

Phytochamistry, Vol. 30, No, 10, pp. 3423 -3427, 1991 Printedin GreatBntain.

FUCOPHLORETHOLS

FROM THE BROWN ALGA CARPQPIIYZLU1M

MASCHALOCARPUM KARL-WERNER GLOMBITZA~

Institut fiir Pharmazeutische Biologie, Universitit (Received in revisedfirm

Key Word Index-Carpphyllum structural elucidation.

masehabcarpum;

and SHU-MING LI

Bonn, NuMlee 6, 5300 Bonn 1, Germany 20 March 1991)

Phaeophyceae; Sargassaceae; phlorotannins; fucophlorethols;

Abstract--Six new phloroglucinol derivatives belonging to the class of fucophlorethols with four to 14 rings were isolated together with the known bisfucotriphlorethol pentadecaacetate and its monodeacetylated derivative from an ethanolic extract of the brown alga Carpophy~~ummffsc~alocurpum using HPLC. By means of spectral analysis, the following compounds were identified: hydroxyfucodip~o~thol undecaacetate, hydroxybisfucot~phlo~thol hexadecaacetate, te~u~opentap~orethol docosaacetate, te~ucohexaphlorethol tetracosaacetate, hydroxyterfucohexaphlorethol pentacosaacetate and quaterfucononaphlorethol triacontaacetate. The last one, containing 14 phloroglucinol units, is the most complex phlorotannin isolated in purified form to date.

INTffODUCl’iON

Recently we reported [l] on the isolation and structural elucidation of 20 phlorotannins belonging to phlorethol, hydroxyphlorethol, and fuhalol classes from Carpop~y~~urnm~ch~lo~a~pum (Turn.). Grev. These compounds consist exclusively of p~oroglucinol units linked by aryl ether bonds. We now describe the isolation and identification of an additional eight phlorotannins from this alga which contain both diary1 and aryl ether bonds. RESULTS

AND DISCUSSION

After extraction of the frozen thallus with ethanol, the ethyl acetate fraction containing phenols was immediately acetylated with acetic anhydride-pyridine. The polymeric acetylated phenols were removed by precipitation with ether-petrol (1: I). More than 30 spots in the low M, fraction could be detected by TLC; they quenched W (254 nm)-induced fluorescence. The compounds described herein became red after spraying with vanillin-H,SO,. Using liquid vacuum CC and HPIC, compounds 1-7 could be isolated from the low+J, fraction. These phloroglucinol derivatives belong to the group of fucophlorethols with one or more 3-phenoxyfated 2,4,6,2’,4’,6’-hexaacetoxybiphenyl units (I). Characteristic signals for this type of structural unit in the ‘HNMR spectrum (CDCl,) indicate at least one proton at C-5 at ea 67.11, protons at C-3’, C-5’ at ca 66.98 and the acetyl group at C-2 at ca 81.78. With the exception of 1, the compounds also contain a 1,2,3_triphenoxylated S-acetoxybenzene ring (II) characterized in their IHNMR spectra (CDCl,) by the signal of two protons at C-4, C-6

*Dedicated to Prof. H. Oelschllger on his 70th birthday. j-Author to whom correspondence should be addressed.

Mim Pklo-Q

shifted upfleld at ea 66.40 and the singlet of an acetyl group at C-5 at ca 62.15. The E&mass spectrum of 1 showed a CM]’ at m/z 976 (C46H40024), from which an 1l-fold ketene elimination series was observed down to m/z 514, the ion of the free phenol. Compound 1 contains four phloroglucinol units, two of them making up one element I characterized by signals in the ‘H NMR spectrum (Table 1) at 57.12,7,01 and 1.9, etc. A singlet at 6.7 and the singlet for two acetyl groups at 6 2.04 are caused by a 1,Cdiphenoxylated 3,5diacetoxybenzene ring (C). A singlet at 66.68 and the signals at 52.22 and 2.23 for two and one acetyl groups, respectively, are attributed to a l-phenoxylated 3,4,5triacetoxybenzene ring (D). It is evident that C-l of ring C is connected to C-3 of the biphenyl unit by an ether bond and that C-4 of ring C is linked to C-l of ring D by an ether bond. Compound 1 is named hydroxyfucodiphlorethol undecaacetate. The [Ml+ of 2, 2a and 3 at m/z 1376 (C,,H,,O,,), 1334 (C,,H,,O,,) and 1434 (CBBH58O35) showed a difference between those of 2 and 2a of 42, and between those of 2 and 3 of 58. Their ‘HNMR spectra showed signals for two substituted biphenyl elements (I), one element II and one phloroglucinol unit linked by ether bonds. Compound 2 is identical with bisfucot~phlorethol pentadecaacetate found in the brown algae Cystoseira baccata [2] and Himanthalia tdongata [33. The ’ H NMR spectrum of 2a (Table 1) differed from that of 2, in particular by an unusual AB system with a distinctive AB effect appearing at ca 66.4, probably caused by a small difference between the substituents at C-l and C-3 of structural element II. In comparison with the spectrum of 2, the singlet at 57.14 represented only one proton, but an additional peak appeared at 66.76. Together with the difference between the M,s above, it is conceivable that one acetoxy group at C-2, C-4 or C-6 of one of the two biphenyl units (I) is deacetylated. After acetylation (Ac,O-pyridine) of an artefact produced by

3423

3424

K.-W. GL~MBITZA and S.-M. Lr Table 1. “HNMR spectral data of compounds 1,2a, 3-7 (90 MHz, CDCI,) 1

H

395 Ac-2, Ac-6 AC-~ 5 Ac-2 AC-~ AC-6

2a

3

5

6

7

Ring A

Ring A/A’

Ring A/A’

Ring A/Al/F

Ring A/Al/H

Ring A/AI/H

Ring A/A’ /AZ/A3

7*01 2.06 2.28 Ring B 7.12 1.90 2.02 2.11

7.00/‘6.96 2.04 2.26/2.27

6.98 2.01 2.25 Ring B/B1

6.98 2.01/1.99 2.27

6.98

6.98 2.02 2.26

Ring B/Bl/G 7.11 1.78 1.97/1.99 2.01/2.05 Ring C/E

Ring B/B*/G

Ring B/B’/B2/B3

7,11/7.10 1.77 1.98 t

7.11 X.76 1.92 1.99

Ring C/E

Ring C/C’/E

6.38 6.3 l/6.41 I[ 2.1512.13

6.375 6.25 2.20/2.l5

Ring D 6.63 t

Ring D/D’ 6.62 2.04

46 AC-S

Ring B/B’ 7.14/6.76 1.79/1.73 1.97 2.01/2.03 Ring C

7.12 1.75 1.97 2.02

6.99 */1.90 2.27 Ring B/Bl/E 7.12/6.65 1.78,‘1.90 * *

Ring C

Ring C

6.41/6&t

6.40

6.38

2.19

2.15

2.16

6.38 6.37/6.30$ 2,13/2.16

Ring D 6.63 *

Ring D 6.64 2.05

Ring c

6.70 Ac-3, AC-~ 2.04 Ring D 26 6.68 AC-~,AC-S 2.22 A*4 2.23 Z6

296 4 AC-~,AC-~

4

2.26

Ring D

Ring F

6.64 2.18 2.18

6.68 2.20 2.20

Ring D 6.523 6.64$ 2.24

Ring G 6.59$ 6.7f $ 2.21

Ring F 6.56$ 6.70% 2.21

Ring F 6.61X 6.68 $ 2.20

*Signals at 6203, 2.02, 2.01, 2.00 and 1.99 (36H). f&pals at 62.04, 2.026 and 2.015 (27H). $ AB, system J = 2.0 Hz. $ AB system J = 2.6 Hz. f AB system J = 2.4 Hz.

deacetylation 2a, a product with a R, value identical to that of 2 with an identical ‘HNMR spectrum was achieved. From the chemical shifts, 2a is a derivative of 2 monodeacetylated at C-4 or C-6. In the ‘H NMR spectrum of 3 (Table I) signals for two structural I units were found at 67.12 (2H), 6.98 (4H), 2.25 (6H), 1.75 (6H), etc.; two singlets of II appeared at 66.4 and 2.15, respectively. Compared to that of 2 the ‘H NMR spectrum of 3 has a singlet at 66.64 instead of an AB, system in the region 66.X-6.70. Taking into account the difference between the M,s of2 and 3 above, ring D of 3 must be acetoxylated at C-4. The ’ 3C NMR data of 3 (Table 2) differed from those of 2 [3], especially in the signals for ring D. Compound 3 is named hydroxybisfucotriphlorethol hexadecaacetate. The FAB mass spectra of 4-6 showed [E/f)+ at rrtfi 2042 (C,sHs20,,), 2250 (C,,,H,,O,,) and 2308 (Cli0H92056), respectively. The difference between the first two amounts to 208 for one diacetoxyhydroxybenzene unit, and between the last two 58 for CH2C02. The 13C NMR spectrum of 5 (Table 2) showed signals for the following structural elements: three of I, two of II, one 1,4-diphenoxylated 3,Sdiacetoxybenzene and one lphenoxylated 3,Sdiacetoxybenzene. Use of the term ‘phenoxylated’ in this paper always includes an appropriate substitution of the aromatic ring by acetoxy groups.

In their ‘H NMR spectra 5 and 6 produced signals for three structural elements I, 4 only for two. C-3 and C-6 of the third biphenyl unit of 4 are diphenoxylated. If an acetoxy group of a phlorotannin is substituted by a phenoxy group, the signals of the proton at the orthopositions are shifted upfield 6 0.13-0.43 (Glombitza, K.-W., unpublished results). The singlet at 66.65 in the spectrum of 4 is, thus, caused by the proton at C-5 of ring E. In addition to the existence of a symmetrically substituted element II in 4-6, determined by the singlets at 66.38 of two protons, 5 and 6 contain another asymmetrically substituted II, featuring an AB system at era 66.4 to 6.3. The singlet of two protons at ca 66.64 and a signal for two acetyl groups in the region 52.05 to 2.0 in the ‘H NMR spectra of 4-6 are caused by a 1,4diphenoxylated 3,5-* diacetoxybenzene ring (D). Ring D may be arranged between the two structural elements II in 5 and 6, or both are connected directly with each other. In the latter case the space around the two highly substituted elements 11 would be narrow, resulting in a great change of chemical shifts of the protons at IX and at other rings around it. However, it was found that the signals of I and II were practically unchanged in comparison to the spectra of 2 and 3. Thus, ring D is arranged between the two II elements. Both 5 and 6 contain 10 phloroglucinol units;

Fucophlorethols from Carpq&4lum maschakocurpum

R

they differ from each other only at C-4 of ring F, d~monst~ted by a singlet at 66.68 of twu protuns (6) instead ofan AB2 system(5) and a singlet at 62.2 for three wetyt groups (6) instead of one at 52.21 for two ace&l groups only (5). Compound 5 is terfucohexaphlorethol tetraeosaa.c&ate and 6 hydrox~~u~h~ap~or~ol pentacosaaeetate. Ring 2) of 4 could be situated either between IIf and the third biphenyl unit or between the third biphenyl unit and the 1-phenoxyfated 3,Sdiaeetoxybenzene ring (ring G), Considering the space factor mentioned above, ring I? is suspected tu be between Ii and the third biphenyl unrt_ compound 4 is te~~u~tap~o~tho~ docosaacetate. Compound 7 is the largest phtorotannin isokted up to now, consisting of 14 ph~oroglu~nol units. The M, of 7 could be deduced Tom an ion at m/z 3524 (C1J0H124075) The compound is a highly s~rnet~&~ mole&e with a relatively simple ‘HNMR spectrum: four identical eiements I praduce signals at 67.11, 6.98, 1.76, etc. Three symmetrkahy substituted II elements, from which two identical ones could be identifkd by signals of four protons at S6.375, two Jdentieaf ~,~~phenoxyl~~ 3,5-

=

OAc

diaetoxybenznes (ring D and IY) with singlets found at 86.62 (4H) and 2.04 (ZZH) and one ~-phenoxy~~t~ 3,5~a~toxy~~ene (ring F) with an AB, system fkom 56-61 to 6.68. It is evident that the four Xelements are cormected with the C-l or C-3 of the two identical II elements respectively, Both II elements are joined, furthermore, either dire&y with C-l and G-3 of the third II ur at first with ring D and II”, and the fast two with C-l and C-3 of the third IL Based on the above space requirements, the latter case is more probable. Ring F could be connected through C-l with C-2 of the third III. The chemical shift of tire protons at C-4,C-6 of ring E appeared shifted upfIeld at 66.25; this may be explained by the narrow space around ring E. Compound 7 is quate~u~no~p~orethol triacontaacetate.

3426

K.-W. Gzmimm

and S.-M. h

7

tim

series: m/i: f473 [M+ K]‘l

1434-4930,

~js~~cut~~~hlorerho~~~t~e~ua~etate (2). Yield 50 mg, ‘NNMR data identical with ref. [2], 4- or 6-Reacetytbis~conipkluretkol tetrudecuacetate (ti). Yield 11.5 mg. FAB-MS ketene elimination series: m/z 1373 1357 EM + Naj * -+1315, 13344830, 1168-+874. tM+KI+, jH NMR sw Table 1. HydToxybisJ”ucotriphtoretkoXhexadecaacetate, 2,tLbis[2,4,6triacetoxy-3-(2,4,6-triacetoxyp~~y~)pkencrxy~-4,3’,4’,5’-tetra~etoxyd~~~~y~~t~~ f3&YieM 85 mg. FAB-MS ketene elimina-

1374+1206,

22684974.

1457 CM-i-N@* -1373% ‘WNMR see Table 1,

S3CNMR in Table 2, T~~o~ta~~~~et~~ docosmetate, 4’-(4_acetoxy-2,&bis[2,4,6-ttiaestoxy-3-~2~4*~-trj~c~toxy~~e~y~rpAenoxygahenoxy~-4-(3,5-d~taxyphenoxyf-3,5,~,6~-tetr~cetoxy-2~2,4,~ triacetoxy~ttanyl)~~~~y~et~ (4). Yield 7 mg, FAB-MS ketene eliminatiuti seties: m& 2081 [M G K] ‘, 2065 [M +NaJ + -+ i981, 2042-*f79U,f94@+1772~ X834-+f666, ‘HNMR see Table 1. T~~~~ohexa~h~oretho~tetracosaucetate, 4’“f4-amtoxy-2,6bis[~~act3~~4,~~toxy~nyi)~xy~~wxy~-2~3,~~et#xyphe~oxy) - 5,2’@- triuizetoxy- 3 I [2S4,6-triaGetoxy-3fz;l,atr~#t~xy~h~y~~~~~y~di~he~y~eth~ (3 Yield 92 mg. FAB-MS ketene elimination series: m/z 2289 [M + RI ‘, 2273 [M+N&J+-+2189, 2250+1934, 2042-+1916. ‘HNMR see Table 1, i “C NMR data in Tabie Z

Fucophlorethob

from Carpophyiium rnaschal~w~m

Table 2. ’ 3CNMR spectral data of compounds 3 and 5 (50 MHz, CDCl,) c

3 Ring A/A”

Ring A/Al/I-I

:6

149.2 114.9

149.1/149.1 114.9/l 14.86

335 4

113.9 150.7

113.9/113.9 150.6,‘150.4

Ring B/B’

Ring B/Bl/G

1 2 3 4 5 6

118.1 143.0 136.5 142.5 116.0 145.1

118.1/118.0 143.1/143.0 136.7/136.5 142.5/142.4 116.0,‘115.9 145.2/145.0

5

Ring C

Ring C/E

193 2 496 5

150.6 130.2 105.4 147.9

151.0/150.67/150.7 130.0/130.1 105.1/105.3/105.7 147.91’147.8

1 2,6 3,5 4

Ring D 1554 108.8 143.8 130.1

Ring F 159.3 106.7 151.4 110.0

1 2,6 3,5 4

C=o:

Ring D 154.9 108.8 144.0 133.1 163.5-169.5 Me: 19.7-20.0

Ran

3427

-‘~R

I

II

Yield 8 mg. FARMS ketene elimination series: m/z 2347 CM+Kl+, 2331 [M + Na] + -2205, 230842224. ‘H NMR see Table I, ~~te~c~no~phl~ret~~ ~~~nt~cet~te, 2,dbis(2&-&wetoxy4[4-4cetoxy-2,bbis(2,4,6-triacetox phenyl)) phenoxy) phenoxy] phenoxy] 4,3’,5’-triacetoxydiphenylether (7). Yield 7.5 mg, FAB-MS ketene elimination series: m/z 3163 [M +K-J+ -*3121, 3147 fM +Na]+-*2937, 3124-2872, 2916. ‘HNMR see Table 1. Acknowledgements-This research was supported by the Deutsche Forschungsgeme ins&aft. We thank Dr Dromgoole, University of Auckland for the introduction to New Zealand algae and the identification of the alga, the Central Analytic Laboratory of the Chemical Institutes, University of Bonn for mass and NMR spectra and Mrs Rebecca Grassau for revising the manuscript. RJJCFERENCES

1. GIombitza, K.-W. and Ii, S.-M. (1991) Phytochenristry (in Press) 2 Glombiw K.-W., Schnabel, C, and Koch, M. (1981) Arch. Phum. (Weinheim) 314,602. 3. Glombitza, K.-W. and Grosse-Dambues, J. (1985) Plantu Ned. 51,42.