WO1990005731A1

WO1990005731A1 - Cytotoxic macromolecules

Info

Publication number: WO1990005731A1
Application number: PCT/AU1989/000505
Authority: WO
Inventors: Clifford J. Hawkins; Diane J. Watters; Martin F. Lavin; David L. Parry; Elizabeth J. Mccaffrey
Original assignee: University Of Queensland
Priority date: 1988-11-24
Filing date: 1989-11-23
Publication date: 1990-05-31

Abstract

A number of cyclic peptides and macrocyclic ethers have been isolated and identified from the marine organisms Lissoclinum patella and Lissoclinum bistratum. These compounds exhibit cytotoxic activity and should be of use in the treatment of cancer and related diseases. The compounds should also be of use as a modulator for a cellular protein (PKC) for the detection and monitoring of cell growth and differentiation. The preferred compounds are lissoclinamide 7, bistratene A and bistratene B.

Description

TITLE: CYTOTOXIC MACROMOLECULES

TECHNICAL FIELD

THE PRESENT INVENTION relates to the isolation, identification and preparation of cytotoxic macromolecules and their application in therapy. In particular, the invention is directed to novel cyclic peptides and macrocyclic ethers which should be of use in the treatment of cancer and related diseases.

BACKGROUND ART

Chemotherapy is now a widely accepted form of cancer treatment, although many forms of cancer are still poorly controlled by drugs. In the search for new pharmaceuticals, it has been recognised that, as terrestrial and marine invertebrates do not have a thymus system for immunological protection, these life forms must have developed sophisticated chemical mechanisms for their protection and, therefore, such chemical protective agents should also prove useful in the treatment of a variety of human medical problems. Recent research has indeed confirmed that marine organisms are a valuable source of organic compounds with antineoplastic activity.

DISCLOSURE OF THE INVENTION

The present inventors have now isolated and identified a number of macrocyclic compounds from the marine organisms Lissoclinum patella and Lissoclinum bistratum which exhibit appreciable cytotoxic activity. In view of the in vitro and in vivo activity of these cyclic compounds, they should find use in the treatment of cancer and related diseases.

Thus, according to a first aspect of the present invention, there is provided a series of compounds- each compound being separably isolable - hereinafter referred to as trisoxazoline, patellamide D, lissoclinamide 4, lissoclinamide 5, lissoclinamide 7, lissoclinamide 8, bistratamide B, bistratamide A, bistratene A and bistratene B, the structures of which are depicted in FIG 1.

Trisoxazoline, bistratamide A, bistratamide B, bistratene A and bistratene B were all isolated from the aplousobranch ascidian Lissoclinum bistratum, and patellamide D, lissoclinamide 4, lissoclinamide 5, lissoclinamide 7 and lissoclinamide 8 were all isolated from the aplousobranch Lissoclinum patella. According to a second aspect of the present invention, there is provided a method for the isolation of the afore-defined series of compounds, said method comprising: 1) homogenising the cleaned ascidian with an alcohol/hydrocarbon solution;

2) filtering the homogenate;

3) extracting the filtrate with an aqueous solution;

4) extracting the aqueous layer with an organic solvent;

5) drying and then evaporating off the organic extract;

6) chromatographing the residue; and

7) isolating the individual compounds. Preferably, (1) the ascidian is homegenised with an alcohol/aromatic hydrocarbon solution, more preferably methanol/toluene solution; (2) the filtrate is extracted with sodium nitrate solution; and (3) the aqueous layer is extracted with chloroform.

The compounds of the present invention have been screened for in vitro and in vivo antitumour activity and positive results have been obtained. Thus the compounds should find a use in the control of cancer and similar diseases.

Preliminary investigations suggest that it is the overall structural conformation of each of these compounds which is the determinant of cytotoxic activity. Therefore, it is likely that derivatives of the afore-defined series of compounds will also exhibit antitumour activity. Thus, according to a third aspect of the present invention, there are provided compounds each with a similar cyclic skeleton to that of the afore-defined series of compounds but which are substituted by differing groups.

In this regard, it will be appreciated that the various compounds referred to above, and any of their derivatives, are chiral and the present invention relates both to the individual stereoisomers and to any mixtures thereof whether these mixtures include enantiomers and/or diastereoisomers.

These derivatives may be prepared by adapting standard procedures well known in the art. Of course, racemization of the compounds of the invention is possible and the present invention thus includes not only all the individual stereoisomers, but also all mixtures of such stereoisomers, including optically inactive racemic mixtures of enantiomers and optically active mixtures in which one enantiomer is present in excess relative to the other enantiomer, as well as mixtures of diastereoisomers. In view of the antineoplastic activity of the compounds of the present invention, according to a third aspect, there is provided a pharmaceutical composition containing one or more of the above-described compounds, or a non-toxic salt thereof, optionally in association with any carrier or diluent which is suitable for its administration.

A non-toxic salt, as used throughout this specification, denotes salts which do not produce undesired side effects when administered at effective dosage levels. Examples of non-toxic acid addition salts include the hydrohalic, sulphuric, nitric, phosphoric, citric, acetic and oxalic acid salts. Also, according to a fourth aspect of the present invention, there is provided a method of therapeutic treatment by the administration of an effective amount of a pharmaceutical composition as hereinbefore described.

The term "effective amount" is understood as meaning a sufficient amount to enable the control of cancer and similar diseases of the patient. However, it will be appreciated that, the doses used can very within wide limits, according to the disease to be treated, the response of the patient and the particular compound employed.

Initial investigations have ascertained that some of the above-described compounds exhibit the ability to activate the enzyme protein kinase C (PKC).

Thus, as a fifth aspect of the present invention, there is provided a method of detecting and monitoring cell growth and differentiation by using one or more of the above-described compounds as a modulator for cellular proteins, such as PKC.

DETAILED DESCRIPTION OF EMBODIMENTS

Specific details of the compounds of the present invention and the methods involved in this invention are illustrated by the following examples. In these examples, all temperatures are in degrees centigrade, and technical terms have the usual meaning in the art. Crude products can be purified by the means described herein, or by other means known in the art.

GENERAL

Structural identification of each compound of the invention was determined by two-dimensional NMR techniques and each compound was tested for cytotoxicity toward human fibroblast and tumour cell lines.

NMR SPECTROSCOPY

Spectra were obtained using a C-5 dual ¹H, ¹³C probe in a JEOL GX400 NMR spectrometer. Compounds were dissolved in deuterated chloroform (Merck) and chemical shifts were derived relative to tetramethylsilane (TMS). For the COSY 45 experiments the following parameters were used: Bistratamide A, spectral width 3076.9 Hzdata matrix 512 x 1024, scans 32, recycle time 3 s, with zero filling in the V₁ domain; bistratamide B, spectral width 3450.7 Hz, data matrix 512 x 2048, scans 32, recycle time 3.23 s with zero filling in the V₁ domain. Sine-bell apodization functions were used. For the heteronuclear correlated experiments the parameters were: bistratamide A, data matrix 128 x 4096, scans 592, recycle time 1.6 s; bistratamide B, data matrix 128 x 4096, scans 2000, recycle time 1.7 s; bistratene A, data matrix 128 x 4096, scans 320, recycle time 1.9 s. Fixed delays of ▲1 and ▲2 (^1/2J and ^1/4J, respectively) were set for J = 138 Hz. Both dimensions were zero filled and multiplied by a sine-bell function before transformation. For long-range ¹H-¹³C shift correlations in bistratene A, two experiments were used with ▲1 and ▲2 (^1/2J and ^1/3J, respectively) set for J = 10 Hz and J = 16.6 Hz.

MASS SPECTROSCOPY

A Kratos MS25 RFA instrument was used. For the FAB-MS an Iontech saddle-field FAB source was used with argon gas. Samples were dissolved in methanol at a concentration of 1 mg/mL and diluted 5 times with glycerol for FAB analysis. High-resolution electron- impact mass spectra (HREIMS) were recorded at 70 keV and a resolving power of 3000.

COLLECTION OF ANIMALS

Specimens of L. patella and L. bistratum were collected from Heron Island Reef on the Great Barrier Reef, Queensland, Australia. Animals were cleared of debris, cut into pieces about 2 x 2 cm and frozen at -20° within four hours of collection.

EXTRACTION OF COMPOUNDS

A typical sample size for extraction, 250g (wet weight) of frozen ascidian, was homogenized in a Waring Blendor with IL of a 3:1 methanol:toluene solution. The homogenate was filtered and the filtrate was extracted with 1M sodium nitrate solution (800 mL). The aqueous layer was extracted with chloroform (6 x 100 mL) and the chloroform dried over anhydrous sodium sulphate. The chloroform was evaporated to dryness yielding a brownish oil.

ACID HYDROLYSIS AND CHIRAL GAS CHROMATOGRAPHY

Peptides (0.3 μmol) were hydrolysed in 6 M HCl (1 mL) and mercaptoethane (100 μL) in vacuo for 24 h at 110°. Mercaptoethane was added to prevent oxidation of cysteine during hydrolysis. After evaporation to dryness, the amino acids were converted to their N- pentafluoropropionyl isopropyl esters and applied to a Chirasil-Val GC column for separation of D and L isomers.

A) TRISOXAZOLINE

A.i Isolation and Characterisation

Frozen L. bistratum was processed following the general extraction procedure described above to yield a dark green-brown oil. Chromatography of the oil on a preparative reverse phase C-18 column (Whatman ODS-3) yielded pure trisoxazoline detected by its absorbance at 210 nm.

Analysis of ¹H and ¹³C NMR spectra and FAB mass spectrometry [(M+H)⁺=547] showed it to be a symmetrical cyclic hexapeptide with the structure depicted in FIG 1.

A.ii Cytotoxicity

Cytotoxicity was examined in vitro using two cell lines - an SV40 transformed fibroblast cell line (MRC5CV1) and a transitional cell carcinoma of the bladder (T24). Cells were incubated for 1 hour with varying concentrations of trisoxazoline and after 5 days the incorporation of [methyl -³H] thymidine into DNA was determined, as a measure of cell viability. The results are shown in FIG.2.

The IC₅₀ value (concentration required to inhibit growth by 50%) was determined as 0.5μg/ml. Cell viability was also determined by measuring the colony formation in agar at various doses of trisoxazoline. From these experiments an IC₅₀ value of 1.2μg/ml was obtained, in good agreement with the value obtained from thymidine incorporation.

A.iii LD₅₀ determination Cytotoxicity in vivo was determined in Quackenbush mice at concentrations up to 200 mg/kg. Even at this highest concentration no mice died. A.iv In vivo antitumor properties

The assay used to assess the in vivo antitumour properties of a single concentration of trisoxazoline was the P-338 system in Balb/C mice as recommended by the Guidelines of the National Cancer Insititute.

Mice (22-24g) were weighed and randomly assorted into three groups. These groups were:-

A: CONTROL (no drug treatment)

B: POSITIVE CONTROL (treatment with 5- fluorouracil)

C: TEST (treatment with trisoxazoline)

Each mouse received 1x10⁶ P388 cells in phosphatebuffered saline by intraperitoneal injection. After 24 h mice were treated with either 5- fluorouracil (a single dose of 200mg/kg) or trisoxazoline (5mg/kg). Trisoxazoline treatment was repeated every second day, with the mice being weighed prior to injection. This was repeated until day 25. The parameters recorded were mean wgt. gain and mean survival time. A T/C index was calculated as follows: T/C (%)=mean survival time (treated) ÷ mean survival time (control) x 100.

The mean weight gains for the three groups were as follows:

CONTROL (no drug) 3.25 g.

FLUOROURACIL TREATED 0.5 g.

TEST (trisoxazoline) 2.75 g. The mean survival time of untreated controls was 9.5 days.

Results:

T/C for trisoxazoline 13/9.5x100 = 137%

T/C for 5-fluorouracil 25/9.5x100 = 263%

According to the NCI Guidelines a T/C index of 125 is considered significant.

B) PATELLAMIDE D, LISSOCLINAMIDES 4, 5, 7 and 8

B.i Isolation and Structure

A total of 7kg of L. patella was processed following the general extraction procedure described above to yield approximately 10g of crude extract. The crude extract was dissolved in methanol:water (77:23) and applied to a Whatman Partisil ODS-3 9mm x 500mm preparative HPLC column. Elution was effected by means of a concave gradient from 77 to 100% methanol over 120 min. and the absorbance of the eluate was monitored using a Waters 990 Photodiode Array detector. Fractions corresponding to the various peaks were pooled, evaporated to dryness, and subjected to further purification, either by rechromatography on the same column under isocratic conditions or by chromatography on Sephadex LH-20 using methanol: Water (80:20).

The structures of the pure compounds were determined by a combination of HREIMS, acid hydrolysis followed by chiral gas chromatography and two-dimensional NMR techniques. Patellamide D

Patellamide D (C₃₈H₄₈N₈O₆S₂) is very similar in structure to the known patellamide B except that a leucine is replaced by an isoleucine at position 6. HREI mass spectroscopy gave a molecular weight of 776.3138 (calc'd 776.3128). Positive ion FAB-MS gave (M+H)⁺ 777. The structure was unambiguously assigned from detailed analysis of ¹H and ¹³C NMR spectra and 2D COSY 45 and ¹H - ¹³C shift correlation experiments.

Lissoclinamides 4, 5, 7 and 8 All four peptides are cyclic heptapeptides which have the same sequence of amino acids around the ring and differ from one another only in their stereochemistry or the number of thiazole and thiazoline rings.

Accurate mass measurements by HREIMS gave a molecular ion mass of 741.2830 for lissoclinamide 4 (C₃₈H₄₁N₇O₅S₂) (calc'd 741.2767) and 739.2636 for lissoclinamide 5 (C₃₈H₄₁N₇O₅S₂) (calc'd 739.2610). Positive ion FAB-MS gave (M+H)⁺ 742 and 740 respectively. The structures were identified from analysis of ¹H, ¹³C, and 2D COSY 45 NMR spectra, and COLOC experiments. HREIMS of lissoclinamide 8 (C₃₈H₄₃N₇O₅S₂) gave a molecular ion mass of 741.2785 (calc'd 741.2767) and 743.2935 (calc'd 743.2927) for lissoclinamide 7 (C₃₈H₄₅N₇O₅S₂). Chiral gas chromatography of the N-pentafluoropropionyl isopropyl esters and analysis of the long range ¹H-¹³C couplings in a COLOC experiment in combination with ¹H-¹H couplings detected by a 2D COSY 45 experiment enabled the respective structures to be determined.

For lissoclinamide 8, the valine residue is at position 31 - the same sequence as occurs in lissoclinamide 4. Therefore, the only difference between lissoclinamides 4 and 8 must reside in the stereochemistry of one or two of the amino acids. The D configuration has been tentatively assigned for lissoclinamide 8.

Lissoclinamides 7 and 8 comprised 0.4% and 0.5% of the dried crude extract respectively. B.ii Cytotoxicity

Patellamide D, lissoclinamides 4 and 5

Two cell lines were used: MRC5CV1 (SV40- transformed human fibroblasts) as control; and T24 (transitional cell carcinoma of the bladder) as a tumor line. Cells were maintained in RPMI-1640 medium (Commonwealth Serum Laboratories) containing 10% foetal calf serum at 37° in a humidified atmosphere of 5% CO₂ in air. Cells were plated at a density of 5 x 10³ cells/16 mm well, 24 h prior to cytotoxicity testing. Initial screening for cytotoxicity was performed by microscopic assessment of cultures 5-7 days after exposure to various concentrations of the compounds for 1 h. Compounds that showed cytotoxicity at this level were studied in more detail by examining their effects on the incorporation of [methyl-³H] thymidine into DNA. Cells were exposed to various concentrations of the compounds for 1 h, and after 5 days were labelled with [methyl-³H] thymidine 10 μCi/mL, 40 Ci/mmol, Amersham) for 4 h, after which they were extracted with cold 10% trichloroacetic acid (TCA). The TCA-insoluble material was collected onto glass fibre filters, and washed with cold 5% TCA followed by 100% ethanol prior to liquid scintillation counting. Each experiment was performed three times with duplicate assays at each point. The statistical significance of differences between mean values was determined using the Student's T-test (FIG 3).

Lissoclinamides 7 and 8

The cytotoxicities of these two compounds were examined in vitro using transitional bladder carcinoma cells (T24), SV40 transformed fibroblasts (MRC5CV1) and normal peripheral blood lymphocytes. Cell viability assays were performed using a colorimetric assay which relies on the conversion of MTT (3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide to a blue formazan dye by live mitochondria. This assay has been shown to give identical results to the [methyl-³H]thymidine incorporation assay.

SV40 transformed fibroblasts (MRC5CV1) and transitional bladder carcinoma cells (T24) were maintained in RPMI 1640 medium (Commonwealth Serum Laboratories) containing 10% foetal calf serum, at 37° in a humidified atmosphere of 5% CO₂ in air. Human lymphocytes were isolated from whole blood using Ficoll Paque (Pharmacia-LKB) as recommended by the manufacturer. Phytohaemagglutinin [PHA, (GIBCO)] was added at 1 μL/mL. the lymphocytes were plated into 96 well microtitre plates at a density of 1x10⁵/mL. After PHA stimulation for 72 h, the lissoclinamides were added in a range from 100 μg/mL to 0.1 μg/mL. After 24 h, the surviving cells were assayed. For cytotoxicity data, MRC5CV1 and T24 cells were plated at a density of 1x10⁴/100 μL in 96 well microtiter plates . After overnight incubation, the cells were exposed to varying concentrations of the lissoclinamides for 1 h. After a 48 h incubation, 10 μL of MTT (5 mg/mL) were added per well and the cultures incubated for 4 h at 37°. Thereafter, 100 μL/well of 0.08 M HCL in isopropanol were added. Samples were mixed until all the blue product was dissolved, and the absorbance at 540 nm determined in a Titertek Multiscan MC instrument using a reference wavelength of 690 nm. The cell survival curves for the three different cell types are shown in FIGS 6, 7 and 8.

Lissoclinamide 7 was the most cytotoxic with an IC₅₀ value of 0.04 μg/mL for a 1 h exposure.

C) BISTRATAMIDE A, BISTRATAMIDE B, BISTRATENE A AND BISTRATENE B

C.i Isolation and Structure Determination A crude brownish oil (0.22 g) was isolated from L. bistratum (250 g) following the general Extraction of Compounds procedure described above. The crude oil was dissolved in methanol:water (77:23) and applied to a Whatman Partisil ODS-3, 9 mm x 50 cm preparative reverse-phase HPLC column. Elution was effected by means of a concave gradient from 77 to 100% methanol over 120 min. Fractions of interest were pooled, evaporated to dryness and subjected to further purification by chromatography on Sephadex LH-20 using methanol: water (77:23) as solvent to yield, inter alia, bistratamide B, bistratamide A, bistratene A and bistratene B. Bistratamides A and B were obtained in approximately equal amounts comprising 0.3% of dried extract each (equivalent to 5mg/kg wet weight). The yields of bistratenes A and B were 10% and 2.8% of the dried extract, respectively (equivalent to 0.56 g/kg and 0.15 g/kg wet weight, respectively). The structures of the compounds were determined unambiguously from detailed analyses of ¹H and ¹³C NMR spectra and from 2D COSY 45 and ¹H-¹³C shift correlation experiments.

C.ii Cytotoxicity Studies Bistratamides A and B

The cytotoxicity of these compounds towards human cell lines, MRC5CV1 fibroblasts and T24 bladder carcinoma cells was determined. The cells were maintained in RPMI-1640 medium (Commonwealth Serum Laboratories) containing 10% foetal calf serum at 37° in a humidified atmosphere of 5% CO₂ in air. Cells were plated at a density of 5x10³ cells/16mm well, 24 h prior to labelling with [methyl-³H] thymidine (10μCi/mL, 40 Ci/mmol, Amersham). The cells were then extracted with cold 10% trichloroacetic acid (TCA), the TCA-insoluble material was collected onto glass fibre filters and washed with cold 5% TCA followed by 100% ethanol prior to liquid scintillation counting (FIG 4). Bistratamide A has an IC₅₀ value of about 60 μg/mL and bistratamide B an IC₅₀ value greater than 100 μg/mL. Their toxicity is similar to that of patellamide D. The conversion of one thiazoline in bistratamide A to a thiazole in bistratamide B results in a less toxic compound.

Bistratenes A and B

The cytotoxicity of these two compounds was determined using procedures similar to those described above for bistratamides A and B.

The bistratenes are among the most cytotoxic compounds yet discovered in the ascidians, in particular, bistratene A with an IC₅₀ value of 0.07 μg/mL. (see FIG 5) and bistratene B, with an IC₅₀ value of 0.09 μg/mL.

A comparison of the cytotoxity of the preferred compounds of the invention, together with that of the known compounds ascidiacyclamide and ulithiacyclamide, is given in Table 1.

Results are reported for T24 Bladder Carcinoma Cells and a 1-hour exposure.

The mechanism of action of the bistratenes has also been examined.

Although not wishing to be bound by theory, the compounds induce, at nanomolar concentrations, the property of differentiation in HL-60, human leukaemia cells along the monocyte/macrophages pathway. They do not have calcium ionophore activity but do activate the enzyme protein kinase C (PKC) at micromolar concentrations.

In more detail, the purity of bistratene B used in the following PKC assays was verified by reverse phase and normal phase thin layer chromatography. TPA, MTT (3, (4, 5-dimethylthiazol-2-yl) -2, 5- diphenyltetrazolium bromide), phosphatidylserine, oleic acid, histone H1 and 1,2-diolein were purchased from Sigma Chemical Co. [ɣ-³²P]ATP was obtained from Bresatec.

A monoclonal antibody against OKM-1 (a macrophage- granulocyte cell surface glycoprotein) was obtained from Ortho Diagnostics. Phycoerythrin labelled antibodies to Leu-M3 (CDw14), the granulocyte marker Leu-lla (CD16), Leu-7 (HNK-1) and HLA-DR were purchased from Becton Dickinson.

HL-60 cells were cultured in RPMI 1640 medium containing 10% foetal calf serum. The cytotoxicity of the bistratenes to HL-60 cells was determined using a colorimetric assay based on conversion of MTT to a blue formazan product by live mitochondria. Adherence was assessed by visual examination after gently shaking the cultures and aspirating non- adherent cells . Cytocentrifuge smears were assayed for nonspecific esterase (NSE) 48 h after treatment with bistratene A. Phagocytosis was determined by incubating differentiated HL-60 cells with 0.8μ polystyrol-latex beads (Boehringer Mannheim).

Supernatants from bistratene-treated cultures were assayed for the presence of interleukin-1 (IL-1), by an ELISA assay using rabbit anti IL-1 as first antibody and an active-site directed anti IL-1 monoclonal antibody (mAb) as second antibody. The presence of surface antigens HLA-DR, OKM-1, Leu-M3, Leu-7 and Leu-lla was determined by Fluorescence Activated Cell Sorting using the appropriate flurorescently labelled mAbs, and in the case of OKM-1, fluorescently labelled goat antimouse IgG as secondary antibody.

PKC was isolated from bovine spleen (1000 g) by sequential chromatographies on DEAE-cellulose, phenyl-Sepharose, threonine-Sepharose and hydroxylapatite. The final preparation yielded 4.3 mg of type II PKC and 2.0 mg of type III PKC in electrophoretically pure form. Assays of enzyme activity were carried out using lysine-rich histone as substrate, and measuring the rate of incorporation of [³²P] phospate from [ -³²PZATP (50 μM, 4 x 10 dpm/assay) into histone (lmg/mL). All assays contained 10 mM MgCl₂ and 5 mM dithiothreitol. The concentrations of calcium ions, phosphatidylserine, oleic acid, diolein, TPA and bistratene included in the assays are given in Table 2.

Phosphatidylserine was added as a sonicated vesicle preparation. Other compounds were added as solutions in either ethanol or methanol. The concentration of organic solvent, which was always less than 2% (v/v), was shown to have no effect on the enzyme activity.

When HL-60, promyelocytic human leukaemia cells, were treated with bistratenes A and B, cytotoxicity was observed at high concentrations, but at lower concentrations dramatic changes in the morphology of these cells were observed, indicative of differentiation. The effect of increasing concentrations of bistratene A on the viability of HL-60 cells is shown in FIG 9. The IC₅₀ value with these cells was 424 nM. At concentrations below this value HL-60 cells undergo differentiation. After 2h of continuous exposure to bistratene A (100 nM), HL-60 cells adhered to the culture plate, and acquired a spindle-shaped morphology and prominent pseudopodia. The cells remained mostly isolated from one another and with time there was an increase in cell size. TPA-treated cells were more strongly adherent and showed greater clumping. The morphology of bistratene-treated HL-60 cells is typical of cells in the process of differentiation along the monocyte/macrophage pathway. The nature of the differentiated cells was investigated in greater detail using a variety of cell surface and other differentiation markers (table 3).

1 All values were determined after 48 h except for Leu-M3 which was determined at 96 h.

² The concentration required to produce half-maximal expression was less than 50 nM for both OKM-1 and Leu-M3. These cells show some of the characteristics of macrophages, for example adherence to plastic and expression of OKM-1 and Leu-M3 antigens, but do not appear to be activated since they lack HLA-DR expression, and nonspecific esterase activity, do not phagocytose and fail to produce IL-1.

The effect of bistratene B on the activity of type II PKC from bovine spleen was examined. Parallel experiments investigating the effects of diolein and TPA on the same enzyme preparation were also undertaken, and the combined results are summarized in Table 2. Bistratene B on its own, at concentrations up to 356 μM, failed to activate the enzyme, and TPA (16 nM) did not enhance the activity in the presence of 35 μM bistratene B. At suboptimal concentrations of calcium ions (10 μM) and phosphatidylserine (10 μg/mL, both TPA and bistratene B enhanced the enzyme activity. In the presence of oleic acid alone (25 μM) (as a replacement for phosphatidylserine), the enzyme is almost completely inactive. In the presence of 25 μM oleic acid and 10 μM diolein, the activity is enhanced to a value which is the same, within experimental error, as that measured in the presence of optimal concentrations of phosphatidylserine and diolein. The present investigation has shown that both TPA and bistratene B similarly enhance the activity of the enzyme in the presence of oleic acid. At 25 μM oleic acid, neither TPA nor bistratene B produces maximal enhancement. However, at higher concentrations of fatty acid (300-600 μM), the enzyme is fully activated in the presence of either 16 nM TPA or 35 μM bistratene B, an enhancement of 30-fold over the activity in the presence of 600 μM fatty acid alone.

It is thus noted that the bistratenes can induce some differentiation of HL-60 cells at nanomolar concentrations and that, like diacylglycerols and phorbol esters, are capable of enhancing the activity of type II PKC from bovine spleen in the presence of suboptimal concentrations of oleic acid.

Preliminary experiments using Indo-1 loaded mouse thymocytes have shown that bistratene A does not cause a change in the intracellular free calcium concentration and is therefore not a calcium ionophore. Thus the mechanism of induction of HL- 60 differentiation by the bistratenes remains to be determined. In summary, the bistratenes induce the incomplete differentiation of HL-60 cells and enhance PKC activity in vitro, thus representing a new type of PKC modulator which should find use in the investigation of the role of PKC in cell growth and differentiation.

In use, as the compounds of the present invention do exhibit appreciable in vitro anti-tumour activity, they are thus potential anti-tumour agents. Accordingly, these compounds should be useful in the treatment of cancer and related diseases.

Those skilled in the art will appreciate that modifications and variations to the invention described above are possible without departing from the present inventive concept as defined in the following claims.

Claims

1. Substantially pure lissoclinamide 7.

2. Substantially pure bistratene A.

3 . Substantially pure bistratene B.

4. Substantially pure trisoxazoline.

5 Substantially pure lissoclinamide 8.

6 Substantially pure lissoclinamide 4.

7. Substantially pure lissoclinamide 5.

8. Substantially pure patellamide D.

9. Substantially pure bistratamide A.

10. Substantially pure bistratamide B.

11. A compound as defined in any one of claims 1 to 10 of a formula wherein at least one radical thereon has been substituted by another group.

12. A method for the isolation of a compound as defined in any one of claims 1 to 10, said method comprising: 1) homogenising a cleaned ascidian with an alcohol/hydrocarbon solution;

2) filtering the homogenate;

3) extracting the filtrate with an aqueous solution;

4) extracting the aqueous layer with an organic solvent;

5) drying and then evaporating off the organic extract;

6) chromatographing the residue; and

7) isolating the individual compounds.

13. A method as defined in claim 12 wherein the ascidian is Lissoclinum patella or Lissoclinum bistratum.

14. A pharmaceutical composition containing at least one of the compounds as defined in any one of claims 1 to 11, or a non-toxic salt thereof, in association with a carrier or diluent.

15. A method of therapeutic treatment by the administration of either an effective amount of a compound as defined in any one of claims 1 to 11 or of a pharmaceutical composition as defined in claim 14.

16. A method as defined in claim 15 wherein the treatment is for cancer or related diseases.

17. A method of detecting and monitoring cell growth and differentiation using a compound as defined in any one of claims 1 to 11 as a modulator for a cellular protein.

18. A method as defined in claim 17 wherein the cellular protein is PKC.