Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
  • C07H19/11—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids containing cyclic phosphate

Definitions

• methylation inhibitors modulation of the hypermethylation of genes involved in the control of cell proliferation by use of methylation inhibitors has been one strategy invoked for cancer therapy, and more specifically, the use of D ⁇ A methylation inhibitors to reactivate antiproliferative, apoptotic, and differentiation- inducing genes in cancer cells.
• fluorodeoxycytidine the compounds typically suffer from one or more characteristics that detract from their use as therapeutic agents, including chemical instability (including in neutral solution), weak potency, short half-life, and generation of toxic metabolites.
• the compounds and compositions herein are useful in treatment or modulation of disease, disease symptoms or conditions in a subject.
• the compounds are useful as DNA methylation inhibitor compounds (and methods thereof).
• the compounds, compositions, and methods thereof are useful in treatment or modulation of cancer, cancer symptoms or cancer conditions in a subject.
• One aspect is an isolated compound of Formula I:
• each X is independently N ⁇ R 2 , or NR ⁇ R 3 + ; each R 1 , R 2 and R 3 is independently H or alkyl; each Y is independently H, OH, or halogen; and each Z is independently a bond or -P(O)(OH)-O-
• Another aspect is the isolated compound of Formula II or pharmaceutically acceptable salt or hydrate thereof:
• Another aspect is the isolated compound of Formula III or pharmaceutically acceptable salt or hydrate thereof,
• Another aspect is the isolated compound of Formula N :
• each X is independently ⁇ R 2 , or ⁇ R*R 2 R 3 + ; each Z is independently a bond or -P(O)(OH)-O-; or pharmaceutically acceptable salt or hydrate thereof.
• Another aspect is the isolated compound of Formula NI or pharmaceutically acceptable salt or hydrate thereof:
• Another aspect is the isolated compound of Formula VIII or pharmaceutically acceptable salt or hydrate thereof:
• aspects are compounds of the formulae herein, wherein Y is halogen; wherein Y is fluoro; wherein Y is hydrogen.
• Otlier aspects are compounds of the formulae herein, wherein X is NH 2 ;. X is (NMe 3 ) + .
• the invention in another aspect, relates to a composition having a therapeutically effective amount of a compound (or pharmaceutically acceptable salt or hydrate thereof) according to any of the formulae herein and a pharmaceutically acceptable carrier.
• the composition can further have an additional therapeutic agent, the additional agent can be an anticancer agent.
• Another aspect is a method of treating a DNA methyl transferase (DNMT) mediated disease, disease symptom or condition that includes administration to a subject in need of such treatment a compound (or pharmaceutically acceptable salt or hydrate thereof) according to any of the formulae herein, or composition thereof.
• the disease, disease symptom or condition can involve hypermethylation of DNA; the administration can be by oral administration.
• Another aspect is a method of assessing the effect of a test compound on methylation of DNA in a cell including: (i) contacting a test compound with a cell that methylation; and measuring the methylation of DNA in the cell; and (iii) comparing the results of step (i) with the results of step (ii).
• the cell can include a hypermethylated nucleic acid molecule; a CpG dinucleotide; and can be a mammalian tumor cell.
• Another aspect is a method of reversing DNA methylation in a cell, including administering to a cell a therapeutically effective amount of a compound of any of the formulae herein.
• the cell can be in a subject or in vitro.
• the invention also relates to a method of treating cancer in a subject including administering an effective amount of a compound according to any of the formulae herein.
• the cancer can be ovarian, breast, colon, rectal, lung, prostate, pancreatic, bladder, solid tumor, or any tumor having a silenced tumor suppressor gene.
• the cancer can be any associated with or exemplified by cancer cell lines, including for example, T24, HCT15, CFPAC-1, SW48, HT-29, PC3, or CALU-1.
• the method can further include administration of an additional anticancer agent; an anti-nausea or an anti-anemia agent.
• the administration of the additional agent(s) can be concurrently or sequentially, and can be individually or in a combined formulation.
• the invention also relates to a kit having a compound of any of the formulae herein and instructions for in vivo or in vitro use of the compound.
• the in vitro use can be screening for demethylation of a hypermethylated DNA.
• the invention also relates to a kit having a compound of any of the formulae herein and instructions for administration to a subject.
• the subject can be in need of treatment for a hypermethylated DNA mediated disease, disease symptom or condition; in need of treatment for a hype ⁇ roliferative disease, disease symptom or condition; in need of treatment for cancer.
• the subject can be a human; or a rat or mouse.
• the administration can be oral;or intravenous or intraperitoneal.
• Another aspect is a method of making a compound of any of the formulae herein, comprising converting a compound of Formula B, wherein Y is H, OH, O-PG, or halo; and PG is a protecting group:
• Formula B to a compound of Formula I herein.
• the process can further include converting the compound of Formula B to the corresponding diphosphate; and further include removal of an oxygen- or nitrogen-protecting group.
• a method of making any of the compounds delineated herein involves one or more reactions and/or reagents as delineated herein.
• the invention relates to a composition comprising a compound of any of the formulae herein, an additional therapeutic agent, and a pharmaceutically acceptable carrier.
• the additional therapeutic agent can be an anticancer agent (e.g., arabinofuranosyl cytosine (ara-C), 5-fiuorouracil (5-FU) and taxol).
• the additional therapeutic agent can also be a histone deacetylase inhibitor: See, e.g., Lemaire et al. Leukemia Lymphoma 2004, 45, 147-154; Leone et al. Clin Immunol. 2003, 109, 89-102; Shaker et al. Leukemia Res 2003, 27, 437-444; Primeau et al. Int. J. Cancer 2003, 103, 177-184.
• Yet another aspect of this invention relates to a method of treating a subject (e.g., mammal, human, horse, dog, cat) having a DNA-methylation-mediated disease or disease symptom (including, but not limited to cancer).
• the method includes administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such
• the invention also relates to a method of making a compound described herein.
• the method includes taking any one of the intermediate compounds described herein and reacting it with one or more chemical reagents in one or more steps to produce a compound described herein.
• the invention relates to a compound made by a process that includes any one, or more, of the reactions delineated herein.
• the process includes the reagent or reagents, or reaction conditions delineated herein.
• the packaged product includes a container, one of the aforementioned compounds in the container, and a legend (e.g., a label or an insert) associated with the container and indicating administration of the compound for treating a disorder associated with DNA- methylation modulation.
• a legend e.g., a label or an insert
• the compounds, compositions, and methods delineated herein are any of the compounds of Table I or Table II (or salts or solvates thereof) herein or methods including them.
• FIG. 1 shows the structures of zebularine and cytidine.
• FIG. 2 illustrates the effects of 5-azacytidine (5-azaC) and zebularine exposure to T24 cell proliferation
• FIG. 3 illustrates radiolabeled metabolites of zebularine incubated with T-24 cells.
• FIG. 4 illustrates the enzymatic characterization of zebularine metabolites.
• FIG. 5 illustrates HPLC radiochromatograms of zebularine metabolites.
• FIG. 6 illustrates formation of phosphorylated zebularine metabolites.
• FIG. 7 profiles zebularine metabolite levels in T-24 cells.
• FIG. 1 shows the structures of zebularine and cytidine.
• FIG. 2 illustrates the effects of 5-azacytidine (5-azaC) and zebularine exposure to T24 cell proliferation
• FIG. 3 illustrates radiolabeled metabolites of zebularine incubated with T-24 cells.
• FIG. 8 illustrates incorporation of zebularine into DNA and RNA of T-24 cells.
• FIG. 9 illustrates the HPLC radiochromatograms of zebularine metabolites in vivo in nude mice in tumor and in normal muscle.
• FIG. 10 illustrates the effect of zebularine on human bladder carcinoma cell proliferation.
• FIG. 11 illustrates a metabolic activation pathway for zebularine.
• halo and halogen refer to any radical of fluorine, chlorine, bromine or iodine.
• alkyl refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C10 indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it.
• lower alkyl refers to a C1-C6 alkyl chain.
• alkenyl refers to an unsaturated hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms.
• alkynyl refers to an unsaturated hydrocarbon chain that may be a straight chain or "ester” refers to a C(O)O-alkyl or C(O)O-aryl group.
• An “amido” is an C(O)NH 2
• an "N-alkyl-substitited amido” is of the formula C(O)N(H)(alkyl).
• cycloalkyl refers to a 6-carbon monocyclic or 10-carbon bicyclic nonaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent.
• cycloalkyl groups include cyclopentyl, cyclohexyl, cyclohexenyl, bicyclo[2.2.1]hept-2-enyl, dihydronaphthalenyl, benzocyclopentyl, and the like.
• aryl refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent.
• aryl groups include phenyl, naphthyl and the like.
• arylalkyl or the term “aralkyl” refers to alkyl substituted with an aryl.
• arylalkoxy refers to an alkoxy substituted with aryl.
• heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent.
• heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, thienyl, indolyl, thiazolyl, and the like.
• heteroarylalkyl or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl.
• heteroarylalkoxy refers to an alkoxy substituted with heteroaryl.
• heterocyclyl refers to a nonaromatic 5-8 membered monocyclic, 8-
• heterocyclyl groups include tetrahydrofuryl, piperidinyl, pyrrolidinyl, morpholinyl, dihydrothiophenyl, dihydrobenzothiophenyl, indolinyl, and the like.
• n 1 or 2; and each R 6 is independently alkyl, alkenyl, aryl, arylalkyl, or heteroarylalkyl, each optionally substituted with 1-4 independent substituents selected from the group hydroxy, mercapto, amino, alkoxy, carboxylic acid, ester, amido, N-alkyl-substitited amido, halo, nitro, and nitrile.
• stable refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic formulations, reagents, kits).
• the compounds produced by the methods herein can be incorporated into compositions, including pills, capsules, gel caps, solutions, tablets, cremes, or ointments for administration to a subject (e.g., human, animal). Acids and bases useful in the methods herein are known in the art.
• Acid catalysts are any acidic chemical, which can be inorganic (e.g., hydrochloric, sulfuric, nitric acids) or organic (e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid) in nature. Acids are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions.
• Bases are any basic chemical, which can be inorganic (e.g., sodium bicarbonate, potassium hydroxide) or organic (e.g., triethylamine, pyridine) in nature. Bases are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions.
• Alkylating agents are any reagent that is capable of effecting the alkylation of the functional group at issue (e.g., oxygen atom of an alcohol, nitrogen atom of an amino group).
• Alkylating agents are known in the art, including in the references cited herein, and include alkyl halides (e.g., methyl iodide, benzyl bromide or chloride), alkyl sulfates (e.g., methyl sulfate), or other alkyl group-leaving group combinations known in the art.
• Leaving groups are any stable species that can detach from a molecule during a reaction (e.g., elimination reaction, substitution reaction) and are known in the art, including in the references cited herein, and include halides carbamates (e.g., N(Me)C(O)Ot-Bu), phosphonates (e.g., -OP(O)(OEt) 2 ), water or alcohols (protic conditions), and the like. Nucleophilic agents are known in the art and are described in the chemical texts and treatises referred to herein.
• the chemicals used in the aforementioned methods may include, for example, solvents, reagents, catalysts, protecting group and deprotecting group reagents and the like.
• the methods described above may also additionally comprise steps, either before or after the steps described specifically herein, to add or remove suitable protecting groups in order to ultimately allow synthesis of the compound of the formulae described herein.
• the methods delineated herein contemplate converting compounds of one formula to compounds of another formula.
• the process of converting refers to one or more chemical transformations, which can be performed in situ, or with isolation of intermediate compounds.
• the transformations can include reacting the starting compounds or intermediates with additional reagents using techniques and protocols known in the art, including those in the references cited herein.
• Intermediates can be used with or without purification (e.g., filtration, distillation, crystallization, chromatography).
• the compounds delineated herein can be synthesized using conventional methods, as illustrated generally in the schemes herein.
• PG is a protecting group (e.g., an oxygen-protecting group, a nitrogen-protecting group).
• protecting group refers to a labile chemical moiety which is known in the art to protect a chemical group (e.g., oxygen- or nitrogen- containing moiety) against undesired reactions during synthetic procedures. After said synthetic procedure(s) the protecting group as described herein may be selectively removed.
• Protecting groups as known in the art are described generally in T.H. Greene and P.G. M.
• oxygen protecting group refers to a labile chemical moiety which is known in the art to protect a hydroxyl group against undesired reactions during synthetic procedures. After said synthetic procedure(s) the hydroxy Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).
• hydroxyl protecting groups include acetyl (Ac or -C(O)CH 3 ), benzoyl (Bz or -C(O)C 6 H 5 ), benzyl (Bn), t-butyldimethylsilyl, (TBDMS), and trimethylsilyl (TMS or-Si(CH 3 ) 3 ).
• nitrogen protecting group refers to a labile chemical moiety which is known in the art to protect a nitrogen-containing group against undesired reactions during synthetic procedures. After said synthetic procedure(s) the nitrogen protecting group as described herein may be select vely removed. Nitrogen protecting groups as known in the are described generally in T.H. Greene and P.G. M.
• Compounds of Formula B can then be converted to the diphosphate compounds of Formula C under standard conditions (e.g., DCC, (t- Bu 3 NO) 3 PO) and then coupled (DCC and choline chloride or N-protected ethanolamine, such as phthalimido-protected ethanolamine) followed by deprotection, if appropriate (e.g., removal of protecting groups, hydrolysis, aqueous acid, ammonia/MeOH, hydrazine), to give compounds of Formula E where Z is -P(O)(OH)-O-.
• standard conditions e.g., DCC, (t- Bu 3 NO) 3 PO
• deprotection if appropriate (e.g., removal of protecting groups, hydrolysis, aqueous acid, ammonia/MeOH, hydrazine), to give compounds of Formula E where Z is -P
• Scheme II illustrates general synthesis of compounds delineated herein.
• Compounds of Formula F can be protected with oxygen protecting groups then coupled (DCC and phosphorylcholine chloride or N-protected ethanolaminephosphoric acid ester derivative, such as phthalimido-protected ethanolaminephosphoric acid ester) followed by deprotection, if appropriate (e.g., removal of protecting groups, hydrolysis, aqueous acid, ammonia/MeOH, hydrazine), if appropriate (e.g., removal of protecting groups, hydrolysis, aqueous acid, ammonia/MeOH, hydrazine), to give compounds of Formula H.
• oxygen protecting groups then coupled (DCC and phosphorylcholine chloride or N-protected ethanolaminephosphoric acid ester derivative, such as phthalimido-protected ethanolaminephosphoric acid ester) followed by deprotection, if appropriate (e.g., removal of protecting groups, hydrolysis, aqueous acid, ammonia
• treating refers to administering a compound described herein to a subject with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a disease, the symptoms of the disease or the predisposition toward the disease.
• An effective amount refers to an amount of a compound, which confers a therapeutic effect on the treated subject.
• the therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).
• an effective amount of the compound described above may range from about 50 mg/Kg to about 200 mg/Kg. Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.
• DNA methylation-modulating compounds can be identified through both in vitro (e.g., cell and non-cell based) and in vivo methods. Representative examples of these methods are described in detail in the Examples. Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds.
• stable as used be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).
• the compounds of this invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention.
• the compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.
• the compounds of this invention including the compounds of formulae described herein, are defined to include pharmaceutically acceptable derivatives or prodrugs thereof.
• a "pharmaceutically acceptable derivative or prodrug” means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention.
• Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.
• Preferred prodrugs include derivatives where a group which enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein.
• the compounds of this invention may be modified by appending appropriate functionalities to enhance selective biological properties.
• Such modifications are known in the art and include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter those derived from pharmaceutically acceptable inorganic and organic acids and bases.
• Suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate
• Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl) 4 salts.
• alkali metal e.g., sodium
• alkaline earth metal e.g., magnesium
• compositions delineated herein include the compounds of the formulae delineated herein, as well as additional therapeutic agents if present, in amounts effective for achieving a modulation of disease or disease symptoms, including ion channel-mediated disorders or symptoms thereof.
• pharmaceutically acceptable carrier or adjuvant refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
• Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d- -tocopherol polyethyleneglycol 1000 succinate, substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
• SEDDS self-emulsifying drug delivery systems
• Cyclodextrins such as ⁇ -, ⁇ -, and ⁇ - cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl- ⁇ -cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.
• the pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection.
• the pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
• the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.
• parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
• the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
• a non-toxic parenterally acceptable diluent or solvent for example, as a solution in 1,3-butanediol.
• acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium employed including synthetic mono- or diglycerides.
• Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
• oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions.
• a long-chain alcohol diluent or dispersant or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions.
• Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
• the pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
• carriers which are commonly used include lactose and corn starch.
• Lubricating agents such as magnesium stearate, are also typically added.
• useful diluents include lactose and dried corn starch.
• the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
• the pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration.
• compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
• suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
• Topical administration of the pharmaceutical compositions of this invention is useful when the desired treatment involves areas or organs readily accessible by topical application.
• the pharmaceutical composition should be formulated with a suitable ointment containing the active petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
• the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents.
• suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
• the pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in this invention.
• the pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation.
• compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
• a composition having the compound of the formulae herein and an additional agent can be administered using an implantable device.
• Implantable devices and related technology are known in the art and are useful as delivery systems where a continuous, or timed-release delivery of compounds or compositions delineated herein is desired. Additionally, the implantable device delivery system is useful for targeting specific points of compound or composition delivery (e.g., localized sites, organs).
• Timed-release technology involving alternate delivery methods can also be used in this invention.
• timed-release formulations based on polymer technologies sustained-release techniques and encapsulation techniques (e.g., polymeric, liposomal) can also be used for delivery of the compounds and compositions delineated herein.
• a patch to deliver active chemotherapeutic combinations herein is also within the invention.
• a patch includes a material layer (e.g., polymeric, cloth, gauze, bandage) and the compound of the formulae herein as delineated herein.
• One side of the material layer can have a protective layer adhered to it to resist passage of the natural or synthetic origin, that when contacted with the skin of a subject, temporarily adheres to the skin. It can be water resistant.
• the adhesive can be placed on the patch to hold it in contact with the skin of the subject for an extended period of time.
• the adhesive can be made of a tackiness, or adhesive strength, such that it holds the device in place subject to incidental contact, however, upon an affirmative act (e.g., ripping, peeling, or other intentional removal) the adhesive gives way to the external pressure placed on the device or the adhesive itself, and allows for breaking of the adhesion contact.
• the adhesive can be pressure sensitive, that is, it can allow for positioning of the adhesive (and the device to be adhered to the skin) against the skin by the application of pressure (e.g., pushing, rubbing,) on the adhesive or device.
• Compounds herein are administered in a dosage ranging from about 10 to about 500 mg/kg of body weight, preferably dosages between 50 mg and 200 mg/dose, every 4 to 120 hours, or according to the requirements of the particular drug.
• the methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect.
• the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
• the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
• a typical preparation will contain from about 5% to about 95% active compound (w/w).
• such preparations contain from about 20% to about 80% active compound.
• Lower or higher doses than those recited above may be required.
• Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician. necessary.
• compositions of this invention comprise a combination of a compound of the formulae described herein and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to
• Prodrug means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of any of the formulae herein.
• prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); and "Hydrolysis In Drug And
• Zeb zebularine
• Zeb-MP zebularine-5 1 - monophosphate
• Zeb-DP zebularine-5'-diphosphate
• Zeb-TP zebularine-5'-triphosphate
• Zeb-DP- EA zebularine-5 '-diphosphoethanolamine
• Zeb-DP-Chol zebularine- 5'- diphosphocholine
• CPEU cyclopentenyl uridine
• IC 50 drug concentration resulting in 50% inhibition of growth
• PDE-1 snake venom phosphodiesterase-1
• AP alkaline phosphatase
• UCK uridine/cytidine kinase
• NCI US National Cancer Institute.
• Compound 100 is reacted with phosphoric acid mono-[2-(l,3-dioxo-l,3- dihydro-isoindol-2-yl)-ethyl] ester in the presence of DCC in methylene chloride to provide the corresponding diphosphate.
• the resulting diphosphate is deprotected (hydrazine; methanolic ammonia) to provide compound 101.
• the 2'-deoxy and 2'- fluoro analogs of compound 101 can be made similarly from available 2'-deoxy and 2' -fluoro analogs of compound 100.
• DCC 1,3-dicyclohexylcarbodiimide
• the diphosphate is hydrolized under methanolic ammonia conditions to provide compound 102.
• the 2'-deoxy and 2'-fluoro analogs of compound 102 can be made similarly from available 2'-deoxy and 2 '-fluoro analogs of compound 102.
• T-24 cell growth Exponentially growing T-24 cells were cultured in 24-well plates (10 5 cells per well) overnight. Cells were then washed with fresh medium and varying concentrations (0-1000 ⁇ M) of zebularine were added. After incubation for 48 h, cells were trypsinized, collected and counted in a ZB 1 Coulter Counter. The cell growth rate was expressed as a percentage of the increase in cell number of the untreated control cultures. The IC 50 was calculated from the linear portion of the growth inhibition curve.
• Reverse-phase HPLC was employed for the separation and identification of zebularine nucleosides in cell extracts, for measuring [2- 14 C]Zeb after enzymatic degradation of its phosphorylated metabolites, and for the DNA and RNA studies. Separations were carried out on a 5- ⁇ m Beckman Ultrasphere C ⁇ 8 column (250 X 4.6 mm) using the following gradient elution program with a flow rate of 2 ml/min throughout: 0-25 min, linear gradient from 1% to 25% methanol; 25-30 min, isocratic with 25% methanol; 30-40 min, linear gradient to 1% methanol, and equilibration. One-minute fractions were collected and radioactivity was determined by liquid 2.5.
• T-24 cells were cultured in DMEM medium supplemented with 10% FCS and 4 mM L-glutamine but without endogenous choline or ethanolamine. The cells so grown exhibited doubling times (24 h) identical to those of T-24 cells in choline replete medium.
• T-24 cells were incubated with 100 ⁇ M [2- 14 C]Zeb (1 ⁇ Ci/ml) alone and in combination with either 28 ⁇ M [methyl- 3 H]choline (10 ⁇ Ci/ml) or 50 ⁇ M [l- 3 H]ethanolamine (10 ⁇ Ci/ml) for 24 h in separate parallel experiments. Thereafter, cells were harvested, washed and extracted with 60% methanol. Zebularine metabolites were determined by gradient ion-exchange HPLC as described above.
• RNA in the aqueous phase
• DNA in the inte ⁇ hase
• the isolated DNA and RNA were hydrolyzed overnight at 37 °C in 1 ml of pH 7.4, 0.1 M HEPES buffer containing 140 ⁇ g of either DNase or Rnase and 0.02 U PDE-1 and 5 U AP.
• EJ6 tumors and straight muscle were removed from mice under ether anesthesia 24 h after drug administration and immediately frozen in liquid N 2 and stored at -70 °C pending sample work-up and analysis.
• Tumors and muscle tissue (100-200 mg) were homogenized at 4 °C in 1.0 ml of 60% methanol using a Polytron homogenizer. The homogenate was heated at 95°C for 3 min and centrifuged at 12,000 X g for 10 min at 4 °C. The supernatant was collected and evaporated under a nitrogen stream. The resulting residue was then dissolved in deionized water and appropriate aliquots subjected to anion-exchange HPLC analysis as described by Noy et al. [D].
• Typical radiochromatograms obtained after zebularine treatment of T-24 cells are shown in Fig. 3 and Fig. 4A.
• the parent drug which elutes close to the void volume (2 min)
• five acidic metabolites were observed.
• the five zebularine metabolites were also observed in these two cell lines after a 6-h incubation with 10 ⁇ M [2- 14 C]Zeb.
• levels of each of the five zebularine metabolites were highest in the MC38 cells with the concentration of Zeb-TP being 6- fold greater than in T-24 or Molt-4 cells.
• Zeb-TP and the Zeb-DP-Chol adduct were the major phosphorylated metabolites in all the cell lines, with the former being highest in Molt-4 and MC38 and the latter greatest in T-24 bladder carcinoma.
• Individual metabolites levels were comparable in the two human cells lines except for the Zeb-DP-Chol adduct which was 3 -fold higher in T-24 cells.
• zebularine metabolites The formation of the five zebularine metabolites was evaluated as a function of zebularine dose in T-24 cells. Cells were incubated with increasing concentrations of [2- 14 C]Zeb (1 - 500 ⁇ M) for 6 h, at which time the levels of zebularine metabolites were measured. As can be seen in Fig. 6, levels of all metabolites increased with increasing zebularine dose. The rates of this dose-dependent formation were the concentrations of these two major metabolites were more than 4-fold greater ttian the other metabolites and approached or exceeded the nanomole per million cell level.
• Zeb- MP, Zeb-DP and Zeb-TP all exhibited an initial rapid rate of intracellular accumulation over the initial 4 h before reaching a more or less constant steady-state level by 8-12 h.
• the rate of accumulation of the phosphodiester adducts was more gradual and sustained, since both the Zeb-DP-EA and Zeb-DP-Chol adducts increased over the entire 24-h period and did not reach a steady-state.
• Zeb-DP and Zeb-TP decayed rapidly with roughly equivalent half-lives of 1.5 h and 1.7 h, respectively.
• the intracellular disappearance of the phosphodiester adducts was much more gradual with estimated half-lives of 6.1 h for the Zeb-DP-EA adduct and 5.4 h for the Zeb-DP-Chol adduct. In contrast to the zebularine 5'-phosphates, these two metabolites could still be detected in the cellular extract 24 h after removal of drug.
• the intracellular elimination rate of Zeb-MP was intermediate to that of the other metabolites with a half-life of 4.1 h.
• Control levels were: Zeb-MP, 5.1; Zeb-DP, 2.2; Zeb-TP,6.7; Zeb-DP-EA Adduct, 2.4; Zeb-DP-Chol Adduct, 22. Average of duplicate measurements. ' Not detectable.
• tissue samples obtained 24 h after drug treatment.
• FIG. 1 Chemical structures of zebularine, cytidine and 5-azacytidine. The asterisk indicates the position of the [ 14 C]radiolabel in zebularine.
• mice were inoculated s.c. witn fcJb tumor cens, w ⁇ ic ⁇ were anuweu ⁇ yi ⁇ w ⁇ weeks. Mice were then treated i.p. with 500 mg/kg [2- 14 C]Zeb (500 ⁇ Ci/kg). Twenty- four hours after treatment, mice were sacrificed and tumors (A) and striated muscle (B) were removed and extracted for analysis. Fig. 10.

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