Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
  • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
  • C07F9/657163—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms the ring phosphorus atom being bound to at least one carbon atom
  • C07F9/657172—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms the ring phosphorus atom being bound to at least one carbon atom the ring phosphorus atom and one oxygen atom being part of a (thio)phosphinic acid ester: (X = O, S)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • 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
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
  • C07F9/6509—Six-membered rings
  • C07F9/6512—Six-membered rings having the nitrogen atoms in positions 1 and 3
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
  • C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
  • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs

Definitions

• the present invention relates to orally administered drugs for treatment of viral infections and certain cancers.
• the present invention relates to metabolically stable alkoxyalkyl esters of phosphonates, nucleoside phosphonates and nucleoside phosphates, compositions containing them, processes for obtaining them, and their use in treating a variety of medical disorders, in particular viral infections, cancers and the like.
• Nucleoside phosphonates have antiviral, antiproliferative and a variety of other therapeutic benefits.
• antiviral nucleoside phosphonates such as, for example, cidofovir, cyclic cidofovir, adefovir, tenofovir, and the like, as well as the 5'-phosphonates and methylene phosphonates of azidothymidine (AZT), ganciclovir, acyclovir, and the like.
• the 5'-hydroxyl of the sugar moiety, or its equivalent in acyclic nucleosides which do not contain a complete sugar moiety, is replaced with a phosphorus-carbon bond.
• a methylene group replaces the 5'- hydroxyl or its equivalent, and its carbon atom is, in turn, covalently linked to the phosphonate.
• nucleoside phosphonate diphosphate which represents the equivalent of nucleoside triphosphates.
• Antiviral nucleoside phosphonate diphosphates are selective inhibitors of viral RNA or DNA polymerases or reverse transcriptases. That is to say, their inhibitory action on viral polymerases is much greater than their degree of inhibition of mammalian cell DNA polymerases ⁇ , ⁇ and ⁇ or mammalian RNA polymerases.
• antiproliferative nucleoside phosphonate diphosphates inhibit cancer cell DNA and RNA polymerases and may show much lower selectivity versus normal cellular DNA and RNA polymerases.
• antiviral and antiproliferative compounds are the antiviral nucleoside phosphonates.
• Two representative structures of this class of compounds, namely CDV and HPMPA, are set forth below:
• Another class of phosphonates is the 5'-phosphonates and methylene phosphonates of azidothymidine, ganciclovir, acyclovir, and the like.
• the 5'-hydroxyl of the sugar moiety, or its equivalent in acyclic nucleosides (ganciclovir, penciclovir, acyclovir), which do not contain a complete sugar moiety, is replaced with a phosphorus-carbon bond.
• a methylene group replaces the 5'-hydroxyl or its equivalent, and its carbon atom is, in turn, covalently linked to the phosphonate.
• nucleoside phosphates such as, acyclovir monophosphate, 2'-O-methyl-guanosine-5'-phosphate, 2 '-O-methyl-cytidine-5 '-phosphate and 2 '-C-methyl-cytidine-5 '-phosphate.
• nucleoside phosphates such as, acyclovir monophosphate, 2'-O-methyl-guanosine-5'-phosphate, 2 '-O-methyl-cytidine-5 '-phosphate and 2 '-C-methyl-cytidine-5 '-phosphate.
• Various substituent groups may be attached to phosphonates and phosphates to produce derivatives having various degrees of pharmacological potency.
• One class of derivative compounds are the alkoxyalkyl esters, such as hexadecyloxypropyl cidofovir (HDP-CDV), which is illustrated by the following general structure:
• HDP-CDV Hexadecyloxypropyl cidof ovir
• CDV itself is not orally active; however esterification of CDV with certain alkoxyalkanols such as hexadecyloxypropanol dramatically increases its antiviral activity and selectivity in vitro and confers a degree of oral bioavailability.
• alkyl chain length of these CDV analogs is related to solubility and the ability of the compounds to associate with biomembranes.
• alkoxyalkyl esters of nucleoside phosphates and phosphonates such as hexadecyloxypropyl-cidofovir (HDP-CDV)
• HDP-CDV hexadecyloxypropyl-cidofovir
• Orally administered drugs are usually taken up from the small intestine into the portal vein, which exposes the drug to potentially rapid lipid metabolism in the enterocytes of the small intestines and in the liver.
• Alkoxyalkyl esters of phosphates and phosphonates can be incorporated into cell membranes where the phosphate or phosphonate is subsequently liberated inside the cell or can be oxidatively metabolized by the cytochrome P450s such as CYP3A4 in the liver or intestine leading to omega oxidation of the alkyl chain followed by beta oxidation. It has recently been determined that alkoxyalkyl esters of phosphates and phosphonates can be oxidized at the terminal end of the alkyl chain by omega oxidation and are further degraded by beta oxidation to short chain inactive metabolites.
• nucleoside monophosphonate HDP-CDV may be very rapid and is deleterious to the intended pharmacologic effect of the compounds.
• the inactive metabolite is water soluble, virologically inactive, and rapidly excreted in the urine. Rapid metabolism by this pathway may lower plasma levels of the prodrug, and reduce the antiviral efficacy of HDP-CDV and alkoxyalkyl esters of phosphonates, nucleoside phosphonates and nucleoside phosphates.
• the present invention includes esters of phosphonates, nucleoside phosphonates and nucleoside phosphates (referred to collectively hereinafter as esters) that are resistant to metabolic inactivation resulting from oxidation of these compounds in the liver. More specifically, the present invention includes terminal or penultimate branched chain, unsaturated and halogen substituted alkoxyalkyl esters of phosphonate compounds, wherein said substituents stabilize these compounds by providing metabolic stability during absorption in the small intestine, first pass liver metabolism and subsequent distribution to peripheral tissues. Included in the present invention are methods for using said esters for treating various diseases and conditions.
• the compounds and methods of this invention are based upon the unique insight that ⁇ -oxidation of lipid esters of phosphonates and phosphates may be slowed by placing a blocking group or groups at or near the penultimate carbon of the alkyl chain.
• Potential blocking groups include, but are not limited to alkyl groups, including, but not limited to methyl, ethyl and propyl, cyclopropyl and halogens.
• Potential blocking groups also include alkenyl groups containing one or more double bonds, including a terminal double bond.
• Phosphonate compounds contemplated for use in accordance with the present invention include those having antiviral and antiproliferative activity.
• nucleoside analogs with antiviral activity against hepatitis C which can be converted to their alkoxyalkyl 5'-phosphates or their alkylglycerol phosphates.
• nucleosides in this class of compounds include, but are not limited to 2'-C-methyl adenosine, 2'-C-methyl guanosine, 7-deaza-2'-methyl adenosine, 2'-C-methyl cytosine.
• Other nucleosides and analogs thereof contemplated for use in accordance with this invention following conversion to their alkoxyalkyl 5'-phosphates or their alkylglycerol phosphates are set forth in the references cited in Table 2.
• nucleoside analogs with antiviral activity against hepatitis B which may be converted to their 5'-phosphates, 5'-phosphonates or 5'-methylene phosphonates.
• exemplary nucleosides in this class of compounds include, but are not limited to 3TC, FTC, DAPD, L-FMAU, entecavir, telbivudine and various ⁇ -L-2'- deoxycytidine, ⁇ -L-2'-deoxyadenine and ⁇ -L-2'-deoxythymidine analogs described by Bryant et al. ((Jan. 2001) Antimicrob Agents Chemother 45[l):229-235).
• Anticancer agents may also be derivatized according to the method of this invention.
• Some subject compounds include but are not limited to (E)-2'-deoxy-2'- fluoromethylene-cytidine (FMdC) and l-(2-deoxy-2-fluoro-4-thio- ⁇ -D- arabinosyl)cytosine (4'-thio-FAC).
• nucleosides may also become active when derivatized according to the invention including, but not limited to Ara-C, Ara-G, 5-fluorouridine, 5-fluoro-deoxyuridine, fludarabine, gemcitabine, decitabine or alkylglycerol phosphate or alkoxyalkyl phosphate esters of taxol.
• Non- nucleoside cancer agents may be similarly derivatized with the metabolically stable alkoxyalkyl groups of the invention including, but not limited to topotecan by phosphorylating and esterifying an available hydroxyl group.
• Etoposide may be derivatized by attaching metabolically stable groups of the invention to the phosphate residue of etoposide.
• Phosphonate and phosphate analogs contemplated for use in accordance with the present invention are selected to improve the bioactivity, selectivity, and/or bioavailability of the antiviral or antiproliferative compounds.
• compositions containing the analogs of the phosphonate compounds described herein there are provided pharmaceutical formulations containing the analogs of the phosphonate compounds described herein.
• therapeutic methods e.g. methods for treating viral infections and methods for treating disorders caused by inappropriate cell proliferation, e.g. cancer and the like.
• Figure 1 illustrates schematically antiviral activation and metabolic inactivation pathways for hexadecyloxypropyl cidofovir (HDP-CDV).
• Figure 2 depicts representative structures of "Metabolism Resistant" lipophilic esters of cidofovir.
• Figure 3 depicts a graph of the % of drug remaining versus time for HDP-
• CDV and 15-methyl-HDP-CDV 15M-HDP-CDV. This figure illustrates that the degradation of branched alkoxyalkyl ester derivative 15M-HDP-CDV by monkey liver fractions is markedly slower than that of the straight chain alkoxyalkyl ester derivative
• the present invention includes chemical methods for synthesizing alkoxyalkyl esters having certain moieties at or near the omega end of the alkyl chain which block or slow degradation and metabolic inactivation.
• the present invention includes terminal or penultimate branched chain, unsaturated and halogen substituted esters of phosphonate compounds, wherein said substituents stabilize these compounds by providing resistance to oxidation.
• Phosphonates, nucleoside phosphonates and nucleoside phosphates having antiviral or anticancer activity are subjects of the invention.
• a or “an” entity refers to one or more of that entity; for example, a phosphonate refers to one or more phosphonates.
• a phosphonate refers to one or more phosphonates.
• the terms “a” or “an”, “one or more” and “at least one” are used interchangeably herein.
• the method of the invention is not limited to any particular isomer and can be extended to the S enantiomer, the R enantiomer or racemic mixtures thereof.
• prodrug refers to derivatives of pharmaceutically active compounds that have chemically or metabolically cleavable groups and become the pharmaceutically active compound by solvolysis or under in vivo physiological conditions.
• purine or pyrimidine base includes, but is not limited to, 6- alkylpurine and N 6 -alkylpurines, N 6 -acylpurines, N 6 -benzylpurine, 6-halopurine, N 6 - acetylenic purine, N -acyl purine, N -hydroxyalkyl purine, 6-thioalkyl purine, N - alkylpurines, 7-deazapurines, N 4 -alkylpyrimidines, N 4 -acylpyrimidines, A- halopyrimidines, N 4 -acetylenic pyrimidines, 4-amino and N 4 -acyl pyrimidines, A- hydroxyalkyl pyrimidines, 4-thioalkyl pyrimidines, thymine, cytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrimidine, uracil, C -alkylpurines, N 6
• ring- expanded and open-ring cogeners of any of the aforementioned purines.
• Functional oxygen and nitrogen groups on the base can be protected as necessary or desired.
• Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl and propionyl, methanesulfonyl, and p- toluenesulfonyl.
• Preferred bases include cytosine, 5-fluorocytosine, uracil, thymine, adenine, guanine, xanthine, 2,6-diaminopurine, 6-aminopurine, 6-chloropurine and 2,6- dichloropurine.
• alkyl refers to a saturated straight or branched hydrocarbon.
• the alkyl group can be optionally substituted with one or more halogens selected from the group consisting of F, Cl, Br or I.
• alkenyl refers to a partially unsaturated straight or branched hydrocarbon.
• the alkenyl group can be optionally substituted with one or more halogens selected from the group consisting of
• protected refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes.
• oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis. Suitable protecting groups are described, for example, in Greene, et al., "Protective Groups in Organic Synthesis,"
• nucleoside phosphonates of the instant invention can be generally represented by the following structures.
• R is selected from the group consisting of -R 1 -O-R 2 , wherein R 1 is selected from the group consisting of an optionally substituted C 1 to C 11 alkyl group and R 2 is selected from the group consisting of a C 6 to Cn alkyl group or a C 6 to Cn alkenyl group; wherein said Ce to Cn alkyl group is substituted with one or more alkyl groups selected from the group including, but not limited to methyl, ethyl, propyl, or cycloalkyl, including, but not limited to cyclopropyl and/or one or more halogens selected from the group consisting of F, Cl, Br and I; and further wherein said C 6 to C 17 alkyl group includes one or more substituents at or near the terminal position of the alkyl group, in particular at the terminal or penultimate position; and wherein said Ce to Cn alkenyl group is optionally-substituted with an alkyl group selected from the group including, but
• B is selected from a purine or pyrimidine base
• A is a counterion selected from the group including, but not limited to H + , Li + , Na + , K + ,
• R is selected from the group of compounds having the general structure:
• R is selected from the group of compounds having the general structure:
• R is selected from the group of compounds having the general structure:
• X is a halogen.
• X is F.
• R is selected from the group consisting of one of the structures set forth in Figure 2.
• derivatized nucleoside phosphonates are analogs of cyclic cidofovir or cidofovir which can be generally represented by the following structures:
• the derivatized nucleoside phosphonates are analogs of 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)-adenine ((S)-HMPMA) which can be generally represented by the following structure:
• cyclic cidofovir, cidofovir and HPMPA included in the present invention include the following compounds: 3-(12-methyltridecyloxy)propyl cyclic cidofovir, 3-(13-methyltetradecyloxy)propyl cyclic cidofovir, 3-(14- methylpentadecyloxy)propyl cyclic cidofovir, 2-(17-methyloctadecyloxy)ethyl cyclic cidofovir, 3-(15-methylhexadecyloxy)propyl cyclic cidofovir, 3-(15- methylhexadecyloxy)ethyl (S)-cyclic HPMPA, 3-(15-methylhexadecyloxy)propyl (S)- cyclic HPMPA, 2-(17-methyloctadecyloxy)ethyl-(S)-cyclic HPMPA, 3-(3(12-methyltride
• nucleoside phosphates and analogs thereof of the instant invention can be generally represented by the following structures.
• R is an alkoxyalkyl group having a structure as defined above and B is a substituted or unsubstituted pyrimidine base or their open ring congeners.
• Representative examples of nucleosides in this group of compounds include, but are not limited to 2'-C-methyl adenosine, 2'-C-methyl guanosine, 7-deaza-2'-methyl adenosine, 2'-C-methyl cytosine.
• Other nucleosides and analogs thereof contemplated for use in accordance with this invention following conversion to their alkoxyalkyl 5'- phosphates or their alkoxyalkylglycerol phosphates are set forth in the references cited in Table 2.
• nucleoside analogs with antiviral activity against hepatitis B which may be converted to their 5'-phosphates, 5'-phosphonates or 5'-methylene phosphonates.
• exemplary nucleosides in this class of compounds include, but are not limited to 3TC, FTC, DAPD, L-FMAU, entecavir, telbivudine and various ⁇ -L-2'- deoxycytidine, ⁇ -L-2'-deoxyadenine and ⁇ -L-2'-deoxythymidine analogs described by Bryant et al. ((Jan. 2001) Antimicrob Agents Chemother 45£l):229-235).
• Phosphates of non-nucleoside antivirals are also subjects of the invention including, but not limited to, zanamivir (Relenza ® ).
• Anticancer agents may also be derivatized according to the method of this invention.
• Some representative compounds include, but are not limited to 2'-deoxy-2'- fluoromethylene-cytidine (FMdC) and l-(2-deoxy-2-fluoro-4-thio- ⁇ -D- arabinosyl)cytosine (4'-thio-FAC).
• nucleosides may also become more metabolically stable when derivatized according to the invention including, but not limited to Ara-C, Ara-G, 5-fluorouridine, 5-fluoro-deoxyuridine, fludarabine, gemcitabine, decitabine or alkylglycerol phosphate or alkoxyalkyl phosphate esters of taxol.
• Non-nucleoside cancer agents may be similarly derivatized with the metabolically stable alkoxyalkyl esters of the invention including, but not limited to topotecan by coupling to an available hydroxyl group.
• Etoposide may be coupled to the metabolically stable alkoxyalkyl esters of the invention by attachment to the phosphate residue of etoposide.
• Tables 1 and 2 provide examples of compounds, which may be subjected to the chemical steps of the invention.
• the references cited in these Tables are hereby incorporated by reference in their entirety.
• Table 1. References citing illustrative phosphonates, nucleoside phosphonates and nucleoside phosphates for use according to the method of this invention
• compositions can be prepared in conventional forms, for example, capsules, tablets, aerosols, solutions, suspensions, or together with carriers for topical applications.
• Pharmaceutical formulations containing compounds of this invention can be prepared by conventional techniques, e.g., as described in Remington's Pharmaceutical Sciences, 1985.
• the pharmaceutical carrier or diluent employed may be a conventional solid or liquid carrier.
• solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, or lower alkyl ethers of cellulose.
• liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene or water.
• the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or distearate, alone or mixed with a wax.
• a solid carrier is used for oral administration, the preparation may be tabletted or placed in a hard gelatin capsule in powder or pellet form.
• the amount of solid carrier will vary widely, but will usually be from about 25 mg to about I g.
• a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule, or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.
• Tablets are prepared by mixing the active ingredient (that is, one or more compounds of the invention), with pharmaceutically inert, inorganic or organic carrier, diluents, and/or excipients.
• excipients which can be used for tablets are lactose, maize, starch or derivatives thereof, talc, stearic acid or salts thereof.
• suitable excipients for gelatin capsules are vegetable oils, waxes, fats, semisolid, and liquid polyols.
• the preparation may contain a compound of the invention dissolved or suspended in a liquid carrier, in particular, an aqueous carrier, for aerosol application.
• a liquid carrier in particular, an aqueous carrier
• the carrier may contain solubilizing agents such as propylene glycol, surfactants, absorption enhancers such as lecithin or cyclodextrin, or preservatives.
• compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or non-aqueous liquids, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
• Suitable excipients for the preparation of solutions and syrups are water, polyols, sucrose, invert sugar, glucose, and the like.
• Suitable excipients for the preparation of injectable solutions are water, alcohols, polyols, glycerol, vegetable oils, and the like.
• the pharmaceutical products can additionally contain any of a variety of added components, such as, for example, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, buffers, coating agents, antioxidants, diluents, and the like.
• the pharmaceutical compositions of the invention may comprise a compound according to the general formula combined with one or more compounds exhibiting a different activity, for example, an antibiotic or other pharmacologically active material. Such combinations are within the scope of the invention.
• This invention provides methods of treating disorders related to viral infections, inappropriate cell proliferation, and the like. The methods particularly comprise administering to a host in need thereof a therapeutically effective amount of the prodrugs of this invention. Thus, in one aspect of the invention there are provided methods for treating disorders caused by viral infections.
• Indications appropriate to such treatment include susceptible viruses including, but are not limited to human immunodeficiency virus (HIV), influenza, herpes simplex virus (HSV), human herpes virus 6 and 8, cytomegalovirus (CMV), hepatitis B and C virus, Epstein-Barr virus (EBV), varicella zoster virus, and diseases caused by orthopox viruses (e.g., variola major and minor, vaccinia, smallpox, cowpox, camelpox, monkeypox, and the like), ebola virus, papilloma virus, and the like, lymphomas, hematological disorders such as leukemia, and the like, and cancers caused by viruses such as cervical cancer which is caused, in most cases, by the high risk subtypes of human papilloma virus.
• HCV human immunodeficiency virus
• HSV herpes simplex virus
• CMV cytomegalovirus
• EBV Epstein-Barr virus
• cancers such as melanoma, lung cancers, pancreatic cancer, stomach, colon and rectal cancers, prostate and breast cancer, the leukemias and lymphomas, and the like.
• Anti-cancer compounds which can be converted to their nucleotide phosphonates or nucleoside-5 '-phosphates for use as compounds of this invention include, but are not limited to, cytarabine (ara-C), fluorouridine, fluorodeoxyuridine (floxuridine), gemcitibine, decitabine, cladribine, fludarabine, pentostatin (2'-deoxycoformycin), 6-mercaptopurine and 6-thioguanine and substituted or unsubstituted ara-adenosine (ara-A), ara-guanosine (ara-G), and ara- uridine (ara-U).
• Anticancer compounds of the invention may be used alone or in combination with other antimetabolites or with other classes of anticancer drugs such as alkaloids, topoisomerase inhibitors, alkylating agents, antitumor antibiotics, and the like.
• the prodrugs of the invention can be administered orally, parenterally, topically, rectally, and through other routes, with appropriate dosage units, as desired.
• parenteral refers to subcutaneous, intravenous, intra-arterial, intramuscular or intravitaeal injection, or infusion techniques.
• topically encompasses administration rectally and by inhalation spray, as well as the more common routes of the skin and mucous membranes of the mouth and nose and in toothpaste.
• “Therapeutic” as used herein, includes treatment and/or prophylaxis. When used, therapeutic refers to humans as well as other animals.
• “Pharmaceutically or therapeutically effective dose or amount” refers to a dosage level sufficient to induce a desired biological result. That result may be the alleviation of the signs, symptoms or causes of a disease or any other alteration of a biological system that is desired.
• a "host” or “patient” is a living subject, human or animal, into which the compositions described herein are administered.
• the "effective amount" is determined with reference to the recommended dosages of the antiviral or anticancer parent compound.
• the selected dosage will vary depending on the activity of the selected compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound(s) at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration, for example, two to four doses per day. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors, including the body weight, general health, diet, time, and route of administration and combination with other drugs, and the severity of the disease being treated.
• the compounds of the present invention are dispensed in unit dosage form comprising 1% to 100% of active ingredient.
• the range of therapeutic dosage is from about 0.01 to about 1,000 mg/kg/day with from about 0.10 mg/kg/day to 100 mg/kg/day being preferred, when administered to patients, e.g., humans, as a drug.
• Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient.
• Compounds of the invention can be prepared in a variety of ways, as generally depicted in Schemes 3 to 7 in Examples 1-9.
• Example 1 (Scheme 1) outlines a general method for the synthesis of branched alkoxyalkanols having the general formula:
• Example 2 (Scheme 2) outlines a general method for the synthesis of branched methylalkoxyalkyl esters from cyclic phosphonates. Cyclic cidofovir was used in this Example for purposes of illustration, however this method can be extended to the use of virtually any cyclic phosphonate of interest.
• Examples 3 and 4 describe the synthesis of two specific branched alkoxyalkyl esters, namely 3-(phytanyloxy)propyl cidofovir and 15-methylhexadecyloxypropyl cidofovir (15-Me HDP-CDV), ammonium, using slight variations of the methods described in Examples 1 and 2.
• Example 5 describes a general method for the synthesis of the branched methylalkoxyalkyl esters of the instant invention from p- toluenesulfonyloxymethyl phosphonates.
• Example 6 (Scheme 5) outlines a general method for the synthesis of alkenyloxyalkyl esters having a terminal double bond. The nucleoside phosphonate cidofovir was used for purposes of illustration resulting in the synthesis of compound 26, hexadec-15-enyl-oxypropyl-cidofovir.
• Examples 7-9 outline general methods for the synthesis of various halogenated alkoxyalkyl esters using CDV and HPMPA for purposes of illustration. Synthesis of the following compounds are exemplified: 3-(15- fluorohexadecyloxy)propyl cidofovir, 3-(15-fluorohexadecyloxy)propyl cyclic cidofovir, 3-(15-fluorohexadecyloxy)propyl-(S)-HPMPA, 3-(15- fluorohexadecyloxy)propyl-(S)-cyclic HPMPA, 3-(16-fluorohexadecyloxy)propyl cidofovir, 3-(16-fluorohexadecyloxy)propyl cyclic cidofovir, 3-(16- fluorohexadecyloxy)propyl-(S)-HPMPA, 3-(16-fluorohexadecy
• Examples 10-12 illustrate the antiviral activity representative penultimate branched methyl alkoxyalkyl esters of CDV and HPMPA.
• the results are set forth in Tables 3-6.
• penultimate branched chain analogs of (S)-HPMPA were highly active against vaccinia and cowpox in vitro and penultimate branched chain alkoxyalkyl cidofovir esters were effective against ectromelia virus in vitro at submicromolar EC50s.
• the branch chain analogs of (S)-HPMPA and CDV were all fully protective against death from lethal poxvirus infection at doses of 5 mg/kg/day or greater. 15M-HDP-(S)-HPMPA appeared to be more active than HDP-(S)-HPMPA.
• FIG. 13 The stability of the compounds of the invention is illustrated in Example 13, the data for which is set forth in Figure 3.
• Figure 3 depicts a graph of the % of drug remaining versus time for HDP-CDV and 15-methyl-HDP-CDV (15M-HDP-CDV) after incubation for various times as indicated in the presence of liver S 9 preparations. This figure illustrates that the degradation of branched alkoxyalkyl ester derivative 15M-HDP-CDV by monkey liver and human liver S 9 fractions is markedly slower than that of the straight chain alkoxyalkyl ester derivative HDP-CDV.
• Reagents a) magnesium, THF; b) Li 2 CuCl 4 , THF; c) methanesulfonyl chloride, triethylamine, CH 2 Cl 2 ; d) 1,2-ethanediol or 1,3-propanediol, NaH, N 5 N-DMF
• 12-methyltridecan-l-ol was prepared from 9-bromononan-l-ol and 3- methylbutylbromide in 51% yield.
• the 1 H NMR and MS-ESI data matched those reported by Yuasa et al. (Yuasa and Tsuruta (2004) Flavour and Fragrance Journal 19:199-204).
• 13-methyltetradecan-l-ol was prepared from 10-bromodecanol and 3- methylbutylbromide in 62% yield.
• the 1 H NMR and MS-ESI data matched those reported by Yuasa et al. (Yuasa and Tsuruta (2004) Flavour and Fragrance Journal
• 14-methylpentadecan-l-ol was prepared from 9-bromononanol and 5- methylhexylbromide in 55% yield.
• the 1 H NMR and MS-ESI data matched those reported by Yuasa et al. (Yuasa and Tsuruta (2004) Flavour and Fragrance Journal
• 15-methylhexadecan-l-ol was prepared from 12-bromo-l-dodecanol and 3- methylbutylbromide.
• the 1 H NMR was identical to that reported by Masuda et al.
• 17-methyloctadecan-l-ol was prepared from 12-bromo-l-dodecanol and 5- methylhexylbromide in 44% yield.
• branched alkoxyalkanols (6) Preparation of branched alkoxyalkanols (6).
• branched alkoxyalkanols were prepared by conversion of the branched methyl alkanols (4) to the corresponding methanesulfonate derivatives (5), followed by reaction with either 1,3-propanediol or 1,2-ethanediol.
• Branched methylalkoxyalkyl esters were prepared from cyclic phosphonates as shown in Scheme 2 using cyclic cidofovir for purposes of illustration. Briefly, the cyclic phosphonates were coupled to branched methylalkoxyalkanols using the Mitsunobu reaction as described by Wan et al. ((2005) Antimicrobial Agents and Chemotherapy 49:656-662) to form the cyclic diesters which were then hydrolyzed to form the branched methylalkoxyalkyl esters.
• Reagents a) triphenylphosphine, diisopropyl azodicarboxylate, N 5 N-DMF; b) 2 M aq. NaOH, 8O 0 C
• reaction mixture was heated at 8O 0 C for 2 hrs at which time the TLC showed complete consumption of 11.
• the reaction was then cooled to room temperature and quenched with dropwise addition of saturated aq. NH 4 Cl. 200 mL of water was added and the target product was extratcted with EtOAc (3x), the combined organic layer was successively washed with water (3x), brine (Ix) and dried over MgSO 4 .
• the solvent was evaporated under reduced pressure and the residue was purified by combiflash using hexane/ethyl acetate as an eluent to furnish 23 g of 12 as colorless oil in 85% yield.
• Branched methyl esters were prepared from the p- toluenesulfonyloxymethylphosphonates 18 as illustrated in Scheme 4, using synthesis of (S)-HPMPA esters for purposes of illustration. The procedure is based on the method reported by Beadle et ah, J. Med. Chem. 49:2010-2015, 2006.
• Reagents a) NaH, N,N-DMF, 50 0 C; b) 80% aq. acetic acid
• (S)-9-[3-trityloxy-2-hydroxypropyl]-N6-trityl- adenine 17 was prepared from adenine and (S)-trityl glycidyl ether (Daiso Co., Ltd., Japan) following the method of Webb ((1989) Nucleosides & Nucleotides 8:619-624). Sodium hydride (24 mg, 1.0 mmol) was added to a stirred solution of (S)-9-[3-trityloxy- 2-hydroxypropyl]-N 6 -trityladenine (640 mg, 0.62 mmol) in dry triethylamine (10 mL).
• 2- bromopropanoic acid 35 is reduced to the alcohol with borane:THF complex solution to provide 2-bromo-l-propanol 36.
• Fluorination of 36 is achieved with l,l,2-trifluoro-2- chloroethyldiethylamine, a mild and safe reagent to convert l-hydroxy-2- halogenoalkanes into the corresponding rearranged fluoride 37.
• Conversion of 37 into a Grignard reagent followed by reaction with 13-bromo-tridecanol in the presence of the catalyst provides 15-fluorohexadecanol 38.
• step a Conversion of alcohol 38 into the methanesulfonate derivative, followed by reaction with 1,3-propanediol provides 3-(15- fluorohexadecyloxy)propan-l-ol 39.
• step b Reaction of 39 with cyclic cidofovir or cyclic (S)- HPMPA as described generally in Example 2 (step a), provides the cyclic esters (40 and 41, respectively) which can then be converted to the desired compounds (42 and 43, respectively) using the general method set forth in Example 2 (step b).
• Reagents a) BH 3 , THF; b) fluoroamine; c) Mg/13-bromo-tridecanol, Li 2 CuCl 4 ; d) methanesulfonyl chloride, pyridine, then 1,3 -propanediol, NaH, N 5 N-DMF; e) cCDV or cHPMPA, DIAD, PPh 3 , N 5 N-DMF; f) 1 M NaOH
• the Grignard reagent prepared from l-bromo-4-fluorobutane and magnesium is reacted with 12-bromododecanol 44 to obtain 16-fluorohexadecanol 45.
• Reaction of 45 with methanesulfonyl chloride followed by reaction with 1,3-propanediol provides 16-fluorohexadecyloxy-l-propanol 46.
• step a Coupling of 46 with cyclic cidofovir or cyclic (S)-HPMPA as described generally in Example 2 (step a), provides the cyclic esters (47 and 48, respectively) which can then be converted to the desired compounds (49 and 50, respectively) using the general method set forth in Example 2 (step b).
• Reagents a) Mg/l-bromo-4-fluorobutane, Li 2 CuCl 4 , THF; b) methanesulfonyl chloride, pyridine; c) 1,3-propanediol, NaH, N 5 N-DMF; d) cCDV or cHPMPA, DIAD, PPh 3 , N 5 N-DMF; e) IM NaOH
• the reaction mixture was stirred for 3 h after which it was diluted with 100 mL of DCM and successively washed with saturated solution of NaHCO 3 , H 2 O and brine, dried over MgSO 4 , filtered and evaporated to dryness.
• the residue was purified using combiflash (120 g silica column) with hexane/ethyl acetate as solvents to furnish 18 g (90%) of the target product 52 as colorless oil.
• reaction was then cooled in an ice bath and quenched with a solution of saturated aq. NH 4 Cl. 300 mL of water was then added and reaction mixture was extracted with DCM (4x). The combined DCM layers were then washed with H 2 O (3x), brine (Ix), dried over MgSO 4 , filtered and the solvent was evaporated under vacuum. The residue was then purified using combiflash (120 g silica column) with hexane/ethyl acetate as eluent to furnish 12.86 g (72%) of the target product 54 as colorless oil.
• Example 10 Evaluation of antiviral activity of penultimate branched methyl alkoxyalkyl analogs of cidofovir (CDV) and 9-(S)-(3-hydroxy-2- phosphonomethoxypropyP-adenine ((S)-HPMPA) against vaccinia virus and cowpox virus in vitro
• Virus pool preparation The vaccinia virus strain, Copenhagen, and cowpox virus, strain Brighton, stock pools were obtained from John Huggins of the U.S. Army Medical Research Institute for Infectious Diseases, Frederick, Md. These pools were prepared in Vero cells and were diluted 1:50 to provide working stocks.
• Plaque reduction assay for efficacy Two days prior to use, HFF cells were plated on six-well plates and incubated at 37°C with 10% CO 2 and 90% humidity. On the day of the assay, the drugs were made up at twice the desired concentration in 2x minimal essential medium (MEM) containing 5% fetal bovine serum (FBS) and antibiotics and diluted serially 1:5 in 2x MEM to provide six concentrations of drug.
• MEM minimal essential medium
• FBS fetal bovine serum
• the initial starting concentration was usually 200 ⁇ M and ranged down to 0.06 ⁇ M.
• the virus to be used was diluted in MEM containing 10% FBS to a desired concentration which would give 20 to 30 plaques per well.
• the medium was then aspirated from the wells, and 0.2 mL of virus was added to each well in triplicate, with 0.2 mL of medium being added to drug toxicity wells.
• the plates were incubated for 1 h with shaking every 15 min. After the incubation period, an equal amount of 1% agarose was added to an equal volume of each drug dilution. This gave final drug concentrations beginning with 100 ⁇ M and ending with 0.03 ⁇ M and a final agarose overlay concentration of 0.5%.
• the medium was aspirated and 125 ⁇ L of drug was added to the first row of wells and then diluted serially 1:5 using the Beckman BioMek liquid-handling system. After the addition of the drug, the plates were incubated for 7 days in a CO 2 incubator at 37°C. At that time, the medium with drug was aspirated, and 200 ⁇ l of 0.01% neutral red in phosphate-buffered saline (PBS)/well was added and incubated for 1 h. The dye was aspirated, and the cells were washed with PBS using a Nunc plate washer. After the PBS was removed, 200 ⁇ L of 50% ethanol-1% glacial acetic acid (in H 2 0)/well was added.
• PBS phosphate-buffered saline
• CDV cidofovir
• HDP-CDV hexadecyloxypropyl-CDV
• 17M-ODE-CDV 17-methyl- octadecyloxyethyl cidofovir
• 15M-HDP-CDV 15-methyl-hexadecyloxypropyl cidofovir
• 14M-PDP- CDV 14-methyl-pentadecyloxypropyl cidofovir
• 13M-TDP-CDV 13-methyl-tetradecyloxypropyl cidofovir
• 12M-TrDP-CDV 12-methyl-tridecyloxypropyl cidofovir
• BS-C-I cells (ATCC CCL 26) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine serum fetal clone III (Hyclone, Logan, UT), 2 mM L-glutamine (GIBCO, Grand Island, NY), 100 U/mL penicillin (GIBCO), and 100 ⁇ g/mL streptomycin (GIBCO).
• DMEM Dulbecco's modified Eagle's medium
• a plaque-purified isolate of the MOS strain of ECTV (ATCC VR-1374) designated MOS-3-P2 was propagated in an African green monkey kidney cell line, BS-C-I. Virus was purified through a sucrose cushion.
• Virus suspensions were serially diluted in PBS + 1% sera, absorbed to monolayers for 1 h at 37°C, and overlayed with a suspension of 1% carboxyl methyl cellulose in DMEM +5% Fetal clone III. After 4 days at 37°C, virus plaques were visualized and virus inactivated by the addition to each well of 0.5 mL of a 0.3% crystal violet/10% formalin solution.
• Plaque reduction assay Plaque reduction assay. CV-I cells were plated in wells of a 24- well cluster plate. Each monolayer was infected with 75 plaque forming units (PFU) of indicator virus in 0.1 mL of DMEM +5% Fetal clone III for 60 min at 37 0 C. Media was removed by aspiration and standard virus overlay media containing no drug or the test drug at concentrations ranging from 0.05 to 50 ⁇ M was added. The plates were incubated at 37°C for 3-4 days for ECTV and 2 days for VACV-WR, monolayers were stained, and plaques counted using a stereomicroscope. The EC 50 concentration for each drug was calculated. The results are set forth in Table 5.
• Example 12 Activity of oral 15M-HDP-(S)-HPMPA in lethal ectromelia virus infection
• mice were injected to effect with an anesthetic cocktail (ketamine 90 mg/kg / xylazine 10 mg/kg), held at 45° C from the vertical on a intubation platform, and inoculated with 5 ⁇ l of virus suspension in each naris for a total challenge dose of 140 PFU (-280 xLD50). Approximately 2.5 min following inoculation of virus, the mice were returned to their cage. Four hours following exposure to ECTV, groups of mice were treated by gavage with 0.1 ml of sterile, distilled water alone or water containing the test compound. This treatment was repeated on days 1, 2, 3, and 4 for a total of five doses. The mice were observed over 21 days for clinical signs of disease (morbidity) and mortality. The results are set forth in Table 6.
• HDP hexadexyloxypropyl
• 15M-HDP 15-methyl-hexadecyloxypropyl
• 13M-TDP 13-methyl-tetradecyloxypropyl
• 14M-PDP 14-methyl-pentadecyloxypropyl.
• MDD Mean day of death
• Target compounds were incubated in pooled S9 liver fractions from monkey and human liver (purchased from a commercial source) at 37 0 C.
• the control sample (immediately quenched) was used to determine the response at time zero.
• the ratio between the response of the incubated samples and time zero indicated the % parent compound remaining.
• Compounds were dissolved in DMSO and serially diluted with buffer to a concentration suitable for the assay (1 to 10 ⁇ M). A portion of a high concentration dilution (-500 mM) was used to determine the LC-MS/MS conditions for analysis (ionization polarity, SRM transition, collision energy). 7-ethoxycoumarin was included as a control.
• Negative controls (without S9) were included to check the stability of the test compounds at incubation conditions. All assays were performed in triplicate and the % parent compound remaining was reported. Incubations were generally performed in microtiter plates with a protein concentration of 3 mg/mL and a compound concentration of 1 ⁇ M. Reactions were sampled at the specified time points and stopped by the addition of a cold acetonitrile/water solution. The quenched plates were centrifuged and subsequently analyzed by fast gradient LC-MS/MS. The results are set forth in Figure 3.

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