WO2005085277A1 - Cyclic hexapeptides, process and use thereof - Google Patents

Abstract

The present invention provides cylclic hexapeptides, microcionamides A and B, isolated from the Philippine marine sponge C/athria (Thalysias) abietina. These new hexapeptides are cyclized via a cystine moiety and have their C-terminus docked by a 2-phenylethylenamine group. The compounds exhibit cytotoxicity against human brean cancer cells lines and displayed inhibitory activity against Mycobacterium tuberculosis. The structure of the compounds was determined to be of formula: (I).

Description

CYCLIC HEXAPEPTIDES, PROCESS AND USE THEREOF

This invention was made with United States Government support under NIH Grant No. CA 67786. The Government has certain rights to this invention.

TECHNICAL FIELD

This invention relates to cyclic hexapeptide, microcionamides A and B of the formula I and to process for preparing thereof. The compounds exhibit significant activity against breast cancer and inhibitory activity against Mycobacterium tuberculosis.

BACKGROUND ART

Cyclic peptides have been isolated from a number of marine phyla, and many show high levels of cytotoxicity. Examples include aplidine (dehydrodidmenin B) from the ascidian Aplidium albicans and dolastatin 16, which was initially isolated from the sea hare Dolabella auricularia, but has more recently been identified in the cyanobacteria, Lyngbya majuscula. Numerous cyclic peptides have been reported from marine sponges to date. However, apart from the eurypamides from Clathria (Thalysias) eurypa, published under its former name Microciona eurypa, no other peptides have been isolated from the genus Clathria or the family Microcionidae.

US Patent No. 6,025,466 discloses Cyclic Hepta-Peptide Derivative with antitumor activity which was isolated from Colonial Ascidians Lissoclinum Specie, collected from the Great Barrier Reef, Austarlia. US Patent No. 5,830,996 discloses Cyclic Peptide with antitumor activity which was isolated from Didedmnum cuculliferum and Polysyncraton lithostrortum specie, collected from Namenalala Island, Fiji.

Several compounds have been isolated from marine o rganisms that belong to the unique peptide structure of sponge cyclic peptides. These include the accidian isolated metabolites, ulithiacyclamide (Ireland, O, et al, J.J. Am. Chem. Soc. 1980, 102,

5688-5691 ); ulithiacyclamides B-G ( Fu, X ., et a l, J . N at. P rod. 1998, 61, 1547-1551; William D.E., et al J. Nat Prod. 1989, 52, 732-739); the marine actinomycete compound thicoraline (Romero, F., et al. J. Antibiot. 1997, 50, 734-737; Perez Baz, J. et al, J. Antibiot. 1997, 50, 738-741). While a large number of cyclic peptides have been reported from marine sponges, none have been reported for the cyclic hexapeptides, microcionamides A and B that contain a cysteine residue.

It is an object of the present invention to provide new compounds, which exhibit antitumor activity.

It is another object of the present invention to provide process of isolating purified cyclic hexapeptides, microcionamides A and B from the species Clathria (Thalysias) abietina.

The present invention provides cyclic hexapeptides, microcionamides A

and B having the formula

or pharmaceutically acceptable salts thereof.

This invention further provides a process for isolating new cyclic hexa-peptides, microcionamides A and B. The compounds of the present invention have been isolated from the Philippine marine sponge Clathria (Thalysias) abietina. These new hexapeptides are cyclized via a cystine moiety and have their C-terminus blocked by a 2-phenylethylenamine group. Their total structures, including absolute stereochemistry, were determined by a combination of standard spectral and chemical methods. Compound I was shown to slowly isomerize about the C-36/C-37 double bond to compound 2 when stored in DMSO.

The purified cyclic hexapeptides, microcionamides A and B of the present invention can be prepared by the following steps: a) collecting specimens of Clathria (Thalysias) abietna; b) extracting said specimens with methanol to yield a methanol extract; c) concentrating under vacuum the extract of step (b) to yield an orange brown gum; d) dissolving the orange brown gum in 90% methanol/10% water and partitioning with 100% hexane; e) adding water to the aqueous phase and partitioning the resulting 30% aqueous methanol fraction with 100% chloroform; f) drying under vacuum the chloroform fraction to yield a bioactive material; g) subjecting the bioactive chloroform soluble material to chromatography on a reverse phase flash column using a 10% stepwise gradient from 100% aqueous trifluoroacetic acid (TFA) (0.5%) to 100% methanol; h) collecting the 20% aqueous trifluoroacetic acid (TFA) (0.5%)/80% methanol fraction; i) purifying the fraction of step (h) by semi-preparative reverse phase high performance liquid chromatography using a linear gradient from 50% aqueous trifluoroacetic acid (TFA) (0.1%)/50% acetonitrile to 100% acetonitrile; j) collecting fractions corresponding to sharp peaks in the chromatogram representing pure cyclic hexapeptides, microcianamides A & B, as their TFA salts. The compounds of the present invention e xhibit s ignificant cytotoxicity a gainst human breast tumor cells lines and inhibitory activity against Mycobacterium tuberculosis. The present invention also provides composition which comprises compounds of the formula I or a pharmaceutical acceptable acid addition salt thereof, and a pharmaceutically acceptable carrier.

Pharmaceutical compositions of this invention include tablets, pills, capsules, granules, etc. or liquid formulations such as solutions, suspension or emulsion for oral, topical, parenteral or further mode of administration. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of a uxiliary substances s uch as wetting agent, b uffers, a nd the I ike can be added.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 : (+)-LRESMS Fragmentation Ions for Peptides 1-3.

DETAILED DESCRIPTION OF THE INVENTION

Isolation and Purification The crude MeOH extract of Clathria (Thalysias) abietina (Lamarck, 1814) (order

Poecilosclerida, family Microcionidae) was concentrated under vacuum then subjected to a solvent partitioning scheme resulting in hexanes and CHCI₃-soluble fractions. The CHCI₃-soluble material was chromatographed on a Cι₃ bonded silica flash column using an aqueous TFA/MeOH gradient. Further purification by C₁₈ HPLC using aqueous TFA and increasing amounts of CH₃CN afforded the TFA salts of microcionamides A ( 27.4 mg) and B ( 19.3 mg). Microcionamide A was isolated as an optically active white solid. The molecular formula of 1 was determined to be C₄₃H₇₀N₈O₇S₂ (13DBE) based on an [ +H]⁺ ion at m/z 875.48494 in the (+)-HRFABMS. The minor metabolite, microcionamide B was isolated as a stable, optically active white solid. The molecular formula C₄₃H₇oN₈O S₂ (13 DBE) was determined by interpretation of the [M+H]⁺ ion at m/z 875.48838 Δ-0.3 ppm) in the (+)-HRFABMS.

Mass Spectometry, NMR and Other Chemical Analysis

Microcionamide A (1) was isolated as an optically active white solid. The molecular formula of 1 was determined to be C ₃H₇₀N₈O₇S₂ based on an [M+H]⁺ ion at m/z 875.48494 in the (+)-HRFABMS. An aromatic chromophore [λ_maχ 284 nm (ε 16 000)] and amide group(s) (v_max 1650 cm^"1) were evident after analysis of the UV and IR spectra, respectively. A positive ninhydrin reaction suggested the presence of an amino moiety. The ¹³C NMR spectrum (Table 1) displayed signals for 41 unique carbons and the DEPT experiment indicated that 1 contained 10 methyls, 6 methylenes, and 17 methine carbons. Seven of the 8 quaternary carbons resonated in the 169-175 ppm region and were consistent for amide moieties. Salient features of the ¹H NMR spectrum of 1 included three aromatic signals at D 7.26 (2H, d, J = 8.0 Hz), 7.22 (2H, dd, J = 8.0, 7.5 Hz) and 7.11 (1 H, dd, J = 7.5, 7.5 Hz) that were assigned to a monosubstituted phenyl ring, two olefinic resonances at D7.35 (1H, d, J = 14.5 Hz) and 6.38 (1 H, d , J = 1 4.5 H z) which belonged to a n i solated trans e thylene m oiety, and seven signals [D3.70 (1 H, d, J = 5.0 Hz), 4.79 (1 H, m), 4.30 (1 H, d, J = 7.0 Hz), 4.13 (1 H, d, J = 8.5 Hz), 4.07 (1 H, d, J = 6.5 Hz), 4.02 (1 H, d, J = 5.5 Hz), 4.60 (1 H, dd, J = 9.0, 4.5 Hz)] that were indicative of α-proton resonances for amino acids. The upfield region of the ¹H NMR spectrum contained a multitude of signals that integrated for 47 protons. A gCOSY experiment allowed several amino acid units to be delineated, which included four isoleucines, one valine and one cystine or two cysteine residues. The gHSQC spectra enabled all the proton signals to be assigned to their directly attached carbons. Several of the above mentioned partial structures were connected using gHMBC data. These included the linkage of the isolated trans ethylene system to the phenyl ring based on three-bond HMBC correlations from D 7.35 (H-36) to the phenyl quaternary carbon at 137.6 (C-38) and from D 6.38 <H-37) to the aromatic methine carbons at 126.6 (C-39). The olefinic proton at D 7.35 (H-36) also showed a ³J_CH correlation to the amide carbonyl at 169.4 ppm and correl ations to this particular carbon were also observed from both the α (H-33) and β (H-34a/H-34b) protons that constituted one of the cysteine residues. The remaining cysteine moiety was linked to the valine residue since both protons [D 3.70 (H-4) and 4.79 CH-6)] shared an HMBC correlation to the amide carbon at 169.6 ppm. The valine-substi tuted cysteine was also attached to an isoleucine based on an HMBC correlation from both α protons [D 4.30 (H-9) and 4.79 (H-6)] to the carbonyl at 172.0 ppm (C-8). Unfortunately, due to insufficient HMBC correlations and crowding in the carbonyl region, no further connectivities across the remaining amide bonds could be made. Analysis of the ROESY spectrum did not provide any extra linkage information. The molecular formula of 1 indicated that microcionamide A contained 13 double bond equivalents (DBE) and with the currently assigned substructures all but one DBE was accounted for, this data established a further ring system that could only be formed by linkage of the two cysteine amino acids to form a cystine. At this stage we could not definitely say whether an isoleucine or valine residue constituted the Λ/-terminus. Edman degradation of 1 followed by sequence analysis revealed that valine was the Λ/-terminal amino acid. This assignment was supported by (+)-LRESMS analysi s of 1 that showed fragmentation ions at /z 802 and 773 (Figure 1). Although definitive positioning for each of the three remaining isoleucine residues in relation to each other could not be determined, by default they h ad to be inserted between C -14 a nd C-33. H ence the planar hexapeptide structure 1 was assigned to microcionamide A. The absolute stereochemistry for the amino acids in 1 was determined by acid hydrolysis of the peptide followed by treatment of the hydrolysate with Marfey's; reagent.¹⁴ Analysis of the mixture of FDAA derivatives by HPLC, using retention tim&s and co-injections with standards revealed the presence of only L-isoleucine and l_-valine. The absolute stereochemistry for the cystine residue was also determined using Marfey's method following the desulfurization of 1 using Raney Ni in refluxing MeOH.^15,16 The linear peptide 36,37-dihydro-desthiomicrocionamide A (3), was shown to contain only L- alanine, hence the cystine system of 1 was also assigned L absolute stereochemistry. No attempt was made to fully characterize 36,37-dihydro-desthiomicrocionamide A (3) due to the small amount of material available, however linear peptide 3 was analyzed by (÷)-LRESMS to confirm reaction success. Analysis of the MS data for 3 showed specific fragmentation ions that provided further proof of the amino acid sequence for 1 (Figure 1). The minor metabolite, microcionamide B (2) was isolated as a stable, optically active white solid. The molecular formula C₄₃H₇₀N₈O₇S₂ (13 DBE) was determined by interpretation of the [M+H]⁺ ion at m/z 875.48838Δ-0.3 ppm) in the (+)-HRFABMS. This data indicated that microcionamide B was an isomer of 1. A positive ninhydrin reaction confirmed the presence of a free Λ/-terminal amino acid. Comparison of the ¹³C NMR data of 2 (Table 2) with 1 (Table 1) showed very few chemical shift discrepancies (< 3.0 ppm). Likewise, the ¹H NMR spectrum for 2 showed the presence of an aromatic ring, an ethylene group and six amino acids units. Indeed, a gCOSY experiment in conjunction with the gHMBC data established the same number and type of amino acid units in 2 as had been identified in 1. Hence four isoleucines, one valine and one cystine residue were attributed to 2. A few notable differences were observed in the ¹H NMR spectrum of 2. These included the chemical shifts and the magnitude (10.0 Hz) of the coupling constant for the ethylene protons, indicating that the C-36/C-37 double bond geometry was cis in 2, plus a broad and complex set of multiplets in the aromatic region that appeared to display non first order characteristics. These aromatic signals were assigned to the phenyl system protons (H-39-H-41) following gHSQC and gHMBC analysis. Linkages for most of the amino acids, ethylene and phenyl systems of 2 were established based on gHMBC analysis. However, in a similar manner to 1, not all of the amide connectivities could be assigned for 2. (+)-LRESMS analysis (Figure 1) and Edman degradation of microcionamide B followed by sequence analysis confirmed that valine was also the Λ/-terminal amino acid in this peptide. Although definitive positioning of each of the three remaining isoleucine residues in relation to each other could not be determined, by default they had to be inserted between C-14 and C-33. Hence the planar structure 2, which was the cis isomer of 1, was assigned to microcionamide B. The absolute stereochemistry for each of the amino acids in microcionamide B (2) was determined using identical methods to those used for 1.¹⁴ L- isoleucine, L-valine and L-cystine were assigned to the constituent amino acids of 2. Biological activity

Microcionamides A and B showed significant cytotoxicity towards human breast tumor cell lines MCF-7 and SKBR-3. Microcionamide A was active against MCF-7 and SKBR-3 cells with IC₅₀ values of 125 and 98 nM, respectively. Microcionamide B displayed similar activity against MCF-7 and SKBR-3 cells with IC₅₀ values of 177 and 172 nM, respectively. These IC50 data were comparable with those of the positive control, doxorubicin (MCF-7, 257 nM; SKBR-3, 33 nM). Furthermore, both compounds were shown to induce apoptosis within 24 h in MCF-7 cells at 5.7 μM. Morphological investigations of the MCF-7 cells 24 hours after drug treatment showed the hallmarks of apoptosis such as cytoplasmic membrane blebbing, cytoplasmic condensation and shrinkage, loss of cell-to-cell contact and formation of membrane bound vesicles(Loo, D. T., et al, Methods Cell Biol. 1998, 57, 251-264). Extensive DNA fragmentation, which is also a significant biochemical marker of apoptotic cells (Robertd, R. Apoptosis in Toxicology: ist ed. Published 1999) was detected in a TUNEL assay (Gratzner, H. G. Science 1982, 218, 474-475) and by Hoescht staining (Goodell, M.A., et al, Exp. Med. 1996, 183, 1797-1806) of peptide treated MCF-7 and SKBR-3 cells, respectively.

The cyclic hexapeptides 1 and 2 were also tested for anti-tuberculosis activity using the microplate alamar blue assay (MABA), [Collins, L.A., et al, Antimicrob. Aegnts Chemother. 1997, 41 , 1004-1009] which uses the avirulent strain Mycobacterium tuberculosis H₃₇Ra. The H₃ Ra strain has been shown to display very similar drug susceptibility profiles as the virulent strain H₃₇Rv. Microcionamides A and B were both shown to display MIC values of 5.7 μM towards M. tuberculosis H₃₇Ra, as compared to the positive control, rifampicin (MIC 1.52 nM).

EXAMPLES

Animal Material. A specimen of Clathria (Thalysias) abietina (190 g wet weig t) was collected i n J uly 1998 using S CUBA (-10 m) at Tigtabon I sland i n Zamboanga,

Southern Mindanao, Philippines. A small portion of the sponge was immediately steeped in MeOH as the voucher specimen, while the rest of the material was frozen. A taxonomic voucher specimen PDZ-|98-10-138 is maintained at the University of Utah, Department of Medicinal Chemistry, Salt Lake City, Utah, USA.

Extraction and Isolation. The MeOH extract from Clathria (Thalysias) abietina (190 g wet weight) was concentrated under vacuum to yield an orange-brown gum.

This material was dissolved in 90% MeOH/10% H₂O (300 mL) and partitioned with

100% hexanes (3 x 300 mL). H₂O (128 mL) was added to the aqueous phase and the resulting 30% aqueous MeOH fraction was partitioned with 100% CHCI₃ (3 x 300 mL).

The hexanes and CHCI₃ fractions were evaporated to dryness under reduced pressure and yielded 361 mg and 690 mg of material, respectively. The bioactive CHCI₃-soluble material was chromatographed on a Cis bonded silica flash column using a 10% stepwise gradient from 100% aqueous TFA (0.5%) to 100% MeOH. The 20% aqueous

TFA (0.5%)/80% MeOH fraction ( 64 m g) was further purified by semipreparative Cι₈

HPLC using a linear gradient from 50% aqueous TFA (0.1%)/50% CH₃CN to 100% CH₃CN in 30 minutes at a flowrate of 1.0 mL/min. This afforded pure microcionamides

A (1 , 27.4 mg, t_R = 28.3 min) and B ( 2,19.3 mg, t_R = 31.1 min) as their TFA salts.

Microcionamide A : stable white solid; [α]_D -36.5° (c 0.273, MeOH); UV (MeOH) λ_max 206 (sh, ε 20 000), 220 (sh, ε 12 000), 284 nm (ε 16 000); IR v_max (NaCI) 3450-3100, 3073, 2964, 2928, 2873, 1650, 1530, 1205, 1185, 1138, 1027, 957, 840, 805, 723, 668 cm^"1; For ¹H and ¹³C NMR data see Table 1; (+)-LRESMS m/z (rel. int.) 756 (12), 773 (37), 802 (5), 875 (100), 897 (20); (+)-LRFABMS m/z (rel. int.) 72 (25), 86 ( 100), 756 ( 10), 773 (5), 875 ( 45); ( +)-HRFABMS m/z 875.48494 ( C₄₃H₇₁ N₈O₇S₂ [M+H]⁺ requires 875.48871 ).

Microcionamide B : stable white solid; [α]_D -40.3° (c 0.327, MeOH); UV (MeOH) λ_maχ 208 (sh, ε 23 000), 222 (sh, ε 15 000), 274 nm (ε 14 000); IR v_maχ (NaCI) 3450-3100, 3073, 2964, 2928, 2873, 1658, 1512, 1203, 1185, 1138, 1027, 846, 801 , 724, 688 cm^"1; For ¹H and ¹³C NMR data see Table 2; (+)-LRESMS tτ?/z (rel. int.) 756 (2), 773 (20), 802 (5), 875 (100), 897 (20); (+)-LRFABMS m/z (rel. int.) 72 (20), 86 (100), 756 (40), 773 (30), 875 (20); (+)-HRFABMS m/z 875.48838 (C₄₃H₇ι N₈O₇S₂ [M+H]⁺ requires 875.48871). Acid Hydrolysis and Stereochemical Determination of Microcionamides A and B.

Each peptide (0.5 mg) was dissolved in 6N HCI (1.0 mL) and heated in an Ar flushed sealed vial at 105 °C for 16 h. The resulting hydrolysate was lyophilized, dissolved in H₂O (25 μL) and 1 N NaHCO₃ (208 μL) then derivatized with 1-fluoro-2,4- dinitrophenyl-5-L-alanine amide (FDAA)¹⁴ (6.2 mg) in acetone (620 μL) at 40 °C for 1 h.

The reaction mixture was neutralized with 2N H CI (104 μL) then analyzed by H PLC using a P henomenex Luna C ι₈(2) column with a linear gradient of triethylammonium phosphate (50 mM, pH 3.0)/MeCN from 90:10 to 60:40 in 40 min, then held at 60:40 for 10 min at a flowrate or 1.0 mL/min. Based on HPLC retention times and co-injection with standard a mino acid derivatives, the h ydrolysate of b oth 1 a nd 2 was s hown to contain L-isoleucine and L-valine. Although we clearly established that all isoleucine residues had L absolute stereochemistry the HPLC conditions were unable to distinguish between L-isoleucine and L-a//o-isoleucine standards due to co-elution.

Desulfurization of Microcionamides A and B. Raney 2800 nickel (50% slurry in H₂O; 3.4 mg, 57 μmol) was added to each cyclic peptide (1.0 mg, 1.1 μmol) in MeOH (1 mL). The resulting black suspension was heated in a sealed vial at 85 °C for 2 h.¹⁵'¹⁶ Upon cooling, the solution was purified on a C₁₈ SPE cartridge using 90:10 MeOH/0.1 % aqueous TFA as eluant. This yielded the pure TFA salt of 36,37-dihydro- desthiomicrocionamide (3, 0.5 mg, 54% yield) for both reactions.

36,37-Dihydro-desthiomicrocionamide (3): stable white s olid; (+)-LRESMS m/z ( rel. int.) 284 (2), 397 (7), 510 (12), 623 (12), 694 (20), 734 (20), 8 16 (23), 838 (100).

Acid Hydrolysis and Stereochemical Determination of 36,37-Dihydro- desthiomicrocionamide.

The linear peptide (3, 0.5 mg) was subjected to the same hydrolysis and derivatization conditions as those used for the cyclic peptides. Based on HPLC retention times and co-injection with standard amino acid derivatives, the hydrolysate of 3 was shown to contain L-isoleucine, L-valine and L-alanine.

Culture of Breast Cancer Cell Lines. MCF-7 and SKBR-3 human breast tumor cells were obtained from the American Type Culture Collection (Rockville, MD). The MCF-7 cells were grown as a monolayer in minimum essential medium (MEM) containing 10% fetal bovine serum (FBS), 1X of antibiotic-antimycotic penicillin- streptomycin and fungizone (PSF) and 0.1 mM non-essential amino acids (NEAA). SKBR-3 cells were grown in McCoy's 5A media containing 1 0% FBS and 1X of antibiotic-antimycotic PSF. Cell cultures were maintained at 37 °C in a humidified 5% CO₂ atmosphere. All reagents were from Gibco Laboratories, Grand Island, NY, except for PBS, which was from Sigma Chemical Co., St. Louis, MO.

Tumor Cytotoxicity Assay. Cytotoxicity towards MCF-7 and SKBR-3 cells was assessed in an MTT-microtiter plate tetrazolium cytotoxicity assay. This assay was originally described by Mossman (Mossman, T.J. Immunol. Methods 1983, 65, 55-63) and has since been modified by others. (Denizot, F.; Lang, R. J. Immunol. Metods 1986, 271-277) MCF-7 and SKBR-3 (20,000 cells/well) were seeded in 200 μL of growth medium in 96-well microtiter plates (Costar) and allowed to attach for 24 h. Cells were treated with the peptides in a 5-fold serial d ilution s tarting from 28.6 μM. The peptides were tested in quadruplicate and were solubilized in 100% DMSO with a final DMSO concentration of 1.0 % or less in each well. The treated cells were incubated for 72 h. The media was removed after 72 h and 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2 --tetrazolium bromide (MTT) solution (15 μL, 5 mg/mL in PBS, pH 7.4) was added followed by a 3 h incubation. MTT is reduced by viable cells to a purple formazan product. Following removal of media the formazan product was solubilized by addition of DMSO (100 μL) to each well. The absorbance for each well at 570 nm was m easured using an S LT Model Tecan S pectra I II ELISA plate reader. T he cell growth in the DMSO control wells was used to determine the zero inhibition growth level for each experiment. The peptides were tested in quadruplicate and were solubilized in 100% DMSO with a final DMSO concentration of 1.0 % or less in each well. Doxorubicin was used as the positive control for both cell lines. Average absorbance for each set of quadruplicate drug-treated wells was compared to the average absorbance of the control wells to determine the percentage of growth inhibition (fractional survival) at a particular drug dose. The fractional survival values were used to compute the IC₅₀ using ICPIN computer software (version 2.01). Apoptosis Assays. MCF-7 human breast tumor cells were plated in a 96-well microtiter plate in triplicate at a density of 20,000 cells/well in 200 μL media. Cells were incubated overnight at 37 °C at 5% CO₂ then treated with the peptides at 5.7 μM for 24 h. After treatment, cells were subjected to a terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay. SKBR-3 human breast tumor cells, plated at the same cell density, were subjected to nuclear staining with Hoechst 33342 fluorescent dye after treatment with the peptides at 2.9 μM for 12 h.

Morphological Investigations. Cellular morphology of the MCF-7 peptide treated cells (5.7 μM) was examined using an inverted Nikon microscope equipped with a Nikon SLR camera. Cell images were captured using Kodak film ASA 400.

TUNEL Assay. DNA fragmentation was investigated using a fluorescein cell death detection kit (Boeringer-Mannheim), also known as the TUNEL assay kit. After drug treatment at 5.7 μM for 24 h, the MCF-7 cells were fixed with fresh paraformaldehyde solution (4% in PBS, pH 7.4) for 30 min at rt then rinsed with PBS [with 1% bovine serum albumin (BSA)]. Cells were incubated with permeabilization solution (0.1 % Triton X-100, 0.1 % sodium citrate) at 4 °C for 2 min then washed twice with PBS (with 1% BSA). TUNEL reaction mixture (50 μL) containing: terminal deoxynucleotidyl transferase enzyme and fluorescein-dUTP was added to the cells and the mixture was incubated at 37 °C in the dark for 1 h, then rinsed three times with PBS (with 1% BSA). Cells were viewed using a Nikon fluorescence microscope equipped with a Nikon SLR camera. Cell images were captured using Kodak film ASA 400.

Nuclear Staining. SKBR-3 human breast tumor cells were seeded at a concentration of 20,000 cells per well in 200 μL media. After overnight incubation at 37

°C with 5% CO₂, each well was treated with 1 μL of 0.57 mM stock solutions of 1 and 2, for a final concentration of 2.9 μM per well. The cultures were incubated with the metabolite for 12 h prior to nuclear staining. Spent media was removed after incubation and replenished with 200 μL of culture medium per well. Then, 1 μL of a 2 mg/mL prepared Hoechst 33342 (Sigma) stock solution was added to each well to give a final concentration of 10 μg/mL. Cells were incubated for 90 min at 27 ° C with 5% CO₂. After washing twice with PBS, 100 μL of PBS was added to each well and the plates stored at 4 °C. The cells were observed under a fluorescence microscope at 480 nm and ordinary light. Photographs were taken at a magnification of 400*.

Mycobacterium Strain and Culture Conditions. Mycobacterium tuberculosis H₃₇Ra, which was a gift from Dr. Scott Franzblau (Institute for Tuberculosis Research, University of Illinois, Chicago, USA), was cultured as described by Collins and Franzblau.²² The following media was prepared for the culturing: 1 ) OADC- supplemented Middlebrook 7H11 agar (100 mL): 1.9 g 7H11 agar powder (Difco, Detroit, Ml) and 0.5 mL glycerol (Fisher Chemicals, Springfield, NJ, USA) were dissolved in 90 mL distilled H₂O, autoclaved for 15 min at 15 psi, allowed to cool, then 10 mL OADC enrichment medium was added. 2) OADC-supplemented Middlebrook 7H9 broth (100 mL): 0.47 g 7H9 broth powder (Difco), 0.1 g casitone (Difco), 0.5 mL glycerol (Fisher) were dissolved in 90 mL distilled water, autoclaved for 15 min at 15 psi, allowed to cool, then 10 mL OADC enrichment medium was added. Twenty μL Tween 80 (Sigma, St. Louis, MO) was added to the culture broth before autoclaving. 3) OADC enrichment media (100 mL): 0.05 g (50 μL) oleic acid (Mallinckrodt Baker, Inc. Paris, Kentucky, USA), 5.0 g BSA fraction V (Sigma), 2.0 g dextrose (Difco), 0.004 g beef catalase (Sigma), and 0.85 g NaCI (Ajax Chemicals), were dissolved in 100 mL distilled water, filter-sterilized and dispensed in 10 mL aliquots. 4) 25% Tween 80 for MABA (20 mL) : 5.0 mL Tween 80 (Sigma) was added to 15.0 mL 7H9 broth, filter- sterilized and 1.0 mL dispensed into 1.5 mL microfuge tubes. M. tuberculosis H₃₇Ra was grown on OADC-supplemented Middlebrook 7H11 agar slants at 35 °C. Inoculum was transferred to OADC-supplemented Middlebrook 7H9 culture broth and grown for 2-3 weeks at 35 °C up to a density of 1-3 x 10 ⁷ cfu/mL. The broth culture was diluted 1 :50 with OADC-supplemented Middlebrook 7H9 broth. Anti-tuberculosis Assay. The MABA assay was performed as described in Collins and Franzblau. Using a 96-well microtiter plate, the plate layout was arranged such that perimeter wells contained only color control set-ups (highest sample dilution without inoculum) or sterile H₂O to prevent dehydration in wells containing samples and inoculum. The final assay volume in each well was 200 μL. Ten μL of the sample (10 mg/mL in DMSO) was placed in wells for color controls. For the test wells, 10 μL of the sample was added for a high dose (e.g., 500 mg /mL) and 1 μL for a low dose (e.g., 50 mg/mL). One hundred ninety μL of 7H9 broth was placed in the color control wells, 90 μL in the high-dose wells and 99 μL in the low-dose wells. DMSO, the negative control, was added in two 10-fold dilutions. Rifampicin, the positive control, was added in at least seven 2-fold dilutions within range of its MIC. Using an 8-channel pipettor, 100 μL of inoculum (H₃₇Ra) was dispensed into all test wells except for color controls. Plates were sealed with parafilm and incubated at 35 °C in a 5% CO₂ incubator for 5 days. Using a combitip and a step pipettor, 10 μL of 25% Tween-80 and 20 μL of Alamar Blue (Alamar B iosciences, Sacramento, CA) was added to the wells. Plates were resealed and incubated overnight. Color change was observed the next day. A blue color in the well was interpreted as no growth or a positive result, and a pink color in the well as growth or a negative result.

Table 1. NMR Data for Microcionamide A (1).^a

Assignments for the four contiguous isoleucine residues may be interchangeable. positio ¹³C (δ) ¹H (δ, mult., J in Hz) COSY HMBC ROESY n 1 17.8 0.98 (d, 7.0) 2 2,3,4 2,4 2 31.6 2.16 (dqq, 5.O. ,7.0, 1,3,4 1,3,4,5 1,3,4 7.0) 3 18.9 1.01 (d, 7.0) 2 1,2,4 2,4 4 59.5 3.70 (d, 5.0) 2 1,2,3,5 1,2,3,6 5 169.6 6 54.5 4.79 (m) 7a, 7b 5,7,8 4,7a 7a 41.4 3.04 (dd, 14.0, ,9.0) 6,7b 6,8 6 7b 3.30 (m) 6,7a 8 8 172.0 9 60.0 4.30 (d, 7.0) 10 8, 10, 11, 12, 10, 11, 12a, 12b 14 10 37.7 1.95 (m) 9, 11, 12a, 9 9, 11, 12a, 12b 12b 11 16.2 0.92 (d, 7.0) 10 9, 10, 12 9, 10, 12a, 12b 12a 25.8 1.20 (m) 10,12b, 13 13 9, 10, 11, 12b, 13 12b 1.51 (m) 10, 12a, 13 9, 10, 11, 12a, 13 13 11.8 0.88 (t, 7.0) 12a, 12b 11, 12 12a, 12b 14 174.5⁶ 15 60.2 4.13 (d, 8.5) 16 16, 17, 18,20 16, 17, 18a, 18b 16 37.1 1.95 (m) 15, 17,18a 15 15, 17, 18a, 18b 17 16.1 0.92 (d, 7.0) 16 15, 16, 18 15, 16, 18a, 18b 18a 26.5 1.17 (m) 16, 18b, 19 19 15, 16, 17, 18b, 19 18b 1.56 (m) 18a, 19 15, 16, 17, 18a, 19 19 11.5 0.88 (t, 7.0) 18a, 18b 17, 18 18a, 18b 20 173.5 21 61.5 4.07 (d, 6.5) 22 22, 23, 24, 26 22, 23, 24a, 24b 22 37.4 2.02 (m) 21,23,24a 21 21 , 23, 24a, 24b 23 16.3° 0.97 (d, 6.5) 22 21,22,24 21,22, 24a, 24b 24a 26.4 1.20 (m) 22, 24b, 25 25 21,22, 24b, 25 24b 1.56 (m) 24a, 25 21,22, 24a, 25 25 11.5^d 0.87 (t, 7.5) 24a, 24b 22,24 24a, 24b 26 174.6^b 27 61.6 4.02 (d, 5.5) 28 29, 30, 32 28, 29, 30a, 30b 28 37.4 2.00 (m) 27, 29, 30a 27 27, 29, 30a, 30b 29 16.4^C 0.95 (d, 7.0) 28 27, 28, 30 27, 28, 30a, 30b 30a 26.7 1.23 (m) 28, 30b, 31 31 27, 28, 29, 30b, 31 30b 1.56 (m) 30a, 31 27, 28, 29, 30a, 31 31 11.2' 0.87 (t, 7.5) 30a, 30b 28,29 30a, 30b 32 173.7 33 55.0 4.60 (dd, 9.0, 4.5) 34a, 34b 35 34a 34a 42.7 3.20 (dd, 13.5 ,9.0) 33, 34b 33,35 33 34b 3.27 (dd, 13.5 ,4.5) 33, 34a 35 35 169.4 36 123.2 7.35 (d, 14.5) 37 35, 37, 38 39 37 116.4 6.38 (d, 14.5) 36 36,39 39 38 137.6 39 126.6 7.26 (d, 8.0) 40 37, 39, 41 36, 37, 40 40 129.7 7.22 (dd, 8.0, 7.5) 39,41 38,40 39,41 41 127.8 7.11 (dd, 7.5, 7.5) 40 39 40 Spectra were recorded in CD₃OD at 26 °C. b- Signals are interchangeable. Table 2. NMR Data for Microcionamide B (2).^a

Assignments for the four contiguous isoleucine residues may be interchangeable. position ¹³C (δ) ¹H(δ, mult.,Ji nHz) COSY HMBC ROESY 1 17.8 0.97 (d, 7.0) 2 2,3,4 2,4 2 31.6 2.13 (dqq, 5.0, 7.0, 7.0) 1,3,4 1,3,4,5 1,3,4 3 18.9 0.99 (d, 7.0) 2 1,2,4 2,4 4 59.5 3.67 (d, 5.0) 2 1,2,3,5 1,2,3 5 169.6 6 54.5 4.80 (m) 7a, 7b 5,7,8 7a 41.7 3.05 (dd, 14.0, 9.0) 6,7b 6,8 7b 3.26 (m) 6,7a 8 172.1 9 60.2 4.26 (d, 7.0) 10 8, 10, 11, 12, 14 10, 11, 12a 10 37.8 1.90 (m) 9,11,12a 9,11,12a, 12b 11 16.1 0.93 (d, 7.0) 10 9, 10, 12 9, 10, 12a, 12b 12a 26.2⁶ 1.19 (m) 10, 12b, 13 13 9, 10, 11,12b, 13 12b 1.50 (m) 12a, 13 9, 10, 11,12a, 13 13 11.4 0.90 (t, 7.0) 12a, 12b 11, 12 12a, 12b 14 174.2° 15 59.5 4.10 (d, 8.5) 16 16, 17,18,20 16, 17, 18a, 18b 16 36.8 1.85 (m) 15, 17, 18a 15, 17, 18a, 18b 17 15.9 0.77 (d, 7.0) 16 15, 16,18 15, 16, 18a, 18b 18a 26.2 1.10 (m) 16, 18b, 19 19 15, 16, 17, 18b, 19 18b 1.50 (m) 18a, 19 15, 16, 17, 18a, 19 19 11.1 0.81 (t, 7.0) 18a, 18b 17, 18 18a, 18b 20 173.9 21 61.5 4.00 (d, 7.0) 22 22, 23, 26 22, 23, 24a, 24b 22 37.0 2.10 (m) 21,23,24a 21,23, 24a, 24b 23 16.3 0.95 (d, 7.0) 22 21,22,24 21,22, 24a, 24b 24a 26.4" 1.14 (m) 22, 24b, 25 25 21,22, 24b, 25 24b 1.50 (m) 24a, 25 21,22, 24a, 25 25 11.6 0.87 (t, 7.0) 24a, 24b 22,24 24a, 24b 26 174.1° 27 61.3 3.92 (d, 6.5) 28 29, 30, 32 28, 29, 30a, 30b 28 37.3 2.00 (m) 27, 29, 30a 27, 29, 30a, 30b 29 16.5 0.91 (d, 6.0) 28 27, 28, 30 27, 28, 30a, 30b 30a 25.9 1.14 (m) 28, 30b, 31 31 27, 28, 29, 30b, 31 30b 1.48 (m) 30a, 31 27, 28, 29, 30a, 31 31 11.6 0.87 (t, 7.0) 30a, 30b 28,29 30a, 30b 32 173.5 33 54.6 4.72 (dd, 9.0, i 5.0) 34a, 34b 34,35 34a 34a 42.2 3.10 (dd, 14.0, 9.0) 33, 34b 33,35 33 34b 3.26 (dd, 14.0, 5.0) 33, 34a 35 35 170.1 36 121.9 6.73 (d, 10.0) 37 35,38 37 37 114.2 5.81 (d, 10.0) 36 36,39 36,39 38 136.6 39 129.5 7.30 (brs) 37,41 37 40 129.9 7.31 (m) 41 38,40 41 128.1 7.19 (brm) 40 39 ' Spectra were recorded in CD₃OD at 26 °C ^: Signals are interchangeable.

Claims

Claims:

1. A cyclic hexapeptides, microcionamides A and B, having the formula

or pharmaceutically acceptable acid addition salt thereof.

2. A cyclic hexa-peptide according to claim 1 , wherein R is

3. A cyclic hexa-peptide according to claim 1 , wherein R is

. A process for preparing a purified cyclic hexa-peptide according to claim 1, 2 or comprising the steps of: a) collecting specimens of clathria (Thalysias) abietna; b) extracting said specimens with methanol to yield a methanol extract; c) concentrating under vacuum the extract of step (b) to yield an orange brown gum; d) dissolving the orange brown gum in 90% methanol/10% water and partitioning with 100% hexane; e) adding water to the aqueous phase and partitioning the resulting 30% aqueous methanol fraction with 100% chloroform; f) drying under vacuum the chloroform fraction to yield a bioactive material; g) subjecting the bioactive chloroform soluble material to chromatography on a reverse phase flash column using a 10% stepwise gradient from 100% aqueous trifluoroacetic acid (TFA) (0.5%) to 100% methanol; h) collecting the 20% aqueous trifluoroacetic acid (TFA) (0.5%)/80% methanol fraction; i) purifying the fraction of step (h) by semi-preparative reverse phase high performance liquid chromatography using a linear gradient from 50% aqueous trifluoroacetic acid (TFA) (0.1%)/50% acetonitrile to 100% acetonitrile; j) collecting fractions corresponding to sharp peaks in the chromatogram representing pure cyclic hexapeptides, microcianamides A & B, as their TFA salts.

5. Use of the cyclic hexapeptides according to claim 1 , 2 or 3 for the manufacture of a medicament for the treatment of breast cancer and tuberculosis.