Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
• • 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
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • 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
  • 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
  • A61P35/00—Antineoplastic agents
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
  • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
  • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/18—Testing for antimicrobial activity of a material
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

• This application relates to prodrugs of methoxyphosphonate nucleotide analogues. In particular it relates to improved methods for making and identifying such prodrugs.
• Prodrugs of methoxyphosphonate nucleotide analogues intended for antiviral or antitumor therapy while known, traditionally have been selected for their systemic effect.
• prodrugs have been selected for enhanced bioavailability, i.e., ability to be absorbed from the gastrointestinal tract and converted rapidly to parent drug to ensure that the parent drug is available to all tissues.
• bioavailability i.e., ability to be absorbed from the gastrointestinal tract and converted rapidly to parent drug to ensure that the parent drug is available to all tissues.
• the objective of this invention is, among other advantages, to produce less toxicity to bystander tissues and greater potency of the parental drug in tissues which are the targets of therapy with the parent methoxyphosphonate nucleotide analogue.
• a screening method for identifying a methoxyphosphonate nucleotide analogue prodrug conferring enhanced activity in a target tissue comprising:
• step (d) determining the relative antiviral activity conferred by the prodrug in the tissues in step (c).
• the target tissue are sites where HIV is actively replicated and/or which serve as an HIV reservoir, and the non-target tissue is an intact animal.
• selecting lymphoid tissue as the target tissue for the practice of this method for HIV led to identification of prodrugs that enhance the delivery of active drug to such tissues.
• a preferred compound of this invention which has been identified by this method has the structure (1),
• Ra is H or methyl
• a preferred compound of this invention has the structure (2)
• R 1 is an oxyester which is hydrolyzable in vivo, or hydroxyl
• B is a heterocyclic base
• R 2 is hydroxyl, or the residue of an amino acid bonded to the P atom through an amino group of the amino acid and having each carboxy substituent of the amino acid optionally esterified, but not both of R 1 and R 2 are hydroxyl;
• E is —(CH 2 ) 2 —, —CH(CH 3 )CH 2 —, —CH(CH 2 F)CH 2 —, —CH(CH 2 OH)CH 2 —, —CH(CH ⁇ CH 2 )CH 2 —, —CH(C ⁇ CH)CH 2 —, —CH(CH 2 N 3 )CH 2 ,
• the broken line represents an optional double bond
• R 4 and R 5 are independently hydrogen, hydroxy, halo, amino or a substituent having 1-5 carbon atoms selected from acyloxy, alkyoxy, alkylthio, alkylamino and dialkylamino;
• R 6 and R 6′ are independently H, C 1 -C 6 alkyl, C 1 -C 6 hydroxyalkyl, or C 2 -C 7 alkanoyl;
• R 7 is independently H, C 1 -C 6 alkyl, or are taken together to form —O— or —CH 2 —;
• R 8 is H, C 1 -C 6 alkyl, C 1 -C 6 hydroxyalkyl or C 1 -C 6 haloalkyl;
• R 9 is H, hydroxymethyl or acyloxymethyl
• the diastereomers of structure (3) are designated the (S) isomers at the phosphorus chiral center.
• Preferred embodiments of this invention are the diastereomerically enriched compounds having the structure (5a)
• R 5 is methyl or hydrogen
• R 6 independently is H, alkyl, alkenyl, alkynyl, aryl or arylalkyl, or R 6 independently is alkyl, alkenyl, alkynyl, aryl or arylalkyl which is substituted with from 1 to 3 substituents selected from alkylamino, alkylaminoalkyl, dialkylaminoalkyl, dialkylamino, hydroxyl, oxo, halo, amino, alkylthio, alkoxy, alkoxyalkyl, aryloxy, aryloxyalkyl, arylalkoxy, arylalkoxyalkyl, haloalkyl, nitro, nitroalkyl, azido, azidoalkyl, alkylacyl, alkylacylalkyl, carboxyl, or alkylacylamino;
• R 7 is the side chain of any naturally-occurring or pharmaceutically acceptable amino acid and which, if the side chain comprises carboxyl, the carboxyl group is optionally esterified with an alkyl or aryl group;
• R 11 is amino, alkylamino, oxo, or dialkylamino
• R 12 is amino or H
• a preferred embodiment of this invention is the compound of structure (6), 9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]propyl]adenine, also designated herein GS-7340
• Another preferred embodiment of this invention is the fumarate salt of structure (5) (structure (7)), 9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]propyl]adenine fumarate (1:1), also designated herein GS-7340-2
• compositions containing pharmaceutically acceptable excipients are used in effective doses in the therapy or prophylaxis of viral (particularly HIV or hepadnaviral) infections.
• a method for the facile manufacture of 9-[2-(phosphonomethoxy)propyl]adenine (hereinafter “PMPA” or 9-[2(phosphonomethoxy)ethyl] adenine (hereinafter “PMEA”) using magnesium alkoxide comprises combining 9-(2-hydroxypropyl)adenine or 9-(2-hydroxyethyl)adenine, protected p-toluenesulfonyloxymethylphosphonate and magnesium alkoxide, and recovering PMPA or PMEA, respectively.
• the methoxyphosphonate nucleotide analogue parent drugs for use in this screening method are compounds having the structure A-OH 2 P(O)(OH) 2 wherein A is the residue of a nucleoside analogue. These compounds are known per se and are not part of this invention. More particularly, the parent compounds comprise a heterocyclic base B and an aglycon E, in general having the structure
• group B is defined below and group E is defined above.
• group E is defined above. Examples are described in U.S. Pat. Nos. 4,659,825, 4,808,716, 4,724,233, 5,142,051, 5,130,427, 5,650,510, 5,663,159, 5,302,585, 5,476,938, 5,696,263, 5,744,600, 5,688,778, 5,386,030, 5,733,896, 5,352,786, and 5,798,340, and EP 821,690 and 654,037.
• the prodrugs for use in the screening method of this invention are covalently modified analogues of the parent methoxyphosphonate nucleotide analogues described in the preceding paragraph.
• the phosphorus atom of the parent drug is the preferred site for prodrug modification, but other sites are found on the heterocyclic base B or the aglycon E.
• Many such prodrugs are already known. Primarily, they are esters or amidates of the phosphorus atom, but also include substitutions on the base and aglycon. None of these modifications per se is part of this invention and none are to be considered limiting on the scope of the invention herein.
• the phosphorus atom of the methoxyphosphonate nucleotide analogues contains two valences for covalent modification such as amidation or esterification (unless one phosphoryl hydroxyl is esterified to an aglycon E hydroxyl substituent, whereupon only one phosphorus valence is free for substitution).
• the esters typically are aryloxy.
• the amidates ordinarily are naturally occurring monoamino acids having free carboxyl group(s) esterified with an alkyl or aryl group, usually phenyl, cycloalkyl, or t-, n- or s- alkyl groups.
• Suitable prodrugs for use in the screening method of this invention are disclosed for example in U.S. Pat. No.
• any prodrug which is potentially believed to be capable of being converted in vivo within target tissue cells to the free methoxyphosphonate nucleotide analogue parent drug, e.g., whether by hydrolysis, oxidation, or other covalent transformation resulting from exposure to biological tissues, is suitable for use in the method of this invention.
• Such prodrugs may not be known at this time but are identified in the future and thus become suitable candidates available for testing in the method of this invention. Since the prodrugs are simply candidates for screening in the methods their structures are not relevant to practicing or enabling the screening method, although of course their structures ultimately are dispositive of whether or not a prodrug will be shown to be selective in the assay.
• pro-moieties bound to the parent drug may be the same or different.
• each prodrug to be used in the screening assay will differ structurally from the other prodrugs to be tested. Distinct, i.e. structurally different, prodrugs generally are selected on the basis of either their stereochemistry or their covalent structure, or these features are varied in combination.
• Each prodrug tested desirably is structurally and stereochemically substantially pure, else the output of the screening assay will be less useful. It is of course within the scope of this invention to test only a single prodrug in an individual embodiment of the method of this invention, although typically then one would compare the results with prior studies with other prodrugs.
• Chiral sites are at the phosphorus atom and are also found in its substituents.
• amino acid used in preparing amidates may be D or L forms, and the phosphonate esters or the amino acid esters can contain chiral centers as well.
• Chiral sites also are found on the nucleoside analogue portion of the molecules, but these typically are already dictated by the stereochemistry of the parent drug and will not be varied as part of the screen.
• the R isomer of PMPA is preferred as it is more active than the corresponding S isomer.
• these diasteromers or enantiomers will be chirally enriched if not pure at each site so that the results of the screen will be more meaningful.
• distinctiveness of stereoisomers is conferred by enriching or purifying the stereoisomer (typically this will be a diastereomer rather than an enantiomer in the case of most methoxyphosphonate nucleotide analogues) free of other stereoisomers at the chiral center in question, so that each test compound is substantially homogeneous.
• substantially homogeneous or chirally enriched we mean that the desired stereoisomer constitutes greater than about 60% by weight of the compound, ordinarily greater than about 80% and preferably greater than about 95%.
• the remaining steps of the screening method of this invention are used to identify a prodrug possessing the required selectivity for the target tissue.
• the prodrugs are labeled with a detectable group, e.g. radiolabeled, in order to facilitate detection later in tissues or cells.
• a label is not required since other suitable assays for the prodrug or its metabolites (including the parent drug) can also be employed. These assays could include mass spectrometry, HPLC, bioassays or immunoassays for instance.
• the assay may detect the prodrug and any one or more of its metabolites, but preferably the assay is conducted to detect only the generation of the parent drug. This is based on the assumption (which may not be warranted in all cases) that the degree and rate of conversion of prodrug to antivirally active parent diphosphate is the same across all tissues tested. Otherwise, one can test for the diphosphate.
• the target tissue preferably will be lymphoid tissue when screening for prodrugs useful in the treatment of HIV infection.
• Lymphoid tissue will be known to the artisan and includes CD4 cells, lymphocytes, lymph nodes, macrophages and macrophage-like cells including monocytes such as peripheral blood monocytic cells (PBMCs) and glial cells.
• Lymphoid tissue also includes non-lymphoid tissues that are enriched in lymphoid tissues or cells, e.g. lung, skin and spleen.
• Other targets for other antiviral drugs of course will be the primary sites of replication or latency for the particular virus concerned, e.g., liver for hepatitis and peripheral nerves for HSV.
• target tissues for tumors will in fact be the tumors themselves. These tissues are all well-known to the artisan and would not require undue experimentation to select.
• target tissue can be infected by the virus.
• Non-target tissues or cells also are screened as part of the method herein. Any number or identity of such tissues or cells can be employed in this regard. In general, tissues for which the parent drug is expected to be toxic will be used as non-target tissues. The selection of a non-target tissue is entirely dependent upon the nature of the prodrug and the activity of the parent. For example, non-hepatic tissues would be selected for prodrugs against hepatitis, and untransformed cells of the same tissue as the tumor will suffice for the antitumor-selective prodrug screen.
• the method of this invention is distinct from studies typically undertaken to determine oral bioavailability of prodrugs.
• the objective is to identify a prodrug which passes into the systemic circulation substantially converted to parent drug.
• the objective is to find prodrugs that are not metabolized in the gastrointestinal tract or circulation.
• target tissues to be evaluated in the method of this invention generally do not include the small intestines or, if the intestines are included, then the tissues also include additional tissues other than the small intestines.
• the target and non-target tissues used in the screening method of this invention typically will be in an intact living animal.
• Prodrugs containing esters are more desirably tested in dogs, monkeys or other animals than rodents; mice and rat plasma contains high circulating levels of esterases that may produce a misleading result if the desired therapeutic subject is a human or higher mammal.
• tissue shall not be construed to require organized cellular structures, or the structures of tissues as they may be found in nature, although such would be preferred. Rather, the term “tissue” shall be construed to be synonymous with cells of a particular source, origin or differentiation stage.
• the target and non-target tissue may in fact be the same tissue, but the tissues will be in different biological status.
• the method herein could be used to select for prodrugs that confer activity in virally-infected tissue (target tissue) but which remain substantially inactive in virally-uninfected cells (corresponding non-target tissue).
• the same strategy would be employed to select prophylactic prodrugs, i.e., prodrugs metabolized to antivirally active forms incidental to viral infection but which remain substantially unmetabolized in uninfected cells.
• prodrugs could be screened in transformed cells and the untransformed counterpart tissue. This would be particularly useful in comparative testing to select prodrugs for the treatment of hematological malignancies, e.g. leukemias.
• tissue selective prodrugs are thought to be selectively taken up by target cells and/or selectively metabolized within the cell, as compared to other tissues or cells.
• the unique advantage of the methoxyphosphonate prodrugs herein is that their metabolism to the dianion at physiological pH ensures that they will be unable to diffuse back out of the cell. They therefore remain effective for lengthy periods of time and are maintained at elevated intracellular concentrations, thereby exhibiting increased potency.
• the mechanisms for enhanced activity in the target tissue are believed to include enhanced uptake by the target cells, enhanced intracellular retention, or both mechanisms working together.
• the manner in which selectivity or enhanced delivery occurs in the target tissue is not important. It also is not important that all of the metabolic conversion of the prodrug to the parent compound occurs within the target tissue. Only the final drug activity-conferring conversion need occur in the target tissue; metabolism in other tissues may provide intermediates finally converted to antiviral forms in the target tissue.
• the degree of selectivity or enhanced delivery that is desired will vary with the parent compound and the manner in which it is measured (% dose distribution or parent drug concentration). In general, if the parent drug already possess a generous therapeutic window, a low degree of selectivity may be sufficient for the desired prodrug. On the other hand, toxic compounds may require more extensive screening to identify selective prodrugs. The relative expense of the method of this invention can be reduced by screening only in the target tissue and tissues against which the parent compound is known to be relatively toxic, e.g. for PMEA, which is nephrotoxic at higher doses, the primary focus will be on kidney and lymphoid tissues.
• the step of determining the relative antiviral activity of a prodrug in the selected tissues ordinarily is accomplished by assaying target and non-target tissues for the relative presence or activity of a metabolite of the prodrug, which metabolite is known to have, or is converted to, a metabolite having antiviral or antitumor activity.
• a metabolite of the prodrug which metabolite is known to have, or is converted to, a metabolite having antiviral or antitumor activity.
• the active metabolite is the diphosphate of the phosphonate parent compounds. It is this metabolite that is incorporated into the viral nucleic acid, thereby truncating the elongating nucleic acid strand and halting viral replication.
• Metabolites of the prodrug can be anabolic metabolites, catabolic metabolites, or the product of anabolism and catabolism together. The manner in which the metabolite is produced is not important in the practice of the method of this invention.
• the method of this invention is not limited to assaying a metabolite which per se possesses antiviral or antitumor activity. Instead, one can assay inactive precursors of the active metabolites.
• Precursors of the antivirally active diphosphate metabolite include the monophosphate of the parent drug, monophosphates of other metabolites of the parent drug (e.g., an intermediate modification of a substituent on the heterocyclic base), the parent itself and metabolites generated by the cell in converting the prodrug to the parent prior to phosphorylation.
• the precursor structures may vary considerably as they are the result of cellular metabolism. However, this information is already known or could be readily determined by one skilled in the art.
• step (d) of the method herein calls for determining the activity, activity can be either measured directly or extrapolated.
• Step (d) only requires assessment of the activity conferred by the prodrug as it interacts with the tissue concerned, and this may be based on extrapolation or other indirect measurement.
• Step (d) of the method of this invention calls for determining the “relative” activity of the prodrug. It will be understood that this does not require that each and every assay or series of assays necessarily must also contain runs with the selected non-target tissue. On the contrary, it is within the scope of this invention to employ historical controls of the non-target tissue or tissues, or algorithms representing results to be expected from such non-target tissues, in order to provide the benchmark non-target activity.
• step (d) The results obtained in step (d) are then used optimally to select or identify a prodrug which produces greater antiviral activity in the target tissue than in the non-target tissue. It is this prodrug that is selected for further development.
• prodrug candidates can be undertaken before the practice of the method of this invention.
• the prodrug will need to be capable of passing largely unmetabolized through the gastrointestinal tract, it will need to be substantially stable in blood, and it should be able to permeate cells at least to some degree. In most cases it also will need to complete a first pass of the hepatic circulation without substantial metabolism.
• Such prestudies are optional, and are well-known to those skilled in the art.
• antiviral activity is applicable to antitumor prodrugs of methoxyphosphonate nucleotide analogues as well.
• these include, for example, prodrugs of PMEG, the guanyl analogue of PMEA.
• cytotoxic phosphonates such as PMEG are worthwhile candidates to pursue as their cytotoxicity in fact confers their antitumor activity.
• a compound identified by this novel screening method then can be entered into a traditional preclinical or clinical program to confirm that the desired objectives have been met.
• a prodrug is considered to be selective if the activity or concentration of parent drug in the target tissue (% dose distribution) is greater than 2 ⁇ , and preferably 5 ⁇ , that of the parent compound in non-target tissue.
• a prodrug candidate can be compared against a benchmark prodrug. In this case, selectivity is relative rather than absolute. Selective prodrugs will be those resulting in greater than about 10 ⁇ concentration or activity in the target tissue as compared with the prototype, although the degree of selectivity is a matter of discretion.
• an improved method for manufacture of preferred starting materials (parent drugs) of this invention PMEA and (R)-PMPA.
• this method comprises reacting 9-(2-hydroxypropyl)adenine (HPA) or 9(2-hydroxyethyl)adenine (HEA) with a magnesium alkoxide, thereafter adding the protected aglycon synthon p-toluene-sulfonyloxymethylphosphonate (tosylate) to the reaction mixture, and recovering PMPA or PMEA, respectively.
• HPA is the enriched or isolated R enantiomer. If a chiral HPA mixture is used, R-PMPA can be isolated from the chiral PMPA mixture after the synthesis is completed.
• the tosylate is protected by lower alkyl groups, but other suitable groups will be apparent to the artisan. It may be convenient to employ the tosylate presubstituted with the prodrug phosphonate substituents which are capable of acting as protecting groups in the tosylation reaction, thereby allowing one to bypass the deprotection step and directly recover prodrug or an intermediate therefore.
• the alkyl group of the magnesium alkoxide is not critical and can be any C 1 -C 6 branched or normal alkyl, but is preferably t-butyl (for PMPA) or isopropyl (for PMEA).
• the reaction conditions also are not critical, but preferably comprise heating the reaction mixture at about 70-75° C. with stirring or other moderate agitation.
• the product is deprotected (usually with bromotrimethylsilane where the tosylate protecting group is alkyl), and the product then recovered by crystallization or other conventional method as will be apparent to the artisan.
• heterocyclic base B is selected from the structures
• R 15 is H, OH, F, Cl, Br, I, OR 16 , SH, SR 16 , NH 2 , or NHR 17 ;
• R 16 is C 1 -C 6 alkyl or C 2 -C 6 alkenyl including CH 3 , CH 2 CH 3 , CH 2 CCH, CH 2 CHCH 2 and C 3 H 7 ;
• R 17 is C 1 -C 6 alkyl or C 2 -C 6 alkenyl including CH 3 , CH 2 CH 3 , CH 2 CCH, CH 2 CHCH 2 , and C 3 H 7 ;
• R 18 is N, CF, CCl, CBr, CI, CR 19 , CSR 19 , or COR 19 ;
• R 19 is H, C 1 -C 9 alkyl, C 2 -C 9 alkenyl, C 2 -C 9 alkynyl, C 1 -C 9 alkyl-C 1 -C 9 alkoxy, or C 7 -C 9 aryl-alkyl unsubstituted or substituted by OH, F, Cl, Br or I, R 19 therefore including —CH 3 , —CH 2 CH 3 , —CHCH 2 , —CHCHBr, —CH 2 CH 2 Cl, —CH 2 CH 2 F, —CH 2 CCH, —CH 2 CHCH 2 , —C 3 H 7 , —CH 2 OH, —CH 2 OCH 3 , —CH 2 OC 2 H 5 , —CH 2 OCCH, —CH 2 OCH 2 CHCH 2 , —CH 2 C 3 H 7 , —CH 2 CH 2 OH, —CH 2 CH 2 OCH 3 , —CH 2 CH 2 OC 2
• R 20 is N or CH
• R 21 is N, CH, CCN, CCF 3 , CC ⁇ CH or CC(O)NH 2 ;
• R 22 is H, OH, NH 2 , SH, SCH 3 , SCH 2 CH 3 , SCH 2 CCH, SCH 2 CHCH 2 , SC 3 H 7 , NH(CH 3 ), N(CH 3 ) 2 , NH(CH 2 CH 3 ), N(CH 2 CH 3 ) 2 , NH(CH 2 CCH), NH(CH 2 CHCH 2 ), NH(C 3 H 7 ), halogen (F, Cl, Br or I) or X wherein X is —(CH 2 ) m (O) n (CH 2 ) m N(R 10 ) 2 wherein each m is independently 0-2, n is 0-1, and
• R 10 independently is
• both R 10 are joined together with N 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 10 groups which is substituted with 1 to 3 halo, CN or N 3 ; but optionally at least one R 10 group is not H;
• R 23 is H, OH, F, Cl, Br, I, SCH 3 , SCH 2 CH 3 , SCH 2 CCH, SCH 2 CHCH 2 , SC 3 H 7 , OR 16 , NH 2 , NHR 17 or R 22 ;
• R 24 is O, S or Se.
• B also includes both protected and unprotected heterocyclic bases, particularly purine and pyrimidine bases.
• Protecting groups for exocyclic amines and other labile groups are known (Greene et al. “Protective Groups in Organic Synthesis”) and include N-benzoyl, isobutyryl, 4,4′-dimethoxytrityl (DMT) and the like.
• DMT 4,4′-dimethoxytrityl
• the selection of protecting group will be apparent to the ordinary artisan and will depend upon the nature of the labile group and the chemistry which the protecting group is expected to encounter, e.g. acidic, basic, oxidative, reductive or other conditions.
• Exemplary protected species are N 4 -benzoylcytosine, N 6 benzoyladenine, N 2 -isobutyrylguanine and the like.
• Protected bases have the formulas Xa.1, XIa.1, XIb.1, XIIa.1 or XIIIa.1
• R 18 , R 20 , R 21 , R 24 have the meanings previously defined;
• R 22A is R 39 or R 22 provided that R 22 is not NH 2 ;
• R 23A is R 39 or R 23 provided that R 23 is not NH 2 ;
• R 39 is NHR 40 , NHC(O)R 36 or CR 41 N(R 38 ) 2 wherein R 36 is C 1 -C 19 alkyl, C 1 -C 19 alkenyl, C 3 -C 10 aryl, adamantoyl, alkylanyl, or C 3 -C 10 aryl substituted with 1 or 2 atoms or groups selected from halogen, methyl, ethyl, methoxy, ethoxy, hydroxy and cyano;
• R 38 is C 1 -C 10 alkyl, or both R 38 together are 1-morpholino, 1-piperidine or 1pyrrolidine;
• R 40 is C 1 -C 1a alkyl, including methyl, eth
• R 39 is present at R 22A or R 23A , both R 39 groups on the same base will generally be the same.
• Exemplary R 36 are phenyl, phenyl substituted with one of the foregoing R 36 aryl substituents, —C 10 H 15 (where C 10 H 15 is 2-adamantoyl), —CH 2 —C 6 H 5 , —C 6 H 5 , —CH(CH 3 ) 2 , —CH 2 CH 3 , methyl, butyl, t-butyl, heptanyl, nonanyl, undecanyl, or undecenyl.
• Specific bases include hypoxanthine, guanine, adenine, cytosine, inosine, thymine, uracil, xanthine, 8-aza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza-8-aza derivatives of adenine, guanine, 2-aminopurine, 2,6-diaminopurine, 2-amino-6chloropurine, hypoxanthine, inosine and xanthine; 1-deaza derivatives of 2aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6chloropurine, hypo
• B is a 9-purinyl residue selected from guanyl, 3-deazaguanyl, 1deazaguanyl, 8-azaguanyl, 7-deazaguanyl, adenyl, 3-deazaadenyl, 1-dezazadenyl, 8-azaadenyl, 7-deazaadenyl, 2,6-diaminopurinyl, 2-aminopurinyl, 6-chloro-2aminopurinyl and 6-thio-2-aminopurinyl, or a B′ is a 1-pyrimidinyl residue selected from cytosinyl, 5-halocytosinyl, and 5-(C 1 -C 3 -alkyl)cytosinyl.
• R 22 independently is halo, oxygen, NH 2 , X or H, but optionally at least one R 22 is X;
• X is —(CH 2 ) m (O) n (CH 2 ) m N(R 10 ) 2 wherein m is 0-2, n is 0-1, and R 10 independently is
• C 2 -C 15 alkyl C 3-15 alkenyl, C 6 -C 15 arylalkenyl, C 3 -C 15 alkynyl, C 7 -C 15 arylalkynyl, C 1 -C 6 -alkylamino-C 1 -C 6 alkyl, C 5 -C 15 aralkyl, C 6 -C 15 heteroalkyl or C 3 -C 6 heterocycloalkyl wherein methylene in the alkyl moiety not adjacent to N 6 has been replaced by —O—,
• both R 10 are joined together with N 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 10 groups are substituted with 1 to 3 halo, CN or N 3 ; but optionally at least one R 10 group is not H; and
• Z is N or CH, provided that the heterocyclic nucleus varies from purine by no more than one Z.
• E groups represent the aglycons employed in the methoxyphosphonate nucleotide analogues.
• the E group is —CH(CH 3 )CH 2 — or —CH 2 CH 2 —.
• the side groups at chiral centers in the aglycon be substantially solely in the (R) configuration (except for hydroxymethyl, which is the enriched (S) enantiomer).
• R 1 is an in vivo hydrolyzable oxyester having the structure —OR 35 or —OR 6 wherein R 35 is defined in column 64, line 49 of U.S. Pat. No. 5,798,340, herein incorporated by reference, and R 6 is defined above.
• R 1 is aryloxy, ordinarily unsubstituted or para-substituted (as defined in R 6 ) phenoxy.
• R 2 is an amino acid residue, optionally provided that any carboxy group linked by less than about 5 atoms to the amidate N is esterified.
• R 2 typically has the structure
• n 1 or 2;
• R 11 is R 6 or H; preferably R 6 ⁇ C 3 —C 9 alkyl; C 3 -C 9 alkyl substituted independently with OH, halogen, O or N; C 3 -C 6 aryl; C 3 -C 6 aryl which is independently substituted with OH, halogen, O or N; or C 3 -C 6 arylalkyl which is independently substituted with OH, halogen, O or N;
• R 12 independently is H or C, —C, alkyl which is unsubstituted or substituted by substituents independently selected from the group consisting of OH, O, N, COOR 11 and halogen; C 3 -C 6 aryl which is unsubstituted or substituted by substituents independently selected from the group consisting of OH, O, N, COOR 11 and halogen; or C 3 -C 6 aryl-alkyl which is unsubstituted or substituted by substituents independently selected from the group consisting of OH, O, N, COOR 11 and halogen;
• R 13 independently is C(O)—OR 13 ; amino; amide; guanidinyl; imidazolyl; indolyl; sulfoxide; phosphoryl; C 1 -C 3 alkylamino; C 1 -C 3 alkyldiamino; C 1 -C 6 alkenylamino; hydroxy; thiol; C 1 -C 3 alkoxy; C 1 -C 3 alkthiol; (CH 2 ) n COOR 11 ; C 1 -C 6 alkyl which is unsubstituted or substituted with OH, halogen, SH, NH 2 , phenyl, hydroxyphenyl or C 7 -C 10 alkoxyphenyl; C 2 -C 6 alkenyl which is unsubstituted or substituted with OH, halogen, SH, NH 2 , phenyl, hydroxyphenyl or C 7 —C 10 alkoxyphenyl; and C 6
• R 14 is H or C 1 -C 9 alkyl or C 1 -C 9 alkyl independently substituted with OH, halogen, COOR 11 , O or N; C 3 -C 6 aryl; C 3 -C 6 aryl which is independently substituted with OH, halogen, COOR 11 , O or N; or C 3 -C 6 arylalkyl which is independently substituted with OH, halogen, COOR 11 , O or N.
• R 11 is C 1 -C 6 alkyl, most preferably isopropyl
• R 13 is the side chain of a naturally occurring amino acid
• n 1
• R 12 is H
• R is H.
• the invention includes metabolites in which the phenoxy and isopropyl esters have been hydrolyzed to —OH.
• the de-esterified enriched phosphonoamidate metabolites of compounds (5a), 5(b) and (6) are included within the scope of this invention.
• Aryl and “O” or “N” substitution are defined in column 16, lines 42-58, of U.S. Pat. No. 5,798,340.
• the amino acids are in the natural or l amino acids. Suitable specific examples are set forth in U.S. Pat. No. 5,798,340, for instance Table 4 and col. 8-10 therein.
• Alkyl as used herein is a normal, secondary, tertiary or cyclic hydrocarbon. Unless stated to the contrary alkyl is C 1 -C 12 . Examples are —CH 3 , —CH 2 CH 3 , —CH 2 CH 2 CH 3 , —CH(CH 3 ) 2 , —CH 2 CH 2 CH 2 CH 3 ), —CH 2 CH(CH 3 ) 2 , —CH(CH 3 )CH 2 CH 3 , —C(CH 3 ) 3 , —CH 2 CH 2 CH 2 CH 2 CH 3 , —CH(CH 3 )CH 2 CH 2 CH 3 , —CH(CH 2 CH 3 ) 2 , —C(CH 3 ) 2 CH 2 CH 3 ), —CH(CH 3 )CH(CH 3 ) 2 , —CH 2 CH 2 CH(CH 3 ) 2 ), —CH 2 CH(CH 3 )CH 2 CH 3 , —CH 2 CH 2 CH 2 , —CH 2 CH(CH
• the prodrug compounds of this invention are provided in the form of free base or the various salts enumerated in U.S. Pat. No. 5,798,340, and are formulated with pharmaceutically acceptable excipients or solvating diluents for use as pharmaceutical products also as set forth in U.S. Pat. No. 5,798,340.
• These prodrugs have the antiviral and utilities already established for the parent drugs (see U.S. Pat. No. 5,798,340 and other citations relating to the methoxyphosphonate nucleotide analogues). It will be understood that the diastereomer of structure (4) at least is useful as an intermediate in the chemical production of the parent drug by hydrolysis in vitro, regardless of its relatively unselective character as revealed in the studies herein.
• bromotrimethylsilane (73.9 g, 0.478 mol) was added and the mixture heated to 77° C. for 3 hours. When complete, the reaction was heated to 80° C. and volatiles were removed via atmospheric distillation. The residue was dissolved into water (120 ml) at 50° C. and then extracted with ethyl acetate (101 ml). The pH of the aqueous phase was adjusted to pH 1.1 with sodium hydroxide, seeded with authentic (R)-PMPA, and the pH of the aqueous layer was readjusted to pH 2.1 with sodium hydroxide. The resulting slurry was stirred at room temperature overnight. The slurry was cooled to 4° C. for three hours.
• a glass-lined reactor was charged with anhydrous PMPA, (I) (14.6 kg, 50.8 mol), phenol (9.6 kg, 102 mol), and 1-methyl-2-pyrrolidinone (39 kg). The mixture was heated to 85° C. and _triethylamine (6.3 kg, 62.3 mol) added. A solution of 1,3dicyclohexylcarbodiimide (17.1 kg, 82.9 mol) in 1-methyl-2-pyrrolidinone (1.6 kg) was then added over 6 hours at 100° C. Heating was continued for 16 hours. The reaction was cooled to 45° C., water (29 kg) added, and cooled to 25° C. Solids were removed from the reaction by filtration and rinsed with water (15.3 kg).
• GS-7171 (III) [0131] GS-7171 (III).
• Scheme 1 A glass-lined reactor was charged with monophenyl PMPA, (II), (9.12 kg, 25.1 mol) and acetonitrile (30.7 kg). Thionyl chloride (6.57 kg, 56.7 mol) was added below 50° C. The mixture was heated at 75° C. until solids dissolved. Reaction temperature was increased to 80° C. and volatiles (11.4 kg) collected by atmospheric distillation under nitrogen. The pot residue was cooled to 25° C., dichloromethane (41 kg) added, and cooled to ⁇ 29° C.
• Half the product solution was purified by chromatography over a 38 ⁇ 38 cm bed of 22 kg silica gel 60, 230 to 400 mesh. The column was eluted with 480 kg acetone. The purification was repeated on the second half of the oil using fresh silica gel and acetone. Clean product bearing fractions were concentrated under reduced pressure to an oil. Acetonitrile (19.6 kg) was charged to the oil and the mixture concentrated under reduced pressure. Acetonitrile (66.4 kg) was charged and the solution chilled to 0 to ⁇ 5° C. for 16 hours.
• the oil was combined with fumaric acid (0.77 g, 6.6 mmol) and acetonitrile (40 mL) and heated to reflux to give a homogeneous solution.
• the solution was cooled in an ice bath and solids isolated by filtration.
• the solid GS-7171 fumarate salt was dried under reduced pressure to 3.7 g.
• the salt (3.16 g, 5.3 mmol) was suspended in dichloromethane (30 mL) and stirred with potassium carbonate solution (5 mL, 2.5 M in water) until the solid dissolved.
• the organic layer was isolated, then washed with water (5 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to afford 2.4 g III as a tan foam.
• Chiralpak AS is a proprietary packing material manufactured by Diacel and sold in North America by Chiral Technologies, Inc. (U.S. Pat. No. 5,202,433, RE 35,919, U.S. Pat. No. 5,434,298, 5,434,299 and 5,498,752).
• Chiralpak AS is a chiral stationary phase (CSP) comprised of amylosetris[(S)- ⁇ -methylbenzyl carbamate] coated onto a silica gel support.
• CSP chiral stationary phase
• the GS-7171 diastereomeric mixture was dissolved in mobile phase, and approximately 1 g aliquots of GS-7171 were pumped onto the chromatographic system.
• the undesired diastereomer, designated GS-7339 was the first major broad (approx. 15 min. duration) peak to elute from the column.
• the mobile phase was immediately switched to 100% methyl alcohol, which caused the desired diastereomer, designated GS-7340 (IV), to elute as a sharp peak from the column with the methyl alcohol solvent front.
• the methyl alcohol was used to reduce the over-all cycle time.
• both diastereomers were collected as a single large fractions containing one of the purified diastereomers (>99.0% single diastereomer).
• the mobile phase solvents were removed in vacuo to yield the purified diastereomer as a friable foam.
• GS-7340 IV.
• GS-7171 (III) 2.8 kg, was purified by simulated moving bed chromatography over 10 cm by 5 cm beds of packing (Chiral Technologies Inc., 20 micron Chiralpak AS coated on silica gel) (1.2 kg). The columns were eluted with 30% methanol in acetonitrile. Product bearing fractions were concentrated to a solution of IV in acetonitrile (2.48 kg). The solution solidified to a crystalline mass wet with acetonitrile on standing.
• GS-7171 (III) was chromatographed by reverse phase HPLC to separate the diastereomers using the following summary protocol.
• Chromatographic column Phenomenex Luna TM C18(2), 5 ⁇ m, 100 ⁇ pore size, (Phenomenex, Torrance, CA), or equivalent Guard column: Pellicular C18 (Alltech, Deerfield, IL), or equivalent
• Mobile Phase A—0.02% (85%) H 3 PO 4 in water: acetonitrile (95:5)
• Equilibration Delay 10 min at 100% mobile phase
• a Flow Rate 1.2 mL/min
• Temperature Ambient Detection: UV at 260 nm
• Sample Solution 20 mM sodium phosphate buffer
• GS-7340 IV.
• a solution of GS7171 (III) in acetonitrile was concentrated to an amber foam (14.9 g) under reduced pressure.
• the foam was dissolved in acetonitrile (20 mL) and seeded with a crystal of IV.
• the mixture was stirred overnight, cooled to 5° C., and solids isolated by filtration.
• GS-7340-02 (V).
• Scheme 1 A glass-lined reactor was charged with GS7340 (IV), (1.294 kg, 2.71 mol), fumaric acid (284 g, 2.44 mol), and acetonitrile (24.6 kg). The mixture was heated to reflux to dissolve the solids, filtered while hot and cooled to 5° C. for 16 hours. The product was isolated by filtration, rinsed with acetonitrile (9.2 kg), and dried to 1329 g (V) as a white powder: mp 119.7-121.1° C.; [ ⁇ ] D 20 -41.7° (c 1.0, acetic acid).
• the oil was purified by chromatography over a 15 ⁇ 13 cm bed of 1.2 kg silica gel 60, 230 to 400 mesh.
• the column was eluted with a gradient of dichloromethane and methanol. Product bearing fractions were concentrated under reduced pressure to afford 211 g VI (Scheme 3) as a tan foam.
• the diastereomeric mixture was purified using the conditions described for GS7171 in Example 3A except for the following: Mobile Phase (Initial) GS-7120 - Acetonitrile Isopropyl Alcohol (98:2) (Final) 100% Methyl Alcohol Elution Profile GS-7341 (diastereomer B) GS-7342 (diastereomer A)
• the diastereomeric mixture was purified using the conditions described for GS-7171 (Example 3A) except for the following: Mobile Phase (Initial) GS-7120 - Acetonitrile Isopropyl Alcohol (95:5) (Final) 100% Methyl Alcohol Elution Profile: GS-7115 (diastereomer B) GS-7114 (diastereomer A)
• GS-7097 Phenyl PMPA, Ethyl L-Alanyl Amidate.
• Phenyl PMPA (15.0 g, 41.3 mmol)
• L-alanine ethyl ester hydrochloride (12.6 g, 83 mmol)
• triethylamine (11.5 mL, 83 mmol) were slurried together in 500 mL pyridine under dry N 2 .
• This suspension was combined with a solution of triphenylphosphine (37.9 g, 145 mmol), Aldrithiol 2 (2,2′-dipyridyl disulfide) (31.8 g, 145 mmol), and 120 mL pyridine.
• the mixture was heated at an internal temperature of 57° C. for 15 hours.
• the complete reaction was concentrated under vacuum to a yellow paste, 100 g.
• the paste was purified by column chromatography over a 25 ⁇ 11 cm bed of 1.1 kg silica gel 60, 230 to 400 mesh.
• the column was eluted with 8 liters of 2% methanol in dichloromethane followed by a linear gradient over a course of 26 liters eluent up to a final composition of 13% methanol. Clean product bearing fractions were concentrated to yield 12.4 g crude (5), 65% theory. This material was contaminated with about 15% (weight) triethylamine hydrochloride by 1 H NMR.
• the contamination was removed by dissolving the product in 350 mL ethyl acetate, extracting with 20 mL water, drying the organic solution over anhydrous sodium sulfate, and concentrating to yield 11.1 g pure GS-7097 as a white solid, 58% yield.
• the process also is employed to synthesize the diastereomeric mixture of GS-7003a and GS-7003b (the phenylalanyl amidate) and the mixture GS-7119 and GS7335 (the glycyl amidate). These diastereomers are separated using a batch elution procedure such as shown in Example 3A, 6 and 7.
• GS7340 shows a 10-fold increase in antiviral activity relative to TDF and a 200-fold increase in plasma stability. This greater plasma stability is expected to result in higher circulating levels of GS7340 than TDF after oral administration.
• GS-7340 results in 10 ⁇ and 30 ⁇ the total intracellular concentration of PMPA species in PBMCs as compared to TDF and PMPA, respectively.
• 84% of the radioactivity is due to intact GS-7340, whereas no TDF is detected at 1 hour. Since no intact TDF is detected in plasma, the 10 ⁇ difference at 1 hour between TDF and GS-7340 is the minimum difference expected in vivo.
• the HPLC chromatogram for all three compounds in PBMCs is shown in FIG. 1. TABLE 2 PMPA Metabolites in Plasma, PBMCs and RBCs After 1 h Incubation of PMPA Prodrugs or PMPA in Human Blood.
• Met. X and Met Y are shown in Table 5. Lower case “p” designates phosphorylation. These results were obtained after 1 hour in human blood. With increasing time, the in vitro differences are expected to increase, since 84% of GS7340 is still intact in plasma after one hour. Because intact GS7340 is present in plasma after oral administration, the relative clinical efficacy should be related to the IC 50 values seen in vitro.
• IC 50 values of tenofovir, TDF, GS7340, several nucleosides and the protease inhibitor nelfinivir are listed. As shown, nelfinavir and GS7340 are 2-3 orders of magnitude more potent than all other nucleotides or nucleosides.
• “Phe-methylester” is the methylphenylalaninyl monoamidate, phenyl monoester of tenofovir; “gly-methylester” is the methylglycyl monoamidate, phenyl monoester of tenofovir.
• isomer A is believed to have the same absolute stereochemistry as GS-7340 (S), and isomer B is believed to have the same absolute stereochemistry that of GS7339.
• reaction mixture 20 ⁇ L
• 60 ⁇ L of methanol containing the internal standard 60 ⁇ L
• the mixture thus obtained was centrifuged at 15,000 G for 5 min and the supernatant was analyzed with HPLC under the conditions described below.
• MT-2 cell extract was prepared from MT-2 cells according to the published procedure [A. Pompon, I. Lefebvre, J.-L. Imbach, S. Kahn, and D. Farquhar, “Antiviral Chemistry & Chemotherapy”, 5:91-98 (1994)] except for using HEPES buffer described below as the medium.
• PBS phosphate-buffered saline, Sigma
• PBS phosphate-buffered saline
• HEPES buffer contains 0.010 M HEPES, 0.05 M potassium chloride, 0.005 M magnesium chloride, and 0.005 M dl-dithiothreitol. pH 7.4 at 37° C.
• TBS tris-buffered saline, Sigma
• TBS contains 0.05 M Tris, 0.0027 M potassium chloride, and 0.138 M sodium chloride. pH 7.5 at 37° C.
• PBMC Peripheral Blood Mononuclear Cell
• Dried samples were reconstituted in 200 ⁇ l derivatization cocktail (0.34% chloroacetaldehyde in 100 mM sodium acetate, pH 4.5), vortexed, and centrifuged. Supernatant was then transferred to a dean screw-cap tube and incubated at 95° C. for 40 min. Derivatized samples were then evaporated to dryness and reconstituted in 100 ⁇ l of water for HPLC analysis.
• derivatization cocktail 0.34% chloroacetaldehyde in 100 mM sodium acetate, pH 4.5
• the HPLC system was comprised of a P4000 solvent delivery system with AS3000 autoinjector and F2000 fluorescence detector (Tlermo Separation, San Jose, Calif.).
• the column was an Inertsil ODS-2 column (4.6 ⁇ 150 mm).
• the mobile phases used were: A, 5% acetonitrile in 25 mM potassium phosphate buffer with 5 mM tetrabutyl ammonium bromide (TBABr), pH 6.0; B, 60% acetonitrile in 25 mM potassium phosphate buffer with 5 mM TBABr, pH 6.0.
• the flow rate was 2 ml/min and the column temperature was maintained at 35° C. by a column oven.
• the gradient profile was 90% A/10% B for 10 min for PMPA and 65%A/35%B for 10 min for the prodrug. Detection was by fluorescence with excitation at 236 nm and emission at 420 nm, and the injection volume was 10 ⁇ l. Data was acquired and stored by a laboratory data acquisition system (PeakPro, Beckman, Allendale, N.J.).
• FIG. 2 shows the time course of GS 7340-2 metabolism summary of plasma and PBMC exposures following oral administration of pure diastereoisomers of the PMPA prodrugs.
• the bar graph in FIG. 2 shows the AUC (0-24 h) for tenofovir in dog PBMCs and plasma after administration of PMPA s.c., TDF and amidate ester prodrugs. All of the amidate prodrugs exhibited increases in PBMC exposure. For example, GS 7340 results in a ⁇ 21-fold increase in PBMC exposure as compared to PMPA s.c. and TDF; and a 6.25-fold and 1.29-fold decrease in plasma exposure, respectively.
• GS7340 isopropyl alaninyl monoamidate, phen

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  • 2016-04-07 BE BE2016C018C patent/BE2016C018I2/fr unknown
  • 2016-04-11 FR FR16C0013C patent/FR16C0013I2/fr active Active
  • 2016-04-12 CY CY2016008C patent/CY2016008I1/el unknown
  • 2016-04-14 LU LU93029C patent/LU93029I2/xx unknown
  • 2016-04-19 NO NO2016006C patent/NO2016006I1/no unknown
  • 2016-04-22 NL NL300803C patent/NL300803I2/nl unknown
• 2017
  • 2017-06-28 CY CY20171100687T patent/CY1119411T1/el unknown
• 2018
  • 2018-03-09 HK HK18103347.4A patent/HK1243711A1/zh unknown
• 2019
  • 2019-05-09 HU HUS1900027C patent/HUS000494I2/hu unknown
• 2023
  • 2023-02-03 NO NO2023006C patent/NO2023006I1/no unknown

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