·~--~---~----~-----~-··-~-
`
`For reprint orders, please contact reprints@future-science.com
`
`Therapeutic potential of
`boron-containing compounds
`
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`Relative to carbon, hydrogen, nitrogen and oxygen, very little is currently known about boron in therapeutics. In
`addition, there are very few boron-containing natural products identified to date to serve as leads for medicinal
`chemists. Perceived risks of using boron and lack of synthetic methods to handle boron-containing compounds
`have caused the medicinal chemistry community to shy away from using the ato~. However, physical, chemical and
`biological properties of boron offer medicinal chemists a rare opportunity to explore and pioneer new areas of
`drug discovery. Boron therapeutics are emerging that show different modes of inhibition against a variety of biological
`targets. With one boron-containing therapeutic agent on the market and several more in various stages of clinical
`trials, the occurrence of this class of compound is likely to grow over the next decade and boron could become
`widely accepted as a useful element in future drug discovery.
`
`The physical, chemical and biological proper(cid:173)
`ties of boron offer 1nedicinal che1nists a rare
`opportunity ro explore and pioneer its utility in
`chemotherapeutics. However, up until che last
`fe\V years, boron has mosdy been overlooked
`by n1edicinal che1nists in cheir design of drug
`molecules. In trying to discern why boron has
`not been \videly considered, \Ve found a con1-
`mon belief within the medicinal chemistry
`community that boron iS toxic. However, as we
`havt: investigated this claim, we have found ic to
`be largely unfounded. The belief chac boron is
`toxic most likely comes from the fact that boric
`acid (Bf0Hl 3
`) is an ingredient of ant poisons.
`However, boric acid, has an LD50 of2660 mg/kg
`(rat, oral), which is similar to regular cable sale
`at 3000 mg/kg (rat, oral) [IOI]. Another source
`of the toxicity concern may have arisen from the
`toxicity ofVdcadeM(49), the only boron-based
`therapeucic currenrly on che market and widely
`prescribed by oncologists. Velcade is approved
`for the treatment of multiple myeloma and
`works through inhibicion of che proceaso1ne.
`Recendy, research has shown that che coxicicy
`of Velcade is due to its mechanism of action
`and not simply because boron is present in the
`1nolecule [11.
`·rhe overwhelming data for the safety of
`boron arc to be noted. Boric acid is the main
`ingredient in 'Goop' the soft semi-solid, often
`brighcly colored toy that children enjoy squeez(cid:173)
`ing through tht:ir fingers; boric acid is used as a
`preservative in eye wash and in vaginal creams;
`it is used as a buffer in biological assay solu(cid:173)
`tions; boron is also found in high concentrations
`
`in fruit, vegetables and nuts. We consume in
`the range of 0.3-4.2 1ng of boron per day [2]
`and it is considered an essencial plant nurrienr,
`although its biological functions are currencly
`unknown. Studies at Anacor Pharmaceuticals
`found background concentracions of boron in
`mouse plasma samples of approximacely 200
`ng/ml [WHEELER c, VNrusL1sttEo DATA]. Therefore,
`it does appear that the body is familiar with
`-boron. The boronic acid group in Vclcadc·has·
`been sho\vn co be metabolized to boric acid and
`the body seems co manage ics metabolism and
`excretion [3]. Paraboronophenylalanine was used
`for boron ncucron-capcurc chcrapy (BNCT) and
`found to be safe in multiple species including
`human. The LD50 values of free base parabo(cid:173)
`ronophenylalanine were determined to be more
`than 3000 mg/kg in rat by intraperitoneal or
`.subcutaneous adtninistration. In repeat dose
`studies in rats, paraboronophenylalanine was
`administered subcutaneously for 28 days and
`che 300-mg/kg group exhibited no signifi(cid:173)
`cant finding when co1npared with the control
`group [4]. In humans, paraboronophenylalanine
`was adminiscrered via infusion as a complex
`with fructose, up to 900 mg/kg of bodyweight
`[;I, and BSH (Na,B,,H,,-SH), another boron
`therapeutic for BNCl~, was administered at the
`JOO-mg/kg bodyweight level [61; both proved to
`be safe and well tolerated. From all these data,
`we have concluded that boron is not an inhcr(cid:173)
`encly toxic element, such as mercury, and can
`be considered by medicinal chemists for use in
`therapeutics. The toxicology question now is not
`what happens to boron, but \vhat happens to the
`
`Stephen J Bakert,
`Charles Z Ding,
`Tsutomu Akama,
`Yong-Kang Zhang,
`Vincent Hernandez & Yi Xia
`tAuthor for correspondence
`Anacor Pharmaceuticals. Inc.,
`1020 East Meadow Circle,
`Palo Alto, CA 94303. USA
`Tel.: +I 650·543 7500
`Fax: +I 650 543 7660
`E-mail: sbaker@anacor.com
`
`EXHIBIT No . .I..
`
`FUTu~D
`SCIENCE
`
`10.4155/FMC.09.71 © 2009 Future Science ltd
`
`Future Med. Chem. (2009) 1(7), 1275-1288
`
`ISSN 1756-8919
`
`1275
`
`CFAD v. Anacor, IPR2015-01776, CFAD EXHIBIT 1056 - Page 1 of 14
`
`

`
`! Baker, Ding, Akama, Zhang, Hernandez & Xia
`
`R-B(OH)-N=N-R', six(cid:173)
`membered heterocycle ring
`containing a boron, two
`nitrogen and three
`carbon atoms
`
`resc of the molecule should boron be eli1ninatcd
`from che parent molecule, a standard question
`for any drug candidate of nonboron origin.
`Why do we not have more US FDA-approved
`boron-containing therapeutics to dace? Our pri(cid:173)
`mary conclusion is that it is because the organo(cid:173)
`boron chen1istry field is still in ics infancy and
`we do not have a very large ponfolio of che1nical
`reactions to introduce boron into organic mol(cid:173)
`t:cules, nor do we have a good understanding of
`the compacibilicy ofboron-concaining molecules
`in common synthesis. Over the lase two decades,
`and since Suzuki-Miyaura coupling reactions
`have becomC more widespread, there has bet:n a
`significanc increase in organoboron chemiscry,
`which has led to the introduction of OC\V cacalysts
`and new methods of incorporating boron into
`organic molecules and has provided nlore insighc
`to the chemical compatibilities of organoboron
`compounds. This chemistry developmcnc is now
`allowing medicinal chemists to build drug-like
`boron-containing 1nolecules to finally explore
`the usefulness of boron in chemotherapeutics.
`Similar to hydrogen, carbon, nitrogen and
`oxygen, boron is, quite simply, another use(cid:173)
`ful atom! Boron can be considered the equal
`and opposite of nicrogen. Nitrogen is a Lewis
`base, boron is a Lewis acid; nitrogen has a
`full p-orbical (lone pair), boron has an empty
`p-orbital;.nitrog.cn is nucleophilic, boron is clec(cid:173)
`tl-ophilic; nitrogen sics to the right of carbon in
`the periodic cable, boron sits co the lefc. Boron
`in organic molecules is most com1nonly present
`), where its
`as a boronic acid group (R-B[OH] 2
`pKa usually ranges from 7 to 9, considerably
`higher than carboxylic acids. This means that, at
`physiological pH, boronic acid is uncharged and
`in the trigonal planar sp2 form (APPENDIX, I). At
`pHs above its pKa, a hydroxy group coordinates
`to che empty p-orbital, forming a dative bond,
`and boron is in a tetrahedral sp3 fonn (2).
`The empty p-orbital can also be occupied by
`a lone pair fro1n ocher nuclcophilcs, including
`alcohols and amines, allowing boron co form a
`dacive bond with biological nucleophiles such as
`enzyme residues, including serine, and hydroxy
`groups fro1n carbohydrates and nucleic acids.
`Boron can form a bond with a therapeutic target
`that is neither ionic nor an irreversible covalent
`bond. Also, the pKa of horon can he tuned hy
`chemical modification of the molecule co make
`it a scrongcr or weaker deccrophile. Modulation
`of electronic and peripheral substitutions can
`allow boron to improve its selectivity towards
`its desired target. _Boronic acids can also fonn
`
`borate esters with alcohols. However, these are
`usually unstable and hydrolyze easily in water.
`Under certain circumstances, the stability of
`chese borate esters can he increased through
`intra1nolecular cyclization, (e.g., forming a dies(cid:173)
`ter with both hydroxyl groups of a 1,2-cis-diol,
`such as ethylene glycol, co form a 5-meinbered
`<lioxahorolane ring). Substituted ci.r-diols arc
`even more stabilized, due to stcric hindrance
`preventing water approaching the boron and
`subsequent hydrolysis. The properties, prepara(cid:173)
`tion and applications ofhoronic acids have been
`comprehensively revie\vcd [7].
`Boron in therapeucics has been n:viewed in
`depth [7-9]. This review is intended co describe
`some of the recent advances since chose earlier
`reports, as well as to describe some older chem(cid:173)
`istry of the·
`'.r·.;·~ and to 1nake a judicial
`prediction about the potential future of boron
`in drug discovery.
`
`Therapeutic areas containing
`boron-based therapeutics
`• Diazaborines & enoyl reductase
`Early history of diazaborines
`One of the first classes of boron-containing co1n(cid:173)
`pounds evaluated as therapeutics was the diaza(cid:173)
`borines [10]. Diazaborines were first synthesized
`by Dewar who was investigating the 'nonhenze(cid:173)
`noid' aro1naticity o£heterocycles [111. Dewar di.cl
`not report their medicinal application, but rather
`chat they \Vere cool compounds to demonstrate
`the potential of replacing the C-C unit in aro(cid:173)
`matic compounds with the isoclcctronic B-N
`bond. However, it \Vas Gronowitz who saw a
`similarity with the hydrazone-containing nitro(cid:173)
`furan antibiotic nitrofurantoin (3); diazaborines,
`he reasoned, contained an internal hydrazone
`and n1ight also share the sa1ne antibiotic activity.
`His work demonstrated the first reported
`antimicrobial activity for a boron-containing
`compound [12,13]. Starting first with structur(cid:173)
`ally si1nilar nitrothiophene (4), Gronowitz
`quickly established that the nitro substitution
`on che ring was not necessary and that the most
`dramatic impact on activity was observed when
`changing the hydrazine component. A strong
`preference for 2-N-sulfonyl substitution in ana(cid:173)
`logs possessing antibacterial activity was noted
`and served as the template for future research.
`Several patents follo\vcd, demonstrating the
`apparent interest in chis area and culminating
`in the largest such evaluation being published
`by Sandoz Pharmaceuticals {14]. In this study,
`the MIC activity of 80 different diazaborincs
`
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`Therapeutic potential of boron-containing compounds l
`
`was consiste1H with previous observations that
`activity was confined almost exclusively to
`Gran1-negative bacteria. Ir was generally escab(cid:173)
`lished that the ranking of potency, relative to
`d1c arene ring, follO'\vcd the order: thienodiaza(cid:173)
`borines, benzodiazaborines, furanodiazaborincs,
`with pyrolodiazaborincs being inactive. In the
`2-N-su lfonyla I k yl- ch ien od iaza ho rine series,
`the effect of homologating the alkyl chain was
`striking, \vich 5 having a high MIC against
`Escherichia coli of more than 50 µg/ml, com(cid:173)
`pared with 6.25 µg/ml for 6. Mcthylation of the
`thiophene ring gave a slight boost in potency to
`provide the 1nosr promising diazaborine reported
`(Sa 84474 [7]) with an MIC of 1.25 µg/ml.
`To better understand
`the
`require(cid:173)
`ments for activity, a nonboron analog,
`4-hyd roxy-3- (p- tolylsu fonony I) isoqu i noli ne,
`was prepared and found to be inactive. It is
`important to note that these synthetic efforts
`were not guided by knowledge of the mechanism
`of action. In face, the target was initially believed
`to be lipopolysaccharide synthesis [15], which was
`consistent with the observation chat activity was
`confined almost exclusively to Gram-negative
`bacteria. Subsequent studies would reveal the
`actual target to be fatty acid biosynthesis, but it
`took another 12 years until the crystal structure
`was reported [16,P].
`· ··Investigation into the structure-activity rela(cid:173)
`tionships of diazaborines during this ti1ne was
`almost exclusively limited to diazaborines con(cid:173)
`taining the sulfonyl side chain. Problems with
`this particular class of diazaborinc arc evident
`from the literature as little progress has been
`made since. This is possibly due to two reported
`cases of toxicity.
`Forbes and Davies reported toxicology studies
`of a furano derivative (ICI 78911 [8]) chac was in
`development for the treatment of Gram-negative
`infections. Toxicology studies in rodents yielded
`no abnormal findings. Ho\vcvcr, corneal ulcer(cid:173)
`ation in dogs was evident follo\ving ad1ninistra(cid:173)
`cion of three daily doses of25 mg/kg [IS] and was
`cited as the reason co stop further development.
`Grassberger et al. cautioned against the
`potential toxicity associated with chis class and
`openly speculated chat boron could be involved
`[14). Ho,vever, no toxicity data were published
`and no proof(or testable hypothesis) chat boron
`was the origin of toxicity was offered. A retro(cid:173)
`spective on Grassberger's work chen misincer(cid:173)
`preted these con1n1ents as proof chat boron can(cid:173)
`not be used clinically because of the 'inherent
`toxicity of boron-containing compounds' [19].
`
`This has had a most unfortunate consequence as
`subsequent articles have referenced chis review
`propagating the notion chat boron is toxic {19].
`More recent studies (20-24] \Vith other classes of
`diazaborines have not mentioned any reports
`of toxicity.
`
`Enoyl reductase is the target for
`N-sulfonyl diazaborines
`Enoyl reductase (ENR) is an enzy1ne involved
`in fatty acid biosynrhesis. ENR is a target of
`a front-line anti-TB drug isoniazid and che
`antimicrobial agent triclosan [9). N-sulfonyl(cid:173)
`substiruted diazaborine inhibitors ofENR form
`a covalent B-0 ester with the 2-hydroxyl group
`of the cofactor nicotinamide adenosine ribose,
`forming an inhibitor-substrate adduct bound
`in the enzyme active site. Co-crystal structures
`for a variety of sulfonyldiazaborines have been
`· published chat explain the preference for the
`sulfonyl group and confirms boron is critical
`to the mechanism of inhibition [25]. In addition
`to accepting an intramolecular hydrogen bond
`from the boron hydroxyl group, the eleccron(cid:173)
`\Vithdrawing nature of the sulfonyl group stabi(cid:173)
`lizes the negative charge on the boron atotn and
`induces a conformational bend into the molecule
`chat orients the sulfonyl substituent into a cavity
`of the active site.
`
`Diazaborines for TB
`Renewed interest in this area followed the pub(cid:173)
`lication of diazaborine's mechanism of action
`\Vith evaluation of new classes of diazaborines
`including the isosteric 2,4,1-benzo[e)diazabo(cid:173)
`rines, targeting Mycobacterium tuberculosis [24].
`Comparisons were made with two front-line TR
`drugs: isoniazid and pyrazinamide.
`While none of the diazaborines tested had
`activity near the potency of isoniazid, two
`derivatives (9 & 10) had MIC values in the
`range of 8-16 pg/ml, \vhich is superior to
`pyrazinamide (-200 µg/ml).
`
`Diazaborines as steroid mimics
`Another potencial application of diazaborines
`is in the desig11 of'ultra-high' fidelity estrogen
`structural mimics (11).
`Crystalographic data for compound 11, which
`contains an intramolecular hydrogen bond, con(cid:173)
`firm the estrogen-like conformation of these
`boron-containing hecerocycles [23]. Screening for
`antiproliferative activity against MCF-7 human
`breast cancer cells demonstrated an IC50 value
`for 11 of approximately 5 ftM [9[.
`
`->-~-~'-"--"-"'
`
`aB;J future science group
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`·-o~
`
`www.future-sc!ence.com
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`
`l Baker, D"ing, Akama, Zhang, Hernandez & Xia
`
`• Boronic acid hepatitis C virus serine
`protease inhibitors
`Hepaticis C virus (HCV) infection is a major
`cause of human liver disease. It is estimated that
`over 200 1nillion people worldwide are chronically
`infected with HCV. HCV was first idendfied by
`molecular cloning in 1989 (26] and is an enveloped
`virus containing a single-strand RNA molecule
`of positive polarity with approximately 9600
`base pairs. ~rhi: HCV serine protease NS3/4A
`is considered co be an essential enzyme for rhe
`replication of the virus and has hecn a clinically
`validated drug target by BILN-2061 [271.
`Pepci<le boronic acid derivacive.s, targeting the
`NS3/4A serine pro cease by trapping the cacalycic
`Ser-139 hydroxyl functional group with its empty
`p-orbital of boron, have been investigated for
`1nore than a decade in the: quest for novd agents
`for the treatment of HCV infection. The func(cid:173)
`tional boronic acid is positioned at the peptide-I
`(Pl) position of the peptidomimetic. Compound
`12 is an exa1nple of the class of peptide boronic
`acids discovered in 1996 and shows an IC50 of
`34 nM against the NS3/4A enzyme [201].
`A less-polar analog, 13, was also n1ade, pre(cid:173)
`sumably with the intc1uion to i1nprove cellular
`penetration. Afterwards, shorter peptide boronic
`acids and their esters \Vith praline scaffold, such
`as 14, 15 and 16, were synthesized [202,203}. The
`(+)-pinancdiol moiety is needed for the chiral
`synthesis of the Pl amino boronic acid and n1ay
`also promote the cellular penetration due to
`its lipophilicity.
`A follow-up study of 14 Pl-variable analogs
`reveals chac compound 17 has a Ki of2 nM against
`NS3 protease, 1000-fold selectivity over clastase
`and 40-fold selectivity over chymotrypsin [28).
`The enzyme potency of 17 is remarkable. The
`large borate ester present at the: Pl sitt: might
`have been hydrolyzed to expose the functional
`boronic acid. More recent examples have quino(cid:173)
`line and isoindoline structures at che P2* posi(cid:173)
`tion. Examples include co1npounds 18 and 19,
`which have borate ester functionalities ?..t the Pl
`site and quinoline and isoindoline at the P2* site,
`respectively. Both inhibitors exhibited increased
`1nolecular interaction with the NS3 protease, as
`reflecced in their potency cnhancen1ent [205.206].
`This also resulced in the lo\ver peptide character
`of the inhibitors compared \.Vith previous com(cid:173)
`pounds, such as 17, and is a progress towards the
`goal of discovering inhibitors for oral use.
`A further advance in this area was the success(cid:173)
`ful synthesis of macrocyclic boronic acid prote(cid:173)
`ase inhibitors, for example 20-22 [207). Although
`
`the biological data have not been disclosed, their
`enzyme potencies are likely to be beccer than the
`corresponding acyclic analogs, as observed wich
`other nonhoronic acid protease inhibitors, due to
`the reduced rotational freedom and the known
`SAR in the HCV protease inhibitor field. le is
`speculated that one of their derivatives might
`have entered clinical development [107].
`Schering-Plough scientists recently pub(cid:173)
`fished tht:ir work on boronic acid derivatives of
`SCH-503034 (23). SCH-503034 is in advanced
`clinical development for the treatment of
`HCV [291.
`As illustrated in the appendices, the replace(cid:173)
`ment of Pl ethyl side chain in 24, 26 and 28
`with cyclobutylmethyl improves enzyme poten(cid:173)
`cies by 50-, 68- and 260-fold, respectively, giv(cid:173)
`ing 25 (Ki = 10 nM), 27 (Ki = 0.5 nM) and 29
`(Ki = 0.2 nM). -Enzyme potencies of boronic
`acids are not significanrly different from their
`pinanediol esters in comparison of 26 with 28
`and 27 with 29. Evaluation of these compounds
`in rhe cell-based replicon assay gave an EC90 of
`in ore than 5 µM. The poor potency suggests
`chat these inhibitors may have very limited
`cell penneability.
`In summary, incorporation of boronic
`acid into HCV serine protease inhibicors has
`been a successful strategy in finding novel
`HCV therapeutics.
`
`• Boronic acid as P-lactamase inhibitors
`P-lactam antibiotics remain the most used
`antibacterial agents in clinical practice. Their
`n1echanism of action consists of interfering
`with cell wall assembly by binding to penicil(cid:173)
`lin-binding proteins that insert the peptidogly(cid:173)
`can precursors into the nascent cell wall and
`inhibiting bacterial growth [.rn]. However, the
`continuous development of resistance repre(cid:173)
`sents a serious threat to the clinical utility of
`p-lacran1s, leading to an urgent requirement for
`ne\v con1pounds (311.
`P-lactamases represent the most common sin(cid:173)
`gle cause of bacterial resistance to P-lactam anti(cid:173)
`biotics, especially in Gram-negative bacteria {32].
`p-lacta1nases ace by catalyzing the hydrolysis of
`the amide bond of the P-lactam ring, thus lead(cid:173)
`ing to biologically inactive products [33). There
`arc more than 450 members of the P-lactamase
`supcrfamily, divided into four classes (A, B, C
`and D). Classes A, C and Dare serine proteases
`and class B is a metallo-P-lactamase. An impor~
`tant strategy that has been successfully utilized
`for overcoming P-lactamasc-mcdiated resistance
`
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`·--~-.--·,~···----··~·----~·· ,._.,,~.-~ ,~ .. ·-··~"''" ,. , .,~·-~~·»· ~,,,, "'~-·~~~~
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`
`,
`
`'
`Therapeutic potential of boron-containing compounds ;
`
`R-B(OH)-OR'. five-membered
`heterocyclic ring containing a
`boron, an oxygen and three
`carbon atoms
`
`to B-lacta111s has been the co-administration of
`the P-lacram antibiotic together with a f3-lacra(cid:173)
`mase inhibitor [34). In these combinations, the
`f3-lactamase inhibitor forms a covalent adduce
`with chc enzyme, preventing it fro1n hydro(cid:173)
`lyzing the f3-lacram antibiotic. ·rhrce widely
`spread clinical f3-lactamasc inhibitors, clavulanic
`acid, tazobaccam and sulhactam, are effective
`only against class A serine P-laccamascs [35).
`·rhen:fore, rhc:re is a clear n1edical need for
`broad-spectrum inhibitors that include activity
`against class C and D enzyines [36].
`Boronic acid dcrivarivcs have proven co be
`pro1nising selective inhibitors of the serine pro(cid:173)
`. tease family of f3-lactamases. The electrophilic
`boron acorn aces as a mimic of che carbonyl car(cid:173)
`bon of che j3-lactam ring and forms a cecrahedral
`adduce with rhe catalytic serine, which closely
`resembles one of che· transition states of che
`hydrolycic 1nechanism [37]. Compounds 30-32
`were discovered as potenc inhibitors of AmpC
`13-lactainase. They were designed co gain inter(cid:173)
`actions with highly conserved residues, such as
`Asn343, in addition to catalytic serine, and to
`hind more cighdy to the enzymes. Compound 30
`has a Ki value of 420 nM in AinpC. The scereo(cid:173)
`concrolled introduction of che phenyl group,
`1nimicking the dihydrochia1.inc ring as well as
`the configuration at the C7 of cephalosporins,
`led to a tenfold improvement in ·affinity (com-·
`pound 31, Ki = 35 nM). Addition of am-car(cid:173)
`boxyphenyl moiety further improved affinity
`against AmpC P-lactamase (32, Ki= 1 nM) [37).
`Another series of glycylboronic acids bearing
`the side chains of cephalosporins and penicil(cid:173)
`lins have proven to be reversible and com(cid:173)
`petitive inhibitors of CTX-M 13-lactamase.
`Compound 33, containing the side chain of naf(cid:173)
`cillin, has Ki values of 1.2 and 3.0 µM again.st
`CTX-M-9 and CTX-M-16, respectively. The
`2-aminochiazole inhibitor 34, containing the
`side chain from cefcazidime, has Ki values of15
`and 4 nM against CTX-M-9 and CTX-M-16,
`respectively. Both 33 and 34 adopted a conforma(cid:173)
`tion in the active site consistent wich acylacion
`transition state analogues [3S}.
`In su1n1nary, the unique ability of the boronic
`acid functionality to accept an active site serine
`into its electophilic p-orbtal has provided a novel
`series ofB-lactamase inhibitors.
`
`chemotype agent.s. A1nino-acyl t-RNA synthe(cid:173)
`cases are crucial for protein synthesis, and cargec(cid:173)
`ing the editing do1nain of this enzyme is a new
`approach co its inhibition. A new class ofboron(cid:173)
`concainingcompounds, known as 1,3-dihydro-l(cid:173)
`hydroxy-2,1-benzoxaboroles, has been identified
`as inhibitors of fungal leucyl t-RNA synthetase
`and have potent antifungal activities with MIC ... o;
`as low as 0.25 ~1g/ml against the major dcrma(cid:173)
`tophytes Trichophyton rubrum and Trichophyton
`mentagrophytes and the yeasts and n1olds Candida
`albicans, Cryptococcus neoformans and Aspergillus
`Jumigatus [39.208). AN2690 (35) and AN2718 (36)
`are two examples of chis class of compounds.
`A penetration study indicated that these
`b-.:nnr<JbDrn!c compounds can effeccively pen(cid:173)
`etrate through human nail plate and reach the
`nail bed in sufficient concentracion to inhibit
`fungal pathogens [40]. AN2690 (35) is currendy
`in clinical developmenc for che topical trcacmenc
`of onychomycosis, a fungal infeccion of the nail
`and nail bed.AN2718 (36) is also in clinical trials
`co neat skin and ocher topical fungal infections.
`Mechanism investigation with AN2690 (35)
`demonstrates that chis compound inhibits yeast
`cytoplasmic leucyl-cRNA synthetase by fonna(cid:173)
`tion of a stable AN2690-tRNA ""adduct (38) in
`the editing site of the enzyme [41). The AN2690-
`cRNA Leu adduct (38) is formed through the boron
`atom of the AN2690 (35) and tho cis-diol on·
`the 3'-terminal adenosine (37) of the tRNA, as
`proposed below.
`The trapping of enzyme-bound tRNA Leu in
`the editing site prevents catalytic turnover, thus
`inhibiting synthesis ofleucyl-cRNALcu and con(cid:173)
`sequentially blocking protein synthesis. This
`result establishes the editing site as a novel cargec
`for aminoacyl-cRNA synthetase inhibitors.
`In sum1nary, che recent discovery of boron
`cherapeutics as amino acyl t-RNA synche(cid:173)
`tase inhibitors, acting by trapping the tRNA
`in the enzyme-editing domain, is expected to
`be a promising field for the discovery of novel
`antifungal therapeutics. The combination of
`the unique boron chemistry, molecular-level
`kno\vledge gained from crystal structure stud(cid:173)
`ies and rational drug design has established a
`powerful drug-discovery machinery to feed the
`development pipeline.
`
`• Amino-acyl tRNA synthetase inhibitors
`There is a clear need to develop new efficacious
`therapeutics to treat fungal infections. One of
`the strategics is to discover and develop novel
`
`• Boron~containing anticoagulants
`"rhro1nbin and Factor Xa have been pro1nising
`targers for anticoagulant agents for more than
`a decade. A number of boro-Lys- and boro(cid:173)
`Arg-bascd (boronic acid analogs of lysine and
`
`. '"--·--~,.·--
`
`mm future science group
`
`. ·-~~~------·---.-·~·--,,~---"-~~~
`
`www.future-science.com
`
`1279
`
`CFAD v. Anacor, IPR2015-01776, CFAD EXHIBIT 1056 - Page 5 of 14
`
`

`
`t i Baker, Ding, Akama, Zhang, Hernandez & Xia
`
`'
`
`arginine) thrombin inhibitors with nano1nolar
`to picomolar potency have been identified [8.9].
`Recendy, a potent thron1bin inhibitor TRl50c
`(39, Ki = IO nM) has heen developed and is in
`clinical trials for the treaunent of chro1nbo(cid:173)
`sis (209,210]. le is formulated for either oral (Ca
`sale: TGNI67) [102] or intravenous (Na sale:
`TGN255) (103) adminimation.
`In addition, phcnylboronic acid derivatives,
`represented by 40, were reported as inhibitors
`of another serine protease in che blood coagula(cid:173)
`tion cascade, Factor Xia (FX!a) [421, While the
`potency o_f 40 is still in the micromolar range,
`it showed a nine- to 31-fold selectivity co FXla
`over FXa and chrombin, respectively, and more
`than 140-fold over trypsin.
`These examples further support the utility of
`boronic acids against a variecy of serine prote(cid:173)
`ases by making use of the ability of the boron co
`form a tetrahedral transition state mimic with
`che active site nucleophile.
`
`• Boron-containing dipeptidyl
`peptidase 4 inhibitors
`Dipeptidyl peptidase 4 (DPP4, also known as
`CD-26) is a serine protease chat specifically
`removesXaa-Pro dipeprides from the N-cerminus
`of polypeptides and proteins (43]. DPP4 is found
`in a variety of mammalian cells and tissues [44].
`However, it was not recognized as an impor(cid:173)
`tant drug target uritil 1995, when glucagon-like
`peptide-I (GLP-1) was identified as one of the
`substrates of DPP4 {45,46]. GLP-l scimulates
`glucose-induced insulin biosynthesis and secre(cid:173)
`tion [47], and increasing blood GLP-1 concl'.ntra(cid:173)
`tion seemed to be a promising approach to treat
`diabetes. However, GLP-l is rapidly inactivated
`by DPP4 in vivo and discovery of DPP4 inhibi(cid:173)
`tors therefore beca1ne a pro1nising concept for
`treatment of diabetes !48). Indeed, the small(cid:173)
`molecule DPP4 inhibitor Januvia® (sitagliptin)
`was approved by che US FDA for the treatment
`ofType 2 diabetes [49].
`Bachovchin et al. reported chat peptide prolyl
`boronic acids arc potent inhibitors of bacterial
`IgAl protcinases [SO]. They applied a similar
`
`strategy to design potent DPP4 inhibitors and,
`following extensive SAR, chey identified Ala(cid:173)
`boroPro (41) and Pro-boroPro (42) as very potent
`inhibitors of DPP4 (2 nM for 41 and 3 nM for
`42) !51.521. 1~he NH 2-P2-boroP1 structure is the
`essenrial pharmacophore for rhe DPP4 inhi(cid:173)
`bition. Simple boroPro (43) and N-Boc-Ala(cid:173)
`boroPro (44) were not active, while the P2 residue
`has some flexibility. As for the P2 residue, Ala,
`Glu, Gly and Pro showed nanomolar co picomo(cid:173)
`lar Ki values. The boroPro moiety can be sub(cid:173)
`stituted \Vith boroAla as well, although boroAla
`derivatives arc less active than boroPro. There
`is a concern of st:lt:ccivicy co DPP4 over DPP8,
`DPP9 and potentially other enzymes for these
`boropeptides. le could be overcome by che struc(cid:173)
`tural modifications of the molecule [53]. Recently,
`in vivo blood glucose-lowering activity of co1n(cid:173)
`pounds 41, 45 and 46 (Glu-boroPro) was reported
`[54] and the safety of these three closely related
`dipeptide boronic acid inhibitors (41, 45 & 46)
`was determined. Just like the nonboron inhibi(cid:173)
`tors of the DPP4 enzyme, their toxicity is related
`co their inhibition of related isozymes DPPS and
`DPP9. A eight correlation was observed between
`intracellular inhibition of DPP9 and the 1naxi(cid:173)
`mum tolerated dose (MTD) (TABLE 1). The Glu(cid:173)
`boroAla (46) is a selective inhibitor of DPP4,
`and it is a safe compound, while the toxicity of
`chc other compounds is· related to their nonsC(cid:173)
`lective nature (TABLE I). ~rherefore, the author
`concluded that boronic acid-based inhibitors of
`l)PP4 do noc exhibit unique or untoward tox(cid:173)
`icities. While Val-boroPro (45, talabostat) is a
`pott:nt DPP4 inhibitor [52], this compound is
`also in clinical trials for colorectal cancer as a
`fibroblast activation protein (FAP) inhibitor {54].
`Very recencly, clinical rrial results of Gly(cid:173)
`boroPro dt:rivative DPP4 inhibitor PHXl 149
`(47) were reporced [55-57.104]. PHXl 149 was given
`orally at the doses of 200 or 400 mg. Patients
`were allowed to continue either metformin or
`d1iazolidinedion, or a co1nbinacion of the two.
`PHX1149 showed srarisrically significant reduc(cid:173)
`tions on hemoglobin Ale (HbAlc) in both 200-
`and 400-mg groups. PHXl 149 also demonstrated
`
`Compound
`Val-boroPro (45)
`! Ala-boroPro (41)
`J Glu-boroAla (46)
`i "Kl is in nM
`: DDP: Dipeptidyl peptidase; MTD: Maximum-tolerated dose.
`
`0.76
`0.53
`2100
`
`360
`7000
`
`0.025
`5.0 "' MTD < 38
`500 "' MTD < 900
`
`0. 18
`0.027
`8.3
`
`1.5
`2.0
`880
`
`'~~~-~~~-~~''"-w-,,~,w~----·--------";-<O~>~-,_
`
`1280
`
`Future Med. Chem. (2009) l (7)
`
`, _ _,~ --<~'A--0< ''"'~-~·-~~~-~--·----
`future science group llJ
`
`CFAD v. Anacor, IPR2015-01776, CFAD EXHIBIT 1056 - Page 6 of 14
`
`

`
`Therapeutic potential of boron-containing compounds ~
`
`statistically significant efficacy on the secondary
`end point including change in fasting and post(cid:173)
`meal blood glucose levels. There was no substan(cid:173)
`tial difference observed between active arms and
`placebo in tenns of safety and tolerability.
`In su1nmary, boronic acid inhibitors ofDPP4
`have n1ade a substantial contribution to chis
`important field.
`
`• Boron-containing
`phosphodiesterase 4 inhibitors
`Phosphodiesterases (PDEs) are a family of
`enzymes responsible for the hydrolysis of sec(cid:173)
`ond-messenger cAMP and cGMP [58]. "rhe PDE
`superfamily comprises at least 11 members,
`including approximately 100 isoforms [59,60].
`Among those, PDE4 specifically catalyzes the
`hydrolysis of cAMP and is che predo1ninanc
`phosphodiesterase enzyme in immune and
`inflammatory cells [61,62]. Therefore, PDE4
`has been considered as a promising therap