Clavularia viridis QUOY and Gaimara

Chlorovulones

The marine prostanoids named clavulones isolated from Stolonifer viridis4750 had received much attention because of their unique structural features and remarkable anticancer activities.51-60 The freeze dried C. viridis (210 g) collected at the coral reef of Ishigaki Island (Okinawa, Japan) were extracted with ether. The ether extract (7.88 g) was chromatographed on a silica gel column using n-hexane-ethyl acetate (5:1) as an eluent to give a mixtures of chlorovulones (100 mg). Chlorovulones were eluted prior to the elution of clavulones. Repeated HPLC [silica gel, n-hexane-ether (2:1)] of the mixture gave chlorovulone-I (40) (colorless oil, 20 mg, C21H29ClO4) and chlorovulone-II (41) (colorless oil, 4 mg, C21H29ClO4) in a ratio of 10:1.2 in order of increasing polarity. Although chlorovulone-IV (42) was not isolated from the chlorovulones mixture in a pure state, its presence was suggested by the isolation of the corresponding acetate 43, when the mixture was subjected to acetylation with acetic anhydride/pyridine to give the easily separable acetates mixture (43, 44, 45 and 46) of chlorovulones which was easily separated by silica gel column and HPLC.

Chlorovulones (I-IV) had the stereochemistry of the carbon-carbon double bonds (5Z, 7E, 14Z), (5E, 7E, 14Z), (5E, 7Z, 14Z) and (5Z, 7Z, 14Z) which corresponds to those of clavulone I, II, III and IV, respectively. The structures of chlorovulones had been assigned by extensive use of mass spectrometry, 1H NMR and 13C NMR spectroscopy. Chlorovulone-I (40) displayed strong antiproliferation and cytotoxic activities in human Promyelocytic leukemia (HL-60) cells in vitro. The IC50 value of (40) in the HL-60 cells was 0.03 M (0.01 g/mL) which is about 13 times stronger than clavulone-I.

Iguchi et al51,60,61 reported the isolation of new halogenated prostanoids (47-51) from the Okinawan soft coral Clavularia viridis. Mainly NMR spectral data were used in order to elucidate the structure of (47) and the relative and absolute configurations of the compound was determined by analysis of NOESY, CD data, chemical conversion, and the modified Mosher's method. The structures of (48-51) were deduced by comparison of their spectral data with those of (47). Hexane extract of 6.83 g crude isolate (out of 14.5 g, freeze-dried soft coral 470 g) was chromatographed on a silica gel column eluted with hexane:AcOEt and AcOEt:MeOH (3: 1 and 1:1) to obtain five fractions. The second fraction (eluted with hexane:AcOEt, 3:1) was further subjected for separation and purification by MPLC and HPLC on normaland reversed-phase columns to obtain compounds (47) (29.8 mg), (48) (2.6 mg), (49) (1.1 mg), (50) (0.3 mg), (51) (0.6 mg) and (40) (0.1 mg).

OH OH

OH OH

Analytical data such as HR-EIMS and 13C NMR indicated that compound (47) possess an iodine-containing molecular formula of C23H33IO6 [472.1109 (M+ -CH 3CO2H), (calcdfor C21H29IO4, 472.1111]. In the UV and IR spectra, the presence of an a, ^-unsaturated carbonyl group [Amax 251 mn], an acetate ester (IR 1732, 1240 cm1 ), and a hydroxyl group (3470 cm1 ) was suggested. Compound (47) showed potent cytotoxic activity.

Clavulones

Iguchi et al62'63 have isolated three new prostanoids named clavulones (I-III) from the Japanese coelenterate, Stolonifer Clavularia viridis QUOY and GAIMAR. Clavulones are the first examples of the prostanoids having oxygen functions at C-4 and C-12 positions and olefins at C-7 and C-14 positions. The methanol extracts of C. viridis (5 kg, wet weight), collected at the coral reefs of Okinawa Japan, was suspended in water and extracted with ethyl acetate. The ethyl acetate extract (30 g) was chromatographed on silica gel column using benzene : ethyl acetate (10 : 1) as eluant to give four fractions. The fraction four was decolorized by passing through a polystyrene gel column using benzene : ethyl acetate (10 : 1) as an eluant to give clavulone-I (870 mg, C25H34O7; [a]D 28.9 ° as a pale yellow oil. Similar separation procedure of the fraction 3 and 2 gave clavulone-II (pale yellow oil, C25H34O7; [a]D +10.9° and clavulone-III (pale yellow oil, 253 mg, C25H34O7; [a]D -15.5 ° respectively. Structure of clavulones-I, III had been determined by UV, IR, 1H NMR, 13C NMR and chemical reaction. The absolute configurations at the C-4 and C-12 positions of clavulones had been determined.64 The clavulones showed a significant antiinflammatory effects at 30 g/ml in the fertile egg test.62

More recently Shen et al65 reported the isolation of seven new clavulone and its derivatives from the CH2Cl2:MeOH extract of Clavularia virdis collect in Tiwan, and designated as 4-deacetoxyl-12-O-deacetylclavulone I (52), 4-deacetoxyl-12-O-deacetylclavulone II (53), bromovulone II (54), iodovulone II (55), 4-deacetoxyl-12-O-deacetylclavulone III (56), bromovulone III (57), and iodovulone III (58).

The dichloromethane and methanol extract of C. viridis was fractionalized between ethyl acetate and water. Silica gel column chromatography and HPLC chromatographic purification yielded seven new compounds. These

COiMe

COiMe

,C02Me

,C02Me

compounds were designated as 4-deacetoxyl-12-O-deacetylclavulone I (52, 1.1 mg), 4-deacetoxyl-12-O-deacetylclavulone II (53, 2.6 mg), bromovulone

II (54, 3.1 mg), iodovulone II (55, 9.1 mg), 4-deacetoxyl-12-O-deacetylclavulone III (56, 2.2 mg), bromovulone III (57, 25 mg), and iodovulone

III (58, 4.4 mg), in addition to seven known prostanoids from the EtOAc soluble fraction. The known compounds were identified as clavulones I (15 mg), II (6 mg), and III (8 mg), 7-acetoxy-7,8-dihydroiodovulone (6 mg), chlorovulones II (17 mg) and III (0.9 mg), by comparison of their spectral data (1H, 13C NMR, MS, and optical rotation) with reported values. Structure of these unknown compounds was determined by the use of 1H NMR, 13C NMR, HRFABMS, and 2D NMR. Pharmacological study revealed that bromoclavulone (57) exhibited potent cytotoxicity against human prostate (PC-3) and colon (HT 29) cancer cells.

Claviridenones

Four new antitumor prostanoids named claviridenone61 (a-d, 59-62) have been isolated from the Okinawan soft coral Clavularia viridis. The acetone extract of the fresh soft coral from Okinawa Japan was partitioned by a EtOAc-water solvent system. Purification of the EtOAc soluble portion by repeated SiO2 column and HPLC (PORASIL) provided claviridenone-a (59), C24H34O5; [ojD-82.2° (CHCl3); claviridenone-b (60), C25H34O7, [a]D+26.7° (CHCl3); claviridenone-c (61) and claviridenone-d (62). The absolute stereo structures of these prostanoids had been elucidated on the basis of chemical and physicochemical evidences which including the application of the CD exciton chirality method to their various benzoyl derivatives having benzoate and conjugated diene chromophores.66 Iguchi et al62 had reported the isolation of three prostanoids, named clavulones-I-III from C. viridis. Subsequent studies by Kitagawa et al66 established that clavulone-I, II and III were identical with those of claviridenone-d (66), claviridenone-c (61) and claviridenone-b (60), respectively.

59, Claviridedone-a

60, Claviridedone-b

59, Claviridedone-a

60, Claviridedone-b

62, Claviridedone-d

63, R = CH2OAc, 20-acetoxy claviridenone

62, Claviridedone-d

C-20 Acetoxy clavulones

Three novel C-20 oxygenated prostanoids named as C-20 acetoxy clavulones had been isolated from C. viridis.61 Silica gel chromatography (benzene-ethyl acetate, 10:1) of the ethyl acetate extract of C. viridis (210 g, wet weight) gave fractions (1-3).

Further, repeated silica gel chromatography and preparative TLC of the fractions gave 20-acetoxy claviridenone-d (62) (13 mg, C25H34O1; [a]D-31.1°, c 0.9, CHCl3); 20-acetoxy claviridenone-c (63) [215 mg, C21H36O9, [a]D 3.1 ° (c, 0.054, CHCl3) and 20-acetoxy claviridenone-b (64) [16 mg,

C25H32O9, [a]D +26.40° (c, 0.86, CHCl3)] as pale yellow oils. The absolute stereostructures of these prostanoids had been elucidated on the basis of chemical and physiochemical evidences.66,67

Labophyton depressum

L. depressum (Alcyonacea, Alcyoniiudae) a soft coral collected in the Gulf of Eilat (the Red Sea) had afforded four PGF derivatives, (15S) PGF2a-11-acetate methyl ester (65); 18-acetoxy derivatives (66) of compound (65) as well as their two corresponding free carboxy acids (67) and (68). The soft coral L. depressum was collected in the Gulf of Eilat and extracted with CH2Cl2. A crystalline compound (C23H38O6) m.p. 55°C; IR (KBr): 3700, 3610, 3510 (OH) 1740, 1730 cm- (OC=O) obtained from the CH2Cl2 was characterized mainly on the basis of its spectral data as methyl-11-acetoxy-9,15 (S)-hydroxy-5 cis-13-trans-prostadienoate (65).

The second compound was obtained as an oil (C25H40O8). The 1H and 13C NMR spectra of the compound were similar to (65), but it had 2-hydroxyl and 2-acetate functionalities. It was characterized as methyl 11,18-diacetoxy-9, 15 (S)-hydroxy 5-cis, 13-trans prostadienoate (67) (the 11,18-diacetate 18-hydroxy PGF2 : methyl ester). The more polar compounds isolated from the crude ethyl acetate extract of L. depressum turned out to the corresponding acetate and diacetate of free acids (67) and (68). Esterification of the acids with CH2N2 gave the corresponding esters (65) and (66), respectively.

Telesto nisei

Scheuer et al68 have isolated unusual halogenated antitumor eicosanoids named punaglandins from the octocoral Telesto riisei. Punaglandins are characterized by C-12 oxygen and unprecedented C-10 chlorine functions. Punaglandin-3 (69) inhibits L1210 leukemia cell proliferation, with an IC50 value of 0.02 g/mL, which represents 15 fold greater activity than displayed by the corresponding clavulone.

It is interesting to note that Telesto riisei a source of punaglandins, is an octocoral that lacks symbiotic algae. The freeze-dried animal (760 g) was refluxed with hexane to yield a (9.5 g) residue. Of which 1.9 g partitioned into 80% MeOH:H2O containing the punaglandins. Column chromatography on Biosil-A (hexane:EtOAc, 7:3) monitored at 254 nm yielded four fractions, punaglandin-IV (70, 0.08% of the freeze-dried animal), punaglandin-III

(69, 1%), punaglandin-II (71, 0.2%) punaglandin-I (72, 0.8%) as colorless oil, each further purified by HPLC (Lichrosorb magnum RP-18, 80% MeOH/H2O). Desorption chemical ionization MS revealed molecular ions and the presence of chlorine, which was confirmed by combustion analysis. The 17,18-dihydro relationship of (72) and (71) and of (69) and (70) was evident from !H and 13C NMR data and from their formula each pair differing by two mass units. The structure of punaglandin-I (72), C27H37ClO10 was deduced as follows as: The 1H NMR and 13C NMR showed the presence of three acetates as three quaternary carbonyl carbons were observed at £171.3, 170.5, 170.4; and three singlet (for three protons each) at £2.11, 2.8, 2.00 ppm and presence of one OMe group was supported by the resesonence at 3.63 ppm in the 1H NMR spectrum (£173.8). An a,^-enone system was supported by the UV absorption at 228 nm. Presence of 3° OH was confirmed by the IR spectroscopy. Extensive decoupling experiments at 500 MHz fully documented all protons from C-2 to C-8 and C-13 to C-20 and, hence, both side chain. A 3° hydroxy at C-12 was supported by the non-equivalence of the C-13 1H NMR signals (£ 2.53, 2.45) and their coupling to H-14, £ 5.30 ppm (J = 7.0 Hz), thereby unequivocally placing chlorine at C-l0. Punaglandin-I (72) loses AcOH when treated with pyridine affording a 3:1 mixture of (Z )-7,8-punaglandin-3 (73 and 70) under the same reaction conditions 71 was transformed into (Z )-7,8-punaglandin-4 (74) and its E isomer, also in a ratio of 3:1. These reactions showed that all four punaglandins belong to the same stereochemical series. The relative stereochemistry of the five centers was deduced by NOE experiments. If one assumes that the punaglandins had the same stereochemistry as other marine eicosanoids, the structures proposed represent the correct absolute stereochemistry.

Recently, Ireland et al69 isolated punaglandins from Telesto riisei. The freeze-dried organism Telesto riisei collected from Hawaii was macerated and subjected to a hexane Soxhlet extraction. The hexane-soluble material

OAc OAc

OAc OAc

70, 17,18-Dihydro of 69

71, 17,18-Dihydro of 72 72

74, 17,18-Dihydro of 73

was extracted with 70% CH3OH: H2O (70:30) resulting in hexane and aqueous CH3OH fractions. The !H NMR spectrum confirmed the presence of punaglandins in the aqueous fraction. The water soluble material was purified by silica gel flash chromatography, yielding three fractions containing mixtures of punaglandins as determined by !H NMR. Each fraction was further purified by reversed-phase C-18 HPLC leading to the following punaglandins: PNG2 (75), PNG4 (76), Z-PNG4 (77), PNG6(78), PNG3 (79). The structures of the punaglandins were confirmed by 1H and 13C NMR and EIMS. Additionally, HMQC, HMBC, and DEPT data were collected for PNG2 (75), Z-PNG4 (77), and PNG6 (78). All data were consistent with literature values. These punaglandins were tested for the ubiquitin-isopeptidase activity. It was anticipated that these punaglandins would inhibit ubiquitin-isopeptidase activity of the proteasome pathway with greater potency due to chlorination at C10. The activity was determined by the use of simple isopeptidase substrate, ubiquitin-PEST (Ub-PEST), a full-length ubiquitin molecule fused with an 18-amino acid C-terminal peptide extension.70,72 The polypeptide extension is rich in Glu (E), Ser (S), Pro (P), and Thr (T) residues called PEST motifs that are responsible for rapid degradation of these and other unstable proteins.71 Ub isopeptidases enzyme specifically cleaves this peptide extension, yielding free, full-length Ub. PNG2 (75), PNG3 (79), and Z-PNG4 (77) were analyzed for isopeptidase activity in vitro in HCT 116 lysates. Isopeptidase activity buffer was used to lyse the cells. and the protein concentration was adjusted to 0.3 mg/mL per sample.

Each sample was subsequently incubated with 2, 6, 20, 60, or 200 ¡M of punaglandins (75), (78) and (79) in addition to 50 ¡g/mL of Ub- PEST. The

negative control cells were treated with 0.5% DMSO (vehicle) and equivalent Ub-PEST. The reaction was terminated after 45 min. The amount of fused Ub-PEST and free Ub product was determined by SDSPAGE fractionation. The vehicle control cells revealed a 1:1 ratio of fused Ub-PEST (10.5 kDa) to free Ub (8.5 kDa) as predicted to occur at a 45 min time interval. The punaglandins were found to inhibit the Ub-PEST degradation. Inhibition was initially observed at 20 |M with complete inhibition occurring at 60 |M, indicating that PNG2 (75), PNG3 (79), and Z-PNG4 (77) are more potent inhibitors of isopeptidase activity in vitro than PGJ2. All of these compounds exhibited potent in vivo isopeptidase activity, and they represent a new chemical class of cancer therapeutics.

79
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