APOPTOSIS-INDUCING COMPOUNDS

Abstract: The invention relates to a pharmaceutical composition comprising an apoptosis-inducing amount of a compound having general formula (I), wherein: (i) R1 represents an unsubstituted C6 or C10 aryl group; or a C6 aryl group substituted with Me or OMe; (ii) A represents O, S or S partially oxidized to sulfoxide; (iii) the cyclic group labelled F represents an unsubstituted C6 or C10 aryl-fused group or a C5 heteroaryl-fused group (nitrogen as heteroatom) or a benzo-fused system substituted with ethoxycarbonyl function; (iv) and Y represents the group (A) wherein R2 and R3 are independently hydrogen; or methyl; or Y represents the group CH3 or a (CH2)2CH3 group; or an unsubstituted C5 heteroaryl group (nitrogen as heteroatom). The compositions may be used for the treatment of tumours or other cancerous conditions, such as CML (chronic myeloid leukaemia), AML, in AIDS-related lymphomas (such as Karpowski's sarcoma, a sub-cutaneous lymphoma) and used as apoptotic agents in the treatment of cancers generally.

Description: Aμopotosis-Inducing Compounds The present invention relates to pyrrolobenzoxazepines, pyrrolobenzthiazepines and related compounds having the ability to induce apoptosis, to pharmaceutical compositions comprising these compounds and to their use as anti-tumour agents Background to the Invention Benzodiazepmes such as Valnim are amongst the most highly prescribed drugs due to their anxiolytic, relaxant and sedative effects Target-specific binding of benzodiazepmes has been observed in many tissues and cell types and can be separated pharmacologically into two classes (see Table 1) The first and more widely studied of the two are the "central-type" binding sites located in brain which mediate the clinical effects of the benzodiazepmes These "central-type" sites are associated with the neuronal postsynaptic gaimna-aminoburyric acid receptor which regulates Cl flux (Costa and Guidotti, 1979) Benzodiazepmes also bind to another distinct binding site (Braeslrup and Squires, 1977, Schoe aker et al , 1981) found in the membranes of many mammalian tissues heart, kidneys, lungs, adrenals, blood platelets and also the brain, termed the peripheral-type benzodiazepine receptor (PBR) Apoptotic cell death can be induced by a variety of drugs with diverse chemical structure and different mechanism of action Among the list of apoptosis inducing agents are a wide range of anti-cancer drugs including camplothecin, etoposide and the antliracyclme antibiotics daunorubicin and doxorubicin Although the mechanism of anti-tumour action of some of these drugs has not been fully elucidated, they ultimately activate the event of apoptosis in cells All of the above anti-cancer drugs have previously been shown to induce apoplosis in cancerous cells derived from the liaemopoietic sjstem such as HL-60 and Jurkat cells Anj novel drugs that can also induce apoptosis in similar model cell lines may have potential as anti-cancer agents One major problem m cancer chemotherapy is inherent or acquired resistance Chronic myeloid leukaemia (CML) is associated with drug resistance, and although anti-cancer agents such as hydroxyurea appear to contain the disease during its chronic phase, progression to a fatal blast crisis is inevitable Resistance to anti-cancer agents in CML has been suggested to be via suppiession of apoptosis It would therefore be useful to develop novel drugs that overcome this apoptosis suppression in CML cell lines Description of the Prior Art The exact function of the PBR is unclear although PBR specific ligands such as Ro5-4864 (4'- chlorodiazepam) and PK11195 ( 1 -(2-chlorophenyl)- 1 ,3 -dih di o- 1 -methyl-propyl) isoquinolme carboxamide) have been shown to elicit a wide variety of effects including alteration in cardiac action potentials and calcium channels, alterations of protooncogene expression modulation of steroidogenesis and alteration of immune function (Zisterer & Williams, 1997 for review) There have been many reports that benzodiazepmes affect cell growth and differentiation in a number of cell types These include induction of differentiation of Friend erytliroleukemia cells (Wang et al , 1984a) and inhibition of cell proliferation (Wang et t , 1984b, Gorman et al , 1989, Ikezaki and Black, 1990, Camins et α/ , 1995) However, because of the high concentrations of PBR ligands necessary to elicit these antφroliferative f effects and the demonstration of similar effects in cell lines which lack the PBR (Gorman et al, 1989), it could be concluded that these antiproliferative effects seem unrelated to a specific interaction of these drugs with this receptor. Recently a novel series of high affinity PBR ligands based on a pyrrolobenzoxazepine skeleton, classified here as PBOX compounds, have been synthesised (Campiani et al, 1996). A recent study (Zisterer et al, 1998) has demonstrated that three of these novel PBR ligands, PBOX 1, 2 and 21 along with the classically used PBR ligands PK 11195 and Ro5-4864 were found to inhibit at micromolar concentrations and in a dose-dependent manner, the proliferation of rat C6 glioma and human 132 1 Nl astrocytoma, without being cytotoxic. This antiproliferative effect was found to be mediated by arrest in the Gl phase of the cell cycle. The present inventors have, while examining the effect of PK 11195, Ro5-4864 and some PBOX compounds on the proliferation of the human cancer cell lines, Jurkal (leukaemic T cell lymphoblast), HL- 60 (promyelocytic leukaemia) HUT 78 (T cell leukemia), LAMA, KYO.1 and K562 cells which are all CML (chronic myeloid lymphoma) cells, Hela (cervix carcinoma), CEM (T lymphoblastoid) cells and MCF-7 (human breast carcinoma) cells, determined that these PBR ligands induce apoptosis, with various potencies, in the nine cell lines. Apoptosis is a cell suicide mechanism invoked in disparate situations, both physiological and pathological, to ablate unwanted, damaged, or potentially neoplaslic cells. Apoptosis is classically defined by a characteristic set of morphological changes in the cell, including membrane blebbing, cell shrinkage, cliromatin condensation, DNA fragmentation and eventual formation of membrane-bound apoptotic bodies. Although a number of reports have identified agents that are capable of inducing apoptosis in a variety of cells and tissues, the mechanisms responsible for the activation of the apoptosis process are poorly understood. International Patent Application No. PCT/1E99/00030 describes pyrrolobenzoxazepines, pyrrolobenzthiazepines and related compounds having the ability to induce apoptosis. The present application relates to compounds selected from the group of compounds disclosed in that international application having especially surprising activity. Summary of the Invention According to the present invention there is provided a pharmaceutical composition comprising an apoptosis-inducing amount of a compound having the general formula (I):- wherein: (i) Ri represents an unsubstituted C 6 or do aryl group; or a C 6 aryl group substituted with Me or OMe; and (ii) A represents O, S or S partially oxidized to sulfoxide; (iii) the cyclic group labelled F represents an unsubstituted Cg or CJQ aryl-fused group or a C5 heleroaryl- fused group (mtrogen as heteroatom) or a benzo-fused system substituted with ethoxycarbonyl function; (iv) and Y represents the group — N wherein R 2 and R 3 are independently hydrogen; or methyl; or Y represents the group CH3 or a (CH 2 ) 2 CH 3 group; or an unsubstituted C heteroaryl group (nitrogen as heteroatom); Suitably Rl is an unsubstituted 1-naphthyl. Suitably F is an unsubstituted benzo-fused group or benzo-fused group substituted by one ethoxycarbonyl function, or unsubstituted 2,3-naphtho- or 2,3-pyrido-fused system. Particularly preferred are compounds in which Ri and F represent a 1-naphthyl group and a 2,3- naphtho-fused group, respectively. Also preferred are compounds in which Y represents a N(Me) 2 or NHMe or a 4-pyridine group. Most preferred compositions are those containing compounds selected from those having the formulae:- PBOX-4, PBOX-6, PBOX-7, PBOX-8, PBOX-9, PBOX-12, PBOX-15, PBOX-16, PBOX-27, PBOX- 28, PBOX-30, PBOX-65, PBOX-68, PBOX-69, PBOX-70, PBOX-71 as defined herein. The pharmaceutical composition may comprise conventional pharmaceutical carriers or excipients. The invention also relates to the use of compounds of formula I defined above as an active pharmaceutical substance and also use of these compounds in the preparation of a medicament for the treatment of tumours or other cancerous conditions, as well as the use of the compounds in methods of medical treatment of humans or animals . The invention also provides novel compounds namely the compounds: PBOX-4, -6, -7, -8, -9, -12, -15, -16, -27, -65, -68, -69, -70, -71 as defined herein. The invention also relates to the use of any of the above-defined compounds in the preparation of a medicament for the treatment of tumours or other cancerous conditions, such as CML (chronic myeloid leukaemia), AML, in AlDS-related lymphomas (such as Karpowski's sarcoma, a sub-cutaneous lymphoma) and to their use as apoptotic agents in the treatment of cancers generally. The invention also provides a method for the synthesis of products with the above mentioned formulae which can be performed following already known methodologies. Starting from the appropriate 2- hydroxyaryla ines, the pyrrole ring is introduced by standard methods. Alkylation of the hydroxy groups with suitable aryl acetic acid ethyl esters followed by saponification provides the corresponding acids which are cyclized under Friedel-Crafts conditions. The ketones thus obtained are functionalized prior transformation in the corresponding potassium enolates. Detailed Description of the Invention The methods are described in Campiani el al, 1996a and 1996b. In greater detail the methods are as follows: Synthesis la - The compounds represented by the general formula (I) according to the present invention wherein A is sulfur can be obtained for example starting from pyrrolylphenyldisulfides, in turn prepared by lite appropriate precursors by standard procedure. Reductive alkylation by means of sodium borohydiide and α-bromoarylacetic acid ethyl esters After hydrolysis to the corresponding acid intermediates, intramolecular cyclization by exposure of the acids to phosphorus pentachloride can provide the thiazepinones. The reaction proceeds in the temperature range of 25 to 85°C for 12 to 30 hours in anhydrous, polar aprotic solvents. Oxidation with methachloroperbenzoic acid at 0°C provided the corresponding sulphoxides Treatment of the corresponding potassium enolates (reaction can be performed at 25°C for 2 to 5 hours) of thiazepinones with selected acyl chlorides and dialkyl carbamoyl chlorides can yield the desired compounds. Synthesis lb - The compounds represented by the general formula (I) according to the present invention wherein A is oxygen can be obtained for example starting from l-(2-hydroxyaryl)pyrroles, 1- (hydroxyheteroaryl)pyrroles by O-alkylation by means of a-bromoarylacetic acid ethyl esters. The reaction is conducted generally in the presence of a base such as potassium carbonate in N.N-dimethylformaπiide as solvent or with sodium hydride in anhydrous tetrahydrofuran at 25 to 0°C for 10 to 30 hours. After hydrolysis to the corresponding acid intermediates, intramolecular cyclization by exposure of the acids to phosphorus pentachloride, in presence or not of tin(lV) chloride, can provide the oxazepinones. The reaction proceeds in die temperature range of 25 to 85°C for 12 to 30 hours in anhydrous, polar aprotic solvents. Treatment of the corresponding potassium enolates (reaction can be performed at 25°C for 2 to 5 hours by means of potassium hydride) of oxazepinones with selected acyl chlorides and dialkyl carbamoyl chlorides can yield the desired compounds. Potassium enolates can be also treated with triphosgene and the resulting acyl chloride can be animated with different amines. The reaction is generally conducted in tetrahydrofuran at 25°C for 12 to 48 hours. Synthesis 2 - The heterocyclic compounds represented by the following general formula according to the present invention, wherein X is CH, R is H, R' is a 1-naphthyl group, A is sulfur and Y is a methyl or a dimethylamino group or a 4-pyridine group, and wherein X is nitrogen, A is oxygen, , R is H R' is a tolyl or a 1-naphthyl group, and Y is a methyl or a 4-pyridine group , and wherein X is CH, A is oxygen, , R is H or an ethoxycarbonyl group, R' is a phenyl or a 1-naphthyl group, and Y is a methyl or a dimethylamino group or a methylainino group, can be produced by the following methods. Synthesis 2a - The compounds wherein X is CH, , R is H R' is a 1-naphthyl or a p-methoxyphenyl group group, A is sulfur and Y is a methyl or a dimethylamino group or a 4-pyridine group can be obtained starting from pyrrolylphenyldisulfide, in turn prepared by the appropriate amine precursor by standard procedure. Reductive alkylation by means of sodium borohydride and α-bromo- 1 -naphtliylacetic acid etliyl esters or a α-bromo-p-methoxyphenyl acetic acid ethyl esters and subsequent hydrolysis to the corresponding acid intermediate, followed by intramolecular phosphorus pentachloride catalyzed cyclization can provide the pyrrolobenzothiazepinone. The reaction proceeds in the temperature range of 25 to 85°C for 12 hours in anhydrous, polar aprotic solvent. Treatment of the corresponding potassium enolates (reaction can be performed at 25°C for 2 hours) of the thiazepinone with acetyl chloride or dimethylcarbamoyl chloride or isonicotinoyl chloride yields the desired compound. The reaction can be conducted in tetrahydrofuran at 2 °C for 30 hours. Synthesis 2b - The compounds wherein X is nitrogen, A is oxygen, , R is H R' is a tolyl or a 1-naphthyl group, and Y is a methyl or a dinielhylamino group or a 4-pyridine can be prepared starting from 3- hydroxy-2 -pyrrol iypyridine, in turn prepared by a Clauson-Kaas reaction on the corresponding amine O- alkylation of the pyrrolyl derivative performed with -bromo-α-(phenyl)acetic acid ethyl ester or with α- bromo-α-toIyl(l-naphthyI)acetic acid etliyl ester in presence of sodium hydride in tetrahydrofuran can provide an ester intermediate which can be hydrolysed, and subjected to a phosphorus pentachloride catalyzed cyclization to obtain the key pyrrolopyridooxazepinone. The reaction can be conducted in 1,2- dichloroethane at 25°C for 15 hours. The reaction of the potassium enolate (reaction can be performed at 25°C for 2 to 5 hours by means of potassium hydride) of the pyrrolopyridooxazepinone with acyl chloride or dimethylcarbamoyl chloride can yield the desired compounds. The reaction is generally conducted in tetrahydrofuran at 25°C for 15 hours. Synthesis 2c- The compounds wherein X is CH, A is oxygen, R is H or an ethoxycarbonyl group, R' is a phenyl or a 1-naphthyl group, and Y is a methyl or a dimethylamino group or a methylamino group can be prepared starting from l-((4-ethoxycarbonyl)2-hydiOxyphenyl)pyrrole, in turn prepared by a Clauson- Kaas reaction on the corresponding aniline. O-alkylation of the pyrrolyl derivative performed with α- bromo-α-(phenyl)acetic acid ethyl ester or with α-bronιo-α-(l-naphthyl)acetic acid ethyl ester in presence of sodium hydride in tetrahydrofuran can provide an ester intermediate which can be hydrolysed, and subjected to a phosphorus pentachloride catalyzed cyclization to obtain the key pyrrolopyridooxazepinone. The reaction can be conducted in 1,2-dichloιoethane at 25"C for 15 hours. The reaction of the potassium enolate (reaction can be performed at 25°C for 2 to 5 hours by means of potassium hydride) of the pyrrolobenzoxazepmone with acetyl chloride or dimethylcarbamo}! chloride can yield the desired compounds In the case of the methylamino derivatives the enolate was exposed to triphosgene and then to methylamine The reaction is generally conducted in tetrahydrofuran at 25°C for 20 hours Synthesis 3 - Compounds PBOX-4, 6, 7, 8, 9, 12, 15, 16, 28, 68, 70, 71 belong to the thiazepine and oxazepine series and are synthesized following synthetic strategies already described above Next, specific examples of the derivatives of the present invention are shown The present invention will be explained in more detail below by way of examples, however is in no way restricted to these examples Examples of the pyrido-fused, naphtho-fused and benzo-fused series Example 1 l-(3-Hydroxy-2-pyridyl)pyrrole. To a solution of 2-amιno-3-hydroxypyπdιne (7 5 g, 68 1 m ol) in 250 niL of glacial acetic acid, heated at 95-100°C, was added 2,5-dιmethoxytetrahydrofuran (9 0 g, 68 1 mmol) in glacial acetic acid (20 L) The mixture was stirred for 30 nun, the solvent was removed in vacitσ, and the residue was extracted with ElOAc The organic layers were washed with 5% NaHCθ3 solution and bπne, dried, and evaporated The crude product was purified by silica-gel column cluoinatography us g dichloromethane/ethyl acetate (8 2) as eluant Fractions contain g the product were combined, dried, and recrystallized by ethyl ether to give analytically pure title compound (4 7 g) as white prisms IR (KBr) 2928 cm ', Η NMR (CD3OD) d 6 08 (m, 2 H), 6 96 (dd, 1H, = 7 9, 4 6 Hz), 7 21 (d, 1 H, J = 79 Hz ), 742 (m, 2 H), 7 75 (d, 1H, ./ = 4 6 Hz) Anal (C H8N2O) C,H,N Example 2 (±)-a-[3-[2-(lH-Pyrrol-l-yl)pyridyJ]oxy]p-toIjlacetic Acid Ethyl Ester Sodium hydride (347 mg, 14 4 mmol) was added to a solution of l-(3-hydroxy-2-pyrιdyl)pyrroIe (2 1 g, 13 1 mmol) in anhydrous THF at room temperature The reaction mixture was stirred for 1 h at room temperature, and then a solution of ethyl α-bromop-tolylacetate (3 3 g, 13 2 mmol) 111 anhydrous THF (15 111L) was added dropwise After 12 h at RT, the solvent was removed in vacuo, and the residue was taken up in dichloromethane The organic layer was washed with bπne, dried and concentrated The residue was purified by flash cluoinatography (dichloromethane/hexane 2 1), to give the title compound as a colorless oil IR (neat) 1748 cm ', Η NMR (CDCI3) d 8 08 (d, 1 H, J = 4 8 HZ), 7 82 (m, 2 H), 7 53 (m, 2 H), 738-741 ( , 3 II), 7 20 (d, 1 H, J= 8 1 Hz), 7 02 (dd, 1 H, J= 8 1, 4 8 Hz), 6 34 (m, 2 H), 5 63 (s, 1 H), 4 15 (m, 2 H), 2 33 (s, 3H), 1 20 (t 3 H, J = 7 1 Hz) Anal (C20H20N2O3) C,H,N Example 3 (±)-α-[3-[2-(lH-Pyrrol-l-yl)pyridyl]oxy]p-tolylacetic Acid. The ester (±)-a-[3-[2-(lH-Pyπol-l-yl)pyπdyl]oxyJp-tolylacelιc acid ethyl ester (0 97 g, 30 mmol) was dissolved in 15 mL of EtOH and 5% aqueous NaOH was added The reaction mixture was stirred at room temperature for 20 h, concentrated, and acidified with IN HO The aqueous phase was extracted with EtOAc, and the organic layer was washed with bπne, dried, and concentrated The residue was reciyslallized to give the title compound as colorless prisms IR (nujol) 1745 cm , H NMR (CDCI3) d 8 11 (d, 1 H, 7= 4 7 Hz), 773 (m, 2 H), 753 (m, 2 H), 740 (m, 3 H), 723 (d,l H, J= 8 1 Hz), 7 10 (dd, 1 H, J = 8 2, 4 6 Hz), 6 35 (m, 2 H), 5 55 (s, 1 H), 2 30 (s, 3 H) Anal (C18H16N2O3) C,H,N Example 4 (±)-6-p-Tolylpyrrolo[l,2-d]pyrido[3,2-bJ[l,4]oxazepin-7(6H)-one. Phosphorus pentachloride (0 55 g, 2 3 mmol) was added to a solution of (-b)-a-[3-[2-(lH-Pyrrol- l-yl)pyridylJoxy]p-toly!acetιc Acid (0 60 g, 2 0 mmol) witlun 20 mm The reaction mixture was stirred al room temperature for 7 h, and then was poiued into crushed ice, basified widi 10% NaOH solution, and extracted with chloroform The organic layers were washed with brine, dried and evaporated The residue was purified by flash chromatography (dichloromethane) and recrystallized (hexane) The title compound was obtained as colorless prisms IR (nujol) 1640 cm "1 , Η NMR (CDCI3) d 8 23 (d, 1 H, J = 4 7 Hz), 8 06 (m, 1 H), 7 59 (m, 1 H), 7 28-736 (m, 5 H), 7 10 (dd, 1 H, J= 8 1, 4 7 Hz), 654 (m, 1 H), 5 66 (s, 1 H), 2 31 (s, 3 H) Anal (C18H14 2O2) C,H,N Example 5 (±)-α-[[2-(l -PyrroI-l-yl)phenyl)oxy|-(l-uaphthyI)acetic Acid Ethyl Ester. Sodium hydride (90 mg, 3 9 nunol) was added to a solution of l-(2-hydroxyphenyl)pyrrole (0 41 g, 2 5 mmol) in anhydrous THF (5 mL) at it The reaction mixture was stirred for 1 h at rt, and then a solution of etliyl α-bromo-α-(l-naphtyl)acetate (1 0 g, 4 0 mmol) in anhydrous THF (5 mL) was added dropwise After 12 h at rt, the solvent was removed in vacuo, and the residue was taken up 111 dichloromethane The organic layers were washed with brine, dried, and evaporated The residue was purified by chromatography (toluene) to give 0 46 g of the title compound as a colorless oil, IR (neat) 1734 cm " ', ! H NMR (CDCI3) δ 1 10 (t, 3 H, J = 7 0 Hz), 4 10 (m, 2 H,), 6 15 (s, 1 H), 6 30 (m, 2 H), 6 35-8 25 ( , 13 H) Example 6 (±)-α-[[2-(lH-Pyrrol-l-yI)phenyIjoxy]-(l-naphthyl)acetic Acid. The (±)-α-f[2-(lH-Pyrrol-l-yl)phenyl]oxy]-(l-naphryl)acetιc Acid Ethyl Ester (0 78 g, 2 1 mmol) was dissolved in 26 mL of EtOH/THF mixture (1 1), and 5% aqueous NaOH (22 4 mL) was slowly added The reaction mixture was stirred at rt for 3 h, concentrated, and acidified with 4N HC1 until pH 3-4 The suspension was extracted with EtOAc and the organic phase was washed with brine, dried, and concentrated The residue was crystallized to give the desired acid (0 67 g) as colorless prisms IR (KBr) 1730 cm " 1 , l H NMR (CDCI3) δ 5 50 (s, 1 H), 6 10 (s, 1 H), 6 32 (m, 2 H), 692-7 15 (m, 4 H), 7 21-7 60 ( , 6 H), 7 84 (m, 2 H), 8 15 (m, 1 H) Example 7 (±)-6-(l-Naphthyl)pyrrolo[2,l-d][l,5jbenzoxazepιn-7(6H)-one. Phosphorus pentachloi ide (04 g, 1 92 mmol) was added to a solution of (±)-α-[[2-(lH-Pyπol-l- yl)phenyl]oxy]-(l-naphthyl)acetιc Acid (0 58 g, 1 68 mmol) in dry 1,2-dιchloroethane (8 5 mL) within 20 m The reaction mixture was stirred at rt for 5 h, then was poured into crushed ice, basified with 10% NaOH solution, and extracted with chloroform The organic layers were washed with bnne, dπed and evaporated The residue was chronialographed (dichloromethane and hexanes 2 1) and recrystallized to yield 300 mg of the oxazepinone as colorless prisms, IR (CHCI3) 1640 cm "1 , Η NMR (CDCI3) δ 6 27 (s, 1 H), 6 55 (m, 1 H), 6 76 (dd, I H, J = 9 0 Hz), 695 (m, I H), 7 09-7 63 (m, 10 H), 8 23 (m, 1 H) Example 8 1 -[2-(α-Ethoxycarbonyl-p-methoxybenzy l)thiopheιιy l]py ri ole. To a solution of sodium (0.46 g, 0.02 g-atom) in 25 mL of anhydrous ethanol, a solution of l-(2- mercaptophenyl)pyrrole (3.5 g, 0.02 mol) in 8 mL of anhydrous ethanol was added dropwise and the mixture was allowed to stirr for 1 h. After evaporation of the solvent the residue was dissolved in anhydrous tetrahydrofuran (25 mL) and ethyl α-bromo-p-methoxyphenylacetate (5.5 g, 0.02 mol) was slowly added. The reaction mixture was heated at 60 °C for 5h and then was cooled and poured onto crushed ice, extracted with ethyl acetate. The organic layer was washed with brine, dried and concentrated. The residue was purified by flash chromatography (dichloromethane as eluant) giving 5.05 g (69%) of the desired product. IR (neat) 1735 cm "1 ; 'HNMR (CDCl3) δ 1.06 (t, 3 H), 3.76 (s, 3 H), 4.07 (q, 2 H), 4.30 (s, 1 H), 6.35 (t, 2 H), 6.70-7.70 (m, 10 H). Example 9 (±)-α-[[2-(lH-Pyrrol-l-yl)phenyl]thio]-p-methoxyphenylacetic Acid. The (±)-α-l[2-(l/ -Pyrrol-l-yl)phenyl]thio]-p-methoxyphenylacetic Acid Ethyl Ester (5.68 g, 15.4 mmol) was dissolved in 35 mL of EtOH/THF mixture (1: 1), and 5% aqueous NaOH (40 mL) was slowly added. The reaction mixture was stirred at RT for 3 h, concentrated, and acidified with 4N HC1 until pH 3-4. The suspension was extracted widi EtOAc and the organic phase was washed with brine, dried, and concentrated. The residue was crystallized to give the acid (4.84 g, 92%) as colorless prisms. IR (KBr): 1710 cm "1 ; 'HNMR (CDC13) d 3.58 (s, 3 II), 4.11 (s, 1 H), 6.11 (m, 2 H), 6.40-7.35 (in, 10 H), 7.67 (s, 1 H). Example 10 (±)-6-p-MethoxyphenylpyrroloI2,l-d][l,5]benzthiazepiu-7(6H)-one. Phosphorus pentachloride (5.48 g, 26.5 nunol) was added to a solution (±)-α-l[2-(l/f -pyrrol- 1- yl)phenyl]lhio]-p-methoxyphenylacetic acid (9.0 g, 26.5 nunol) in dry 1,2-dichloroethane (8.5 mL) within 20 min. The reaction mixture was stirred at RT for 6 h, then was poured into crushed ice, basified with 10% NaOH solution, and extracted with chloroform. The organic layers were washed with brine, dried and evaporated. The residue was chromatographed (dichloromethane as eluant) and recrystallized to yield 3.78 g (44%) of the thiazepinone as colorless prisms; IR (CHCI3) 1638 cm "1 ; ] H NMR (CDCI3) δ 3.65 (s, 3 H), 5.10 (s, 1 H), 6.51 (to, 1 H), 6.73 (m, 2 H), 6.91-7.80 ( , 8 H). Example 11 7-Acetoxy-6-(l-naphthyI)pyrrolo[2,l-dl[l,5]benzoxazepine (PBOX-4). To a suspension of potassium hydride (0.185 g, 1.65 nunol, 35% in oil) in anhydrous THF (2 0 mL) was added the ketone (±)-6-(l-naphtyl)pyrrolo[2,l-d][l,5]benzoxazepin-7(6 f)-one (0.54 g, 1.65 nunol) dissolved in anlvydrous THF (2 mL). The reaction mixture was stirred at rt for 2 h, and then acetyl chloride (120 μL, 1.65 mmol) was slowly added. After stirring for 12 h at rt, the solvent was removed in vacuo and the residue was dissolved in EtOAc. The organic layer was washed with brine, dried and concentrated. The residue was chromatographed (dichloromethane and hexane 1 : 1) to give the PBOX-4 in 73% yield. IR (neat) 1765 cm "1 ; 1 HNMR (CDCl3) δ 1.83 (s, 3 H), 6.40 (m, 1 H), 6.86 ( , 1 H), 7.10-8.11 (111, 12 H). Example 12 7-((Dimethylcarbamoyl)oxy)-6-(l-naphthyl)pyrrolo[2,l-d][l,5] benzoxazepine (PBOX-6). To a suspension of potassium hydride (0 030 g, 0 27 nunol, 35% in oil) in anhydrous THF (1 0 L) was added the ketone (±)-6-(l-naphthyl)pyrrolo[2,l-d][l,5]benzoxazepιn-7(6/ )-one (90 mg, 0 27 mmol) dissolved in anhydrous THF (1 mL) The reaction mixture was stirred at rt for 2 h, and then dimethylcarbamoyl chloride (20 μL, 0 27 mmol) was slowly added After stirring for 18 h at 30 °C, the solvent was removed in vacuo and the residue was dissolved in EtOAc The organic layer was washed with brine, dned and concentrated The residue was chromatographed (dichloromethane and hexane 1 3) to give the PBOX-6 in 53% yield IR (neat) 1725 cm ', 'HNMR (CDCI3) δ 2 61 (s, 3 H), 2 71 (s, 3 H), 6 41 ( , 1 H), 6 88 (m, 1 H), 7 13-8 16 (m, 12 H) Example 13 7-[(MethyIcarbamoyl)oxy]-6-(l-naphthyl)pyrrolo[2,l-d][l,5]-benzoxazepine (PBOX- 7). To a suspension of potassium hydride (0 13 g, 1 1 mmol, 35% in oil) anhydrous THF (1 5 mL) was added the ketone (±)-6-(l-naphtyl)pyπolo[2,l-d][l,5]benzoxazepm-7(67/)-one (0 36 g, 1 1 mmol) dissolved in anhydrous THF (1 mL) The reaction mixture was stirred at rt for 2 h, and then tπphosgene (102 mg, 0 34 mmol) was slowly added within 30 m After stirring for additional 5 mm at rt, methylamine (2 2 nunol) and dusopropylethylannne (2 2 mmol) were added and stirring was continued for 10 mm The leaction was quenched with 5% sodium carbonate solution and the mixture was extracted with EtOAc The oiganic layer was washed with brine, dried, and concentrated 1 he residue was chromatographed (dichloromethane and hexanes 1 1) to give PBOX-7 as a colorless oil, IR (CHCI3) 1678 cm ', Η NMR (CDCI3) δ 2 61 (s, 3 H), 5 71 (br s, 1 H), 6 41 (m, 1 H), 6 88 (m, 1 H), 7 13-8 16 (m, 12 H) Example 14 7-((Dimethylcarbamoyl)oxy)-6-(l-naphthyl)pyrrolo[2,l-d][l,5j benzothiazepine (PBOX-8). lo a suspension of potassium hydride (0 167 g, 1 46 nunol, 35% in oil) in anhydrous THF (4 0 mL) was added the the (±)-6-(l-naphthyl)pyτrolo[2,l-d][l,5Jbenzothιazepιn-7(6H)-one (500 mg, 1 46 nunol) dissohed in anhydrous THF (5 mL) The reaction mixture was stirred at rt for 2 h, and then dimethylcarbamoyl chloride (0 181 g, 1 68 mmol) was slowly added After stirring for 8 h at 30 °C, the solvent was remoΛed in vacuo and the residue was dissolved 111 EtOAc The organic layer was washed with brine, dried and concentrated The residue was chromatographed (dichloromethane) lo give the PBOX-8 m 82% yield IR (neat) 1725 cm ', "HNMR (CDCI3) δ 2 43 (s, 3 H), 2 58 (s, 3 H), 6 45 (m, 1 H), 6 64 (in, 1 H), 7 18-7 57 (in, 10 H), 7 77-7 86 (m, 2 H) Example 15 7-Acetoxy-6-(l-naphthyl)pyrrolo[2,l-d)ll,5]benzothiazepine (PBOX-9). To a suspension of potassium hydride (0 303 g, 2 64 mmol, 35% in oil) in anhydrous THF (5 0 mL) was added the (±)-6-(l-naphthyl)pyrrolo[2,l-dJ[l,5]benzothιazepιn-7(6H)-one (0 9 g, 2 64 mmol) dissolved in anhydrous THF (7 mL) The reaction mixture was stirred at rl for 2 h, and then acetyl chloride (0 239 g, 3 04 mmol) was slowly added After stirring for 12 h at rt, the solvent was removed in vacuo and the residue was dissolved in chloroform The orgamc layer was washed with bruie, dried and concentrated The residue was chromatographed (dichloromethane and hexane 1 1) to give the PBOX-9 in 86% yield IR (neat) 1762 cm ', 'HNMR (CDCI3) δ 1 70 (s, 3 H), 6 45 (m, 1 H), 6 63 (m, 1 H), 7 19-7 55 (m, 10 H), 7 78-7 87 (m, 2 H) Example 16 7-Acetoxy-6-(l-naphthyl)pyι rolo[l,2-d]pyrido[3,2-b][l,4]oxazepιne (PBOX-12). To a suspension of potassium hydride (0 245 g, 2 14 mmol, 35% in oil) in anhydrous THF (7 0 mL) was added the (±)-6-(l-naphthyl)pyrrolo[l,2-d|pyπdo[3,2-b][l,4]oxazepιn-7(6H)-one (0 7 g, 2 14 mmol) dissolved in anhydrous THF (7 mL) The reaction mixture was stirred at rt for 2 h, and then acetyl chloride (0 193 g, 246 mmol) was slowly added After stirπng for 12 h at rt, the solvent was removed in vacuo and the residue was dissolved in chloroform The orgamc layer was washed with brine, diied and concentrated The residue was chromatographed (dichloromethane and hexane 1 1) to give the PBOX-12 in 75% yield IR (neat) 1760 cm ', 'HNMR (CDCI3) δ 1 83 (s 3 H), 6 43 (m, 2 H), 7 06-7 21 (m, 2 H), 741-7 53 (m, 4 H), 7 77-8 10 (m, 4 H), 8 26 (m, 1 H) Example 17 4-Acetoxy-5-(l-naphthyl)naphtho[2,3-b]pyriOlo[l,2-d)[l,4]oxazcpine (PBOX-15). To a suspension of potassium hydride (0 035 g, 0 31 mmol, 35% in oil) in anhydrous THF (2 0 mL) was added the ketone (±)-5-(l-naphthyl)naphtho[2,3-b]pyrrolo[l,2-d][l,4Joxazepιn-4(5/ )-one (0 31 mmol) dissolved 111 anhydrous THF (2 mL) The reaction mixture was stirred at it for 2 h, and then acetyl chloride (22 5 μL, 0 31 nunol) was slowly added After stirring for 6 h at rt, the solvent was removed in vacuo and the residue was dissolved in chloroform Ihe organic layer was washed with brine, dried and concentrated The residue was chromatographed (dichloromethane and hexane 1 4) to give the PBOX- 15 in 60% yield IR (neat) 1745 cm ', 'HNMR (CDCI3) δ 2 18 (s, 3 H), 6 46 (m, 1 H), 7 26 (m, 1 H), 7 38- 8 20 (m, 14 H) Example 18 4-[(Diinethylcarbamoyl)oxy]-5-(l-naphtyl)naphtho[2,3-b]pyrrolo[l,2-dJ[l,4| oxazepine (PBOX- 16). To a suspension of potassium hydride (0 102 g, 0 92 nunol, 35% 111 oil) m anhydrous THF (4 0 mL) was added the ketone (±)-5-(l-naphthyl)naphtha[2,3-b]pyτιolo[l,2-dJ[l,5]oxazepιn-4(5 )-one (0 92 mmol) dissolved in anhydrous THF (3 mL) The reaction mixture was stirred at rt for 2 h, and then dimethylcarbamoyl chloride (68 μL, 0 92 mmol) was slowly added After stirruig for 5 h at 30 °C, the solvent was removed 111 vacuo and the residue was dissolved in EtOAc The organic layer was washed with bnne, dried and concentrated The residue was chromatographed (dichloromethane and hexane 1 5) to give the PBOX-16 in 50% yield IR (neat) 1715 cm ', 'HNMR (CDCI3) δ 2 82 (s, 3 H), 3 09 (s, 3 H), 6 46 (m, 1 H), 7 25-8 24 (m, 15 H) Example 19 7-IsonicotinoyIoxy-6-p-methoxyphenylpyriOlo[2,l-d][l,5]- benothiazepine (PBOX- 28). To a suspension of potassium hydride (0 124 g, 3 1 mmol, 35% in oil) in anhydrous 1 HF (5 0 mL) was added the ketone (±)-6-(p-metlιoxyphenyl)pyrrolo[2,l-d][l,5]benzothιazepm-7(6/7)-one (1 0 g, 3 1 mmol) dissolved in anhydrous THF (5 mL) Hie reaction mixture was stirred at rt for 2 h, and then isonicotinoyi chloride hydrochlonde (0 55 g, 3 1 mmol) and TEA (0 31 g, 3 1 mmol) in 7 L of anhydrous tetrahydrofman was slowly added After stirring for 12 h at rt, the solvent was removed in vacuo and the residue was dissolved in EtOAc The organic layer was washed with brine, dried and concentrated The residue was chromatographed (dichloromethane and hexane 1 1) to give the PBOX-28 in 95% yield (1 25 g) IR (neat) 1738 cm "1 , *HNMR (CDCI3) δ 3 75 (s, 3 H), 6 40 (m, 1 H), 6 62 (m, 1 H), 6 81 (m, 2 H), 7 1-9 3 (m, 11 H) Example 20 7-Acetoxy-6-p-tolylpyrrolo|l,2-d]pyrido[3,2-bJ[l,4]oxazepine (PBOX-68). To a suspension of potassium hydride (0 13 g, 1 1 nunol, 35% in oil) in anhydrous THF (1 5 mL) was added the ketone (±)-6-p-tolylpyιrolo[l,2-d]pyrιdo[3,2-b][l,4]oxazepιn-7(6H)-one (0 36 g, 1,1 mmol) dissolved m anhydrous THF (2 mL) The reaction mixture was stirred at rt for 2 h and then acetyl chloride (68 5 μL, 1 0 nunol) was slowly added After stirring for f 2 h at rt, the solvent was removed in vacuo and the residue was taken up 111 EtOAc The organic layer was washed with brme, dried, and concentrated The residue was chromatographed (dichloromethane and hexane 1 3) to give the title compound as a colorless oil (69%), IR (CHCI3) 1765 cm ', Η NMR (CDCI3) d 2 20 (s, 3 H), 6 50 (111, 1 H), 7 06-740 (m, 6 H), 8 05-8 20 (m, 3 H) Example 21 3-(Ethoxycarbonyl)-7-((dimethylcarbamoyl)oxy)-6-(l-naphthyl)pyrrolo[2,l-dJ [1,5] bcnzoxazepinc (PBOX-70). To a suspension of potassium hydride (0 252 g, 2 20 mmol, 35% in oil) in anhydrous THF (5 0 mL) was added the the (±)-3-(ethoxycarbonyl)-6-(l-naphtlιyI)pyrrolo[2,l-d][l,5]benzothιazepιn-7(6H)- one (2 20 mmol) dissolved in anhydrous THF (5 mL) Hie reaction mixture was stirred at rt for 2 h, and then dimethylcarbamoyl chloride (0 303 g, 2 82 mmol) was slowly added After stirring for 8 h at rt, the solvent was removed 111 vacuo and the residue was dissolved in EtOAc The organic layer was washed with brine, dried and concentrated The lesidue was chromatographed (dichloromethane) to give the PBOX-70 111 80% yield IR (neat) 1730 cm ', 'HNMR (CDCI3) δ 1 23 (t, 3 H, J = 7 0 Hz), 2 82 (s, 3 H), 3 11 (s, 3 H), 4 10 ( , 2 H), 6 45 ( , 1 H), 6 62 (m, 1 H), 7 19- 6 (111, 9 H), 7 82 (m, 2 H) Example 22 4-Isoπicotinoyloxy-5-phenylnaphtho[2,3-b]pyrrolo[l,2-d][l,4]oxa7epine (PBOX-71). To a suspension of polassium hydride (0 306 g, 2 76 mmol, 35% in oil) in anhydrous THF (6 0 mL) was added the kelone (±)-5-phenyInaphtho[2,3-bJpyπolo[l,2-dJLl,4Joxa7epιn-4(5//)-one (2 7 mmol) dissolved in anhydrous THF (10 mL) The reaction mixture was stirred at rt for 2 h, and then isonicotinoyi chloride (3 0 nunol) was slowly added After stirring for 5 h at rt, the solvent was removed in vacuo and the residue was dissolved in chloiofoπn The organic layei was washed with brme, dried and concentrated The residue was chromatographed (EtOAc) to give the PBOX-71 in 58% yield IR (neat) 1715 cm ', I I 'HNMR (CDCI3) δ 6 38 (m, 1 H), 6 62 (m, 1 H), 7 11-7 42 (m, 9 H), 7 60-774 (m, 3 H), 8 0-8 20 (m, 4 H) Example 23 7-(Butyryloxy)-6-(p-methoxyphenyl)pyrrolo[2,l-dJ[l,5]benzothiazepine-5-oxide (PBOX30). To a well stirred and cooled (2 °C) solution of (±)-6-(p-methoxyphenyl)pyrrolo[2,l-d][l,5]benzothιazepιn- 7(6H)-oae (0 85 g, 2 64 mmol) in 10 mL of dry chloroform was added 3-chloroperbenzoιc acid (2 64 mmol) dissolved 111 die sam4e solvent (20 mL) dropwise over 1 h after stirring for 12 h at 2 °C the reaction mixture was filtered and the filter cake was rinsed with chloroform The combined organic solutions were washed with brine, dried and concentrated to give (±)-6-(p-melhoxyphenyl)pyrrolo[2,l-dl[l,5]benzothιazepιπ- (85% yield) which was recrystallized from ligroine Η NMR (CDC1 3 ) δ 3 71 (s, 3 H), 4 78 (s, 1 H), 6 47 (m, 1 H), 6 68 (d, 2 H, J = 8 8 Hz), 6 95 (d, 2 H, J = 8 7 Hz), 7 12-7 80 (m, 6 H) Starting from 1 05 g (3 26 mmol) of the above mentioned sulfoxide, by treatment with potassium hydride and butyryl chloride (3 3 mmol) the title coπipouind was obtamed in 44 % yield PBOX30 was recrystallized from ethyl acetate as colorless prisms mp 113-115 °C Η NMR (CDC1 3 ) δ 0 75 (t, 3 H, J = 74 Hz), 1 47 (m, 2 H), 2 17 (t, 2 H, J - 73 Hz), 3 81 (s, 3 H), 6 47 (m, 1 H), 6 64 (m, 1 H), 6 90 (d, 2 H, J = 8 9 Hz), 7 11 (d, 2 H, J = 8 9 Hz), 7 22-7 87 (m, 5 H) IR (nujol) 1759 cm ' Anal Cald for C 23 H 2 ,N0 4 S C,H,N Example 24 7-[(Methylcarbamoyl)oxj ]-6-phenylpyrrolo[2,l-d][l,5]-benzoxazepine (PB0X-27). To a suspension of potassium hydride (0 26 g, 2 2 mmol, 35% 111 oil) in anhydrous THF (3 mL) was added the ketone (±)-6-phenj lpyrrolo[2,l-dJ[l,5]ben/oxazepιn-7(6/ϊ)-one (2 2 mmol) dissolved in anhydrous THF (2 L) The reaction mixture was stirred at rt for 2 h, and then tπphosgcne (204 mg, 0 68 nunol) was slowly added within 30 mm After stirring for additional 5 nun at rt, methylamme (4 4 mmol) and dιιsoprop>lethylamιne (4 4 mmol) were added and stirring was continued for 1 nun The reaction was quenched with 5% sodium carbonate solution and the mixture was extracted widi EtOAc The organic layer was washed with brine, dried, and concentrated The residue was cliromatographed (dichloromethane and hexanes 2 1) to give PB0X-27 as a colorless oil, IR (CHCI3) 1675 cm' 1 , l H NMR (CDCI3) δ 2 59 (s, 3 H), 5 70 (br s, 1 H), 6 36 (m, 1 H), 6 87 (m, 1 H), 7 10-7 70 (m, 10 H) Anal Calcd for C 2 oHι 6 N 2 0 3 C,H,N Repiesentative compounds included 111 the piesent invention are as follows - Stnicture Foπnula Molecular weiglit For comparative piuposes reference is made herein to PBOX compounds which are not within the scope of the present invention. These compounds are PBOX-2: 7-[(dimethylcarbamoyl)oxy]-6-p-tolylpyrrolo[2,l-d][l,5]benzoxazepine PBOX- 1 : 7-[(dimethylcarbamoyl)oxy]-6-phenylpyrrolo[2, 1 -d] [1 ,5]benzoxazepine PBOX-5: 4-[(dimethylcarbamoyl)oxy]-5-phenylnaphtho[2,3-b]pyrrolo[l,2-d][l,4]oxazepitie PBOX-3: 4-Acetoxy-5-phenylnaphtho[2,3-b]pyrrolo[l,2-d][l,4]oxazepine. MATERIALS AND METHODS Materials HL 60 (ECACC No 881 12501), Jurkat T (ECACC No 88042803), HUT 78 (ECACC No 88041901 ), Hela (ECACC No 85060701) and MCF-7 (ECACC No 86012803) cells were obtained from the European Collection of Animal Cell Cultures, Porton Down, Salisbury, U K The LAMA 84, KYO 1 and K562 cells were the gift of Dr Mark Lawlor of St James' Hospital, Dublin. [ 3 H] PK 11195 (85 8 Ci/mmol) was from Amersham Phaπnacia Biotech (Aylesbury, UK) CEM cells, neo and overexpressmg Bcl2 were kindly provided by Dr Seamus Martin, Dept of Genetics, Trinity College Dublin The benzodiazepine, Ro 5-4864 [7-chloro-5-(4-chlorophenyl)-l,3-dιhydro-l-methyl-2-H-l,4- benzodιazepιn-2-oneJ was obtained from Fluka, Cheniie AG, Buchs, Switzerland PK-11195 [l-(2- chlorophenyl)-N-methyl-N-(l methylpropyl)-3-ιsoquιnolιnecarboxamιde] was a gift from Dr Alan Doble, Pharmuka Laboratories, Gennevilhers, France The pyrrolobenzoxazepine derivatives (PBOX compounds) were synthesized by Prof Giuseppe Campiani, University of Siena, Italy The RapiDiffTM kit was obtained from Diagnostic Developments, Burscough, Lancashire, U K The 22 bp oligonucleotide containing the NF kappa B consensus sequence was from Promega (Madison, Wl, USA) Anti-procaspase 3 was a monoclonal antibody against caspase 3/CPP32D, raised against a 24 7 kDa fragment corresponding to amino acids 1-219 of human caspase 3 and was obtained from Transduction Laboratories (Lexington, KY, USA) The enhanced chemiluminescence (ECL) kit was from Amersham Coip (U K ) All other reagents, including RPMI 1640, fetal calf serum, TNF, slaurosporine and the antioxidant compounds, were supplied from Sigma (Poole, Dorset, U K ) Diazepam and Clonazepam, N-acetyl-cysteine and butylaled hydroxyanisole, TNF alpha, PDTC (pyrollodithiocarbamate) and TEMPO (TM) were obtained fiom Sigma (Poole, Dorset, U K ) Tyrophostm was obtained from Calbiochem-Novabiochem, whereas, geiustein, eloposide, herbimycin A, tetrapropylammonium hydroxide and [ 3 H] Thymidme were obtamed from Sigma The caspase 3 fluorogenic substrate [Ac-DEVD-AMCJ was obtamed from Alexis (Nottingham, UK) The caspase 3 inhibitor, z-DEVD-fhik, and the anli c-abl antibody was supplied by Calbiochem- Novabiochem (Nottingham, UK) Anti-Cytochrome C was a mouse monoclonal antibody obtained from PharMuigen (San Diego, CA, USA) Λnti-PARP antibody was a gift from Mark Dempsey, Dept of Pharmacology, University College Dublin, Ireland The antι-p38, antι-p42-44 and anti-JNK phospho and total antibodies were purchased from New England Biolabs The Topo II inhibitor, ICRF 187, the lethal toxin and the plasmid expressing JTP were all obtained from Prof L O'Neill, Biochemistry Dept , Trinity College Dublin Antibodies were obtained from the following souices anti-BCL-2 from Santa Ciuz, anti -BCL- XL from Transduction Labs, Phospho specific ATF-2 and c-jun antibodies from New England Biolabs The DMR1E reagent was purchased from Gibco OPNG B-Gal substate was from Sigma Methods Cell culture and cytospin analysis of apoptosis HL-60 human promyelocytic leukaemia cells were maintained as logarithmically growing cultures in 80% RPM1 1640 medium and 20% heat-inactivated fetal calf serum (FCS) in a humidified incubator widi 95% air, 5% C0 2 at 37°C. The HUT 78 human T cell lymphoma, the Jurkat human leukaemic T cell lymphoblast cells, the CEM cells, and die three CML cells were grown under the same conditions as above with the exception of the FCS which was at 10%. HeLa cells were maintained as logarithmically growing cultures in 90% DMEM (Sigma) and 10% FCS in a humidified incubator with 95% air and 5% CO 2 at 37°C. These cells adhere to the culture flask and were removed from the surface of the flask by incubation with trypsin (Sigma) (0.025% vv/v) in DMEM for approximately 1 niin. Growth medium (10ml) was then added to inhibit the action of trypsin. The cells were reseeded into culture flasks at the appropriate density. MCF-7 cells were maintained in minimum essential medium supplemented with non-essential amino acids (1% v/v), glutamine (ImM), FCS (10%) and sub-cultured by the protocol described for Hela cells. In all experiments performed, cells were seeded at a density of 3x10-* cells/nil into 24-welI plates (1 ml well) and immediately treated with the indicated compound. All drugs were diluted from freshly prepared ethanol stock solutions. The final concentration of ethanol in the wells was always 1% (vol/vol); a concentration which by itself had no effect on the cells tested. Following exposure to the appropriate drug, the cells (lOOμL) were cytospiui onto glass slides pre-coated with poIy-L-lysine using a Cytospin 3 *™ cytocentrifuge (Shandon). They were then stained using the Rapi-Diff kit (eosinmelhylene blue) under the conditions described by the manufacturer. The percent apoptosis and necrosis was deleπnined by counting approximately 300 cells under a light microscope with the aid of a graticule inserted into the eyepiece. At least 3 fields of view per slide, with an average of approximately 100 cells per field, were counted. Assay for NF kappa B binding activity - Cells were fractionated, and nuclear extracts were prepared as described by Slylianou et al. (1992). Briefly, cells were added to ice-cold phosphate-buffered saline and centrifuged using a swing out rotor al 136 X g. They were washed in buffer A (10 mM Hepes, pH 7.9, 1.5 mM MgCl 2 , 10 mM KC1, 10 mM phenylmethylsulfonyl fluoride [PMSF]) and centrifuged at 10 000 X g for 10 niin. They were treated wilh buffer A + 0 1% (vol/vol) Nonidet P40 on ice for 10 niin. They were then centrifuged at 10 000 X g for 10 min and the pellet was heated with buffer C (20 mM Hepes, pH 7.9, 420 mM NaCl, 1.5 mM MgCI 2 , 2 mM EDTA, 20% [vol/vol] glycerol, 0.5 mM PMSF) on ice for 15 min. This material was centrifuged at 10 000 X g for 10 min and die supernatant was added to 75ul of buffer D [lOmM Hepes, pH 7.9, 50 mM KC1, 0.2 M EDTA, 20% (vol/vol) glycerol, 0.5 mM PMSFJ per sample. The protein concentration in this crude nuclear extract was determined using the method of Bradford (1976). Protein (4 μg) was incubated with 10 000 cpm of a 32 P-labelled oligonucleotide, containing the consensus sequence for NF kappa B, with binding buffer [40% (vol vol) glycerol, 1 mM EDTA, 10 mM Tris, pH 7.5, lOOmM NaCl, 0.1 mg/ml nuclease free BSA] and 2 μg of poly(dl-dC) at room temperature for 30 min. Samples were resolved on a 5% native polyacrylamide gel which was subsequently dried and autoradiographed at -70°C overnight. Assay for caspase 3 using Western blotting - Whole cell extracts from approximately 1X10? cells were isolated as follows. Cells were centrifuged at 600 X g for 5 min and the resulting pellet washed in sterile PBS (1ml) and transferred into a minifuge tube. The sample was again centrifuged at 600 X g for 5 min and the supernatant carefully removed. The pellet was resuspended in 0.2 ml of ice-cold RIPA [PBS, 1% (vol/vol) Nonidet P40, 0.5% (wt vol) sodium deoxycholate, 0. 1% (wt/vol) sodium dodecyl sulphate (SDS), lOOμM PMSF, 3% (vol vol) aprotinin and 100 μM sodium orthovanadate] and incubated on ice for 30 min. The cells were then homogenised by passage through a 21G needle (5-7 tunes) on ice. Following a further incubation on ice for 30 min, die sample was centrifuged in a minifuge at 4 C C for 20 min. Supernatant (150μl) was removed into fresh minifuge tubes (whole cell lysate) and the protein concentration determined by the method of Bradford (1976). Extracts were frozen at -70°C until use. The volume of each thawed sample was adjusted with RIPA so diat equivalent amounts of protein were present in each sample. Samples were resolved on a 15 % polyacrylamide gel containing 0. 1% (wt/vol) SDS and immediately transfeπed onto nitro-celhilose membrane. The membranes were blocked overnight in PBST (PBS containing 0.05% [vol/vol] Tween-20) containing 5% (wt/vol) powdered milk (Marvel), incubated for 1 h with anti-procaspase 3 diluted (1: 1000) in 5% (wt/vol) Marvel/TBST and then washed 3 times with PBST for 5 min each. This was followed by the incubation of the membranes in goat anti-mouse horseradish peroxidase-coupled secondary antibody at a dilution of 1: 1000 in 5% (wt/vol) Marvel/TBST, for 45 min. Enhanced chemiluminescence (ECL) was used to visualise the cross-reacting bands, following a protocol described by the manufacturer. Measurement of Cytochrome C by Western Blot - Cells (15 x 10 6 ) were harvested by centrifugalion at 1800g for 10 min at 4°C. After being washed once with ice-cold phosphate-buffered saline, the cell pellet was suspended in lOOμl of ice-cold buffer A (20mM Hepes, pH 7.5, lOmM KC1, 1.5ιnM MgCl 2 , l M sodium EDTA, ImM sodium EGTA, ImM dithiotreitol and 0.1 mM phenylinethylsulfonyl fluoride) supplemented with protease inhibitors (5μg/ml pepstatin A, lOμtg/ml leupeptin and 2μg/ml of aprotinin). The cells were left to sit on ice for 15 min and then centrifuged at 20,000g for 20 min. The resulting supernatant was stored at -70°C until measurement of cytochrome C. Protein determination was measured by using the Bradford assay. Equal amounts of protein were resolved by SDS-polyacrylatnide gel electropho esis in 15% gels and transferred onto nitro-cellulose. Membranes were blocked with phosphate-buffeied saline/5% (w/v) dry milk and probed with anti- cytochrome C antibody. Blots were washed, incubated with goat anti-mouse IgG peroxidase conjugate and developed by ECL according to the manufacturer's recommendations. Measurement of protein tyrosine phosphoi ylatiυn by Western Blotting - Jurkat and CML cells (5 x 10 s ) were harvested by centrifugation at 1800g for 10 in at 4°C. After being washed once wiUi ice- cold phosphate-buffered saline, the cell pellet was suspended in lOOμl of ice-cold lysis buffer (50mM Tris-HCl, pH 8.0, 150mM NaCI, 0.1%. SDS, 1% Triton X-100, ImM sodium ImM phenylniethylsulfonyl, lOng/ml leupeptin and lOng/ml of aprotinin). The cells were left to sit on ice for 10 min and then centrifuged at 20,000g for 20 min. The resulting supernatant was stored at -70°C until measurement of tyrosine phosphorylation. Protein deteπnination was measured by using the Bradford assay. Equal amounts of protein were resolved by SDS -polyacrylamide gel electrophoresis in 15% gels and transferred onto polyvinyhdene difluoride (PVDF) membranes. Membranes were blocked with Tris- buffered saline with Tween 20 (lOmM Tris-HCl, pH 8.0, 150mM NaCI, 0.05% Tween 20) containing 1% w/v BSA and probed with anti-phosphotyrosine antibody. Blots were washed, incubated with goat anti-mouse IgG peroxidase conjugate and developed by ECL according to the manufacturer's recommendations. Radioligand Binding Assays - HL-60 cells were harvested by centrifugation at 600g for 5 min and washing in phosphate-buffered saline. The resulting cell pellet was homogenised in 50mM Tiis/HCl buffer, pH 7.4 (2ml), using an Ultraturrax homogeniser (lOsec) and then passed 5 times dirough a 21 gauge needle. Cell homogenates (50μg protein) were incubated with 0.5-50nM [ 3 H] PK 11195 in 50nιM Tris/HCI buffer, pH 7 4 (incubation buffer), in a total volume of 0.5ml on ice. Total and non- specifϊc/non-saturable binding in each case was determined in the absence and presence of lOμM unlabelled PK 11195 respectively. All samples were incubated in triplicate for 60 min. The incubation mixtures were then filtered and counted as previously described (O'Beirne, et al., 1988). When testing the potency of a compound to inhibit [ 3 H] PK 11195 binding, samples were incubated with 2nM [ 3 H] PK 11195 and various concentrations (0.1 nM to lμM) of compound and subsequently treated exactly as above. The resulting Ki values were then generated by the use of the computer programs EBDA and LIGAND. Caspase 3 Fluorogenic Assay - Cells (5 x 10 6 cells) were harvested by centrifugation, washing in ice- cold phosphate-buffered saline and the pellets resuspended in 200μl of harvesting buffer (20mM Hepes, pH 7.5, 10% (w/v) sucrose, 0.1% (w/v) CHAPS, 2mM dithiotreitol, 0.1% (v/v) Nonidet NP40, ImM sodium EDTA and ImM phenylniethylsulfonyl fluoride) supplemented with protease iidiibilors (Iμg/ml pepstatin A and lμg/ l leupeptin). Following incubation on ice for 10 min, samples were passed up and down 10 times tluough a 21 gauge needle. Following a further incubation on ice for 10 min the homogenates were centrifuged at 20,000g for 20 min and the resulting supernatants used to measure caspase 3 activity. This enzyme activity was determined by a fluorometiic assay using the substrate Ac- DEVD-AMC, which is cleaved by caspase 3 to release the fluorescent leaving group, amino-4-methyl couniarin (AMC). Enzyme extracts (50μg) were incubated with lϋOmM Hepes, pH 7.5, containing 10% (w/v) sucrose, 0.1% (w/v) CHAPS, lOmM dithiotreitol and 20μM substrate in a total reaction volume of 3ml Following incubation for 60 min at 25°C, fluorescence was monitored continuously using a spectrofluorimeter (excitation wavelength 380nm, emission wavelength 460nm). The amount of AMC released was deteπuined by comparison with a standard curve generated with known amounts of AMC. Agarose gel electrophoresis to detect DNA ladders - CML cells were seeded at lxl 0 7 cells in 20ml of growth medium and treated with ethanol (1% v/v) or PBOX-6 for the required time period at 37°C. Cells were centrifuged at 500g for 5 minutes and the supernatant removed. The pellet was resuspended in lysis buffer (1ml) (20mM EDTA, lOOmM Tris pH 8.0, 0.8% (w/v) sodium lauryl sarcosinate) and incubated at 37°C for t hour. After this incubation, RNAse A was added to each tube (0.5mg/ml), and left at 37°C for a further 2-2.5 lirs. Proteinase K (6mg/ml) was then added to each sample and tubes were incubated overnight at 37°C 45μL of each sample was mixed with 5μl of loading dye and samples were run on a 1.5% agarose gel in TBE for 4 hours at 55V. lOμl of Ethidium bromide (lOmg ml) was added into lOOml agarose. DNA ladders were visible under UV light. Western Blot analysis of BCR-abl - Whole cell extracts from approximately 7xl0 6 cells were isolated as follows. Cells were centrifuged at 500g for 5 minutes and die resulting pellet washed in lOmls of PBS. The sample was again centrifuged at 500g for 5 minutes. The supernatant was removed and the pellet was resuspended in 0.2ml of harvesting buffer (20mM Hepes pH 7.5, 10% (w/v) sucrose, 0.1% (w/v) CHAPS, 2mM DTT, ImM EDTA, ImM PMSF, lμg/ l leupeptin, lμg/ml pepstatin) and transferred to a minifuge lube. Samples were incubated on ice for 10 min. The cells were homogenised by passing through a 21G needle 12 times, and samples were incubated on ice for a further 5 nun. Samples were then centrifuged at 20,000g for 10 min at 4 C. The supernatant was carefully removed to a fresh tube. Protein concentration was determined by the Bradford method. Extracts were frozen at -20 C until use. The volume of each sample was adjusted with harvesting buffer to obtain equal amounts of protein in each sample. Protein was resolved on an 8% polyacrylamide gel and transferred onto PVDF membrane overnight at 4 C using wet transfer apparatus. Following transfer, the membrane was blocked for 21ιr at room temperature in PBS containing 5% dry milk. The membrane was then washed 2 2 niin in PBST followed by 2hr incubation at room temperature with anti-c-abl, 1 : 100 dilution in PBST containing 1% dry milk. The membrane was then washed for 20 min in PBST. Following washing, the membrane was incubated for llir with goat anti mouse horseradish peroxidase coupled secondary antibody at 1: 1000 dilution in PBST containing 1% dry milk. ECL was used lo visualise cross reacting bands, following the manufacturer's instructions. Western Blot analysis of PARP (poly ADP-Ribose polymerase) cleavage - CML cells were seeded at 2xl0 6 cells in 5mls of growth mediiun, and treated widi lOμM PBOX-6 for 16 and 24 hr. Hela cells were seeded at lx lO 6 cells per 25 cm 2 flask. Following a 24 hour incubation, the cells were treated with PBOX-3 for a further 48 hours and the cells were scraped up. Cells were then cenlrifuged at 500g for 5min, the supernatant was removed and the pellet was resuspended in 200μl of PARP sample buffer (62.5 mM Tris/HCl pH 6.8, 6M urea, 10% (w/v) glycerol, 2% (w/v) SDS, 0.00125% bromophenol blue, and 5% (v/v) β-mercaploelhanol was added immediately before use). Samples were sonicated for 15 sec before aliquoting into 15μl volumes. Samples were stored at -20°C until use. 10-15μl of thawed samples were resolved directly on a 10% polyacrylamide gel and transferred onto PVDF membrane at room temperature using wet transfer apparatus. After transfer, the membrane was blocked overnight in PBS containing 5% dry milk (Marvel). After blocking, the membrane was washed in PBST for 2 x 2 nύnules before incubating for lhr with anti-PARP antibody (2μg/ml in PBST containing 1% dry milk). Following incubation the membrane was washed for 20 minutes in PBST and incubated with goat anti mouse HRP conjugated secondary antibody (1 1000 dilution in PBST containing 1% dry milk). After washing in PBST for 30 minutes, ECL was used to visualise the proteins. The full enzyme is represented by a 119kDa band while the cleavage products are repiesenled by 29kDa and 87kDa bands. Measurement of DNA strand breaks. Alkaline elution assay to measure DNA strand breaks - This assay was set up as described by et al., 1976 with the following modifications. Cells were seeded at 0.4x10 6 cells/ml and were in die logarithmic phase of growth. The doubling time was approximately 16 hours. Cells harvested for the experiments were at a density of 2xl0 6 cells/ml. Cells (IxlO 6 ) were labelled with [ 3 HJ thymidine (sp. act. 2Ci mmol) (0.2μCi ml) and lμM unlabelled thymidine, and incubated for 20 hr. Cells were harvested by centrifugation at 500xg for 5 mins following by two washes in PBS and the pellet was resuspended in 3ml of PBS. Cells were aliquoted into 1ml amounts in a 24 well plate and allowed to recover for 1 hr at 37°C before treating with indicated compounds. Following treatment, cells were washed once in PBS and resuspended in 250μl of PBS. An aliquot of cells (200μl) was loaded directly onto a 25 mm diameter glass fibre filler, pore size of 0.2μM. The cells were lysed on the filter by the addition of 5ml lysis buffer (2% (w/v) SDS, lOmM disodium EDTA (pH 10) supplemented with 0.5mg/ml proteinase K. The lysis buffer was allowed to flow slowly through the filter without suction. Filters were washed using 3ml wash solution (0.02M Na 3 EDTA pH 10) allowing it to flow without suction. Filters were eluted in the dark with an alkaline solution (0.04M EDTA plus tetrapropylammonium hydroxide (10% in H 2 0) added in the amount required to give pH 12.1) The elution apparatus consisted of a filter funnel with output tubing passing through a peristaltic pump to a collector. Elution took place overnight at a flow rate of 0.05 ml/niin. Following elution, filters were treated with 0.4ml HC1 (IN) for 1 hr at 70°C followed by 2 5ml NaOH (0.4N) for 30 min at room temp Scintillation fluid (10ml) was added and radioactivity remaining on the filters was determined by scintillation counting. Liquid scintillation counting - The [ 3 H] iadioactivity was measured using eitlier a Packard Tricarb 300 or a Packard 1500 scintillation counter. The sciπtillant cocktail used was the commercial scintillant Ecoscint. The average counting efficiency for [ 3 H] counting in the Packard Tricarb 300 was calculated to be 45 %, based on a quciich-correction curve relating counting efficiency to channels ratio. The same counting efficiency was achieved in the 1500 model scintillation counter and this was calculated from a quench curve relating counting efficiency to the spectral index of die external standard. Western blot to detect activation of MAPK's - Cells (6xl0 6 ) were harvested by centrifugation at 500xg for 5 min and pellets were washed with ice cold phosphale buffered saline containing sodiiun orthovanadate (Na 3 V0 ) (ImM). The pellets were resuspended in ice cold lysis buffer (150mM NaCI, SOniM Tris/Cl pH 8.0, 0.1% (w/v) SDS, 1% (w/v) Triton X-100, ImM sodium orthovanadate, lμg/ml leupeptin, ImM phenylniethylsulfonyl fluoride and lOμg/ml aprotinin). Samples were incubated on ice for 20 min followed by centrifugation at 20,000xg for 10 min at 4°C. The resultant supeπialants were removed, stored at -70°C until use and used to measure activation of the phosphorylated form of eitlier JNK, p38 or p42-44. A Bradford assay was used to determine protein concentration, and equal amounts of protein was resolved on a 10% SDS-polyacrylamide gel. Proteins were transferred onto PVDF membrane, and blocked overnight with Tris buffered saline (lOmM Tris/Cl pH8.0, 150mM NaCI) (TBS) containing 5% (w/v) fatty acid free BSA. Membranes were washed with TBS for 5 ruins and probed widi eidier anti-JNK phospho, anti-p38 phospho, or anti-p42-44 phospho antibodies, diluted 1: 1000 in TBS containing 0.5% Tween 20 (TBST) and 5% (w/v) BSA. Membranes were washed in TBST for 30 min and incubated widi goat anti-rabbit horseradish peroxidase coupled secondary antibody, diluted 1:2000 in TBST containing 5% (w/v) BSA. Membranes were washed for 1 hour and ECL was used to visualise cross-reacting bands. Membranes which were probed with anti-JNK phospho were subsequently stripped by incubating for 30 min at 50°C in a stripping buffer (62.5mM Tris pH 7.8, l OnM β-mercaptoethanol and 2% SDS (w/v)). Membranes were tiien blocked for 1 hour and incubated with anti-JNK total antibody as a loading control. Bone Marrow colony assay - Bone Marrow samples from donor patients were suspended in RPMI and heparin. Approximately 5mls of Lympoprep was poured into a sterilin and tiie bone marrow sample was gently layered on top. The sample was centrifuged at 1800g for 25 inin, brake at 2. Following centrifugation, die buffy coat layer which contained the mononucleur fraction was gently removed using a sterile pasteur pipette and was transferred to a fresh tube. Approximately lO ls of RPMI was added and the sample was centrifuged at 1800g for 10 minutes, brake at 9. The supernatant was removed and the wash repeated. The pellet was resuspended in approximately 2mls of RPMI. 20μl was removed from the sample, mixed widi 380μl of ethidium bromide/acridine orange nιix.(each at lOOμg/ml in PBS, 1 lv/v). Ethidium bromide and acridine orange are fluorescent dyes which bind DNA. Acridine orange intercalates into DNA giving it a green appearance. Ethidium bromide is taken up by non-viable cells only. This dye also intercalates into the DNA, making it appear orange. The cells were counted under a fluorescent microscope and the volume of cells was adjusted to obtain 4x10 cells per ml. Cells were divided into 2 flasks and treated with 0.5% (v/v) ethanol or lOμM PBOX-6. lOOμl was taken from each flask and added to 1.9mls of culture media (0.58ml methylcellulose, 0.55ml FCS, 0.18ml BSA, 0.21ml RPMI, 5U/ml of granulocytoe monocyte-colony stimulating factor (GM-CSF) and 20U/ml of erylhropoietin (EPO). Tubes were mixed by vortexing and allowed to stand for 1 min before adding 0.5ml into each of Uuee wells of a 4 well dish. 0.5ml of sterile water was added to the fourth well. Plates were incubated at 37°C for 14 days. After 14 days, colonies were scored. A colony is scored as a cluster containing >40 cells, which originates fonn one stem cell. Western blot to detect phosphoi ylation of BCL-2 and BCL-XL. Cells (4xl0 6 ) were harvested by centrifugation at 500xg for 5 min followed by washing in ice-cold phosphate-buffered saline (PBS). Pellets were resuspended in lOOμl of ice-cold lysis buffer (50mM Tris- HCl, pH 8.0, 150mM NaCI, 0 1% (w/v) SDS, 1% (w/v) Triton X-100, ImM sodium orthovanadate, Iμg/ml leupeptin, lniM phenylniethylsulfonyl fluoride and 10 μg/ml aprotinin). Cells were incubated on ice for 10 min followed by passage 12 times through a 21-gauge needle. Following a further 10 in incubation on ice the homogenates were centrifuged at 20,000xg for 20 min at 4°C. Supeπiatants were removed and protein concentration was determined using a Bradford assay. Equal amounts of protein were resolved on a 10% SDS-polyacrylamide gel and transferred onto PVDF. Following transfer, the membranes were blocked in PBS containing 5% (w/v) dried milk and probed with anti-BCL-2 or anti- BCL-XL antibodies. Blots were washed and probed with horseradish peroxidase coupled secondary antibody and developed usmg ECL detection according to manufacturer's instructions. Western blot to detect phosphorylation of ATF-2 and c-jun. K562 cells were seeded at 4x10 cells per sample and treated with the relevant compounds. Cells were harvested by centrifugation at 500xg for 5 mins and pellet was resuspended in lOOμl of SDS lysis buffer (62.5 mM Tris (pH 6.8), 2% w/v SDS, 10% glycerol, 50mM DTT, 0.1% w/v bromophenol blue). Extracts were sonicated for 5 sec and samples were heated to 95-100 c C for 5 mins followed by centrifugation for 5 in in a minifuge. An aliquot (20μl) was loaded directly onto an SDS-PAGE gel. Proteins were transferred onto PVDF and blocked for 1-3 hrs at room temp in PBS containing 5% (w/v) dried milk and probed overnight at 4°C with phospho- ATF-2 or phospho c-jun antibodies. Blots were washed and probed with horseradish peroxidase coupled secondary antibody and developed using ECL detection according to manufactiuer's instructions. Transformation of E.coli cells and extraction of plasmid DNA Competent E coli SCS-1 cells were incubated with DNA from the relevant plasmids (0.5μg), and incubated on ice for 2-5 hours. Cells were heat shocked for 2 min, at 37°C and tubes placed back on ice.L Broth (1% w/v tryptone, 0.5% w/v yeast extract, 0.5% w/v NaCI) (0.8ml) without antibiotic was added to each tube and incubated at 37°C for 60-90 min. Cells were plated onto selective agar plates, and incubated at 37°C overnight. A colony was selected from the plates and lOOmls of sterile L broth containing the appropriate antibiotic for selection, was inoculated and incubated overnight in a 37°C shaker. Plasmid DNA was extracted using Sigma Genelute Maxiprep kit according lo manufactiuer's instructions. The resulting high quality DNA was stored at -20°C until use Transfection ol K562 cells using DMRIE-C. K562 cells were transfected in 24 well plates using a cationic lipid reagent (DMRIE-C). 4μl of reagent was mixed with lOOμl of RPMI medium (serum- and antibiotic-free). 400ng of total DNA was added in lOOμl RPMI, mixed gently and allowed to stand at room temp for 15-45 mins to allow foπnation of Iipid- DNA complexes. Cells (4x10 5 ) were added to die plate in 40μl RPMI, mixed gently and incubated at 37°C for 4-5 hours RPMI containing 20% FCS (400μl) was added to each well, and cells were left overnight at 37°C to recover. Cells were treated with either vehicle (1% ethanol) or PBOX-6 (lOμM). Measurement of Luciferase activity Lysales were prepared by harvesting cells by centrifugation at 500xg for 5 min followed by a wash with PBS. Pellets were then lysed in 50μl of lx Passive lysis solution (Promega), and samples were incubated at room temp on a shaker for 15 mins. An aliquot (20μl) was removed to a 96 well plate, 40μl of luciferase assay mix added and activity measured using a luminometer. Measurement of B-Galactosidase activity. K562 cells were traπsfected witii a plasmid expressing the lac Z gene and B-Galactosidase activity was measured by adding 40μl OPNG substrate (O-nitiophenyl-B-D-galactopyraiioside) and 140μl B-Gal buffer (23mM 77mM Na 2 HP0 4 , 0. ImM MnCl 2 , 2mM MgS0 , 40mM B-mercaptoethanol pH 7.3) to 20μl of cell lysate prepared as for luciferase activity (above), in a 96 well plate. B-Gal activity was measured at 405nm using a spectophotometer. RESULTS A number of pyrrolobenzoxazepines and pyrrolobenziazepines tested were found to induce apoptosis in HL-60 cells (see Table 2 for a summary of apoptosis). The characteristic morphological effects of apoptosis i.e. shrinkage of cells, extensive membrane blebbing, condensation of cliromatin and DNA fragmentation, were observed in these cells (Fig. 1). In order to make a direct comparison of potencies, all the PBR ligands weie tested in HL-60 cells (human pro yelotic leukaemia) in the same experiment • at a single concentration (lOμM). Of the PBOX compounds tested in HL-60 cells PBOX numbers 4, 6, 7, 8, 9, 15, 16, 65, 70 and 71 were found to be die most potent apoptotic inducers (Figs. 2A and 2C). After treatment of HL-60 cells for 16 horns with a final concentration of lOμM of these drugs, the cells exhibited between 38 and 55% apoptosis. The degree of necrosis obsei ved under the same conditions was negligible. At the same time odier members of the PBOX series such as PBOX-1 and 2 elicited no effect on cell viability even at the highest concentration tested (50mM, limits of solubility). Some of the more widely known PBR ligands, PK 11195 and Ro5-4864 also induced apoptosis in HL 60 cells, albeit at a higher concentration (lOOμM) (fig. 2B) These compounds did not induce apoptosis at a concentration of lOμM (fig. 2B). Many of the subsequent experiments were performed using PBOX-6 as a representative "model" apoptotic pyrrolo-l ,5-benzoxazepine. PBOX-6 caused a dose- (Fig. 3A) and time-dependent (Fig. 3B) induction of apoptosis in HL- 60 cells. The nioiphological signs of apoptosis became apparent at 6 hours and increased linearly up to 16 hours. Apoptosis was negligible below a final concentration of 5μM of the drug. PBOX-6-induced apoptosis also resulted in DNA fragmentation. When die DNA from HL-60 cells incubated with PBOX- 6 for 24 hours was analysed by agarose gel electrophoresis it generated a characteristic "ladder pattern" of discontinuous DNA fragments (Fig. 3C). Several studies have demonstrated activation of caspases in different pathways of apoptosis (Polverino and Patterson, 1997) To directly address the involvement of caspase 3 -like proteases in PBOX-6-mediated apoptosis, we studied caspase 3 -like activity in HL-60 cells following PBOX-6 treatment. Cytosolic extracts from HL-60 cells treated with PBOX-6 were incubated with the fluorogenic caspase 3 -like substrate DEVD-AMC As shown in Figure 4A, treatment of cells with PBOX-6 caused a dose-dependent activation of caspase 3-like proteases. This protease activity became evident at 6 hours and increased linearly up to 16 hours (Fig. 4B). This dose-dependent and time- dependent activation of caspase 3-like proteases directly parallels the observed morphological effects of apoptosis induced by PBOX-6, as determined from cytospinning and staining of cells. This result was confirmed by Western blotting, demonstrating that PBOX-6 induces, in a dose-dependent manner, the processing of pro-caspase 3, as monitored by the disappearance of die 32kDa form of the enzyme (Fig. 5), and this correlates with the appearance of the morphological signs of apoptosis. Caspases are specifically inhibited in vitro and in vivo by cell-peπneable tetrapeptides designed lo mimic cleavage sites of their respective substrates (Nicholson et al, 1995). Pre-treatment of HL-60 cells for 1 hour with a caspase 3-like protease inhibitor, z-DEVD-fmk, followed by treatment for a further 8 hours with PBOX-6, inhibited bodi the activity of caspase 3-like proteases (Fig. 4C), and die appearance of the morphological signs of apoptosis (Fig. 4D). This would suggest tiiat activity of caspase 3-like proteases is an essential part of the mechanism by which PBOX-6 induces apoplosis in HL-60 cells Previous studies have shown that accumulation of Cytochrome C in Ihe cytosol occurs in response to multiple apoptotic stimuli and that this released Cytochrome C in turn activates caspase 3, thus playing an important part in inducing apoptosis (Kluck et al., 1997). The effect of PBOX-6 treatment of HL-60 cells on accumulation of Cytochrome C in the cytosol was analysed. Treatment of HL-60 cells with PBOX-6 under conditions of induction of apoptosis (Fig. 6) caused an accumulation of Cytochrome C in the cytosol, suggesting that its release may be important in the mechanism by which PBOX-6 induces apoptosis. Several observations suggest an involvement of reactive oxygen intermediates (ROI) in the signal transduction pathways leading to apoptosis, and that they lie upstream of Cytochrome C release and caspase 3 activation. (Jacobson, 1996). In order to determine whether the induction of apoptosis in HL-60 cells by PBOX-6 involved the production of ROI, these cells were pie-treated with either a commonly used antioxidant, N-acetylcysteine (5mM), or the free radical scavenger, TEMPO ( 1 μM), for 30 min prior to incubation widi PBOX-6 for a further 8 hours (Fig. 7). Bodi NAC and TEMPO, at the concentrations utilised, have previously been shown to prevent the foπnation of ROI in HL-60 cells (Kakeya et at, 1998). Neither of these compounds were found to protect against PBOX-6 induced apoptosis, suggesting that die mechanism by which this compound causes apoptosis in HL-60 cells does not involve the production of ROI. During apoplosis, Cytochrome C has been reported to exit the mitochondria tluough the mitocliondrial porin channel (also called the voltage-dependent anioπ channel or VDAC) (Shimizu et al, 1999). As VDAC is associated with the PBR in the mitocliondrial outer membrane (McEnery et al, 1992), and as PBOX-compounds can bind with high affinity to the PBR we examined whether this receptor was involved in the mechanism by which these compounds cause apoptosis. HL-60 cell homogenates displayed saturable, high affinity binding of [ 3 H] PK 11195, a selective ligand for the PBR, yielding Kd and Bmax values of 17.7 ± 5.0nM and 9.5 ± 1.5 p ol/mg of prolein, respectively. All of the PBOX compounds tested were shown to inhibit [ 3 H] PK 11195 binding to HL-60 homogenates with Ki values between l-7nM (Table 3), yet micromolar concentrations of these compounds were required to induce apoptosis. All the ligands totally inhibit [ 3 H]PK 11195 binding at high concentrations showing them to be fully competitive inhibitors. In addition, although PBOX-1 and PBOX-2 bind to the PBR with Ki values in die nM range, these drugs did not induce apoptosis. Furtheπnore, the effect of PBOX compounds on a human T Jurkat cell line was examined, a cell line previously shown to the lack the PBR (Carayon et al., 1996), an observation confirmed by us by ligand binding studies (results not shown). Figure 8 shows that apoptosis is induced in Jurkat cells treated with PBOX compounds, with similar potency to that observed in treated HL-60 cells. These results suggest that the mechanism by which these PBOX compounds induce apoptosis does not involve their interaction with the peripheral- type benzodiazepine receptor. NF B activation has been implicated in induction of resistance of tumour cells to apoptosis (Giri and Aggarwai, 1998). Pre-treatment of HL-60 cells with TNF for 30 min, at a concentration which activated NFKB but did not induce apoptosis, followed by treatment with PBOX-6 for 16 hours, afforded no protection against PBOX-6-induced apoptosis (Fig. 9A). We dien examined a human Sezary lymphoma cell line, Hul-78, which constitutively expresses NFKB, and as such reportedly causes these cells to be resistant to apoptosis (Giri and Aggarwai, 1998). The five PBOX drugs tested induced apoplosis in Hul-78 cells with similar potency to that observed in HL-60 cells (Fig. 9B). In addition, PBOX-6 had no effect on NFKB in these cells (Fig. 9D). These results would ύnply that activation of NFKB does not necessarily cause resistance to apoptosis by all apoptotic inducing drugs. The activation of the transcription factor, NFKB, is initiated by a wide variety of stress stimuli such as cei amide and H 2 0 2 , which themselves cause apoptosis (Baeuerle and Henkel, 1994). We tested whether PBOX-6 itself could affect NFKB expression in HL-60 cells. We used TNF treatment as a positive control for activation of NFKB in tiiese cells. Although TNF, in agreement with other reports, was shown to activate NFKB in HL-60 cells, as was demonstrated by the detection of protein-DNA complexes in nuclear extracts, PBOX-6 failed to affect NFKB expression (fig 9C). These results would suggest that PBOX-6 induced apoptosis employs an NFicB-independent mechanism The mitogen activated protein kinase family (MAPK's), which includes JNK have previously been shown to be activated during signal transduction in many cells in response lo various foπns of stress and has recently been implicated in apoptosis (Denhardt, 1996). In order to determine the effect of PBOX-6 on the activation of JNK, a Western blot assay was developed. Irradiation of HL-60 cells with UV light has previously been shown to activate JNK and was thus used as a positive control in this assay. HL-60 cells were treated with vehicle or PBOX-6 for various lengdis of time It was found that PBOX-6 induces a transient activation of 2 JNK isofoπns, JNK1 and JNK2, which peaks at 1 hour (Fig.10). Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 can prevent cells from undergoing apoplosis in response to a variety of stimuli. PBOX-6 caused a dose-dependent induction of apoptosis in human CEM (T lymphoblastoid) cells, both in normal (Fig. 11) and in CEM cells overexpressing the Bcl-2 protein (Fig. 12). Some benzoxazepine compounds were found to induce apoptosis in three CML (chronic myeloid leukaemia) cell lines, K562, KYO. l and Lama 84. A series of these compounds were tested at lOμM and it was found that after a 16 hour treatment PBOX-3, 4, 5, 6 and 7 were the most potent of those tested, inducing up to 50 % apoptosis (Fig. 13A). Some other benzoxazepines such as PBOX-1 and 2 failed to induce apoptosis in these cells at the same concentration. It was also found tiiat some widely known PBR ligands such as PK 11195 and Ro5-4864 failed to induce apoptosis in CML cells at a similar concentration, however, apoptosis was induced, although not to die same extent, at the higher concentration of lOOμM (Fig. 13B). PBOX-6 induced a time (Fig. 14A) and dose-dependent (Fig. 14B) induction of apoptosis in the three CML cell lines, K562, KYO.l and Lama 84. The morphological features of apoptosis, which include cell shrinkage, chromalin condensation, DNA fragmentation and the production of apoptotic bodies were visible in all cell lines. DNA fragmentation is considered to be the hallmark of apoptosis, and produces 180-200 bp internucleosomal DNA fragments which are visible as "DNA ladders" on a gel. This DNA laddering pattern was visible on an agarose gel when DNA was extracted from all three CML cell lines following treatment widi PBOX-6 (Fig. 15). The effects of PBOX dosage on the ability of these compounds to induce apoptosis in K562 cells was examined. After treatment for 16 hours widi a range of concentrations of PBOX compounds, PBOX-15 and PBOX-16 were found to be the most potent of the compounds tested in these CML cells, inducing over 60% apoptosis at a final concentration of 0 5 μM (Table 5) Expression of the BCR-abl fusion protein in CML cells has been suggested to be responsible for die resistance of CML cells to many apoptotic agents. To investigate the effect of PBOX-6 on BCR-abl expression, levels of BCR-abl were deteπnined by Western analysis of protein extracts isolated from control and PBOX-6 treated CML cells In K562 and KYO. l cells, downregulation of BCR-abl was detected, but not until 24 hours after PBOX-6 treatment (Fig.16 A and B), where approximately 50% of cells had undergone apoplosis. Downregulation of BCR-abl was not detected in Lama 84 cells up to 72 hours after treatment with PBOX-6 (Fig. 16C). Detection of β-actin was used as a loading control. These results suggest that downregulation of BCR-abl is not involved in the initial upstream events associated with PBOX-6 induced apoptosis. Release of cytochrome C from mitochondria has been suggested to be important in triggering apoplosis. To determine the role of cytochrome C in PBOX-6-mediated apoptosis in CML cells, K562, KYO. l and Lama 84 cells were exposed to die compound for 16 hours, and its effect on the accumulation of cytochrome C in the cytosol was deteπnined. Cytosolic fractions were isolated and analysed for levels of cytochrome C by Western blotting It has previously been shown thai staurospoπne treatment of HL-60 cells causes the release of cytochrome C into the cytosol Therefore, treatment of HL-60 cells widi 1 μM slaurospoπne for 6 hours was used as a positive control in setting up the assay (Fig 17A) ft was found that cytochrome C levels in die cytosol were unchanged in CML cells after a 16 hour treatment with PBOX-6 (Fig 17 B, C, D) Cytochrome C was evident in die cytosol of untreated CML cells probably due to non-specific release diuing sample preparation In order to ensure that cytochrome C was not all non-specifically released during preparation of the cytosohc fractions, the remaining pellet was solubhsed and analysed for Cytochrome C levels Western blots showed that the majority of cytochrome C remained in the pellet (Fig 18) These results indicate that the release of cytochrome C from mitochondria may be important for triggering apoptosis in some cells in response to certain agents but does not occur during PBOX-6 mduced apoptosis m CML cells Caspase 3 is one of the most intensely studied cysteine proteases and is believed to play a role of most downstream executioner m many apoptotic pathways Control and apoptotic cell lysates from CML cells were isolated and examined for activated caspase 3 status, using a fluorogenic assay in which active caspase 3 will cleave a substrate (z-DEVD-AMC) and release the fluorogenic group (AMC), which can subsequently be measured using a spectrofluormieter The amount of AMC released was calculated from a standard curve of AMC, prepared usmg known concentrations In the current study' it has been shown diat after 16 horns treatment with PBOX 6, caspase 3 is activated m two CML cell lines, K562 and Lama 84 (Fig 19A) Caspase 3 activation coπelates with die appearance of the morphological features of apoptosis as deteπnined using the RapiDiff kil Caspase 3 activation was not detected in KYO 1 cells following treatment vsilh PBOX-6 (Fig 19 A), although apoptosis was induced to the same extent in this cell line Certain cell lines eg MCF-7 human breast carcinoma cells, have been shown to lack pro-caspase 3, and thus during apoptosis caspase 3 cannot become activated To ensure that KYO 1 cells contain pro- caspase 3, a Western blot using an antibody directed against this pioenzyme was set up Results showed the piesence of this proenzyme in all three CML cell lines (Fig 19B) CML cells were pretieated with a caspase 3 inhibitor, z-DEVD-fmk (200μM), for 1 hr followed by treatment with PBOX-6 for a further 8 houis Results show that prelrealment of K562 and Lama 84 cells with the caspase 3 inhibitor prevented the activation of caspase 3, as shown using the fluorogenic assay (Fig 20A), but failed to prevent the induction of apoptosis by PBOX-6 (Fig 20B) These results would suggest that although during apoptosis caspase 3 activation was detecled in two of Ihe CML cell lines, this activation is not an essential step m the pathway under which PBOX-6 induces apoptosis in CML cells There have been conflicting reports regarding the requirement of caspase 3 lo cleave various death substrates (Martins el al , 1997, Mc Gowan et al , 1996, Jamcke et al , 1998) Having established that activation of caspase 3 is not necessarily required for the induction of apoptosis in CML cells, control and apoptotic cell lysates from CML cells were examined for evidence of PARP (poly ADP- πbose polymerase) cleavage PARP (116kDa) is cleaved dining apoptosis into its 87kDa and ?9kDa fragments which were detected by Western blot analysis of cell lysates. Results show tiiat PARP cleavage occurs in K562 and Lama 84 cells following treatment widi PBOX-6 for 24 and 16 hours respectively (Fig. 21A and 21B). In addition, tiie induction of apoptosis by PBOX-6 in KYO. l cells, which does not involve caspase 3 activation, was also shown to result in PARP cleavage after 24 hours (Fig. 21C). These results would suggest that during apoptosis induced by PBOX-6 in CML cells, caspase 3 activation is not a requirement for PARP cleavage. Treatment of Hela cells with a representative PBOX compound, PBOX-3, induced PARP cleavage, demonstrating that these PBOX compounds can also induce apoptosis in a human cervix carcinoma cell line (Fig. 2 ID). Several observations suggest an involvement of reactive oxygen intermediates (ROI) in the signal transduction pathway leading to apoptosis (Jacobson, 1996). In order to determine whether the induction of apoplosis in K562 cells by PBOX-6 involved the production of ROI, cells were pretreated with Vitamin E or N-acelylcysteine for 1 hour or 24 hours respectively, prior to treatment widi PBOX-6 for a further 16 hours. These compounds failed to protect against PBOX-6 induced apoptosis (Fig. 22 A and B) suggesting that the production of ROI is not involved in the mechanism by which PBOX-6 induces apoptosis in K562 cells. Tyrosine phosphorylation is involved in the regulation of some apoptotic padiways. Protein tyrosine kinases and phosphatases jointly maintain the tyrosine phosphorylation status of cellular proteins. In order to determine the effect of PBOX-6 on the tyrosine phosphorylation status of proteins in K562 cells, a Western blot assay was set up using an antibody directed against the phosphorylated form of tyrosine residues. Whole cell extracts from K562 cells were resolved by SDS-PAGE and membranes were probed with the antibody. Tyrosine phosplioiylated proteins were observed. In this study, treatment of K562 cells with PBOX-6 for 5, 15 or 30 min, had no effect on the phosphorylation status of proteins (Fig. 23A). However, an increase in die phosphorylation of two proteins was detected in K562 cells following treatment with PBOX-6 for 16 hours (Fig. 23B). These proteins, as yet unidentified, are approximately 63 kDa and 71 kDa in size. Pretreatmenl of K 62 cells with the broad range phosphotyrosine kinase inhibitor, genistein (lOOμM in DMSO), for 1 lir prior to PBOX-6 treatment, prevented die phosphorylation of these two proteins (Fig. 24A) and completely inhibited the induction of apoplosis (Fig. 24B). Pretreat ent with herbimycin A (5μM in DMSO) and tyrophostin (200μM in DMSO), also commonly used tyrosine kinase inhibitors, reduced levels of apoptosis induced by PBOX-6 in K562 cells (Fig. 25), although they are themselves somewhat cytotoxic to the cells. Herbimycin A and tyrophostin alone induced approximately 30% and 10% apoplosis respectively. Etoposide and miloxanthrone are commonly used chemolherapeutic agents. These compounds are topoisomerase II inhibitors, also known as poisons, which stabilise the Topo II-DNA complex causing the accumulation of DNA strand breaks (Ritke et al, 1995). In agreement with previous reports (Martins et al, 1997), treatment of K562 cells with these compounds does not induce apoptosis (Fig. 26). In the present study, il has been found diat pretreatment of K562 cells with these compounds for 1 hour prior to treatment witii PBOX-6 for a further 16 hours protects against apoptosis. (Fig. 26). It could be suggested that PBOX-6 induces apoptosis by acting as a Topo II inhibitor, and that pretreatment of cells with other Topo II inhibitors, etoposide and mitoxanduone, reduces the amount of available target for PBOX-6, thus preventing apoptosis. To investigate whether PBOX-6 functions as a topoisomerase II poison in these cells, an alkaline elution assay was developed which measures DNA single strand breaks. The procedure is based on a previously reported finding that DNA from mammalian cells lysed on filters is eluted only very slowly by alkaline solutions, and that substantial increases in elution rates are produced by a relatively small extent of DNA strand breaks (Kohn et al, 1974) Values obtained from control cells which had undergone alkaline elution were taken as 100% and values obtained from (he treatment of cells with various compounds were compared directly to diis. As a positive control for the development of the assay to measure DNA strand breaks, Jurkat cells were treated with etoposide for 1 hour and cell extracts were lysed and eluted overnight with an alkaline solution. Results showed tiiat etoposide induced a significant increase in DNA strand breaks (Fig. 27A). It was found that K562 cells, treated with PBOX-6 for 3 hours, did not show any significant increase in DNA strand breaks (Fig. 27B). These results would suggest lhat PBOX-6 does not function as a Topo II poison in K562 cells. A series of compounds called ICRF compounds, including ICRF 154 and its derivative ICRF 193, have been shown lo inhibit DNA topoisomerase 11 activity, but without intercalation into the DNA or the induction of cleavable complexes. These are catalytic inhibitors of Topo II as opposed to Topo II poisons which were described earlier (Ishida et al, 1991) It has been reported that tiiese ICRF compounds inhibited Topo II mediated DNA breaks induced by etoposide (Tanabe el al, 1991) by reducing die amount of available target for etoposide. In the present study, pretreatment of Jurkat cells with ICRF 187 (200μM) prevented etoposide-induced DNA strand breaks (Fig. 28A) and also inhibited apoptosis induced by eloposide, as shown by RapiDiff staining (Fig. 28B). Pretreatment of K562 cells with ICRF 187 (200μM) for 1 hour followed by PBOX-6 (lOμM) for a further 16 hours reduced but did not completely inhibit PBOX-6 induced apoplosis (Fig. 29). In order to determine if PBOX-6 operates in a similar way to these ICRF compounds, Jurkat cells were pretreated with a sub-cytotoxic concentration of PBOX-6 (0 μM) for 1 hour followed by treatment with etoposide (2. μM) for a further horn. Results showed that PBOX-6 did not prevent the induction of DNA strand breaks by eloposide (Fig. 30A). Jurkal cells were pretreated with PBOX-6 for 1 hour prior to treatment with eloposide for a further 16 hours and cells were spun onto a slide. Results from RapiDiff staining showed that PBOX-6 does not protect against eloposide induced apoplosis (Fig. 30B). These results suggest that PBOX-6 does not function in a manner similar to the topo II poisons or the catalytic inhibitors of topo II, however, results suggest that PBOX-6 may require topo II to function. The mitogen activated protein kinase family (MAPK's), which includes p42-44, JNK and p38 have been shown to be activated during signal transduction in many cells in response to mitogens, growth factors, and various forms of stress (Denhardt, 1996). In order to deteπnine the effect of PBOX-6 on the activation of MAPK's, a Western blot assay was developed. K562 cells were treated with vehicle or PBOX-6 for various lengths of time. It was found that PBOX-6 induces a transient activation of 2 JNK isoforms, JNK1 and JNK2, which first becomes visible after 15 min and peaks at 30-45 min (Fig. 31 A). A gradual decrease in JNK activation was visible from 1 hour up to 8 hours after PBOX-6 treatment (Fig. 3 IB). In Lama 84 and KYO.l cells treated with PBOX-6 for 45 minutes, JNK1 and JNK2 were also found to be activated (Fig 32). A Western blot assay to detect activation of ρ38 was set up in which K562 cells were treated with vehicle or PBOX-6 and whole cell extracts were prepared. UV treatment of Jurkat cells was previously shown to activate p38, so was used as a positive control. Results showed that PBOX-6 failed to activate p38 following treatment periods of 5 min up to 8 hours (Fig. 33A and B). A similar time course was set up lo determine whether the induction of apoptosis by PBOX-6 involved the activation of p42-44. K562 cells treated with PMA for 30 minutes was used as a positive control, and results showed that P42-44 was not activated in response to PBOX-6 treatment (Fig. 34 A and B). Small GTP-binding proteins, which include cdc42, Rac, Rho, Rap and Ras, function as molecular switches that promote the activity of effector molecules in response to exchanging GDP for GTP. These G proteins have been shown to regulate a wide variety of cellular activities including signal transduction pathways. G proteins are reportedly involved in the activation of M APK, including JNK, some through specific ligand-receplor interactions. The small GTP-binding proteins Racl and Cdc42 have been implicated in the regulation of the JNK signalling pathway (Coso et al, 1995). In an attempt to investigate events upstream of JNK activation a western blot was set up in which K562 cells were prclreated for 1 hour with a lethal toxin which inhibits the activity of Rac 1 , and cells were then treated with PBOX-6 for a further 45 minutes Results showed that pretreatment with the lethal toxin did not inhibit PBOX-6-induced JNK activation (Fig. 35A) and failed to inhibit die induction of apoptosis as determined by RapiDiff staining (Fig. 35B). In the present study, it has been shown that the induction of apoptosis in K562 cells by PBOX-6 involves the transient activation of two JNK isoforms, JNK1 and JNK2. It has also been shown that pretreatment of these cells widi the Topo II irdiibitors, etoposide and ICRF 187 either prevents or reduces the extent of apoptosis induced by PBOX-6. Results suggest that PBOX-6 may require active Topo II to function. In order to deteπnine whether a possible requirement for Topo II lies upstream of JNK activation, a wcsleπi blot assay was set up in which K562 cells were pretreated with either etoposide or ICRF 187 prior to treatment with PBOX-6 and extracts were prepared and analysed for levels of JNK activation. Results show that pretreatment with etoposide (Fig. 36A) or ICRF 187 (Fig. 36B) failed to prevent against PBOX-6 induced activation of JNK, suggesting that a requirement for Topo II lies downstream of JNK activation in the pathway by which PBOX-6 induces apoptosis in K562 cells In order to determine the effect of PBOX-6 on normal bone maπow cells, samples were obtained from donors and cultures were set up to detect CFU-GM progenitors. Cells were treated with vehicle (0.5% ethanol, v/v) or PBOX-6 (lOμM) for 72 hours. Mean results from colony assays performed on 10 normal bone marrow samples was 16.9 for edianol treated and 17 for PBOX-6 treated samples (Table 4). Statistical analysis of these data gave a p value of 0.9578, which is considered not significant. PBOX-6 also induced apoptosis in MCF-7 cells, a human breast carcinoma cell line, in a dose- dependent manner. The morphological features of apoptosis as identified by cytospiiining and staining of cells, was first observed and at a concentration of 5μM (Fig. 37). The anti-apoptotis potential of BCL-2 protein is well established and it has been shown that phosphorylation of anti-apoptotic BCL-2 family members causes inactivation of activity resulting in apoptosis (Haider et al., 1995). In the present study we have found that in all three CML cell lines, PBOX-6 treatment results in phosphorylation of BCL-2, as shown by delayed migration during SDS- PAGE (Fig. 38 A), In K562 cells we have shown that phosphorylation of BCL-2 occurs in a time (Fig 38 B) and dose-dependent (Fig. 38C) manner. Another anti-apoplotic member of the BCI-2 family, BCL-XL, is phosphorylated in a time-dependent manner in K562 cells in response to PBOX-6 (Fig. 38D). In HL-60 cells, a time- and dose-dependent phosphorylation of BCI-2 was also observed following treatment with PBOX-6 (Fig 39A and B). Pretreatment of HL-60 cells with the caspase-3-like protease inhibitor, z- DEVD-Dnk, for 1 hour prior to treatment with PBOX-6 failed to prevent the phosphorylation of BCL-2 (Fig.39C) suggesting that phosphorylation of BCL-2 occurs upstream of caspase 3 protease activation in the pathway in which PBOX-6 induces apoptosis in these cells. ATF-2 and c-jun are transcription factors which are widely reported to be downstream targets of the JNK signalling pathway. Their transcriptional activity is regulated by phosphorylation. Using phospho-specific antibodies we have shown that Ihcse two transcription factors become phosphorylated in * response to PBOX-6 (Fig 40) in K562 cells The ATF-2 reporting system consists of (1) a trans-activator plasmid which contains the CMV promoter to drive the constitutive expression of ATF-2, (2) a Luciferase reporter plasmid which controls expression of the luciferase gene. Direct or indirect phosphorylation of ATF-2 causes the binding of the DNA binding domain of the AFT-2 protein to the reporter plasmid at the Gal 4 binding site, thus activating transcription of the luciferase gene from the reporter plasmid. Expression levels of luciferase reflect the activation status of the signalling events. ATF-2 is activated by JNK in many signal transduction pathways and its activity reflects the in vivo activation of JNK. K562 cells were transfected with die ATF-2 reporting system followed by treatment with vehicle or PBOX-6, and luciferase activity was measured. Results are expressed as fold stimulation over untreated cells. Cells were co-transfected with a plasmid expressing the Lac-Z gene and B-gal activity was measured and used to normalise transfection efficiency. Results from tliis study have shown Uiat PBOX-6 stimulates a 5 fold increase in expression of die luciferase gene (Fig 41) K562 cells were also transfected with a plasmid expressing an inhibitor of die JNK signalling pathway, JIP. Results show that co-expression of the JIP plasmid blocks PBOX-6 induced phosphorylation of ATF-2 (Fig 41). GTP binding proteins As mentioned above, G proteins are reportedly involved in the activation of MAPK, including JNK. K562 cells, transfected with the ATF2-Luc reporting system, were co-lransfected with a number of dominant negative mutant G-proteins, and their effect on PBOX-6-induced stimulation of ATF-2 phosphorylation was analysed. A dominant negative mutant of cdc42 appears to further stimulate PBOX- 6 activation of ATF2 suggesting that cdc42 may have an inhibitory effect on PBOX-6 activity (Fig.42). On the other hand, co-transfection of cells with dominant negative Rac depresses PBOX-6-induced phosphorylation of ATF-2, suggesting that Rac may have a positive stimulatory role during PBOX-6- induced apoptosis. Dominant negative Rap had no effect on PBOX-6 activity (Fig.42). Structure Activity Relationship Background. Recently we reported the synthesis and the pharmacological evaluation of a series of pyrrolobenzoxazepines as potent ligands for ihe peripheral-type benzodiazepine receptor (PBR) (Campiani et al ,1996) Using these probes to study the physiological role of the PBR, we found out that, along with die classically used PBR ligands PK11195 and Ro 5-4864, three pyrrolobenzoxazepines inhibited the proliferation of rat C6 glioma and 1321N1 astrocytoma cells, and die antiproliferative effect was found to be mediated by aπest in ihe Gl phase of the cell cycle (Zisterer et al 1998). However, we proved diat the antiproliferative activity of such compounds was not mediated by a specific interaction of these ligands with the PBR. While examining the effect of pyrrolobenzoxazepines on the proliferation of a range of human cancer ceil lines we observed tiiat some of them could induce apoplosis with various potencies. We detail herein the development of new oxa(lhia)zepine (PBOXs) derivatives and their SARs ' for apoptotic activity associated widi variation of the substituents on the heterocyclic system. Our SAR study led to identification of non-cytotoxic oxa(thia)zepiπes that induce apoptosis, with various potencies and in a dose and time-dependent manner, in several human cancer cell lines such as HL-60, HUT 78, Jurkat, a cell line that does not express peripheral benzodiazepine receptor, and NCI-H460. Representative compounds were also investigated in three CML cell lines such as KYO.1 , Lama 84 and K562 which are known to be highly resistant lo chemolherapeutic agents such as slaurosporine, ara-C, and etoposide. SAR Studies. Although a receptor target has not been yet identified, the apoptotic activity of the members of the new series of potential antitumor agents is strictly dependent upon their straclure. PBOX 68 is characterized by RI = p-lolyl and displayed low apoptotic activity. The potency could be highly improved by the replacement of the aromatic group RI by a 1-naphthyl group. Interestingly, derivatives characterized by RI = 2-naphlhyl are devoid of apoptotic activity, while PBOX-4,6,7, and 70 characterized by a 1-naphthyl group at C-6 (F = benzo or ethoxycarbonylbenzo), represent potent apoptotic compounds. The higher apoptotic activity of C-6 1-naphthyl derivatives PBOX-4,6,7 and 70 with respecl to PBOX-68, suggests that at the corresponding site of the receptor binding cleft there should be a hydrophobic pocket capable of accommodating large lipophilic groups, in the direction of a 1- πaphthyl system, characterized by high electron density. In the 1-naphthyl series the activity is also influenced by the substituent Y. hi fact in the carbamoyl series (Y = NR2R3), while the potent apoptotic agent PBOX-7 (Y = NHMe) showed a comparable activity to PBOX-6 (Y = NMe 2 ), their superior homologue (Y = NEt 2 ) was found poorly active. On die contrary, introduction of a heteroaromatic system at C-7 in place of the dimethyl amino group is well tolerated (Y = 4-pyridyl, PBOX-65,71=PBOX-6) indicating that a further hydrophobic interaction does not destabilize the complex widi a potential receptor, occupying a receptor pocket capable of tolerating an aromatic ring or a dimetiiylcarbamoyl group, but not longer alkyl chains as ethyl. In general the dimethylcarbamoyl derivatives are more potent dian the coπesponding methyl esters (PBOX-6 and 7 vs PBOX-4) (NHMe=NMe 2 >Me). Increasing of the length of the alkyl chain of PBOX-4 is deleterious for apoptotic activity. These SAR data are consistent with the presence of a small lipophilic pocket in die hypotiietical receptor binding site that does not tolerate bulky alkyl chains at position 7. Replacement of the benzo-fused ring (F = benzo) with a pyrido-fused system (F = pyrido) (PBOX-12) led to an apoptotic agent almost as potent as PBOX-4 and 6, indicating diat the potency is only slightly influenced by replacement of a CH with a nitrogen atom. Substitution of the oxygen-bridged atom with a sulfur (A = S) is not critical for potency. In fact PBOX-8 and 9 are found to be as potent as PBOX-4 and 6 in the cell lines used . Although PBOX-68 (RI =/ otyl, Y = Me) is characterized by low apoptotic activity, maintaining F = pyrido or benzo, the potency could be also improved by replacing the alkyl group at C-7 (Y = Me) with an heteroaromatic group (Y = 4-pyridyl) as in PBOX-28 and 69. This fact could be explained assuming a different binding mode of PBOX-28 and 69 (and PBOX-71) at the hypothetical binding site, with the 4-pyridyl group oriented against die binding cleft occupied by the 1- naphlhyl of PBOX-6. For mapping the hypothetical receptor binding site, starting from die apoptotic agents PBOX-4 ,6 and 7 (RI = 1-naphtyl), PBOX- 15 and 16 were synthesized, being characterized by a second large conjugated system bound to the oxazepine skeleton, directed to the opposite site of the group Y (F = [2,3]naphtho, RI = 1-naphthyl). Introduction of a [2,3]naphtho -fused system in place of the benzo-fused group provided potent apoptotic agents, active in the nanomolar range, and PBOX- 15 and 16 are among the most potent of the series. Even in this case the results are consistent with the existence of a receptor binding site that can accommodate hindered lipophilic groups. Taking into account the SAR studies, for optimal apoptotic activity the following phaπnacophoric groups are required: i) a naphtho- fused (F) system as a group with high electron density for hydrophobic interaction, ii) a 1-naphthyl group al C-6 (RI), and iii) a dimethylcarbamoyl group at C-7 (Y). DISCUSSION The inventors have demonstrated that a new class of apoptotic agents, pyrrolo-1 ,5- benzoxazepines and pyrrolo-l,5-benzthiazepines, induce cell death in a nmnber of human cancerous cell lines, HL-60, Jurkat, Hut-78, LAMA 84, KYO. l, K562, CEM, Hela and MCF-7 cells. The morphological characteristics associated with apoptosis using previously defined criteria (Ken el al, 1972) such as cell shrinkage, chromatin condensation, membrane blebbing and DNA fragmentation was observed. Of all diese compounds screened in HL-60 promyeloctic leukaemia cells PBOX-4, 6, 7, 8, 9, 15, 16, 65, 70 and 71 were found be most potent (much more potent than PK 11195 or Ro5-4864) whereas PBOX-1 and 2 had no effect. In K562 chromic myeloid leukaemia cells, PBOX-4, 6, 7, 15 and 16 were found induce apoptosis in a concentration-dependent manner, with PBOXs 15 and 16 being by far the most potent of die compounds tested. PBOX-6 induces a time-and dose-dependent induction of apoptosis in HL-60 cells and in die 3 chrome myeloid leukaemia cells. Apoptosis was first observed after 4hrs and with 5μM PBOX-6. The induction of apoptosis by PBOX-6 was confirmed in all 4 cell lines by demonstrating the appearance of DNA Ladders, after a 48hr treatment. DNA laddering is a hallmark of apoptosis and is a result of internucleosomal DNA degradation. The ultimate DNA fragments are multiples of 180-200bp nucleosomal units which give the appearance of DNA laddering in standard agarose gels. Multiple lines of evidence indicate that apoptosis can be triggered by the activation of a set of death effector cysteine proteases called caspases widi specificity for Asp-X bonds. Some experimental observations would however suggest that a caspase-independent mechanism for commitment to cell death also exists. For example, overexpression of a proapoptotic protein such as Bax in mammalian cells can induce DNA condensation and membrane alterations leading to apoplosis, witiiout any caspase activation (Xiang et al, 1996). In the present study we determined whether caspase-3 like proteases were involved in cell death induced by PBOX-6 in HL-60 cells. PBOX-6 caused a dose- and timc- dependent activation of caspase 3-like proteases which directly correlated with the observed morphological effects of apoptosis induced by this compound. The caspase 3-like protease inhibitor, z- DEVD-fmk, prevented bodi the activity of caspase 3-like proteases and the appearance of the morphological signs of apoptosis. This would suggest the involvement of caspase 3-like proteases in the mechanism by which PBOX-6 induces apoptosis in HL 60 cells. In CML cells, caspase 3 was activated in response to PBOX-6 in only two out of the three cell lines whereas PBOX-6 induced apoplosis to a similar extent in all 3 cell lines Pre-treatment of CML cells with a caspase 3 inhibitor, DEVD-fink, did not protect against PBOX-6-induced apoptosis. This would indicate that activation of caspase 3 is not necessarily required for induction of apoptosis by PBOX-6 in these CML cells. Overexpression of Bcl-2 can prevent cells from undergoing apoptosis in response to a variety of stimuli (Adams & Cory, 1998). PBOX-6 caused a dose-dependent induction of apoptosis in human CEM (T lymphoblastoid) cells, both in normal and in CEM cells overexpressiπg the Bcl-2 protein. This suggests that this pro-survival protein camiot protect against PBOX-6 induced apoptosis. The BCI-2 family of cytoplasmic proteins consists of pro- and anti-apoptotic members Anti- apoptotic members such as BCL-2 and BCL-XL are targets for phosphorylation, suggesting one potential mechanism of control (Maundrell et al, 1997). Some uncertainty exists whether phosphorylation increases or decreases the anti-apoptolic function of BCL-2. However, most data support the hypodiesis that phosphorylation inactivates BCL-2, thus promoting apoptosis (Fan et al., 2000). In die present study we have shown that BCL-2 becomes phosphorylated in all tiiree CML cell lines in response to PBOX-6. In K562 and HL-60 cells, phosphorylation of BCL-2 occurs in a time and dose-dependent manner. We have previously shown that in HL-60 cells, activation of caspase 3 is essential during PBOX-6 induced apoptosis. Pretreatment of HL-60 cells with a caspase 3 inhibitor, z-DEVD-fmk, failed to prevent PBOX- 6 induced phosphorylation of BCL-2 suggesting that this phosphorylation lies upstream of caspase 3 activation in the pathway in which PBOX-6 induces apoptosis in these cells. A similar result was found widi K562 cells although we have previously shown tiiat caspase 3 activation is not essential during PBOX-6 induced apoptosis in K562 cells. Results have also shown that another member of the anti- apoptotic group, BCL-XL, also becomes phosphorylated following PBOX-6 treatment in a time-dependent manner, in K562 cells. These results suggest dial during apoptosis, PBOX-6 causes phosphorylation and thus inactivation of die anli-apoptotic BCL-2 family members, BCL-2 and BCL-XL. Cytochrome C is a milochondrial protein that induces apoptosis when accumulated in die cytosol in response to diverse stress inducers (Kluck et al, 1997; Yang et al, 1997). This protein has also been shown lo cause apoptosis when added to cell free extracts (Liu et al, 1996). In some cell lines however, such as multiple myeloma cells, there are at least two different pathways that lead to apoplosis, one involving and one not involving Cytochrome C release from mitochondria (Chauhan et al, 1997). These workers studied the role of Cytochrome C in dexamethasone-, anti-Fas niAb- and ionizing radiation- induced apoptosis and demonstrated that while ionizing radiation-induced apoptosis is associated with an increase in cytosolic Cytochrome C levels, apoptosis induced by the two other agents had no detectable effect on Cytochrome C release. In addition, there are many reports that during apoptosis, accumulation of Cytochrome C in die cytosol results in the activation of caspase 3-like proteases (Kluck et al, 1997; Chu et al, 1997) although pathways leading to caspase 3 activation without Cytochrome C release have also been described (Chauhan et al, 1997). In the present stud)', PBOX-6-induced apoptosis in HL 60 cells was associated with an accumulation of Cytochrome C in the cytosol. This result indicates that release of Cytochrome C from the mitochondria may be important for triggering apoplosis in response to PBOX-6 This was not the case with the CML cells. PBOX-6 did not cause an increase in the release of cytocluonie C into the cytosol of CML cells. This would suggest that cytochrome C is not involved in the mechanism in which PBOX-6 induces apoptosis in CML cells. Apoptosis is sometimes associated with increases in iπtracellular ROI levels and addition of exogenous antioxidants such as N-acetylcysteine (NAC) and vitamin E can inhibit apoptosis (Butke and Sandstroni, 1994). The specific molecular mechanisms involved however remain to be elucidated. In the present work it has been shown dial PBOX-6-induced apoptosis in HL-60 cells and in K562 cells was unaffected by the presence of a number of antioxidants. This would suggest that PBOX-6-induced apoptosis is not mediated by ROI. This is in agreement with recent reports which have indicated that ROI are not necessarily a requirement for apoptosis. For example, progranmied cell death induced by the Fas ligand or by staurosporine do not appear to require the generation of ROI, and are not inhibited by the use of antioxidants (Jacobson and Raff, 1995). Recently there has been some suggestion that the PBR may be involved in apoptosis A group of workers have shown that PK 11195, a prototypic ligand of the PBR, facilitates the induction of apoptosis by a variety of stimuli in a number of cell types including thymocytes and the T-cell leukaemia CEM cells (Hirsch et al 1998) However, PK 11195 by itself had no apoptotic effect In addition, it has been recently reported dial during apoptosis Cytochrome C can exit the mitochondria tlirough VDAC (Slumizu et al , 1999), which is itself associated with the PBR in die mitochondπal outer membrane (McEnery el al , 1992) In die present study we describe how PK 11195 and some PBOX compomids induce apoptosis by themselves, independently of other apoptosis-mducing stimuli It is unlikely howevei that the PBR is involved in the mechanism by which these PBOX compounds duce apoptosis Much higher concenb ations of the compomids were required to induce apoptosis than were necessary to saturate the receptor Furthermore, some of the compounds e g PBOX-1 and PBOX-2, did not mduce apoptosis yet all of these compounds bmd lo die leceptor with similar affinity Finally, we have shown that some PBOX compounds duce apoptosis in Jurkat cells which have previously been shown to be devoid of the PBR (Carayon et al , 1996) These studies demonstrate that the apoptotic effects of the PBR ligands are incompatible w itlτ PBR mvolvement Several recent papers have shown that activation of die transcription factor NFKB IS linked to apoptosis (Beg and Baltimore, 1996, Gin and Aggarwai, 1998) There have been some suggestions that this factor once activated plays an anti-apoptolic role, most likely by inducing expression of gene products, such as clAP2 (cellular inhibitor for apoptosis) (Chu et al , 1997), that inhibit die apoptotic pathway However, a general role for NFKB as a transcription factoi that pi events apoptosis is far from established The actuation of NFKB is initiated by a wide variety of stress stimuli, such as TNF, ceramidc and daunonibicin, which themselves cause apoptosis (Boland et al , 1997) In this case NFKB activation may dien cause cell death Thus the role of NFKB as a promoter or inhibitor of cell death may depend on bodi the cell type and Ihe apoptosis-inducmg stimulus In the present study we have shown that while PBOX-6 mduced apoptosis in both HL-60 and Hut-78 cells, this compound did not affect NFKB levels Hut-78 cells conslitutively express NFKB and as such have been reported to be resistant to a range of stress stimuli including TNF, hpopolysachaπde, H 2 0 2 , cerannde and okadaic acid (Gin and Aggarwai, 1998) In this work the PBOX compounds mduced apoplosis in both HL-60 and Hut-78 cells, with similar potency Furtheπiiore, pre-treatment of HL-60 cells with TNF at a concentration which activates NFKB, afforded no protection against PBOX- 6-mdιιced apoptosis It can thus be concluded that PBOX-6-ιnduced apoptosis most likely employs an NFκB-ιndependent mechanism Furthermore this study argues against a general role for NFi B as a transcription factor that prevents cell death Several anli-cancer dnigs such as camptotiiec (Piret and Pielte, 1996), etoposide (Perez et al , 1997) and the anthracycline antibiotics, daunorubicm and doxonibicm (Das and White, 1997), activate NFKB in addition to inducing cell death Daunonibicin is widely used in cancer chemotherapy and although its mechanism of anti-tumour action has not been fully elucidated, it ultimately induces apoptosis in cells. The concomitant activation of NFKB may counteract the therapeutic effects of tiiese chemotherapeutic compomids. Therefore anti-cancer drugs which do not activate NFKB may result in more effective anti-cancer treatments. It is evident that apoptosis can be induced by a variety of drugs with diverse chemical structure and different mechanism of action. Among the list of apoptosis inducing agents are a wide range of anti- cancer drugs including inhibitors of the mitotic spindle apparatus, such as vinca alkaloids, inhibitors of DNA synthesis such as aphidicolin and drugs like campothecin that cause protein-associated DNA strand breaks mediated by DNA topoisomerase I (Sen and D'lncalci, 1992). All diese drugs have been shown to induce apoptosis in cancerous cells derived from the haeinopoietic system such as HL-60 cells (Sen and D'lncalci, 1992). We propose that diese pyπolo- 1,5 -benzoxazepines may be potential anti- cancer drugs. The observation that these compounds do not activate NFKB may also result in them being more effective anti-cancer agents. The BCR-abl fusion gene transcribes a novel 8.6 kb mRNA which in turn encodes a p210 kDa protein which, unlike normal c-abl protein, is phosphorylated on tyrosine residues and has increased intrinsic tyrosine kinase activity. It has been suggested that p210 bcr-abl tyrosine kinase may have an important role in the palhogenesis of CML (Clarkson el al, 1997). The BCR-abl fusion gene is probably the usual sole causative event and is responsible for all the manifestations of die early chronic phase of die disease (Clarkson et al , 1997) In this study, it has been shown tiiat BCR-abl is down regulated in two of the tluee CML cell lines after treatment with PBOX-6. However, down regulation was not detected before 241u treatment with 1 OμM PBOX-6 at which time apoptotic levels reach approximately 50%. Therefore, it is unlikely that downregulation of die BCR-abl fusion protein is the initial target of the drug. The cleavage of PARP in all three CML cell lines was demonstrated after treatment with PBOX-6 for 24hr. During apoptosis PARP has been suggested to be cleaved by caspases 3 and/or 7 (Cohen, 1997). The induction of PARP cleavage in the KYO. l cells, where caspase 3 is not activated, would suggest that caspase 3 is not required for PARP cleavage in these cells upon induction of apoptosis by PBOX-6. These results are in agreement with a recent report in which it was shown that MCF-7 breast carcinoma cells, which lack caspase 3, underwent PARP cleavage after treatment with the apoptotic inducer staurospoπne (Janicke et al, 1998). Phosphorylation and dephosphorylalion of cellular proteins is implicated in many biologically important processes such as cell growth and differentiation (Weng el at., 1 98). Protein tyrosine kinases (PTK's) and phosphalases (PTPases) jointly maintain the tyrosine phosphoiylation of cellular proteins in homeostasis (Yousefi et al, 1994). Alteration in die tyrosine phosphorylation status of proteins has previously been implicated in apoptotic cell death (Uckun et al, 1992). There also appears lo be conflicting reports as to whether an increase or decrease in the tyrosine phosphoiylation status of proteins results in apoplosis (Bergamaschi et al, 1993; Johnson et al, 1996). In this study it has been shown that treatment of K562 CML cells with PBOX-6 induces an increase in the tyrosine phosphorylation status of two proteins, approximately 63 kDa and 71 kDa in size. Pretreatment of cells with the general tyrosine kinase inhibitor, genistein, blocked the phosphorylation of these proteins by PBOX-6 and also prevented the induction of apoptosis. In conclusion, an increase in die tyrosine phosphorylation of two, as yet unidentified proteins, has been observed in K 62 cells following treatment with PBOX-6. This increased phosphoiylation did not become apparent until 16 hours following the addition of PBOX-6 where approximately 40-50% of cells had undergone apoptosis. Therefore, from diese results it can be suggested that increased tyrosine phosphorylation of these two proteins, following treatment with PBOX-6, does not trigger the induction of apoptosis but rather accompanies apoptosis induced in K562 cells. Treatment of Jurkat cells with PBOX-6 causes a time and dose-dependent decrease in protein tyrosine phosphorylation which coπelates with the appearance of die morphological signs of apoptosis. As this downregulation does not precede the appearance of die morphological signs of apoptosis, it is unlikely that it is an upstream event in the apoptotic pathway in which PBOX-6 causes cell death. The DNA binding protein, topoisomerase II, is a target for several clinically effective anticancer drugs, among which are mitoxanthrone and etoposide. These compounds stabdise the DNA-Topo II complex preventing the religation of cleaved DNA strands which results in the accumulation of strand breaks (Perez et al, 1997). In the present study it has been demonstrated that treatment of K562 cells with these Topo II inhibitors protects against PBOX-6 induced apoptosis. It could possibly be suggested that PBOX-6 also acts as a Topo II poison and that pretreatment of cells with another poison such as etoposide reduces the amount of available target for PBOX-6, thus reducing the extent of apoptosis. Hoλvever, it is unlikely that PBOX-6 functions in a similar manner to diese Topo II poisons as demonstrated by its failure to induce DNA strand breaks. Anodier series of compomids, Bis 2, 6-dioxopiρerazine derivatives, which include ICRF 154 and ICRF 193 have been shown to be new anli-tumour agents and are cuπently under clinical trial. These drugs inhibit topoisomerase II activity, but not topoisomerase I. They do not intercalate into die DNA or induce the cleavable complex, but rather block the catalytic activity of the enzyme by directly intercalating with the enzyme and not with DNA (Tanabe et al, 1991). They are thus called catalytic Topo II inhibitors. ICRF- 193 has also been shown to inhibit DNA stand breaks induced by other cleavable complex-forming agents. Thus ICRF-193 antagonized etoposide or ainsacrine-stimulated DNA stiand cleavage, and Ihe resulting apoplosis suggesting that ICRF-193 and related compounds block at some earlier step in the catalytic cycle of the enzyme reaction (Tanabe et al, 1991). These observations were confirmed in the present study by the demonstration that ICRF 187 prevented ctoposide-induced apoptosis in Jurkal cells and prevented etoposide-mediated DNA strand breaks. Although ICRF 187 caused a reduction in levels of PBOX-6 induced apoptosis in K562 cells, results suggest that PBOX-6 does not function in a manner similar to these ICRF compomids as it does not protect against etoposide-induced DNA strand breaks or etoposide-induced apoptosis in Jurkat cells. In agreement with previous reports (Ritke et al, 1994) it was observed that the treatment of K562 cells with etoposide (25μM) induced DNA strand breaks. However, unlike Jurkat cells, K562 cells failed to undergo apoplosis up to 24 hours following treatment with etoposide. A possible explanation may be die ability of K562 cells to repair DNA damage induced by etoposide. Further studies need to be carried out to investigate a possible DNA repair mechanism in K562 cells. Receptor tyrosine kinases (RTKs) have been shown lo activate a number of intracellular signaling pathways during signal transduction Three kinases, p38, p42-44, and JNK, known as MAPK's, are found in the nucleus upon activation and serve to link signals from the cell surface to cytoplas ic and nuclear events. Many reports suggest a role for MAPK's, including JNK and p38, in the cellular response to various extracellular stimuli and in the induction of apoplosis. For example, in Jurkat cells, JNK and ρ38 activation have been coupled to Fas-induced apoptosis (Juo et al, 1997). The induction of apoptosis in U937 cells by etoposide or camptothecin resulted in the transient activation of JNK 1 which in turn led to transient expression of c-jun (Seimiya et al, 1997). However, a number of reports suggest tiiat the specific signal transduction pathways activated during apoptosis may vary in response to different stimuli and between cell types (Osbom and Chambers, 1996). Similar observations have been reported for p38 activation. For example, in human neutrophils, apoptosis induced by stress stimuli resulted in activation of ρ38, but not JNK, within 30 minutes following stimulation. In contrast, apoptosis mduced by Fas ligation failed to activate either p38 or JNK in the same cells (Frasch et al, 1998). In the present study, transient activation of JNK was observed in K5 2 cells following a 15 minute treatment with PBOX-6 and induction of apoptosis followed. JNK activation peaked following 30-45 minutes treatment with PBOX-6 and levels of activation slowly declined over an 8 hour period. Transient activation of JNK was also observed in HL-60 cells following treatment with PBOX-6. Induction of apoplosis by PBOX-6, in K562 cells, failed to activate p38 or p42-44 up to 8 hours after treatment In an attempt to identify proteins activated upstieam of JNK , K562 cells were pretreated with a lethal toxin which inhibits Rac 1. Rac 1, a member of a group of small GTP-binding proteins, has previously been implicated upstream of JNK (Coso et al, 1995). However, inhibition of Rac 1 failed to prevent PBOX-6 induced activation of JNK in K562 cells indicating that activation of Racl does not lie upstream of JNK in the pathway in which PBOX-6 induces apoptosis in K562 cells Further preliminary data from transfection assays have shown tiiat small GTPases may play a role in regulating PBOX-6-induced apoptosis in K562 cells. In the present study it has been shown that PBOX-6 induces transient activation of two JNK isofoπns, JNK 1 and JNK 2 during apoptosis in K562 cells. It has also been shown that pretreatment of K562 cells with the Topo II inhibitors, eloposide and ICRF 187 either inhibited or reduced the extent of apoptosis induced by PBOX-6. These results suggest that PBOX-6 requires active Topo II for the induction of apoptosis. Using western blot analysis it has been shown that activation of JNK lies upstream of a requirement for Topo II in the pathway by which PBOX-6 induces apoptosis in K562 cells. The transcription factors ATF-2 and c-jun have been shown to be phosphorylated downstream of JNK in a number of signalling pathway. We have shown tiiat ATF-2 and c-jun are phosphorylated in K562 cells following treatment with PBOX-6. Following transfection of K562 cells with an ATF2 -luciferase reporting system, we have shown that PBOX-6 stimulates a five fold increase in luciferase activity in these cells. This activity reflects die in vivo activation of JNK. When K562 cells were co-transfected with JIP, an inhibitor of the JNK signalling padiway (Yasuda el al., 1999), PBOX-6 mediated phosphorylation of ATF2 was blocked. These results suggest that JNK activation is essential in the apoptotic signalling pathway activated by PBOX-6 in K562 cells In die present study it has been shown that K562 cells, which are resistant to most anti-cancer agents, readily undergo apoptosis when treated with die novel potential anti-cancer agent, PBOX-6. To ensure that PBOX-6 does not elicit a general toxic effect, normal bone marrow samples were received from 10 donors. Mononucleur cells were isolated from the samples and subsequently treated widi PBOX-6. Stem cell progenitor cultures were prepared and results indicated that PBOX-6 has no significant effect on normal bone marrow stem cells. Whether other components acting in parallel pathways ate also required, in addition to the activation of JNK and caspase 3-like proteases, for the induction of apoptosis by PBOX-6 has not been deteπnined. The mechanism(s) of activation of the caspase(s) and the potential targets involved in this process will need to be identified. In conclusion, it has been demonstrated that the compomids of the present invention can induce apoptosis in cancerous cell lines, indicating the potential of these compounds as anti-tumour drugs. SUMMARY A range of pyπolobenzoxazepine and pyrrolobenzthiazepines derivatives (PBOX compomids) have been shown to induce apoplosis, with various potencies, in some or all of the following nine human cancer cell lines: HL 60 (promyelocytic leukaemia), HUT 78 (T cell Lymphoma), Jurkat (leukaemic T cell Lymphoblast) and LAMA 84, KYO.l, K562 (clironic myeloid leukemia cells), Hela (cervix carcinoma) CEM (T lymphoblastoid) and MCF-7 (breast carcinoma) cells. The induction is botii dose and lime- dependent. It does not involve the specific interaction of these ligauds with the peripheral-type benzodiazepine receptor (PBR), to which they bind with high affinity, as much higher concentrations of these compomids were required to induce apoptosis than were necessary to saturate ihe receptor. In addition, some of the PBOX compounds induce apoplosis in Jurkat cells, which have previously been reported to lack the PBR. PBOX compound-induced apoptosis was unaffected by the presence of antioxidants such as N- acelylcysleine, suggesting that the mechanism by which these compounds cause apoptosis does not involve the production of reactive oxygen intermediates. Activation of Ihe transcription factor NF kappa B has previously been suggested to protect against apoplosis. Five selected PBOX compounds induced apoplosis in HUT 78 cells which contain constitutively active NF kappa B, albeit with reduced potency to that observed in HL 60 cells. In addition, activation of NF Kappa B by the cytokine TNF-alpha in HL 60 cells prior to the addition of PBOX-6 did not protect diese cells against apoptosis Furthermore, PBOX-6-ιnduced apoplosis did not effect die levels of NF kappa B activity in either HUT 78 or HL 60 cells These results would suggest that PBOX-6-ιnduced apoptosis employs an NF kappa B-independent mechanism Caspases are a closely related family of cysteine proteases that play a key role in apoptosis In this study it was shown that caspase 3 becomes activated during PBOX-6-ιnduced apoptosis in HL60 cells In addition, pre-treatment of HL 60 cells with a specific inhibitor of caspase 3, z-DEVD-fmk, inhibits apoptosis induced by PBOX-6 These results indicate tiiat caspase 3 plays an important role in apoptosis m response to PBOX-6 in HL60 cells PBOX-6-mduced apoptosis m HL-60 cells was associated with an accumulation of the mitochoiidrial protein cytochrome C in the cytosol, which may be important for tπggeπng apoptosis This was not the case with the CML cells Results would indicate that activation of caspase 3 and release of cytocluome C is not required for induction of apoptosis by PBOX-6 in diese CML cells PBOX-6 caused a dose-dependent induction of apoptosis m human CEM (T lymphoblastoid) cells, both in normal and in CEM cells overexpressuig the Bcl-2 protein This suggests tiiat this pro- survival protein cannot protect against PBOX-6 induced apoptosis PBOX-6 caused a time- and dose-dependent phosphorylation of BCL-2 in K562 and HL-60 cells BCL-XL also became phosphorylated in a time-dependent manner m K562 cells following PBOX-6 treatment Pretreatment with a caspase 3 inhibitor did not protect against PBOX-6 induced phosphoiylation of BCL-2 in HL-60 cells These results suggest that during apoptosis in CML and HL- 60 cells, PBOX-6 phosphorylates and inactivates these anti-apoptolic BCL-2 family members and m HL-60 cells phosphorylation of BCL-2 occurs upstream of caspase 3 activation in die apoptotic pathway The BCR-abl fusion prote is probably responsible for die manifestation of CML It is unlikely, however, that the BCR-abl fusion protein is die initial target of the PBOX-6, as its down regulation in response lo PBOX-6 in two of the CML cell lines does not occur until after 24 hours Recent reports suggest that signalling through protein tyiosme phosphorylation is required foi the induction and/or inhibition of apoptosis Treatment of Jurkat cells and the CML cells with PBOX-6 resulted in changes in protein tyrosine phosphorylation levels Pre-treatment of Jurkat cells and K562 cells with the topoisomerase II inhibitors etoposide, genisten and mitoxantrone reduced the levels of apoptosis by PBOX-6 This may suggest that PBOX-6 could also be working at the level of die DNA and that the topoisomerase II inhibitors and PBOX-6 are competing with one another for binding to the DNA It was found that both Topo II poisons and catalytic inhibitors of Topo II either reduced or completely inhibited PBOX-6 induced apoptosis in K562 However, it seems unlikely that PBOX-6 functions in a manner similar to eithei the Topo II poisons or catalytic inhibitors , but that PBOX-6 requires active Topo II to function PBOX-6 mduced apoplosis ui CML and HL-60 cells involved the transient activation of JNK The small GTP-binding protein Rac 1 does not seem to be activated upstream of JNK. However, preliminary data from transfection assays have shown tiiat small GTPases may play a role in regulating PBOX-6 induced apoptosis in K562 cells. Activation of JNK lies upstream of a requirement for Topo II in the patiiway by which PBOX-6 induces apoptosis in K562 cells. The transcription factors ATF-2 and c-jun become phosphorylated in K562 cells in response lo PBOX-6. PBOX-6 causes phosphorylation of ATF2 transcription factor in K562 cells and this stimulation is blocked by the co-tiansfection of JIP, an inhibitor of the JNK signalling patiiway. This suggests that JNK activation is essential in the apoptotic signalling pathway induced by PBOX-6. The induction of apoptosis in K562 cells did not involve the activation of the MAPK's p38 or p42-44 following up to 8 hours treatment with PBOX-6. Finally PBOX-6 was found to have no effect on normal myeloid cells. The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used lo specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. 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Claims: Claims 1 A pharmaceutical composition comprising an apoptosis-induc g amount of a compound having the general foπnula (I) - wherein (1) Ri represents an unsubstituted Cg or C JQ aiyl group, or a Cg aryl group substituted with Me or OMe, (n) A represents 0, S or S partially oxidized to sulfoxide, (in) the cyclic group labelled F represents an misubstituted Cg or CJQ aryl-fυsed group or a C5 heteroaryl-fused group (nitrogen as heteroatom) or a benzo-fused system substituted with ethoxycarbonyl function, (IV) and Y represents the group — N ^3 wheicin R 2 and R 3 are independently hydrogen, or methyl, or Y represents the group CH3 or a (CH 2 ) 2 CH 3 group, or an misubstituted C5 heteroaryl group (nitrogen as heteroatom) 2 A phaπnaceutical composition as claimed in claim 1 wherein Rj is an unsubstituted 1-naphthyl 3 A phaπnaceutical composition as claimed in claim 1 or claim 2 wherein F is an unsubstituted benzo-fused group or benzo-fused group substituted by one ethoxycarbonyl fmiction, or misubstituted 2,3-naphtho- or 2,3-pyπdo-fused system 4 A pharmaceutical composition as claimed in any preceding claim wherein R| and F lepresent a 1-naphthyl group and a 2,3-naplιtho-fused group, respectively 5 A pharmaceutical composition as claimed any preceding claim wherein Y represents a (Me)2 or NHMe or a 4-pyπdιne group 6. A pharmaceutical composition as claimed in any preceding claim wherein the compounds are selected from tiiose having die formulae:- PBOX-4, PBOX-6, PBOX-7, PBOX-8, PBOX-9, PBOX-12, PBOX-15, PBOX-16, PBOX-27, PBOX- 28, PBOX-30, PBOX65, PBOX68, PBOX69, PBOX70, PBOX71 as defined herein. 7. Use of compomids of formula I wherein: (i) RI represents an unsubstituted C β or C j Q aryl group; or a C aryl group substituted with Me or OMe; (ii) A represents O, S or S partially oxidized to sulfoxide; (iii) ihe cyclic group labelled F represents an unsubshtuled Cg or Ci Q aiyl-fused group or a C5 heteroaryl-fused group (nitrogen as heteroatom) or a benzo-fused system substituted with ethoxycarbonyl function; (iv) and Y represents the group — N wherein R 2 and R 3 are independently hydrogen; or methyl; or Y represents the group CH3 or a (CH 2 ) 2 CH 3 group; or an misubstituted C5 heteroaryl group (nitrogen as heteroatom), as an active pharmaceutical substance. 8. Use of compomids selected from diose having the formulae: - PBOX-4, PBOX-6, PBOX-7, PBOX-8, PBOX-9, PBOX-12, PBOX-15, PBOX-16, PBOX-27, PBOX- 28, PBOX-30, PBOX65, PBOX68, PBOX69, PBOX70, PBOX71 as defined herein, as an active pharmaceutical substance. 9. Use of compounds of formula I wherein: (i) RI represents an unsubstituted Cg or C ] ø aryl group; or a Cg aryl group substituted with Me or OMe; (ii) A represents O, S or S partially oxidized to sulfoxide; (iii) die cyclic group labelled F represents an unsubstituted Cg or Cι Q aryl-fused group or a C5 heteroaryl-fiised group (nitrogen as heteroatom) or a benzo-fused system substituted widi ethoxycarbonyl function; (iv) and Y represents the group — N V R 3 wherein R 2 and R 3 are independently hydrogen; or methyl; or Y represents the group CH3 or a (CH 2 ) 2 CH 3 group; or an unsubstituted C5 heteroaryl group (nitrogen as heteroatom), in the preparation of a medicament for the treatment of tumours or other cancerous conditions. 10. Use of compounds selected from those having the formulae:- PBOX-4, PBOX-6, PBOX-7, PBOX-8, PBOX-9, PBOX-12, PBOX-15, PBOX-16, PBOX-27, PBOX- 28, PBOX-30, PBOX65, PBOX68, PBOX69, PBOX70, PBOX71 as defined herein,, in the preparation of a medicament for the treatment of tumours or other cancerous conditions. 11. A method of medical treatment comprising administering a phaπnaceulically effective amount of a compound of formula I. wherein (1) RI represents an unsubstituted Cg or CJ aryl group, or a Cg aryl group substituted with Me or OMe, (u) A represents 0, S or S partially oxidized to sulfoxide, (in) the cyclic group labelled F represents an misubstituted Cg or Cir j aryl-fused group or a C5 heteroaryl-fused group (nitrogen as heteroatom) or a benzo-fused system substituted with ethoxycarbonyl function, (IV) and Y represents the group — N N R 3 wherein R 2 and R 3 are independently hydrogen, or methyl, or Y represents the group CH3 or a (CH 2 ) 2 CH 3 group, or an unsubstituted C5 heteroaryl group (nitrogen as heteroatom) 12 A method of medical treatment comprising administering a pharmaceutically effective amount of a compound selected fiom those having the formulae - PBOX-4, PBOX-6, PBOX-7, PBOX-8, PBOX-9, PBOX-12, PBOX-15, PBOX-16, PBOX-27. PBOX- 28, PBOX-30, PBOX65, PBOX68, PBOX69, PBOX70, PBOX71 as defined herein 13 The compounds PBOX-4, -6, -7, -8, -9, -12, -15, -16, -65, -68, -69, -70, -71 as defined herein

Inventors: CAMPIANI GIUSEPPE, NACCI VITO, WILLIAMS DAVID CLIVE, ZISTERER DANIELA M, CAMPIANI, GIUSEPPE, NACCI, VITO, WILLIAMS, DAVID, CLIVE, ZISTERER, DANIELA, M.
Applicants: CAMPIANI GIUSEPPE, NACCI VITO, TRINITY COLLEGE DUBLIN, UNI DI SIENA, WILLIAMS DAVID CLIVE, ZISTERER DANIELA M, CAMPIANI, GIUSEPPE, NACCI, VITO, THE PROVOST, FELLOWS AND SCHOLARS OF THE COLLEGE OF THE HOLY AND UNDIVIDED TRINITY OF QUEEN ELIZABETH NEAR DUBLIN, UNIVERSITA' DI SIENA, WILLIAMS, DAVID, CLIVE, ZISTERER, DANIELA, M.
Application year: 2001
Main classification: C07D498
IPC codes: A61P35, C07D498, C07D513