WO2006114065A2 - Use of compounds to inhibit neoplasia - Google Patents

Use of compounds to inhibit neoplasia Download PDF

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WO2006114065A2
WO2006114065A2 PCT/CZ2006/000024 CZ2006000024W WO2006114065A2 WO 2006114065 A2 WO2006114065 A2 WO 2006114065A2 CZ 2006000024 W CZ2006000024 W CZ 2006000024W WO 2006114065 A2 WO2006114065 A2 WO 2006114065A2
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alkyl
compound
prodrug
alkynyl
alkenyl
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PCT/CZ2006/000024
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WO2006114065A3 (en
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Miroslav Hajek
Nadezda Matulova
Ivan Votruba
Antonin Holy
Eva Tloustova
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Institute Of Organic Chemistry And Biochemistry A Cademy Of Sciences Of The Czech Republic
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Publication of WO2006114065A3 publication Critical patent/WO2006114065A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • telomerase Human telomerase is a large cellular ribonucleoprotein complex (Morin GB, Cell, 1989, 59, 521-9; Wenz C, et al., EMBO J 2001;20:3526-34; and Collins K,
  • telomere telomerase As a functional catalytic reverse transcriptase prompted studies of inhibiting telomerase with established HIV reverse transcriptase inhibitors, such as the chain terminating 3'- azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) (Nakamura TM, et al., Science, 1997, 277, 955-9; Blackburn EH, Annu Rev Biochem, 1992, 61, 113-29; and Cech TR, et al., Biochemistry (Moscow), 1997, 62, 1202-5).
  • ZTT chain terminating 3'- azido-3'-deoxythymidine
  • ddC 2',3'-dideoxycytidine
  • telomeres are transformed in vivo in the form of triphosphates, which inhibit reverse transcriptases by acting as competitive substrates for the enzyme and terminate DNA synthesis de novo, AZT-TP inhibits the activity of telomerase to ⁇ 50% at 30 ⁇ mol/L.
  • Other nucleotide-based inhibitors both purine and pyrimidine derivatives, have been evaluated against telomerase with effective IC 50 values in the micro-molar range.
  • Fletcher reported the use of deazadeoxypurines as inhibitors of telomerase with one compound, 6-thio-7-deaza-2'- deoxyguanosine 5 '-triphosphate showing an IC 50 value of 60 nmol/L.
  • telomere activity has previously been shown to inhibit telomerase activity (see Morin GB, Cell, 1989, 59, 521-9; Strahl C, Blackburn EH., MoI Cell Biol, 1996, 16, 53-65; Pai RB, et al., Cancer Res, 1998, 58, 1909-13; Fletcher TM 5 et al., Bioorg Chem, 2001, 29, 36-55; Tendian SW, Parker WB., MoI Pharmacol, 2000, 57, 695-9; Strahl C, Blackburn EH., Nucleic Acids Res, 1994, 22, 893-900; Fletcher TM, et ⁇ Biochemistry, 1996, 35, 15611-7; and Raymond E, et al,Curr Opin
  • S- HPMPC cidofovir, CDV, Vistide ®
  • ⁇ -PMPA tenofovir, TDV, Viread ®
  • PMEA adefovir, ADV, Hepsera ®
  • the invention provides the use of an acyclic nucleoside purine of formula I:
  • X is OH or -N(R ⁇ 2 wherein R 1 independently is, H;
  • C 2 -C 15 alkyl C 3 -C 15 alkenyl, C 6 -C 15 arylalkenyl, C 3 -C 15 alkynyl, C 7 -C 15 arylalkynyl, Ci-Ce-alkylamino-Ci-Ce alkyl, C 5 -C 15 aralkyl, C 6 -C 15 heteroalkyl or C 3 -C 6 heterocycloalkyl wherein methylene in an alkyl moiety not adjacent to NH has been replaced by ⁇ O— ; or
  • R 1 are joined together to form a saturated or unsaturated C 2 -C 5 heterocycle containing one or two N heteroatoms and optionally an additional O or S heteroatom; wherein one of the foregoing R 1 groups can be substituted with halo, -CN or -N 3 ;
  • W is H or NH 2 ;
  • Y independently is, OH, -OR 2 , -OCH(R 3 )OC(O)R 2 , a monophosphate, a diphosphate, an amino acid amidate, a polypeptide amidate, -NHR 2 , or -N(R 2 ) 2 ;
  • R 2 independently is unsubstituted alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl wherein H is substituted by halo, carboxy, hydroxyl, cyano, nitro, N- morpholino, or amino; or alkyl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl in which a -CH 2 - moiety has been substituted by NH, S, or O;
  • Z is C 1 -C 6 alkyl, optionally substituted with one or more hydroxy; and R 3 is H or R 2 ; or a prodrug, diphosphate, pharmaceutically acceptable salt, or other phosphorus- substituted derivative thereof, in the manufacture of a medicament for inhibiting an enzyme that causes neoplasia inan animal (e.g. a human).
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I, or a prodrug, pharmaceutically acceptable salt, or other phosphorus-substituted derivative thereof, and a pharmaceutically acceptable excipient.
  • the invention also provides the use of PMEODAPy or a prodrug, diphosphate, pharmaceutically acceptable salt, or other phosphorus-substituted derivative thereof in the manufacture of a medicament for inhibiting an enzyme that causes neoplasia in an animal.
  • the invention also provides a method for inhibiting an enzyme that causes neoplasia comprising contacting the enzyme (in vitro or in vivo) with a compound of formula I or a prodrug, pharmaceutically acceptable salt, or other phosphorus- substituted derivative thereof.
  • the invention also provides a method for treating neoplasia in an animal comprising administering an effective amount of a compound of formula I or a prodrug, pharmaceutically acceptable salt, or other phosphorus-substituted derivative thereof to the animal.
  • the invention also provides novel compounds of formula I described herein as well as prodrugs and pharmaceutically acceptable salts thereof.
  • Figure 1 Illustrates the structure of PME, PMEO, PMP, and HPMP purines and pyrimidines.
  • Figure 2 Illustrates the inhibition of HL-60 telomerase by PMEGpp as measured in Example 1.
  • Figure 3 Illustrates the enhancement of telomerase processivity by 6- Me 2 PMEDAPpP (A) and (S)-PMP App measured in Example 1.
  • Figure 4 Illustrates inhibition of HL-60 telomerase by guanine derivatives PMEGpp (1), (£)-PMPGpp (2), (i?)-HPMPGpp (3), (S)-PMPGpp (4) and (B) illustrates inhibition by adenine and 2,6-diaminopurine derivatives PMED APpp (5), OS)-HPMPAp ⁇ (6), PMEO-DAPypp (7) and (i?)-6-cyprPMPDAPpp (8).
  • the enzyme activity was measured in HL-60 cell extract in the presence of 125 ⁇ mol/L dNTPs.
  • Diphosphates of the antiviral acyclic nucleoside phosphonates were evaluated in telomeric repeat amplification protocol (TRAP) (Kim NW, et al., Science, 1994, 266, 2011-5; Wright WE, Nucleic Acids Res, 1995, 23, 3794-5; and Kim NW, Wu F., Nucleic Acids Res, 1997, 25, 2595-7) for their ability to inhibit the extension of telomeres by human telomerase, using extracts from human leukaemia HL-60 cells as a source of the enzyme. Extracts from human leukaemia HL-60 cells were used as a source of the enzyme.
  • ANP acyclic nucleoside phosphonate
  • ANPpp ANP diphosphate
  • TRAP telomeric repeat amplification protocol
  • PMEG 9-[2-(phos ⁇ honomethoxy)ethyl]guanine
  • i ⁇ -PMPG, (i?)-9-[2- (phosphonomethoxy)propyl] -guanine
  • i.)-HPMPG (R)-9-[(3 -hydroxy-2- phosphonomethoxy)propyl]guanine
  • PMEDAP 2,6-diamino-9-[2-(phosphono- methoxy)ethyl]purine
  • S)-PMPG (S)-9-[2-(phosphonomethoxy)propyl]guanine
  • S)- HPMPA (S)-9-[(3-hydroxy-2-phosphonomethoxy)propyl]adenine
  • PMEO-DAPy 2,4- diamin
  • prodrug refers to any compound that when administered to a biological system generates the drug substance, i.e. active ingredient, as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), photolysis, and/or metabolic chemical reaction(s).
  • a prodrug is thus a covalently modified analog or latent form of a therapeutically-active compound.
  • Prodrug moiety refers to a labile functional group which separates from the active inhibitory compound during metabolism, systemically, inside a cell, by hydrolysis, enzymatic cleavage, or by some other process (Bundgaard, Hans, “Design and Application of Prodrugs” in A Textbook of Drug Design and Development (1991), P.
  • Enzymes which are capable of an enzymatic activation mechanism with the phosphonate compounds include, but are not limited to, amidases, esterases, microbial enzymes, phospholipases, cholinesterases, and phosphases.
  • Prodrug moieties can serve to enhance solubility, absorption and lipophilicity to optimize drug delivery, bioavailability and efficacy.
  • a prodrug moiety may include an active metabolite or drug itself.
  • the acyloxyalkyl ester was first used as a prodrug strategy for carboxylic acids and then applied to phosphates and phosphonates by Farquhar et al. (1983) J Pharm. Sd. 72: 324; also US Patent Nos. 4816570, 4968788, 5663159 and 5792756.
  • acyloxyalkyl ester was used to deliver phosphonic acids across cell membranes and to enhance oral bioavailability.
  • a close variant of the acyloxyalkyl ester, the alkoxycarbonyloxyalkyl ester (carbonate), may also enhance oral bioavailability as a prodrug moiety in the compounds of the combinations of the invention.
  • Aryl esters of phosphorus groups are reported to enhance oral bioavailability (De Lombaert et al. (1994) J. Med. Chem. 37: 498). Phenyl esters containing a carboxylic ester ortho to the phosphate have also been described (Khamnei and Torrence, (1996) J. Med. Chem. 39:4109-4115). Benzyl esters are reported to generate the parent phosphonic acid. In some cases, substituents at the ort/zo-or p ⁇ r ⁇ -position may accelerate the hydrolysis.
  • Benzyl analogs with an acylated phenol or an alkylated phenol may generate the phenolic compound through the action of enzymes, e.g., esterases, oxidases, etc., which in turn undergoes cleavage at the benzylic C-O bond to generate the phosphoric acid and the quinone methide intermediate.
  • enzymes e.g., esterases, oxidases, etc.
  • this class of prodrugs are described by Mitchell et al. (1992) J. Chem. Soc. Perkin Trans. II 2345; Glazier WO 91/19721.
  • Still other benzylic prodrugs have been described containing a carboxylic ester-containing group attached to the benzylic methylene (Glazier WO 91/19721).
  • Thio-containing prodrugs are reported to be useful for the intracellular delivery of phosphonate drugs.
  • These proesters contain an ethylthio group in which the thiol group is either esterified with an acyl group or combined with another thiol group to form a disulfide. Deesterification or reduction of the disulfide generates the free thio intermediate which subsequently breaks down to the phosphoric acid and episulfide (Puech et al. (1993) Antiviral Res., 22: 155-174; Benzaria et al. (1996) J. Med. Chem. 39: 4958). Cyclic phosphonate esters have also been described as prodrugs of phosphorus-containing compounds (Erion et al., US Patent No. 6312662).
  • Examples of pharmaceutically acceptable salts include salts derived from an appropriate base, such as an alkali metal (for example, sodium), an alkaline earth (for example, magnesium), ammonium and NX 4 + (wherein X is C 1 -C 4 alkyl).
  • an alkali metal for example, sodium
  • an alkaline earth for example, magnesium
  • ammonium and NX 4 + (wherein X is C 1 -C 4 alkyl).
  • Pharmaceutically acceptable salts of a compound having an amino group include salts of organic carboxylic acids such as acetic, benzoic, lactic, fumaric, tartaric, maleic, malonic, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids; and inorganic acids, such as hydrochloric, sulfuric, phosphoric and sulfamic acids.
  • organic carboxylic acids such as acetic, benzoic, lactic, fumaric, tartaric, maleic, malonic, malic, isethionic, lactobionic and succinic acids
  • organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids
  • inorganic acids such as
  • salts of a compound having a hydroxy group include the anion of said compound in combination with a suitable cation such as Na + and NX 4 + (wherein X is independently selected from H or a C 1 -C 4 alkyl group).
  • a suitable cation such as Na + and NX 4 + (wherein X is independently selected from H or a C 1 -C 4 alkyl group).
  • salts of active ingredients will typically be pharmaceutically acceptable, i.e. they will be salts derived from a pharmaceutically acceptable acid or base.
  • Alkyl is branched or unbranched hydrocarbon containing normal, secondary, or tertiary carbon atoms. Examples are methyl (Me,-CH3), ethyl (Et,-CH2CH3), 1- propyl (n-Pr, n-propyl,-CH2CH2CH3), 2-pro ⁇ yl (i-Pr, i-propyl,-CH(CH3)2), 1 -butyl (n-Bu, n-butyl,-CH2CH2CH2CH3), 2-methyl-l -propyl (i-Bu, B>utyl,-CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl,-CH(CH3)CH2CH3), 2-methyl-2- propyl (fcBu, t-butyl,-C(CH3)3), 1-pentyl (n-pentyl,-CH2CH2CH2CH3), 2-pentyl (-CH(CH(CH)CH
  • Aryl means a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system.
  • Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like.
  • Alkyl refers to an alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an aryl radical.
  • Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan- 1-yl, , naphthylmethyl, 2-naphthylethan-l-yl, naphthobenzyl, 2-naphthophenylethan-l- yl and the like.
  • the arylalkyl group comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.
  • Arylalkenyl refers to an alkenyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an aryl radical.
  • Arylalkynyl refers to an alkynyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an aryl radical.
  • Heterocycle or “Heterocyclo” as used herein includes by way of example and not limitation these heterocycles described in Paquette, Leo A.; Principles of Modern Heterocyclic Chemistry (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs” (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.
  • heterocycle includes a “carbocycle” as defined herein, wherein one or more (e.g. 1, 2, 3, or 4) carbon atoms have been replaced with a heteroatom (e.g. O, N, or S).
  • a heteroatom e.g. O, N, or S
  • heterocycles include by way of example and not limitation pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,
  • treatment or "treating,” to the extent it relates to a disease or condition includes preventing the disease or condition from occurring, inhibiting the disease or condition, eliminating the disease or condition, and/or relieving one or more symptoms of the disease or condition.
  • Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane- polarized light.
  • the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s).
  • the prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory.
  • a compound prefixed with (+) or d is dextrorotatory.
  • these stereoisomers are identical except that they are mirror images of one another.
  • a specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
  • racemic mixture A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
  • racemic mixture and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
  • Protecting groups are available, commonly known and used, and are optionally used to prevent side reactions with the protected group during synthetic procedures, i.e. routes or methods to prepare the compounds. For the most part the decision as to which groups to protect, when to do so, and the nature of the chemical protecting group "PG" will be dependent upon the chemistry of the reaction to be protected against (e.g., acidic, basic, oxidative, reductive or other conditions) and the intended direction of the synthesis. The PG groups do not need to be, and generally are not, the same if the compound is substituted with multiple PG. In general, PG will be used to protect functional groups such as carboxyl, hydroxyl, thio, or amino groups and to thus prevent side reactions or to otherwise facilitate the synthetic efficiency.
  • Suitable amino acids whose residues can be represented by Y in Formula I include the following: Glycine; Aminopolycarboxylic acids, e.g., aspartic acid, ⁇ -hydroxyaspartic acid, glutamic acid, ⁇ -hydroxyglutamic acid, ⁇ -methylaspartic acid, ⁇ -methylglutamic acid, ⁇ , ⁇ -dimethylaspartic acid, ⁇ -hydroxyglutamic acid, ⁇ , ⁇ - dihydroxy glutamic acid, ⁇ -phenylglutamic acid, ⁇ -methyleneglutamic acid, 3- aminoadipic acid, 2-aminopimelic acid, 2-aminosuberic acid and 2-aminosebacic acid; Amino acid amides such as glutamine and asparagine; Polyamino- or polybasic- monocarboxylic acids such as arginine, lysine, ⁇ -aminoalanine, ⁇ -aminobutyrine, ornithine, citruline, homoarg
  • Diaminodicarboxylic acids such as ⁇ , ⁇ '-diaminosuccinic acid, ⁇ , ⁇ '-diaminoglutaric acid, ⁇ , ⁇ '-diaminoadipic acid, ⁇ , ⁇ '-diaminopimelic acid, ⁇ , ⁇ '-diamino- ⁇ -hydroxypimelic acid, ⁇ , ⁇ '-diaminosuberic acid, ⁇ , ⁇ '-diaminoazelaic acid, and ⁇ , ⁇ '-diaminosebacic acid; Imino acids such as proline, hydroxyproline, allohydroxyproline, ⁇ -methylproline, pipecolic acid, 5-hydroxypipecolic acid, and azetidine-2-carboxylic acid; A mono- or di-alkyl (typically Ci-Cs branched or normal) amino acid such as alanine, valine, leucine, allylglycine, butyrine, norvaline, nor
  • Aliphatic ⁇ -amino- ⁇ -hydroxy acids such as serine, ⁇ -hydroxyleucine, ⁇ -hydroxynorleucine, ⁇ -hydroxynorvaline, and ⁇ -amino- ⁇ -hydroxystearic acid; ⁇ -Amino, ⁇ -, ⁇ -, ⁇ - or ⁇ -hydroxy acids such as homoserine, ⁇ -hydroxynorvaline, ⁇ -hydroxynorvaline and ⁇ -hydroxynorleucine residues; canavine and canaline; ⁇ -hydroxyornithine; 2-hexosaminic acids such as D-glucosaminic acid or D- galactosaminic acid; ⁇ -Amino- ⁇ -thiols such as penicillamine, ⁇ -thiolnorvaline or ⁇ -thiolbutyrine; Other sulfur containing amino acid residues including cysteine; homocystine, ⁇ -phenylmethionine, methionine, S-allyl-L-c
  • Polypeptides are polymers of amino acids in which a carboxyl group of one amino acid monomer is bonded to an amino or imino group of the next amino acid monomer by an amide bond.
  • Polypeptides include dipeptides, low molecular weight polypeptides (about 1500-5000 MW) and proteins. Proteins optionally contain 3, 5, 10, 50, 75, 100 or more residues, and suitably are substantially sequence-homologous with human, animal, plant or microbial proteins. They include enzymes (e.g., hydrogen peroxidase) as well as immunogens such as KLH, or antibodies or proteins of any type against which one wishes to raise an immune response. The nature and identity of the polypeptide may vary widely.
  • Peptidolytic enzymes for cleaving polypeptide conjugates are well known, and in particular include carboxypeptidases.
  • Carboxypeptidases digest polypeptides by removing C-terminal residues, and are specific in many instances for particular C-terminal sequences.
  • Such enzymes and their substrate requirements in general are well known.
  • a dipeptide (having a given pair of residues and a free carboxyl terminus) is covalently bonded through its ⁇ -amino group to the phosphorus or carbon atoms of the compounds herein.
  • Suitable dipeptidyl groups include AA, AR, AN, AD, AC, AE, AQ, AG, AH, AI, AL 5 AK, AM, AF, AP, AS 5 AT, AW 5 AY 5 AV 3 RA, RR, RN, RD, RC 5 RE, RQ 5 RG 5 RH, RI 5 RL 5 RK 5 RM 5 RF 5 RP, RS 5 RT 5 RW, RY 5 RV 5 NA, NR 5 NN 5 ND 5 NC 5 NE, NQ, NG 5 NH, NI 5 NL 5 NK, NM 5 NF 5 NP, NS 5 NT, NW 5 NY, NV, DA, DR 5 DN 5 DD, DC 5 DE, DQ
  • Tripeptide residues are also useful as protecting groups.
  • Dipeptide or tripeptide species can be selected on the basis of known transport properties and/or susceptibility to peptidases that can affect transport to intestinal mucosal or other cell types.
  • Dipeptides and tripeptides lacking an ⁇ -amino group are transport substrates for the peptide transporter found in brush border membrane of intestinal mucosal cells (Bai, J.P.F., (1992) Pharm Res. 9:969-978).
  • Transport competent peptides can thus be used to enhance bioavailability of the compounds.
  • Dior tripeptides having one or more amino acids in the D configuration are also compatible with peptide transport and can be utilized in the compounds.
  • Amino acids in the D configuration can be used to reduce the susceptibility of a di- or tripeptide to hydrolysis by proteases common to the brush border such as aminopeptidase N.
  • di- or tripeptides alternatively are selected on the basis of their relative resistance to hydrolysis by proteases found in the lumen of the intestine.
  • tripeptides or polypeptides lacking asp and/or glu are poor substrates for aminopeptidase A
  • di- or tripeptides lacking amino acid residues on the N-terminal side of hydrophobic amino acids are poor substrates for endopeptidase
  • peptides lacking a pro residue at the penultimate position at a free carboxyl terminus are poor substrates for carboxypeptidase P.
  • Similar considerations can also be applied to the selection of peptides that are either relatively resistant or relatively susceptible to hydrolysis by cytosolic, renal, hepatic, serum or other peptidases.
  • Such poorly cleaved polypeptide amidates are immunogens or are useful for bonding to proteins in order to prepare immunogens.
  • a specific acyclic nucleoside purine is a compound of formula 1 :
  • n 2 or 3;
  • R 1 independently is H
  • Y independently is, OH,-OR 2 ,-OCH(R 3 )OC(O)R 2 , a monophosphate, a diphosphate, an amino acid amidate, a polypeptide amidate,-NHR 2 , or-N(R 2 ) 2 ;
  • R 2 independently is unsubstituted alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl wherein H is substituted by halo, carboxy, hydroxyl, cyano, nitro, N- morpholino, or amino; or alkyl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl in which a-CH 2 - moiety has been substituted by NH, S, or O;
  • R 3 is H or R 2 ; or a prodrug, diphosphate, or other phosphorus-substituted derivative thereof.
  • Another specific acyclic nucleoside purine of formula I is a compound of the following formula:
  • Another specific acyclic nucleoside purine is a compound of the following formula:
  • each R 1 independently is H, C 1 -C 15 alkyl, C 2 -C 15 alkenyl, C 2 -C 15 alkynyl, or C 3 -C 8 cycloalkyl; or optionally both R 1 are joined together to form a saturated or unsaturated C 2 -C 5 heterocycle containing one or two N heteroatoms and optionally an additional O or S heteroatom.
  • R 1 independently is H, methyl, or cyclopropyl.
  • a specific value for each R 1 independently is H.
  • a specific value for each R 1 independently is methyl.
  • a specific value for one R 1 is H and the other R 1 is cyclopropyl.
  • a specific value for W is H.
  • a specific value for W is NH 2 .
  • a specific value for X is OH.
  • a specific value for Z is -CH 2 -CH 2 -, -CH 2 -CH(CH 3 )-, Or-CH 2 -CH(CH 2 OH)-.
  • a specific value for n is 2.
  • n 3
  • a specific compound of formula I is PMEGpp or a pharmaceutically acceptable salt or prodrug thereof.
  • a specific compound of formula I is (R)-PMPGpp or a pharmaceutically acceptable salt or a prodrug thereof.
  • a specific compound of formula I is (S)-PMPGpp or a pharmaceutically acceptable salt or a prodrug thereof.
  • a specific compound of formula I is PMED APpp or a pharmaceutically acceptable salt or a prodrug thereof.
  • a specific compound of formula I is 6-cypr-(R)-PMPDAPpp or a pharmaceutically acceptable salt or a prodrug thereof.
  • the compounds may have chiral centers, e.g., chiral carbon or phosphorus atoms.
  • the compounds thus include racemic mixtures of all stereoisomers, including enantiomers, diastereomers, and atropisomers.
  • the compounds include enriched or resolved optical isomers at any or all asymmetric, chiral atoms.
  • the chiral centers apparent from the depictions are provided as the chiral isomers or racemic mixtures.
  • racemic mixtures are separated into their individual, substantially optically pure isomers through well-known techniques such as, for example, the separation of diastereomeric salts formed with optically active adjuncts, e.g., acids or bases followed by conversion back to the optically active substances.
  • optically active adjuncts e.g., acids or bases
  • the desired optical isomer is synthesized by means of stereospecific reactions, beginning with the appropriate stereoisomer of the desired starting material.
  • the compounds can also exist as tautomeric isomers in certain cases. All though only one delocalized resonance structure may be depicted, all such forms are contemplated within the scope of the invention.
  • ene-amine tautomers can exist for purine, pyrimidine, imidazole, guanidine, amidine, and tetrazole systems and all their possible tautomeric forms are within the scope of the invention.
  • the invention also provides salts of compounds of Formula (I), especially pharmaceutically acceptable non-toxic salts containing, for example, Na + , Li + , K + > Ca + ⁇ and Mg + ⁇ .
  • Such salts may include those derived by combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium and quaternary amino ions with an acid anion moiety, typically a carboxylic acid.
  • Monovalent salts are preferred if a water soluble salt is desired.
  • Metal salts typically are prepared by reacting the metal hydroxide with a compound of this invention.
  • metal salts which are prepared in this way are salts containing Li + , Na + , and K + .
  • a less soluble metal salt can be precipitated from the solution of a more soluble salt by addition of the suitable metal compound.
  • compositions herein comprise compounds in their un-ionized, as well as zwitterionic form, and combinations with stoichiometric amounts of water as in hydrates.
  • the salts formed with one or more amino acids are suitable, especially the naturally-occurring amino acids found as protein components, although the amino acid typically is one bearing a side chain with a basic or acidic group, e.g., lysine, arginine or glutamic acid, or a neutral group such as glycine, serine, threonine, alanine, isoleucine, or leucine.
  • the compounds of this invention can be formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like.
  • Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. All formulations will optionally contain excipients such as those set forth in the Handbook of Pharmaceutical Excipients
  • Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like.
  • the pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10. While it is possible for the active ingredients to be administered alone it may be preferable to present them as pharmaceutical formulations.
  • the formulations, both for veterinary and for human use, of the invention comprise at least one active ingredient, as above defined, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.
  • the formulations include those suitable for the foregoing administration routes.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be administered as a bolus, electuary or paste.
  • a tablet can be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingredient therefrom.
  • the formulations are preferably applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/w such as
  • the active ingredients may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredients may be formulated in a cream with an oil-in-water cream base.
  • the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e.
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas.
  • dermal penetration enhancers include dimethyl sulphoxide and related analogs.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat.
  • the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax
  • the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate.
  • the choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties.
  • the cream should preferably be a non-greasy, non- staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di- isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils are used.
  • compositions according to the present invention comprise one or more compounds together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents.
  • Pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration.
  • tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation.
  • Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable.
  • excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc.
  • inert diluents such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate
  • granulating and disintegrating agents such as maize starch, or alginic acid
  • binding agents such as cellulose, microcrystalline cellulose, starch,
  • Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
  • Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example calcium phosphate or kaolin
  • an oil medium such as peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate).
  • a suspending agent such as sodium carb
  • the aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p- hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives such as ethyl or n-propyl p- hydroxy-benzoate
  • coloring agents such as ethyl or n-propyl p- hydroxy-benzoate
  • flavoring agents such as sucrose or saccharin.
  • sweetening agents such as sucrose or saccharin.
  • Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
  • Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
  • the pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions.
  • the oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these.
  • Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.
  • the emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
  • compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • a sterile injectable preparation such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile fixed oils may conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic
  • a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weight: weight).
  • the pharmaceutical composition can be prepared to provide easily measurable amounts for administration.
  • an aqueous solution intended for intravenous infusion may contain from about 3 to 500 ⁇ g of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.
  • Formulations suitable for administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.
  • the active ingredient is preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% particularly about 1.5% w/w.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
  • Formulations suitable for intrapulmonary or nasal administration have a particle size for example in the range of 0.1 to 500 microns (including particle sizes in a range between 0.1 and 500 microns in increments microns such as 0.5, 1, 30 microns, 35 microns, etc.), which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs.
  • Suitable formulations include aqueous or oily solutions of the active ingredient.
  • Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment of inflammation as described below.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations are presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • sterile liquid carrier for example water for injection
  • Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
  • the invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefore.
  • Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.
  • Compounds can also be formulated to provide controlled release of the active ingredient to allow less frequent dosing or to improve the pharmacokinetic or toxicity profile of the active ingredient. Accordingly, the invention also provided compositions comprising one or more compounds formulated for sustained or controlled release.
  • Effective dose of active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against existing inflammation, the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using conventional dose escalation studies. It can be expected to be from about 0.0001 to about 100 mg/kg body weight per day. Typically, from about 0.01 to about 10 mg/kg body weight per day. More typically, from about .01 to about 5 mg/kg body weight per day. More typically, from about .05 to about 0.5 mg/kg body weight per day.
  • the daily candidate dose for an adult human of approximately 70 kg body weight will range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, and may take the form of single or multiple doses.
  • One or more compounds can be administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, aasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that the preferred route may vary with for example the condition of the recipient.
  • An advantage of the compounds of this invention is that they are orally bioavailable and can be dosed orally.
  • Active ingredients of the invention can also be used in combination with other active ingredients. Such combinations are typically selected based on the condition to be treated, cross-reactivities of ingredients and pharmaco-properties of the combination. It is also possible to combine any compound with one or more other active ingredients in a unitary dosage form for simultaneous or sequential administration to a patient.
  • the combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.
  • the combination therapy may provide "synergy" or a "synergistic effect", i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately.
  • a synergistic effect may be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen.
  • a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., in separate tablets, pills or capsules, or by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
  • the invention includes compounds produced by a process comprising contacting a compound of this invention with a mammal for a period of time sufficient to yield a metabolic product thereof.
  • Such products typically are identified by preparing a radiolabeled (e.g., C ⁇ O r H ⁇ ) compound, administering it parenterally in a detectable dose (e.g., greater than about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples.
  • a detectable dose e.g., greater than about 0.5 mg/kg
  • an animal such as rat, mouse, guinea pig, monkey, or to man
  • sufficient time for metabolism to occur typically about 30 seconds to 30 hours
  • isolating its conversion products from the urine, blood or other biological samples typically isolating its conversion products from the urine, blood or other biological samples.
  • the metabolite structures are determined in conventional fashion, e.g., by MS or NMR analysis.
  • the invention also relates to methods of making the novel compounds herein.
  • the compounds are prepared by any of the applicable techniques of organic synthesis. Many such techniques are well known in the art. However, many of the known techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6, Michael B. Smith; as well as March, J., Advanced Organic Chemistry, Third Edition, (John Wiley & Sons, New York, 1985), Comprehensive Organic Synthesis.
  • reaction conditions such as temperature, reaction time, solvents, work-up procedures, and the like, will be those common in the art for the particular reaction to be performed.
  • the cited reference material, together with material cited therein, contains detailed descriptions of such conditions.
  • temperatures will be -100°C to 200 0 C
  • solvents will be aprotic or protic
  • reaction times will be 10 seconds to 10 days.
  • Work-up typically consists of quenching any unreacted reagents followed by partition between a water/organic layer system (extraction) and separating the layer containing the product.
  • a single stereoisomer, e.g., an enantiomer, substantially free of its stereoisomer may be obtained by resolution of the racemic mixture using a method such as formation of diastereomers using optically active resolving agents (Stereochemistry of Carbon Compounds. (1962) by E. L. Eliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3) 283-302).
  • Racemic mixtures of chiral compounds can be separated and isolated by any suitable method, including: (1) formation of ionic, diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with chiral derivatizing reagents, separation of the diastereomers, and conversion to the pure stereoisomers, and (3) separation of the substantially pure or enriched stereoisomers directly under chiral conditions.
  • diastereomeric salts can be formed by reaction of enantiomerically pure chiral bases such as brucine, quinine, ephedrine, strychnine, ⁇ - methyl- ⁇ -phenylethylarnine (amphetamine), and the like with asymmetric compounds bearing acidic functionality, such as carboxylic acid and sulfonic acid.
  • the diastereomeric salts may be induced to separate by fractional crystallization or ionic chromatography.
  • the substrate to be resolved is reacted with one enantiomer of a chiral compound to form a diastereomeric pair
  • a diastereomeric pair Eliel, E. and Wilen, S. (1994) Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., p. 322).
  • Diastereomeric compounds can be formed by reacting asymmetric compounds with enantiomerically pure chiral derivatizing reagents, such as menthyl derivatives, followed by separation of the diastereomers and hydrolysis to yield the free, enantiomerically enriched xanthene.
  • a method of determining optical purity involves making chiral esters, such as a menthyl ester, e.g., (-) menthyl chloroformate in the presence of base, or Mosher ester, ⁇ -methoxy- ⁇ -(trifluoromethyl)phenyl acetate
  • a racemic mixture of two enantiomers can be separated by chromatography using a chiral stationary phase (Chiral Liquid Chromatography (1989) W. J. Lough, Ed. Chapman and Hall, New York; Okamoto, (1990) J. ofChromatogr. 513 :375-378).
  • Enriched or purified enantiomers can be distinguished by methods used to distinguish other cliiral molecules with asymmetric carbon atoms, such as optical rotation and circular dichroism.
  • the eluate was evaporated at 30 0 C, dissolved in 0.05 M triethylammonium bicarbonate and purified by chromatography on POROS ® 50HQ anion exchanger (Applied Biosystems, Foster City, CA, USA) in the linear concentration gradient of triethylammonium bicarbonate (0.05 - 0.4 mol/L). Peak corresponding to ANPpp (triethylammonium salt) was collected, evaporated in vacuo at room temperature and then converted to the ANPpp sodium salt on DOWEXTM 50X8 (Na + ) (SERVA Electrophoresis GmbH, Heidelberg, Germany). All other chemicals and materials were commercial products, e.g.
  • activated charcoal N,N'-dicyclohexylcarbodiimide, morpholine, tert-butanol, Celite ® , dimethylsulfoxide, ddGTP, streptomycin, penicillin G, CHAPS 5 ⁇ -mercaptoethanol, RNase A, proteinase K, PBS and RPMI 1640 medium (Sigma- Aldrich, St.
  • Human acute promyelocytic leukaemia HL-60 cells (ATCC CCL 240) were cultured in RPMI- 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, antibiotics (200 ⁇ g/mL of streptomycin and 200 units/mL of penicillin G), 10 niM ⁇ -mercaptoethanol, and vitamin B 12 at 37 0 C in a humidified atmosphere containing 5% CO 2 . After harvesting in log-phase growth, cells were pelleted, washed in PBS, and frozen at-70 0 C.
  • Extracts with telomerase activity were prepared and analyzed as described by Kim NW, et al., Science, 1994, 266, 2011-5, with some modifications. Briefly, the cells were thawed and incubated on ice for 30 min in CHAPS lysis buffer containing 0.5% CHAPS, 10 mM HEPES-NaOH (pH 7.5), 1 mM MgCl 2 , 1 niM EGTA, 5 mM ⁇ -mercaptoethanol, 2 mM Pefabloc-SC, 10% glycerol plus 1 ⁇ L (40 U) of Protector RNase inhibitor. 200 ⁇ L of CHAPS lysis buffer was used to lyse one million cells.
  • telomerase activity was determined using the TRAP assay as described by Kim NW, Wu F., Nucleic Acids Res, 1997, 25, 2595-7 with the modifications described below.
  • telomere assay In the present study, the sensitivity of the TRAP assay was increased by prolongation of incubation time from 10 to 15 min and increasing the number of PCR cycles from 27 to 33.
  • An aliquot of 800 pmol of TS substrate primer (5'- AATCCGTCGAGCAGAGTT-3') (SEQ ID NO:1) was labeled in 100 ⁇ L reaction mixture containing 60 ⁇ Ci [ ⁇ - 32 P]ATP (60 mCi/mL, 7000 Ci/mmol), T4 Polynucleotide kinase buffer and 40 U T4 polynucleotide kinase.
  • TRAP reactions contained Taq polymerase reaction buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25 0 C), 1.5 mM MgCl 2 , 0.1% Triton X ® -100), dNTPs (30, 60 and 125 ⁇ mol/L), 18 pmol of end-labeled TS substrate primer, an appropriate amount of studied ANPpp, ANPp and ANP respectively.
  • Reaction was started by cell extract addition (0.15 ⁇ g protein). Each TRAP reaction mixture was placed in a thermocycler block preheated to 30 0 C and incubated at 30 0 C for 15 min and then heated at 95 0 C for 2 min (for one cycle) to stop telomerase reaction. After addition of 10 ⁇ L of mixture containing 6 pmol ACX reverse primer (5'-GCGCGG[CTTACC] 3 CTAACC-3') (SEQ ID NO:2), 3 pmol NT internal control primer, 0.01 amol TSNT internal control and 1.25 U Taq DNA polymerase, the reaction was cycled 33 times at 94 0 C for 20 s, 52 0 C for 30 s, 72 0 C for 20 s.
  • ACX reverse primer 5'-GCGCGG[CTTACC] 3 CTAACC-3'
  • telomerase activity was very dependent on telomerase itself.
  • a number of inactivation experiments were performed. Aliquot of cell lysate was incubated with RNase A (50 ⁇ g/mL) at 37 0 C for 30 min. Proteinase K and heat-inactivated cell extracts were prepared by incubating of the cell extract with proteinase K (50 ⁇ g/mL) at 37 0 C for 30 min and heating 10 ⁇ L extract at 75 0 C for 10 min prior to assaying 3 ⁇ L by TRAP assay respective.
  • HL-60 cell extract showed telomerase activity with the characteristic primer extension-binding pattern on the autoradigraphs.
  • telomere products were analyzed on a denaturating 15% polyacrylamide-7 M urea sequencing gel at 1 ,900 V for 2 h with Tris-borate-EDTA. Dried gels were exposed to a Phosphorlmager storage screen and the amount of reaction products was evaluated using TYPHOONTM 9410 imagerMmageQuantTM software (Molecular Dynamics, Sunnyvale, CA, USA).
  • TYPHOONTM 9410 imagerMmageQuantTM software Molecular Dynamics, Sunnyvale, CA, USA.
  • the TRAP assay signals of the telomerase ladders were normalized to the signal of the corresponding internal standard after background substraction. The signal intensity of the bands from ANPpp, ANPp and ANP- treated samples was expressed as a percentage of the signal intensity detected in the control. Their relative intensities were calculated with the ImageQuant software. All results were expressed as mean ⁇ SD of the four independent determinations.
  • the guanine derivative PMEGpp is the most potent telomerase inhibitor among all acyclic nucleotide analogues studied with the IC 50 12.7 ⁇ 0.5 ⁇ mol/L at 125 ⁇ M dNTPs (Table 1, Fig. 2). Its inhibitory potency towards telomerase is comparable to that of ddGTP (IC 50 8.1 ⁇ 0.4 ⁇ mol/L at 125 ⁇ M dNTPs), which is known to be one of the most effective nucleotide analogue based telomerase inhibitors.
  • PMEGpp inhibits telomerase activity by 50% when it is present in the range 0.07 to 0.11 of the dGTP concentration only, depending on the dNTPs concentration. The PMEG monophosphate and PMEG itself do not show at 125 ⁇ M dNTPs any effect on telomerase activity up to the concentration of 300 ⁇ M PMEG and/or PMEGp, respectively.
  • PMEApp with pyrimidine derivatives PMETpp and PMECpp, which inhibit retroviral reverse transcriptases show no significant inhibitory potency towards telomerase.
  • the inhibition is observed with PMEO-DAPypp; this open ring ANP is considered to be PMEDAPpp analogue (Fig.
  • the most potent inhibitor among the PMP type analogues is the guanine derivative (i ⁇ )-PMPGpp, which inhibits the enzyme activity with the IC 50 at 5 to 8 times lower concentration compared to that of the natural substrate dGTP.
  • (S)- PMPGpp is dramatically less inhibitory than its (i?)-enantiomer. its IC 50 exceeds almost 5 times that of (iR)-PMPGpp. This indicates that absolute configuration plays a significant role in inhibition of telomerase and that the enzyme can distinguish between the (Ry and (S)-enantiomers.
  • (S)-PMP App increases processivity of the enzyme (Fig.
  • telomerase active site has been shown to be related to that of other reverse transciptases (Nugent CI 5 Lundblad V., Genes Dev, 1998, 12, 1073-85), (i ⁇ )-PMPApp and PMEApp, nucleotide analogues known to be chain-terminating inhibitors of retroviral reverse transcriptases (Holy A., Curr Pharm Des, 2003, 9, 2567-92), do not inhibit telomerase activity.
  • RNA template of one subunit could be used primarily for substrate binding, while the second template would be copied during telomere repeats addition (Kelleher C, et al., Trends Biochem Sci, 2002, 27, 572-9).
  • So-called DNA anchor site which is distinct from the catalytic site, could be affected by both (S)-PMP App and/or 6-Me 2 PMED APpp and might thereby facilitate processivity during the synthesis of telomere repeats.
  • All literature and patent citations herein are hereby expressly incorporated by reference at the locations of their citation. Specifically cited sections or pages of the above cited works are incorporated by reference with specificity.
  • the invention has been described in detail sufficient to allow one of ordinary skill in the art to make and use the subject matter of the following claims. It is apparent that certain modifications of the methods and compositions of the following claims can be made within the scope and spirit of the invention.

Abstract

The invention provides the use of acyclic nucleoside phosphonates and their derivatives for the manufacture of a medicament for inhibiting an enzyme that causes neoplasia in an animal (e.g. a human) and enhancing the processivity of a telomerase.

Description

USE OF COMPOUNDS TO INHIBIT NEOPLASIA
Background of the Invention
Human telomerase is a large cellular ribonucleoprotein complex (Morin GB, Cell, 1989, 59, 521-9; Wenz C, et al., EMBO J 2001;20:3526-34; and Collins K,
Mitchell JR., Oncogene, 2002, 21, 564-79) that is responsible for adding the telomeric repeats onto the 3' ends of chromosomes YGreider CW, Blackburn EH., Cell, 1985, 43, 405-13) and thus compensating for the telqmere loss that accompanies chromosomal replication and cell division. Telomerase is ύpregulated in almost 90% of all malignancies (Kim NW, et al., Science, 1994, 266, 2011-5; Shay JW, Bacchetti S3 Eur J Cancer, 1997, 33, 787-91; and Hiyama E, Hiyama K, Oncogene, 2002, 21, 643-9). Hence, it is thought to be very promising not only as a tumor-specific marker but also as a target for anticancer treatment (Lichtsteiner SP, et al., Ann N YAcadSci, 1999, 555, 1-11). The identification of the hTERT component of telomerase as a functional catalytic reverse transcriptase prompted studies of inhibiting telomerase with established HIV reverse transcriptase inhibitors, such as the chain terminating 3'- azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) (Nakamura TM, et al., Science, 1997, 277, 955-9; Blackburn EH, Annu Rev Biochem, 1992, 61, 113-29; and Cech TR, et al., Biochemistry (Moscow), 1997, 62, 1202-5).
These parent nucleosides are transformed in vivo in the form of triphosphates, which inhibit reverse transcriptases by acting as competitive substrates for the enzyme and terminate DNA synthesis de novo, AZT-TP inhibits the activity of telomerase to ~50% at 30 μmol/L. Other nucleotide-based inhibitors, both purine and pyrimidine derivatives, have been evaluated against telomerase with effective IC50 values in the micro-molar range. In a more recent study, Fletcher reported the use of deazadeoxypurines as inhibitors of telomerase with one compound, 6-thio-7-deaza-2'- deoxyguanosine 5 '-triphosphate showing an IC50 value of 60 nmol/L. Series of the other nucleotide analogues (ddGTP, ddATP, ddTTP, d4TTP, deazadGTP, deazadATP, thiodGTP, CBV-TP, araGTP, and FaraTTP) have previously been shown to inhibit telomerase activity (see Morin GB, Cell, 1989, 59, 521-9; Strahl C, Blackburn EH., MoI Cell Biol, 1996, 16, 53-65; Pai RB, et al., Cancer Res, 1998, 58, 1909-13; Fletcher TM5 et al., Bioorg Chem, 2001, 29, 36-55; Tendian SW, Parker WB., MoI Pharmacol, 2000, 57, 695-9; Strahl C, Blackburn EH., Nucleic Acids Res, 1994, 22, 893-900; Fletcher TM, et ^Biochemistry, 1996, 35, 15611-7; and Raymond E, et al,Curr Opin Biotechnol, 1996, 7, 583-91).
Numerous ANPs possess excellent antiviral activities against a broad spectrum of DNA viruses and retroviruses as well as a significant antiproliferative potency. (S)- HPMPC (cidofovir, CDV, Vistide®), (φ-PMPA (tenofovir, TDV, Viread®), PMEA (adefovir, ADV, Hepsera®) were approved for treatment of cytomegalovirus retinis in AIDS patients, HIV infection, and chronic hepatitis B, respectively. In cells, ANPs are activated by conversion to their diphosphates, active antimetabolites, which inhibit viral replicases and terminate nascent DNA chain (Holy A., Curr Pharm Des, 2003, 9, 2567-92).
Summary of the Invention
The invention provides the use of an acyclic nucleoside purine of formula I:
Figure imgf000003_0001
wherein;
X is OH or -N(R^2 wherein R1 independently is, H;
C2-C15 alkyl, C3-C15 alkenyl, C6-C15 arylalkenyl, C3-C15 alkynyl, C7-C15 arylalkynyl, Ci-Ce-alkylamino-Ci-Ce alkyl, C5-C15 aralkyl, C6-C15 heteroalkyl or C3-C6 heterocycloalkyl wherein methylene in an alkyl moiety not adjacent to NH has been replaced by~O— ; or
C1-C15 alkyl, C2-C15 alkenyl, C6-C15 arylalkenyl, C6-C15 arylalkynyl, C2- C15 alkynyl, Ci-Ce-alkylamino-Q-Cδ alkyl, C5-C15 aralkyl, C6-C15 heteroaralkyl, C4-C6 aryl, C3 -C8cycloalkyl, or C2-C6 heterocycloalkyl; or optionally both R1 are joined together to form a saturated or unsaturated C2-C5 heterocycle containing one or two N heteroatoms and optionally an additional O or S heteroatom; wherein one of the foregoing R1 groups can be substituted with halo, -CN or -N3;
W is H or NH2;
Y independently is, OH, -OR2, -OCH(R3)OC(O)R2, a monophosphate, a diphosphate, an amino acid amidate, a polypeptide amidate, -NHR2, or -N(R2)2 ;
R2 independently is unsubstituted alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl wherein H is substituted by halo, carboxy, hydroxyl, cyano, nitro, N- morpholino, or amino; or alkyl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl in which a -CH2- moiety has been substituted by NH, S, or O;
Z is C1-C6 alkyl, optionally substituted with one or more hydroxy; and R3 is H or R2; or a prodrug, diphosphate, pharmaceutically acceptable salt, or other phosphorus- substituted derivative thereof, in the manufacture of a medicament for inhibiting an enzyme that causes neoplasia inan animal (e.g. a human).
The invention also provides a pharmaceutical composition comprising a compound of formula I, or a prodrug, pharmaceutically acceptable salt, or other phosphorus-substituted derivative thereof, and a pharmaceutically acceptable excipient.
The invention also provides the use of PMEODAPy or a prodrug, diphosphate, pharmaceutically acceptable salt, or other phosphorus-substituted derivative thereof in the manufacture of a medicament for inhibiting an enzyme that causes neoplasia in an animal.
The invention also provides a method for inhibiting an enzyme that causes neoplasia comprising contacting the enzyme (in vitro or in vivo) with a compound of formula I or a prodrug, pharmaceutically acceptable salt, or other phosphorus- substituted derivative thereof.
The invention also provides a method for treating neoplasia in an animal comprising administering an effective amount of a compound of formula I or a prodrug, pharmaceutically acceptable salt, or other phosphorus-substituted derivative thereof to the animal. The invention also provides novel compounds of formula I described herein as well as prodrugs and pharmaceutically acceptable salts thereof.
Brief Description of the Figures Figure 1 Illustrates the structure of PME, PMEO, PMP, and HPMP purines and pyrimidines.
Figure 2 Illustrates the inhibition of HL-60 telomerase by PMEGpp as measured in Example 1.
Figure 3 Illustrates the enhancement of telomerase processivity by 6- Me2PMEDAPpP (A) and (S)-PMP App measured in Example 1.
Figure 4 (A) Illustrates inhibition of HL-60 telomerase by guanine derivatives PMEGpp (1), (£)-PMPGpp (2), (i?)-HPMPGpp (3), (S)-PMPGpp (4) and (B) illustrates inhibition by adenine and 2,6-diaminopurine derivatives PMED APpp (5), OS)-HPMPApρ (6), PMEO-DAPypp (7) and (i?)-6-cyprPMPDAPpp (8). The enzyme activity was measured in HL-60 cell extract in the presence of 125 μmol/L dNTPs.
Detailed Description
Diphosphates of the antiviral acyclic nucleoside phosphonates (ANPs) were evaluated in telomeric repeat amplification protocol (TRAP) (Kim NW, et al., Science, 1994, 266, 2011-5; Wright WE, Nucleic Acids Res, 1995, 23, 3794-5; and Kim NW, Wu F., Nucleic Acids Res, 1997, 25, 2595-7) for their ability to inhibit the extension of telomeres by human telomerase, using extracts from human leukaemia HL-60 cells as a source of the enzyme. Extracts from human leukaemia HL-60 cells were used as a source of the enzyme. Data show that the most effective compound studied was the guanine derivative PMEGpp (IC50 12.7 ± 0.5 μmol.L"1 at 125 μmol.L"1 dNTPs). The inhibitory effects of other PME, PMP and HPMP diphosphates on telomerase reverse transcriptase decreased in the order: (φ-PMPGpp > (i?)-HPMPGpp > PMED APpp > (S)-PMPGpp > (S)-HPMPApp > PMEO-DAPypp > (i?)-6-cyprPMPDAPpp > (R)- PMPApp > (i?)-PMPDAPpρ > PMEApp > PMECpp > PMETpp > (S)-PMP App ~ 6- Me2PMEDAPpP . These results are consistent with the observed antineoplastic activities of the parental guanine (PMEG) and 2,6-diaminopurine (PMEDAP) PME- derivatives. Moreover, structure-activity relationship indicates enantioselectivity of some of these human telomerase inhibitors: (i?)-isomers of the PMP-derivatives possess stronger affinity towards the enzyme than (S)-isomers. In accordance with human telomeric sequence, the adenine derivatives are typically less effective inhibitors than their guanine counterparts.
The following abbreviations are used herein: ANP, acyclic nucleoside phosphonate; ANPpp, ANP diphosphate; TRAP, telomeric repeat amplification protocol; PMEG, 9-[2-(phosρhonomethoxy)ethyl]guanine; (iϊ)-PMPG, (i?)-9-[2- (phosphonomethoxy)propyl] -guanine; (i.)-HPMPG, (R)-9-[(3 -hydroxy-2- phosphonomethoxy)propyl]guanine; PMEDAP, 2,6-diamino-9-[2-(phosphono- methoxy)ethyl]purine; (S)-PMPG, (S)-9-[2-(phosphonomethoxy)propyl]guanine; (S)- HPMPA, (S)-9-[(3-hydroxy-2-phosphonomethoxy)propyl]adenine; PMEO-DAPy, 2,4- diamino-6-[2-(phosphonomethoxy)-ethoxy]pyrimidine; (i?)-6-cyprPMPDAP, (R)-2- amino-6-(cyclopropylamino)-9-[2-(phosphonomethoxy)propyl]purine; (i?)-PMPA, (i?)-9-[2-(phosphonomethoxy)propyl]-adenine; (i?)-PMPDAP, (i?)-2,6-diamino-9-[2- (phosphonomethoxy)propyl]purine; PMEA, 9-[2-(phosphonomethoxy)ethyl]adenine; PMEC, l-[2-(phosphonomethoxy)ethyl]cytosine; PMET, l-[2-(phosphonomethoxy)- ethyl]thymine; (S)-PMPA, (S)-9-[2-(phosphonomethoxy)-propyl]adenine; 6- Me2PMEDAP, 2-amino-6-(dimethylamino)-9-[2-(phosphonomethoxy)-ethyl]purine; hTERT, human telomerase reverse transcriptase; AZT-TP, 3'-azido-3'- deoxythymidine 5 '-triphosphate; ddC, 2',3'-dideoxycytidine; d4TTP, 2',3'-didehydro- 2',3'-deoxythymidine 5 '-triphosphate; deazadGTP, 7-deaza-2'-deoxyguanosine 5'- triphosphate; deazadATP, 7-deaza-2'-deoxyadenosine 5 '-triphosphate; thiodGTP, 6- thio-2'-deoxyguanosine 5 '-triphosphate; CBV-TP, 2',3'-didehydro-2',3'- dideoxyguanosine 5 '-triphosphate; araGTP, 9-β-D-arabinofuranosylguanine 5'- triphosphate; FaraTTP, 2'-fluoro-2'-deoxy-β-arabinofuranosylthymine 5'- triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate; and TS, telomerase substrate (nontelomeric oligonucleotide).
Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:
The term "prodrug" as used herein refers to any compound that when administered to a biological system generates the drug substance, i.e. active ingredient, as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), photolysis, and/or metabolic chemical reaction(s). A prodrug is thus a covalently modified analog or latent form of a therapeutically-active compound. "Prodrug moiety" refers to a labile functional group which separates from the active inhibitory compound during metabolism, systemically, inside a cell, by hydrolysis, enzymatic cleavage, or by some other process (Bundgaard, Hans, "Design and Application of Prodrugs" in A Textbook of Drug Design and Development (1991), P. Krogsgaard-Larsen and H. Bundgaard, Eds. Harwood Academic Publishers, pp. 113- 191). Enzymes which are capable of an enzymatic activation mechanism with the phosphonate compounds include, but are not limited to, amidases, esterases, microbial enzymes, phospholipases, cholinesterases, and phosphases. Prodrug moieties can serve to enhance solubility, absorption and lipophilicity to optimize drug delivery, bioavailability and efficacy. A prodrug moiety may include an active metabolite or drug itself.
Exemplary prodrug moieties include the hydrolytically sensitive or labile acyloxymethyl esters -CH2OC(=O)R9 and acyloxymethyl carbonates -CH2OC(=O)OR9 where R9 is C1-C6 alkyl, C1-C6 substituted alkyl, C6-C20 aryl or C6-C20 substituted aryl. The acyloxyalkyl ester was first used as a prodrug strategy for carboxylic acids and then applied to phosphates and phosphonates by Farquhar et al. (1983) J Pharm. Sd. 72: 324; also US Patent Nos. 4816570, 4968788, 5663159 and 5792756. Subsequently, the acyloxyalkyl ester was used to deliver phosphonic acids across cell membranes and to enhance oral bioavailability. A close variant of the acyloxyalkyl ester, the alkoxycarbonyloxyalkyl ester (carbonate), may also enhance oral bioavailability as a prodrug moiety in the compounds of the combinations of the invention. An exemplary acyloxymethyl ester is pivaloyloxymethoxy, (POM) -CH2OC(=O)C(CH3)3. An exemplary acyloxymethyl carbonate prodrug moiety is pivaloyloxymethylcarbonate (POC) -CH2OC(=O)OC(CH3)3. Aryl esters of phosphorus groups, especially phenyl esters, are reported to enhance oral bioavailability (De Lombaert et al. (1994) J. Med. Chem. 37: 498). Phenyl esters containing a carboxylic ester ortho to the phosphate have also been described (Khamnei and Torrence, (1996) J. Med. Chem. 39:4109-4115). Benzyl esters are reported to generate the parent phosphonic acid. In some cases, substituents at the ort/zo-or pαrø-position may accelerate the hydrolysis. Benzyl analogs with an acylated phenol or an alkylated phenol may generate the phenolic compound through the action of enzymes, e.g., esterases, oxidases, etc., which in turn undergoes cleavage at the benzylic C-O bond to generate the phosphoric acid and the quinone methide intermediate. Examples of this class of prodrugs are described by Mitchell et al. (1992) J. Chem. Soc. Perkin Trans. II 2345; Glazier WO 91/19721. Still other benzylic prodrugs have been described containing a carboxylic ester-containing group attached to the benzylic methylene (Glazier WO 91/19721). Thio-containing prodrugs are reported to be useful for the intracellular delivery of phosphonate drugs. These proesters contain an ethylthio group in which the thiol group is either esterified with an acyl group or combined with another thiol group to form a disulfide. Deesterification or reduction of the disulfide generates the free thio intermediate which subsequently breaks down to the phosphoric acid and episulfide (Puech et al. (1993) Antiviral Res., 22: 155-174; Benzaria et al. (1996) J. Med. Chem. 39: 4958). Cyclic phosphonate esters have also been described as prodrugs of phosphorus-containing compounds (Erion et al., US Patent No. 6312662).
Examples of pharmaceutically acceptable salts include salts derived from an appropriate base, such as an alkali metal (for example, sodium), an alkaline earth (for example, magnesium), ammonium and NX4 + (wherein X is C1-C4 alkyl).
Pharmaceutically acceptable salts of a compound having an amino group include salts of organic carboxylic acids such as acetic, benzoic, lactic, fumaric, tartaric, maleic, malonic, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids; and inorganic acids, such as hydrochloric, sulfuric, phosphoric and sulfamic acids.
Pharmaceutically acceptable salts of a compound having a hydroxy group include the anion of said compound in combination with a suitable cation such as Na+ and NX4 + (wherein X is independently selected from H or a C1-C4 alkyl group). For therapeutic use, salts of active ingredients will typically be pharmaceutically acceptable, i.e. they will be salts derived from a pharmaceutically acceptable acid or base.
"Alkyl" is branched or unbranched hydrocarbon containing normal, secondary, or tertiary carbon atoms. Examples are methyl (Me,-CH3), ethyl (Et,-CH2CH3), 1- propyl (n-Pr, n-propyl,-CH2CH2CH3), 2-proρyl (i-Pr, i-propyl,-CH(CH3)2), 1 -butyl (n-Bu, n-butyl,-CH2CH2CH2CH3), 2-methyl-l -propyl (i-Bu, B>utyl,-CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl,-CH(CH3)CH2CH3), 2-methyl-2- propyl (fcBu, t-butyl,-C(CH3)3), 1-pentyl (n-pentyl,-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (- C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3 -methyl- 1 -butyl (-CH2CH2CH(CH3)2), 2-methyl-l -butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3))5 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl- 3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3.
"Alkenyl" is branched or unbranched hydrocarbon containing normal, secondary, or tertiary carbon atoms with at least one site of unsaturation, i.e. a carbon- carbon, sp2 double bond. Examples include, but are not limited to, ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7), 5-hexenyl (-CH2 CH2CH2CH2CH=CH2), and 2,5-hexadienyl (-CH2CH=CHCH2CH=CH2).
"Alkynyl" is a branched or unbranched hydrocarbon containing normal, secondary, or tertiary carbon atoms with at least one site of unsaturation, i.e. a carbon- carbon, sp triple bond. Examples include, but are not limited to, acetylenic (-C=CH), propargyl (-CH2C≡CH), and 2,5-hexadiynyl (-CH2C=CH CH2C≡CH)
"Aryl" means a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like.
"Aralkyl" refers to an alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan- 1-yl, , naphthylmethyl, 2-naphthylethan-l-yl, naphthobenzyl, 2-naphthophenylethan-l- yl and the like. The arylalkyl group comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.
"Arylalkenyl" refers to an alkenyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl radical.
"Arylalkynyl" refers to an alkynyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl radical. "Heterocycle" or "Heterocyclo" as used herein includes by way of example and not limitation these heterocycles described in Paquette, Leo A.; Principles of Modern Heterocyclic Chemistry (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs" (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. In one specific embodiment of the invention "heterocycle" includes a "carbocycle" as defined herein, wherein one or more (e.g. 1, 2, 3, or 4) carbon atoms have been replaced with a heteroatom (e.g. O, N, or S). Examples of heterocycles include by way of example and not limitation pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H- 1,2,5- thiadiazinyl, 2H,6H-l,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, lH-indazoly, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH- carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, isatinoyl, and bis-tetrahydrofuranyl:
Figure imgf000010_0001
The term "treatment" or "treating," to the extent it relates to a disease or condition includes preventing the disease or condition from occurring, inhibiting the disease or condition, eliminating the disease or condition, and/or relieving one or more symptoms of the disease or condition. Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane- polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
Protecting groups are available, commonly known and used, and are optionally used to prevent side reactions with the protected group during synthetic procedures, i.e. routes or methods to prepare the compounds. For the most part the decision as to which groups to protect, when to do so, and the nature of the chemical protecting group "PG" will be dependent upon the chemistry of the reaction to be protected against (e.g., acidic, basic, oxidative, reductive or other conditions) and the intended direction of the synthesis. The PG groups do not need to be, and generally are not, the same if the compound is substituted with multiple PG. In general, PG will be used to protect functional groups such as carboxyl, hydroxyl, thio, or amino groups and to thus prevent side reactions or to otherwise facilitate the synthetic efficiency. The order of deprotection to yield free, deprotected groups is dependent upon the intended direction of the synthesis and the reaction conditions to be encountered, and may occur in any order as determined by the artisan. A very large number of protecting groups and corresponding chemical cleavage reactions are described in Protective Groups in Organic Synthesis, Theodora W. Greene (John Wiley & Sons, Inc., New York, 1991, ISBN 0-471-62301-6) ("Greene"). See also Kocienski, Philip J.; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994). Examples of suitable amino acids whose residues can be represented by Y in Formula I include the following: Glycine; Aminopolycarboxylic acids, e.g., aspartic acid, β-hydroxyaspartic acid, glutamic acid, β-hydroxyglutamic acid, β-methylaspartic acid, β-methylglutamic acid, β, β-dimethylaspartic acid, γ-hydroxyglutamic acid, β, γ- dihydroxy glutamic acid, β-phenylglutamic acid, γ-methyleneglutamic acid, 3- aminoadipic acid, 2-aminopimelic acid, 2-aminosuberic acid and 2-aminosebacic acid; Amino acid amides such as glutamine and asparagine; Polyamino- or polybasic- monocarboxylic acids such as arginine, lysine, β-aminoalanine, γ-aminobutyrine, ornithine, citruline, homoarginine, homocitrulline, hydroxylysine, allohydroxylsine and diaminobutyric acid; Other basic amino acid residues such as histidine;
Diaminodicarboxylic acids such as α, α'-diaminosuccinic acid, α, α'-diaminoglutaric acid, α, α'-diaminoadipic acid, α, α'-diaminopimelic acid, α, α'-diamino- β-hydroxypimelic acid, α, α'-diaminosuberic acid, α, α'-diaminoazelaic acid, and α,α'-diaminosebacic acid; Imino acids such as proline, hydroxyproline, allohydroxyproline, γ-methylproline, pipecolic acid, 5-hydroxypipecolic acid, and azetidine-2-carboxylic acid; A mono- or di-alkyl (typically Ci-Cs branched or normal) amino acid such as alanine, valine, leucine, allylglycine, butyrine, norvaline, norleucine, heptyline, α-methylserine, α-amino-α-methyl-γ-hydroxyvaleric acid, α-amino- α-methyl-δ-hydroxyvaleric acid, α-amino- α-methyl-ε-hydroxycaproic acid, isovaline, α-methylglutamic acid, α-aminoisobutyric acid, α-aminodiethylacetic acid, α-aminodiisopropylacetic acid, α-aminodi-n-propylacetic acid, α- aminodiisobutylacetic acid, α-aminodi-n-butylacetic acid, α-aminoethylisopropylacetic acid, α-amino-n-propylacetic acid, α-aminodiisoamyacetic acid, α-methylaspartic acid, α-methylglutamic acid, 1-arninocyclopropane-l-carboxylic acid, isoleucine, alloisoleucine, tert-leucine, β-methyltryptophan and α-amino- β-ethyl-β- phenylpropionic acid; β-phenylserinyl;
Aliphatic α-amino-β-hydroxy acids such as serine, β-hydroxyleucine, β-hydroxynorleucine, β-hydroxynorvaline, and α-amino-β-hydroxystearic acid; α-Amino, α-, γ-, δ- or ε-hydroxy acids such as homoserine, δ-hydroxynorvaline, γ-hydroxynorvaline and ε-hydroxynorleucine residues; canavine and canaline; γ-hydroxyornithine; 2-hexosaminic acids such as D-glucosaminic acid or D- galactosaminic acid; α-Amino-β-thiols such as penicillamine, β-thiolnorvaline or β-thiolbutyrine; Other sulfur containing amino acid residues including cysteine; homocystine, β-phenylmethionine, methionine, S-allyl-L-cysteine sulfoxide, 2- thiolhistidine, cystathionine, and thiol ethers of cysteine or homocysteine; Phenylalanine, tryptophan and ring-substituted α-amino acids such as the phenyl- or cyclohexylamino acids α-aminophenylacetic acid, α-aminocyclohexylacetic acid and α-amino-β-cyclohexylpropionic acid; phenylalanine analogues and derivatives comprising aryl, lower alkyl, hydroxy, guanidino, oxyalkylether, nitro, sulfur or halo- substituted phenyl (e.g., tyrosine, methyltyrosine and o-chloro-, p-chloro-, 3,4- dichloro, o-, m- or p-meikyl-, 2,4,6-trimethyl-, 2-ethoxy-5-nitro-, 2-hydroxy-5-nitro- and p-nitro-phenylalanine); furyl-, thienyl-, pyridyl-, pyrimidinyl-, purinyl- or naphthyl-alanines; and tryptophan analogues and derivatives including kynurenine, 3- hydroxykynurenine, 2-hydroxytryptophan and 4-carboxytryptophan; α- Amino substituted amino acids including sarcosine (N-methylglycine), N-benzylglycine, N-methylalanine, N-benzylalanine, N-methylphenylalanine, N-benzylphenylalanine, N-methylvaline and N-benzylvaline; and α-Hydroxy and substituted α-hydroxy amino acids including serine, threonine, allothreonine, phosphoserine and phosphothreonine.
Polypeptides are polymers of amino acids in which a carboxyl group of one amino acid monomer is bonded to an amino or imino group of the next amino acid monomer by an amide bond. Polypeptides include dipeptides, low molecular weight polypeptides (about 1500-5000 MW) and proteins. Proteins optionally contain 3, 5, 10, 50, 75, 100 or more residues, and suitably are substantially sequence-homologous with human, animal, plant or microbial proteins. They include enzymes (e.g., hydrogen peroxidase) as well as immunogens such as KLH, or antibodies or proteins of any type against which one wishes to raise an immune response. The nature and identity of the polypeptide may vary widely. Peptidolytic enzymes for cleaving polypeptide conjugates are well known, and in particular include carboxypeptidases. Carboxypeptidases digest polypeptides by removing C-terminal residues, and are specific in many instances for particular C-terminal sequences. Such enzymes and their substrate requirements in general are well known. For example, a dipeptide (having a given pair of residues and a free carboxyl terminus) is covalently bonded through its α-amino group to the phosphorus or carbon atoms of the compounds herein. This peptide can be cleaved by the appropriate peptidolytic enzyme, leaving the carboxyl of the proximal amino acid residue to autocatalytically cleave the phosplionoamidate bond. Suitable dipeptidyl groups (designated by their single letter code) include AA, AR, AN, AD, AC, AE, AQ, AG, AH, AI, AL5 AK, AM, AF, AP, AS5 AT, AW5 AY5 AV3 RA, RR, RN, RD, RC5 RE, RQ5 RG5 RH, RI5 RL5 RK5 RM5 RF5 RP, RS5 RT5 RW, RY5 RV5 NA, NR5 NN5 ND5 NC5 NE, NQ, NG5 NH, NI5 NL5 NK, NM5 NF5 NP, NS5 NT, NW5 NY, NV, DA, DR5 DN5 DD, DC5 DE, DQ5 DG5 DH5 DI5 DL5 DK, DM, DF5 DP5 DS5 DT, DW, DY, DV, CA, CR, CN5 CD5 CC5 CE5 CQ5 CG5 CH5 CI5 CL5 CK5 CM5 CF5 CP5 CS5 CT5 CW, CY5 CV, EA, ER5 EN, ED5 EC5 EE5 EQ5 EG, EH, EI5 EL, EK, EM5 EF, EP, ES5 ET, EW5 EY, EV5 QA5 QR5 QN5 QD5 QC5 QE5 QQ, QG, QH, QI5 QL5 QK5 QM5 QF5 QP5 QS5 QT, QW5 QY5 QV5 GA, GR, GN, GD, GC5 GE5 GQ, GG5 GH5 GI5 GL5 GK, GM, GF5 GP5 GS, GT5 GW5 GY5 GV5 HA5 HR5 HN5 HD, HC5 HE, HQ, HG, HH, HI, HL, HK, HM, HF5 HP5 HS, HT, HW5 HY5 HV5 IA5 IR, IN5 ID5 IC, IE5 IQ5 IG, IH, II, IL, IK5 IM5 IF5 IP, IS5 IT5 IW, IY5 IV, LA, LR, LN5 LD5 LC5 LE, LQ, LG5 LH5 LI5 LL, LK, LM5 LF, LP, LS, LT5 LW, LY5 LV, KA, KR, KN, KD, KC, KE, KQ, KG, KH, KI, KL, KK, KM, KF, KP, KS, KT, KW, KY, KV5 MA5 MR5 MN5 MD, MC, ME, MQ5 MG, MH5 MI, ML, MK5 MM5 MF, MP, MS, MT5 MW5 MY5 MV5 FA5 FR5 FN5 FD5 FC, FE, FQ, FG, FH, FI, FL, FK, FM, FF, FP, FS, FT, FW, FY, FV, PA, PR, PN5 PD5 PC, PE5 PQ5 PG5 PH5 PI5 PL, PK, PM, PF5 PP5 PS5 PT, PW, PY, PV, SA5 SR5 SN, SD5 SC5 SE, SQ5 SG5 SH, SI5 SL, SK, SM, SF, SP, SS, ST, SW, SY5 SV, TA, TR5 TN5 TD, TC, TE, TQ, TG, TH5 TI5 TL, TK, TM, TF, TP, TS, TT, TW, TY, TV, WA, WR5 WN, WD, WC, WE5 WQ, WG, WH, WI, WL5 WK5 WM5 WF5 WP5 WS5 WT5 WW5 WY, WV, YA, YR, YN, YD, YC, YE, YQ5 YG, YH, YI5 YL5 YK, YM, YF, YP5 YS5 YT5 YW, YY, YV5 VA5 VR5 VN5 VD5 VC5 VE5 VQ, VG, VH, VI, VL, VK, VM, VF, VP, VS, VT, VW, VY and VV. Tripeptide residues are also useful as protecting groups. Dipeptide or tripeptide species can be selected on the basis of known transport properties and/or susceptibility to peptidases that can affect transport to intestinal mucosal or other cell types. Dipeptides and tripeptides lacking an α-amino group are transport substrates for the peptide transporter found in brush border membrane of intestinal mucosal cells (Bai, J.P.F., (1992) Pharm Res. 9:969-978). Transport competent peptides can thus be used to enhance bioavailability of the compounds. Dior tripeptides having one or more amino acids in the D configuration are also compatible with peptide transport and can be utilized in the compounds. Amino acids in the D configuration can be used to reduce the susceptibility of a di- or tripeptide to hydrolysis by proteases common to the brush border such as aminopeptidase N. In addition, di- or tripeptides alternatively are selected on the basis of their relative resistance to hydrolysis by proteases found in the lumen of the intestine. For example, tripeptides or polypeptides lacking asp and/or glu are poor substrates for aminopeptidase A, di- or tripeptides lacking amino acid residues on the N-terminal side of hydrophobic amino acids (leu, tyr, phe, val, trp) are poor substrates for endopeptidase, and peptides lacking a pro residue at the penultimate position at a free carboxyl terminus are poor substrates for carboxypeptidase P. Similar considerations can also be applied to the selection of peptides that are either relatively resistant or relatively susceptible to hydrolysis by cytosolic, renal, hepatic, serum or other peptidases. Such poorly cleaved polypeptide amidates are immunogens or are useful for bonding to proteins in order to prepare immunogens.
Specific Embodiments of the Invention
Specific values described for radicals, substituents, and ranges, as well as specific embodiments of the invention described herein, are for illustration only; they do not exclude other defined values or other values within defined ranges.
A specific acyclic nucleoside purine is a compound of formula 1 :
Figure imgf000015_0001
wherein; n is 2 or 3;
X is-N(R1)2 wherein
R1 independently is H;
C2-C15 alkyl, C3-C15 alkenyl, C6-C15 arylalkenyl, C3-C15 alkynyl, C7-C15 arylalkynyl, CrCe-alkylamino-d-Cδ alkyl, C5-C15 aralkyl, C6-C15 heteroalkyl or C3-C6 heterocycloalkyl wherein methylene in an alkyl moiety not adjacent to NH has been replaced by— O— ; or C1-C15 alkyl, C2-C15 alkenyl, C6-C15 arylalkenyl, C6-C15 arylalkynyl, C2- C15 alkynyl, Ci-Cό-alkylamino-Ci-Ce alkyl, C5-C15 aralkyl, C6-C15 heteroaralkyl, C4-C6 aryl, C2-C6 heterocycloalkyl; or optionally both R1 are joined together to form a saturated or unsaturated C2-C5 heterocycle containing one or two N heteroatoms and optionally an additional O or S heteroatom; wherein one of the foregoing R1 groups can be substituted with halo,- CN or-N3, but either one or two R1 groups are not H;
Y independently is, OH,-OR2,-OCH(R3)OC(O)R2, a monophosphate, a diphosphate, an amino acid amidate, a polypeptide amidate,-NHR2, or-N(R2)2 ;
R2 independently is unsubstituted alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl wherein H is substituted by halo, carboxy, hydroxyl, cyano, nitro, N- morpholino, or amino; or alkyl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl in which a-CH2- moiety has been substituted by NH, S, or O;
R3 is H or R2; or a prodrug, diphosphate, or other phosphorus-substituted derivative thereof.
Another specific acyclic nucleoside purine of formula I is a compound of the following formula:
Figure imgf000016_0001
or a prodrug or pharmaceutically acceptable salt thereof.
Another specific acyclic nucleoside purine is a compound of the following formula:
Figure imgf000017_0001
or a prodrug thereof.
A specific value for each R1 independently is H, C1-C15 alkyl, C2-C15 alkenyl, C2-C15 alkynyl, or C3-C8cycloalkyl; or optionally both R1 are joined together to form a saturated or unsaturated C2-C5 heterocycle containing one or two N heteroatoms and optionally an additional O or S heteroatom.
A specific value for each R1 independently is H, methyl, or cyclopropyl.
A specific value for each R1 independently is H.
A specific value for each R1 independently is methyl. A specific value for one R1 is H and the other R1 is cyclopropyl.
A specific value for W is H.
A specific value for W is NH2.
A specific value for X is OH.
A specific value for Z is -CH2-CH2-, -CH2-CH(CH3)-, Or-CH2-CH(CH2OH)-. A specific value for n is 2.
A specific value for n is 3.
A specific compound of formula I is PMEGpp or a pharmaceutically acceptable salt or prodrug thereof.
A specific compound of formula I is (R)-PMPGpp or a pharmaceutically acceptable salt or a prodrug thereof.
A specific compound of formula I is (S)-PMPGpp or a pharmaceutically acceptable salt or a prodrug thereof.
A specific compound of formula I is PMED APpp or a pharmaceutically acceptable salt or a prodrug thereof. A specific compound of formula I is 6-cypr-(R)-PMPDAPpp or a pharmaceutically acceptable salt or a prodrug thereof.
The compounds may have chiral centers, e.g., chiral carbon or phosphorus atoms. The compounds thus include racemic mixtures of all stereoisomers, including enantiomers, diastereomers, and atropisomers. In addition, the compounds include enriched or resolved optical isomers at any or all asymmetric, chiral atoms. In other words, the chiral centers apparent from the depictions are provided as the chiral isomers or racemic mixtures. Both racemic and diastereomeric mixtures, as well as the individual optical isomers isolated or synthesized, substantially free of their enantiomeric or diastereomeric partners, are all within the scope of the invention. The racemic mixtures are separated into their individual, substantially optically pure isomers through well-known techniques such as, for example, the separation of diastereomeric salts formed with optically active adjuncts, e.g., acids or bases followed by conversion back to the optically active substances. In most instances, the desired optical isomer is synthesized by means of stereospecific reactions, beginning with the appropriate stereoisomer of the desired starting material.
The compounds can also exist as tautomeric isomers in certain cases. All though only one delocalized resonance structure may be depicted, all such forms are contemplated within the scope of the invention. For example, ene-amine tautomers can exist for purine, pyrimidine, imidazole, guanidine, amidine, and tetrazole systems and all their possible tautomeric forms are within the scope of the invention.
The invention also provides salts of compounds of Formula (I), especially pharmaceutically acceptable non-toxic salts containing, for example, Na+, Li+, K+> Ca+^ and Mg+^. Such salts may include those derived by combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium and quaternary amino ions with an acid anion moiety, typically a carboxylic acid. Monovalent salts are preferred if a water soluble salt is desired.
Metal salts typically are prepared by reacting the metal hydroxide with a compound of this invention. Examples of metal salts which are prepared in this way are salts containing Li+, Na+, and K+. A less soluble metal salt can be precipitated from the solution of a more soluble salt by addition of the suitable metal compound.
In addition, salts may be formed from acid addition of certain organic and inorganic acids, e.g., HCl, HBr, H2SO4s H3PO4 or organic sulfonic acids, to basic centers, typically amines, or to acidic groups. Finally, it is to be understood that the compositions herein comprise compounds in their un-ionized, as well as zwitterionic form, and combinations with stoichiometric amounts of water as in hydrates.
Also included within the scope of this invention are the salts formed with one or more amino acids. Any of the amino acids described above are suitable, especially the naturally-occurring amino acids found as protein components, although the amino acid typically is one bearing a side chain with a basic or acidic group, e.g., lysine, arginine or glutamic acid, or a neutral group such as glycine, serine, threonine, alanine, isoleucine, or leucine. The compounds of this invention can be formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. All formulations will optionally contain excipients such as those set forth in the Handbook of Pharmaceutical Excipients
(1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like. The pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10. While it is possible for the active ingredients to be administered alone it may be preferable to present them as pharmaceutical formulations. The formulations, both for veterinary and for human use, of the invention comprise at least one active ingredient, as above defined, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.
The formulations include those suitable for the foregoing administration routes. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary or paste.
A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingredient therefrom.
For administration to the eye or other external tissues e.g., mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/w such as
0.6% w/w, 0.7% w/w, etc.), preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulphoxide and related analogs.
The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
Emulgents and emulsion stabilizers suitable for use in the formulation of the invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties. The cream should preferably be a non-greasy, non- staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di- isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils are used. Pharmaceutical formulations according to the present invention comprise one or more compounds together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. Pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p- hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.
Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. The pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.
The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.
The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weight: weight). The pharmaceutical composition can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.
Formulations suitable for administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% particularly about 1.5% w/w.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
Formulations suitable for intrapulmonary or nasal administration have a particle size for example in the range of 0.1 to 500 microns (including particle sizes in a range between 0.1 and 500 microns in increments microns such as 0.5, 1, 30 microns, 35 microns, etc.), which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment of inflammation as described below.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
The formulations are presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents. The invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefore.
Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.
Compounds can also be formulated to provide controlled release of the active ingredient to allow less frequent dosing or to improve the pharmacokinetic or toxicity profile of the active ingredient. Accordingly, the invention also provided compositions comprising one or more compounds formulated for sustained or controlled release.
Effective dose of active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against existing inflammation, the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using conventional dose escalation studies. It can be expected to be from about 0.0001 to about 100 mg/kg body weight per day. Typically, from about 0.01 to about 10 mg/kg body weight per day. More typically, from about .01 to about 5 mg/kg body weight per day. More typically, from about .05 to about 0.5 mg/kg body weight per day. For example, the daily candidate dose for an adult human of approximately 70 kg body weight will range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, and may take the form of single or multiple doses.
One or more compounds (herein referred to as the active ingredients) can be administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, aasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that the preferred route may vary with for example the condition of the recipient. An advantage of the compounds of this invention is that they are orally bioavailable and can be dosed orally.
Active ingredients of the invention can also be used in combination with other active ingredients. Such combinations are typically selected based on the condition to be treated, cross-reactivities of ingredients and pharmaco-properties of the combination. It is also possible to combine any compound with one or more other active ingredients in a unitary dosage form for simultaneous or sequential administration to a patient. The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. The combination therapy may provide "synergy" or a "synergistic effect", i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., in separate tablets, pills or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
Also falling within the scope of this invention are the in vivo metabolic products of the compounds described herein. Such products may result for example from the oxidation, reduction, hydrolysis, amidation, esterification and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the invention includes compounds produced by a process comprising contacting a compound of this invention with a mammal for a period of time sufficient to yield a metabolic product thereof. Such products typically are identified by preparing a radiolabeled (e.g., C^ Or H^) compound, administering it parenterally in a detectable dose (e.g., greater than about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples. These products are easily isolated since they are labeled (others are isolated by the use of antibodies capable of binding epitopes surviving in the metabolite). The metabolite structures are determined in conventional fashion, e.g., by MS or NMR analysis. In general, analysis of metabolites is done in the same way as conventional drug metabolism studies well-known to those skilled in the art. The conversion products, so long as they are not otherwise found in vivo, are useful in diagnostic assays for therapeutic dosing of the compounds even if they possess noanti- inflammatory activity of their own.
The invention also relates to methods of making the novel compounds herein. The compounds are prepared by any of the applicable techniques of organic synthesis. Many such techniques are well known in the art. However, many of the known techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6, Michael B. Smith; as well as March, J., Advanced Organic Chemistry, Third Edition, (John Wiley & Sons, New York, 1985), Comprehensive Organic Synthesis.
Selectivity, Strategy & Efficiency in Modern Organic Chemistry. In 9 Volumes, Barry M. Trost, Editor-in-Chief (Pergamon Press, New York, 1993 printing).
Generally, the reaction conditions such as temperature, reaction time, solvents, work-up procedures, and the like, will be those common in the art for the particular reaction to be performed. The cited reference material, together with material cited therein, contains detailed descriptions of such conditions. Typically the temperatures will be -100°C to 2000C, solvents will be aprotic or protic, and reaction times will be 10 seconds to 10 days. Work-up typically consists of quenching any unreacted reagents followed by partition between a water/organic layer system (extraction) and separating the layer containing the product.
A single stereoisomer, e.g., an enantiomer, substantially free of its stereoisomer may be obtained by resolution of the racemic mixture using a method such as formation of diastereomers using optically active resolving agents (Stereochemistry of Carbon Compounds. (1962) by E. L. Eliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3) 283-302). Racemic mixtures of chiral compounds can be separated and isolated by any suitable method, including: (1) formation of ionic, diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with chiral derivatizing reagents, separation of the diastereomers, and conversion to the pure stereoisomers, and (3) separation of the substantially pure or enriched stereoisomers directly under chiral conditions.
Under method (1), diastereomeric salts can be formed by reaction of enantiomerically pure chiral bases such as brucine, quinine, ephedrine, strychnine, α- methyl-β-phenylethylarnine (amphetamine), and the like with asymmetric compounds bearing acidic functionality, such as carboxylic acid and sulfonic acid. The diastereomeric salts may be induced to separate by fractional crystallization or ionic chromatography. For separation of the optical isomers of amino compounds, addition of chiral carboxylic or sulfonic acids, such as camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid can result in formation of the diastereomeric salts.
Alternatively, by method (2), the substrate to be resolved is reacted with one enantiomer of a chiral compound to form a diastereomeric pair (Eliel, E. and Wilen, S. (1994) Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., p. 322). Diastereomeric compounds can be formed by reacting asymmetric compounds with enantiomerically pure chiral derivatizing reagents, such as menthyl derivatives, followed by separation of the diastereomers and hydrolysis to yield the free, enantiomerically enriched xanthene. A method of determining optical purity involves making chiral esters, such as a menthyl ester, e.g., (-) menthyl chloroformate in the presence of base, or Mosher ester, α-methoxy-α-(trifluoromethyl)phenyl acetate
(Jacob III. (1982) J. Org. Chem. 47:4165), of the racemic mixture, and analyzing the NMR spectrum for the presence of the two atropisomeric diastereomers. Stable diastereomers of atropisomeric compounds can be separated and isolated by normal- and reverse-phase chromatography following methods for separation of atropisomeric naphthyl-isoquinolines (Hoye, T., WO 96/15111).
By method (3), a racemic mixture of two enantiomers can be separated by chromatography using a chiral stationary phase (Chiral Liquid Chromatography (1989) W. J. Lough, Ed. Chapman and Hall, New York; Okamoto, (1990) J. ofChromatogr. 513 :375-378). Enriched or purified enantiomers can be distinguished by methods used to distinguish other cliiral molecules with asymmetric carbon atoms, such as optical rotation and circular dichroism.
The invention will now be illustrated by the following non-limiting Examples.
Example 1 Materials and methods
The following compounds were evaluated (Fig. 1): (S)-HPMP App, (R)- HPMPGpp, PMEApp, PMECpp, PMEGpp, PMETpp, PMED APpp, PMEO-D APypp, 6-Me2PMEDAPpp, (i?)-6-cyprPMPDAPpp, (iϊ)-PMPDAPpp, (i?)-PMPApp, (S)-
PMP App, (i?)-PMPGpp, and (S)-PMPGpp. (S)-HPMPA and (R)-HPMPG diphosphates were synthesized according to Otmar M, Masojidkova M, Votruba I, Holy A., Collect Czech Chem Commun, 2001, 66, 500-6. Other ANPs diphosphates were synthesized by the modified morpholidate method. In a typical experiment, a mixture of ANP (free acid, 1 mmol ), NjN'-dicyclohexylcarbodiimide (1.3 g) and morpholine (2 mL) in 80% aqueous tert-butanol (20 mL) was refluxed under stirring for 6-8 hours and evaporated in vacuo. The residue in water (100 mL) was filtered over Celite®, the filtrate extracted with ether (3 x 50 mL) and the aqueous phase was taken down in vacuo. The residue was transferred into 100-mL flask, evaporated, codistilled with ethanol (2 x 20 mL) and dried overnight at 15 Pa over phosphorus pentoxide. Bis(tributylammonium) monophosphate or tris(tributylammonium) diphosphate solution in dry dimethylsulfoxide (1 mol/L, 2.5 mL) was added and the mixture was stirred at ambient temperature in a tightly closed flask for 4- 6 days. Reaction mixture was then acidified with 6 M HCl to pH 3 and an appropriate amount of activated charcoal was added. After an exhaustive washing of pelleted activated charcoal with HPLC water, desalted nucleotides were eluted by 5% NH4OH in 50% methanol. The eluate was evaporated at 30 0C, dissolved in 0.05 M triethylammonium bicarbonate and purified by chromatography on POROS® 50HQ anion exchanger (Applied Biosystems, Foster City, CA, USA) in the linear concentration gradient of triethylammonium bicarbonate (0.05 - 0.4 mol/L). Peak corresponding to ANPpp (triethylammonium salt) was collected, evaporated in vacuo at room temperature and then converted to the ANPpp sodium salt on DOWEX™ 50X8 (Na+) (SERVA Electrophoresis GmbH, Heidelberg, Germany). All other chemicals and materials were commercial products, e.g. activated charcoal, N,N'-dicyclohexylcarbodiimide, morpholine, tert-butanol, Celite®, dimethylsulfoxide, ddGTP, streptomycin, penicillin G, CHAPS5 β-mercaptoethanol, RNase A, proteinase K, PBS and RPMI 1640 medium (Sigma- Aldrich, St. Louis, MO, USA), fetal calf serum (PAA Laboratories GmbH, Pasching, Austria), vitamin B12 (Leciva a.s., Prague, Czech Republic), Pefabloc-SC, Protector RNase inhibitor (Roche Diagnostics GmbH, Mannheim, Germany), [γ-32P]ATP (MP Biomedicals GmbH, Germany), T4 Polynucleotide Kinase Buffer and T4 Polynucleotide Kinase (TaKaRa Bio, Inc., Shiga, Japan), HEPES, deoxynucleotide triphosphates (dNTPs), Taq polymerase reaction buffer, Taq DNA polymerase (Promega, Madison, WI, USA) and TS, ACX, NT, TSNT primers (Invitrogen Ltd, Paisley, United Kingdom).
Human acute promyelocytic leukaemia HL-60 cells (ATCC CCL 240) were cultured in RPMI- 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, antibiotics (200 μg/mL of streptomycin and 200 units/mL of penicillin G), 10 niM β-mercaptoethanol, and vitamin B12 at 37 0C in a humidified atmosphere containing 5% CO2. After harvesting in log-phase growth, cells were pelleted, washed in PBS, and frozen at-70 0C.
Extracts with telomerase activity were prepared and analyzed as described by Kim NW, et al., Science, 1994, 266, 2011-5, with some modifications. Briefly, the cells were thawed and incubated on ice for 30 min in CHAPS lysis buffer containing 0.5% CHAPS, 10 mM HEPES-NaOH (pH 7.5), 1 mM MgCl2, 1 niM EGTA, 5 mM β-mercaptoethanol, 2 mM Pefabloc-SC, 10% glycerol plus 1 μL (40 U) of Protector RNase inhibitor. 200 μL of CHAPS lysis buffer was used to lyse one million cells. In order to ensure lysis of HL-60 cells, these cells were subjected to two freeze/thaw cycles during the incubation. This additional step did not affect telomerase activity in the HL-60 cells. Cell debris was pelleted for 20 min at 16 000 x g at 40C. The supernatant was removed, aliquoted, frozen on dry ice, and stored at -70 0C. The protein concentration of the supernatant was determined by the Bradford assay. Telomerase activity was determined using the TRAP assay as described by Kim NW, Wu F., Nucleic Acids Res, 1997, 25, 2595-7 with the modifications described below. In the present study, the sensitivity of the TRAP assay was increased by prolongation of incubation time from 10 to 15 min and increasing the number of PCR cycles from 27 to 33. The dependence of the amount of amplified telomerase product versus number of PCR cycle was linear in the range of 30 to 35 cycles (data not shown). An aliquot of 800 pmol of TS substrate primer (5'- AATCCGTCGAGCAGAGTT-3') (SEQ ID NO:1) was labeled in 100 μL reaction mixture containing 60 μCi [γ-32P]ATP (60 mCi/mL, 7000 Ci/mmol), T4 Polynucleotide kinase buffer and 40 U T4 polynucleotide kinase. After incubation for 30 min at 37 0C and then 2 min at 85 0C, an excess of unincorporated [γ-32P]ATP was removed from reaction mixture on MicroSpin™ G-25 Column (Amersham Biosciences, Piscataway, NJ, USA). Forty microlitre TRAP reactions contained Taq polymerase reaction buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25 0C), 1.5 mM MgCl2, 0.1% Triton X®-100), dNTPs (30, 60 and 125 μmol/L), 18 pmol of end-labeled TS substrate primer, an appropriate amount of studied ANPpp, ANPp and ANP respectively. Reaction was started by cell extract addition (0.15 μg protein). Each TRAP reaction mixture was placed in a thermocycler block preheated to 30 0C and incubated at 30 0C for 15 min and then heated at 95 0C for 2 min (for one cycle) to stop telomerase reaction. After addition of 10 μL of mixture containing 6 pmol ACX reverse primer (5'-GCGCGG[CTTACC]3CTAACC-3') (SEQ ID NO:2), 3 pmol NT internal control primer, 0.01 amol TSNT internal control and 1.25 U Taq DNA polymerase, the reaction was cycled 33 times at 94 0C for 20 s, 52 0C for 30 s, 72 0C for 20 s. To ensure that the observed telomerase activities were really dependent on telomerase itself, a number of inactivation experiments were performed. Aliquot of cell lysate was incubated with RNase A (50 μg/mL) at 370C for 30 min. Proteinase K and heat-inactivated cell extracts were prepared by incubating of the cell extract with proteinase K (50 μg/mL) at 37 0C for 30 min and heating 10 μL extract at 75 0C for 10 min prior to assaying 3 μL by TRAP assay respective. HL-60 cell extract showed telomerase activity with the characteristic primer extension-binding pattern on the autoradigraphs. We considered the sample as positive for telomerase activity if the signal had disappeared after RNase A treatment and if no signal was detected with the lysis buffer alone (negative control). RNase A and proteinase K treatments abolished the PCR product ladder bands and confirmed both the protein and RNA dependence of the enzyme activity.
The amplified telomerase products were analyzed on a denaturating 15% polyacrylamide-7 M urea sequencing gel at 1 ,900 V for 2 h with Tris-borate-EDTA. Dried gels were exposed to a Phosphorlmager storage screen and the amount of reaction products was evaluated using TYPHOON™ 9410 imagerMmageQuant™ software (Molecular Dynamics, Sunnyvale, CA, USA). To compare relative telomerase activity in the presence of inhibitors, the TRAP assay signals of the telomerase ladders were normalized to the signal of the corresponding internal standard after background substraction. The signal intensity of the bands from ANPpp, ANPp and ANP- treated samples was expressed as a percentage of the signal intensity detected in the control. Their relative intensities were calculated with the ImageQuant software. All results were expressed as mean ± SD of the four independent determinations.
Results and Discussion
The approximate IC50 values for the PME-derivatives are shown in Table 1, where they are listed in the order of their telomerase inhibitory potency: PMEGpp > PMED APpp > PMEO-D APypp > PMEApp > PMECpp > PMETpp > 6- Me2PMEDAPρp.
Table 1
Inhibition of HL-60 telomerase by PME type of ANPpp (IC50)a dNTPs, [μmol/L]
Compound 125 60 30
PMEGpp 12.7 + 0.5 7.1 + 1.7 2.30 ± 0.04
PMEDAPpp 76.0 ± 13.5 41.6 + 10.1 23.3 + 3.2
PMEO-DAPypp 133 + 15 N.D.b N.D.
PMEApp 380 + 28 N.D. 110 + 11
PMECpp N.I.° N.D. 106 + 16
PMETpp N.I. N.D. N.I.
6-Me2PMEDAPpp P.E.d N.D. N.D. a Values are means ± SD of the four independent determinations; b not determined; cno inhibition; processivity enhancement.
The guanine derivative PMEGpp is the most potent telomerase inhibitor among all acyclic nucleotide analogues studied with the IC50 12.7 ± 0.5 μmol/L at 125 μM dNTPs (Table 1, Fig. 2). Its inhibitory potency towards telomerase is comparable to that of ddGTP (IC50 8.1 ± 0.4 μmol/L at 125 μM dNTPs), which is known to be one of the most effective nucleotide analogue based telomerase inhibitors. PMEGpp inhibits telomerase activity by 50% when it is present in the range 0.07 to 0.11 of the dGTP concentration only, depending on the dNTPs concentration. The PMEG monophosphate and PMEG itself do not show at 125 μM dNTPs any effect on telomerase activity up to the concentration of 300 μM PMEG and/or PMEGp, respectively.
PMED APpp, which selectively inhibits DNA polymerase δ (Holy A., Curr Pharm Des, 2003, 9, 2567-92) and exerts significant cytostatic effects, inhibits the activity of telomerase with ICS0 76 ± 13.5 μmol/L (at 125 μM dNTPs). Surprisingly, N6- dimethyl derivative 6-Me2PMEDAPpP increases processivity of the enzyme (Fig. 3A). Nonphosphorylated form 6-Me2PMEDAP has no effect on telomerase ladder pattern. PMEApp with pyrimidine derivatives PMETpp and PMECpp, which inhibit retroviral reverse transcriptases (Holy A., Curr Pharm Des, 2003, 9, 2567-92), show no significant inhibitory potency towards telomerase. The inhibition is observed with PMEO-DAPypp; this open ring ANP is considered to be PMEDAPpp analogue (Fig.
1). Inhibitory potency of PMEGpp and PMEDAPpp towards telomerase is consistent with the capability to induce apoptosis, strong cytostatic efficiency, and anticancer activity of their parental compounds PMEG and PMEDAP (Holy A., Curr
Pharm Des, 2003, 9, 2567-92).
The approximate IC50 values for the diphosphates of PMP-derivatives, listed in the order of their efficacy as telomerase inhibitors, are shown in Table 2: (i?)-PMPGpp
> (S)-PMPGpp > (i?)-6-cyprPMPDAPpp > (i?)-PMPApp > (i?)-PMPDAPpp > (S)-
PMP App.
Table 2 Inhibition of HL-60 telomerase by PMP type of ANPpp (IC50)a dNTPs, [μmol/L]
Compound 125 60 30
(i?)-PMPGpp 17.3 ± 2.8 11.3 ± 1.6 3.6 ± 1.0
(S)-PMPGpp 81.7 ± 19.6 N.D.b N.D.
(i?)-6-cyprPMPDAPpp 152 ± 3 N.D. N.D.
(JT)-PMP App 224 ± 30 125 ± 24 51.3 ± 6.7
(i?)-PMPDAPpp 376 ± 24 N.D. N.D.
(5)-PMPAρρ P.E.C N.D. N.D. a Values are means ± SD of the four independent determinations; bnot determined; c processivity enhancement.
The most potent inhibitor among the PMP type analogues is the guanine derivative (iϊ)-PMPGpp, which inhibits the enzyme activity with the IC50 at 5 to 8 times lower concentration compared to that of the natural substrate dGTP. (S)- PMPGpp is dramatically less inhibitory than its (i?)-enantiomer. its IC50 exceeds almost 5 times that of (iR)-PMPGpp. This indicates that absolute configuration plays a significant role in inhibition of telomerase and that the enzyme can distinguish between the (Ry and (S)-enantiomers. Alike, (S)-PMP App increases processivity of the enzyme (Fig. 3B), while no significant inhibition of telomerase activity is found for (i?)-PMPApp (IC50 224 ± 30 μmol/L at 125 μM dNTPs), which is very efficient as a chain-terminating inhibitor of retroviral reverse transcriptases (Holy A., Curr Pharm Des, 2003, 9, 2567-92). These results are consistent with the observation of Pai RB, et al., Cancer Res, 1998, 58, 1909-13 on discrimination between the D and L enantiomers of FaraTTP, and Tendian SW, Parker WB., MoI Pharmacol, 2000, 57, 695-9, concerning inhibition efficiency difference in the pair of D and L enantiomers of CBV-TP. Structure-activity relationship study also indicates enantioselectivity of some of these ANP-type inhibitors of human telomerase. In general, (i?)-enantiomers of the PMP-derivatives possess stronger affinity towards the enzyme than (S)- enantiomers.
The approximate IC50 values of the diphosphates of purine HPMP derivatives are shown in Table 3. In accordance with the PME and PMP series, the adenine derivative (S)-HPMP App is less inhibitory than the guanine derivative (i?)-HPMPGpp. Table 3
Inhibition of HL-60 telonierase by HPMP type of ANPpp (IC50)1 a dNTPs, [μmol/L]
Compound 125 60 30
(φ-HPMPGpp 43.6 + 5.9 28.3 + 9.5 23.3 + 5.9 (,S)-HPMPApp 104 ± 15 61 ± 25 N.D.b a Values are means + SD of the four independent determinations; bnot determined.
The previously reported inhibitory effect of ddGTP on telomerase activity (see Pai RB, et al, Cancer Res, 1998, 55, 1909-13) is comparable with inhibitory potency of the most efficient ANP based compounds PMEGpp and (i?)-PMPGpp. (R)- HPMPGpp, PMEDAPpp, (S)-PMPGpp, (iO-6-cyprPMPDAPpp, PMEO-DAPypp and (S)-HPMPApp have been shown as moderate inhibitors with IC50 values comparable to the concentrations of natural substrates. Although the telomerase active site has been shown to be related to that of other reverse transciptases (Nugent CI5 Lundblad V., Genes Dev, 1998, 12, 1073-85), (iϊ)-PMPApp and PMEApp, nucleotide analogues known to be chain-terminating inhibitors of retroviral reverse transcriptases (Holy A., Curr Pharm Des, 2003, 9, 2567-92), do not inhibit telomerase activity.
The data in Table 1, 2 and 3 show that the IC50 values for inhibition of telomerase activity by all guanine derivatives studied [PMEGpp, (i-)-PMPGpp, (S)- PMPGpp and (β)-HPMPGpp] are from 2 to 10 times lower than the dGTP concentration in the assay. Thus, the affinities of these nucleotide analogues towards telomerase are considerably higher than that for the natural substrate dGTP (Fig. 4A). In accordance with human telomeric sequence, adenine and/or 2,6-diarninopurine ANPpps are less efficient inhibitors (Fig. 4B).
Interestingly, two compounds from the studied series differ very substantially from the other ANPpp molecules: (S)-PMP App and 6-Me2PMEDAPpp do not inhibit telomerase, however, they increase the processivity of telomerase at 125 μM dNTPs concentration (Fig. 3). We can speculate that both (S)-PMP App and 6- Me2PMEDAPpp might interfere in some way with telomerase reaction cycle similarly to dGTP, which is supposed to facilitate telomerase RNA template and DNA strand separation and increase repeat addition processivity (Hammond PW, Cech TR., Biochemistry, 1998, 37, 5162-72; and Hardy CD3 et al.,J Biol Chem, 2001, 276, 4863- 71) in cooperating two-subunits of telomerase complex (see Wenz C, et al., EMBOJ, 2001, 20, 3526-34).
According to the "anchor site model", the RNA template of one subunit could be used primarily for substrate binding, while the second template would be copied during telomere repeats addition (Kelleher C, et al., Trends Biochem Sci, 2002, 27, 572-9). So-called DNA anchor site, which is distinct from the catalytic site, could be affected by both (S)-PMP App and/or 6-Me2PMED APpp and might thereby facilitate processivity during the synthesis of telomere repeats. All literature and patent citations herein are hereby expressly incorporated by reference at the locations of their citation. Specifically cited sections or pages of the above cited works are incorporated by reference with specificity. The invention has been described in detail sufficient to allow one of ordinary skill in the art to make and use the subject matter of the following claims. It is apparent that certain modifications of the methods and compositions of the following claims can be made within the scope and spirit of the invention.

Claims

Claims
1. The use of an acyclic nucleoside purine of formula I
Figure imgf000037_0001
wherein;
X is OH or -N(R^2 wherein R1 independently is,
H; C2-Ci5 alkyl, C3-Ci5 alkenyl, C6-C15 arylalkenyl, C3-C15 alkynyl, C7-Ci5 arylalkynyl, Ci-Q-alkylamino-Q-Cδ alkyl, C5-C15 aralkyl, C6-Ci5 heteroalkyl or C3-C6 heterocycloalkyl wherein methylene in an alkyl moiety not adjacent to NH has been replaced by~O~; or
C1-Ci5 alkyl, C2-Ci5 alkenyl, C6-C15 arylalkenyl, C6-Ci5 arylalkynyl, C2- Ci5 alkynyl, Ci-Ce-alkylamino-CrCδ alkyl, C5-Ci5 aralkyl, C6-Ci5 heteroaralkyl, C4-C6 aryl, C3 -Cgcycloalkyl, or C2-C6 heterocycloalkyl; or optionally both R1 are joined together to form a saturated or unsaturated C2-C5 heterocycle containing one or two N heteroatoms and optionally an additional O or S heteroatom; wherein one of the foregoing R1 groups can be substituted with halo,-
CN or-N3;
W is H or NH2;
Y independently is, OH,-OR2,-OCH(R3)OC(0)R2, a monophosphate, a diphosphate, an amino acid amidate, a polypeptide amidate,-NHR , or-N(R )z I R2 independently is unsubstituted alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl wherein H is substituted by halo, carboxy, hydroxyl, cyano, nitro, N- nαorpholino, or amino; or alkyl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl in which a-CH2- moiety has been substituted by NH, S, or O;
Z is C1-C6 alkyl, optionally substituted with one or more hydroxy; and
R3 is H or R2; or a prodrug, diphosphate, pharmaceutically acceptable salt, or other phosphorus- substituted derivative thereof in the manufacture of a medicament for inhibiting an enzyme that causes neoplasia in an animal.
2. The use of claim 1 wherein the acyclic nucleoside purine is a compound of formula I
Figure imgf000038_0001
wherein; n is 2 or 3;
X Is-N(R^)2 wherein
R1 independently is H;
C2-C15 alkyl, C3-C15 alkenyl, C6-Ci5 arylalkenyl, C3-Ci5 alkynyl, C7-C15 arylalkynyl, Ci-Cβ-alkylamino-Ci-Cβ alkyl, C5-C15 aralkyl, C6-C15 heteroalkyl or C3-C6 heterocycloalkyl wherein methylene in an alkyl moiety not adjacent to NH has been replaced by~O— ; or
C1-C15 alkyl, C2-C15 alkenyl, C6-Ci5 arylalkenyl, CO-CI5 arylalkynyl, C2- C15 alkynyl, Ci-C6-alkylamino-d-C6 alkyl, C5-C15 aralkyl, C6-C15 heteroaralkyl, C4-C6 aryl, C2-Cg heterocycloalkyl; or optionally both R1 are joined together to form a saturated or unsaturated C2-C5 heterocycle containing one or two N heteroatoms and optionally an additional O or S heteroatom; wherein one of the foregoing R1 groups can be substituted with halo,- CN or-N3, but either one or two R1 groups are not H;
Y independently is, OH,-OR2,-OCH(R3)OC(O)R2, a monophosphate, a diphosphate, an amino acid amidate, a polypeptide amidate,-NHR2, or-N(R2)2 ;
R2 independently is unsubstituted alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl wherein H is substituted by halo, carboxy, hydroxyl, cyano, nitro, N- morpholino, or amino; or alkyl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl in which a-CH2- moiety has been substituted by NH, S, or O;
R3 is H or R2; or a prodrug, diphosphate, or other phosphorus-substituted derivative thereof in the manufacture of a medicament for enhancing the processivity of a telomerase or inhibiting an enzyme that causes neoplasia in an animal.
3. The use of claim 1 wherein the acyclic nucleoside purine of formula I is a compound of the following formula:
Figure imgf000039_0001
or a prodrug or pharmaceutically acceptable salt thereof.
4. The use of claim 2 wherein the acyclic nucleoside purine is a compound of the following formula:
Figure imgf000040_0001
or a prodrug thereof.
5. The use of claim 1 or 3 wherein X is OH.
6. The use of any one of claims 1-4 wherein each R1 independently is H, methyl, or cyclopropyl.
7. The use of any one of claims 1-4 wherein each R1 independently is H.
8. The use of any one of claims 1-4 wherein each R1 independently is methyl.
9. The use of any one of claims 1-4 wherein one R1 is H and the other R1 is cyclopropyl.
10. The use of claim 1 or 3 wherein W is H.
11. The use of claim 1 or 3 wherein W is NH2.
12. The use of claim 1, 3, or 10 wherein Z is -CH2-CH2-, -CH2-CH(CH3)-, or- CH2-CH(CH2OH)-.
13. The use of claim 2 or 4 wherein n is 2.
14. The use of claim 2 or 4 n is 3.
15. The use of claim 1 , 3 , or 10 wherein the compound of formula I is an R- enantiomer.
16. The use of claim 1 wherein the compound of formula I is PMEGpp or a pharmaceutically acceptable salt or prodrug thereof.
17. The use of claim 1 wherein the compound of formula I is (R)-PMPGpp or a pharmaceutically acceptable salt or a prodrug thereof.
18. The use of claim 1 wherein the compound of formula I is (S)-PMPGpp or a pharmaceutically acceptable salt or a prodrug thereof.
19. The use of claim 1 wherein the compound of formula I is PMED APpp or a pharmaceutically acceptable salt or a prodrug thereof.
20. The use of claim 1 wherein the compound of formula I is 6-cypr-(R)- PMPD APpp or a pharmaceutically acceptable salt or a prodrug thereof.
21. The use of PMEODAPy or a prodrug, diphosphate, pharmaceutically acceptable salt, or other phosphorus-substituted derivative thereof in the manufacture of a medicament for inhibiting an enzyme that causes neoplasia in an animal.
22. A pharmaceutical composition comprising a compound as described in any one of claims 1-21 and a pharmaceutically acceptable excipient.
23. A method for inhibiting an enzyme that causes neoplasia comprising contacting the enzyme with a compound as described in any one of claims 1-22.
24. A method for treating neoplasia in an animal comprising administering an effective amount of a compound as described in any one of claims 1-22 to the animal.
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