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WO1996032388A1 - Antheliatin, zahavin a and zahavin b: new cytotoxic xenicane diterpenes - Google Patents

Antheliatin, zahavin a and zahavin b: new cytotoxic xenicane diterpenes Download PDF

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Publication number
WO1996032388A1
WO1996032388A1 PCT/GB1996/000903 GB9600903W WO9632388A1 WO 1996032388 A1 WO1996032388 A1 WO 1996032388A1 GB 9600903 W GB9600903 W GB 9600903W WO 9632388 A1 WO9632388 A1 WO 9632388A1
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Prior art keywords
zahavin
antheliatin
ppm
xenicane
glauca
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PCT/GB1996/000903
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French (fr)
Inventor
Amira Rudi
Yoel Kashman
Dolores Garcia Gravalos
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Pharma Mar, S.A.
Ruffles, Graham, Keith
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Priority to AU53389/96A priority Critical patent/AU5338996A/en
Publication of WO1996032388A1 publication Critical patent/WO1996032388A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/94—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems condensed with rings other than six-membered or with ring systems containing such rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

Definitions

  • the present invention relates to new cytotoxic xenicane diterpenes.
  • Anthelia belongs to the Xeniidae along with other Indo- Pacific genera of this family such as Cespituluria, Effatounaria, Heteroxemia and Xenia, all of which associate with symbiotic algae (zooxanthellae).
  • Anthelia is characterized by having individual, non-retractile polyps united by a thin membrane and in this respect it differs from the related genera. Anthelia colonies tend to grow among other soft corals mainly of the Xeniidae family (Reference 14: Y. Benayahu, Proc. 5th Int.
  • A. glauca (Lammarck, 1816) is the most common species of the genus and has a wide zoogeographical distribution throughout the Indo- Pacific region. On the coral reefs of Sodwana Bay, South Africa, A. glauca is sporadic in its abundance and appears on the reefs in small clusters of colonies at a depth range of 14-22 meters.
  • a recent survey of the soft coral fauna of Sodwana Bay (Reference 12: Y. Benayahu, Invest. Rep. Oceanogr. Res. Inst. (Durban), No. 67, 1 (1993)) lists eight xeniid species. Unlike other reef areas such as the Red Sea (Reference 14), at Sodwana Bay, A. glauca is the most abundant xeniid.
  • Alcyonium belongs to the family Alcyoniidae (Reference 15: J. Verseveldt and F.M. Bayer, Zool. Verhand Leiden, 245, 1 (1988)), and Alcyonium comprises species recorded within a large latitudinal range and found in various habitats along a wide depth gradient (Reference 16: J. Verseveldt, Zool. Med Leiden, 39, 153 (1964); Reference 17: J. Verseveldt, Amer. Mus. Nov., 2282, 1 (1967); Reference 18: J. Verseveldt, Zool. Verhand Leiden, 117, 1 (1971); Reference 19: J. Verseveldt, Zool.
  • aurea is an azooxanthellated species collected at Sodwana Bay (Reference 13: Y Benayahu and M. Schleyer, in press). A. aurea was found at a depth of 28-36 meters, where almost no soft corals associated with endosymbiotic algae appear. There is no doubt that at Sodwana Bay A. aurea belongs to communities living below the euphotic zone.
  • the present invention provides three new compounds extracted and isolated from soft corals.
  • the compounds are antheliatin, zahavin A and zahavin B, the following formulae (I) and (II):
  • the compounds of the present invention exhibit antitumor activity.
  • the present compounds exhibit antitumour activity against cell lines derived from human tumors, such as P-388 mouse lymphoma, A-549 human lung carcinoma, HT-29 human colon carcinoma and MEL-28 human melanoma.
  • the present invention also provides a method of treating a mammal affected by a malignant tumor sensitive to antheliatin, zahavin A, or zahavin B , which comprises administering a therapeutically effective amount of antheliatin, zahavin A, and zahavin B, or a pharmaceutical composition thereof.
  • the present invention further provides pharmaceutical compositions which contain as active ingredient the compound antheliatin, zahavin A or zahavin B, as well as a process for their preparation.
  • a further aspect of the invention is a method for preparing the compounds, which comprises extraction and isolation from the respective soft coral, Anthelia glauca for antheliatin or Alcyonium aurea for zahavin a and zahavin B.
  • pharmaceutical compositions include any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) with suitable formulation for oral, topical or parenteral administration, and they may contain the pure compound or in combination with any carrier or other pharmacologically active compounds. These compositions may need to be sterile when administered parenterally.
  • a pharmaceutical composition comprising antheliatin, zahavin A or zahavin B, will vary according to the pharmaceutical formulation, the mode of application, and the particular situs, host and tumor being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose.
  • the antitumor cells employed were P-388 (suspension culture of a lymphoid neoplasm from DBA/2 mouse), A-549 (monolayer culture of a human lung carcinoma), HT-29 (monolayer culture of a human colon carcinoma) and MEL-28 (monolayer culture of a human melanoma).
  • P-388 cells were seeded into 16 mm wells at 1 x 10 cells per well in 1 ml aliquots of MEM 5FCS containing the indicated concentration of drug. A separate set of cultures without drug was seeded as control growth to ensure that cells remained in exponential phase of growth. All determinations were carried out in duplicate. After three days of incubation at 37°C, 10% CO in a 98% humid atmosphere, an approximate IC50 was determined by comparing the growth in wells with drug to the growth in wells control.
  • HT-29 and MEL-28 cells were seeded into 16 mm wells at 2 x 10 4 cells per well in 1 ml aliquots of MEM 10FCS containing the indicated concentration of drug.
  • a separate set of cultures without drug was seeded as control growth to ensure that cells remained in exponential phase of growth. All determinations were carried out in duplicate. After three days of incubation at 37°C, 10% CO2 in a 98% humid atmosphere, the wells were stained with 0.1% Crystal Violet. An approximate IC50 was determined by comparing the growth in wells with drug to the growth in wells in control.
  • Antheliatin (1) The soft coral Anthelia glauca was collected in Sodwana Bay South Africa, in May 1994 by divers using scuba A voucher (TASA 293) is deposited in the Zoological Department at Tel Aviv University
  • TASA 217 A voucher (TASA 217) is deposited in the Zoological Department at Tel Aviv University.
  • the gum was chromatographed first over a Sephadex LH-20 column eluted with MeOH- CHCl 3 -hexane, 1; 1;2) and then several times over Si gel columns eluted with hexane- EtOAc (8:2) to afford 2 (30 mg); Rf 0.7 (EtOAc-hexane, 1.1), and 3 (5 mg) Rf 0.65.
  • the Sodwana Bay A. glauca in contrast to the Red Sea species, was found to contain a single diterpenoid designated antheliatin (1), a crystalline material, m.p. 165°C (MeOH), [ ⁇ ] D + 3.5° (c, 1.2, CHC1 3 ), 0.2 % dry wt.
  • the four additional carbon atoms were assigned to (a) a polarized double bond ( ⁇ 13c 138.2 ppm(d) and 113.4 p ⁇ m( ⁇ )), (b) a lactol-methine ( ⁇ 13c 91.9 ppm (d), ⁇ , 5.77 ppm(d)) and (c) an additional aliphatic methine ( ⁇ J3c 38.4 ppm (d)).
  • the low-field nmr signal of the lactol-proton ( ⁇ 1H , 5.77 ppm) suggested that the third acetate was attached to this position (7).
  • HMBC CH-Hetero correlations in the nmr spectrum of 1, between (a) and C-atom of the methine C-4a, ( ⁇ 13C 38.9 ppm), the allylic pair H-6 and 6' and protons H-5 and 5' of B, the other end-methine of moiety B (H-l la), and H-3, and (b) between C-4 ( ⁇ 13C 1 13.4 ppm), one of the polarized double bond carbon atoms, and protons H-5 and 5' of site B and H-12 of A ( ⁇ 1H 5.69 ppm), suggested C-4 and C-4a to be the link between moieties A and B, a connection which was further extended by correlations between C-12 and H-3.
  • the location of this OH-group was suggested mainly on the basis of the following CH-correlations, deduced from an HMBC experiment: C-8/H-10; C- lO/H-19; C-1 la/H-19 as well as from homo-COSY correlations i.e. H-l 0/9,9' and H- 88/9,9'.
  • the chemical shifts of C-9 and C-1 la also support the C'lO location of the OH-group; (If the OH group was on C-9 the line of C-10 should have been at ca.
  • Zahavin B (3) is very unstable both under mild basic and mild acidic conditions; the compound decomposes readily under acetylation conditions (Ac 2 O, pyridine, CH 2 C1 2 ) or in a CDC1 3 solution.

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Abstract

Antheliatin, zahavin A and zahavin B, three novel xenicane diterpenoids, have been isolated from two Indo-Pacific reef-inhabiting soft corals Anthelia glauca and Alcyonium aurea. The structures of the three compounds have been established on the basis of 1-D and 2-D nmr data.

Description

Antheliatin, Zahavin A and Zahavin B: New Cytotoxic Xenicane Diterpenes
The present invention relates to new cytotoxic xenicane diterpenes.
Background of the Invention
Marine organisms, especially soft corals, sponges and tunicates, provide many secondary metabolites and exhibit a varying degree of biological activity (Reference 1 : D.J. Faulkner, Nat. Prod Reports., 11, 355 (1994) and references cited therein). In the framework of our (where "our" indicates at least one of the present inventors, possibly with co-workers) continuing studies of the chemistry of compounds found in soft corals, we investigated the Indo-Pacific soft corals Anthelia glauca (Reference 1, Reference 2: D.Green, S. Carmely, Y. Benayahu and Y. Kashman, Tetrahedron Lett., 29, 1605 (1988); and Reference 3; A.B. Smith III, P.J. Carroll, Y. Kashman and D. Green, Tetrahedron Lett. , 30, 3363 (1989)) and Alcyonium aurea.
Soft corals of the families Alcyoniidae and Xeniidae are widespread in many coral reefs. The genus Anthelia belongs to the Xeniidae along with other Indo-Pacific genera of this family such as Cespituluria, Effatounaria, Heteroxemia and Xenia, all of which associate with symbiotic algae (zooxanthellae). Anthelia is characterized by having individual, non-retractile polyps united by a thin membrane and in this respect it differs from the related genera. Anthelia colonies tend to grow among other soft corals mainly of the Xeniidae family (Reference 14: Y. Benayahu, Proc. 5th Int. Coral Reef Congress, Tahiti, 6, 255 (1985)). A. glauca (Lammarck, 1816) is the most common species of the genus and has a wide zoogeographical distribution throughout the Indo- Pacific region. On the coral reefs of Sodwana Bay, South Africa, A. glauca is sporadic in its abundance and appears on the reefs in small clusters of colonies at a depth range of 14-22 meters. A recent survey of the soft coral fauna of Sodwana Bay (Reference 12: Y. Benayahu, Invest. Rep. Oceanogr. Res. Inst. (Durban), No. 67, 1 (1993)) lists eight xeniid species. Unlike other reef areas such as the Red Sea (Reference 14), at Sodwana Bay, A. glauca is the most abundant xeniid.
Earlier investigations of Anthelia glauca, from the entrance to the Gulf of Suez in the northern part of the Red Sea, led to the isolation of eight compounds (six xenicanes and two antheliolides) that have a nonacyclic ring, fused to one or four other rings in common (Reference 4; D.J. Vanderah, P. A. Steudler, L S. Clereszko, F.J Schmitz, J.D. Extrand and D.Van der Helm, J.Am.Chem. Soc, 99, 5780 (1977); Reference 5: A. Groweiss and Y. Kashman, Tetrahedon Lett., 2205 (1978); Reference 6: Y. Kashman and A Groweiss, Tetrahedron Lett., 4833 (1978); Reference 7: Y. Kashman and A Groweiss, J.Org.Chem., 45, 3814 (1980); Reference 8: A. Groweiss and Y. Kashman, Tetrahedron 39, 3385 (1983) and references cited therein; Reference 9: G.M. Kόnig, A.D. Wright and O. Sticher, Tetrahedron, 47, 1399 (1991)). The acetoacetylated C24-diterpenoids, antheliolide A and B which seem to be closely related biogenetically to the xenicanes (References 2, 3), have not been seen before.
The genus Alcyonium belongs to the family Alcyoniidae (Reference 15: J. Verseveldt and F.M. Bayer, Zool. Verhand Leiden, 245, 1 (1988)), and Alcyonium comprises species recorded within a large latitudinal range and found in various habitats along a wide depth gradient (Reference 16: J. Verseveldt, Zool. Med Leiden, 39, 153 (1964); Reference 17: J. Verseveldt, Amer. Mus. Nov., 2282, 1 (1967); Reference 18: J. Verseveldt, Zool. Verhand Leiden, 117, 1 (1971); Reference 19: J. Verseveldt, Zool. Med Leiden, 46, 457 (1973); and Reference 20: G.C. Williams, Annals of the South African Museum, 100, 249 (1992). These studies also indicate that the Alcyonium species are highly variable with respect to their gross morphology and the shape or size of the sclerites. In addition, some of the coral-reef inhabiting species (i.e. A. flaccidum and A. urinomii) possess zooxanthellae, while others are azooxanthellated (i.e. A. digitatum and A. palmatum) (Reference 21 : Y. Benayahu, personal observations). A. aurea is an azooxanthellated species collected at Sodwana Bay (Reference 13: Y Benayahu and M. Schleyer, in press). A. aurea was found at a depth of 28-36 meters, where almost no soft corals associated with endosymbiotic algae appear. There is no doubt that at Sodwana Bay A. aurea belongs to communities living below the euphotic zone.
Summary of the Invention
The present invention provides three new compounds extracted and isolated from soft corals. The compounds are antheliatin, zahavin A and zahavin B, the following formulae (I) and (II):
Figure imgf000005_0001
Antheliatin
Figure imgf000006_0001
Zahavin A R = H Zahavin B R = OH
The compounds of the present invention, antheliatin, zahavin A, and zahavin B (also herein identified as 1, 2 and 3 respectively), exhibit antitumor activity. In particular, the present compounds exhibit antitumour activity against cell lines derived from human tumors, such as P-388 mouse lymphoma, A-549 human lung carcinoma, HT-29 human colon carcinoma and MEL-28 human melanoma.
The present invention also provides a method of treating a mammal affected by a malignant tumor sensitive to antheliatin, zahavin A, or zahavin B , which comprises administering a therapeutically effective amount of antheliatin, zahavin A, and zahavin B, or a pharmaceutical composition thereof.
The present invention further provides pharmaceutical compositions which contain as active ingredient the compound antheliatin, zahavin A or zahavin B, as well as a process for their preparation.
A further aspect of the invention is a method for preparing the compounds, which comprises extraction and isolation from the respective soft coral, Anthelia glauca for antheliatin or Alcyonium aurea for zahavin a and zahavin B. Preferred Embodiments of the Invention Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) with suitable formulation for oral, topical or parenteral administration, and they may contain the pure compound or in combination with any carrier or other pharmacologically active compounds. These compositions may need to be sterile when administered parenterally.
The correct dosage of a pharmaceutical composition comprising antheliatin, zahavin A or zahavin B, will vary according to the pharmaceutical formulation, the mode of application, and the particular situs, host and tumor being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose.
Antitumour Activity
Cells were maintained in logarithmic phase of growth in Eagle's Minimum Essential Medium, with Earle's Balanced Salts, with 2.0 mM L-glutamine, with non- essential amino acids, without sodium bicarbonate (EMEM/neaa); supplemented with 10% Fetal Calf Serum (FCS), 10" M sodium bicarbonate and 0.1 g/1 penicillin-G + streptomycin sulfate.
A screening procedure was carried out to determine and compare the antitumor activity of these compounds, using an adapted form of the method described by Bergeron et. al. (Reference A: Raymond J. Bergeron, Paul F. Cavanaugh, Jr., Steven J Kline, Robert G. Hughes, Jr., Gary T. Elliot and Carl W. Porter. Antineoplastic and antiherpetic activity of spermidine catecholamide iron chelators. Biochem. Bioph. Res. Co m. 1984, 121(3), 848-854)). The antitumor cells employed were P-388 (suspension culture of a lymphoid neoplasm from DBA/2 mouse), A-549 (monolayer culture of a human lung carcinoma), HT-29 (monolayer culture of a human colon carcinoma) and MEL-28 (monolayer culture of a human melanoma).
P-388 cells were seeded into 16 mm wells at 1 x 10 cells per well in 1 ml aliquots of MEM 5FCS containing the indicated concentration of drug. A separate set of cultures without drug was seeded as control growth to ensure that cells remained in exponential phase of growth. All determinations were carried out in duplicate. After three days of incubation at 37°C, 10% CO in a 98% humid atmosphere, an approximate IC50 was determined by comparing the growth in wells with drug to the growth in wells control.
A-549, HT-29 and MEL-28 cells were seeded into 16 mm wells at 2 x 104 cells per well in 1 ml aliquots of MEM 10FCS containing the indicated concentration of drug. A separate set of cultures without drug was seeded as control growth to ensure that cells remained in exponential phase of growth. All determinations were carried out in duplicate. After three days of incubation at 37°C, 10% CO2 in a 98% humid atmosphere, the wells were stained with 0.1% Crystal Violet. An approximate IC50 was determined by comparing the growth in wells with drug to the growth in wells in control.
The results are given in the following table:
IC50(μg/ml)
P-388 A-549 HT-29 MEL-28 antheliatin 1.0 1.0 0.12 1.2 zahavin A 1.2 5 5 5 zahavin B 0.5 0.5 0.125 1.0
Extraction and Isolation Ir spectra were recorded on a Nicolet 205 Ft-ir spectrophotometer Low- resolution mass spectra were recorded on a Finnigan-4021 mass spectrometer Hrms were taken on a VG70 VSEQ instrument 1H and 1 C-nmr spectra were recorded on Bruker AMX-360 and ARX-500 spectrometers All chemical shifts are reported with respect to TMS (δ=0) Optical rotations were measured on a Perkin-Elmer Model 141 polarimeter using 1-cm microcell All percentages reported herein, unless otherwise specified, are present by weight All temperatures are expressed in degrees Celsius All incubations are carried out at 28°C and flasks are shaken in an orbital shaker at 250 rpm All media and recipients are sterile and all culture processes aseptic
As part of our investigation of soft corals (References 2 and 7, and Reference 10 D Green, Y Benayahu and Y Kashman, J. Nat.Prod, 55, 1 186 (1992)), we were interested in comparing Red Sea soft corals with their Indo-Pacific counterparts We now report the results of investigation of the two Indo-Pacific soft corals, Anthelia glauca (Reference 12) and Alcyonium aurea (Reference 13), from Sodwana Bay, South Africa giving antheliatin, zahavin A and zahavin B
Antheliatin (1) - The soft coral Anthelia glauca was collected in Sodwana Bay South Africa, in May 1994 by divers using scuba A voucher (TASA 293) is deposited in the Zoological Department at Tel Aviv University
The freshly collected soft coral was immediately frozen at -25°C The freeze dried coral, a single animal (5 g), was then extracted with EtOAc to give a brown gum (90 mg) The gum was chromatographed first over a Sephadex LH 20 column eluted with MeOH-CHCl3-hexane (1 1 2) and then over Si gel eluted with hexane-EtOAc 1 1 to afford antheliatin 1 (10 mg), m p 165° (methanol), [α]20 D + 3 5 ° (c = 1 2, CHC13), ir (neat) υ ma 3485, 2950, 1740, 1710, 1451 cm"1 , Hreims 597 2707 (MIT, C33H41O10) (Calcd 597 2700) For nmr data see Table 1 Table 1. Nmr Data for antheliatin {1}*
H# δ 13Cb δ 'H m J(Hz) COSY.TOCSY HMBC (H to C#) nOe
1 91.9 5.77 d 1.9 11a 3-l la.19.19" 11a
-> 138.3 6.45 s 12
4 113.4 - 3.4a.5.11a.l2
4a 38.4 2.05 m 1.3.5.5'.6.6'.11a
5 28.7 2.00 m 4a,5',6,6' 11a
5' 1.55 m 4a.5',6)6'
6 40.8 2.32 brd 12.1 6',5,5' 18,19.19'
6' 2.10 m 6\5.5'
7 137.3 5 ,18
8 122.0 5.00 d 9.2 9, 18 6,9.10,18 9-OAc
9 76.9 5.42 d 9.2 8.10 10 10
10 83.1 4.12 brs 9,OH 9,l la.l9.19' 1.9.19'
11 149.1 1.9.10.11a. l8
11a 43.1 2.30 brs 1,19' 19' 1
12 71.6 5.69 d 4.2 3 3
13 71.9 5.86 dd 9,3,4,2 12, 14
14 118.5 5.34 dd 9.3.0.9 13,16,17 12.13,16,17
15 140.8 16,17
16 18.9 1.79 s 14 14,17
17 25.9 1.71 s 14 14,16
18 18.0 1.78 s 8 8 9, 11a.
13
19 115.2 4.95 s 19' 10,11a 19'
19' 4.90 s l la,19 10, 19
1' 165.6 3',7.13
2' 130.1 3'.7' y,τ 129.6 7.95 d 7.7 4'.6' 4\5'.6'
4*,6' 128.4 7.32 t 7.7 3',5* 3',5\T
5' 133.0 7.50 t 7.7 3',4' 3\7
1-OAc 169.7 - l.OCOHj
20.1 1.92 s
9-OAc 170.9 9. OCOCH3
20.9 2.05 s
12-OAc 169.8 12. OCOCH Q3
21.2 2.10
a CDC13; 500 MHz for 1H, 125 MHz for 13C. Carbon resonances assigned by a
HMQC experiment. b The carbon chemical shifts of xenicin are: δ (m,C#): 91.7 (d,l), 142.6 (d,3),
113.5 (s,4), 37.1 (d,4a), 30.5 (t,5), 40.8 (t,6), 134.3 (s,7), 126.2 (d,8), 70.6 (d,9), 42.9 (t,10), 146.5 (s,l l), 43.1 (d,l la), 76.4 (d,12), 69 (d,13), 119.6 (d, 14), 140.7 (s,15), 18.9 (q, 16), 25.6 (q,17), 17.5 (q, 18), 116.1 (t,19). Zahavin A (2) and B (3). - Alcyonium aurea was collected in Sodwana Bay, South Africa, in May 1994 by divers using scuba. A voucher (TASA 217) is deposited in the Zoological Department at Tel Aviv University. The freeze dried soft coral, a single animal (9 g), was extracted with EtOAc to give a yellowish gum (155 mg). The gum was chromatographed first over a Sephadex LH-20 column eluted with MeOH- CHCl3-hexane, 1; 1;2) and then several times over Si gel columns eluted with hexane- EtOAc (8:2) to afford 2 (30 mg); Rf 0.7 (EtOAc-hexane, 1.1), and 3 (5 mg) Rf 0.65.
Zahavin A (2), - A viscous oil [α]20 D + 7.3° (c = 1.8, CHC13); ir (neat υmax 2950, 1740, 1372, 1238 cm"1. Hreims 461.2546 (MH\ C26H37O7) (Calcd 461.2540) For nmr see Table 2.
Zahavin B (3), - A viscous oil; [α]20 D + 4.8° (c = 0.7, CHC13); ir (neat) υπ 3460, 2925, 1743, 1372, 1238 cm"1. Hreims 477.2493 (MH^, C26H37Og) (Calcd.
477.2489). For nmr sec Table 2.
Table 2. NMR Data for zahavin A and B {2 & 3}B
zahavin A(2) zahavin B (3)
H# δ 13C δ Η m(J,Hz) COSY HMBC δ 1Jc δ 'H m
1 92.0 5.79 11a 3.4a,19.19' 3.4a.l9.19' 92.8 5.79 d(2.3) J -» 137.0 6.33 s 1,12 137.0 6.33 s
4 117.2 3.12.13,13' 117.1
4a 37.5 1.90 m 1.3.6,6" 37.7 2.40 m
5 30.1 1.83 m 4a,5\6.6' 11a 29.6 1.92 m
5' 1.49 m 4a.5.6,6' 1.60 m
6 40.2 2.19 m 5,5',6' 8.18 40.6 2.28 m
6' 1.92 m 5.5 6 2.05
7 135.6 18 137.3
8 124.5 5.30 m 9.9'.18 5,18 121.2 5.17 dd(7.10)
9 25.2 2.41 m 8.9.10.10' 8.19' 35.2 2.50 m
9' 2.05 m 8.9, 10,10' 2.50 m
10 35.4 2.00(2H) m 9.9', 10' 19.19' 76.2 4.67 dd(7.2)
11 150.0 1,9.10. 152.3 10M9.19'
Ha 49.5 1.94 m 5.5 -10.10 .19.19' 42.9 2.47 m
12 68.9 5.28 m 3 68.9 5.34 dd(10.4)
13 38.5 1.92 m 13',14 38.6 1.92 m
13' 1.88 m 13,14 1.92 m
14 73.2 5.16 dd(8.5,3. 13,13* 16,16',17 73.2 5.23 dd(9.4) 5)
15 142.9 14.16.17 142.9
16 112.7 4.91 bs 16',17 13.13U4.17 112.8 4.99 s
16' 4.84 bt(1.5) 16.17 4.95 s
17 18.3 1.69 s 16,16' 18.3 1.55 s
18 16.8 1.69 s 8 8 17.2 1.72 s
19 113 4 4.23 bs 113.4 5.06 s
4.77 bd(l. l) 5.02 s
1 OAc (170.1, ; 21.0, 2.05s)b 1. CH3 (170.0. 21.0. 2.0s)
12 OAc (169.8. , 21.0, 2.05s; >b 12, CH3 (169.8. 21.0, 2.0s)
14 OAc (169.7. . 21.0, 1.98s; )b 14, CH3 (169.0, 21.0. 2.0s)
a. CDC13; 500 MHz for 1H, 125 MHz for 13C Carbon resonances assigned by a HMQC experiment. b. May be interchanged. The two studied species, --. glauca and A. aurea, belong to different families of reef- inhabiting soft corals.
The Sodwana Bay A. glauca, in contrast to the Red Sea species, was found to contain a single diterpenoid designated antheliatin (1), a crystalline material, m.p. 165°C (MeOH), [α]D + 3.5° (c, 1.2, CHC13), 0.2 % dry wt. The molecular formula C33H40O10 was established by high-resolution ms (M"596.2627. Calcd. MW = 596.2622) and 13C nmr data. Twelve of the 14 degrees of unsaturation implied by the molecular formula were due to four double bonds, three acetates and one benzoate (Table 1), and the molecule was thus considered bicyclic. The ir data (3450 cm'1) and the preparation of a mono acetate by micro acetylation of 1, (shift of H-9 from 8, 4.12 ppm, in 1, to bx 5.25 ppm in the mono-acetate) confirmed the presence of a hydroxyl function.
Interpretation of nmr experiments (DEPT, COSY, TOCSY, nOe, HMQC and HMBC - see Table 1) suggested two moieties, A and B, which, together with the third acetate, accounted for 29 out of the 33 atoms of the molecule.
(CH3)2C=CHCH(OCOC6H5)CH(OAc)- -CH2CH2C(CH3)=CHCH(OAc)CH(OHC(=CH2)CI<^
B
The four additional carbon atoms were assigned to (a) a polarized double bond (δ13c 138.2 ppm(d) and 113.4 pρm(δ)), (b) a lactol-methine (δ13c 91.9 ppm (d), δ, 5.77 ppm(d)) and (c) an additional aliphatic methine (δJ3c 38.4 ppm (d)). The low-field nmr signal of the lactol-proton (δ1H, 5.77 ppm) suggested that the third acetate was attached to this position (7). CH-Hetero correlations (HMBC) in the nmr spectrum of 1, between (a) and C-atom of the methine C-4a, (δ13C 38.9 ppm), the allylic pair H-6 and 6' and protons H-5 and 5' of B, the other end-methine of moiety B (H-l la), and H-3, and (b) between C-4 (δ13C 1 13.4 ppm), one of the polarized double bond carbon atoms, and protons H-5 and 5' of site B and H-12 of A (δ1H 5.69 ppm), suggested C-4 and C-4a to be the link between moieties A and B, a connection which was further extended by correlations between C-12 and H-3. All the latter correlations suggested the 2- oxabicyclo[7,4,0]tridecane system. At this point in the data analysis it was evident that antheliatin (1) had many structural similarities with the known xenicanes and other xenialactols. Detailed comparison of the C nmr data for compound 1 and those for xenicin (Table 1, note b) and other xenialactols clearly pointed to 1 being a xenialactol, i.e., 10-hydroxy-13-desacetyl-13-benzoylxenicin. CH-Correlations of each one of the four ester carbonyls to their carrying methine protons (H-l, -9, -12 and -13) established their positions.
The 13C chemical shifts had already proved to be a delicate probe for the structure and stereochemical assignments of 2-oxabicyclo[7,4,0]tridecane systems (7,8). Thus, based on the C spectrum of 1, a trans ring junction between the two rings was suggested. The latter conclusion was further confirmed from the almost zero coupling constant between 1 l-4a and H-l la which, in the trans ring junction, are at a ca 90° angle to each other (Dreiding model). A similar observation for a different xealcane was recently reported by Kόnig (Reference 9). The appearance of the H- 11 a resonance as a singlet also determined the configuration of the lactol carbon, that is the H-lα configuration (ΨH -n- H .lα~80°). The E configuration of the C(7)C(8) double bond was determined from the carbon nmr spectrum, from the characteristic 18 ppm chemical shift of Me- 18 (against ca. 26 ppm for the Z isomer), due to a γ-effect. The configurations of C-9 and C-10 (i.e. OAc-9α and OH-lOβ) were determined from the 8,9 and 9, 10 coupling constants, i.e. J8α-9β = 9.2 Hz in agreement with ΨH-8O. H -9β ~ 80° All the configurations of the chiral centers of the bicyclic ring system were also unequivocally confirmed by d-nOe measurements. nOe correlations were observed between H-l/H-1 la; H-4a/H-8α, -1 la(β); Me-18/H-l la and H-9β; H-9β/H-10α and H- 10c /H-19, all being in full agreement with a proper Dreiding model in the "αβ- configuration" (Reference 22A H. Shirahama, E. Osawa, B R. Chabra, T. Shimokawa, Y. Yokono, T. Kanaiwa, T. Amiya and T. Matsumoto, Tetrahedron Lett., 22, 1527 (1981)). that is the C-l 1(19) methylene inclining towards the α direction and Me- 18 towards the β one.
As for the configuration of C-12 and C-13, due to the large benzoxy group it could be expected that the side chain would adopt a different conformation from the one found in xenicin (Reference 4) or 13-epi-9-desacetylxenicin (Reference 23: J.C Braekman, D. Daloze and B. Tursch, Bull. Soc. Che . Belg, 71 (1979)) for which the 12, 13 -configurations were determined by X-ray diffraction analysis. This analysis confirmed the suggested structure from the nmr spectra and also determined the stereochemistry of C-12 and C-13 (12S* and 13R*) as shown. The coupling constant of 4.2 Hz between H-12and H-13 measured for 1, versus 9.5 Hz and 7 Hz, respectively, for other xenicanes suggested a different conformation..
From the second studied Indo-Pacific soft coral, Alcyonium aurea, we isolated two additional xenicanes designated zahavin A and B, compounds 2 and 3 respectively. The structure elucidation of the two followed the same route described for compound 1. The xenicane skeleton of zahavin A, m/z 460.2462, C26H36O7 was unequivocally determined by comparison of the C-chemical shifts of the bicyclic system of 2 with those of 9, 13-desacetoxyxenicin, earlier isolated by us from Xenia obscuronata (Reference 8). The complete nmr line assignments were achieved from 2D-nmr experiments (HMQC, COSY, HMBC). The structure of the side chain [- CH(OAc)CH2CH(OAc)C(CH3)=(CH2] was suggested on the basis of its nmr data and particularly the COSY experiment (Table 2). Because of the conformational mobility of the side chain we could not determine the relative stereochemistry of C-12 and C-14. The small available amount of compound, and especially its instability, precluded chemical transformations which could shed light on the stereochemistry of C-12 and C- 14. Zahavin B (3), m/z 476.241 1, C26H36O8, is closely related to zahavin A. It possesses the bicyclo [7,4,0] tridecane system and the same side chain. Zahavin B, however, carries an additional sec alcohol group at C-10 (δnc 76.2 ppm, δ1H 4.67 ppm(dd), J=7,2 Hz) The location of this OH-group was suggested mainly on the basis of the following CH-correlations, deduced from an HMBC experiment: C-8/H-10; C- lO/H-19; C-1 la/H-19 as well as from homo-COSY correlations i.e. H-l 0/9,9' and H- 88/9,9'. In addition, the chemical shifts of C-9 and C-1 la also support the C'lO location of the OH-group; (If the OH group was on C-9 the line of C-10 should have been at ca. 46 ppm as in xenicin (and not at 35.2 ppm). The up-field shift of 6 ppm of C-1 la, to 42.9 ppm, due to a strong γ-effect of the 10-OH group is in full agreement with the later position. Zahavin B (3) is very unstable both under mild basic and mild acidic conditions; the compound decomposes readily under acetylation conditions (Ac2O, pyridine, CH2C12) or in a CDC13 solution.
The confirmation of the bicyclic system of 3 as well as the stereochemistry of the sec alcohol group were determined from d-nOe measurements.
An nOe correlation between CH3-18 and H-l la(β) and H-6(β)(διH2.28 ppm) determined the conformation of the 18-methyl group to be on the same side as H-l la. The later nOe correlations, together with the measurable proton coupling constants, require the 11(19) double bond to be in the α orientation Thus zahavin B has the same "αβ-conformation" (Reference 22), as in antheliatin, see above.
Another nOe between H- 19(a) and H-10 determined the H-lOα configuration (OH-lOβ). The latter configuration is also in full agreement with the above mentioned γ-effect of the alcohol on C-1 la. References
1 D J Faulkner, Nat. Prod. Reports., 1 1, 355 (1994) and references cited therein
2 D Green, S Carmely, Y Benayahu and Y Kashman, Tetrahedron Lett., 29, 1605 (1988)
3 A B Smith III, P J Carroll, Y Kashman and D Green, Tetrahedron Lett., 30, 3363 (1989)
4 D J Vanderah, P A Steudler, L S Clereszko, F J Schmitz, J D Extrand and D Van der Helm, J.Am.Chem. Soc, 99, 5780 (1977)
5 A Groweiss and Y Kashman, Tetrahedon Lett., 2205 (1978)
6 Y Kashman and A Groweιss,Tetrahedron Lett , 4833 (1978)
7 Y Kashman and A Groweiss, J.Org.Chem., 45, 3814 (1980)
8 A Groweiss and Y Kashman, Tetrahedron 39, 3385 (1983) and references cited therein
9 G M Konig, A D Wright and O Sticher, Tetrahedron, 47, 1399 (1991)
10 D Green, Y Benayahu and Y Kashman, J. Nat.Prod, 55, 1186 (1992)
11 Z Kinamoni, A Groweiss, S Carmely, Y Kashman and Y Loya, Tetrahedron, 39, 1643 (1983)
12 Y Benayahu, Invest. Rep. Oceanogr. Res. Inst. (Durban), No 67, 1 (1993)
13 Y Benayahu and M Schleyer, in press
14 Y Benayahu, Proc. 5th Int. Coral Reef Congress, Tahiti, 6, 255 (1985)
15 J Verseveldt and F M Bayer, Zool. Verhand Leiden, 245, 1 (1988)
16 J Verseveldt, Zool Med Leiden, 39, 153 (1964)
17 J Verseveldt, Amer. Mus. Nov., 2282, 1 (1967)
18 J Verseveldt, Zool. Verhand Leiden, 117, 1 (1971)
19 J Verseveldt, Zool. Med. Leiden, 46, 457 (1973)
20 G C Williams, Annais of the South African Museum, 100, 249 ( 1992)
21 Y Benayahu, personal observations
22 H Shirahama, E Osawa, B R Chabra, T Shimokawa, Y Yokono, T Kanaiwa, T Amiya and T Matsumoto, Tetrahedron Lett., 22, 1527 (1981) 23 J C Braekman, D Daloze and B Tursch, Bull. Soc. Chem. Belg, 71 (1979)
24 S J Coval, P J Scheuer, G.K. Matsumoto and J. Clardy, Tetrahedron, 40, 3823 (1984)
A Raymond J. Bergeron, Paul F. Cavanaugh, Jr., Steven J. Kline, Robert G
Hughes, Jr , Gary T. Elliot and Carl W. Porter Antineoplastic and antiherpetic activity of spermidine catecholamide iron chelators. Biochem. Bioph. Res. Comm. 1984, 121(3).
848-854
B Alan C. Schroeder, Robert G. Huges, Jr. and Alexander Bloch Effects of
Acyclic Pyrimidine Nucleoside Analogues. J. Med Chem. 1981, 24 1078-1083.

Claims

CLAIMS:
1 A compound chosen from antheliatin, zahavin A or zahavin B, of the following formulae (I) or (II):
Figure imgf000019_0001
Antheliatin
Figure imgf000019_0002
Zahavin A R = H Zahavin B R = OH
2. A method of treating an mammal affected by a malignant tumor sensitive to antheliatin, zahavin A, or zahavin B , which comprises administering to the affected individual a therapeutically effective amount of antheliatin, zahavin A, and zahavin B, or a pharmaceutical composition thereof.
3 A pharmaceutical preparation which contains as active ingredient the compound antheliatin, zahavin A or zahavin B, as defined in claim 1.
4. A process for preparing a pharmaceutical composition for use in the treatment of a tumor, wherein an active ingredient is admixed with a pharmaceutically acceptable carrier, characterized in that antheliatin, zahavin A or zahavin B, as defined in claim 1 , is employed as active ingredient.
5. A method for preparing a compound according to claim 1, which comprises extraction and isolation from the respective soft coral, Anthelia glauca for antheliatin or Alcyonium aurea for zahavin A or zahavin B.
PCT/GB1996/000903 1995-04-13 1996-04-15 Antheliatin, zahavin a and zahavin b: new cytotoxic xenicane diterpenes WO1996032388A1 (en)

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WO2011111804A1 (en) * 2010-03-11 2011-09-15 学校法人早稲田大学 Antiprotozoal compound originating in coelenterata
CN113402391A (en) * 2021-05-07 2021-09-17 宁波大学 Diterpenoid compound derived from Balanophora japonica, preparation method and application thereof

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KR100350891B1 (en) * 2000-03-08 2002-09-05 한국해양연구원 Acalycixeniolide c, d, e and f
WO2011111804A1 (en) * 2010-03-11 2011-09-15 学校法人早稲田大学 Antiprotozoal compound originating in coelenterata
CN102812015A (en) * 2010-03-11 2012-12-05 学校法人早稻田大学 Antiprotozoal compounds from coelenterates
EP2546245A1 (en) * 2010-03-11 2013-01-16 Waseda University Antiprotozoal compound originating in coelenterata
EP2546245A4 (en) * 2010-03-11 2013-05-29 Univ Waseda ANTIPROTOZOAN COMPOUND FROM COELENTERATES
US8722909B2 (en) 2010-03-11 2014-05-13 Waseda University Antiprotozoal compound derived from coelenterata
JP5721186B2 (en) * 2010-03-11 2015-05-20 学校法人早稲田大学 Coelenterate-derived antiprotozoan compound
CN113402391A (en) * 2021-05-07 2021-09-17 宁波大学 Diterpenoid compound derived from Balanophora japonica, preparation method and application thereof
CN113402391B (en) * 2021-05-07 2022-04-26 宁波大学 Diterpenoids derived from soft coral Coral Pollens and their preparation method and use

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