I lllll llllllll II llllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US 20040259833Al
`
`(19) United States
`(12) Patent Application Publication
`Benkovic et al.
`
`(10) Pub. No.: US 2004/0259833 Al
`Dec. 23, 2004
`(43) Pub. Date:
`
`(54) DNA METHYL TRANSFERASE INHIBITORS
`
`Related U.S. Application Data
`
`(75)
`
`Inventors: Stephen J. Benkovic, State College, PA
`(US); Lucille Shapiro, Stanford, CA
`(US); Stephen J. Baker, State College,
`PA (US); Daphne C. Wahnon, State
`College, PA (US); Mark Wall, State
`College, PA (US)
`
`Correspondence Address:
`Kevin E. Noonan
`McDonnell Boehnen Hulbert & Berghoff LLP
`32nd Floor
`300 S. Wacker Drive
`Chicago, IL 60606 (US)
`
`(63) Continuation of application No. 09/578,991, filed on
`May 25, 2000, now abandoned.
`
`(60) Provisional application No. 60/174,256, filed on Jan.
`3, 2000. Provisional application No. 60/154,582, filed
`on Sep. 17, 1999. Provisional application No. 60/135,
`870, filed on May 25, 1999.
`
`Publication Classification
`
`(51)
`
`Int. Cl.7 ..................... A61K 31/7076; C07H 19/19;
`A61K 31/52
`(52) U.S. Cl. ..................... 514/46; 514/263.4; 514/263.2;
`544/277; 536/27.4
`
`(73) Assignees: The Board of Trustees of the Leland
`Stanford Junior University; The Penn
`State Research Foundation
`
`(57)
`
`ABSTRACT
`
`(21) Appl. No.:
`
`10/877,729
`
`(22) Filed:
`
`Jun.25,2004
`
`This invention provides broad-spectrum antibiotics that are
`inhibitors of bacterial adenine DNA methyltransferases.
`
`CFAD v. Anacor, IPR2015-01776, CFAD EXHIBIT 1053 - Page 1 of 26
`
`

`
`Patent Application Publication Dec. 23, 2004 Sheet 1 of 2
`
`US 2004/0259833 Al
`
`Figure 1 - Proposed Active Site of Adenine DNA Methyltransferase.
`
`CFAD v. Anacor, IPR2015-01776, CFAD EXHIBIT 1053 - Page 2 of 26
`
`

`
`Patent Application Publication Dec. 23, 2004 Sheet 2 of 2
`
`US 2004/0259833 Al
`
`Figw:e ~ Mechanism of Adenine DNA methyltransferase
`
`SAM
`Binding
`NH2
`'t~) Site
`co;.·
`N ~a~5~
`~/) NH~
`--···
`r
`,
`•• HO
`.OH
`.. ,
`a
`..
`H,, • .H
`tf_;·····
`
`-··
`
`RO~o~ _N
`)-.:../ . DNA .
`RO
`Binding .
`.............. Site
`
`. . . 1111 __ _
`
`.
`
`CFAD v. Anacor, IPR2015-01776, CFAD EXHIBIT 1053 - Page 3 of 26
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`US 2004/0259833 Al
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`Dec. 23, 2004
`
`1
`
`DNA METHYL TRANSFERASE INHIBITORS
`
`BACKGROUND OF THE INVENTION
`
`[0001] 1. Field of the Invention
`
`[0002] The present invention relates to the field of antibi(cid:173)
`otics and particularly antibacterial compounds. The inven(cid:173)
`tion specifically relates to antibiotics targeted to DNA modi(cid:173)
`fication
`enzymes,
`in
`particular
`adenine DNA
`methyltransferases, that are the components of a broad
`variety of different bacterial pathogens including those that
`are essential for bacterial cell growth. The invention par(cid:173)
`ticularly provides inhibitors of such adenine DNA methyl(cid:173)
`transferases having little or no inhibitory effects on cytosine
`methyltransferases, and hence having limited antibiotic
`effect on eukaryotic, particularly mammalian, cells. Meth(cid:173)
`ods for preparing and using the adenine DNA methyltrans(cid:173)
`ferase inhibitors of the invention, and pharmaceutical com(cid:173)
`positions thereof, are also provided.
`
`[0003] 2. Background of the Invention
`
`[0004] One hallmark of the modern era of medicine has
`been the decline in morbidity and mortality associated with
`bacterial infections. The development of a variety of anti(cid:173)
`biotic drugs in the early and middle parts of the twentieth
`century provided medical practitioners for the first time with
`effective treatments for a variety of infectious diseases.
`
`[0005] However, misuse of conventional antibiotics and
`natural selection of the infectious bacterial population has
`resulted in the development of varying degrees of drug
`resistance by most bacterial infectious agents to most anti(cid:173)
`biotic agents. In severe cases, such as MRSA Multidrug(cid:173)
`Resistant StaphA, one or only a few antibiotics are currently
`effective. In addition, the existence of immunodeficiency
`syndromes results in additional incidence of opportunistic
`infections requiring intensive antibiotic treatment.
`
`[0006] Thus, there is an increasing need in the art for
`novel, more effective antibiotic compounds for treating
`bacterial infections that are resistant to currently available
`therapies.
`
`[0007] Most bacteria modify their genomic DNA by
`methylation of specific nucleotide bases. DNA methylation
`is critical to gene regulation and repair of mutational lesions
`(see Jost & Soluz, 1993, DNA METHYLATION,
`MOLECULAR BIOLOGY AND BIOLOGICAL SIGNIFI(cid:173)
`CANCE, Birhauser Verlag: Basel, Switzerland; Palmer &
`Marinus, 1994, Gene 143: 1-12; Dryden, 1999, "Bacterial
`DNA Methyltransferases," in S-ADENOSYLMETHION(cid:173)
`INE-DEPENDENT METHYLTRANSFERASES: STRUC(cid:173)
`TURES AND FUNCTIONS, X. Cheng and R. M. Blumen(cid:173)
`thal (eds.), World Scientific Publishing, p.283-340 for
`review). DNA methylation is catalyzed by a class of
`enzymes having different sequences specificities. There are
`those DNA methyltransferases for example (dam) that
`methylate adenine residues in GATC sequences or cytosine
`(dcm) residues in CCAGG or CCTGG sequences which are
`not contained in the recognition site of a cognate restriction
`enzyme. There are those DNA methyltransferases that
`methylate residues contained in the recongnition site of a
`cognate restriction enzyme (for example, ApaI, Avail, Bell,
`ClaI, Dpnll, EcoRI, HaeIII, HhaI, MboI, and MspI; see,
`Marinus & Morris, 1973, J. Bacterial. 114: 1143-1150; May
`& Hatman, 1975, J. Bacterial. 123: 768-770; Heitman,
`
`1993, Genet. Eng. 15: 57-108). In addition, the instant
`inventors have discovered an adenine DNA methyltrans(cid:173)
`ferase from Caulobacter cresentus that methylates the
`adenine residue in the sequence GANTC, as disclosed in
`International Application Publication No. W098/12206.
`This methyltransferase is cell-cycle regulated and essential
`for successful bacterial cell growth; inhibition of the enzyme
`makes the bacteria non-viable. Similar methyltransferases
`have also been discovered in Brucella abortus, Helicobacter
`pylori, Agrobacterium tumefaciens and Rhizobium meliloti.
`In contrast with bacterial cells, DNAmethylation in eukary(cid:173)
`otic, and particularly mammalian cells, is limited to cytosine
`methylation at sites comprising the sequence CpG (Razin &
`Riggs, 1980, Science 210: 604-610; Jost & Bruhat, 1997,
`Prag. Nucleic Acid Res. Malec. Biol. 57: 217-248).
`
`[0008] Thus, the existence of DNA methylation, in par(cid:173)
`ticular, the cell-cycle regulated adenine DNA methyltrans(cid:173)
`ferase found by the inventors in certain bacterial species,
`addresses the need in the art for novel targets for antibiotic
`activity.
`
`SUMMARY OF THE INVENTION
`
`invention provides antibiotic compounds
`[0009] The
`capable of inhibiting adenine DNA methyltransferases in
`bacterial cells. The antibiotic compounds of the invention
`specifically inhibit adenine-specific bacterial DNA methyl(cid:173)
`transferases, and do not inhibit bacterial or eukaryotic,
`particularly mammalian and most particularly human,
`cytosine-specific DNA methyltransferases. The compounds
`of the invention also inhibit adenine-specific DNA methyl(cid:173)
`transferases in plants. The antibiotic compounds are also
`provided as pharmaceutical compositions capable of being
`administered to an animal, most preferably a human, for
`treatment of a disease having a bacterial etiology, or an
`opportunistic infection with a bacteria in an animal, most
`preferably a human, in an immunologically compromised or
`debilitated state of health.
`
`[0010] The invention also provides methods for preparing
`the antibiotic compounds and pharmaceutical compositions
`thereof, and methods of using said antibiotics therapeuti(cid:173)
`cally. Kits and packaged embodiments of the antibiotic
`compounds and pharmaceutical compositions of the inven(cid:173)
`tion are also provided.
`
`[0011] Specific preferred embodiments of the present
`invention will become evident from the following more
`detailed description of certain preferred embodiments and
`the claims.
`
`DESCRIPTION OF THE DRAWINGS
`
`[0012] FIGS. 1 and 2 depict schematic diagrams of the
`"active site" of bacterial adenine DNA methyltransferases.
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`
`[0013] This invention provides antibiotics, and specifi(cid:173)
`cally antibacterial compounds, that are inhibitors of bacterial
`adenine DNA methyltransferases. The compounds of the
`invention exhibit antibacterial, growth-inhibitory properties
`against any bacterial species that produces an adenine DNA
`methyltransferase. These include adenine DNAmethyltrans(cid:173)
`ferases that are components of bacterial restriction/modifi-
`
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`2
`
`cation systems as understood in the art, as well as cell-cycle
`regulated adenine DNA methyltransferases (CcrM), such as
`those disclosed in International Application Publication No.
`W098/12206, incorporated by reference. Thus, inhibitors of
`adenine DNA methyltransferases are particularly provided
`by the invention.
`
`[0014] The adenine DNA methyltransferase inhibitors of
`the invention comprise a novel class of broad-spectrum
`antibiotics. Most bacterial species possess a DNA methyl(cid:173)
`transferase that is part of a modification apparatus, typically
`associated with a restriction enzyme, that preserves the
`integrity of cellular DNA while providing a defense against
`foreign (most typically viral) DNA In addition, certain
`bacteria produce an adenine DNA methyltransferase that is
`essential for bacterial cell growth. Medically-important bac(cid:173)
`terial species that provide appropriate targets for the anti(cid:173)
`bacterial activity of the inhibitors of the invention include
`gram-positive bacteria, including cocci such as Staphylo(cid:173)
`coccus species and Streptococcus species; bacilli, including
`Bacillus species, Corynebacterium species and Clostridium
`species; filamentous bacteria, includingActinomyces species
`and Streptomyces species; gram-negative bacteria, including
`cocci such as Neisseria species; bacilli, such as Pseudomo(cid:173)
`nas species, Brucella species, Agrobacterium species, Bor(cid:173)
`detella species, Escherichia species, Shigella species, Yers(cid:173)
`inia species, Salmonella species, Klebsiella species,
`Enterobacter species, Hemophilus species, Pasteurella spe(cid:173)
`cies, and Streptobacillus species; spirochetal species,
`Campylobacter species, Vibrio species; and intracellular
`bacteria including Rickettsiae species and Chlamydia spe(cid:173)
`cies.
`
`[0015] Specific bacterial species that are targets for the
`adenine DNA methyltransferase inhibitors of the invention
`include Staphylococcus aureus; Staphylococcus saprophyti(cid:173)
`cus; Streptococcus pyrogenes; Streptococcus agalactiae;
`Streptococcus pneumoniae; Bacillus anthracis; Corynebac(cid:173)
`terium diphtheria; Clostridium perfringens; Clostridium
`botulinum; Clostridium
`tetani; Neisseria gonorrhoeae;
`Neisseria meningitidis;
`Pseudomonas
`aeruginosa;
`Legionella pneumophila; Escherichia coli; Yersinia pestis;
`Hemophilus influenzae; Helicobacter pylori; Campylo(cid:173)
`bacter fetus; Vibrio cholerae; Vibrio parahemolyticus; Tre(cid:173)
`pomena pallidum; Actinomyces
`israelii; Rickettsia
`prowazekii; Rickettsia rickettsii; Chlamydia trachomatis;
`Chlamydia psittaci; Brucella abortus and Agrobacterium
`tumefaciens.
`
`[0016]
`It is an important property of the adenine DNA
`methyltransferase inhibitors of the invention that the level of
`activity of these substances with cytosine-specific DNA
`methyltransferases is low. This is because cytosine-specific
`DNAmethyltransferases occur in mammalian, most particu(cid:173)
`larly human, cells, and it is an advantageous property of the
`adenine DNA methyltransferases of the invention to have
`little or no inhibitory activity against mammalian methyl(cid:173)
`transferases. This property confers upon the molecules pro(cid:173)
`vided by the invention the beneficial property of being
`bacterial cell specific, and having little antibiotic activity
`against mammalian, most preferably human, cells. Prefer(cid:173)
`ably, the IC50 of these compounds for cytosine-specific DNA
`methyltransferases is greater than 500 µM.
`
`[0017] The inhibitory compounds provided by the inven(cid:173)
`tion are represented by Formula I:
`
`[0018] where R1, R2 and R3 are the same or different and
`are independently hydrogen, lower alkyl, aryl or substituted
`aryl, lower alkoxy, lower alkoxyalkyl, or cycloalkyl or
`cycloalkyl alkoxy, where each cycloalkyl group has from
`3-7 members, where up to two of the cycloalkyl members
`are optionally hetero atoms selected from oxygen and nitro(cid:173)
`gen, and where any member of the alkyl, aryl or cycloalkyl
`group is optionally substituted with halogen, lower alkyl or
`lower alkoxy, aryl or substituted aryl, and where R3 can be
`ribose, deoxyribose or phosphorylated derivatives thereof,
`including phosphorothioates, phosphoramidites and similar
`derivatives known in the art, provided that R 1
`, R 2 and R 3 are
`not all hydrogen, and where R3 is ribose, deoxyribose or
`phosphorylated derivatives thereof, R1 and R2 are not both
`hydrogen. In preferred embodiments, R1
`is H, R2
`is
`(2-diphenylborinic ester)ethyl or diphenylpropyl, and R3 is
`H, 2-( 4-morpholinyl)-ethyl, 3-(N-phthaloyl)-aminopropyl,
`2-(2-(2-hydroxyethoxy)ethoxy)ethyl, or ethyl-2-( acrylate)(cid:173)
`methyl. In additional preferred embodiments, R1 is H, R2 is
`(S-homocysteinyl)methyl and R3 is ribose, 5'phosphorylri(cid:173)
`bose, deoxyribose or 5' phosphoryl deoxyribose. In other
`preferred embodiments, R3 is Hand R1 and R2 are together
`2-( diphenylmethyl)cyclopentyl or 2-( diphenylhydroxym(cid:173)
`ethyl)cyclopentyl. In further preferred embodiments, R 1 is
`H1 . R2 is alanylbutyl ester, 2-carboximido-2-aminoethyl,
`2-aminoethyl or mono- or bisubstituted 2-amino ethyl, and
`R3 is 2-(4-morpholinyl)-ethyl.
`[0019] The invention also provides compounds of For(cid:173)
`mula II:
`
`bond 2
`
`[0020] wherein bonds 1 and 2 can be double or single, Ar1
`and Ar2 can be the same or different and are each indepen(cid:173)
`dently aryl or heteroaryl, or aryl or heteroaryl substituted at
`one or a plurality of positions with halogen, nitro, nitroso,
`lower alkyl, aryl or substituted aryl, lower alkoxy, lower
`alkoxyalkyl, or cycloalkyl or cycloalkyl alkoxy, where each
`cycloalkyl group has from 3-7 members, where up to two of
`the cycloalkyl members are optionally hetero atoms selected
`from sulfur, oxygen and nitrogen, and where any member of
`the alkyl, aryl or cycloalkyl group is optionally substituted
`with halogen, lower alkyl or lower alkoxy, aryl or substituted
`
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`3
`
`aryl, halogen, nitro, nitroso, aldehyde, carboxylic acid,
`amide, ester, or sulfate, and R", Rb and Rc are the same or
`different and are independently hydrogen, halogen, nitro,
`nitroso, lower alkyl, lower alkoxy, lower alkoxyalkyl, or
`cycloalkyl or cycloalkyl alkoxy, or aryl or heteroaryl, or aryl
`or heteroaryl substituted at one or a plurality of positions
`with lower alkyl, lower alkoxy, lower alkoxyalkyl, or
`cycloalkyl or cycloalkyl alkoxy, where each cycloalkyl
`group has from 3-7 members, where up to two of the
`cycloalkyl members are optionally hetero atoms selected
`from sulfur, oxygen and nitrogen, and where any member of
`the alkyl, aryl or cycloalkyl group is optionally substituted
`with halogen, lower alkyl or lower alkoxy, aryl or substituted
`aryl, halogen, nitro, nitroso, aldehyde, carboxylic acid,
`amide, ester, or sulfate, or wherein R", Rb and Rc may be
`connected by aromatic, aliphatic, heteroaromatic, het(cid:173)
`eroaliphatic ring structures or substituted embodiments
`thereof.
`
`[0021] The invention also provides combinatorial chemi(cid:173)
`cal libraries of purine derivatives. In one preferred embodi(cid:173)
`ment, 6-chloropurine is converted into adenine derivatives
`by amination of the C6 position of the purine ring; these
`libraries are termed "N6 libraries" herein. In other preferred
`embodiments, unsubstituted adenine or 6-chloropurine is
`derivatized at the N9 position of the purine ring; these
`libraries are termed "N9 libraries" herein. In still further
`embodiments, both the C6 and N9 positions are derivatized,
`with the C6 position being aminated with an amine or
`substituted amino group; these libraries are termed "N6/N9
`libraries" herein.
`
`[0022]
`In the preparation of the N6 or N9 libraries, the
`starting purine ring structure is reacted in individual "pots"
`or reaction mixtures with each of a plurality of amines or
`substituted amines (for N6 libraries) or halides (for N9
`libraries). These libraries thus are provided as collections of
`separate products of the reaction between the starting mate(cid:173)
`rials. For N6/N9 libraries, most preferably the N9 position is
`first derivatized followed by reaction at the C6 position. In
`these libraries, reaction is typically performed using a single
`halide (resulting in uniform substitution at the N9 position)
`and a plurality of amines (preferably 2 to 5 amines, most
`preferably 3 different amines), thereby providing a mixture
`of compounds. In addition, regioisomers (including the Nl,
`N3, and N7 isomers) can be produced according to the
`methods of the invention. Typically, reaction mixtures are
`also provided lacking the purine starting material, to monitor
`for reactions between the halides and the different amines.
`
`[0023] The invention also provides so-called "rational
`design" adenine DNAmethyltransferase inhibitors, based on
`an understanding of the putative active site of an adenine
`DNA methyltransferase enzyme, shown in FIG. 1. As sche(cid:173)
`matically depicted in the Figure, the enzyme has a binding
`site for the adenine residue in a DNA strand, and an
`S-adenosylmethionine binding site, which provides the
`donor methyl group as shown. So-called "rational design"
`inhibitors mimic the configuration of the molecules in the
`binding site of the enzyme, as shown in FIG. 2. These
`compounds in general comprise an adenosine residue, with
`or without a 5' phosphate group, covalently linked through
`a methylene bridge to a homocysteine moiety. The invention
`also provides adenine DNAmethyltransferase inhibitors that
`are derivatives of borinic acid, most preferably diphenyl or
`substituted diphenyl borinic acid, and most preferably
`
`diphenyl or substituted diphenyl borinic acid alkylamine
`esters thereof. In preferred embodiments, the invention
`provides compounds including di-(p-fiuorophenyl)borinic
`acid 8-hydroxyquiniline ester, di-(p-chlorophenyl)borinic
`acid 8-hydroxyquiniline ester, diphenylborinic acid 8-hy(cid:173)
`droxyquiniline ester, di-(p-fiuorophenyl)borinic acid etha(cid:173)
`nolamine ester, and di-(p-chlorophenyl)borinic acid ethano(cid:173)
`lamine ester.
`
`[0024] The invention also provides adenine DNA meth(cid:173)
`yltransferase inhibitors synthesized using solid phase chem(cid:173)
`istry, most preferably using resins comprising a residue
`(such as an amine or halide) as provided herein for substi(cid:173)
`tution at the C6 or N9 positions of the purine ring. In
`preferred embodiments, these resins are provided whereby
`the substituent is covalently linked to the resin using a
`covalent bond that can be specifically cleaved to liberate the
`compound from the resin after solid phase synthesis is
`complete. Preferably, the substituent is presented on the
`resin with an activated group, such as an amine or halide,
`accessible to a purine contacted with the resin. After reac(cid:173)
`tion, the purine is linked to the resin through the substituent,
`and the reaction product can then be worked up and removed
`from the resin using methods well known in the art. See, for
`example, Bunin, 1998, THE COMBINATORIAL INDEX,
`Academic Press.
`
`[0025]
`In certain situations, compounds of the invention
`may contain one or more asymmetric carbon atoms, so that
`the compounds can exist in different stereoisomeric forms.
`These compounds can be, for example, racemates or opti(cid:173)
`cally active forms. In these situations, the single enanti(cid:173)
`omers, i.e., optically active forms, can be obtained by
`asymmetric synthesis or by resolution of the racemates.
`Resolution of the racemates can be accomplished, for
`example, by conventional methods such as crystallization in
`the presence of a resolving agent, or chromatography, using,
`for example a chiral HPLC column.
`
`[0026] Advantageously, solid phase chemistry employing
`resins as described above can be useful for determining
`whether a substituent exhibits chirality or stereospecificity
`that has a bearing on antibacterial activity. In these embodi(cid:173)
`ments, compounds are prepared for screening using a race(cid:173)
`mic mixture of optically-active species, such as an amino
`acid. Upon finding the resulting compound has adenine
`DNA methyltransferase inhibitory activity, optically-pure
`preparations of each of the stereoisomers can be used to
`prepare the corresponding optically-pure isomers of the
`adenine DNA methyltransferase inhibitory compound, to
`determine whether there is any difference in biological
`activity between the isomers. This approach is advantageous
`over the alternative, separating the racemic mixture into its
`stereoisomeric components.
`
`[0027] Regardless of how a putative adenine DNA meth(cid:173)
`yltransferase is prepared according to the invention, the
`compound is analyzed for both adenine and cytosine-spe(cid:173)
`cific DNA methyltransferase activity. Susceptible bacteria
`(known to express an adenine DNA methyltransferase) are
`grown in the presence and absence of the inhibitory com(cid:173)
`pound, and the extent of growth inhibition in the presence of
`the compound is determined relative to growth in the
`absence of the compound. The mechanism of action (i.e.,
`inhibition of adenine DNAmethyltransferase) is verified for
`each growth-inhibitory compound by filter-binding radioas-
`
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`4
`
`tritiated S-adenosyl
`say using hemimethylated DNA,
`methionine (C3 H3 ) and a purified adenine DNAmethyltrans(cid:173)
`ferase according to International Application Publication
`No. W098/12206.
`
`lower alkylthio, trifiuoromethyl, lower acyloxy, aryl, het(cid:173)
`eroaryl, and hydroxy. Preferred aryl groups include phenyl
`and naphthyl, each of which is optionally substituted as
`defined herein.
`
`[0036] By "heteroaryl" is meant one or more aromatic ring
`systems of 5-, 6-, or 7-membered rings containing at least
`one and up to four heteroatoms selected from nitrogen,
`oxygen, or sulfur. Such heteroaryl groups include, for
`example, thienyl, furanyl, thiazolyl, imidazolyl, (is)ox(cid:173)
`azolyl, pyridyl, pyrimidinyl, (iso )quinolinyl, napthyridinyl,
`benzimidazolyl, benzoxazolyl. Preferred heteroaryls are
`thiazolyl, pyrimidinyl, preferrably pyrimidin-2-yl, and
`pyridyl. Other preferred heteroaryl groups include 1-imida(cid:173)
`zolyl, 2-thienyl, 1-, or 2-quinolinyl, 1-, or 2-isoquinolinyl,
`1-, or 2-tetrahydro isoquinolinyl, 2- or 3-furanyl and 2-tet(cid:173)
`rahydrofuranyl.
`
`[0037] The bacterial growth inhibitory, adenine DNA
`methyltransferase inhibiting compounds of the invention are
`provided either from combinatorial libraries, solid phase
`synthesis, "rational" drug design, or conventional synthesis
`as described herein.
`
`[0038] Construction of Combinatorial Libraries
`
`[0039] Combinatorial libraries are prepared according to
`methods understood by those with skill in the art. For the
`single substitution libraries (N6 and N9), the individual
`substituents are used in separate reaction mixtures to pro(cid:173)
`duce each of the purine derivatives described herein. In the
`combination libraries (N6/N9 herein), on the other hand, one
`position (typically N9) is typically reacted with a particular
`substituent, and then a mixture of substituents (most pref(cid:173)
`erably 3) used to derivatize the other reaction position
`(typically C6).
`
`[0040] The reactions are performed on a scale adapted to
`economically producing sufficient product for testing. Pref(cid:173)
`erably, reactions are performed in parallel, for example
`using a 96-well plate with each well having a sufficiently
`small volume (100-500 µL) to minimize the amount of
`reagents required. The use of this type of reaction vessel also
`facilitates parallel handling and analysis, including auto(cid:173)
`mated versions of such processes.
`
`[0041]
`
`a. N6 Libraries
`
`[0042] The following conditions were developed to syn(cid:173)
`thesize analogues of adenine substituted at the N-6 position.
`In one reaction scheme, 6-chloropurine is reacted at 85° C.
`overnight with a primary or secondary amine in triethy(cid:173)
`lamine in n-butanol. Alternatively, these reagents are reacted
`in potassium carbonate in dimethylformamide at 85° C.
`overnight. This synthesis is described in Reaction Scheme 1:
`
`[0028] Compounds of the invention can exist as tautomers
`in solution. When structures and names are given for one
`tautomeric form the other tautomeric form is also included
`in the invention.
`
`[0029] Representative compounds of the present invention
`include, but are not limited to the compounds disclosed
`herein and their pharmaceutically acceptable acid and base
`addition salts. In addition, if the compound of the invention
`is obtained as an acid addition salt, the free base can be
`obtained by basifying a solution of the acid salt. Conversely,
`if the product is a free base, an addition salt, particularly a
`pharmaceutically acceptable addition salt, may be produced
`by dissolving the free base in a suitable organic solvent and
`treating the solution with an acid, in accordance with con(cid:173)
`ventional procedures for preparing acid addition salts from
`base compounds.
`
`[0030] The present invention also encompasses the acy(cid:173)
`lated prodrugs of the compounds of the invention. Those
`skilled in the are will recognize various synthetic method(cid:173)
`ologies which may be employed to prepare non-toxic phar(cid:173)
`maceutically acceptable addition salts and acylated prodrugs
`of the inventive compounds.
`[0031] By "alkyl", "lower alkyl", and "C1-C6 alkyl" in the
`present invention is meant straight or branched chain alkyl
`groups having 1-6 carbon atoms, such as, methyl, ethyl,
`propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl,
`2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and
`3-methylpentyl.
`
`[0032] By "alkoxy", "lower alkoxy", and "C1-C6 alkoxy"
`in the present invention is meant straight or branched chain
`alkoxy groups having 1-6 carbon atoms, such as, for
`example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,
`sec-butoxy, tert-butoxy, pentoxy, 2-pentyl, isopentoxy, neo(cid:173)
`pentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.
`
`[0033] By the term "halogen" in the present invention is
`meant fluorine, bromine, chlorine, and iodine.
`
`[0034] By "cycloalkyl", e.g., C3 -C7 cycloalkyl, in the
`present invention is meant cycloalkyl groups having 3-7
`atoms such as, for example cyclopropyl, cyclobutyl, cyclo(cid:173)
`pentyl, cyclohexyl, and cycloheptyl. In the C3 -C7 cycloalkyl
`groups, preferably in the C5 -C7 cycloalkyl groups, one or
`two of the carbon atoms forming the ring can optionally be
`replaced with a hetero atom, such as sulfur, oxygen or
`nitrogen. Examples of such groups are piperidinyl, piper(cid:173)
`azinyl, morpholinyl, pyrrolidinyl, imidazolidinyl, oxazolidi(cid:173)
`nyl, azaperhydroepinyl, oxazaperhydroepinyl, oxepanyl,
`oxazaperhydroinyl, and oxadiazaperhydroinyl. C3 and C4
`cycloalkyl groups having a member replaced by nitrogen or
`oxygen include aziridinyl, azetidinyl, oxetanyl, and oxira(cid:173)
`nyl.
`
`[0035] By "aryl" is meant an aromatic carbocyclic group
`having a single ring (e.g., phenyl), multiple rings (e.g.,
`biphenyl), or multiple condensed rings in which at least one
`is aromatic, (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl,
`anthryl, or phenanthryl), which is optionally mono-, di-, or
`trisubstituted with, e.g., halogen, lower alkyl, lower alkoxy,
`
`N~N\_
`lJ-1
`
`Cl
`
`N
`
`(1)
`
`or
`R11NHR 111
`
`N
`H
`
`CFAD v. Anacor, IPR2015-01776, CFAD EXHIBIT 1053 - Page 7 of 26
`
`

`
`US 2004/0259833 Al
`
`Dec. 23, 2004
`
`5
`
`-continued
`R"
`HN/
`
`CC>°'
`
`~
`
`N
`
`(2)
`
`(3)
`
`[0043] R" and R"' are lower alkyl, hetero atom-substituted
`lower alkyl, aryl, heteroaryl or substituted aryl or heteroaryl,
`as exemplified by the compounds set forth below. Any
`primary or secondary amine can be used in this reaction.
`Preferred embodiments of primary or secondary amines
`used in these reactions is as follows:
`
`[0044] histamine dihydrochloride
`
`[0045] norphenylephrine hydrochloride
`
`[0046] 1,2-diaminopropane
`
`[0047] 5-amino-1,3,3-trimethylcyclohexanemethyl(cid:173)
`amme
`
`[0048] 3-isopropoxypropylamine
`
`[0049] diphenylborinic acid, ethanolamine ester
`
`[0050] 2-(2-aminoethylamino )-ethanol
`
`[0051]
`
`tetrahydrofurfurylamine
`
`[0052]
`
`5-methyltryptamine hydrochloride
`
`[0053]
`
`3,3-diphenylpropylamine
`
`[0054]
`
`[0055]
`
`1-(3-aminopropyl)-2-pyrrolidinone
`
`2-(2-aminoethyl)-1-methylpyrrolidine
`
`[0056] 2-(aminomethyl)benzimidazole dihydro-chlori(cid:173)
`dehyrate
`
`[0057] 2,2,2-trifiuoroethylamine hydrochloride
`
`[0058] L-carnosine
`
`[0059]
`
`(R)-(-)-1-amino-2-propanol
`
`[0060] 2-(1-cyclohexenyl)ethylamine
`
`[0061] 4-(trifiuoromethyl)benzylamine
`
`[0062] 2,5-dichloroamylamine hydrochloride
`
`[0063]
`
`( +/-)-4-amino-3-hydroxybutyric acid
`
`[0064] N ,N-dimethy le thy lenediamine
`
`[0065] 3,3-dimethylbutylamine
`
`[0066] 1,4-diamino-2-butanone dihydrochloride
`
`[0067]
`
`aminomethylbenzoic acid
`
`[0068]
`
`aminohydroxymethylpropane dial
`
`[0069] 2-( aminoethyl)pyridine
`
`[0070]
`
`aminobutanol
`
`[0071]
`
`adamantamine
`
`[0072]
`
`aminohexanoic acid
`
`[0073] N-benyzylethanolamine
`
`[0074] ethyl-6-aminobutyrate hydrochloride
`[0075] ethylenediamine
`[0076] 2-cyclohex-1-enylethylamine
`[0077] Some of these amines produce different regioiso(cid:173)
`mers, i.e., for some compounds the amine can be added to
`the C6 position of 6-chloropurine in different orientations,
`depending on which reactive moiety comprising the amine
`covalently bonds to C6. However, the occurrence of these
`regioisomers is not deleterious, since it merely increases the
`number of candidate compounds in the library.
`[0078] b. N9 Libraries
`[0079] N9 libraries were prepared using the following
`reaction schemes. It was found that Path A of Reaction
`Scheme 2 did not yield product with all organic halides
`(Rivx or R4 X); alternative Path B was found to form product
`throughout the range of organic halides tested. In each
`alternative, the organic halide was reacted with purine
`(either adenine or 6-chloropurine) at 45° C. overnight in
`potassium carbonate in dimethylformamide. In Path B,
`however, the N9-derivatized 6-chloropurine was converted
`to N9-derivatized adenine by reaction of the product of the
`first reaction with ammonium hydroxide at 85° C. overnight.
`Both reactions are performed sequentially in the same
`reaction mixture.
`
`Reaction Scheme 2
`
`Path A
`
`Path B
`
`(4)
`
`(5)
`
`Rivx -45° c.
`
`(1)
`
`NH40H -85° c.
`
`(6)
`
`(5)
`
`[0080] Riv is lower alkyl, hetero atom-substituted lower
`alkyl, aryl, heteroaryl or substituted aryl or heteroaryl, as
`exemplified by the compounds set forth below.
`[0081] The products of Path B were analyzed by HPLC
`and found to be a mixture of N-9 and N-7 substituted
`adenine analogues; there may also be N-1 and N-3 substi(cid:173)
`tuted analogues in certain reaction mixtures. As discussed
`above, the advantage of these side products is that their
`existence simply increases the number of candidate mol(cid:173)
`ecules in the library.
`
`CFAD v. Anacor, IPR2015-01776, CFAD EXHIBIT 1053 - Page 8 of 26
`
`

`
`US 2004/0259833 Al
`
`Dec. 23, 2004
`
`6
`
`[0082] Any organic halide can be used in this reaction.
`Preferred embodiments of organic halides used in these
`reactions is as follows:
`
`Reaction Scheme 3
`
`[0083]
`
`methyl 4-iodobutyrate
`
`[0084]
`
`l-bromo-3-phenylpropane
`
`[0085]
`
`cinnamyl bromide
`
`[0086] 2-chloroethylphosphonic acid
`
`[0087] ethyl 2-(2-chloroacetamido )-4-thiazole-acetate
`
`[0088] 4-(2-chloroethyl)morpholine hydrochloride
`
`[0089]
`
`(2-bromoethyl)trimethylammonium bromide
`
`[0090] 4-chlorophenyl 2-bromoethyl eth