Patent Application
20090029972
This invention relates to dibenzodiazepinone analogues having improved properties, which are chemical derivatives of Compound 1. The invention further relates to their pharmaceutically acceptable salts, solvates and prodrugs, and to methods for obtaining the compounds. One method of obtaining the derivatives involves post-biosynthesis chemical modification of Compound 1. The present invention further relates to the use of dibenzodiazepinone analogues, and their pharmaceutically acceptable salts, solvates 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 a dibenzodiazepinone analogue, or a pharmaceutically acceptable salt, solvate or prodrug thereof.
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. Pat. 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 (Compound 1) and Micromonospora sp. strains 046-ECO11 and [S01]046 are disclosed in CA 2,466,340. Its use for the treatment of cancer is disclosed in PCT/CA2005/000751.
Synthetic dibenzodiazepinone analogs were disclosed in the published Canadian patent application 2,248,820 as having anti-histamine properties.
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 methods to generate these novel compounds by post-biosynthetic chemical modifications.
In one aspect of the invention, the dibenzodiazepinone analogue is represented by a compound of Formula I as defined below, or a hydrogenated or hydroalkoxylated farnesyl derivative, or a pharmaceutically acceptable salt, solvate or prodrug of a compound of Formula I. In another embodiment, the dibenzodiazepinone analogue is represented by any one of Compounds 2 to 27 as defined below, or a pharmaceutically acceptable salt, solvate or prodrug, or salt of a prodrug of any one of Compounds 2 to 25. In a further embodiment, the dibenzodiazepinone analogue is represented by any one of Compounds 2 to 5, 7, 13, 14, 15, 18, 21, and 22 as defined below, or a pharmaceutically acceptable solvate or prodrug of any one of Compounds 2 to 5, 7, 13, 14, 15, 18, 21, and 22.
In yet another aspect of the invention, the dibenzodiazepinone analogue is represented by any one of Compounds 23 to 25 as defined below, or a pharmaceutically acceptable solvate or prodrug of any one of Compounds 23 to 25.
The invention further encompasses a dibenzodiazepinone analogue obtained by a method comprising the steps of: (a) providing an isolated form of ECO-4601, (b) chemically modifying Compound 1. In another embodiment, the dibenzodiazepinone analogue is a compound of Formula I, or a hydrogenated or hydroalkoxylated farnesyl derivative, or a pharmaceutically acceptable salt, solvate or prodrug thereof. In another embodiment the chemical modification step (b) is one or more steps selected from N-alkylation, O-triacetylation, aryl bromination, and farnesyl hydrogenation or hydroalkoxylation. In another embodiment the dibenzodiazepinone analogue is selected from Compounds 2 to 27. In another embodiment the dibenzodiazepinone analogue is selected from Compounds 2 to 5, 7, 13, 14, 15, 18, 21, and 22. In another embodiment the dibenzodiazepinone analogue is selected from Compounds 23 to 25.
The invention further encompasses a method for making a dibenzodiazepinone compound, comprising chemically modifying the farnesyl dibenzodiazepinone Compound 1, and optionally isolating and purifying the dibenzodiazepinone compound produced. In one embodiment, the chemical modification step comprises at least one step selected from N-alkylations, O-acylations, aryl bromination and modifications of the double bonds of the farnesyl side chain including, hydrogenation, and hydroalkoxylation. In a subclass of this embodiment, the farnesyl side chain modification reaction is partial (one or two double bonds modified) or complete (all three double bonds modified).
The invention further encompasses the use of a compound of Formula I or a hydrogenated or hydroalkoxylated farnesyl derivative, or a pharmaceutically acceptable salt, solvate 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 selected from Compounds 2 to 27. In another embodiment, the compound is selected from Compounds 2 to 5, 7, 13, 14, 15, 18, 21, and 22. In another embodiment the compound is selected from Compounds 23 to 25.
The invention further encompasses the use of a compound of Formula I or a hydrogenated or hydroalkoxylated farnesyl derivative, or a pharmaceutically acceptable salt or prodrug thereof as an antineoplastic agent for the treatment of a proliferative disorder in a mammal. In one embodiment, the compound is selected from Compounds 2 to 27. In another embodiment, the compound is selected from Compounds 2 to 5, 7, 13, 14, 15, 18, 21, and 22. In another embodiment the compound is selected from Compounds 23 to 25.
The invention further encompasses the use of a compound of Formula I or a hydrogenated or hydroalkoxylated farnesyl derivative, 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 selected from Compounds 2 to 27. In another embodiment, the compound is selected from Compounds 2 to 5, 7, 13, 14, 15, 18, 21, and 22. In another embodiment the compound is selected from Compounds 23 to 25.
The invention further encompasses a commercial package comprising a compound of Formula I or a hydrogenated or hydroalkoxylated farnesyl derivative, or a pharmaceutically acceptable salt or prodrug thereof, together with instructions for use in the treatment of a neoplasm or a pre-cancerous or cancerous condition. In one embodiment, the compound is selected from Compounds 2 to 27. In another embodiment, the compound is selected from Compounds 2 to 5, 7, 13, 14, 15, 18, 21, and 22. In another embodiment the compound is selected from Compounds 23 to 25.
In one embodiment, the cancer cell, neoplastic, pre-cancerous or cancerous condition, in the above-mentioned uses, is selected from leukemia, melanoma, breast cancer, lung cancer, pancreatic cancer, ovarian cancer, renal cancer, colon or colorectal cancer, prostate cancer, and CNS cancer. In another embodiment, the cancer cell, and pre-cancerous or cancerous condition, in the above-mentioned methods and uses, is selected from leukemia, breast cancer, prostate cancer, and CNS cancer.
The present invention relates to novel dibenzodiazepinone analogues having improved properties, herein referred as the compounds of Formula I and hydrogenated or hydroalkoxylated farnesyl derivatives, and Compounds 2 to 27. The invention further relates to pharmaceutically acceptable salts, solvates and prodrugs of dibenzodiazepinone compounds. The invention further relates to processes of obtaining the analogs by chemical modification of Compound 1. Compound 1 is isolated from strains of actinomycetes, Micromonospora sp. 046-ECO 11 (also as 046(ECO11)) or [S01]046, as described in PCT/CA04/000069.
The invention also relates to a method for producing novel dibenzodiazepinone analogs, by chemical modification of the farnesyl dibenzodiazepinone obtained from fermentation and isolation. In a subclass of this embodiment, the compound produced is a compound of Formula I or a hydrogenated or hydroalkoxylated farnesyl derivative thereof, or a compound selected from Compounds 23 to 27.
The present invention also relates to pharmaceutical compositions comprising a compound selected from the compounds of Formula I, and Compounds 2 to 27, and their pharmaceutically acceptable salts, solvates and derivatives. The compounds are useful as pharmaceuticals, in particular for use as inhibitors of neoplastic cell growth.
The following detailed description discloses how to make and use the dibenzodiazepinone analogs and compositions containing these compounds to inhibit tumor growth.
Accordingly, certain aspects of the present invention relate to pharmaceutical compositions comprising the dibenzodiazepinone compounds of the present invention together with a pharmaceutically acceptable carrier, and methods of using the pharmaceutical compositions to treat pre-cancererous or cancerous conditions.
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. 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 Compound 1, namely 10-farnesyl-4,6,8-trihydroxy-5,10-dihydrodibenzo[b,e][1,4]diazepin-11-one, also referred to as ECO-4601.
As used herein, the terms “compound(s) of the invention”, “dibenzodiazepinone analogue(s)”, “dibenzodiazepinone compound(s)”, and equivalent expressions refer to a class of dibenzodiazepinone compounds containing a farnesyl moiety or being derived from a farnesyl moiety, and pharmaceutically acceptable salts, solvates and prodrugs thereof. The term includes a compound of Formula I, a compound selected from Compounds 2 to 27, or the exemplified compounds of the present invention, Compounds 2 to 5, 7, 13, 14, 15, 18, and 21 to 25, or a pharmaceutically acceptable salt, solvate or prodrug of any of the above compounds. As used herein, the term “dibenzodiazepinone analogues” includes compounds of this class that can be used as intermediates in chemical syntheses and variants containing different isotopes than the most abundant isotope of an atom (e.g, D replacing H, 13C replacing 12C, etc). The compounds of the invention are also sometimes referred as “active ingredients”.
As used herein, the term “chemical modification” refers to one or more steps of modifying a dibenzodiazepinone compound, referred to as “starting material”, by chemical synthesis. Preferred compound used as starting materials in a chemical modification process is Compound 1. Examples of chemical modification steps include N-alkylations, O-acylations, aromatic bromination, and modifications of the double bonds of the farnesyl side chain including, hydrogenation and hydroalkoxylation. Farnesyl side chain modification reaction can be partial (one or two double bonds modified) or complete (three double bonds modified). Chemical modification steps are also defined in the Schemes of Section IIIB, and exemplified in Examples 4 to 8.
The term “ester” refers to a dibenzodiazepinone analogue obtained by the replacement of a hydrogen atom from an alcohol by a C(O)R″ replacement group by an O-acylation reaction as defined in Scheme 1(b) below, wherein C(O)R″ can also be a C-coupled amino acid. The term ester also encompasses ester equivalents including, without limitation, carbonate, carbamate, and the like. More particularly, the term “ester” encompasses esters of the alcohols in positions 4, 6, and 8 (see Examples 3-8 for atom numbering).
The term “N-alkylated derivative” refers to a dibenzodiazepinone analogue obtained by the replacement of a hydrogen atom of a nitrogen atom by an R replacement group by an N-alkylation reaction as defined in Scheme 2(a) below. More particularly, the term “N-alkylated derivative” encompasses substituted derivatives at the nitrogen in position 5 (see Examples 3-8 for atom numbering).
As used herein, the term “hydrogenated or hydroalkoxylated farnesyl derivative” refers to a compound having a modified farnesyl side chain at one to three positions by either saturation (addition of two hydrogen atoms) or by addition of a molecule of alcohol (H and OC1-6alkyl) produced respectively by the procedures generally defined in Schemes 3(a) and (b) of Section IIIB, and more specifically in Examples 4 and 7.
As used herein, abbreviations have their common meaning. Unless otherwise noted, the abbreviations “Ac”, “Me”, “Et”, “Pr”, “i-Pr”, “Bu”, “Bz”, “Bn” and “Ph”, respectively refer to acetyl, methyl, ethyl, propyl (n- or iso-propyl), iso-propyl, butyl (n-, iso-, sec- or tert-butyl), benzoyl, benzyl and phenyl. Abbreviations in the specification correspond to units of measure, techniques, properties or compounds as follows: “RT” and “Rt” mean retention time, “min” means minutes, “h” means hour(s), “μL” means microliter(s), “mL” means milliliter(s), “mM” means millimolar, “M” means molar, “mmole” means millimole(s), “eq” means molar equivalent(s). “High Pressure Liquid Chromatography” or “High Performance Liquid Chromatography” are abbreviated HPLC.
The term “alkyl” refers to linear, branched or cyclic, saturated hydrocarbon groups. Examples of alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl, heptyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, 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 “C1-nalkyl”, 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 C1-nalkyl can be cyclic or a straight or branched chain.
The term “linear C1-nalkyl”, wherein n is an integer from 2 to 10, refers to an alkyl group having from 1 to the indicated “n” number of carbons and being linear, i.e. not cyclic or branched in the vicinity of the attached atom (herein the nitrogen). The C1-nalkyl can optionally be substituted with groups such as amino, cyano, halo, hydroxyl, nitro, thio, and alkoxy.
The term “alkenyl” refers to linear, branched or cyclic unsaturated hydrocarbon groups containing, 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 portion(s) of the unsaturated hydrocarbon chain may be either in the cis or trans configuration.
The term “C2-nalkenyl”, 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-nalkenyl can be cyclic or a straight or branched chain.
The term “alkynyl” refers to linear, branched or cyclic unsaturated hydrocarbon groups containing 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-nalkynyl”, 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-nalkynyl can be cyclic or a straight or branched chain.
The term “cycloalkyl” or “cycloalkyl ring” refers to an alkyl group, as defined above, further comprising a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to fifteen ring members. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopenten-1-yl, cyclopenten-2-yl, cyclopenten-3-yl, cyclohexyl, cyclohexen-1-yl, cyclohexen-2-yl, cyclohexen-3-yl, cycloheptyl, bicyclo[4,3,0]nonanyl, norbornyl, and the like. Cycloalkyl 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 and formyl.
The term “C3-ncycloalkyl”, wherein n is an integer from 4 to 15, refers to a cycloalkyl ring or ring system or 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, further comprising one to four hetero atoms (e.g. N, O, S, P) or hetero groups (e.g. NH, NRX, PO2, SO, SO2) in a single or fused heterocyclic ring system having from three to fifteen ring members (e.g. tetrahydrofuranyl has five ring members, including one oxygen atom). Examples of a heterocycloalkyl, heterocyclic or heterocycloalkyl ring include, without limitation, pyrrolidino, tetrahydrofuranyl, tetrahydrodithienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3,1,0]hexanyl, 3-azabicyclo[4,1,0]heptanyl, 3H-indolyl, and quinolizinyl. The foregoing heterocycloalkyl groups, as derived from the compounds listed above, may be C-attached or N-attached where such is possible. Heterocycloalkyl, heterocyclic or heterocycloalkyl 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-nheterocycloalkyl”, wherein n is an integer from 4 to 15, refers to an heterocycloalkyl group having from 3 to the indicated “n” number of atoms in the cycle and at least one hetero group as defined above.
The terms “halo” or “halogen” refers to bromine, chlorine, fluorine or iodine substituents.
The term “aryl” or “aryl ring” refers to common aromatic groups having “4n+2” π(pi) 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 may be directly attached, or connected via a C1-3alkyl group (also referred to as aralkyl). Examples of aryl include, without limitation, phenyl, benzyl, phenethyl, 1-phenylethyl, tolyl, naphthyl, biphenyl, terphenyl, and the like. Aryl groups may optionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, azido, alkylthio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.
The term “C5-naryl”, 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-naryl can be mono or polycyclic.
The term “heteroaryl” or “heteroaryl ring” refers to an aryl ring, as defined above, further containing one to four heteroatoms selected from oxygen, nitrogen, sulphur or phosphorus. Examples of heteroaryl include, without limitation, pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isooxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrollyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl groups. Heteroaryl may optionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, azido, alkylthio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl. Heteroaryl may be directly attached, or connected via a C1-3alkyl group (also referred to as heteroaralkyl). The foregoing heteroaryl groups, as derived from the compounds listed above, may be C-attached or N-attached where such is possible.
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-nheteroaryl 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, tryptophan, methionine, glycine, serine, threonine, cysteine, asparagine, glutamine, tyrosine, histidine, lysine, arginine, aspartic acid, glutamic acid, desmosine, ornithine, 2-aminobutyric acid, cyclohexylalanine, dimethylglycine, phenylglycine, norvaline, norleucine, hydroxylysine, allo-hydroxylysine, hydroxyproline, isodesmosine, allo-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 optical 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, including chiral chromatography (e.g. HPLC), immunoassay techniques, or the use of covalently (e.g. Mosher's esters) or non-covalently (e.g. chiral salts) bound chiral reagents to respectively form a diastereomeric ester or salt, which can be further separated by conventional methods, such as chromatography, distillation, crystallization or sublimation. The chiral ester or salt is then cleaved or exchanged by conventional means, to recover the desired isomer(s).
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 mixture by weight, 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 term “pharmaceutically acceptable salt” refers to nontoxic salts synthesized from a compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, methanol, ethanol, isopropanol, or acetonitrile are preferred. Another method for the preparation of salts is by the use of ion exchange resins. The term “pharmaceutically acceptable salt” includes both acid addition salts and base addition salts, either of the parent compound or of a prodrug or solvate thereof. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Exemplary acids used in acid addition salts include, without limitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, sulfonic, phosphoric, formic, acetic, citric, tartaric, succinic, oxalic, malic, glutamic, propionic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, β-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, such as those 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 (1977), Journal of Pharmaceutical Sciences, vol 66, no 1, pp 1-19, the content of which is incorporated herein by reference in its entirety.
The term “solvate” refers to a physical association of a compound of this invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Exemplary solvates include hydrates, ethanolates, methanolates, hemiethanolates, and the like.
The term “pharmaceutically acceptable prodrug” means any pharmaceutically acceptable ester, salt of an ester or any 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 a biologically active metabolite or residue thereof. Particularly favored salts or prodrugs are those with improved properties, such as solubility, efficacy, or 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. As used herein, a prodrug is a drug having one or more functional groups covalently bound to a carrier wherein metabolic or chemical release of the drug occurs in vivo when the drug is administered to a mammalian subject. Pharmaceutically acceptable prodrugs of the compounds of this invention include derivatives of hydroxyl groups such as, without limitation, acyloxymethyl, acyloxyethyl and acylthioethyl ethers, esters, amino acid esters, phosphate esters, sulfonate and sulfate esters, and their metal salts, and the like.
In one aspect, the invention relates to novel dibenzodiazepinone analogues, referred to herein as the compounds of the invention, and to pharmaceutically acceptable salts, solvates and prodrugs thereof.
The compounds of the invention, Compounds 2 to 5, 7, 13, 14, 15, 18, and 21 to 25, may be characterized by any one of their physicochemical and spectral properties, such as mass and NMR spectra, detailed in Example 4 through Example 8.
In another aspect, compounds of the invention are characterized by Formula I:
wherein,
R1 is a linear C1-10alkyl;
or a farnesyl derivative thereof, wherein said farnesyl derivative has one, two or three hydrogenated or hydroalkoxylated double bonds;
or a pharmaceutically acceptable salt or prodrug thereof.
In one embodiment, R1 is a linear C1-10alkyl, 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, amino and carboxy, or pharmaceutically acceptable salt thereof. In another embodiment, R1 is methyl. In another embodiment, R1 is ethyl. In another embodiment, R1 is n-propyl. In another embodiment, R1 is n-butyl. In another embodiment, R1 is n-pentyl. In another embodiment, R1 is n-hexyl. In another embodiment, R1 is methyl, and the farnesyl is fully hydrogenated (i.e. saturated). In another embodiment, R1 is methyl, and one double bond of the farnesyl is hydrogenated. In another embodiment, R1 is methyl, and two double bonds of the farnesyl are hydrogenated. In another embodiment, R1 is a linear C1-10alkyl, and one double bond of the farnesyl is hydrogenated. In another embodiment, R1 is a linear C1-10alkyl, and two double bonds of the farnesyl are hydrogenated. In another embodiment, R1 is a linear C1-10alkyl, and the farnesyl is fully hydrogenated (i.e. saturated). In another embodiment, R1 is a linear C1-10alkyl, and one double bond of the farnesyl is hydromethoxylated. In another embodiment, R1 is a linear C1-10alkyl, and two double bonds of the farnesyl are hydromethoxylated. In another embodiment, R1 is methyl, and one double bond of the farnesyl is hydroalkoxylated. In another embodiment, R1 is methyl, and two double bonds of the farnesyl are hydroalkoxylated. In another embodiment, R1 is ethyl, and one double bond of the farnesyl is hydroalkoxylated. In another embodiment, R1 is ethyl, and two double bonds of the farnesyl are hydroalkoxylated. The invention encompasses all esters or N-alkylated derivatives, and pharmaceutically acceptable salts, solvates 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:
or a pharmaceutically acceptable salt, solvate of prodrug of any one of Compounds 2 to 27.
The invention further provides esters and N-alkylated derivatives of any of the foregoing compounds. Certain embodiments expressly exclude one or more of the compounds of Formula I.
Prodrugs of the compounds of the invention include compounds wherein one or more of the 4, 6 and 8-hydroxy groups, or any other hydroxyl group on the molecule is bounded to any group that, when administered to a mammalian subject, is cleaved to form the free hydroxyl group. Examples of prodrugs include, but are not limited to, acetate, formate, hemisuccinate, benzoate, dimethylaminoacetate and phosphoryloxycarbonyl derivatives of hydroxy functional groups; dimethylglycine esters, aminoalkylbenzyl esters, aminoalkyl esters or carboxyalkyl esters of hydroxy functional groups. Carbamate and carbonate derivatives of the hydroxy groups are also included. Derivatizations of hydroxyl groups also encompassed, are (acyloxy)methyl and (acyloxy)ethyl ethers, wherein the acyl group contains an alkyl group optionally substituted with groups including, but not limited to, ether, amino and carboxylic acid functionalities, or where the acyl group is an amino acid ester. Also included are phosphate and phosphonate esters, sulfate esters, sulfonate esters, which are in alkylated (such as bis-pivaloyloxymethyl (POM) phosphate trimester or —P(O)O2Et2) or in the salt form (such as sodium phosphate ester (—P(O)O−2Na+2)). For further examples of prodrugs used in anticancer therapy and their metabolism, see Rooseboom et al (2004), Phamacol. Rev., vol 56, 53-102, incorporated herein by reference. When the prodrug contains an acidic or basic moiety, the prodrug may also be prepared as its pharmaceutically acceptable salt.
The compounds of this invention may be formulated into pharmaceutical compositions comprised of a compound of the invention, in combination with a pharmaceutically acceptable carrier, as discussed in Section IV below.
A. Fermentation
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.
Compound is produced by isolation of the fermentation broth of Micromonospora strains 046-ECO11 and [S01]046 as described in U.S. Ser. No. 10/762,107, incorporated herein by reference in its entirety. It is to be understood that the production of Compound 1 is not limited to the use of the particular strains 046-ECO11 and [S01]046. Rather, other ECO-4601 producing organisms may be used, such as mutants or variants of 046-ECO11 and [S01]046 that can be derived from this organism by known means such as X-ray irradiation, ultraviolet irradiation, treatment with nitrogen mustard, phage exposure, antibiotic resistance selection and the like; or through the use of recombinant genetic engineering techniques. For examples, see Manual of Industrial Microbiology and biotechnology, Demain and Solomon, American Society for Microbiology, Washington D.C., 1986; Hesketh et al. (1997), J. Antibiotics, vol 50, no 6, 532-535; and Hosoya et al. (1998), Antimicrobial Agents and Chemotherapy, vol 42, no 8, 2041-2047), the content of which are incorporated herein by reference in their entirety.
The farnesyl dibenzodiazepinone compound may be biosynthesized by various microorganisms. Microorganisms that may synthesize the farnesyl dibenzodiazepinone compound 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, Actinoplanes, 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. The content of which references is incorporated by reference in their entirety.
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.
The culture media inoculated with a farnesy dibenzodiazepinone-producing microorganism may be aerated by incubating the inoculated culture media with agitation, for example, shaking on a rotary shaker, a shaking water bath, or in a fermentor. 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 compound can be extracted and isolated from the cultivated culture media by techniques known to a person skilled in the art and/or disclosed herein, including for example centrifugation, chromatography, adsorption, filtration. For example, the cultivated culture media can be optionally acidified and mixed with a suitable organic solvent such as methanol, ethanol, n-butanol, ethyl acetate, n-butyl acetate or 4-methyl-2-pentanone. The organic layer can be separated from the mycelial cake for example, by centrifugation and decantation or filtration. The mycelial cake is further optionally extracted with an organic solvent, and the organic extracts combined. The organic layer is further optionally treated, for example by: aqueous washings, precipitation, filtration and the like, followed the removal of the solvent, for example, by evaporation to dryness under vacuum. The resulting residue can optionally be reconstituted with for example water, ethyl ether, ethanol, ethyl acetate, methanol or a mixture thereof, and re-extracted in a two-phase system with a suitable organic solvent such as hexane, carbon tetrachloride, methylene chloride or a mixture thereof. After removal of the solvent, the compound can be further purified by the use of standard techniques such as normal and reverse-phase liquid chromatography, crystallization, sublimation, adsorption, mass exclusion chromatography, and the like.
B. Chemical Modifications:
The farnesyl dibenzodiazepinone Compound 1 is biosynthesized by microorganisms and isolated as described herein, and in Canadian patent 2,466,340 (and PCT/CA04/000069). Compound 1 is subjected to random and/or directed chemical modifications to form compounds that are derivatives or structural analogues. Such derivatives or structural analogues having similar functional activities are within the scope of the present invention. The farnesyl dibenzodiazepinone may be modified by one or more chemical modification steps, using methods known in the art and described herein. Examples of chemical modifications procedures are also provided in Examples 4 to 8.
Dibenzodiazepinone analogues that are derivatives of Compound 1, for example those identified herein as the compounds of Formula I and their derivatives, and Compounds 2 to 27, 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,” 4th Edition by Jerry March (1992), Wiley-Interscience, USA, incorporated herein by reference in its entirety. 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, 3rd Edition, John Wiley & Sons, New York (1999), incorporated herein by reference in its entirety.
Alcohols and phenols are protected, if necessary 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, if necessary, 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 the invention. 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.
wherein, R″ is an acetate.
In Scheme 1, phenolic alcohols are converted to esters when reacted with activated carboxylic acids (R″C(O)X) such as an acid halide, anhydride, N-hydroxysuccinimide ester, or a carboxylic acid activated by a coupling agent (e.g.: EDC (1-(3-dimethylaminopropyl)-3-dïïsopropylethylcarbodiimide hydrochloride); or HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate)) with a base (e.g., pyridine or N,N-diisopropylethylamine (DIPEA)) and optional acatalysts such HOBt (1-hydroxybenzotriazole hydrate) and/or DMAP (4-(dimethylamino)pyridine). The same reactions may be accomplished on alcohols formed by farnesyl modification reactions (Scheme 3).
Scheme 1 is used to obtain, for example, Compounds 25 from Compound 1; and to produce esters of the Compounds of the invention.
wherein, R is selected an optionally substituted linear C1-10alkyl.
In Scheme 2, amine group in position 5 (for position, see Example 3) is alkylated. In Scheme 2(a), an amine is alkylated using an RX alkylating agent such as dialkyl sulfates and alkyl halides, preferably in the presence of a base (e.g., sodium bicarbonate, pyridine and the like).
Scheme 2 is used to prepare, for example, Compounds 2 to 13 from Compound 1, and Compound 14 from Compound 23; and to produce any of the Compounds of Formula I comprising an N-alkyl group.
wherein Rz is OC1-6alkyl.
In Scheme 3, double bond is modified by: (a) hydrogenation; and (b) hydroalkoxylation. In (a) hydrogenation is carried out using a hydrogen source (e.g. hydrogen, formic acid) and a catalyst (such as rhodium, platinum, or palladium). In (b), electrophilic addition to the double bond is achieved by the formation of a carbocation from addition of a proton in acidic conditions (e.g., p-toluene sulfonic acid, alkyl sulfate/NaHCO3/MeOH, and the like), and trapping of the carbocation with an alcohol (C1-6alkyl alcohol, hydroalkoxylation).
Scheme 3 is used to obtain, for example: in (a) Compound 23 from Compound 1, and Compounds 14 to 16 from Compound 2; in (b) Compounds 17 to 19 from Compound 2, Compounds 20 to 22 from Compound 3. Schemes 3 (a) and (b) are also used to produce any hydrogenated and hydroalkoxylated farnesyl derivative of the Compounds of Formula I.
wherein, X is Br.
In Scheme 4, the aryl group is modified by aromatic substitutions (bromination). brominating agents include bromine, N-haloamides (e.g, N-bromosuccinimide (NBS), tetraalkylammonium polyhalides).
Scheme 4 is used to prepare Compound 24 from Compound 1, Compound 26 from Compound 2 and Compound 27 from Compound 14.
Prodrugs are prepared by routine chemical modifications such as described in Jerry March, supra, including esterification (Scheme 1) and alkylation reactions, i.e., use of activated acids or mixed anhydrides (acyl halides, use of coupling reagents, etc), and by the use of alkylating agents (R—X, wherein X is a leaving group, such as diazo, and R is the desired group). Phosphate prodrugs are prepared by phosphorylation, for example, with dialkyl phosphites using procedure such as described in Silverberg et al. (1996), Tet. Lett., Vol. 37, 711-774, U.S. Pat. No. 5,561,122 (Pettit et a) and in Hwang and Cole (2004), Org. Lett., vol 6, no 10, 1555-1556 ((POM)2phosphate triester from (POM)2phosphoryl chloride), the content of which is incorporated herein by reference in their entirety.
The invention provides a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof, in combination with a pharmaceutically acceptable carrier. The pharmaceutical composition comprising a dibenzodiazepinone analogue is useful for treating diseases and disorders associated with uncontrolled cellular growth and proliferation, such as a neoplastic condition. The pharmaceutical composition is also useful in treating other diseases and disorders, including inflammation, autoimmune diseases, infections, neurodegenerative diseases and stress. The pharmaceutical composition comprising a dibenzodiazepinone analogue may be packaged into a convenient commercial package providing the necessary materials, such as the pharmaceutical composition and written instructions for its use in treating a neoplastic condition, in a suitable container.
The compounds of the present invention, or pharmaceutically acceptable salts, solvates or prodrugs thereof, can be formulated for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, topical or parenteral administration for the therapeutic or prophylactic treatment of neoplastic and proliferative diseases and disorders. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracerebral or intracranial, intraspinal, intracisternal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of drug formulations can be used to effect such administration. For oral and/or parental administration, compounds of the present invention can be mixed with conventional pharmaceutical carriers and excipients and used in the form of solutions, emulsions, tablets, capsules, soft gels, elixirs, suspensions, syrups, wafers and the like. The compositions comprising a compound of the 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. (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.; and Goodman and Gilman, Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, N.Y., the contents of which are incorporated herein by reference, for a general description of the methods for administering various agents for human therapy).
As used herein, the term “unit dosage” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of dibenzodiazepinone analogue calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutically acceptable carriers. In one embodiment, the unit dosage contains from 10 to 3000 mg of active ingredient. In another embodiment, the unit dosage contains 20 to 1000 mg of active ingredient. 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 are described in U.S. Pat. No. 4,452,775 (issued to Kent), U.S. Pat. No. 5,039,660 (issued to Leonard), and U.S. Pat. No. 3,854,480 (issued to Zaffaroni), incorporated herein by reference in their entirety.
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. Pharmaceutically acceptable carriers include, for example, solvents, vehicles or medium such as saline, buffered saline, dextrose, water, glycerol, ethanol, propylene glycol, polysorbate 80 (Tween-80™), poly(ethylene) glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (E.g. Cremophor EL), poloxamer 407 and 188, hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phopholids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes. The term specifically excludes cell culture medium.
Excipients or additives included in a formulation have different purposes depending, for example on the nature of the drug, and the mode of administration. Examples of generally used excipients include, without limitation: stabilizing agents, solubilizing agents and surfactants, buffers, antioxidants and preservatives, tonicity agents, bulking agents, lubricating agents, emulsifiers, suspending or viscosity agents, inert diluents, fillers, disintegrating agents, binding agents, wetting agents, lubricating agents, antibacterials, chelating agents, sweetners, perfuming agents, flavouring agents, coloring agents, administration aids, and combinations thereof.
The compositions may contain common carriers and excipients, such as cornstarch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. The compositions may contain crosarmellose sodium, microcrystalline cellulose, sodium starch glycolate and alginic acid.
Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions, suspensions or fat emulsions, comprising a compound of this invention, or a pharmaceutically acceptable salt or prodrug thereof. The parenteral form used for injection must be fluid to the extent that easy syringability exists. These solutions or suspensions can be prepared from sterile concentrated liquids, powders or granules. The compounds can be dissolved in a carrier such as a solvent or vehicle, for example, polyethylene glycol, propylene glycol, ethanol, corn oil, benzyl alcohol, glycofurol, N,N-dimethylacetamide, N-methylpyrrolidone, glycerine, saline, dextrose, water, glycerol, hydrophobic carriers, and combinations thereof.
Excipients used in parenteral preparations also include, without limitation, stabilizing agents (e.g. carbohydrates, amino acids and polysorbates), solubilizing agents (e.g. cetrimide, sodium docusate, glyceryl monooleate, polyvinylpyrolidone (PVP) and polyethylene glycol (PEG)) and surfactants (e.g. polysorbates, tocopherol PEG succinate, poloxamer and Cremophor™), buffers (e.g. acetates, citrates, phosphates, tartrates, lactates, succinates, amino acids and the like), antioxidants and preservatives (e.g. BHA, BHT, gentisic acids, vitamin E, ascorbic acid and sulfur containing agents such as sulfites, bisulfites, metabisulfites, thioglycerols, thioglycolates and the like), tonicity agents (for adjusting physiological compatibility), suspending or viscosity agents, antibacterials (e.g. thimersol, benzethonium chloride, benzalkonium chloride, phenol, cresol and chlorobutanol), chelating agents, and administration aids (e.g. local anesthetics, anti-inflammatory agents, anti-clotting agents, vaso-constrictors for prolongation and agents that increase tissue permeability), and combinations thereof.
Parenteral formulations using hydrophobic carriers include, for example, fat emulsions and formulations containing lipids, lipospheres, vesicles, particles and liposomes. Fat emulsions include in addition to the above-mentioned excipients, a lipid and an aqueous phase, and additives such as emulsifiers (e.g. phospholipids, poloxamers, polysorbates, and polyoxyethylene castor oil), and osmotic agents (e.g. sodium chloride, glycerol, sorbitol, xylitol and glucose). Liposomes include natural or derived phospholipids and optionally stabilizing agents such as cholesterol.
In another embodiment, the parenteral unit dosage form of the compound can be a ready-to-use solution of the compound in a suitable carrier in sterile, hermetically sealed ampoules or in sterile pre-loaded syringes. The suitable carrier optionally comprises any of the above-mentioned excipients.
Alternatively, the unit dosage of the compound of the present invention can be in a concentrated liquid, powder or granular form for ex tempore reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery. In addition the above-mentioned excipients, powder forms optionally include bulking agents (e.g. mannitol, glycine, lactose, sucrose, trehalose, dextran, hydroxyethyl starch, ficoll and gelatin), and cryo or lyoprotectants.
For example, in intravenous (IV) use, a sterile formulation of the compound of formula I and optionally one or more additives, including solubilizers or surfactants, can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Intravenous fluids include, without limitation, physiological saline, phosphate buffered saline, 5% glucose or Ringer's™ solution.
In another example, in intramuscular preparations, a sterile formulation of the compound of the present invention or suitable soluble salts or prodrugs 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 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, tablets, capsules, suspensions or liquid syrups or elixirs, wafers and the like. For general oral administration, excipient or additives include, but are not limited to inert diluents, fillers, disintegrating agents, binding agents, wetting agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.
The oral pharmaceutical composition is preferably made in the form of a unit dosage 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: inert diluents (e.g., sodium and calcium carbonate, sodium and calcium phosphate, and lactose), binding agents (e.g., acacia gum, starch, gelatin, sucrose, polyvinylpyrrolidone (Providone), sorbitol, or tragacanth methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and ethylcellulose), fillers (e.g., calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose), lubricants or lubricating agents (e.g., magnesium stearate or other metallic stearates, stearic acid, polyethylene glycol, waxes, oils, silica and colloical silica, silicon fluid or talc), disintegrants or disintegrating agents (e.g., potato starch, corn starch and alginic acid), flavouring, coloring agents, or acceptable wetting agents. Carriers may also include coating excipients such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
Oral liquid preparations, generally in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs, 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 both liquid and solid oral preparations, flavoring agents such as peppermint, oil of wintergreen, cherry, grape, fruit flavoring 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. 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.
In one aspect, the invention relates to a method for inhibiting growth and/or proliferation of cancer cells in a mammal. In another aspect, the invention provides a method for treating neoplasms in a mammal. Mammals include ungulates (e.g. sheeps, goats, cows, horses, pigs), and non-ungulates, including rodents, felines, canines and primates (i.e. human and non-human primates). In a preferred embodiment, the mammal is a human.
The dibenzodiazepinone analogues of the present invention may bind to or interact with other cancer-associated proteins and polypeptides, including, without limitation, polypeptides encoded by oncogenes, polypeptides that induce angiogenesis, proteins involved in metastasizing and/or invasive processes, and proteases that regulate apoptosis and the cell cycle. Regardless of the mechanism of action, the dibenzodiazepinone analogues of the invention have been demonstrated to exhibit anti-cancer activity both in vitro and in vivo. Based on these discoveries, applicants have developed methods for treating neoplasms.
As used herein, the terms “neoplasm”, “neoplastic disorder”, “neoplasia” “cancer,” “tumor” and “proliferative disorder” refer to cells having the capacity for autonomous growth, i.e., an abnormal state of condition characterized by rapidly proliferating cell growth which generally forms a distinct mass that show partial or total lack of structural organization and functional coordination with normal tissue. The terms are meant to encompass hematopoietic neoplasms (e.g. lymphomas or leukemias) as well as solid neoplasms (e.g. sarcomas or carcinomas), including all types of pre-cancerous and cancerous growths, or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Hematopoietic neoplasms are malignant tumors affecting hematopoietic structures (structures pertaining to the formation of blood cells) and components of the immune system, including leukemias (related to leukocytes (white blood cells) and their precursors in the blood and bone marrow) arising from myeloid, lymphoid or erythroid lineages, and lymphomas (relates to lymphocytes). Solid neoplasms include sarcomas, which are malignant neoplasms that originate from connective tissues such as muscle, cartilage, blood vessels, fibrous tissue, fat or bone. Solid neoplasms also include carcinomas, which are malignant neoplasms arising from epithelial structures (including external epithelia (e.g., skin and linings of the gastrointestinal tract, lungs, and cervix), and internal epithelia that line various glands (e.g., breast, pancreas, thyroid). Examples of neoplasms that are particularly susceptible to treatment by the methods of the invention include leukemia, and hepatocellular cancers, sarcoma, vascular endothelial cancers, breast carcers, central nervous system cancers (e.g. astrocytoma, gliosarcoma, neuroblastoma, oligodendroglioma and glioblastoma), prostate cancers, lung and bronchus cancers, larynx cancers, esophagus cancers, colon cancers, colorectal cancers, gastro-intestinal cancers, melanomas, ovarian and endometrial cancer, renal and bladder cancer, liver cancer, endocrine cancer (e.g. thyroid), and pancreatic cancer.
The dibenzodiazepinone analogue is brought into contact with or introduced into a cancerous cell or tissue. In general, the methods of the invention for delivering the compositions of the invention in vivo utilize art-recognized protocols for delivering therapeutic agents with the only substantial procedural modification being the substitution of the dibenzodiazepinone analogue of the present invention for the therapeutic agent in the art-recognized protocols. The route by which the dibenzodiazepinone analogue is administered, as well as the formulation, carrier or vehicle will depend on the location as well as the type of the neoplasm. A wide variety of administration routes can be employed. The dibenzodiazepinone analogue may be administered by intravenous or intraperitoneal infusion or injection. For example, for a solid neoplasm that is accessible, the compound of the invention may be administered by injection directly into the neoplasm. For a hematopoietic neoplasm the compound may be administered intravenously or intravascularly. For neoplasms that are not easily accessible within the body, such as metastases or brain tumors, the compound may be administered in a manner such that it can be transported systemically through the body of the mammal and thereby reach the neoplasm and distant metastases for example intrathecally, intravenously or intramuscularly or orally. Alternatively, the compound can be administered directly to the tumor. The compound can also be administered subcutaneously, intraperitoneally, topically (for example for melanoma), rectally (for example colorectal neoplasm) vaginally (for example for cervical or vaginal neoplasm), nasally or by inhalation spray (for example for lung neoplasm).
The dibenzodiazepinone analogue is administered in an amount that is sufficient to inhibit the growth or proliferation of a neoplastic cell, or to treat a neoplastic disorder. The term “inhibition” refers to suppression, killing, stasis, or destruction of cancer cells. The inhibition of mammalian cancer cell growth according to this method can be monitored in several ways. Cancer 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 50% or more at 100 micromolar, is indicative of cancer cell inhibition (see Anticancer Drug Development Guide: preclinical screening, clinical trials and approval; B. A. Teicher and P. A. Andrews, ed., 2004, Humana Press, Totowa, N.J.). Alternatively, cancer cell inhibition can be monitored by administering the compound to an animal model of the cancer of interest. Examples of experimental non-human animal cancer models are known in the art and described below and 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 by least a 58%) in animals treated with the compound relative to tumors in control animals not treated with the compound is indicative of significant tumor growth inhibition (see Anticancer Drug Development Guide: preclinical screening, clinical trials and approval; B. A. Teicher and P. A. Andrews, ed., 2004, Humana Press, Totowa, N.J.).
The term “treatment” refers to the application or administration of a dibenzodiazepinone analogue to a mammal, or application or administration of a dibenzodiazepinone analogue to an isolated tissue or cell line from a mammal, who has a neoplastic disorder, a symptom of a neoplastic disorder or a predisposition toward a neoplastic disorder, with the purpose to cure, heal, alleviate, relieve, alter, ameliorate, improve, or control the disorder, the symptoms of disorder, or the predisposition toward disorder. The term “treating” is defined as administering, to a mammal, an amount of a dibenzodiazepinone analogue sufficient to result in the prevention, reduction or elimination of neoplastic cells in a mammal (“therapeutically effective amount”). The therapeutically effective amount and timing of dosage will be determined on an individual basis and may be based, at least in part, on consideration of the age, body weight, sex, diet and general health of the recipient subject, on the nature and severity of the disease condition, and on previous treatments and other diseases present. Other factors also include the route and frequency of administration, the activity of the administered compound, the metabolic stability, length of action and excretion of the compound, drug combination, the tolerance of the recipient subject to the compound and the type of neoplasm or proliferative disorder. In one embodiment, a therapeutically effective amount of the compound is in the range of about 0.01 to about 750 mg/kg of body weight of the mammal. In another embodiment, the therapeutically effective amount is in the range of about 0.01 to about 300 mg/kg body weight per day. In yet another embodiment, the therapeutically effective amount is in the range of 10 to about 50 mg/kg body weight per day. The therapeutically effective doses of the above embodiments may also be expressed in milligrams per square meter (mg/m2) in the case of a human patient. Conversion factors for different mammalian species may be found in:Freireich et al, Quantitative comparison of toxicity of anticancer agents in mouse, rat, dog, monkey and man, Cancer Chemoth. Report, 1966, 50(4): 219-244, incorporated herein by reference in its entirety. When special requirements may be needed (e.g. for children patients), the therapeutically effective doses described above may be outside the ranges stated herein. Such higher or lower doses are within the scope of the present invention.
To monitor the efficacy of tumor treatment in a human, tumor size and/or tumor morphology is measured before and after initiation of the treatment, and treatment is considered effective if either the tumor size ceases further growth, or if the tumor is reduced in size, 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). Prolongation of survival, time-to-disease progression, partial response and objective response rate are surrogate measures of clinical activity of the investigational agent. Tumor shrinkage is considered to be one treatment-specific response. This system is limited by the requirement that patients have visceral masses that are amenable to accurate measurement. Methods of determining the size 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 certain types of cancer, evaluation of serum tumor markers are also used to evaluate response (eg prostate-specific antigen (PSA) for prostate cancer, and carcino-embryonic antigen (CEA), for colon cancer). Other methods of monitoring cancer growth include cell counts (e.g. in leukemias) in blood or relief in bone pain (e.g. prostate cancer).
The dibenzodiazepinone compound may be administered once daily, or the compound may be administered as two, three, four, or more sub-doses at appropriate intervals throughout the day. In that case, the dibenzodiazepinone compound contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dibenzodiazepinone compound over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. The effective dose can be administered either as a single administration event (e.g., a bolus injection) or as a slow injection or infusion, e.g. over 30 minutes to about 24 hours. The compound may be administered as a treatment, for up to 30 days. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments (e.g., a four-week treatment repeated 3 times, with a 2 months interval between each treatment). Estimates of effective dosages, toxicities and in vivo half-lives for the dibenzodiazepinone compounds encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model.
The dibenzodiazepinone compound may be administered in conjunction with or in addition to known other anticancer treatments such as radiotherapy, or other known anticancer compounds or chemotherapeutic agents. Such agents include, but are not limited to, 5-fluorouracil, mitomycin C, methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone, anthracyclines (Epirubicin and Doxurubicin), etopside, pregnasome, platinum compounds such as carboplatin and cisplatin, taxanes such as Paclitaxel™ and Docetaxel™; hormone therapies such as tamoxifen and anti-estrogens; antibodies to receptors, such as herceptin and Iressa; aromatase inhibitors, progestational agents and LHRH analogues; biological response modifiers such as IL2 and interferons; multidrug reversing agents such as the cyclosporin analogue PSC 833.
Toxicity and therapeutic efficacy of dibenzodiazepinone compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Therapeutic efficacy is determined in animal models as described above and in the examples herein. Toxicity studies are done to determine the lethal dose for 10% of tested animals (LD10). Animals are treated at the maximum tolerated dose (MTD): the highest dose not producing mortality or greater than 20% body weight loss. The effective dose (ED) is related to the MTD in a given tumor model to determine the therapeutic index of the compound. A therapeutic index (MTD/ED) close to 1.0 has been found to be acceptable for some chemotherapeutic drugs, a preferred therapeutic index for classical chemotherapeutic drugs is 1.25 or higher.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions of the invention will generally be within a range of circulating concentrations that include the MTD. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC.
Animal models to determine antitumor efficacy of a compound are generally carried out in mice. Either murine tumor cells are inoculated subcutaneously into the hind flank of mice from the same species (syngeneic models) or human tumor cells are inoculated subcutaneously into the hind flank of severe combined immune deficient (SCID) mice or other immune deficient mice (nude mice) (xenograft models).
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases including cancer. The MMHCC (Mouse models of Human Cancer Consortium) web page (emice.nci.nih.gov), sponsored by the National Cancer Institute, provides disease-site-specific compendium of known cancer models, and has links to the searchable Cancer Models Database (cancermodels.nci.nih.gov), as well as the NCI-MMHCC mouse repository. Mouse repositories can also be found at: The Jackson Laboratory, Charles River Laboratories, Taconic, Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Network and at the European Mouse Mutant Archive. Such models may be used for in vivo testing of dibenzodiazepinone compounds, as well as for determining a therapeutically effective dose.
In addition to the compounds of the invention, pharmaceutically acceptable salts, solvates or prodrugs of said compounds may also be employed in compositions to treat or prevent the above-identified disorders.
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 500™ Spectrometer (1H NMR at 500 MHz, 13C NMR at 125 MHz). UV and mass spectra were collected by Waters 2690™ HPLC using a photodiode array detector (PDA, 210-400 nm) coupled to a Waters Micromass™ ZQ™ mass detector.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, molar equivalents (eq), percentage of binding and/or inhibition, GI50, IC50 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 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 analyses and such.
In the following section, examples describe in detail the chemical synthesis of representative compounds of the present invention. The procedures are illustrations, and the invention should not be construed as being limited by chemical reactions and conditions they express. No attempt has been made to optimize the yields obtained in these reactions, and it would be obvious to one skilled in the art that variations in reaction times, temperature, solvent and/or reagents could increase the yields.
In addition, the materials, methods, and examples, including in vitro and in vivo efficacy, bioavailability, toxicity and pharmacological properties are illustrative only and not intended to be limiting. 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. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Micromonospora sp. (deposit accession number IDAC 070303-01) was maintained on agar plates of ISP2 agar (Difco Laboratories, Detroit, Mich.). An inoculum for the production phase was prepared by transferring the surface growth of the Micromonospora sp. from the agar plates to 125-mL flasks containing 25 mL of sterile medium comprised of glucose 10 g, potato dextrin type IV (Sigma) 20 g, yeast extract 5 g, N Z Amine-A 5 g, 1 g CaCO3 made up to one liter with tap water (pH 7.0). The culture was incubated at about 28° C. for approximately 70-72 hours on a rotary shaker set at 250 rpm. Following incubation, 10 mL of culture was transferred to a 2 L baffled flask containing 600 mL of sterile production medium containing 20 g/L potato dextrin type IV (sigma), 30 g/L glycerol, 2.5 g/L Bacto-peptone, 8.34 g/L yeast extract, 3 g/L CaCO3, pH 7.0. Fermentation broth was prepared by incubating the production culture at 28° C. in a rotary shaker set at 250 rpm for 5 days.
The fermentation was accomplished as a 1×10 L batch in a 14.5 L fermentor (BioFlo 110™ Fermentor, New Brunswick Scientific, Edison, N.J., USA) using an improved procedure described in CA patent application 2,466,340, filed Jan. 21, 2004.
Micromonospora sp. (deposit accession number IDAC 070303-01) was maintained on agar plates of ISP2 agar (Difco Laboratories, Detroit, Mich.). 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 CaCO3 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 (Chem Service), 0.05 ml Proflo Oil™ (Traders protein) and 3 g CaCO3 made to one liter with distilled water and adjusted to pH 7.0. The culture was incubated at 28° C., with dissolved oxygen (dO2) controlled at 25% in a cascade loop with agitation varied between 320-600 RPM and aeration set at a fixed rate of 0.5 v/v/m.
In addition to the above medium, other preferred media for the production of Compound 1 by fermentation are provided in Table 1 (QB, MA, KH, RM, JA, FA, CL). Any one of Micromonospora sp. 046-ECO 11 or [S01]046 may be used in these exemplified methods.
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 MgSO4 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 Büchner funnel and the filtrate was discarded. The adsorbed HP-20 resin was then washed successively with 2×125 mL of 50% acetonitrile in water, 2×125 mL of 75% acetonitrile in water and 2×125 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 Chromatography (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 60F254 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 (10 μm), 19×150 mm, Waters Co., Milford, Mass.), with a 9 mL/min flow rate and UV peak detection at 300 nm. The column was eluted with acetonitrile/buffer (5 mM of NH4HCO3) according to the following gradient shown in Table 2.
Fractions containing Compound 1 were combined, concentrated and lyophilized to give a yield of 3.8 mg compound.
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/H2O 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. HP-20 resin was washed successively with 2×500 mL of 50% MeOH/H2O, 2×500 mL of 75% MeOH/H2O and 2×500 mL 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 μm), 19×150 mm, Waters Co., Milford, Mass.) with a flow rate of 9 mL/min and peak detection at 300 nm.
The column was eluted with acetonitrile/buffer (5 mM of NH4HCO3) according to gradient shown in Table 3.
Fractions containing Compound 1 were combined, concentrated and lyophilized to yield about 33.7 mg of compound.
10 liters of the whole broth from Example 1 was extracted twice with equal volumes of ethyl acetate and the two extracts were combined and concentrated to dryness. The dried extract was weighed, and for every gram of dry extract, 100 mL of MeOH—H2O (2:1 v/v) and 100 mL of hexane was added. The mixture was swirled gently but well to achieve dissolution. The two layers were separated and the aqueous layer is washed with 100 mL of hexane. The two hexane layers were combined and the combined hexane solution was washed with 100 mL methanol:water (2:1, v/v). The two methanol:water layers were combined and treated with 200 mL of EtOAc and 400 mL of water. The layers were separated and the aqueous layer extracted twice further with 200 mL portions of EtOAc. The EtOAc layers are combined and concentrated. The residue obtained (220 mg) was suitable for final purification, either by HSCC or by HPLC as described above. This extraction process achieved a ten-fold purification when compared with the extraction protocol used in (a) or (b).
The calculated molecular weight of the major isotope (462.25) and formula (C28H34N2O4) of Compound 1 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 461.2 and positive ionization gave an (M+H)+ molecular ion of 463.3. UVmax was determined to be 230 nm with a shoulder at 290 nm.
Proton and carbon NMR spectral analysis is shown in Table 4. NMR data were collected dissolved in MeOH-d4 including proton, carbon and multidimensional pulse sequences gDQCOSY, gHSQC, gHMBC, and NOESY. A number of cross peaks in the 2D spectra of Compound 1 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. Assignment of proton and carbon signals are shown in Table 4.
1H and 13C NMR (δH, ppm) Data of Compound 1 in MeOH-D4
1H
13C
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.
Compound 2, namely 10-farnesyl-4,6,8-trihydroxy-5-methyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, and
Compound 18, namely 10-(7-methoxy-3,7,11-trimethyldodeca-2,10-dienyl)-4,6,8-trihydroxy-5-methyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, were prepared and identified as follows:
Compound 1 (500.0 mg) was dissolved in methanol (MeOH, 20 mL) and stirred with dimethyl sulfate (0.5 mL) and NaHCO3 (250 mg) at room temperature for 48 hrs. The reaction mixture was diluted to 200 mL by adding water and extracted with ethyl acetate (EtOAc, 300 mL×3). The organic layer was separated and dried under vacuum, re-dissolved in MeOH and filtered through a 0.45 μm 13 mm Acrodisc™ GHP syringe filter. The filtrate was subjected for isolation on a Waters HPLC coupled to a photodiode array detector. Compound 18 (12.1 mg) and Compound 2 (308.5 mg) were isolated by the multiple injections on Nova-Pack™ HR C18 6 μm 25×200 mm column (20 mL/min, H2O/CH3CN gradient 80:20-30:70, 0-8 min; 30:70-0:100, 8-18 min), eluting at 14.5 and 16.8 min, respectively.
The calculated molecular weight for the major isotope (476.27) and formula (C29H36N2O4) of Compound 2 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 475.6 and positive ionization gave an (M+Na)+ molecular ion of 499.4. Proton and carbon NMR spectral analysis is shown in Table 5. Signals were easily assigned based on Compound 1 structure knowledge. The calculated molecular weight for the major isotope (508.29) and formula (C30H40N2O5) of Compound 18 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 507.3 and positive ionization gave an (M+H)+ molecular ion of 509.3. The characteristic N-methyl (signal 5), methoxy (signal 7′-OMe) and the methylene group (6′), from the addition of methanol on the farnesyl chain were easily assigned as shown in Table 5.
1H
13C
1H
aCH in Compound 2, CH2 in Compound 18
bSignals for 4′, 5′, 8′ and 9′ are very close; assignment was based on Compound 1
Compound 3:1 0-farnesyl-4,6,8-trihydroxy-5-ethyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one;
Compound 21: 10-(11-methoxy-3,7,11-trimethyl-2,6-dodecadienyl)-4,6,8-trihydroxy-5-ethyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one;
Compound 22:10-(7,11-dimethoxy-3,7,11-trimethyl-2-dodecenyl)-4,6,8-trihydroxy-5-ethyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one;
Compound 1 (85.3 mg) was stirred for 72 hrs at room temperature in a mixture of diethyl sulfate (2.0 mL) and NaHCO3 (99.9 mg) in MeOH (2 mL). The resulting mixture was filtered through a 0.45 μm 13 mm Acrodisc™ GHP syringe filter. The solution was purified by preparative HPLC (multiple injections on a NovaPack™ HR C-18 25×200 mm column (20 mL/min, H2O/CH3CN gradient 80:20-30:70, 0-8 min; 30:70-0:100, 8-18 min) to give four major peaks: Compound 3 (20.0 mg with some impurities, RT: 16.6 min), Compound 21 (17.54 mg, RT: 14.3 min) and Compound 22 (7.82 mg, RT: 12.6 min) were obtained. Fractions containing non-ethylated analogs of Compounds 21 and 22 were also isolated respectively in 5.65 mg (RT: 11.6 min) and 2.20 mg (RT: 10.3 min) quantities. The fraction containing Compound 3 was further purified by HPLC using the same column (20 mL/min, H2O/CH3CN gradient 80:20-30:70, 0-8 min; 30:70-0:100, 8-18 min, curve 7), to give substantially pure Compound 3 (13.85 mg, RT: 17.9 min).
The calculated molecular weight of the major isotope (490.28) and formula (C30H38N2O4) of Compound 3 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 489.3 and positive ionization gave an (M+H)+ molecular ion of 491.3. Proton NMR signals were easily assigned based on Compounds 1 and 2 structures knowledge. The characteristic N-ethyl group (5-N-Et) was easily assigned as shown in Table 6.
The calculated molecular weight of the major isotope (522.31) and formula (C31H42N2O5) of Compound 21 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 521.3 and positive ionization gave an (M+H)+ h molecular ion of 523.5, and a fragment having an (M+H—HOCH3)+ molecular ion of 491.3. The characteristic N-ethyl group (5-N-Et), and the methoxy (11′-OMe) and methylene (10′) groups from the addition of methanol on the farnesyl chain were easily assigned as shown in Table 6.
The calculated molecular weight of the major isotope (554.34) and formula (C32H46N2O6) of Compound 22 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 553.4 and positive ionization gave an (M+H)+ molecular ion of 555.4, and fragments having respectively (M+H—HOCH3)+ and (M+H—HOCH3—HOCH3)+ molecular ion of 523.5 and 491.3. The characteristic N-ethyl group (5-N-Et), and methoxy (7′-OMe and 11′-OMe) groups from the addition of two molecules of methanol on the farnesyl chain were easily assigned as shown in Table 6. The methylene groups (5′, 6′, 8′, 9′ and 10′) were all found to have similar chemical shifts, which is consistent with the saturated alkyl group.
1H NMR (δH, ppm) Data of Compounds 3, 21 and 22 in MeOH-D4
aCH in Compounds 3 and 21, CH2 in Compound 22
bCH in Compound 3, CH2 in Compounds 21 and 22
Compound 4, namely 10-farnesyl-4,6,8-trihydroxy-5-n-propyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, was prepared and identified as follows:
Compound 1 (46.7 mg) was stirred for 72 hrs at room temperature in a mixture of dipropyl sulfate (0.5 mL) and NaHCO3 (46.3 mg) in MeOH (3 mL). The resulting mixture was filtered through a 0.45 μm 13 mm Acrodisc™ GHP syringe filter. The solution was purified by preparative HPLC (multiple injections on a NovaPack™ HR C-18 25×200 mm column: 20 mL/min, H2O/CH3CN gradient 80:20-30:70, 0-8 min; 30:70-0:100, 8-18 min) to give substantially pure Compound 4 (18.0 mg, RT: 17.3 min).
The calculated molecular weight of the major isotope (504.30) and formula (C31H40N2O4) of Compound 4 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 503.4 and positive ionization gave an (M+H)+ molecular ion of 505.5. Proton NMR signals were easily assigned based on Compounds 1 and 2 structures knowledge. The characteristic N-Propyl group (5-N-Pr (C1 to C3)) was easily assigned as shown in Table 7 below.
Compound 13: 10-Farnesyl-4,6,8-trihydroxy-5-(trideuteriomethyl)-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, was prepared and identified according to the following procedure:
Compound 1 (121.3 mg) was dissolved in MeOH (3.0 mL), dimethyl sulfate-d6 (150 μL, CDN isotopes Inc.) and NaHCO3 (58.1 mg) were added, and the reaction was stirred at room temperature overnight. The reaction mixture was filtered and the filtrate was subjected to Waters HPLC purification (multiple injections on Nova Pack™ HR C-18 25×200 mm column: 20 mL/min, H2O/CH3CN gradient 70:30-20:80, 0-4 min; 20:80-0:100, 4-9 min, 100% CH3CN, 9-12 min) to give Compound 13 (82.7 mg, RT 9.4 min).
The calculated molecular weight of the major isotope (479.29) and formula (C29H33D3N2O4) of Compound 13 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 478.5, and positive ionization gave an (M+H)+ molecular ion of 480.6. The structure was further confirmed by proton NMR spectrum as shown in Table 7 below.
1H NMR (δH, ppm) Data of Compounds 4 and 13 in MeOH-D4
aCH2 in Compound 4, CD3 in Compound 13.
Compound 5, namely 10-farnesyl-4,6,8-trihydroxy-5-n-butyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, was prepared and identified as follows:
Compound 1 (43.5 mg) was stirred in 1-bromobutane (2.0 mL) with pyridine (50 μL) at 80° C. overnight. The reaction mixture was diluted with MeOH (1.0 mL), filtered and subjected for Waters HPLC as described above (in Example 4(c)) to give a semi-purified Compound 5 (RT: 18.1 min). The semi-purified compound was further purified using the same conditions (except with curve 7) to give substantially pure Compound 5 (10.5 mg, RT: 17.9 min).
The calculated molecular weight of the major isotope (518.31) and formula (C32H42N2O4) of Compound 5 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 517.4 and positive ionization gave an (M+H)+ molecular ion of 519.5. The characteristic N-n-butyl group (5-N-alkyl (C1-C4)) was easily assigned as shown in Table 8 below.
Compound 7, namely 10-farnesyl-4,6,8-trihydroxy-5-n-hexyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, was prepared and identified as follows:
Compound 1 (39.2 mg) was stirred in 1-bromohexane (2.0 mL) with pyridine (50 μL) at 80° C. overnight. The reaction mixture was diluted with MeOH (1.0 mL), filtered and subjected for Waters HPLC (multiple injections on a NovaPack™ HR C-18 25×200 mm column: 20 mL/min, H2O/CH3CN gradient 80:20-30:70, 0-8 min; 30:70-0:100, 8-18 min; isocratic CH3CN 18-24 minutes) to give substantially pure Compound 7 (14.0 mg, RT: 20.1 min).
The calculated molecular weight (546.35) and formula (C34H46N2O4) of Compound 7 was confirmed by mass spectral analysis: negative ionization gave an (M−H)— molecular ion of 545.6 and positive ionization gave an (M+H)+ molecular ion of 547.6. Proton NMR signals were easily assigned based on Compounds 1 and 2 structures knowledge. The characteristic N-n-hexyl group (5-N-alkyl (C1-C6)) was easily assigned as shown in Table 8 below.
Compounds 2 to 4, 6, and 8 to 12 are produced by the procedure described in a) and b), by substituting the alkylating agent used in these procedures respectively by the following alkylating agents: iodomethane, bromoethane, 1-bromopropane, 1-bromopentane, 1-bromoheptane, 1-chlorooctane, trifluoromethyl iodide, heptafluoro-1-iodopropane, and 2-iodo-1,1,1-trifluoroethane.
1H NMR (δH, ppm) Data of Compounds 5 and 7 in MeOH-D4
b
aSignals at 4′, 5′, 8′ and 9′ are very close; assignment was based on Compound 1.
bCH3 in Compound 5, CH2 in Compound 7.
Compound 25: 4,6,8-triacetoxy-10-farnesyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, was prepared and identified according to the following procedure:
Compound 1 (120.5 mg) was stirred overnight with acetic anhydride (720 μL, 29 eq, 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 HR™ C18, 6 μm, 60A 25×200 mm column (20 mL/min H2O/CH3CN 80:30-70:75, 0-8 min; 30:70-0:100, 8-18 min, 100% CH3CN, 18-20 min) gave Compound 25 (91.2 mg) with a retention time of 18.5 min. The 4,8-diacetoxy (11.4 mg), 4,6-diacetoxy (9.2 mg), and 6,8-diacetoxy (11.4 mg) compounds were also isolated at retention times of 16.2, 17.6, and 18.0 respectively.
The calculated molecular weight of the major isotope (588.28) and formula (C34H40N2O7) of Compound 25 were confirmed by mass spectral (MS) analysis. MS of Compound 25 gave a (M−H)− molecular ion of 587.6 by negative ionization and a (M+Na)+ molecular ion of 611.5 by positive ionization. Proton NMR spectral analysis for Compound 25 is shown in Table 9. Signals were easily assigned based on comparison with the spectra of Compound 1.
1H NMR (δH, ppm) Data of Compound 25 in CDCl3
aSignals of 4′, 5′, 8′ and 9′ are very close; assignment was based on Compound 1.
Compound 14, namely 10-(3,7,11-trimethyldodecyl)-4,6,8-trihydroxy-5-methyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, was prepared and identified as follows:
A solution of Compound 2 (23.7 mg) in MeOH (2.0 mL) was stirred under hydrogen gas overnight in presence of platinum oxide (PtO2, 10 mg, catalyst) as a catalyst. The reaction mixture was filtered and concentrated in vacuo to give 21.6 mg of Compound 14.
The calculated molecular weight (482.31) and formula (C29H42N2O4) of Compound 14 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 481.3 and positive ionization gave an (M+H)+ molecular ion of 483.3. The farnesyl olefinic protons on the NMR spectra were replaced by aliphatic proton signals in the region of around 0.76-1.86 ppm, integrating for 17 protons, 3CH, 7CH2. The characteristic N-methyl group (5-N-Me) was easily assigned as shown in Table 10 below.
Compound 15, namely 10-(3,7,11-trimethyl-2-dodecenyl)-4,6,8-trihydroxy-5-methyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, was prepared and identified as follows:
A solution of Compound 2 (308.5 mg) in MeOH (220 mL) was stirred under hydrogen gas overnight in presence of platinum oxide (PtO2, 10 mg) as catalyst. The reaction mixture was filtered and purified by HPLC according to the procedure described in Example 4(c) to give pure Compound 15 (82.3 mg, RT: 17.0 min).
The calculated molecular weight (480.30) and formula (C29H40N2O4) of Compound 15 was confirmed by mass spectral analysis: negative ionization gave an (M−H)− molecular ion of 479.3 and positive ionization gave an (M+H)+ molecular ion of 481.6. The NMR spectrum analysis was based on the structural elucidation of Compounds 2 and 14, and the results presented in Table 10. The farnesyl olefinic protons of positions 6′-7′ and 10′-11′ on the NMR spectra were replaced by aliphatic proton signals and, together with the aliphatic protons of positions 5′, 8′ and 9′, are observed in the region of about 1.07 to 1.51 ppm, integrating for 12 protons, 2CH, 5CH2. The farnesyl olefinic proton at position 2′(CH) was shown on the NMR to have a chemical shift of 5.41 ppm. The methylene group at positions 4′ was shown at 2.03 ppm. The characteristic 5-N-methyl group was also easily assigned to a chemical shift of 2.93 ppm.
Compound 23: 10-(3,7,11-trimethyldodecyl)-4,6,8-trihydroxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, was prepared and identified according to the following procedure:
A solution of Compound 1 (51.1 mg in 3.0 mL MeOH) was stirred under hydrogen gas overnight in presence of platinum oxide (PtO2, 10 mg, 0.4 eq) as a catalyst. The reaction mixture was filtered and purified by direct preparative HPLC using a Phenomenex Synergi™ MAX RP 21.2×200 mm column (20 mL/min, H2O/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 23.
Calculated molecular weight of the major isotope (468.30) and formula (C28H40N2O4) were confirmed by mass spectral analysis. Compound 23 mass spectra gave a (M−H)− molecular ion of 467.4 by negative ionization and a (M+H)+ molecular ion of 469.4 by positive ionization. Proton NMR spectral analysis of Compound 23 is shown in Table 10 below. Signals were easily assigned based on Compound 1 structure knowledge. As expected, aliphatic proton signals at positions 2′-11′ all have very close chemical shifts ranging from about 1 to 1.75 ppm (integrating for 17 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 2 diastereomers of positions 3′ and 7′ are present in the mixture, and in different proportions. Labile protons were not observed since NMR was done in deuterated methanol.
1H NMR (δH, ppm) Data of Compounds 14, 15 and 23 in CD3OD
c
c
d
d
aSignals are very close.
bMixture of isomers.
cCompounds 14 and 23: 3CH, 7CH2 (17H); Compound 15: 2′(CH) at 5.41 ppm, 4′(CH2) at 2.03 ppm, 5′-11′ (12H) at 1.07-1.51 ppm.
dCompounds 14 and 23: 4CH3 (12H); Compound 15: 1″ (CH3) at 1.75 ppm, 2″, 3″ and 12′ (9H) at 0.88-0.80 ppm.
Compound 24: 10-(farnesyl)-7-bromo-4,6,8-trihydroxy-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, was prepared and identified according to the following procedure:
Compound 1 (116.0 mg) and N-bromosuccinimide (NBS, 45.5 mg) were dissolved in tetrahydrofuran (THF, 3.0 mL) and stirred at room temperature for 4 days. The reaction mixture was filtered and subjected to Waters HPLC purification (Nova-Pack™ HR C-18 25×200 mm column: 20 mL/min, H2O/CH3CN gradient 80:20-30:70, 0-8 min; 30:70-0:100, 8-18 min) to give Compound 24 (13.6 min) together with some impurities. The semi-purified sample was further purified by HPLC (Symmetry™ C-18 25×100 mm column: 20 mL/min, H2O/CH3CN gradient 70:30-30:70, 0-15 min), to give pure Compound 24 (9.5 mg, RT 13.0 min).
The calculated molecular weight of the major isotopes (540.16 and 542.16) and formula (C28H33BrN2O4) of Compound 24 was confirmed by mass spectral analysis: negative ionization gave (M−H)− molecular ions of 539.2 and 541.1, and positive ionization gave (M+H)+ molecular ions of 541.3 and 543.2. The presence of the two molecular ions in each mass spectrum confirmed the presence of a bromine group in the molecule. The structure was further confirmed by the absence of the aromatic (position 7) signal in the proton NMR spectrum as shown in Table 11 below.
1H NMR (δH, ppm) Data of Compound 24 in CD3OD
Compound 2 and N-bromosuccinimide are dissolved in tetrahydrofuran and stirred at room temperature for 4 days (conditions as described in (a)). The reaction mixture is filtered and subjected to HPLC purification to provide pure Compound 26.
Compound 14 and N-bromosuccinimide are dissolved in tetrahydrofuran and stirred at room temperature for 4 days (conditions as described in (a)). The reaction mixture is filtered and subjected to HPLC purification to provide pure Compound 27.
In vitro cytotoxic activities of exemplified Compounds are shown in Table 12, along with hemolytic activity of each compound. Compounds were tested in four cell lines: HT-29 (colorectal carcinoma), SF268 (CNS), MDA-MB-231 (mammary gland adenocarcinoma) and PC-3 (prostate adenocarcinoma). Procedures used for each series of tests are described below.
aResults obtained by method (a) below
bResults obtained by method (b) below
cAverage GI50 values in μM.
All compounds described in Table 12 were shown to have better anticancer activity than Compound 1. These compounds include Compounds 2 to 5, 7, 13, 14, 15, 18, and 21 to 25. Compounds Compounds 2 to 5, 7, 13, 14, 15, 18, 21, and 22 are N-linear alkyl derivatives of Compound 1, encompassed by Formula I, and their hydrogenated and hydromethoxylated farnesyl derivatives. The hydrogenated farnesyl derivative Compound 23, brominated aryl derivative Compound 24, and triacetylated Compound 25 were also found to be generally more active than Compound 1.
Cytotoxic activities were determined in vitro for Compounds 1, 23 and 25 to determine the concentration of each compound needed to obtain a 50% inhibition of cell proliferation (GI50). The GI50 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)], GI50 is calculated as [(Ti-Tz)/(C-Tz)]×100=−50, which is the drug concentration resulting in a 50% reduction in the net DNA content intreated relative to control cells during the drug incubation.
Compounds were dissolved at 10 mM in DMSO. Dilution in vehicle to concentrations of 30, 10, 3, 1 and 0.3 μM were prepared immediately before assays. Depending on the cell line's growth characteristics, 4000-10000 cells were plated in two 96-wells pates (day 0) and incubated for 16 hours. The following day, propidium iodide was 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). GI50 results were calculated using the formula above and are shown in Table 12.
In vitro cytotoxic activities (GI50) of Compounds 1, 2 to 5, 7, 13, 14, 15, 18, 21, 22, and 24 were determined using propidium iodide (PI) as being the concentration of drug resulting in 50% growth inhibition, and by using the following method.
Two 96-well plates were seeded in duplicate with each cell line at the appropriate inoculation density (HT29: 3,000; SF268: 3,000; PC-3: 3,000; and MDA-MB-231: 7,500 cells) and according to the technical data sheet of each cell line (rows A-G, 75 μL of media per well). Row H was filled with medium only (150 μL, negative control-medium).
The plates were incubated at appropriate temperature and CO2 concentration for 24 hrs.
Test Compounds were prepared as 15× stock solutions in appropriate medium and corresponding to 450, 45, 0.45, 0.045, and 0.0045 μM (prepared the day of the experiment). An aliquot of each was diluted 7.5-fold in appropriate test medium to give a set of six 2× concentration solutions (60, 6, 0.6, 0.06, 0.006, and 0.0006 μM). A 75 μL aliquot of each concentration was added to each corresponding well (rows A to F) of the second plate. Row G was filled with 75 μL of medium/0.6% DMSO (negative control-cells). The second plate was incubated at appropriate temperature and CO2 concentration for 96 hrs.
First Plate: PI (30 μL, 50 μg/mL) was added to each well of the first plate without removing the culture medium. The plate was centrifuged (Sorvall Legend-RT, swinging bucket) at 3500 rpm/10 min. Fluorescence intensity (Thermo, Varioskan, λex: 530 nm; λem: 620 nm) was measured to give the first measurement, dead cells (DC at T0; before freezing). Two round of Freeze (−80° C.)/Thaw (37° C.) were done. Fluorescence intensity was determined to give the second measure, total cells (TC at T0; after freeze/thaw)
Second plate was processed as the first one, except there were three rounds of freeze/thaw instead of two. First measurement gave the treated dead cells value (TDC), and the second measurement gave the treated total cells value (TTC). Both values were collected for each treated well and control (CTC and CDC).
Each value (DC, TC, TDC, TTC, CTC and CDC) was corrected by removing the background value (medium only) to give the value (FUDC(T=0), FUTC(T=0), FUTDC, FUTTC, FUCTC and FUCDC) used in the calculation of the T/C (%) (Treated/Control) for each concentration. T/C (%) for each concentration is calculated using the following formula:
The GI50 value emphasizes the correction for the cell count at time zero for cell survival. The T/C values are transposed in a graph to determine GI50 values, the concentration at with the T/C is 50%.
Culture conditions and activity evaluations of Compound 2 against 36 cancer cell lines were done as described in Method(a) of Example 9(a), except that results were expressed as the concentration of drug which inhibits 50% of the cell growth (IC50, calculated using the formula: [Ti/C]×100=−50). The low micromolar to nanomolar levels of IC50 values shown in Table 13 demonstrated a pharmacologically relevant cytotoxic activity of Compound 2 against a variety of 36 tumor types including melanomas, pancreatic, lung, colon, gastric, bladder, renal, CNS, head and neck, prostate, uterus, ovarian and breast carcinomas.
The aim of this study was to test whether Compounds 1 and 2 prevent or delay 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 comité d′éthique du CNRS, juillet 2003) and the English guidelines for the welfare of animals in experimental neoplasia (WORKMAN, P., TWENTYMAN, P., BALKWILL, F., et al. (1998). United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Guidelines for the welfare of animals in experimental neoplasia (Second Edition, July 1997; British Journal of Cancer, 77:1-10). 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 (23±2° C.), humidity (45±5%), 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 labeled with a specific code.
Tumor Cell Line: The C6 cell line was cloned from a rat glial tumor induced by N-nitrosomethyurea (NMU) according to 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. 1975 Feb. 13; 381(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% CO2, 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%), PEG 400 (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 (Compound 1). 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 (D0). On D0, mice received a superficial intramuscular injection of C6 tumor cells (5×105 cells) in 0.1 mL of DMEM complete medium into the upper right posterior leg.
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 μL of test or control articles by the 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 Compound 1 for 16 days. Mice of group 4 were treated every two days with 30 mg/kg of Compound 1 and received 8 treatments. Mice of group 5 were treated every three days with 30 mg/kg of Compound 1 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 14 and
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 μL of Compound 1 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 Compound 1. Mice of group 4 were treated daily with 30 mg/kg of Compound 1. 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 15 and
Histological analysis of tumor sections showed pronounced morphological changes between tumors from Compound 1-treated mice and those from mice in the control groups. In tumors from mice treated with Compound 1 (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 tumors from vehicle-treated mice (
Antitumor efficacy of Compound 2 against rat glioblastoma tumor (C6) xenografts in female swiss nude mice was accomplished as described above. The results and dosage regimen are summarized in
Compounds 1 and 2 were separately dissolved in ethanol (5%), Polysorbate 80 (15%), PEG 400 (5%) and dextrose (5%) at a final concentration of 6 mg/ml (for all parenteral administration routes). For oral z, Compound 1 was solubilized in Cremophor® EL/Ethanol (50%:50%) at a final concentration of 6 mg/ml. Prior to dosing, animals (female Crl: CD1 mice; 6 weeks of age, 22-24 g) were weighed, randomly selected and assigned to the different treatment groups. Compound 1 was administered by the intravenous (iv), subcutaneous (sc), intraperitoneal (ip), or oral (po) route to the assigned animals. Compound 2 was administered by the intravenous (iv), or intraperitoneal (ip) route to the assigned animals. The dosing volume of Compounds 1 and 2 was 5 mL per kg body weight. Animals were anesthetized with 5% isoflurane prior to bleeding. Blood was collected into microtainer tubes containing the anticoagulant K2EDTA by cardiac puncture from each of 4 animals per bleeding timepoint (2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h and 8 h). Following collection, the samples were centrifuged and the plasma obtained from each sample was recovered and stored frozen (at approximately −80° C.) pending analysis. At the 5 min and 30 min time points, the following organs were harvested from each animal: brain, lungs, skeletal muscle, fat tissue, kidneys, spleen, thymus and liver. Tissues were frozen (at approximately −80° C.) pending analysis. Samples were analysed by LC/MS/MS. Standard curve ranged from 25 to 2000 ng/mL with limit of quantitation (LOQ)≦25 ng/mL and limit of detection (LOD) of 10 ng/mL.
Plasma values of Compounds 1 and 2 falling below the limit of quantitation (LOQ) were set to zero. Mean concentration values and standard deviation (SD) were calculated at each timepoints of the pharmacokinetic study (n=4 animals/timepoint).
The following pharmacokinetic parameters were calculated: area under the plasma concentration versus time curve from time zero to the last measurable concentration time point (AUC0-t), area under the plasma concentration versus time curve extrapolated to infinity (AUCinf), maximum observed plasma concentration (Cmax), time of maximum plasma concentration (tmax), apparent first-order terminal elimination rate constant (kel), apparent first-order terminal elimination half-life will be calculated as 0.693/kel (t1/2). The systemic clearance (CL) of Compound 1 after intravenous administration was calculated using Dose/AUCinf. Pharmacokinetic parameters were calculated using Kinetica™ 4.1.1 (InnaPhase Corporation, Philadelphia, Pa.).
Results:
Mean plasma concentrations of Compound 1 following intravenous (iv), intraperitoneal (ip), subcutaneous (sc), and oral (po) administrations at 30 mg/kg are presented in
Mean (±SD) plasma concentrations of Compound 1 following I.V. administration of a 30 mg/kg dose declined rapidly in a biexponential manner resulting in very short half lives (t1/2 α and β of 4.6 min and 2.56 h, respectively). The pharmacokinetics of Compound 1 following intraperitoneal and subcutaneous administrations, and Compound 2 following intraperitoneal and intravenous administration, showed a PK profile suggestive of slow release. With these routes of administration, the compound plasma concentration was sustained and maintained at therapeutically relevant levels for over 8 hours. Compound 2 showed a half life (t1/2) of more than 40 hours following both IP and IV administrations. Oral administration of Compound 1 resulted in moderate but sustained drug levels. These data indicated that Compound 1 was orally bioavailable at a 5-8% level.
Mean tissue concentrations of Compound 1, 30 min after intravenous (iv), intraperitoneal (ip) or subcutaneous (sc) administrations at 30 mg/kg are presented in
Acute toxicity studies in CD-1 nu/nu mice for Compound 2, using the same formulation, gave an MTD ≧50 mg/kg (ip, NOAEL: 30 mg/kg) and ≧100 mg/kg (iv, NOAEL: 75 mg/kg), with weight losses of about 7% for several days post-injection. Compound 1 had an MTD of 150 mg/kg when administered iv. Acute toxicity studies with Compound 46 gave an MTD of 30 mg/kg (ip).
All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10/591436 | Sep 2004 | US | national |
| 2,497,031 | Feb 2005 | CA | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA05/01467 | 9/26/2005 | WO | 00 | 2/25/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60625653 | Nov 2004 | US | |
| 60647381 | Jan 2005 | US | |
| 60701472 | Jul 2005 | US |
