CA2497031A1 - Farnesyl dibenzodiazepinones, processes for their production and their use as pharmaceuticals - Google Patents

Abstract

The present invention relates to dibenzodiazepinones of Formula I, their pharmaceutically acceptable salts and prodrugs, to methods for obtaining such compounds, to pharmaceutical compositions comprising them, and to their use as pharmaceuticals, in particular as inhibitors of cancer, and mammalian lipoxygenase, or as a PBR binding agent.

Description

FARNESYL DIBENZODIAZEPINONES, PROCESSES FOR THEIR PRODUCTION
AND THEIR USE AS PHARMACEUTICALS
FIELD OF THE INVENTION
This invention relates to derivatives of a naturally produced farnesylated dibenzodiazepinone, referred to as Compound 1, their pharmaceutically acceptable salts and prodrugs, and to methods for obtaining the compounds. One method of obtaining Compound 1 is by cultivation of a strain of Micromonospora sp., i.e., 046-EC011 or [S01 ]046. One method of obtaining the derivatives involves post-biosynthesis chemical modification of Compound 1. The present invention further relates to the use of Compound 1 derivatives, and their pharmaceutically acceptable salts and prodrugs as pharmaceuticals, in particular to their use as inhibitors of cancer cell growth, mammalian lipoxygenase, and for treating acute and chronic inflammation, and to pharmaceutical compositions comprising Compound 1 derivatives, or pharmaceutically acceptable salts or prodrugs thereof.
BACKGROUND OF THE INVENTION
The euactinomycetes are a subset of a large and complex group of Gram-positive bacteria known as actinomycetes. Over the past few decades these organisms, which are abundant in soil, have generated significant commercial and scientific interest as a result of the large number of therapeutically useful compounds, particularly antibiotics, produced as secondary metabolites. The intensive search for strains able to produce new antibiotics has led to the identification of hundreds of new species.
Many of the euactinomycetes, particularly Streptomyces and the closely related Saccharopolyspora genera, have been extensively studied. Both of these genera produce a notable diversity of biologically active metabolites. Because of the commercial significance of these compounds, much is known about the genetics and physiology of these organisms.

Another representative genus of euactinomycetes, Micromonospora, has also generated commercial interest. For example, U.S. Patent No. 5,541,181 (Ohkuma et al.) discloses a dibenzodiazepinone compound, specifically 5-farnesyl-4,7,9-trihydroxy-dibenzodiazepin-11-one (named "BU-4664L"), produced by a known euactinomycetes strain, Micromonospora sp. M990-6 (ATCC 55378). ECO-4601 (herein referred to as Compound 1 ) and novel Micromonospora sp. strains 046-EC011 and [S01 ]046 are disclosed in CA 2,466,340. ECO-4601 was also disclosed as Diazepinomicin in Charan et al (2004), J. Nat. Prod., vol 67, 1431-1433.
Compound 1 Although many biologically active compounds have been identified from bacteria, there remains the need to obtain novel compounds with enhanced properties.
Thus, there exists a considerable need to obtain pharmaceutically active compounds in a cost-effective manner and with high yield. The present invention solves these problems by providing new therapeutic compounds and to methods to generate these novel compounds by post-biosynthesis chemical modifications.
SUMMARY OF THE INVENTION
In another aspect, the invention relates to derivatives of Compound 1 and to pharmaceutical compositions comprising a derivative of Compound 1 or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
In a further aspect, the invention relates to a derivatve of Compound 1 represented by Formula I:

Formula 1 wherein, W', W2 and W3 are each independently selected from H ~ H3 ~ H3 H i Hs -~- ; Or R5 R6 ~ O
the chain from the tricycle terminates at W3, W2 or W' with W3, W2 or W' respectively being either -CH=O or -CH20H;
R' is selected from H, C1_,oalkyl, C2_~oalkenyl, C2_~oalkynyl, C6_,oarYl, C5_ ,oheteroaryl, C3_~ocycloalkyl, C3_loheterocycloalkyl, C(O)Ci_~oalkyl, C(O)C2_loalkenyl, C(O)C2_,oalkynyl, C(O)Cs_ioaryl, C(O)C5_,oheteroaryl, C(O)C3_locycloalkyl;
C(O)C3_ ,oheterocycloalkyl or a C-coupled amino acid;
R2, R3, and R4 are each independently selected from H, C,_loalkyl, C2_ioalkenyl, C2-loalkynyl, C6_,oaryl, C5_~oheteroaryl, C3_locYcloalkyl, C3_~oheterocycloalkyl, C(O)H, C(O)C,_~oalkyl, C(O)C2_,oalkenyl, C(O)C2_~oalkynyl, C(O)Cs_,oaryl, C(O)C5_,oheteroaryl, C(O)C3_,ocycloalkyl; C(O)C3_,oheterocycloalkyl or a C-coupled amino acid;
R5 and R6 are each independently selected from H, OH and OC1_salkyl;
wherein, when any of R', R2, R3, R4, R5 and R6 comprises an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group, then the alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group is optionally substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, C1_salkyl, C2_~alkenyl, C2_~alkynyl, C3_ iocycloalkyl, C3_~oheterocycloalkyl, C6_~oaryl, C5_loheteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, oxo, guanidino and formyl; and with the proviso that when W', W2 and W3 are all -CH=C(CH3)-, and R2, R3 and R4 are all H, then R' is not H;

or a pharmaceutically acceptable salt or prodrug thereof.
In one embodiment, R' is H, and all other groups are as previously disclosed.
In another embodiment, R' is -CH3, and all other groups are as previously disclosed. In another embodiment, R' is C,_~oalkyl, and all other groups are as previously disclosed.
In a subclass of this embodiment, the alkyl group is optionally substituted with a substituent selected from halo, fluoro, C6_~oaryl, and C5_~oheteroaryl. In another embodiment, R' is -C(O)Ci.~oalkyl, and all other groups are as previously disclosed. In another embodiment, R2 is H, and all other groups are as previously disclosed.
In another embodiment, R3 is H, and all other groups are as previously disclosed.
In another embodiment, R4 is H, and all other groups are as previously disclosed.
In another embodiment, R2, R3 and R4 are each H, and all other groups are as previously disclosed. In another embodiment, one of R2, R3 and R4 is CH3, the others being each H, and all other groups are as previously disclosed. In another embodiment, two of R2, R3 and R4 are CH3, the other being H, and all other groups are as previously disclosed.
In another embodiment, R2, R3 and R4 are each CH3, and all other groups are as previously disclosed. In another embodiment, R2, R3 and R4 are each H, and W' is -CH=C(CH3)-, and all other groups are as previously disclosed. In another embodiment, R2, R3 and R4 are each H, and W2 is -CH=C(CH3)-, and all other groups are as previously disclosed. In another embodiment, R2, R3 and R4 are each H, and W3 is -CH=C(CH3)-, and all other groups are as previously disclosed. In another embodiment, R' is H and R2, R3 and R4 are each H, and all other groups are as previously disclosed.
In another embodiment, R' is H, each of W', W2, and W3 is -CH=C(CH3)-, and all other groups are as previously disclosed. In another embodiment, R' is H, each of W', W2, and W3 is -CH2CH(CH3)-, and all other groups are as previously disclosed. In another embodiment, if each of W', W2 and W3 are -CH=C(CH3)-, and each of R2, R3, and are H, then R' is not H. In further embodiment, if each of W', W2 and W3 are -CH=C(CH3)-, and each of R2, R3, and R4 are H, then R' is not CH3. In further embodiment, if each of W', W2 and W3 are -CH=C(CH3)-, and each of R2, R3, and are H, then R' is neither H nor CH3. The invention encompasses all pharmaceutically acceptable salts and prodrugs of the foregoing compounds.

The following are exemplary compounds of the invention:

N / / /
/ ~ ~ OH
OH H
HO
Compound 1; Compound 2;

N / / /
O
/ N ~ ~ OH \ N /
OH
H / N ~ ~ OH
OMe HO
Compound 3; Compound 4;
/ / / /
Compound 5; Compound 6;
Compound 7; Compound 8;

Compound 9; Compound 10;
i I i Compound 11; Compound 12;
Compound 13; Compound 14;
Compound 15; Compound 16;
i i Compound 17; Compound 18;

o / / o Compound 19; Compound 20;
Compound 21; Compound 22;
Compound 23; Compound 24;
Compound 25; Compound 26;

Compound 27; Compound 28;

o N
OH H
Compound 29; Compound 30;
Compound 31; Compound 32;

Compound 33; Compound 34;
Compound 35; Compound 36;
~i i Compound 37; Compound 38;

Compound 39; Compound 40;
/ /
Compound 41; Compound 42;

N I / N
OH ~ OH
Compound 43; Compound 44;
Compound 45; Compound 46;
/ ,o / off Compound 47; Compound 48;

~o Compound 49; Compound 50;
Compound 51; Compound 52;
O OH O
N / / ~ N ~ /
OH OH
OH
N ~ OH ~ / N ~ ~ OH
OH H OH
HO HO
Compound 53; Compound 54;
Compound 55; Compound 56;
Compound 57; Compound 58;

off OH ~ OH ~ OH
OH OH
Compound 59; Compound 60;
Compound 61; Compound 62;
Compound 63; Compound 64;
i i i Compound 65; Compound 66;

Compound 67; Compound 68;
Compound 69; Compound 70;
Compound 71; Compound 72;
i i i i Compound 73; Compound 74;

Compound 75; and Compound 76;
or a pharmaceutically acceptable salt or prodrug of any one of Compounds 1 to 76.
In one embodiment, the compounds comprise a pharmaceutically acceptable carrier.
The invention further encompasses a farnesyl dibenzodiazepinone obtained by a method comprising: a) cultivating Micromonospora sp. strain [S01 ]046, wherein the cultivation is performed under aerobic conditions in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms;
and b) isolating a farnesyl dibenzodiazepinone from the bacteria cultivated in step (a).
The invention further encompasses a farnesyl dibenzodiazepinone obtained by a method comprising: a) cultivating Micromonospora sp. strain selected from strains [S01 ]046 and 046-EC011, wherein the cultivation is performed under aerobic conditions in a riutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms; b) isolating a farnesyl dibenzodiazepinone from the bacteria cultivated in step (a) and c) chemically modifying the compound isolated in (b).
In one embodiment the farnesyl dibenzodiazapinone is selected from Compounds 2 to 76. In another embodiment the farnesyl dibenzodiazapinone is selected from Compounds 2 to 12, 17, 18 and 46.
The invention further encompasses a process for making a farnesyl dibenzodiazapinone compound, comprising cultivation of Micromonospora sp.
strain 046-EC011, in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms, isolation and purification of the compound, and optional chemical modification of the isolated compound..
The invention further encompasses a process for making a farnesyl dibenzodiazepinone compound comprising cultivation of Micromonospora sp.
strain [S01 ]046, in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms, isolation and purification of the compound, and optional chemical modification of the isolated compound.
In one embodiment, the cultivation occurs under aerobic conditions.
In another embodiment, the carbon atom and nitrogen atom sources are chosen from the components shown in Table 1.
In another embodiment, the cultivation is carried out at a temperature ranging from 18°C to 40°C. In a further embodiment, the temperature range is 18°C to 29°C.
In another embodiment, the cultivation is carried out at a pH ranging from 6 to 9.
The invention further encompasses the Micromonospora sp. having IDAC
Accession No. 231203-01.
The invention further encompasses a method of treating a diseases selected from pre-cancerous or cancerous conditions, inflammation, autoimmune diseases, infections, neurodegenerative diseases and stress, the method comprising administering a compound of Formula I to a mammal in need of such treatment.
In one embodiment, the compound is a compound selected from Compounds 2 to 76. In another embodiment, the invention provides a method for treating a diseases selected from pre-cancerous or cancerous conditions, inflammation, autoimmune diseases, infections, neurodegenerative diseases and stress, the method comprising administering a compound of Formula I to a mammal in need of such treatment, with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
The invention further encompasses a method of inhibiting the growth of a cancer cell, the method comprising contacting the cancer cell with a compound of Formula I, such that growth of the cancer cell is inhibited. In one embodiment, the compound is a compound selected from Compounds 2 to 76. In another embodiment, the invention provides a method of inhibiting the growth of a cancer cell, the method comprising contacting the cancer cell with a compound of Formula I such that growth of the cancer cell is inhibited, with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
The invention further encompasses a method of inhibiting the growth of a cancer cell in a mammal, the method comprising administering a compound of Formula I
to a mammal comprising a cancer cell, such that growth of the cancer cell is inhibited in the mammal. In one embodiment, the compound is a compound selected from Compounds 2 to 76. In another embodiment, the invention provides a method of inhibiting the growth of a cancer cell in a mammal, the method comprising administering a compound of Formula I to the mammal comprising a cancer cell such that growth of the cancer cell is inhibited, with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
The invention further encompasses a method of treating a pre-cancerous or cancerous condition in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of a compound of Formula I, such that a pre-cancerous or cancerous condition is treated. In one embodiment, the compound is a compound selected from Compounds 2 to 76. In another embodiment, the invention provides a method of treating a pre-cancerous or cancerous condition in a mammal, the method comprising the step of administering to the mammal a therapeutically effctive amount of a compound of Formula I such that a pre-cancerous or cancerous condition is treated, with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
The invention further encompasses the use of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof as an antitumor agent for the treatment of a pre-cancerous or cancerous condition in a mammal. In one embodiment, the compound is a compound selected from Compounds 2 to 76. In another embodiment, the invention provides use of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof as an antitumor agent for the treatment of a pre-cancerous or cancerous condition in a mammal, with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.

The invention further encompasses the use of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof in the preparation of a medicament for the treatment of a pre-cancerous or cancerous condition in a mammal. In one embodiment, the compound is a compound selected from Compounds 2 to 76. In another embodiment, the invention provides use of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof in the preparation of a medicament for the treatment of a pre-cancerous or cancerous condition in a mammal, with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
The invention further encompasses the use of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof as a peripheral benzodiazepine receptor (PBR) binding agent for the treatment of a condition involving PBR.
The peripheral benzodiazepine receptor (PBR) is a well-characterized receptor known to be directly involved in diseases states. PBR is involved in the regulation of immune responses. These diseases states include tumors, inflammatory diseases (such as rheumatoid arthritis and lupus), parasitic infections and neurodegenerative diseases (such as Alzheimer, Huntington and Multiple Sclerosis). See, for example, Casellas et al (2003), Current Med. Chem., vol 10, 1563-1572; Miettinen et al (1995), Cancer Res., vol 55, 2691; Junck et al (1989), J. Ann. Neurol., vol 26, 752; Gavish et al (1993), Clin.
Neuropharm., vol 16, no 5, 401-417; Papadopoulos et al (2003), Ann. Pharm.
Fr., vol 61, 30-50; Vowinckel et al (1997), J. Neurosci. Res., vol 50, 345; Gelhert et al (1997), Neurochem. Int., vol 31, 705; Lacor et al (1999), Brain Res., vol 815, 70). In one embodiment, the compound for use in the treatment of a condition involving PBR
is a compound selected from Compounds 2 to 76. In another embodiment, the compound for use in the treatment of a condition involving PBR is a compound of Formula I with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
The invention further encompasses a method of reducing inflammation in a mammal, comprising administering to a mammal having inflammation a therapeutically effective amount of a compound of Formula I, such that the inflammation is reduced. In one embodiment, the compound is a compound selected from Compounds 2 to 76. In another embodiment, the compound for use as an anti-inflamatory is a compound of Formula I with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
The invention further encompasses the use of a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof as a 5-Lipooxygenase (5-LO) inhibitor for the treatment of a condition involving the 5-LO enzyme. 5-Lipoxygenase (5-LO) catalyzes the oxidative metabolism of arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE), the initial reaction leading to formation of leukotrienes. Eicosanoids derived from arachidonic acid by the action of lipoxygenases or cycloxygenases have been found to be involved in acute and chronic inflammatory diseases (i.e. asthma, multiple sclerosis, rheumatoid arthritis, ischemia, edema) as well in neurodegeneration (Alzheimer disease), aging and various steps of carcinogenesis, including tumor promotion, progression and metastasis. Soberman et al (2003), J. Clin.
Invest, vol 111, no 8, 1107-1113. In one embodiment, the compound for use in the treatment of a condition involving the 5-LO enzyme is a compound selected from Compounds 2 to 76. In another embodiment, the compound for use as in the treatment of a condition involving the 5-LO enzyme is a compound of Formula I with the proviso that the compound of Formula i is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
Leukotriene, Cysteinyl (CysLT1) receptor is involved in inflammation and CysLT~-selective antagonists are used as treatment for bronchial asthma. CysLT~ and 5-LO
were known to be upreguiated in colon cancer. See, for example, Lynch et al (1999), Nature, 399(67387), 789; Nielsen et al (2003), Adv. Exp. Med. Biol., vol 525, 201-4. In one embodiment, the invention provides a compound of Formula I for treatment of a condition involving the Leukotriene, Cysteinyl (CysLT~) receptor. In one embodiment, the compound is a compound selected from Compounds 2 to 76. In another embodiment, the compound for use in the treatment of a condition involving CysLT1 receptor is a compound of Formula I with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
Cyclooxygenase (COX-2) enzyme is known to be produced only in response to injury or infection. It produces prostaglandins involved in inflammation and the immune response. Elevated levels of COX-2 in the body have been linked to cancer.
See, for example, W ikstrom et al (2003), Biochem. Biophys. Res. Commun, vol 302, no 2, 335. In one embodiment, the invention provides a compound of Formula I for treatment of a condition involving the COX-2 enzyme. In one embodiment, the compound is a compound selected from Compounds 2 to 76. In another embodiment, the compound for use as in the treatment of a condition involving the COX-2 enzyme is a compound of Formula I with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
AcyICoA-Cholesterol Acyltransferase, Hepatic (AcoA-AT) is known to convert cholesterol to colesteryl esters and is involved in the development of artherioscerosis.
See, for example, Kusunoki et al (2001 ), Circulation, 2604-2609. In one embodiment, the invention provides a compound of Formula I for treatment of a condition involving AcyICoA-Cholesterol Acyltransferase. In one embodiment, the compound is a compound selected from Compounds 2 to 76. In another embodiment, the compound for use as in the treatment of a condition involving AcyICoA-Cholesterol Acyltransferase is a compound of Formula I with the proviso that the compound of Formula I is not Compound 1, or alternatively is not Compound 2, or alternatively is neither Compound 1 nor 2.
BRIEF DESCRIPTION OF THE FIGURES
FIGURE 1 shows'H NMR data for Compound 1 dissolved in MeOH-d4.
FIGURE 2 shows the in vitro anti-inflammatory activity of ECO-4601. Graph shows percent inhibition of 5-lipoxygenase activity plotted against the Log NM
concentration of ECO-4601 and NDGA. Graph shows the ECSO of ECO-4601 to be 0.93NM.
FIGURE 3 shows inhibition of tumor growth resulting from administration of 10 to 30 mg/kg of ECO-4601 to glioblastoma-bearing mice one day after tumor cell inoculation.
FIGURE 4 shows inhibition of tumor growth resulting from administration of 20-mg/kg of ECO-4601 to glioblastoma-bearing mice ten days after tumor cell inoculation.
FIGURE 5 shows micrographs of tumor sections from mice bearing glioblastoma tumors and treated with saline or ECO-4601. The cell density of tumor treated with ECO-4601 appears decreased and nuclei from tumor cells are larger and pynotic suggesting a cytotoxic effect.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel farnesyl dibenzodiazepinone herein referred as the compounds of Formula I, which are derivatives of "ECO-4601"
(Compound 1 ) isolated from strains of actinomycetes, Micromonospora sp. 046-and strain [S01 ]046. These organisms were deposited on March 7, 2003, and December 23, 2003, respectively, with the International Depository Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, under Accession Nos. IDAC 070303-01 and IDAC
231203-01, respectively.
The invention further relates to pharmaceutically acceptable salts and derivatives of ECO-4601, and to methods for obtaining such compounds. One method of obtaining the compound is by cultivating Micromonospora sp. strain 046-EC011, or a mutant or a variarot thereof, under suitable Micromonospora culture conditions, preferably using the fermentation protocol described hereinbelow, and by optional chemical modification of the compound obtained by isolation from the fermentation procedure.
The invention also relates to a method for producing novel farnesyl dibenzodiazepinone compounds, by chemical modification of the farnesyl dibenzodiazepinone obtained from fermentation and isolation.
The present invention also relates to pharmaceutical compositions comprising a compound of Formula I and its pharmaceutically acceptable salts and derivatives.
Compounds of Formula I are useful as a pharmaceutical, in particular for use as an inhibitor of cancer cell growth, and mammalian lipoxygenase.
The following detailed description discloses how to make and use the compounds of Formula I and compositions containing these compound to inhibit microbial growth and/or cancer and/or specific disease pathways.
Accordingly, certain aspects of the present invention relate to pharmaceutical compositions comprising the farnesylated dibenzodiazepinone compounds of the present invention together with a pharmaceutically acceptable carrier, and methods of using the pharmaceutical compositions to treat diseases, including cancer, and chronic and acute inflammation, autoimmune diseases, and neurodegenerative diseases.
Definitions For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below.
As used herein, the term "farnesyl dibenzodiazepinone" refers to a class of dibenzodiazepinone compounds containing a farnesyl moiety or being derived from a farnesyl moiety. The term includes, but is not limited to, a compound of Formula I, a compound selected from Compounds 2 to 76, or the exemplified compounds of the present invention, Compounds 2 to 12, 17, 18 and Compound 46. As used herein, the term "farnesyl dibenzodiazepinone" includes compounds of this class that can be used as intermediates in chemical syntheses.
As used herein, the term "compound(s) of the invention" refers to a farnesyl dibenzodiazepinone as defined above and to pharmaceutically acceptable salts or prodrugs thereof.
The term "receptor" refers to a protein located on the surface or inside a cell that may interact with a different molecule, known as a ligand, to initiate or inhibit a biological response.
As used herein, the term "ligand" refers to a molecule or compound that has the capacity to bind to a receptor and modulate its activity.

As used herein, the terms "binder", "receptor binder" or "binding agent"
refers to a compound of the invention acting as a ligand. The binding agent can act as an agonist, or an antagonist of the receptor. An agonist is a drug which binds to a receptor and activates it, producing a pharmacological response (e.g. contraction, relaxation, secretion, enzyme activation, etc.). An antagonist is a drug which attenuates the effects of an agonist, or a natural ligand. Antagonism can be competitive and reversible (i.e. it binds reversibly to a region of the receptor in common with the agonist.) or competitive and irreversible (i.e. antagonist binds covalently to the agonist binding site, and no amount of agonist can overcome the inhibition). Other types of antagonism are non-competitive antagonism where the antagonist binds to an allosteric site on the receptor or an associated ion channel.
As used herein, the term "enzyme inhibitor" or "inhibitor" refers to a chemical that disables an enzyme and prevents it from performing its normal function.
As used herein, abbreviations have their common meaning. Unless otherwise noted, the abbreviations "Ac", "Me", "Et", "Pr", "i-Pr", "Bu", and "Ph", respectively refer to acetyl, methyl, ethyl, propyl (n- or iso-propyl), iso-propyl, butyl (n-, iso-or tert-butyl) and phenyl.
The term "alkyl" refers to linear, branched or cyclic, saturated or unsaturated hydrocarbon groups. Examples of saturated alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl, heptyl, cyclopentyl, cyclohexyl, cyclohexymethyl, 1-propane-2-yl, 1-butane-4-yl, 1-propyne-3-yl, 1-butyne-4-yl, cyclopent-2-yl, and the like. Alkyl groups may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, oxo, guanidino and formyl.
The term "C,."alkyl", wherein n is an integer from 2 to 12, refers to an alkyl group having from 1 to the indicated "n" number of carbons. The C,_"alkyl can be cyclic or a straight or branched chain.
The term "alkenyi" refers to an alkyl group, as defined above, having from one to six carbon-carbon double bonds. Examples of alkenyl groups include, without limitation, vinyl, 1-propene-2-yl, 1-butene-4-yl, 2-butene-4-yl, 1-pentene-5-yl and the like. Alkenyl groups may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, formyl, oxo and guanidino. The double bond portions) of the unsaturated hydrocarbon chain may be either in the cis or trans configuration.
The term "C2_~alkenyl", wherein n is an integer from 3 to 12, refers to an alkenyl group having from 2 to the indicated "n" number of carbons. The C2_"alkenyl can be cyclic or a straight or branched chain.
The term "alkynyl" refers to an alkyl group, as defined above, having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propyne-3-yl, 1-butyne-4-yl, 2-butyne-4-yl, 1-pentyne-5-yl and the like.
Alkynyl groups may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, formyl, oxo and guanidine.
The term "C2_~alkynyl", wherein n is an integer from 3 to 12, refers to an alkynyl group having from 2 to the indicated "n" number of carbons. The C2_"alkynyl can be a straight or branched chain.
The term "cycloalkyl" or "cycloalkyl ring" refers to an alkyl group, as defined above, comprising a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to fifteen ring members. A
cycloalkyl ring may include from 1 to 4 heteroatoms or groups selected from: O, N, NH, NRx, P02, S, SO or S02. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, oxiranyl, morpholinyl, tetrahydropyranyl and tetrahydrofuranyl. Cycloalkyl groups may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, vitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

The term "C3.ncycloalkyl", wherein n is an integer from 4 to 15, refers to a cycloalkyl group having from 3 to the indicated "n" number of carbons.
The term "heterocycloalkyl", "heterocyclic" or "heterocycloalkyl ring" refers to a cycloalkyl group, as defined above, containing one to four hetero atoms or hetero groups selected from O, N, NH, NRX, P02, S, SO or S02 in a single or fused heterocyclic ring system having from three to fifteen ring members. Examples of a heterocycloalkyl, heterocyclic or heterocycloalkyl ring include, without limitation, morpholinyl, piperidinyl, and pyrrolidinyl. Heterocycloalkyl, heterocyclic or heterocycloaikyl ring may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, oxo, thiocarbonyl, imino, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.
The term "C3."heterocycloalkyl", wherein n is an integer from 4 to 15, refers to an heterocycloaikyl group having from 3 to the indicated "n" number of atoms in the cycle and at least one hetero group as defined above.
The term "halo" refers to bromine, chlorine, fluorine or iodine substituents.
The term "aryl" or "aryl ring" refers to common aromatic groups having "4n+2"
electrons, wherein n is an integer from 1 to 3, in a conjugated monocyclic or polycyclic system and having from five to fourteen ring atoms. Aryl ring may include from 1 to 4 heteroatoms such as nitrogen, oxygen and sulphur atoms. Examples of aryl include, without limitation, phenyl, naphthyl, biphenyl, terphenyl, furyl, pyrrollyl, thienyl, pyridyl, oxazolyl, imidazolyl, pyrazolyl and indolyl groups. Aryl may optionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, azido, alkythio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thin, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.
The term "C5_~aryl", wherein n is an integer from 5 to 14, refers to an aryl group having from 5 to the indicated "n" number of atoms, including carbon, nitrogen, oxygen and sulfur. The C5_~aryl can be mono or polycyclic.

The term "heteroaryl" or "heteroaryl ring" refers to an aryl ring, as defined above, containing one to tour heteroatoms such as nitrogen, oxygen and sulphur atoms.
Examples of heteroaryl include, without limitation, furyl, pyrrollyl, thienyl, pyridyl, oxazolyl, imidazolyl, pyrazolyl and indolyl groups. Heteroaryl may optionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, azido, alkythio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, vitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.
The term "C5_nheteroaryl", wherein n is an integer from 5 to 14, refers to an heteroaryl group having from 5 to the indicated "n" number of atoms, including carbon, nitrogen, oxygen and sulphur atoms. The C5_naryl can be mono or polycyclic.
The term "amino acid" refers to an organic acid containing an amino group. The term includes both naturally occurring and synthetic amino acids; therefore, the amino group can be but is not required to be, attached to the carbon next to the acid. A C-coupled amino acid substituent is attached to the heteroatom (nitrogen or oxygen) of the parent molecule via its carboxylic acid function. C-coupled amino acid forms an ester with the parent molecule when the heteroatom is oxygen, and an amide when the heteroatom is nitrogen. Examples of amino acids include, without limitation, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophane, methionine, glycine, serine, threonine, cysteine, asparagines, glutamine, tyrosine, histidine, lysine, arginine, aspartic acid, glutamic acid, desmosine, ornithine, 2-aminobutyric acid, cyclohexylalanine, dimethylglycine, phenylglycine, norvaline, norleucine, hydroxylysine, alto-hydroxylysine, hydroxyproline, isodesmosine, alto-isoleucine, ethylglycine, beta-alanine, aminoadipic acid, aminobutyric acid, ethyl asparagine, and N-methyl amino acids. Amino acids can be pure L or D isomers or mixtures of L and D isomers.
The compounds of the present invention can possess one or more asymmetric carbon atoms and can exist as optics! isomers forming mixtures of racemic or non-racemic compounds. The compounds of the present invention are useful as single isomers or as a mixture of stereochemical isomeric forms. Diastereoisomers, i.e., nonsuperimposable stereochemical isomers, can be separated by conventional means such as chromatography, distillation, crystallization or sublimation. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes.
The invention encompasses isolated or purified compounds. An "isolated" or "purified" compound refers to a compound which represents at least 10%, 20%, 50%, 80% or 90% of the compound of the present invention present in a mixture, provided that the mixture comprising the compound of the invention has demonstrable (i.e.
statistically significant) biological activity including cytostatic, cytotoxic, enzyme inhibitory or receptor binding action when tested in conventional biological assays known to a person skilled in the art.
The terms "farnesyl dibenzodiazepinone-producing microorganism" and "producer of farnesyl dibenzodiazepinone," as used herein, refer to a microorganism that carries genetic information necessary to produce a farnesyl dibenzodiazepinone compound, whether or not the organism naturally produces the compound. The terms apply equally to organisms in which the genetic information to produce the farnesyl dibenzodiazepinone compound is found in the organism as it exists in its natural environment, and to organisms in which the genetic information is introduced by recombinant techniques.
Specific organisms contemplated herein include, without limitation, organisms of the family Micromonosporaceae, of which preferred genera include Micromonospora, Actinoplanes and Dactylosporangium; the family Streptomycetaceae, of which preferred genera include Streptomyces and Kitasatospora; the family Pseudonocardiaceae, of which preferred genera are Amycolatopsis and Saccharopolyspora; and the family Actinosynnemataceae, of which preferred genera include Saccharothrix and Actinosynnema; however the terms are intended to encompass all organisms containing genetic information necessary to produce a farnesyl dibenzodiazepinone compound. A preferred producer of a farnesyl dibenzodiazepinone compound includes microbial strain 046-EC011, a deposit of which was made on March 7, 2003, with the International Depository Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, under Accession No. IDAC 070303-01.
As used herein, the term "treatment" refers to the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder, e.g., a disease or condition, a symptom of disease, or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of disease, or the predisposition toward disease.
As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a farnesyl dibenzodiazepinone and a pharmaceutically acceptable carrier. As used herein, "pharmacologically effective amount,"
"therapeutically effective amount" or simply "effective amount" refers to that amount of a farnesyl dibenzodiazepinone effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25%
reduction in that parameter.
The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc.
If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

The term "pharmaceutically acceptable salt" refers to both acid addition salts and base addition salts. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Exemplary acid addition salts include, without limitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulphuric, phosphoric, formic, acetic, citric, tartaric, succinic, oxalic, malic, glutamic, propionic, glycolic, gluconic, malefic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, ~i-hydroxybutyric, malonic, galactaric, galacturonic acid and the like. Suitable pharmaceutically acceptable base addition salts include, without limitation, metallic salts made from aluminium, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, procaine and the like. Additional examples of pharmaceutically acceptable salts are listed in Berge et al., Journal of Pharmaceutical Sciences (1977) 66:2, 1-19. All of these salts may be prepared by conventional means from a farnesyl dibenzodiazepinone by treating the compound with the appropriate acid or base.
The term "pharmaceutically acceptable salt or prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an biologically active metabolite or residue thereof. Particularly favored salts or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Pharmaceutically acceptable prodrugs of the compounds of this invention include, without limitation, esters, amino acid esters, phosphate esters, metal salts and sulfonate esters.
Unless otherwise indicated, all numbers expressing quantities of ingredients and properties such as molecular weight, reaction conditions, MIC and Gl~o values, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of significant figures and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the examples, tables and figures are reported as precisely as possible. Any numerical values may inherently contain certain errors resulting from variations in experiments, testing measurements, statistical analysis and such.
II. Compounds of the invention In one aspect, the invention relates to novel farnesyl dibenzodiazepinones, referred to herein as Compound 1 derivatives and to pharmaceutically acceptable salts and prodrugs thereof.
The Compounds of the invention may be characterized as chemically modified Compound 1 by: O and N acylation and alkylation, farnesyl side chain hydrogenolysis, epoxidation, and dihydroxylation. Compounds 2 to 12, 17, 18 and 46 may be characterized by any one of their physicochemical and spectral properties, such as mass and NMR, detailed in Examples 4 through 8.
In another aspect, the invention relates to derivatives of Compound 1, represented by Formula I:
Formula I

wherein, W', W2 and W3 are each independently selected from H I _ _I _ _H I__ I ( ~~ . ~ H~C~ ' ~~ ~ ~C ~ ' Or R5 Rs ~ 0 the ctoain from the tricycle terminates at W3, W2 or W' with W3, W2 or W' respectively being either -CH=O or -CH20H;
R' is selected from H, C~_~oalkyl, C2.~oalkenyl, C2_,oalkynyl, Cs.~oaryl, C5_ ~oheteroaryl, C3.iocycloalkyl, C3.~oheterocycloalkyl, C(O)Cl.~oalkyl, C(O)C2.~oalkenyl, C(O)C2_,oalkynyl, C(O)C6_~oaryl, C(O)C5_,oheteroaryl, C(O)C3_,ocycloalkyl;
C(O)C3_ ,oheterocycloalkyl or a C-coupled amino acid;
R2, R3, and R4 are each independently selected from H, C1_,oalkyl, C2_,oalkenyl, C2_,oalkynyl, C6.,oaryl, C5_ioheteroaryl, Cs_locycloalkyl, C3.ioheterocycloalkyl, C(O)H, C(O)C,_,oalkyl, C(O)C2_ioalkenyl, C(O)C2_ioalkynyl, C(O)C6_,oaryl, C(O)CS.ioheteroaryl, C(O)C3_locycloalkyl; C(O)C3_,oheterocycloalkyl or a C-coupled amino acid;
R5 and R6 are each independently selected from H, OH and OC,_salkyl;
wherein, when any of R', R2, R3, R4, R5 and R6 comprises an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group, then the alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group is optionally substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thin, C,_salkyl, C2_,alkenyl, Cz_~alkynyl, C3.
iocycloalkyl, C3.loheterocycloalkyl, Cg.loaryl, C5_loheteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, oxo, guanidino and formyl; and with the proviso that when W', W2 and W3 are all -CH=C(CH3)-, and R2, R3 and R4 are all H, then R' is not H;
or a pharmaceutically acceptable salt or prodrug thereof.
In one embodiment, R' is H, and all other groups are as previously disclosed.
In another embodiment, R' is -CHs, and all other groups are as previously disclosed. In another embodiment, R' is C,_loalkyl, and all other groups are as previously disclosed.
In a subclass of this embodiment, the alkyl group is optionally substituted with a substituent selected from halo, fluoro, Cs.~oaryl, and C5_,oheteroaryl. In another embodiment, R' is -C(O)C1_,oalkyl, and all other groups are as previously disclosed. In another embodiment, R2 is H, and all other groups are as previously disclosed.
In another embodiment, R3 is H, and all other groups are as previously disclosed.
In another embodiment, R4 is H, and all other groups are as previously disclosed.
In another embodiment, R2, R3 and R4 are each H, and all other groups are as previously disclosed. In another embodiment, one of R2, R3 and R4 is CH3, the others being each H, and all other.groups are as previously disclosed. In another embodiment, two of R2, R3 and R4 are CH3, the other being H, and all other groups are as previously disclosed.
In another embodiment, R2, R3 and R4 are each CH3, and all other groups are as previously disclosed. In another embodiment, R2, R3 and R4 are each H, and W' is -CH=C(CH3)-, and all other groups are as previously disclosed. In another embodiment, R2, R3 and R4 are each H, and W2 is -CH=C(CH3)-, and all other groups are as previously disclosed. In another embodiment, R2, R3 and R4 are each H, and W3 is -CH=C(CH3)-, and all other groups are as previously disclosed. In another embodiment, R' is H and R2, R3 and R° are each H, and all other groups are as previously disclosed.
In another embodiment, R' is H, each of W', W2, and W3 is -CH=C(CH3)-, and all other groups are as previously disclosed. In another embodiment, R' is H, each of W', W2, and W3 is -CH2CH(CH3)-, and all other groups are as previously disclosed. In another embodiment, if each of W', W2 and W3 are -CH=C(CH3)-, and each of R2, R3, and R°
are H, then R' is not H. in further embodiment, if each of W', W2 and W3 are -CH=C(CH3)-, and each of R2, R3, and R4 are H, then R' is not CH3. In further embodiment, if each of W', W2 and W3 are -CH=C(CH3)-, and each of R2, R3, and are H, then R' is neither H nor CH3. The invention encompasses all pharmaceutically acceptable salts and prodrugs of the foregoing compounds.
The following are exemplary compounds of the invention, such named compounds are not intended to limit the scope of the invention in any way:

Compound 1; Compound 2;
Compound 3; Compound 4;
Compound 5; Compound 6;
i i I i i Compound 7; Compound 8;

Compound 9; Compound 10;
Compound 11; Compound 12;
Compound 13; Compound 14;
i i i i Compound 15; Compound 16;
i i Compound 17; Compound 18;
i , i Compound 19; Compound 20;

Compound 21; Compound 22;
Compound 23; Compound 24;
Compound 25; Compound 26;
Compound 27; Compound 28;

Compound 29; Compound 30;
i Compound 31; Compound 32;

Compound 33; Compound 34;
Compound 35; Compound 36;
Compound 37; Compound 38;

Compound 39; Compound 40;
Compound 41; Compound 42;
Compound 43; Compound 44;
Compound 45; Compound 46;
Compound 47; Compound 48;

Compound 49; Compound 50;
Compound 51; Compound 52;
Compound 53; Compound 54;
Compound 55; Compound 56;
Compound 57; Compound 58;

Compound 59; Compound 60;

i i i OH
N >-=Y
OH
HO
Compound 61; Compound 62;
Compound 63; Compound 64;
Compound 65; Compound 66;
Compound 67; Compound 68;

Compound 69; Compound 70;
Compound 71; Compound 72;
i i i i Compound 73; Compound 74;
Compound 75; and Compound 76;
or a pharmaceutically acceptable salt of prodrug of any one of Compounds 1 to 76.
Certain embodiments expressly exclude one or more of the compounds of Formula I. In one embodiment, Compound 1 is excluded. In another embodiment, Compound 2 is excluded. In a further embodiment, both Compound 1 and Compound are excluded.
The compounds of this invention may be formulated into pharmaceutical compositions comprised of compounds of Formula I in combination with a pharmaceutically acceptable carrier, as discussed in Section IV below.
ill. Method of Making a Farnesyl Dibenzodiazepinone A. Fermentation In one embodiment, ECO-4601 is obtained by cultivating a novel strain of Micromonospora, namely Micromonospora sp. strain 046-EC011. Strain 046-EC011 was deposited on March 7, 2003, with the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, under Accession No. 070303-01. The deposit of the strain was made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure.
The deposited strains will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strains are provided merely as convenience to those skilled in the art and are not an admission that a deposit is required for enablement.
It is to be understood that the present invention is not limited to use of the particular strain 046-EC011. Rather, the present invention contemplates the use of other ECO-4601 producing organisms, such as mutants or variants of 046-EC011 that can be derived from this organism by known means such as X-ray irradiation, ultraviolet irradiation, treatment with nitrogen mustard, phage exposure, antibiotic selection and the like; or through the use of recombinant genetic engineering techniques, as described in Section IV below.
The farnesyl dibenzodiazepinone compounds of the present invention may be biosynthesized by various microorganisms. Microorganisms that may synthesize the compounds of the present invention include but are not limited to bacteria of the order Actinomycetales, also referred to as actinomycetes. Non-limiting examples of members belonging to the genera of Actinomycetes include Nocardia, Geodermatophilus, Actinopianes, Micromonospora, Nocardioides, Saccharothrix, Amycolatopsis, Kutzneria, Saccharomonospora, Saccharopolyspora, Kitasatospora, Streptomyces, Microbispora, Streptosporangium, and Actinomadura. The taxonomy of actinomycetes is complex and reference is made to Goodfellow, Suprageneric Classification of Actinomycetes (1989); Bergey's Manual of Systematic Bacteriology, Vol. 4 (Williams and Wilkins, Baltimore, pp. 2322-2339); and to Embley and Stackebrandt, "The molecular phylogeny and systematics of the actinomycetes," Annu. Rev. Microbiol. (1994) 48:257-289, for genera that may synthesize the compounds of the invention.
Farnesyl dibenzodiazepinone-producing microorganisms are cultivated in culture medium containing known nutritional sources for actinomycetes. Such media having assimilable sources of carbon, nitrogen, plus optional inorganic salts and other known growth factors at a pH of about 6 to about 9. Suitable media include, without limitation, the growth media provided in Table 1. Microorganisms are cultivated at incubation temperatures of about 18 °C to about 40 °C for about 3 to about 40 days.
TABLE 1: PREFERRED MEDIA COMPOSITION FOR PRODUCTION OF ECO-4601 Com~~onent pg MA KH RM JA FA

pH*5 7.2 7.57 6.857.3 7.0 Glucose 12 10 10 10 Sucrose 100 Lactose Cane molasses 15 Corn starch 30 Soluble starch10 25 Potato dextrin 20 40 Corn steep 5 solid Corn steep 5 15 liquor Dried yeast 2 Yeast extract 5 Malt extract 35 PharmamediaT""10 15 Glycerol NZ-Amine 5 10 Soybean powder 15 Component pB. MA KH RM JA FA

Soybean flour Meat extract Bacto-peptone MgS04.7H20 1 MgCl2. 6H20 CaC03 4 1 2 2 NaCI 5 (NHa)z SOa 2 KZ S04 0.25 MnC12.4H20 MgC12.6H20 10 FeC12.4H20 ZnCl2 Na2HP04 3 Thiamine Casamino 0.1 acid Proflo oil 4 Trace element 2 solution *3 ml/L

Unless otherwise indicated all the ingredients are in gm/L.
*3Trace elements solution contains: ZnCl2 40 mg; Fe CI36H20 (200 mg); CuCl2 2H20 (10 mg);
MnC12.4H20; Na2B40,.10Hz0 (l0mg); (NH4) 6 MO~OZ4.4H20 (10 mg) per litre.
*5 The pH is to adjusted as marked prior to the addition of CaC03.
The culture media inoculated with the farnesyl dibenzodiazepinone-producing microorganisms may be aerated by incubating the inoculated culture media with agitation, for example, shaking on a rotary shaker, or a shaking water bath.
Aeration may also be achieved by the injection of air, oxygen or an appropriate gaseous mixture to the inoculated culture media during incubation. Following cultivation, the farnesyl dibenzodiazepinone compounds can be extracted and isolated from the cultivated culture media by techniques known to a skilled person in the art and/or disclosed herein, including for example centrifugation, chromatography, adsorption, filtration. For example, the cultivated culture media can be mixed with a suitable organic solvent such as n-butanol, n-butyl acetate or 4-methyl-2-pentanone, the organic layer can be separated for example, by centrifugation followed by the removal of the solvent, by evaporation to dryness or by evaporation to dryness under vacuum. The resulting residue can optionally be reconstituted with for example water, ethanol, ethyl acetate, methanol or a mixture thereof, and re-extracted with a suitable organic solvent such as hexane, carbon tetrachloride, methylene chloride or a mixture thereof.
Following removal of the solvent, the compounds may be further purified by the use of standard techniques, such as chromatography.
8. Chemical Modifications:
The farnesyl dibenzodiapezinones biosynthesized by microorganisms are subjected to random and/or directed chemical modifications to form compounds that are derivatives or structural analogs. Such derivatives or structural analogs having similar functional activities are within the scope of the present invention.
Farnesyl dibenzodiapezinone compounds may be modified using methods known in the art and described herein. Examples of chemical modifications are shown in Examples 4 to 8 and Example 15.
Derivatives of the ECO-4601 molecule, for example those identified herein as the compounds of Formula I and Compounds 2 to 76, are generated by standard organic chemistry approaches. General principles of organic chemistry required for making and manipulating the compounds described herein, including functional moieties, reactivity and common protocols are described, for example, in "Advanced Organic Chemistry,"
4t" Edition by Jerry March (1992), Wiley-Interscience, USA. In addition, it will be appreciated by one of ordinary skill in the art that the synthetic methods described herein may use a variety of protecting groups, whether or not they are explicitly described. A "protecting group" as used herein means a moiety used to block one or more functional moieties such as reactive groups including oxygen, sulfur or nitrogen, so that a reaction can be carried out selectively at another reactive site in a polyfunctional compound. General principles for the use of protective groups, their applicability to specific functional groups and their uses are described for example in T.
H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3'~
Edition, John Wiley & Sons, New York (1999).
Alcohols and phenols are protected with, for example: silyl ethers (TMS:
trimethylsilyl, TIPS: triisopropylsilyl), acetals (MOM: methyloxymethyl, BOM:
benzyloxymethyl), esters (acetate, benzoyl) and ethers (Bn: benzyl). Alcohols are deprotected by conditions such as: TBAF (tetrabutylammonium fluoride) for silyl ethers, aqueous acid catalysis for acetals and esters, saponification for esters, and hydrogenolysis for Bn and BOM. Amine is protected using standard amino acid protecting groups, for example, carbamates (such as t-butyl (BOC) and benzyl (CBZ)), fluorene derivatives (such as FMOC: N (9-fluorenylmethoxycarbonyl)-), etc.
Amine is deprotected by conditions such as: acid hydrolysis for BOC, hydrogenolysis for CBZ, or base treatment for FMOC. All protection and deprotection conditions are demonstrated in the Greene et al reference above.
Those skilled in the art will readily appreciate that many synthetic chemical processes may be used to produce derivatives of Compound 1. The following schemes are exemplary of the routine chemical modifications that may be used to produce compounds of Formula I. Any chemical synthetic process known to a person skilled in the art providing the structures described herein may be used and are therefore comprised in the present invention.
Scheme 1: Phenolic alcohol(s) modifications OH Rs_X ~z~
s Rs~~O~X ~ O R
O
wherein, R5 and Rs are each selected from alkyl, alkalene, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and X is a suitable leaving group.
In Scheme 1, Phenols are independently alkylated or esterified. Allkylation is accomplished with an alkylating agent such as RX is a diazoalkane, or with a RX
reagent, wherein X is a suitable leaving group such as Br, I and trifluoromethane sulfonate in the presence of a base, preferably, a diazoalkane is used. A
phenolic alcohol is converted to ester when reacted with an activated carboxylic acid such as acid halides, anhydrides and N-hydroxysuccinimide esters or with carboxylic acids and coupling agents in the presence of a base like diisopropylethylamine. Scheme 1 is used to obtain Compounds 4 to 12 and 35 to 39 from Compound 1, and Compound 15 from Compound 13.
Scheme 2: Amine modifications ~R
R_x ~N
~NH
RC(O)X ~
~N R
wherein, R is selected from alkyl, alkene, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; and X is a suitable leaving group.
In Scheme 2, amine group is optionally alkylated or acylated. Amine is alkylated using an RX alkylating agent such as dialkyl sulfates and alkyl halide, preferably RX is an alkyl halide and a base is used. An amine is also acylated when reacted with an activated carboxylic acid such as acid halides, anhydrides and N-hydroxysuccinimide esters or with carboxylic acids and coupling agents in the presence of a base like diisopropylethylamine. Scheme 2 is used to obtain Compounds 2, 3, 13, 14 and 60 to 76 from Compound 1.
Scheme 3: Double bonds) modifications H
~w / 1ol..i ~ H+
O OH
H Oa H H OH
O H~ ~ ~ OH
H
H H
In Scheme 3, double bond is modified by hydrogenation, epoxidation and ozonolysis.
Hydrogenation is carried out using a hydrogen source in the presence of a catalyst (such as rhodium, platinum, palladium, etc). Epoxide is obtained from the reaction of a double bond with an oxidizing agent such as a peracid. The epoxide obtained is opened in aqueous conditions, preferably acidic, to produce a diol. An aldehyde is obtained form the reaction of the double bond with a controlled quantity of ozone. The aldehyde obtained is reduced to alcohol by a reducing agent [H] such as sodium borohydride (NaBH4), sodium cyanoborohydride (NaBH3CN) or LAH. Scheme 3 is used to obtain Compounds 16 to 22 and 40 to 46 from Compound 1, Compounds 53 to 59 respectively from Compounds 16 to 22, Compounds 23, 24 and 26 from Compound 42, Compounds 25, 27 and 29 from Compound 41, Compounds 28, 30 and 31 from Compound 40, Compounds 32, 33 and 34 respectively from Compounds 45, 44 and 43, Compounds 47, 49 and 51 from Compound 1, and Compounds 48, 50 and 52 respectively from Compounds 47, 49 and 49.
IV. Pharmaceutical compositions comhrisinp the compounds of the invention In another embodiment, the invention relates to a pharmaceutical composition comprising a farnesyl dibenzodiazepinone, as described in the preceding section, and a pharmaceutically acceptable carrier, as described below. The pharmaceutical composition comprising the farnesyl dibenzodiazepinone is useful for treating a variety of diseases and disorders, including cancer, inflammation, autoimmune diseases, infections, neurodegenerative diseases and stress.
The compounds of the present invention, or pharmaceutically acceptable salts thereof, can be formulated for oral, intravenous, intramuscular, subcutaneous, topical or parenteral administration for the therapeutic or prophylactic treatment of diseases, particularly acute and chronic inflammation, cancer, autoimmune diseases, infections, neurodegenerative diseases and stress. For oral or parenteral administration, compounds of the present invention can be mixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers and the like. The compositions comprising a compound of this present invention will contain from about 0.1 % to about 99.9%, about 1 % to about 98%, about 5% to about 95%, about 10% to about 80% or about 15% to about 60% by weight of the active compound.
The pharmaceutical preparations disclosed herein are prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent, or eliminate cancer or inflammation, autoimmune diseases, infections, neurodegenerative diseases or stress. (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA; and Goodman and Gilman, Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, NY, for a general description of the methods for administering various antimicrobial agents for human therapy). The compositions of the present invention can be delivered using controlled (e.g., capsules) or sustained release delivery systems (e.g., bioerodable matrices).
Exemplary delayed release delivery systems for drug delivery that are suitable for administration of the compositions of the invention (preferably of Formula I) are described in U.S. Patent Nos 4,452,775 (issued to Kent), 5,039,660 (issued to Leonard), 3,854,480 (issued to Zaffaroni).
The pharmaceutically acceptable compositions of the present invention comprise one or more compounds of the present invention in association with one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants and/or excipients, collectively referred to herein as "carrier" materials, and if desired other active ingredients. The compositions may contain common carriers and excipients, such as corn starch or gelatin, lactose, sucrose, microcrystailine cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. The compositions may contain crosarmellose sodium, microcrystalline cellulose, sodium starch glycolate and alginic acid.
Tablet binders that can be included are acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Providone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose.
Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicon fluid, talc, waxes, oils and colloidal silica.
Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or the like can also be used. It may also be desirable to add a coloring agent to make the dosage form more aesthetic in appearance or to help identify the product comprising a compound of the present invention.
For oral use, solid formulations such as tablets and capsules are particularly useful. Sustained released or enterically coated preparations may also be devised. For pediatric and geriatric applications, suspension, syrups and chewable tablets are especially suitable. For oral administration, the pharmaceutical compositions are in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a therapeutically-effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, the tablets and capsules which can contain, in addition to the active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica or talc: disintegrants, for example, potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations generally are in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs and may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.
For intravenous (iv) use, compounds of the present invention can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Intravenous fluids include, without limitation, physiological saline or Ringer's solution.
Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions or suspensions can be prepared from sterile powders or granules having one or more of the carriers mentioned for use in the formulations for oral administration.
The compounds can be dissolved in polyethylene glycol, propylene glycol, ethanol, corn oil, benzyl alcohol, sodium chloride, and/or various buffers.
For intramuscular preparations, a sterile formulation of compounds of the present invention or suitable soluble salts forming the compound, can be dissolved and administered in a pharmaceutical diluent such as Water-for-Injection (WFI), physiological saline or 5% glucose. A suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.
For topical use the compounds of present invention can also be prepared in suitable forms to be applied to the skin, or mucus membranes of the nose and throat, and can take the form of creams, ointments, liquid sprays or inhalants, lozenges, or throat paints. Such topical formulations further can include chemical compounds such as dimethylsulfoxide (DMSO) to facilitate surface penetration of the active ingredient.
For application to the eyes or ears, the compounds of the present invention can be presented in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.

For rectal administration the compounds of the present invention can be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, wax or other glyceride.
Alternatively, the compound of the present invention can be in powder form for reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery. In another embodiment, the unit dosage form of the compound can be a solution of the compound or a salt thereof in a suitable diluent in sterile, hermetically sealed ampoules.
The amount of the compound of the present invention in a unit dosage comprises a therapeutically-effective amount of at least one active compound of the present invention which may vary depending on the recipient subject, route and frequency of administration. A recipient subject refers to a plant, a cell culture or an animal such as an ovine or a mammal including a human.
According to this aspect of the present invention, the novel compositions disclosed herein are placed in a pharmaceutically acceptable carrier and are delivered to a recipient subject (including a human subject) in accordance with known methods of drug delivery. In general, the methods of the invention for delivering the compositions of the invention in vivo utilize art-recognized protocols for delivering the agent with the only substantial procedural modification being the substitution of the compounds of the present invention for the drugs in the art-recognized protocols.
The compounds of the present invention provide a method for treating pre-cancerous or cancerous conditions, and acute or chronic inflammatory disease.
As used herein, the term "unit dosage" refers to a quantity of a therapeutically effective amount of a compound of the present invention that elicits a desired therapeutic response. As used herein, the phrase "therapeutically effective amount" means an amount of a compound of the present invention that prevents the onset, alleviates the symptoms, or stops the progression of pre-cancerous or cancerous condition.
The term "treating" is defined as administering, to a subject, a therapeutically effective amount of at least one compound of the present invention, both to prevent the occurrence of inflammation or pre-cancer or cancer condition, or to control or eliminate inflammation or pre-cancer or cancer condition. The term "desired therapeutic response refers to treating a recipient subject with a compound of the present invention such that a pre-cancer or cancer condition is reversed, arrested or prevented in a recipient subject.
The compounds of the present invention can be administered as a single daily dose or in multiple doses per day. The treatment regime may require administration over extended periods of time, e.g., for several days or for from two to four weeks. The amount per administered dose or the total amount administered will depend on such factors as the nature and severity of the disease condition, the age and general health of the recipient subject, the tolerance of the recipient subject to the compound and the type of the inflammatory disorder, or type of cancer.
A compound according to this invention may also be administered in the diet or feed of a patient or animal. The diet for animals can be normal foodstuffs to which the compound can be added or it can be added to a premix.
The compounds of the present invention may be taken in combination, together or separately with any known clinically approved antibiotic, inflammation or anti-cancer agent to treat a recipient subject in need of such treatment.
V. Method of Inhibiting Tumor Growth In another embodiment, the present invention relates to a method of inhibiting tumor growth. Compounds as described herein can possess anti-tumor activity.
The compounds are effective against mammalian tumor cells such as leukemia cells, melanoma cells, breast carcinoma cells, lung carcinoma cells, pancreatic carcinoma cells, ovarian carcinoma cells, renal carcinoma cells, colon carcinoma cells and glioma cells. The antitumor method of the invention results in inhibition of tumor cells. The term "inhibition", when used in conjunction with the antitumor method refers to suppression, killing, stasis, or destruction of tumor cells. The antitumor method preferably results in prevention, reduction or elimination of invasive activity and related metastasis of tumor cells. The term "effective amount" when used in conjunction with the antitumor cell method refers to the amount of the compound sufficient to result in the inhibition of mammalian tumor cells.
The inhibition of mammalian tumor growth according to this method can be monitored in several ways. First, tumor cells grown in vitro can be treated with the compound and monitored for growth or death relative to the same cells cultured in the absence of the compound. A cessation of growth or a slowing of the growth rate (i.e., the doubling rate), e.g., by 10% or more, is indicative of tumor cell inhibition.
Alternatively, tumor cell inhibition can be monitored by administering the compound to an animal model of the tumor of interest. Examples of experimental animal tumor models are known in the art and described in the examples herein. A cessation of tumor growth (i.e., no further increase in size) or a reduction in tumor size (i.e., tumor volume) or cell number (e.g., at least a 10% decrease in either) in animals treated with a compound as described herein relative to tumors in control animals not treated with the compound is indicative of tumor growth inhibition.
To monitor the efficacy of tumor treatment in a human, tumor size or tumor cell titer is measured before and after initiation of the treatment, and treatment is considered effective if either the tumor size or titer ceases further growth, or if the tumor is reduced in size or titer, e.g., by at least 10% or more (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100%, that is, the absence of the tumor). Methods of determining the size or cell titer of a tumor in vivo vary with the type of tumor, and include, for example, various imaging techniques well known to those in the medical imaging or oncology fields (MRI, CAT, PET, etc.), as well as histological techniques and flow cytometry.
For the anti-tumor method of the invention, a typical effective dose of the compounds given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about 300 mg/kg of body weight of the subject.
VI. Method of Treating Cancer In another embodiment, the present invention relates to a method of treating a precancerous or cancerous condition in a mammal, comprising the steps of administrating a pharmaceutical composition, containing a dibenzodiazepinone compound of the present invention in combination with a physiologically acceptable carrier. In one embodiment, the compound is represented by Formula i. As described elsewhere in the application, the compounds of the present invention can have antitumor activity. Specifically, the compounds are effective against mammalian tumor cells such as leukemia cells, melanoma cells, breast carcinoma cells, lung carcinoma cells, pancreatic carcinoma cells, ovarian carcinoma cells, renal carcinoma cells, colon carcinoma cells, prostate carcinoma cells and glioma cells.
In addition, the compound of Formula I is bioavailable via the oral, intraperitoneal, subcutaneous and intravenous administration. Furthermore, as is described within the Examples section, the compounds of the present invention penetrate into brain tissues, and therefore can be useful in the treatment of numerous pre-cancerous and cancerous conditions of the brain.
The cancer treatment method preferably results in prevention, reduction or elimination of invasive activity and related metastasis of tumor cells. The term "therapeutically effective amount" when used in conjunction with the cancer treatment method refers to the amount of the compound sufficient to result in the prevention, reduction or elimination tumors or cancer in a mammal.
The treatment of cancer according to this method can be monitored in several ways. To monitor the efficacy of tumor treatment in a human, tumor size or tumor cell titer is measured before and after initiation of the treatment, and treatment is considered effective if either the tumor size or titer ceases further growth, or if the tumor is reduced in size or titer, e.g., by at least 10% or more (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100%, that is, the absence of the tumor). Methods of determining the size or cell titer of a tumor in vivo vary with the type of tumor, and include, for example, various imaging techniques well known to those in the medical imaging or oncology fields (MRI, CAT, PET, etc.), as well as histological techniques and flow cytometry.
For the cancer treatment method of the invention, a typical effective dose of the compounds given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about 300 mg/kg of body weight of the subject. Alternatively, compounds can be given intravenously, intraperitoneally or subcutaneously at a range from about 5 to about 300 mg/kg.
In addition to the compounds of this invention, pharmaceutically acceptable derivatives or prodrugs of the compounds of this invention may also be employed in compositions to treat or prevent the above-identified disorders.
A "pharmaceutically acceptable derivative or prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an inhibitorily active metabolite or residue thereof. Particularly favored derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.
Pharmaceutically acceptable prodrugs of the compounds of this invention include, without limitation, esters, amino acid esters, phosphate esters, metal salts and sulfonate esters.
VII. Method of Inhibiting Lipoxygenase In another embodiment, the present invention also provides for a method of treating diseased states, in particular inflammation, caused by the 5-lipoxygenase system and/or by the synthesis of the Leukotrienes C4, D4, E4 and F4 as well as Leukotriene B4 in mammals, especially in human subjects. This method comprises administering to a subject an effective amount of a compound of Formula I. The compound of Formula I may be used alone or in combination with other anti-inflammatory compounds to treat or prevent disease states related to inflammation including pulmonary conditions, inflammation, cardiovascular conditions, central nervous system conditions or skin conditions. More specific diseases include gastritis;
erosive esophagitis; inflammatory bowel disease; ethanol-induced hemorrhagic erosions; hepatic ischemia; ischemic neuronal injury; noxious agent induced damage or necrosis of hepatic, pancreatic, renal, neuronal or myocardial tissue; liver parenchyma) damage caused by hepatoxic agents such as CC14 and D-galactosamine; ischemic renal failure; disease-induced hepatic damage; trauma- or stress-induced cell damage;
asthma, multiple sclerosis, ischemic reperfusion, edema, rheumatoid arthritis, viral encephalitis; bacterial pneumonia, neurodegeneration, Alzheimer's disease and glycerol-induced renal failure.
For the method of the invention related to the 5-lipoxygenase system and/or the biosynthesis of Leukotrienes, a typical effective unit dose of a compound of Formula I, or an analog given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about 300 mg/kg of body weight of the subject.
The inhibition of lipoxygenase enzymes is monitored using methods well known in the art and as described in the examples herein. A decrease in enzyme activity by at least 10%, relative to the activity in the absence of a compound as described herein is indicative of effective inhibition of lipoxygenase activity.
Farnesyl dibenzodiazepinone compounds useful according to the invention can be used to reduce or prevent inflammation. Among the hallmarks of local acute inflammation are heat, redness, swelling, pain and loss of function. These changes are induced largely by changes in vascular flow and caliber, changes in vascular permeability and leukocyte exudation (Robbins et al., "Pathologic Basis of Disease", 6tn Ed., W.B. Saunders Co., Philadelphia, PA). Anti-inflammatory therapy performed using compounds useful according to the invention can be monitored for success by tracking any of these changes. For example, a decrease in swelling (e.g., at least 10%
decrease following treatment) or reported pain (e.g., a sustained decrease of 1 point or more on a 1-10 scale reported by the patient, with 10 being the worst pain experienced in association with this disorder prior to treatment, and 0 being no pain) can be used to indicate successful treatment.
Other measurable hallmarks of inflammation include leukocyte infiltration and inflammatory cytokine levels. These hallmarks can be monitored by biopsy of the affected tissue. A decrease of 10% or more in leukocyte infiltration in fixed, stained tissue relative to infiltration in similar tissue prior to treatment can be used to indicate successful treatment, as can a decrease of 10% or more in the level of any given inflammatory cytokine, relative to the level before treatment. Those skilled in the art can readily assay for inflammatory cytokine levels in tissue, blood, or other fluid samples. Alternatively, the level of systemic indicators of inflammation such as C
reactive protein levels and erythrocyte sedimentation rate can be monitored.
Each of these has established normal ranges in medicine, and treatment is considered successful if one or more of such indicators goes from outside the normal range to inside the normal range after the initiation of treatment.
VIII. Method of Bindin tq o Peripheral-type Benzodiazepine Receptor PBR) PBR receptor is a mitochondria) protein, involved in the regulation of cholesterol transport for the outer to the inner mitochondria) membrane, the rate-determining step in steroid biosynthesis. PBR is a critical component of the mitochondria) permeabilitytransition pore (MPTP), which is intimately involved in the initiation and regulation of apoptosis. PBR overexpression is also found to be a prognostic indicator of the aggressive phenotype at least in breast, colorectal and prostate cancer. PBR
expression is also correlated with the quality of kidney preservation.
Overexpression is also found to have correlation with, inflammation and autoimmune diseases, viral infections, Alzheimer's disease and other neurodegenerative diseases.
The method of treatment of a subject with a condition which needs administration of a PBR binding agent is accomplished using methods known to the art (for example see: cancer treatment: Starosta-Rubinstein et al (1987), Proc.
Nat). Acad.
Sci. USA, vol 84, no 3, 891; Junck et al (1989), Ann. Neurol.,vol 26, 752;
Maaser et al (2002), Clin. Cancer Res., vol 8, 3205; Alzheimer's disease : Owen ef al (1983), Br.

Res., vol 273, 373; Diorio et al (1991 ), Neurobiology of Aging, vol 12, 255;
Huntington's disease : Messmer et al (1998), Neuroscience Letters, vol 241, 53; Multiple sclerosis Debruyne et al (2002), Acta Neul. 8e1., vol 102, no 3, 127) . The method comprises administering to a subject an effective amount of a compound of Formula I. The compound may be used alone or in combination with other compounds to treat or prevent disease states related to cancer, inflammation/auto-immune diseases (such as rheumatoid arthritis, lupus, skin or pulmonary inflammation), infections (antiparasitic), neurodegenerative diseases (Alzeihmer's disease, Huntington's disease and multiple sclerosis) and stress.
For the method of the invention related to the binding of PBR, a typical effective unit dose of the compound of the invention given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about 300 mg/kg of body weight of the subject.
The binding of PBR is monitored using methods well known in the art and as described in the examples herein. For example, cancer treatment is monitored by correlation with tumor grade and patient survival. Imaging is used to monitor efficacy of the compounds in neurodegenerative diseases. Farnesyl dibenzodiazepinone compounds useful according to the invention can be used to reduce or prevent inflammation. Inflammation treatment is monitored as in Section VII above.
In addition to the compounds of this invention, pharmaceutically acceptable derivatives or prodrugs of the compounds of this invention may also be employed in compositions to treat or prevent the above-identified disorders.
A "pharmaceutically acceptable derivative or prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an inhibitorily active metabolite or residue thereof. Particularly favored derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.
Pharmaceutically acceptable prodrugs of the compounds of this invention include, without limitation, esters, amino acid esters, phosphate esters, metal salts and sulfonate esters.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Unless otherwise noted, all reagents were purchased from Sigma Chemical Co.
(St. Louis, MO), Aldrich.
All NMR spectra were collected in deuterated solvent on a Varian 500T""
Spectrometer ('H NMR at 500 MHz,'3C NMR at 125 MHz). UV and mass spectra were collected by Waters 2690T"~ HPLC using a photodiode array detector (PDA, 210-400nm) coupled to a Waters MicromassT"" ZQTM mass detector.
EXAMPLE 1: PREPARATION OF PRODUCTION CULTURE
Micromonospora spp. (deposit accession number IDAC 070303-01 ) was maintained on agar plates of ISP2 agar (Difco Laboratories, Detroit, MI). An inoculum for the production phase was prepared by transferring the surface growth of the Micromonospora spp. from the agar plates to 125-mL flasks containing 25 mL of sterile medium comprised of 24 g potato dextrin, 3 g beef extract, 5 g Bacto-casitone, 5 g glucose, 5 g yeast extract, and 4 g CaCOs made up to one liter with distilled water (pH
7.0). The culture was incubated at about 28°C for approximately 60 hours on a rotary shaker set at 250 rpm. Following incubation, 10 mL of culture was transferred to a 2L
baffled flask containing 500 mL of sterile production medium containing 20 g/L
potato dextrin, 20 g/L glycerol, 10 g/L Fish meal, 5 g/L Bacto-peptone, 2 g/L CaC03, and 2 g/L
(NH4)2S04, pH 7Ø Fermentation broth was prepared by incubating the production culture at 28°C in a rotary shaker set at 250 rpm for one week.
Alternate procedure:
The fermentation was accomplished as a 1 x 10L batch in a 14.5 L fermentor (BioFlo 110T"~ Fermentor, New Brunswick Scientific, Edison, NJ, USA) using an improved procedure described in CA patent application 2,466,340, filed January 21, 2004.
Micromonospora sp. (deposit accession number IDAC 070303-01) was maintained on agar plates of ISP2 agar (Difco Laboratories, Detroit, MI). An inoculum for the production phase was prepared by transferring the surface growth of the Micromonospora sp. from the agar plates to 2-L flasks containing 500 mL of sterile medium comprised of 10 g glucose, 20 g potato dextrin, 5 g yeast extract, 5 g NZ-Amine A, and 1 g CaC03 made up to one liter with tap water (pH 7.0). The culture was incubated at about 28°C for approximately 70 hours on a rotary shaker set at 250 rpm.
Following incubation, 300 mL of culture was transferred to a 14.5 L fermentor containing 10 L of sterile production medium. Each liter of production medium was composed of 20 g potato dextrin, 30 g glycerol, 2.5 g Bacto-peptone, 8.34 g yeast extract, 0.3 mL Silicone defoamer oil CChem Service), 0.05 ml Proflo oilT""
(Traders protein) and 3 g CaC03 made to one liter with distilled water and adjusted to pH 7Ø
The culture was incubated at 28 °C, with dissolved oxygen (d02) controlled at 25% in a cascade loop with agitation varied between 150-450 RPM and aeration set at a fixed rate of 0.5 V/V/M.
Other preferred media for the production of Compound 1 by fermentation are provided in Table 1.

EXAMPLE 2: ISOLATION OF COMPOUND 1 500 mL ethyl acetate was added to 500 mL of fermentation broth prepared as described in Example 1 above. The mixture was agitated for 30 minutes on an orbital shaker at 200 rpm to create an emulsion. The phases were separated by centrifugation and decantation. Between 4 and 5 g of anhydrous MgS04 was added to the organic phase, which was then filtered and the solvents removed in vacuo.
An ethyl acetate extract from 2 L fermentation was mixed with HP-20 resin (100 mL; Mitsubishi Casei Corp., Tokyo, Japan) in water (300 mL). Ethyl acetate was removed in vacuo, the resin was filtered on a Buchner funnel and the filtrate was discarded. The adsorbed HP-20 resin was then washed successively with 2 x 125 mL
of 50% acetonitrile in water, 2x125 mL of 75% acetonitrile in water and 2 x 125 mL of acetonitrile.
Fractions containing Compound 1 were evaporated to dryness and 100 mg was digested in the 5 mL of the upper phase of a mixture prepared from chloroform, cyclohexane, methanol, and water in the ratios, by volume, of 5:2:10:5. The sample was subjected to centrifugal partition chromatography using a High Speed Countercurrent (HSCC) system (Kromaton Technologies, Angers, France) fitted with a 200 mL cartridge and prepacked with the upper phase of this two-phase system.
The HSCC was run with the lower phase mobile and Compound 1 was eluted at approximately one-half column volume. Fractions were collected and Compound 1 was detected by TLC of aliquots of the fractions on commercial Kieselgel 60F2sa plates.
Compound could be visualized by inspection of dried plates under UV light or by spraying the plates with a spray containing vanillin (0.75%) and concentrated sulfuric acid (1.5%, v/v) in ethanol and subsequently heating the plate. Fractions contained substantially pure Compound 1, although highly colored. A buff-colored sample could be obtained by chromatography on HPLC as follows.

6 mg of sample was dissolved in acetonitrile and injected onto a preparative HPLC column (XTerra ODS (l0pm), 19x150mm, Waters Co., Milford, MA), with a 9 mUmin flow rate and UV peak detection at 300 nm. The column was eluted with acetonitrile/buffer (5 mM of NH4HC03) according to the following gradient shown in Table 2.
Table 2 Time min Water % Acetonitrile 0 50 _ 0 _ Fractions containing the Compound 1 were combined, concentrated and lyophilized to give a yield of 3.8 mg compound.
Alternative Protocol 1 Compound 1 was also isolated using the following alternative protocol. At the end of the incubation period, the fermentation broth from the baffled flasks of Example 1 was centrifuged and the supernatant decanted from the pellet containing the bacterial mycelia. 100 mL of 100% MeOH was added to the mycelial pellet and the sample was stirred for 10 minutes and centrifuged for 15 minutes. The methanolic supernatant was decanted and saved. 100 mL of acetone was then added to the mycelial pellet and stirred for 10 minutes then centrifuged for 15 minutes. The acetonic supernatant was decanted and combined with the methanolic supernatant. Finally, 100 mL of 20%
MeOH/H20 was added to the mycelial pellet, stirred for 10 minutes and centrifuged for 15 minutes. The supernatant was combined with the acetonic and methanolic supernatants.
The combined supernatant was added to 400 ml of HP-20 resin in 1000 mL of water and the organics were removed in vacuo. The resulting slurry was filtered on a Buchner funnel and the filtrate was discarded. Adsorbed HP-20 resin was washed successively with 2x500mL of 50% MeOH/H20, 2x500mL of 75% MeOH/H20 and 2x500mL of MeOH.
The individual washes were collected separately and analyzed by TLC as described above. Those fractions containing Compound 1 were evaporated to near dryness and lyophilized. The lyophilizate was dissolved in methanol and injected onto a preparative HPLC column (Xterra ODS (10~,m), 19x150mm, Waters Co., Milford, MA) with a flow rate of 9 mUmin and peak detection at 300 nm.
The column was eluted with acetonitrile/buffer (5 mM of NH4HC03) according to gradient shown in Table 3.
Teble 3 Time min Buffer % Acetonitrile Fractions containing Compound 1 were combined, concentrated and lyophilized to yield about 33.7 mg of compound.
Alternative Protocol 2 liters of the whole broth from Example 1 are extracted twice with equal volumes of ethyl acetate and the two extracts are combined and concentrated to dryness. The dried extract is weighed, and for every gram of dry extract, 100 mL of MeOH-H20 (2:1 v/v) and 100 mL of hexane is added. The mixture is swirled gently but well to achieve dissolution. The two layers are separated and the aqueous layer is washed with 100 mL of hexane. The two hexane layers are combined and the combined hexane solution is washed with 100 mL methanol:water (2:1, v/v). The two methanol:water layers are combined and treated with 200 mL of EtOAc and 400 mL
of water. The layers are separated and the aqueous layer is extracted twice more with 200 mL portions of EtOAc. The EtOAc layers are combined and concentrated. The residue obtained will be suitable for final purification, either by HSCC or by HPLC as described above. This extraction process achieves a ten-fold purification when compared with the extraction protocol used above.

EXAMPLE 3: ELUCIDATION OF THE STRUCTURE OF COMPOUND 1 1" 2" 3"
O
1' 2 ~1 a 11 1N 2~3, 4. 5, g~~, g~ 9, 1 ~ ~
~4a 4 5N C28H34N2~4 Mol. Wt.: 462.58 H
The structure of Compound 1 (ECO-4601 ) was derived from spectroscopic data, including mass, UV, and NMR spectroscopy. Mass was determined by electrospray mass spectrometry to be 462.6, UVmax 230nm with a shoulder at 290 nm. Mass data confirmed a calculated molecular weight of 462.58 and a molecular formula of C28H34N2O4. NMR data were collected dissolved in MeOH-d4 including proton (Figure 1 ), and multidimensional pulse sequences gDQCOSY, gHSQC, gHMBC, and NOESY.
A number of cross peaks in the 2D spectra of ECO-4601 are key in the structural determination. For example, the farnesyl chain is placed on the amide nitrogen by a strong cross peak between the proton signal of the terminal methylene of that chain at 4.52 ppm and the amide carbonyl carbon at 170 ppm in the gHMBC experiment.
This conclusion is confirmed by a cross peak in the NOESY spectrum between the same methylene signals at 4.52 ppm and the aromatic proton signal at 6.25 ppm from one of the two protons of the tetra substituted benzenoid ring. Assignement of proton and carbon signals are shown in Table 4.
Based on the mass, UV and NMR spectroscopy data, the structure of the compound was determined to be the structure of Compound 1 shown above.
Table 4 'H and'3C NMR (8H, ppm) Data of Compound 1 in MeOH-D4 Assignment 'H '3C Group 1 7.15 122.3 CH

1 a - 125.0 C

2 6.74 121.0 CH

Table 4 'H and'3C
NMR (8H, ppm) Data of Compound 1 in MeOH-D4 Assignment'H '3C Group 3 6.83 116.9 CH

4 - 146.0 C-OH

4a - 142.0 C

6 - 153.0 C-OH

6a - 126.0 C

7 6.20 100.0 CH

8 - 148.2 C-OH

9 6.25 100.1 CH

9a - 135.0 C

11 - 170.0 C(O) 1' 4.52 48.7 CH2 2' 5.35 i 21.1 CH

3' - 138.5 C

4' 1.95 39.6 CH2 5' 2.02 26.7 CHz 6' 5.09 124.1 CH

T - 135.0 C

8' 2.03 39.5 CH2 9' 2.08 26.3 CH2 10' 5.06 124.4 CH

11' - 130.9 C

12' 1.55 16.5 CH3 1" 1.72 15.5 CH3 2" 1.59 14.9 CH3 3" 1.64 24.8 CH3 EXAMPLE 4: SYNTHESIS AND ELUCIDATION OF COMPOUND 2 Compound 2, namely 10-Farnesyl-4,6,8-trihydroxy-5-methyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, was prepared and identified as follows:

.~° ,2~

Preparation:
Compound 1 (61.3 mg) was stirred overnight with NaHCOs (60.9 mg, Aldrich) and dimethyl sulfate (360 NI, Sigma) in MeOH (3.0 mL). The resulting mixture was filter through a 0.45 Nm 13 mm Acrodisc GHP syringe filter and subjected to HPLC
separation. Purification by multiple injection on a Waters RCM Nova-Pak HR C18 6Nm 60A 25 x 200 mm column (20 mUmin H20/CH3CN 80:30-70:75, 0-8 min; 30:70-0:100, 8-18 min) gave Compound 2 (37.5 mg) with retention time of 13.5 min.
Compounds 60 to 76 are also prepared via this procedure, by reaction of Compound 1 with the appropriate dialkyl sulfate.
Structure elucidation:
The calculated molecular weight (476.61 ) and formula (C29HssN20a) of Compound 2 was confirmed by mass spectral analysis: negative ionization gave an (M-H)~ molecular ion of 475.55 and positive ionization gave an (M+Na)+ molecular ion of 499.35. Proton NMR spectral analysis is shown in Table 5. Signals were easily assigned based on Compound 1 structure knowledge.
Table 5 'H NMR (8H, ppm) Data of Compound 2 in MeOH-D4 Assignment Compound 2 Group 1 7.21 CH

2 7.12 CH

3 7.01 CH

2.92 N-CH3 7 6.22 CH

9 6.34 CH

1' 4.82, 4.58 CH2 2' 5.44 C H

4' a 1.94 CHz 5' a 2.06 CHZ

6' 5.07 CH

8' a 2.03 CH2 9' a 2.11 CHZ

10' 5.07 CH

Table 5 'H NMR (8H, ppm) Data of Compound 2 in MeOH-D4 Assignment Compound 2 Group 12' 1.54 CH3 1" 1.76 CH3 2" 1.59 CH3 3" 1.64 CH3 a. Signals at 4', 5', 8' and 9' are very close; assignement was based on Compound 1 EXAMPLE 5: SYNTHESIS AND ELUCIDATION OF COMPOUND 3 1.
11 10 2~3, 4~ 5. 6~~, g' 9 1 ~ ~, 12' \a ~N
9a 9 ~4a ~ ~ B OH
4 5 N 6a C3sHaoN2~a off J1~' ~ Mol. Wt.: 552.70 2~'~
3"' Compound 3, namely 5-Benzyl-10-farnesyl-4,6,8-trihydroxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one was prepared and identified as follows:
Preparation:
Compound 1 (60.5 mg) was stirred 84 hrs with benzyl chloride (1.8 mL, Sigma) in presence of two drops of pyridine (Aldrich). The resulting mixture was directly subjected to HPLC separation. Purification by multiple injection on a Waters RCM Nova-Pak HR
C18 6Nm 60A 25 x 200 mm column (20 mL/min H20/CH3CN 80:30-70:75, 0-8 min;
30:70-0:100, 8-18 min) gave Compound 3 (46.0 mg) with retention time of 17.5 min.
Compounds 60 to 76 are also prepared via this procedure, by reaction of Compound 1 with the appropriate alkyl halide.
Structure elucidation:
The calculated molecular weight (552.70) and formula (C~H~N204) of Compound 3 was confirmed by mass spectral analysis: negative ionization gave an (M-H)- molecular ion of 551.65 and positive ionization gave an (M+Na)+ molecular ion of 575.45. Proton NMR spectral analysis is shown in Table 6. Signals were easily assigned based on Compound 1 structure knowledge.
Table 6 'H NMR (8H, ppm) Data of Compound 3 in MeOH-D4 Assignment Compound 3 Group 1 7.19 CH

2 7.09 CH

3 6.96 CH

7 6.16 CH

9 6.31 CH

1' 4.55, 4.48 CH2 2' 5.44 CH

4' a 1.94 CH2 5' a 2.08 CH2 6' 5.10 CH

8' a 2.01 CHZ

9' a 2.13 CH2 10' 5.04 CH

12' 1.52 CH3 1 " 1.79 CH3 2" 1.56 CH3 3" 1.64 CH3 1 "' 4.34, 4.25 CH2 3"' 7.20 CH (2H) 4"' 7.23 CH (2H) 5"' 7.23 CH

a. Signals at 4', 5', 8' and 9' are very close; assignement was based on Compound 1 EXAMPLE 6: SYNTHESIS AND ELUCIDATION OF COMPOUNDS 4. 5L6. 7 AND 8 1" ~ 2..

2~3' 4' S' ~ T 8' 9. 1 X11' 12' g 4-OMe6-OH8-OH
~
~
~
\

x Compound 4:

a N Compound 5: 4-OH 6-OMe8-OH

4-OH,6-OMe8-OMe a Compound 6:

x 6 7 Compound 7: 4-OMe6-OMe8-OH

Compound 4-OMe6-OMe8-OMe 8:

x Monomethylated: Compounds 4 and 5: 10-Farnesyl-6,8-dihydroxy-4-methoxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one and 10-Farnesyl-4,8-dihydroxy-6-methoxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, have a calculated molecular weight of 476.61 g/mol and a formula of C2sHssN204.
Dimethylated: Compounds 6 and 7: 10-Farnesyl-4-hydroxy-6,8-dimethoxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one and 10-Farnesyl-8-hydroxy-4,6-dimethoxy-5,10-dihydro-dibenzo(b,e][1,4]diazepin-11-one, have a calculated molecular weight of 490.63 g/mol and a formula of C3oH38N204.
Trimethylated: Compound 8: 10-Farnesyl-4,6,8-trimethoxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, has a calculated molecular weight of 504.66 g/mol and a formula of C3, H4oN204.
All 0-methylated compounds (4 to 8) were prepared and identified according to the following procedure:
Preparation:
Compound 1 (20.0 mg) in MeOH (2.0 mL) was treated with excess of CH2N2 in diethyl ether and the mixture stirred overnight. The resulting mixture was separated by preparative TLC (Merck Silica gel 60 F254), using 2.5% MeOH in CHC13 as eluent. A
mixture of Compounds 4 and 5 (1.0 mg), Compound 6 (0.5 mg), Compound 7 (5.5 mg) and Compound 8 (3.0 mg) were isolated with Rf value of 0.09, 0.35, 0.39 and 0.92 respectively.
Structure elucidation The calculated molecular weights (mono: 476.61, di: 490.63 and tri: 504.66) and formulae (mono: C29H36N2O4, di: C~oH~N204 and tri: C3lH~oN2O4) respectively of mono methylated (Compounds 4 and 5), dimethylated (Compounds 6 and 7) and trimetylated (Compound 8) were confirmed by mass spectral (MS) analysis. Compounds 4 and 5 MS gave a (M-H)- molecular ion of 475.45 by negative ionization and a (M+Na)+
molecular ion of 499.35 by positive ionization. Compound 6 MS gave a (M-H)-molecular ion of 489.45 by negative ionization and a (M+Na)+ molecular ion of 513.35 by positive ionization. Compound 7 MS gave a (M-H)- molecular ion of 489.45 by negative ionization and a (M+Na)+ molecular ion of 513.35 by positive ionization.
Compound 8 gave a (M-H)- molecular ion of 503.55 by negative ionization and a (M+Na)+ molecular ion of 527.35 by positive ionization. Proton NMR spectral analysis for Compounds 4 to 8 is shown in Table 7. Signals were easily assigned based on Compound 1 structure knowledge.
Table 7 'H NMR (SH, ppm) Data of Compounds 4, 5, 6, 7 and 8 in MeOH-D4 Assignment 4 5 6 7 8 Group 1 7.28 7.17 7.19 7.28 7.29 CH

2 6.91 6.78 6.86 6.92 6.92 CH

3 7.03 6.86 6.79 7.03 7.03 CH

4 3.94 N/D N/D 3.93 3.94 C-X a N/D N/D N/D N/D 6.95 NH

6 N/D 3.87 3.91 3.87 3.89 C-X b 7 6.28 6.23 6.46 6.33 6.45 CH

8 N/D N/D 3.75 N/D 3.74 C-X

9 6.38 6.34 6.52 6.38 6.57 CH

1' 4.56 4.56 4.61 4.57 4.60 CH2 2' 5.36 5.36 5.38 5.34 5.31 CH

4' d 1.96 1.96 1.95 1.95 1.93 CH2 5' d 2.05 2.05 2.05 2.05 2.04 CHZ

6' 5.09 5.09 5.07 5.08 5.06 CH

8' d 2.04 2.04 2.04 2.04 2.03 CH2 9' d 2.10 2.10 2.09 2.09 2.08 CHZ

10' 5.09 5.09 5.07 5.08 5.06 CH

12' 1.57 1.57 1.55 1.57 1.55 CH3 1 " 1.74 1.74 1.75 1.73 1.73 CH3 2" 1.60 1.60 1.60 1.59 1.59 CH3 3" 1.66 1.66 1.65 1.66 1.65 CH3 N/D: Not determined - not observed a. X is OCH3 in Compounds 4, 7 and 8; X is OH in Compounds 5 and 6 b. X is OCH3 in Compounds 5, 6, 7 and 8; X is OH in Compound 4 c. X is OCH3 in Compounds 6 and 8; X is OH in Compounds 4, 5 and 7 d. Signals at 4', 5', 8' and 9' are very close; assignement was based on Compound 1 EXAMPLE 7: SYNTHESIS AND ELUCIDATION OF COMPOUNDS 9. 10. 11 AND 12 2~ 3' a~ 5~~~9'~ 1~~ 2~

P
x ~aa ~ ~ ~ x Compound 9: 4-OH 6-OAc 8-OAc a 5N sa~ Compound 10: 4-OAc 6-OAc 8-OH
x ~~g-=--'~ Compound 11: 4-OAc 6-OH 8-OAc Com ound 12: 4-OAc 6-OAc 8-OAc Diacetylated: Compounds 9, 10 and 11: 6,8-Diacetoxy-10-farnesyl-4-hydroxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one; 4,6-Diacetoxy-10-farnesyl-8-hydroxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one and 4,8-Diacetoxy-10-farnesyl-6-hydroxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, have a calculated molecular weight of 546.65 g/mol and a formula of Cs2H38N20s.
Triacetylated: Compound 12: 4,6,8-Triacetoxy-10-farnesyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, has a calculated molecular weight of 588.69 g/mol and a formula of C34HaoN2~~.
All acetylated compounds (9 to 12), were prepared and identified according to the following procedure:
Preparation:
Compound 1 (120.5 mg) was stirred overnight with acetic anhydride (720 NL, Aldrich) in presence of 6 drops of pyridine (Aldrich). The reaction mixtures submitted to HPLC separation. Purification by multiple injection on a Waters RCM Nova-Pak HRT""
C18, 6Nm, 60A 25 x 200 mm column (20 mUmin H20/CH3CN 80:30-70:75, 0-8 min;
30:70-0:100, 8-18 min and HPLC run for 20 min) gave Compound 11 (11.4 mg), Compound 10 (9.2 mg), Compound 9 (11.4 mg), Compound 12 (91.2 mg) with retention time of 16.2, 17.6, 18.0 and 18.5 min, respectively.
Structure elucidation:
The calculated molecular weights (di: 546.65 and tri: 588.69) and formulae (di:
C32H38N206 and tri: C34H40N2O7) respectively of diacetylated (Compounds 9, 10 and 11 ) and triacetylated (Compound 12) were confirmed by mass spectral (MS) analysis.

Compound 9 MS gave a (M-H)- molecular ion of 545.55 by negative ionization and a (M+Na)+ molecular ion of 569.35 by positive ionization. Compound 10 MS gave a (M-H)-molecular ion of 545.55 by negative ionization and a (M+Na)+ molecular ion of 569.45 by positive ionization. Compound 11 MS gave a (M-H)- molecular ion of 545.45 by negative ionization and a (M+Na)+ molecular ion of 569.35 by positive ionization.
Compound 12 gave a (M-H)- molecular ion of 587.55 by negative ionization and a (M+Na)+ molecular ion of 611.45 by positive ionization. Proton NMR spectral analysis for Compounds 9 to 12 is shown in Table 8. Signals were easily assigned based on Compound 1 structure knowledge.
Table 8 'H NMR (8H, ppm) Data of Compounds 9, 10, 11 and 12 in CDCI3 Assignment 9 10 11 12 Group 1 7.27 7.72 7.75 7.75 CH

2 6.67 6.99 7.02 7.10 CH

3 6.67 7.15 7.15 7.16 CH

4 N/D 2.42 2.42 2.41 C-X a 6.45 5.90 6.29 6.15 NH

6 2.36 2.41 N/D 2.40 C-X b 7 6.82 6.52 6.15 6.96 CH

8 2.28 N/D 2.25 2.27 C-X

9 6.95 6.67 6.55 6.84 CH

1' 4.60 4.57 4.57 4.58 CH2 2' 5.43 5.38 5.41 5.42 CH

4' d 1.98 1.97 1.98 1.98 CH2 5' d 2.08 2.06 2.05 2.06 CHZ

6' 5.10 5.09 5.09 5.10 CH

8' d 2.08 2.06 2.05 2.06 CH2 9' d 2.09 2.08 2.09 2.09 CH2 10' 5.10 5.09 5.09 5.10 CH

12' 1.60 1.60 1.60 1.60 CH3 1 " 1.72 1.70 1.71 1.72 CH3 2" 1.61 1.60 1.60 1.61 CH3 3" 1.69 1.69 1.69 1.68 CH3 N/D: Not determined - not observed a. X is OAc in Compounds 10, 11 and 12; X is OH in Compound 9 b. X is OAc in Compounds 9, 10 and 12; X is OH in Compound 11 c. X is OAc in Compounds 9, 11 and 12; X is OH in Compound 10 d. Signals at 4', 5', 8' and 9' are very close; assignement was based on Compound 1 EXAMPLE 8: FARNESYL SIDE CHAIN MODIFICATIONS
8.1 S~inthesis and elucidation of Compound 46 i1" i 1' 2' 3, 4. 5, g' ~, g' g, 10' 11 N
9a g ----- C28H40N2~4 ~h H ~ Mol. Wt.: 468.63 Compound 46: 10-(3,7,11-trimethyldodecanyl)-4,6,8-trihydroxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one.
Hexahydro Compound 46, was prepared and identified according to the following procedure:
Preparation:
A solution of Compound 1 (51.1 mg in 3.0 mL MeOH) was stirred under hydrogen gas overnight in presence of platinum oxide (Pt02,10 mg) as a catalyst. The reaction mixture was filtered and purified by direct preparative HPLC using Phenomenex MAX PR 21.2 x 200 mm column (20 mUmin, H20/CH3CN gradient 30:70-30:70, 0-2 min; 30:70-0:100, 2-20 min). Fractions having a retention time of 12.8 min were combined to give 45.2 mg of Compound 46.
Structure elucidation:
Calculated molecular weight (468.63) and formulae (C2sH4oN2O4) were confirmed by mass spectral analysis. Compound 46 mass spectra gave a (M-H)- molecular ion of 467.35 by negative ionization and a (M+H)+ molecular ion of 469.43 by positive ionization. Proton NMR spectral analysis of Compound 46 is shown in Table 9.
Signals were easily assigned based on Compound 1 structure knowledge. As expected, aliphatic proton signals at positions 1'-11' all have very close chemical shifts ranging from about 1 to 1.75 ppm (integrating for 19 protons), methyl protons at positions 12' and 1 "-3" are all very close as well (shifts 0.8-0.95 ppm, integrating for 12 protons).
These signals are also complex from the fact that 4 diastereomers of positions 3', 7 and 11' are present in the mixture, and in different proportions. Labile protons were not observed since NMR was done in deuterated methanol.
Table 9 'H NMR (8H, ppm) Data of Compound 46 in CD30D

Assignment 8H, ppm Group 1 7.15 CH

2 6.76 CH

3 6.84 CH

7 6.24 CH

9 6.26 CH

1'-11' a - 1.00-1.753CH, 8CH2 (19H) 12' and 1"-3" -- 0.8-0.954CH3 (12H) a a. Signals are very close 8.2 Synthesis and elucidation of Com,nounds 17 and 18 1" 2" 3"
1' 2~ 4' S' 6' $' g' 1 ~ 12' -~~OH
C28H34N2~5 Mol. Wt.: 478.58 I' Ho Compound 17 1" 2" 3"
1' 2~ 4' S' 6~ 8' 9. 10' 12' -~~OH
C28H34N2~5 Mol. Wt.: 478.58 Compound 18 Compound 17: 10-(3,7,11-trimethyl-6,7-epoxydodeca-2,10-dienyl)-4,6,8-trihydroxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one.
Compound 18: 10-(3,7,11-trimethyl-10,11-epoxydodeca-2,10-dienyl)-4,6,8-trihydroxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one.
Monoepoxide Compounds 17 and 18, were prepared and identified according to the following procedure:
Preparation:
A mixture of Compound 1 (24.0 mg) and 3-chloroperbenzoic acid (mCPBA, 7.8 mg) in THF (1.0 mL) were stirred overnight at room temperature. The reaction mixture was diluted with MeOH (1.0 mL) and subjected to purification on Waters HPLC
using a Photodiode Array detector. The mixture was purified by multiple injections on a Nova PackT"" C-18 25 x 200 mm column (20 mUmin, H20/CH3CN gradient 80:20-30:70, 0-8 min; 30:70-0:100, 8-20 min). Pure Compound 17 (2.11 mg) and Compound 18 (1.68 mg) were obtained by concentration in vacuo of the combined fractions respectively having retention time 11.2 min and 10.6 min.
Structure elucidation:
Calculated molecular weights (478.58) and formulae (C2sH~N205) were confirmed by mass spectral analysis. Compound 17 mass spectra gave a (M-H)-molecular ion of 477.34 by negative ionization and a (M+H)+ molecular ion of 479.44 by positive ionization. Compound 18 mass spectra gave a (M-H)- molecular ion of 477.34 by negative ionization and a (M+H)+ molecular ion of 479.40 by positive ionization.
Proton NMR spectral analysis of Compounds 17 and 18 is shown in Table 10.
Signals were easily assigned based on Compound 1 structure knowledge. As expected, epoxide protons signals were shifted upfield, compared to the alkene protons of Compound 1 (from 5.09 to 2.75 ppm for Compound 17, and from 5.06 to 2.73 ppm for Compound 18). Labile protons were not observed since NMR was done in deuterated methanol.
Table 10 'H NMR (8H, ppm) Data of Compounds 17 and 18 in CD30D

Assignment Compound 17 Compound 18 Group 1 7.17 7.18 CH

2 6.77 6.77 CH

3 6.85 6.86 CH

7 6.22 6.23 CH

9 6.27 6.27 CH

1' 4.61, 4.54 4.55 CHz 2' 5.42 5.37 CH

4' 2.17 2.08 a CH2 5' 1.62, 1.42 2.13 a CH2 6' 2.75 5.16 CH

8' 1.62, 1.42 2.08 a CH2 9' 2.09 1.60 CH2 10' 5.09 2.73 CH

12' 1.60 1.20 CH3 1 " 1.77 1.74 CH3 2" 1.26 1.64 CH3 3" 1.67 1.26 CH3 a. Signals are very close, and are interchangeable EXAMPLE 9: PHARMACOLOGICAL ACTIVITY PROFILE
Compound 1 and analogs of Compounds 1 referred to herein as Compounds 2 to 12 and Compound 46 were tested for binding against a variety of enzymes and/or receptors. These enzymes or receptor used for these assays were known to be involved in anticancer activity of known compounds, as well as other diseases.
Data were also compared to inhibition of GABAA Central Benzodiazepine Receptor, to show selectivity.
A. Enzymes and Receptors:
5-Lipoxygenase (5-LO) catalyzes the oxidative metabolism of arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE), the initial reaction leading to formation of leukotrienes. Eicosanoids derived from arachidonic acid by the action of lipoxygenases or cycloxygenases have been found to be involved in acute and chronic inflammatory diseases (i.e. asthma, multiple sclerosis, rheumatoid arthritis, ischemia, edema) as well in neurodegeneration (Alzheimer disease), aging and various steps of carcinogenesis, including tumor promotion, progression and metastasis. The aim of this study was to determine whether ECO-4601, is able to block the formation of leukotrienes by inhibiting the enzymatic activity of human 5-LO.
AcyICoA-Cholesterol Acyltransferase, Hepatic (AcoA-AT) converts cholesterol to colesteryl esters and is involved in the development of artherioscerosis.
Cyclooxygenase (COX-2) enzyme is made only in response to injury or infection.
It produces prostaglandins involved in inflammation and the immune response.
Elevated levels of COX-2 in the body have been linked to cancer.
The peripheral benzodiazepine receptor (PBR or PBenzR) is a well-characterized receptor known to be directly involved in diseases states. PBR
is involved in the regulation of immune responses. These diseases states include tumors, inflammatory diseases (such as rheumatoid arthritis and lupus), parasitic infections and neurodegenerative diseases (such as Alzheimer, Huntington and Multiple Sclerosis).
Leukotriene, Cysteinyl (CysLT~) is involved in inflammation and CysLTi-selective antagonists are used as treatment for bronchial asthma. CysLT, and 5-LO were found to be upregulated in colon cancer.
GABAA Central Benzodiazepine Receptor (CBenzR or CBR) binding is involved in anxiolitic activities.
B. General Procedures:
The procedures used were based on known assays: AcoA-AT (from rat; Ref:
Largis et al (1989), J. Lipid. Res., vol 30, 681-689), COX-2 (human; Ref:
Riendeau et al (1997), Can. J. Physiol. Pharmacol., vol 75, 1088-1095 and Warner ef al (1999), Pro.
Natl. Acad Sci. USA, vol 96, 7563-7568), 5-LO (human; Ref: Carter et al (1991 ), J.
Pharmacol. Exp. Ther., vol 256, no 3, 929-937, and Safayhi et al (2000), Planta Medica, vol 66, 110-113), PBenzR (from rat; Le Fur et al (1983), Life Sci. USA, vol 33, 449-457), CvsLT, (human; Martin et al (2001 ), Biochem. Pharmacol., vol 62, no 9, 1193-1200) and CBenR (from rat; Damm et al (1978), Res. Comm. Chem. Pathol. Pharmacol., vol 22, 597-600 and Speth et al (1979), Life Sci., vol 24, 351-357).
C. Binding assay of Compound 1 on 5-LO:
Human peripheral blood mononuclear cells (PMNs) were isolated through a Ficoll-Paque density gradient. PMNs were stimulated by addition A23187 (30 NM
final concentration). Stimulated PMNs were adjusted to a density of 5 x106 cells/mL
in HBBS
medium and incubated with the vehicle control (DMSO), ECO-4601 (at final concentrations of 0.1, 0.5, 1, 2.5, 5 and 10 NM) and NDGA as positive control (at final concentrations of 3, 1, 0.3, 0.1 and 0.03 NM) for 15 minutes at 37°C.
Following incubation, samples were neutralized with NaOH and centrifuged. Leukotriene B4 content was measured in the supernatant using an Enzyme Immunosorbant Assay (EIA) assay. The experiment was performed in triplicate.
Results shown in Figure 2 demonstrated that ECO-4601 inhibited the activity of human 5-LO with an apparent IC~o = 0.93 NM (versus 0.1 NM for the positive control NDGA) and therefore displays anti-inflammatory properties.
D. Percentage inhibition or binding of Compound 1-12 and 46:
Binding assays were done for each of Compounds 1-12 and 46 using AcoA-AT, COX-2, 5-LO, PBenzR and CysLTi enzymes. The procedures used are based on the respective references mentioned above and the conditions are summarized in Tables 11 (enzyme assays) and 12 (radioligand receptor assays).
Table 11 Enzyme Assays Conditions Source Substrate Pre-I a I
ACoA AT ° Wistar rat hepatic 12.7 NM ['4C)palmitoyl CoA
l5min/37°C l0min/37°C
microsomes COX-2 d Human recombinant 0.3 uM arachidonic acid l5miN37°C
5miN37°C

Table 11 Enzyme Assays Conditions Source Substrate Pre-I a I b insect Sf21 cells 5-LO a Human PBML cells Arachidonic acid l5miN37°C l5miN37°C
a. Pre-Incubation TimelTemperature b. Incubation Time/Temperature c. Incubation buffer: 0.2 M phosphate buffer (pH 7.4 at 25°C); Method:
Quantitation of ['4C]cholesterol ester by column chromatography.
d. Incubation buffer: 100mM Tris-HCI, pH 7.7, 1mM glutathione, 1NM hematin, 500NM phenol;
Method: EIA quantitation of PGE2.
e. Incubation buffer: HBSS (Hank's balanced salt solution); Method: EIA
quantitation of LTB4.
Table 12 Radioligand Binding Assays Conditions a Source Ligand I b Non-spec ligand PBenzR ° Wistar rat heart 0.3 nM [3H]PK-11195 l5min/25°C
Dipyridamole f CysLT, d Human recombinant 0,3 nM [3H]leukotriene D4 30miN25°C
Leukotriene D4 9 CHO-K1 cells CBenzR a Wistar rat brain 1 nM [3H]flunitrazepam 60min/25°C
Diazepam "
a. Quantitation Method: Radioligand binding b. Incubation Time/Temperature c. Incubation buffer: 50mM Tris-HCI, pH 7.5, lOmM MgCl2 at 25°C.
d. Incubation buffer: 50mM Tris-HCI, pH 7.4, 5mM CaCl2, 5mM MgCl2, 100NglmL
bacitracin, 1mM
bezamidine, 0.1 mM PMSF.
e. Incubation buffer: 50mM Na-K phosphate, pH 7.4 at 25°C.
f. Non specific ligand: 100NM, Kp: 2.3nM, 8",a,~: 0.17 pmoUmg protein, Specific binding: 90%
g. Non specific ligand: 0.3NM, Kp: 0.21 nM, Bm~: 3 pmol/mg protein, Specific binding: 93%
h. Non specific ligand: 10NM, Kp: 4.4nM, Bmax~ 1.2 pmol/mg protein, Specific binding: 91 Binding Assays were done at constant concentration of the compound, in 1 DMSO as vehicle, and are specified below each enzyme/receptor type in Table 13. The results are expressed in Table 13 as percentage inhibition. Significance was obtained when a result was ?50% binding or inhibition (underlined).
Table 13 Percentage of inhibition or binding activity Compound ACoA-AT COX-2 5-LO PBenzR CysLT, CbenzR
(10 NM) (4 NM) (4 NM) (1 NM) (4 NM) (10 NM) 1 90 9~ 99 80 92 39 Table 13 Percentage of inhibition or binding activity ACoA-AT COX-2 5-LO PBenzR CysLT, CbenzR

Compound (10 NM) (4 NM) (4 NM) (1 NM) (4 NM) (10 NM) All of the exemplified Compounds 1-12 and 46 possessed inhibition and/or binding activity. None of them significantly binded the central benzodiazepine receptor (CBenzR), which showed selectivity was present.
Cytotoxic activity (Gl5o) of these compounds are shown in Example 10.
EXAMPLE 10. IN VITRO CYTOTOXIC ACTIVITY OF COMPOUNDS 1-12 AND 46 Cytotoxic activities were determined in vitro for exemplified Compounds 1-12 and 46 to determine the concentration of each compound needed to obtain a 50%
inhibition of cell proliferation (GlSO). The GlSO value emphasizes the correction for the cell count at time zero and, using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], Gl5o is calculated as [(Ti-Tz)/(C-Tz)] x 100 = -50, which is the drug concentration resulting in a 50% reduction in the net DNA increase in control cells during the drug incubation.
Compounds were tested in four cell lines: HT-29 (colorectal carcinoma), SF268 (CNS), MDA (mammary gland adenocarcinoma) and PC-3 (prostate adenocarcinoma).
Compounds were dissolved at 10 mM in DMSO. Dilution in vehicle to concentrations of 30, 10, 3, 1 and 0.3 NM were prepared immediately before assays.
Depending on the cell lines growth characteristics, 4000-i 0000 cells were plated in two 96-wells pates (day 0) and incubated for i 6 hours. The following day, propidium iodide ws added to one of the two plates and fluorescence measured (Tz). Test compounds were added to the second plate, as well as vehicle control, and cells further incubated for 96 hours. Each compound was tested at each concentration and in triplicates. The equivalent cell number was determined after adding propidium iodide by measuring the signal by fluorescence (C for control). GlSO results were calculated using the formula above and are shown in Table 14.
Table 14 Cytotoxic Activities of Compounds 1-12 and 46 (G1~ NM) Compound HT-29 SF268 PC-3 MDA-231 1 9.538 2.134 2.223 2.143 2 0.457/0.821*<0.3/0.014*<0.3/0.046*<0.3/0.3*

3 5.945 5.419 5.701 5.745 4 14.934 7.4 6.671 16.545 19.420 14.615 17.984 18.003 6 > 30 21.775 24.231 > 30 7 12.765 19.283 17.945 24.463 8 > 30 > 30 > 30 > 30 9 11.699 2.417 1.605 9.115 12,606 2.602 1.944 2.007 11 15.565 6.656 5.868 7.349 12 7.533 2.252 0.935 1.442 46 2.633 0.808 0.937 0.711 *Result of a second test, using lower concentrations.
Compound 8 was not active at 30 pM against these four cell lines, but showed a 55% binding to the peripheral benzodiazepine receptor in Example 9. It is expected that this compound is active at higher concentration levels or on different strains than the four strains used.
Compound 2 has an unexpected increase in cytotoxic activity compared to parent Compound 1. A fifty fold increase of activity was observed against HT-29 cell line. Cytotoxic activity (GlSO) of Compound 2 for the other three strains was outside the expected range of concentrations used in the first tests. The second test showed nanomolar activities for Compound 2, a 100-fold increase in potency.
EXAMPLE 11. IN VITRO ANTICANCER ACTIVITY OF COMPOUND 1 AGAINST
HUMAN AND ANIMAL TUMOR CELL LINES FROM VARIOUS TISSUES
Culture conditions: The cell lines listed in Table 15 were used to characterize the cytotoxicity of ECO-4601 against human and animal tumor cell lines. These cell lines were shown to be free of mycoplasma infection and were maintained in the appropriate media (Table 15) supplemented with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin, under 5% C02 at 37°C. Cells were passaged two to three times per week. Viability was examined by staining with 0.25% trypan blue and only flasks where cell viability was >95% were used for this study.
Cell lines amplification and platinq: Tumor cells were seeded (1-3 x 103 cells per 100 ~L) in 96-well flat bottom microtiter plates and incubated at 37°C and 5% C02 for 16 hrs before treatment in drug-free medium supplemented with 10% serum.
Evaluation of inhibitory activity on cell proliferation: Cells were incubated for 96 hrs with 6 logo-fold concentrations of the test substance starting at lONg/ml (20 NM).
The test substance stock solution (5 mg/mL) was initially diluted at 1/70 fold in medium supplemented with serum. Other concentrations were then obtained from 1/10 fold successive dilutions in the same supplemented medium. Cell survival was evaluated 96 h later by replacing the culture media with 150 pL fresh medium containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4. Next, 50 NL of 2.5 mg/mL
of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate buffer solution, pH 7.4, was added. After 3-4h of incubation at 37°C, the medium and soluble MTT was removed, and 200 NL of dimethyisulfoxide was added to dissolve the precipitate of reduced MTT followed by addition of 25 pL glycine buffer (0.1 M
glycine plus 0.1 M NaCI, pH 10.5). The absorbance was determined at 570 nm with a microplate reader. Results were expressed as the concentration of drug which inhibits 50% of the cell growth (IC5o). The low micromolar levels of ICSO values shown in Table 15 demonstrated a pharmacologically relevant cytotoxic activity of ECO-4601 against a variety of tumor types such as leukemias, melanomas, pancreatic and breast carcinomas.
Table 15 ICso Cell linesType Origin Source Culture medium (NgImL) K562 Leukemia Human ATCC RPM11640 8.6 myelogeneous P388 Leukemia Mouse ATCC RPM11640 10.9 183 Leukemia Human ATCC RPM11640 2.7 B16 (F10) Melanoma Mouse ATCC RPMI 1640 11.4 SK-II~EL Melanoma Human ATCC RPMI 1640 14.0 SK-MEL Melanoma Human ATCC RPMI 1640 14.3 28~

(expressing VEGF) SK-MEL-1 Melanoma Human ATCC EMEM 1 % non-essential14.1 amino acid 1% Sodium puryvate Panc 96 Pancreatic Human ATCC RPMI 1% Sodium 12.5 carcinoma puryvate _ Pancreatic Human ATCC RPMI 1% Sodium 14.2 Panc 10.05 carcinoma puryvate Insulin MCF-7 Breast Human ATCC RPMI1640 9.7 adenocarcinoma EXAMPLE 12. IN VITRO ANTICANCER ACTIVITY OF COMPOUND 1 AGAINST
VARIOUS HUMAN TUMOR CELL LINES FROM THE U.S. NCI PANEL
A study measuring the in vitro antitumor activity of ECO-4601 was performed by the National Cancer Institute (National Institutes of Health, Bethesda, Maryland, USA) against panel of human cancer cell lines in order to determine the ECO-4601 concentrations needed to obtain a 50% inhibition of cell proliferation (GlSO).
The operation of this unique screen utilizes 50 different human tumor cell lines, representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney.
Culture conditions and platina: The human tumor cell lines of the cancer-screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM
L-glutamine. For a typical screening experiment, cells were inoculated into 96 well microtiter plates in 100 NL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines (Table 16). After cell inoculation, the microtiter plates were incubated at 37°C, 5% COz, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line were fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz).
Evaluation of inhibitory activity on cell proliferation: ECO-4601 was provided as a lyophilized powder with an estimated purity of 90+%. The compound was stored at -20°C until day of use. ECO-4601 was solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50Ng/mL gentamicin. Additional four, 10-fold or ~h log serial dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 NI of these different drug dilutions were added to the appropriate microtiter wells already containing 100 NI of medium, resulting in the required final drug concentrations (8.0 x 10-5 M to 8.0 x 10-9 M).
Following drug addition, the plates were incubated for an additional 48 h at 37°C, 5% C02, 95% air, and 100% relative humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 NI of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for minutes at 4°C. Supernatants were discarded, and the plates were washed five times with tap water and air-dried. Sulforhodamine B (SRB) solution (100 NI) at 0.4%
(w/v) in 1 % acetic acid was added to each well, and plates were incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1 % acetic acid and the plates were air-dried. Bound stain was subsequently solubilized with 10 mM trizmaT"~ base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 NI of 80% TCA (final concentration, 16% TCA).
The growth inhibitory activity of ECO-4601 was measured by NCI utilizing the GlSO value, rather than the classical ICSO value. The GlSO value emphasizes the correction for the cell count at time zero and, using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)j, Gl5o is calculated as [(Ti-Tz)/(C-Tz)j x 100 = -50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation.
Result: ECO-4601 shows a significant antitumor activity against several types of tumor as revealed by the NCI screening. Results of the screen are shown in Table 16, and more detailed results of activity against gliomas are shown in Example 13 (Table 17).
Table 16 Inoculation Density Cell Line NameType Origin Glso (NM) (cells/well) CCRF-CEM Leukemia Human 40,000 1.08 K-562 Leukemia Human 5,000 1.43 RPMI-8226 Leukemia Human 20,000 3.15 A549/ATCC Non-Small Cell Human 7,500 9.10 Lung EKVX Non-Small Cell Human 20,000 0.23 Lung HOP-62 Non-Small Cell Human 10,000 8.29 Lung NCI-H226 Non-Small Cell Human 20,000 2.00 Lung NCI-H23 Non-Small Cell Human 20,000 2.02 Lung NCI-H460 Non-Small Cell Human 7,500 13.60 Lung NCI-H522 Non-Small Cell Human 20,000 3.44 Lung COLO 205 Colon Human 15,000 12.70 HCT-116 Colon Human 5,000 2.92 HCT-15 Colon Human 10,000 9.73 HT29 Colon Human 5,000 20.70 SW-620 Colon Human 10,000 2.72 SF-268 CNS Human 15,000 4.94 SF-295 CNS Human 10,000 12.70 SF-539 CNS Human 15,000 0.0075 Table 16 Inoculation Density Cell Line NameType Origin Gl~o (IrM) (cells/well) SNB-7 9 CNS Human 15,000 2.90 SNB-75 CNS Human 20,000 7.71 U251 CNS Human 7,500 2.19 LOX IMVI Melanoma Human 7,500 4.53 M14 Melanoma Human 15,000 4.57 SK-MEL-2 Melanoma Human 20,000 25.0 SK-MEL-28 Melanoma Human 10,000 11.6 SK-MEL-5 Melanoma Human 10,000 7.80 UACC-257 Melanoma Human 20,000 2.31 UACC-62 Melanoma Human 10,000 1.55 IGR-OV1 Ovarian Human 10,000 3.11 OVCAR-3 Ovarian Human 10,000 13.50 OVCAR-4 Ovarian Human 15,000 9.67 OVCAR-5 Ovarian Human 20,000 2.81 OVCAR-8 Ovarian Human 10,000 2.65 SK-OV-3 Ovarian Human 20,000 4.00 786-0 Renal Human 10,000 6.99 A498 Renal Human 25,000 22.30 ACHN Renal Human 10,000 3.10 CAKI-1 Renal Human 10,000 15.20 RXF 393 Renai Human 15,000 7.71 SN12C Renal Human 15,000 3.85 UO-31 Renal Human 15,000 19.70 DU-145 Prostate Human 10,000 3.56 MCF7 Breast Human 10,000 10.10 NCI/ADR-RES Breast Human 15,000 18.30 MDA-MB-231/ATCCBreast Human 20,000 2.72 HS 578T Breast Human 20,000 2.76 MDA-MB-435 Breast Human 15,000 15.30 BT-549 Breast Human 20,000 0.11 T-47D Breast Human 20,000 0.77 The results indicate that ECO-4601 was effective against most of the human tumor cell lines that have been assayed in the NCI screening panel suggesting a broad anticancer activity against several types of human cancer.
EXAMPLE 13: IN VITRO ANTIPROLIFERATIVE STUDY AGAINST A PANEL OF
GLIOMA CELL LINES
The anticancer activity of ECO-4601 was evaluated using a panel of glioma cancer cell lines shown in Table 17, and the 50% inhibition of cell proliferation (ICSO) was determined.
Culture conditions: The cell lines listed in Table 17 were shown to be free of mycoplasma infection and were maintained on DMEM medium supplemented with 10%
heat-inactivated fetal bovine serum and 1 % penicillin-streptomycin, under 5%
C02 at 37°C. Cells were passaged once a week. Prior to use the cells were detached from the culture flask by treating with trypsin for five to ten minutes. The cells were counted with a Neubauer glass slide and viability assessed by 0.25% trypan blue exclusion.
Only flasks with >95% cell viability, were used in the study.
Cell lines amplification and plating: Cells, 5 x 103 cells per well in 100 NL
drug-free medium supplemented with 10% serum, were plated in 96-well flat bottom microtiter plates and incubated at 37°C for 48 hrs before treatment.
Evaluation of inhibitor~r activity on cell proliferation: Cells (in triplicate wells) were incubated 96 hrs with medium containing different concentrations of ECO-4601, starting at 5.0 Ng/ml (10 NM). The compound was used in a solution of 1 % DMSO in D-MEM
or RPMI media (or other equivalent media). The concentrations of ECO-4601 were as follows: 10 NM (5.0 Ng/ml), 1 NM (0.50 Ng/ml), 0.5 NM (0.25 Ng/ml), 0.1 NM
(0.050 Ng/ml), 0.5 NM (0.025 Ng/ml), 0.01 NM (0.0050 Ng/ml), 0.001 NM (0.00050 Ng/ml).
Negative controls were cells treated with vehicle alone (1 % DMSO in culture medium).
Positive controls were cells treated with 4 to 6 increasing concentrations of cisplatin (CDDP) (data not shown). The optical density was measured before incubation (time 0) and following 96 hrs of incubation with test compound in order to measure the growth rate of each cell line.
At the end of the cell treatment, cell culture media was replaced with 150 NI
of fresh medium containing 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4. Then 50 NI of 2.5 mg/ml of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide in PBS pH 7.4, were added to each well and the culture plates incubated for 4 hrs at 37°C. The resulting supernatant was removed and formazan crystals were dissolved with 200 NI of DMSO followed by 25 NI of glycine buffer (0.1 M glycine plus 0.1 M NaCI, pH 10.5). The optical density was read in each well using a single wavelength spectrophotometer plate reader at 570 nm.
Results were expressed as the concentration of drug, which inhibits 50% of the cell growth (ICSO). Each of the cell lines was tested in at least 3 independent experiments.
Results shown in Table 17 confirmed the activity of ECO-4601 against different brain cancer cell lines including gliosarcoma, which is the most malignant form of type IV glioblastoma multiform. Gliosarcomas are a mixture of glial and endothelial cells and are resistant to any chemotherapy.
Table 17 Cell linesType Origin Source IC5o (x 10'~
M) 9L Gliosarcoma Rat ATCC 6.82 t 2.90 GHD Astrocytoma Human ATCC 6.29 t 2.98 U 373 Astrocytoma Human ATCC 3.83 t 1.37 GL26 Glioblastoma Human ATCC 8.93 t 1.10 C6 Glioblastoma Rat ATCC 4.28 t 2.82 DN OligodendrogliomaHuman ATCC 3.26 t 0.93 GHA OligodendrogliomaHuman ATCC 1.78 t 0.84 EXAMPLE 14: IN VIVO EFFICACY IN A GLIOMA MODEL
The aim of this study was to test whether ECO-4601 administered by i.p. route prevents or delays tumor growth in C6 glioblastoma cell-bearing mice, and to determine an effective dosage regimen.
Animals: A total of 60 six-week-old female mice (Mus musculus nude mice), ranging between 18 to 25 g in weight, were observed for 7 days before treatment.
Animal experiments were performed according to ethical guidelines of animal experimentation (Charte du comite d'ethique du CNRS, juillet 2003) and the English guidelines for the welfare of animals in experimental neoplasia ( UVORKMAN, P., 71NENT'YMAN, P., BALKI~VILL, F., et al. ()998). United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Guidelines for the welfare of animals in experimental neoplasia (Second Edition, July )997; British Journal of Cancer 77: )-)0). Any dead or apparently sick mice were promptly removed and replaced with healthy mice. Sick mice were euthanized upon removal from the cage. Animals were maintained in rooms under controlled conditions of temperature (2312°C), humidity (4515%), photoperiodicity (12 hrs light / 12 hrs dark) and air exchange. Animals were housed in polycarbonate cages (5/single cage) that were equipped to provide food and water. Animal bedding consisted of sterile wood shavings that were replaced every other day. Food was provided ad libitum, being placed in the metal lid on the top of the cage. Autoclaved tap water was provided ad libitum. Water bottles were equipped with rubber stoppers and sipper tubes. Water bottles were cleaned, sterilized and replaced once a week. Two different numbers engraved on two earrings identified the animals. Each cage was labelled with a specific code.
Tumor Cell Line: The C6 cell line was cloned from a rat glial tumor induced by N-nitrosomethyurea (NMU) by Premont et al. (Premont J, Benda P, Jard S., ~3H]
norepinephrine binding by rat glial cells in culture. Lack of correlation between binding and adenylate cyclase activation. Biochim Biophys Acta. )975 Feb )3;38)(2):368-76.) after series of alternate culture and animal passages.
Cells were grown as adherent monolayers at 37°C in a humidified atmosphere (5%
C02, 95% air). The culture medium was DMEM supplemented with 2 mM L-glutamine and 10% fetal bovine serum. For experimental use, tumor cells were detached from the culture flask by a 10 min treatment with trypsin-versen. The cells were counted in a hemocytometer and their viability assessed by 0.25% trypan blue exclusion.
Preparation of the Test Article: For the test article, the following procedure was followed for reconstitution (performed immediately preceding injection). The vehicle consisted of a mixture of benzyl alcohol (1.5%), ethanol (8.5%), propylene glycol (27%), (27%), dimethylacetamide (6%) and water (30%). The vehicle solution was first vortexed in order to obtain a homogeneous liquid. 0.6 mL of the vortexed vehicle solution was added to each vial containing the test article (ECO-4601 ). Vials were mixed thoroughly by vortexing for 1 minute and inverted and shaken vigorously.
Vials were mixed again prior to injection into each animal.
Animal Inoculation with tumor cells: Experiment started at day 0 (Do). On Do, mice received a superficial intramuscular injection of C6 tumor cells (5 x 105 cells) in 0.1 mL
of DMEM complete medium into the upper right posterior leg.
Treatment regimen and Results In a first series of experiments, treatment started 24 hrs following inoculation of C6 cells. On the day of the treatment, each mouse was slowly injected with 100 NL of test or control articles by i.p. route. For all groups, treatment was performed until the tumor volume of the saline-treated mice (group 1 ) reached approximately 3 cm3 (around day 16). Mice of group 1 were treated daily with a saline isosmotic solution for 16 days.
Mice of group 2 were treated daily with the vehicle solution for 16 days. Mice of group 3 were treated daily with 10 mg/kg of ECO-4601 for 16 days. Mice of group 4 were treated every two days with 30 mg/kg of ECO-4601 and received 8 treatments.
Mice of group 5 were treated every three days with 30 mg/kg of ECO-4601 and received 6 treatments. Measurement of tumor volume started as soon as tumors became palpable (>100 mm3; day 11 post-inoculation) and was evaluated every second day until the end of the treatment using callipers. As shown in Table 18 and Figure 3, the mean value of the tumor volume of all ECO-4601 treated groups (6 mice/group) was significantly reduced as demonstrated by the one-way analysis of variance (Anova) test followed by the non-parametric Dunnett's multiple comparison test comparing treated groups to the saline group. An asterisk in the P value column of Table 18 indicates a statistically significant value, while "ns" signifies not significant.
Table 18 Treatment Treatment Tumor volume (mm) % InhibitionP value regimen (mean t SEM) Saline Q1 x 16 3,004.1 t 249.64 - -Vehicle solution Q1 x 16 2,162.0 t 350.0 28.0% >0.05 ns ECO-4601 (10 mg/kg)Q1 x 16 1,220.4 t 283.46 59.4% <0.01 ECO-4601 (30 mg/kg)Q2 x 8 1,236.9 t 233.99 58.8% <0.01 ECO-4601 (30 mg/kg)Q3 x 6 1,184.1 t 221.45 60.6% <0.01 In a second series of experiments, treatment started at day 10 following inoculation of C6 cells when tumors became palpable (around 100 to 200 mm3).
Treatment was repeated daily for 5 consecutive days. On the day of the treatment, each mouse was slowly injected with 100 pL of ECO-4601 by i.p. route. Mice of group 1 were treated daily with saline isosmotic solution. Mice of group 2 were treated daily with the vehicle solution. Mice of group 3 were treated daily with 20 mg/kg of ECO-4601.
Mice of group 4 were treated daily with 30 mg/kg of ECO-4601. Mice were treated until the tumor volume of the saline-treated control mice (group 1 ) reached around 4 cm3.
Tumor volume was measured every second day until the end of the treatment using callipers. As shown in Table 19 and Figure 4, the mean value of the tumor volume of all ECO-4601 treated groups (6 mice/group) was significantly reduced as demonstrated by the one-way analysis of variance (Anova) test followed by the non-parametric Dunnett's multiple comparison test comparing treated groups to the saline group. An asterisk in the P value column of Table 19 indicates a statistically significant value, while "ns"
signifies not significant.
Histological analysis of tumor sections showed pronounced morphological changes between ECO-4601-treated tumors and control groups. In tumors treated with ECO-4601 (20 - 30 mg/kg), cell density was decreased and the nuclei of remaining tumor cells appeared larger and pycnotic while no such changes were observed for vehicle-treated mice (Figure 5).
Table 19 Treatment Treatment Tumor volume (mm % InhibitionP value ) regimen (mean t SEM) Saline Q1 x 5 4,363.1 t 614.31 - -Vehicle solution Q1 x 5 3,205.0 632.37 26.5% >0.05 ns ECO-4601 (20 mg/kg)Q1 x 5 1,721.5 t 374.79 60.5% <0.01 ECO-4601 (30 mg/kg)Q1 x 5 1,131.6 t 525.21 74.1 % <0.01 EXAMPLE 15: COMPOUND 1 DERIVATIVES GENERAL PROCEDURES
15.1: Eeoxidation The epoxide compounds of the present invention (e.g., compounds according to exemplary Compounds 16-22 are made from Compound 1 (ECO-4601 ) by treatment with any of a number of epoxidizing reagents such as perbenzoic acid, monoperphthalic acid or more preferably by m-chloroperbenzoic acid in an inert solvent such as dichloromethane or 1,2-dichloroethane. It will be appreciated by one of ordinary skill in the art that slightly greater than one molecule equivalent of epoxidizing agent will result in the maximal yield of mono-epoxides, and that the reagent, solvent, concentration and temperature of the reaction will dictate the ratio of specific mono-epoxides formed. It will also be appreciated that the mono-epoxides will be enantiomeric mixtures, and that the di-epoxides and the tri-epoxide can be prepared as diastereomers and that the conditions of the reaction will determine the ratios of the products. One skilled in the art will appreciate that under most conditions of reactions the product will be a mixture of all possible epoxides and that these may be separated by standard methods of chromatography. Exemplary approaches to the generation of mono-, di-, and tri-epoxides are provided below.
A) Mono-epoxides Compounds 16, 17 and 18 prepared by epoxidation of Compound 1:

O
i i \ / N O \ / N O
/ N / \
Ho H _ \ off Compound 16 Ho H _ off Compound 17 HO
i i \ / N O
HO
\ pH Compound 18 H
To a solution of Compound 1 dissolved in tetrahydrofuran (THF) is added 1.1 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0 °C for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of SephadexT"" LH-20 to isolate a mixture of predominantly Compounds 16, 17 and 18, contaminated with some unchanged starting material and some di- and tri-epoxides.
Compounds 16, 17 and 18 are separated and purified by HPLC using the system described in Example 2 for the purification of Compound 1. In a typical experiment yields of 15% to 25% are obtained for each of Compounds 16, 17 and 18.
B) Synthesis of Compounds 19, 20 and 21 by di-epoxidation of Compound 1:
\ / N O O / \ / N / O
/ /
HO HO _ \ OH Compound 19 Ho H - \ off Compound 20 \ / N / O O
HO H /_ \ off Compound 21 HO
To a solution of Compound 1 dissolved in tetrahydrofuran (THF) is added 2.3 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0 °C for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of SephadexT"" LH-20 to isolate a mixture of predominantly Compounds 19, 20 and 21, contaminated with traces of unchanged starting material and some mono- and tri-epoxides. Compounds 19, 20 and 21 are separated and purified by HPLC using the system described in Example 2 for the purification of Compound 1. In a typical experiment, yields of 15% to 20% are obtained for each of Compounds 19, 20 and 21.
C) Synthesis of Compound 22 by tri-epoxidation of Compound 1:
\ / N o Compound 22 To a solution of Compound 1, dissolved in tetrahydrofuran (THF), is added 3.5 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0 °C for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of SephadexT"" LH-20 to isolate Compound 22 in an essentially pure form in a yield of 80+%.
D) Syntheses of Compounds 23 and 24 by epoxidation of Compound 42.
\ / 'N O \ /
OH Compound 23 HO H / ~ OH Compound 24 HO
To a solution of Compound 42 dissolved in tetrahydrofuran (THF) is added 1.1 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0 °C for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of SephadexT"" LH-20 to isolate a mixture of predominantly Compounds 23 and 24, contaminated with some unchanged starting material and some diepoxide.

Compounds 23 and 24 are separated and purified by HPLC or HSCC using one of the systems described in Example 2 for the purification of Compound 1. In a typical experiment yields of 35% to 40% are obtained for each of Compounds 23 and 24.
E) Synthesis of Compound 28 by epoxidation of Compound 40.
~ 'N
H N ~ \ H Compound 28 H
H
To a solution of Compound 40 dissolved in tetrahydrofuran (THF) is added 2.2 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0 °C for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of SephadexT"" LH-20 to isolate essentially pure Compound 28 in good yield.
15.2: N-Aceylation Synthesis of Compound 13 by N-acetylation of Compound 1.
N i i i HO N / \ OH Compound 13 Ac --HO
To a solution of Compound 1 dissolved in tetrahydrofuran (THF) is added 1.2 equivalents of acetic anhydride and a few drops of triethylamine. The reaction mixture allowed to stand at room temperature for 1-2 hours and then evaporated to dryness under reduced pressure to obtain Compound 13 in an essentially pure form in an almost quantitative yield 15.3: N-alk !ay tion Syntheses of Compounds 2, 3 and 14 by N-alkylation of Compound 1.

i ~ i Compound 2 R = methyl Compound 3 R = benzyl Ho Compound 14 R = ethyl To a solution of Compound 1 dissolved in terachloroethylene is added 1.2 equivalents of the appropriate alkyl bromide (iodomethane for Compound 2, benzyi bromide for Compound 3 or ethyl bromide for Compound 14). The reaction mixture the reaction mixture is heated under reflux for 1-2 hours and then evaporated to dryness under reduced pressure to obtain Compound 2, 3 or 14 respectively, in an essentially pure form in an almost quantitative yield.
Compounds 60 to 76 are also prepared via this procedure, by reaction of Compound 1 with the appropriate alkyl halide or dialkyl sulfate.
15.4: H~roqenolysis A) Syntheses of Compounds 40, 41 and 42 by hydrogenolysis of Compound 1.
Compound 40 Ho ~N--~ ~~, , Compound 41 Compound 42 H
A solution Compound 1 (462 mg) in ethanol (200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenation apparatus in an atmosphere of hydrogen.
The uptake of hydrogen by the reaction is measured carefully and at the point where one millimole of hydrogen has been consumed, shaking is stopped, the vessel is rapidly evacuated and the atmosphere is replaced with nitrogen. The catalyst is removed by filtration and the filtrate is concentrated to obtain a crude mixture of Compounds 40, 41 and 42 contaminated by unreacted starting material and minor amounts of over reduced products. The desired products may be separated and purified by HPLC
or HSCC chromatography using the systems as described in Example 2 above, to obtain approximately 100 mg of each of Compounds 40, 41 and 42.
B) Syntheses of Compounds 43, 44 and 45 by hydrogenolysis of Compound 1.
Compound 43 Ho ~N--~~ ~~~ Compound 44 H
Compound 45 H
A solution of Compound 1 (462 mg) in ethanol (200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenation apparatus in an atmosphere of hydrogen. The uptake of hydrogen by the reaction is measured carefully and at the point where two millimoles of hydrogen has been consumed, shaking is stopped, the vessel is rapidly evacuated and the atmosphere is replaced with nitrogen. The catalyst is removed by filtration and the filtrate is concentrated to obtain a crude mixture of Compounds 43, 44 and 45 contaminated by trace amounts unreacted starting material and minor amounts of under and over reduced products. The desired products may be separated and purified by HPLC or HSCC chromatography using the systems as described in Example 2 above, to obtain approximately 100 mg of each of Compounds 43, 44 and 45.
C) Syntheses of Compound 46 by hydrogenolysis of Compound 1 (alternate procedure).

Compound 46 A solution of Compound 1 (462 mg) in ethanol (200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenation apparatus in an atmosphere of hydrogen. The uptake of hydrogen by the reaction is measured carefully and at the point where three millimoles of hydrogen has been consumed, shaking is stopped, the vessel is rapidly evacuated and the atmosphere is replaced with nitrogen. The catalyst is removed by filtration and the filtrate is concentrated to obtain an essentially pure sample of Compound 46.
15.5: Peracet~rlation Syntheses of Compound 15 by peracetylation of Compound 1.
N ~
A N / \ OAc Compound 15 Act Ac0 A solution of Compound 1 (100 mg) in acetic anhydride (5 ml) is treated with pyridine (250 u1). The reaction mixture is allowed to stand overnight at room temperature and is then diluted with toluene (100 ml). The toluene solution is washed well with aqueous 5% sodium bicarbonate solutions, then with water and is finally concentrated under reduced pressure to give an essentially pure sample of Compound 15 in almost quantitative yield.
15.6: Eiooxide opening Syntheses of Compound 53 by opening the epoxide of Compound 16.

i i \ I N f..pH
HO N ~ \ Compound 53 H , OH
H
A solution of Compound 16 (100 mg) in tetrahydrofuran (50 ml) is treated with aqueous hydrochloric acid (5 ml). The reaction mixture is stirred overnight at room temperature and is then diluted with toluene (100 ml) and water (200 ml). The toluene layer is separated and the aqueous layer is extracted with a further 100 ml of toluene.
The combined toluene layers are washed once more with water (50 ml) and the separated and dried under vacuum to give the vicinal glycol Compound 53.
15. 7: Ozonolvsis Syntheses of Compounds 47, 49 and 51 by ozonolysis of Compound 1.
O
N i i ~O ' N i ~O
\ I \ I
Compound 47 N ~ \ Compound 49 HO H ._ OH HO H , OH
HO H
I NCO
\ N / ~ Compound 51 HO H OH
HO
A solution of Compound 1 (462 mg) in dry ethyl acetate (200 ml) in an ozonolysis apparatus is cooled to below -20°C. A stream of ozone containing oxygen is passed into the solution from an ozone generator, which has been precalibrated such that the rate of ozone generation is known. To obtain predominantly Compound 47 the passage of ozone is halted after 0.9 millimole have been generated. To obtain predominantly Compound 49 the ozone passage is halted after 2 millimoles have been generated and to obtain Compound 51 as the predominant product 3.3 millimoles of ozone are generated.
At the completion of the ozonolysis, the reaction mixture is transferred to an hydrogenation apparatus, 5°/a palladium on calcium carbonate catalyst (0.2 g) is added to the reaction mixture which is maintained at less than -20°C and is hydrogenated.
When hydrogen uptake is complete the hydrogen atmosphere is replaced with nitrogen and the reaction mixture is allowed to come to room temperature, filtered to remove catalyst and the filtrate is concentrated. The crude product may be purified by chromatography using either HPLC or HSCC with the systems as described in Example 2 to give, dependent on the amount of ozone used, Compounds 47, 49 and 51.
15.8: Reduction Synthesis of Compound 48 by reduction of the aldehyde of Compound 47.
/ N i i OH
Ho N / \ Compound 48 H , OH
H
A solution of Compound 47 (50 mg) in isopropanol (5 ml) is cooled in an ice-salt bath and sodium borohydride (10 mg) is added and the mixture is stirred for 20 minutes. It is then diluted with water (20 ml) and extracted twice with toluene (10 ml portions) at ambient temperature. The combined toluene extracts are filtered and the filtrate is concentrated to give Compound 48.
15.8: Esterification Syntheses of Compounds 35, 36 and 37 by esterification of Compound 1.
i i i \ / N i i i Ac0 N ~ \ Compound 35 HD N / \ Compound 36 OH H , OH
HO Ac0 N i i HO H / \ oAc Compound 37 H
To a solution of Compound 1 dissolved in toluene (9 parts) tetrahydrofuran (1 part), cooled in an ice-bath is added 1.1 equivalents of acetic anhydride and two drops of boron trifluoride etherate. The reaction is maintained cool in an ice bath and stirred at 0 °C for 1-2 hours. The reaction mixture is then poured into aqueous 5%
sodium bicarbonate solution shaken and the toluene layer is removed. The aqueous laer is re-extracted with toluene and the combined toluene layers are concentrated to a mixture of predominantly Compounds 35, 36 and 37, contaminated with some unchanged starting material and some diacetates. Compounds 35, 36 and 37 are separated and purified by HPLC or HSCC using one of the systems described in Example 2 for the purification of Compound 1. In a typical experiment yields of 25% to 30% are obtained for each of Compounds 35, 36 and 37.
15.9: Alk~rlation Syntheses of Compounds 6, 7 and 38 by methylation of Compound 1.
O
N i i i \ / N i i i Me0 N ~ \ Compound 6 Hp N ~ \ off Compound 7 OH
HO Me0 N i i i HO N ~ \ Compound 38 H , OMe H
A solution of Compound 1 (1 g) in tetrahydrofuran 50 (ml) is titrated with exactly one equivalent of sodium methoxide, allowed to stand for 30 minutes at room temperature and then treated with 1.2 equivalents of dimethylsulphate. Heat the mixture under reflux for one hour, cool to room temperature and pour into a mixture of toluene (200 ml) and water (200 ml). The layers are separated and the aqueous layer is extracted once more with an equal portion of toluene. The combined toluene layers are washed once with 1 N aqueous acetic acid and then concentrated to s crude product, which is predominantly a mixture of Compounds 6, 7 and 38 with some unchanged starting material and traces of over-methylated derivatives. The desired products may be separated and purified by HPLC or HSCC chromatography using the systems as described in Example 2 above, to obtain approximately 200 mg of each of Compounds 6, 7 and 38.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims (34)

1. A compound of Formula I:

wherein, W1, W2 and W3 are each independently selected from the chain from the tricycle terminates at W3, W2 or W1 with W3, W2 or W1 respectively being either -CH=O or -CH2OH;
R1 is selected from H, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C6-10aryl, C5-10heteroaryl, C3-10cycloalkyl, C3-10heterocycloalkyl, C(O)C1-10alkyl, C(O)C2-10alkenyl, C(O)C2-10alkynyl, C(O)C6-10aryl, C(O)C5-10heteroaryl, C(O)C3-10cycloalkyl;
C(O)C3-10heterocycloalkyl or a C-coupled amino acid;
R2, R3, and R4 are each independently selected from H, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C6-10aryl, C5-10heteroaryl, C3-10cycloalkyl, C3-10heterocycloalkyl, C(O)H, C(O)C3-10alkyl, C(O)C2-10alkenyl, C(O)C2-10alkynyl, C(O)C6-10aryl, C(O)C5-10heteroaryl, C(O)C3-10cycloalkyl; C(O)C3-10heterocycloalkyl or a C-coupled amino acid;
R5 and R6 are each independently selected from H, OH and OC1-6alkyl;
wherein, when any of R1, R2, R3, R4, R5 and R6 comprises an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group, then the alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group is optionally substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, C1-6alkyl, C2-7alkenyl, C2-7alkynyl, C3-10cycloalkyl, C3-10heterocycloalkyl, C6-10aryl, C5-10heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, oxo, guanidino and formyl; and with the proviso that when W1, W2 and W3 are all -CH=C(CH3)-, and R2, R3 and R4 are all H, then R1 is not H;
or a pharmaceutically acceptable salt or prodrug thereof.

2. The compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof, wherein said R1 is H.

3. The compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof, wherein said R1 is CH3.

4. The compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof, wherein said R1 is C1-10alkyl.

5. The compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof, wherein said R1 is C1-10 alkenyl.

6. The compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof, wherein said R2 is H.

7. The compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof, wherein said R3 is H.

8. The compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof, wherein said R4 is H.

9. The compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof, wherein said R2, R3 and R4 are each H.

10. The compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof, wherein two of R2, R3 and R4 are CH3.

11. The compound of claim 1, or a pharmaceutically acceptable salt or prodrug thereof, wherein each of W1, W2, and W3 is -CH2CH(CH3)-.

12. The compound of claim 1, wherein said compound is selected from:

or a pharmaceutically acceptable salt or prodrug thereof.

13. The compound of claim 12, wherein said compound is Compound 2, or a pharmaceutically acceptable salt or prodrug thereof.

14. The compound of claim 12, wherein said compound is selected from Compounds 17 and 18, or a pharmaceutically acceptable salt or prodrug thereof.

15. The compound of claim 12, wherein said compound is selected from Compounds 3 to 7, 9 to 12 and 46, or a pharmaceutically acceptable salt or prodrug thereof.

16. The compound of claim 12, wherein said compound is a selected from Compounds 4 to 7, 10 and 46, or a pharmaceutically acceptable salt or prodrug thereof.

17. The compound of claim 12, wherein said compound is a compound selected from Compounds 3 and 60 to 76, or a pharmaceutically acceptable salt or prodrug thereof.

18. The compound of claim 12, wherein said compound is Compound 8, or a pharmaceutically acceptable salt or prodrug thereof.

19. A process for making a compound of any one of claims 1-18, comprising cultivation of a Micromonospora sp. strain, in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms, isolation and purification of the compound, and chemical modification of the isolated compound.

20. A pharmaceutical composition comprising a compound of any one of claims 1 to 11, together with a pharmaceutically acceptable carrier.

21. A pharmaceutical composition comprising a compound of claim 12, together with a pharmaceutically acceptable carrier.

22. A pharmaceutical composition comprising a compound of claim 13, together with a pharmaceutically acceptable carrier.

23. A pharmaceutical composition comprising a compound of any one of claims 14-18, together with a pharmaceutically acceptable carrier.

24. Use of a compound of any one of claims 1-12 as an antitumor agent.

25. Use of a compound of claim 13 as an antitumor agent.

26. Use of a compound of any one of claims 14-17 as an antitumor agent.

27. Use of a compound of any one of claims 1-12 for the treatment of precancerous or cancerous conditions.

28. Use of a compound of claim 13 for the treatment of precancerous or cancerous conditions.

29. Use of a compound of any one of claims 14-17 for the treatment of precancerous or cancerous conditions.

30. Use of a compound of any one of claims 1-12, in the manufacture of a medicament for the treatment of precancerous or cancerous conditions.

31. Use of a compound of claim 13, in the manufacture of a medicament for the treatment of precancerous or cancerous conditions.

32. Use of a compound of any one of claims 14-17, in the manufacture of a medicament for the treatment of precancerous or cancerous conditions.

33. Use of a compound of any one of claims 1-18 as a peripheral benzodiazepine receptor (PBR) binding agent for the treatment of a condition involving PBR.

34. Use of a compound of any one of claims 1-17 as a 5-Lipooxygenase (5-LO) inhibitor for the treatment of a condition involving the 5-LO enzyme.