))2J.
`l
`
`IH4U
`
`J Mal. Chcm. 2007,50, 1840-1849
`
`Application of the Phosphoramidate ProTide Approach to 4'-Azidouridine Confers
`Sub-micromolar Potency versus Hepatitis C Virus on an Inactive Nucleoside
`
`Plinio Perrone! Giov~nna M. Luoni.' Mary Rose Kelleher/ Felice Daverio,t Annette Angell/ Sinead Mulready,t
`Costantino Congiatu,l Sonal Rajyuguru,i: Joseph A. Martin) Vincent Leveque,: Sophie Le Pogam,: Isabel Najera,:
`Klaus Klumpp.: David 13. Smith,: and Christopher McGuigan•·t
`
`1-1-'elsh School o(Pharma<:v. Cardiff University. /,'in~ Edward VII At'C'/11/e. CardiffCFIO JXF, UK. and Roche Palo Alto, 343/ Hillview Ammue,
`·
`Palo Alto. Calijiwnia 943114
`
`We report the application of our phosphoramidatc ProTide technology to the 1ibonucleoside analogue 4'(cid:173)
`azidouridinc to generate novel antiviral agents for the inhibition of hepatitis C virus (I-ICY). 4'-Azidouridinc
`did not inhibit I-ICY, although 4'-azidocytidine was a potent inhibitor of HCY replication under similar
`assay conditions. However 4'-azidouridine triphosphate was a potent inhibitor of RNA synthesis by I-ICY
`polymerase, raising the question as to whether our phosphoramidate ProTide approach could effectively
`deliver 4'-azidouridinc monophosphate to HCY replicon cells and unleash the antiviral potential of the
`triphosplmtc. Twenty-two phosphoramidates were prepared. including variations in the aryl, ester, and amino
`acid regions. A number of compounds showed sub-micromolar inhibition of HCY in cell culture without
`detectable cytotoxicity. These results confirm that phosphoramidate ProTides can deliver monophosphates
`of ribonucleoside analogues and suggest a potential path to the generation of novel antiviral agents against
`HCV intcction. The generic message is that ProTide synthesis from inactive parent nuclcosidcs may be a
`warranted drug cliscove1y strategy.
`
`Introduction
`The hepatitis C vims (HCV") was idemitied for the first time
`in 19R9 as a singl~-stranded positive sense RNA virus of the
`Flaviviridae t'amily. 1 According to the World 1-lcalth Organila(cid:173)
`lion (WilD). more than 170 million people are estimated to be
`chronically int'ected by this virus, which is a major cause of
`severe liver disease. 2
`At present, treatment options comprise immunotherapy using
`recombinant interferon (often pegylated) in ~ombination with
`ribavirin. The clinical benefit of this treatment is limited, and a
`vaccine has not yet been developed. The development of
`selective inhibitors of ess~ntial viral enzymes such as the serine
`protease NS3 or the RNA-dependent RNA polymerase NS5b
`are expected to improve the pot.:ncy and tolerability of future
`treatment options for HCV infected paticnts.J.4
`Nuclco~ide analogues have already been validated as an
`important class of polymerase inhibitors of other viral targets,
`such as HCMV. HSV, HIV, and HBV.5 All antiviral agents
`acting via a nudeoside analogue mode of action need to be
`their corresponding 5'(cid:173)
`phosphmylatcd, most of them
`to
`tt·iphosphatcs. by cellular andlor viral enzymes. The nucleotide
`tt·ipho:;phate analogues will then inhibit the requisite polymerase
`and/or compete with natural nucleotide triphosphates as sub(cid:173)
`strates for incor-pot·ation into viral nucleic acid during viral
`replication. 5
`Recently, 4'-azidocylidine was discovered a'i a polent inhibitor
`of HCV replication in c'ell culture. The corresponding 5'-
`
`.., Con·t'sponding aulhor: Tel. +44 29 20874537; fitx: +4419 20874537;
`mcguigan~~t:ardi ff.nc. u k.
`'Cardiff Univcrsily.
`! Roch~ Palo A ho.
`{/ Abbr~vif'tlons: NS3, nonstruclural protein 3; NS5B. nonstructural
`protein SB: HSV. herpes simplex vin1s; HCV, hepatitis C virus; HIV. hum£ln
`hnmunod<liciency virus: HBV. heputitis B virus: d4Aid4T. 2',3'-dideoxy(cid:173)
`:!:'}'~didchydroac.lcuu:-;int.:!thymidine; ddA. 2'.3'-didcoxy~1dcnosin~; LCd4A.
`(I R,cis)-4-(6·Aminu-9H-purin-9-yl)-2-cyclopentenc-l-tm:lluuwl; BVdU, £-5-
`C!-bromovinyl)<'!' -dcoxyuridint::: HCMV. human cytomegalovirus; AZU.
`:~' -azidouridine; NM(. N-mcthylimidaznl~.
`
`triphosphate was desc1ibed as a competitive inhibitor of
`cytidylate incorporation by HCV polymerase and a potent
`inhibitor of native, membrane-associated HCY replicase in
`vitro."
`Interestingly, the corresponding uridine analogue, 4'-azido(cid:173)
`uridinc (1 ), was inactive as an inhibitor of HCV replication in
`the cell-based.replicon system7
`It was hypothesized that (l) (1, Figure I) may be a poor
`substrate for phosphorylation by cellular enzymes. The first
`phosphorylation step to produce the 5' -monophosphate has often
`been found to be the rate-limiting step in the pathway to
`intracellular nucleotide triphosphate formation, suggesting that
`nucleoside monophosphate analogues could be usefttl antiviral
`agents. However. as unmodified agents, nucleoside monophos(cid:173)
`phatcs arc unstable in biological media and they also show poor
`membrane permeation because of the associated negative
`charges at physiological pH.8.9
`Our atyloxy phosphoramid•tte ProTide approach allows
`bypass of the initial kinase dependence by intracellular delivery
`of the monophosphorylatcd nucleoside analogue as a membrane
`p.::nneable "ProTide" form. 10•11 This teclmology greatly increases
`the lipophilicity of the nucleoside monophosphate analogue with
`a consequent increase of membrane permeation and intracellular
`availability. Previously we have demonstrated the success of
`our approach with the aryloxy-phosphoramidate derivatives of
`ddA, 1o d4T, 1L 12 LCd4A.U and d4A. 10•14 These nucleotide
`monophosphate analogues were shown to exhibit greatly
`enhanced activity against HIY compared to the parent nucleoside
`analogues in vitro. In contrast to the parent nucleosides, full
`antiviral activity of the monophospbatc analogues was retained
`in kinase-deficient cell lin~s, which was consistent with an
`efficient bypass of the tirst phosphorylation step in HIY infected
`cells. Aryloxy-phosphoramidates are considered to be efficient
`lipophilic prodrugs of the corresponding 5'-monophosphate
`spcci<Js in which the two masking groups are represented by an
`mnino ac:id ester and an aryl moiety. AHer passive diffusion
`
`:1) 2007 Am<rican Chemicul Society
`l O.tfJ2l;jm0613370 CCC: $37.00
`Published on Web 03/1712007
`
`IPR2018-00126
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`

`

`Phosphoramidare ProTide Approach to 4'-Azidouridine
`
`Joumal of lvfedicinal Chemi.wy, 2007, Vol. 50, No. 8
`
`t 8~ I
`
`comptlunds were always isolated as mixture;; of tw(> dia~tc reo­
`isontel·s. Th<;: pres~ n ce ol· these diasterco isomers in the i·inal
`preparations was conlirmcd by .llp (two peaks), 111 , and "C
`NMR. A totalof22 phcnyi phosphoramidatcs were synthesized
`as reported in Table 1.
`\Vc have pfl;v iously H;porlcd t:xlcusivc slrw..:lure-·a..:tivi ty·
`relationship (SAR) studies of anti-Il l V phosphoramidntcs
`cxploring lite amino acid r~giun, including natura l am ino "''Jd
`variati('ll,21 un-natural a.,a-dialkyls;~2 stcrcocltcntical variation.1.l
`and amino acid extensions2• and replacerncnts 25 In general,
`L-nlanin• and the unnatural am ino acid a .u-d imethylglyci ne
`showed the best activity for the d4T parent molecule versus
`HIV. It.ll
`Using the previously described method (St:hemc I}, we
`syn thcsi7.cd the t-:\luninc Cl2), fHI-d imelhylglycinc ( !8),
`cycl opcntylgly~i nc (20}, L-pltenylalanine (22), I.-leucine (27),
`L-mcthioninc (29), ethyl I.-glutamate (31), attd L-prolinc (28) / '
`phenylphosphoramidatcs of I. each bearing an ethyl e,;tcr.
`Furthe1· inw:;t igations on the amint> acid voriatit>n wer~ cun(cid:173)
`ductcd on a sctics of benzyl t.:s!(r:;: L-a l u ni n~ (17), U,(t(cid:173)
`d i methylglycin~: ( 19). cydopentylg!ycinc (21), 1.-phcny!a laninc
`(B), I.-valine (24), and glyc ine (25). We further compared the
`importance or the skreocltcmistry ut the mninn add pos ition·
`by preparing a D-alanine benzyl ester phosphmamidutc (26).
`On the ba:;i:; of the t.-alanine phenyl phosphoratnidalc backbone.
`Wt' also explored the SAR of dilfcn:nt esters includ ing 111dhyl
`(11 ). erhyl (12), butyl ( 13 ), 2-butyl ( 14), isopropyl ( 1 5), !err-
`butyl (16), and benzyl (17). In ord~ r to have an indirect proal·
`or phenyl pho~phnramidute metabo lism, we synthesized the
`N-mt:lhylglycine (30) and (3-alaninc (32) analogues. which wt:re
`consitlerccl unfavNabk substrates according to the ~ l•:>t u l atc:cl
`me~hanism ol· activ:~tion. 1 ;
`Recently we noted an increase of in uitm poten<:y o l· a
`I-i1aphthyl-phosphormnidatc analogue compared to the CCJ ITC(cid:173)
`sponding phenyl derivative while investigating the anticancer
`activity of BVdU phosphorumidatesX• Therefore. similar phos(cid:173)
`ph<)ramidate analogues w~rc al~<! genera t.:d for ( 1). The
`synthesis of the !-naphthyl plm~phorumidatc (33) was pcrfonned
`by reacting 1-mtphthol with pho:>pl10rus o . ..:yc:hloridc in an almost
`quanritativc rcac:tion to give the correspondi ng phusphoro(cid:173)
`di-:h l oritlal~ (Scheme 3"1, which was then coup le-d to an antino
`a.: it! ester and thc nu~koside analogue acctlrding to ou1· standard
`procedu res. In this case, the separation or tltc: twt1 phosphate
`diastcrcoisomers (34 and 35) was at:hicved hy using a semi(cid:173)
`preparative HPLC purilication with elution conditions t>f 7()'~.-;,
`watcr!JO'Yo acet(>nitri!c. The 31 P NtviR spc<:trtttn sltowcd the
`pres<!nct: or only one peak t'or the fi rst of the two fmctio ns
`sepurmcd, and tlte 1fl NMR spectrum supported the sugges tion
`of" single diastt'l"<:oisomcr in litis ca,e. The second fract i<m
`contained an excess of the second <.J iastcrcoiso lll~r to\!cthcr wi th
`a mi nor proportion (estimated at 7'!'o by ·"P NMR i~tcgrati(>lll
`of' the first diustercoi:;omcr (sec Supporting lnfonnatiun ror
`data).
`·
`Antiviral Activity, The pheny I phospltoramidales described
`above (II - 32) w.:re characterized in ~'ilro as inhibitors of HCV
`replication in a HCV r<:plicon assay as previously reported.i'·'
`Da ta arc prcS(ntcd in Table I as EC;o values (representing the
`concentration ot"compounds r~duci11g HCV replicntiun l>y 50"•;.)
`and CC..,, values (rcprc;;cnting the concen tration of com pound~
`reducing eel! viability by 50% ) as dt:tcnn incd using the WST
`a,;say. All compounds :;hawed CC;,, values greater tlwn I (ll) ,11 M.
`The parent conipmtnd I did not inhibit I·ICV rcp!ica1ion
`>ignilicatHly in the n:plicun system <ECu '' 100 p<vl ). In
`contrast. a nu mber of ph,»phoramidate derivatives ~howed
`
`,r--..
`
`AZU(l)
`Figure 1. Structure of AZU and its corresponding pheny!-phos(cid:173)
`phoramidate ProTide.
`
`through cell membranes, the suggested activation pathway15
`involves initial enzyme-catalyzed ckavagc of the carbo,~ylic
`ester, followed by the internal nucleophilic 8trHck of the acid
`residue on the phosphorus center, displacing the aryloxy group.
`The putative transient, cyclic mixed-anhydride is then rnpid ly
`hydrolyzed to the cotTesponding amint' acid phosphomonocster.
`Last, a sttggested phosphoramida,;e activity catalyz~s the d~a v­
`age of the P-N bond to free the nudeoside monophosphatc
`intracellu!arly. In the cum:nt study we tested the possibility to
`apply our ProTide approach to the inactive 4' -azidouridine ( 1)
`in order to achieve bypass of the first phosphotylation step an<l
`thereby generate novel antiviral agents (2) with potent act ivity
`against HCV.
`
`Results und Discussion
`
`Cheiuistry. The synthe:;is of 1 has been prev iuu:;ly dc(cid:173)
`scribed. 16 To prepare monophosphate prodrugs (2) of I we
`initially followed the previously described phosplwrochloridate
`chemistly for the synthesis of· ProTides developed in uur
`laboratory, using 1-mcthylimidazok: (NMIJ as the coupling
`agent. 14· 17. l ~ Several attempts were performed using dit"!erent
`condition; (different amino acid esters, di fferent rca~tion
`conditions) without successfLd isolution of the .:orrespondi ng
`aryloxy-phosphoratnidate. These initi<il unsuccessful attempts
`might be explained considering the presence of a hul ky group
`(azido) at the 4'-position adjacent to the coupling site at the
`5'-position; in all previously published ProTide exampks the
`4'-position was unsubsti tuted.
`The method of Uchiyama. was investigated ne.xt.t'• This
`approach is based on the treatment of a nucleoside with I equiv
`of a strong organometal lic base, such as a solution of ler/(cid:173)
`butylmagnesium chloride (tBuMgCl), to form th.: corr~spond ing
`metal alkoxide. !n the case or (I), this n::a~ t ion was observed
`to be very rapid and gave yields between 3% and 20% of desired
`products. In the first instance, we synthesi zed 4'-azidou ridine
`phosphoramidates starti ng from an unpmtectcd nucleosid<:. The
`apparent reactivity at the 2'- and 3'-positions was low, suggesting
`high regioselectivity for the reaction at the 5'-position. In this
`way it was also possible to synthesize compounds 13, 21. and
`26. In order to achieve higher solubility in the rea•tion solvent
`(tetrahydroti.1ran) and increase reactivity at the 5' posit ion, the
`2'- and 3'-positions of l we r~ protected with a cyclopentyl
`group.2o The !ina! synthetic pathway (Scheme I) involves the
`coupling of phenyl dichlorophosphate with uifTerent amino acid
`ester salts (5) to give the corresponding phcnylt•.xy-phuspho(cid:173)
`rochloridates (6), which were purified by fla.~ h chromatography
`and then coupled with the 2'3' -0,0-cyd opentylidene derivative
`7 of (IJ in the presence of tBuMgC I (I M sol ution in THF).
`The dcprotection step was perform eel with a solution of SO%
`fotmic acid in water for 4 h at room tcmpcratttre (Sch;,me 2).
`Due to the stereochemistry at th~ phosphorus center, the tina!
`
`IPR2018-00126
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`I-MAK 1012
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`

`

`1842 Journal of Medicinal Chemisll)', 2007, Val. 50, No.8
`
`Perrone et al.
`
`Scheme 1. General Synthetic Pathway for the Synthesis of AZU Aryloxy-phosphoramidatcs
`0
`II
`Ar-0-P-CI
`I
`Cl
`
`0
`0
`Ar-O-~-~- )l ,Rz
`y.R.-0
`I
`Cl
`R 'R,
`
`5
`
`EtJN. CH2CI2 dry
`-78°C to r.t
`4h
`
`tBuMgCI
`THFdry
`t7 h
`
`R3= H, AZU (1)
`R3= cyclopentylidene (7)
`
`Ar= phenyl (11·32)
`Ar= t -naphthyl (33, 34, 35)
`
`8
`
`28
`
`30
`
`32
`
`Scheme 2. General Synthetic Pathway for the Dcprotcction of 2'·3'-cyclopcntylidcnc AZU Phcnyl-phosphoramidatcs
`
`· . .._,.,..
`
`80% HCOOH
`
`room remperarure, 4 h
`
`potent inhibition o f H CV replication. Assttming that 4'·
`~zidou ridine- 5'- triphosphatc is the active 1-!CV poly me rase
`inhibitor, these results support lhe notion that the active
`phosphoramidatcs successfully ddiv~ered 4' -azidouritline mono(cid:173)
`phospht\1~ intr"cellularly, that 4'-azidourid ine (I) is inefftcic nlly
`phosphOI) 'Iatcd to !he mo nClphosphate in replicon cells, and that
`4'-~zi do midinc I IHJr~t> phosphatc can be phosphorylated to th~
`5'-triphosphatc in rcplicon cells. As shown in Tabk 2, 4'(cid:173)
`azido uridine triphosphate notably inhibited rccom binanl HC'V
`rol ymcras~ NSSb in vilro, and did so with s im ilar sub(cid:173)
`micrmnolar potency, li ke rhat of the previously de>cribed NSSb
`inhib ito r R 1479-TP (4'-az idocytidine triphosphate)" Therefore.
`the applicatio n of our phosphoramidate approa~h shown to be
`a successful tool in overcoming the phosphorylation block o r I
`and converting an inactive nucleoside analogue to a pote.nt
`
`inhibitor of HCV replication, thus accessing the full potential
`o f the 4' -azidouridine triphosphate.
`As shown in Table I , \..-alanine derivatives represented a
`series o r active antiviral p hospho ramidates ( 11- 17). Low or
`sub-micromol ar activity was noted in marked contrast to the
`i nacli l!e nucleoside parent (I ). The len-buty l ester (16) was the
`k ast active of the set·ies, whic h was in agree ment with the SARs
`previously obtained in the d4T serieii and may relate to the
`relative stabil ity of tertiary ~sters to enzyme-mediated hydrolysis.
`The isopm pyl ester (15) showed hig h potency and represented
`one of the most active phosphoramidatcs prepared. Simila rly,
`th~ 2-butyl cs t~r (14) w as highly active in our a>say in contrast
`to previous observatio ns with other nucleoside a nalogues.
`Together with the benzyl analogue ( 1 7), these three esters
`prov ided the most potent compound s or HCV replication
`
`IPR2018-00126
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`I-MAK 1012
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`

`Phosphoramidate ProTide Approach to 4'-Azidouridine
`
`Journal qf Medicinal Chemistry, 2007. Vol. 50, No. 8 184J
`
`Tnblc I. Anti HCV Activity and Cytotoxicity Datn tor ( ll and Phenyl
`Pbosphoramidate Nucleotide An::alogues
`
`Table 3. Anti HCV Activity and Cytotoxicity Data for (I) and
`1-Nnphthyl Nucleotide Analogues
`
`compound
`
`mnino t~cid
`
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`31
`32
`4'-azidouridinc (I)
`
`L-Aia
`L-Ab
`L-Ain
`L-Ale
`L-Ab
`L-Ain
`L-AI8
`\k2Giy
`\1e2Giy
`cPntGiy
`cPntGiy
`Phe
`Phe
`Val
`Gly
`u-Aia
`Leu
`Pro
`~·let
`N-MeGiy
`EtGiu
`{3-Aia
`
`ester
`Me
`Et
`81!
`2-Bu
`iPr
`tBu
`Dn
`Et
`Bn
`E!
`Bn
`E!
`Bn
`Bn
`Bn
`Bn
`Et
`Et
`Et
`Et
`Et
`Et
`
`EC;o(.uM)
`
`_tl
`1.]
`!.2
`0.63
`0.77
`:\.1
`ll.fll
`10 . .1
`3.4
`>100
`<100
`U7
`<lOll
`<.J()(J
`l.tJ
`1.2
`2..3
`6.0
`14
`>100
`>lOU
`> lilO
`'"100
`
`cc,,cuMJ
`> J[JIJ
`''illll
`'·[[![!
`>Jill)
`>·J(l(l
`,_ IUO
`> l!l!l
`>-100
`,_ 100
`> 100
`>[()()
`>J(](J
`>I!)[)
`''100
`>J(J(J
`>11)0
`> 100
`--100
`>ltlll
`>100
`''1()0
`>lOll
`"100
`
`Scheme 3. Synthetic l'othway for the Synthesis of
`1-naphthylphosphorodichloridatc
`
`~
`~ Et3N, Uicthyl dht:r
`
`-78° C to r.t.
`3 h
`
`0
`II
`CI-P-CI
`I
`0
`
`~
`VJ
`
`4
`
`Table 2. Inhibition of HCV Polymerase (NS5B) Activity in Vitro
`
`IC,o[uM]
`
`enzyme
`
`NS58570n-BK
`NS58570-Con 1
`
`RI479-TP (4'-azido CTP)
`0.29 ± 0.13
`0.32 ± 0.11
`
`4' -azido UTP
`0.23 ± 0.01
`0.22 ± 0.02
`
`inhibitors in the I.-alanine series, all lwving ,11M inhibition of
`HCV. The antiviral activity of these three phosphoramidates
`was exceptional if compared to rhe parent compound l (EC;o
`> 100 ,uM), providing strong support tor the notion ofPruTicle(cid:173)
`mecliatecl kinase bypass.
`In the benzyl ester family, L-alunine (17) provided the most
`active compound with D-alaninc (26) and glycine (25) being
`only slightly kss potent. These results were striking when
`compared to the 60-70 fold reduction in anti-111\i potency for
`d4T ProTides with an L-alanine to glycine replacement and a
`20-40 fold reduction l'or the corresponding abaeavir Pru(cid:173)
`Tides21.27 This reinforces our earlier conclusion that a separate
`ProTide motif optimization process is needed for each nucleo(cid:173)
`side analogue versus a given target. It may be that cell line
`dependent enzyme expressi.on may cletermin~ different pho~­
`phoramidate SARs.
`The presence of a methyl (D- and L-abninc, 26 and 17) or
`o.,a-dimethyl (19) enhanced the activity if compared to larger
`and hydrophobic amino acid side chain residues such as L-valine
`(24), L-phenylalanine (23), and cyclopentylglycinc (21). which
`were weakly active in the replicon assay.
`An unexpected con·clation was found b.:twecn amino acid
`and ester function. While the L-phenylalaninc derivative was
`substantially inactive as a benzyl ester (23). the corresponding
`ethyl ester (22) showed a significantly increus<3d antiviral
`
`compotJnd
`
`33
`3J
`35
`17 (Phenyl ProTide)
`4'-azidouridine ( 1)
`
`phosphorus
`t:uniiguraliun
`
`SIR
`R
`s
`SIR
`
`amino
`acid
`
`L·Aia
`L-.A.!<~
`L-Aia
`L-Aia
`
`ester
`
`Bn
`8!1
`Bn
`Bn
`
`EC,o
`(.uivl)
`
`cc5(l
`(ltM)
`
`> 100
`0.22
`0.39 >!00
`> 100
`0.43
`> 100
`0.61
`> 100
`> 100
`
`activity, displaying an EC,11 value of 3.4 ,ll M. Therefore, matri.~­
`bascd optimization c>f amino acid and ester liatctions may be
`prclcrrcd over stepwise approaches.
`The inactivity of the f)-alanine (32) and ,,f th~ ;V-mctliyl
`glyc·inc (30) compounds might undcdinc the presence ol' an
`a-amino acid and a li'cc NH m; a minimum requircm~111 in the
`amino acid structure to enable the 1netabolic activatiDn or
`aryloxy-phosphoramidates. l-!owewr, the proline compound
`(with a blocked NH) did show modest (28) activity. pointing
`to a complex amino acid SAR.
`In conclusion, ester variation was widely tolerated except for
`the len-butyl which gave a slight rcduetiDn in potency in the
`the benzyl
`in
`the case ol· tlu;
`t.-alaninc series ( 16) and
`i.-phenylalanine derivative (23). I.-Alanine remained the most
`ei'l'ective amino acid, with glycine and D-alaninc shu wing oni\·
`slightly reduced potency. Dimctbylglycinc, t.-lcucinc, and I.(cid:173)
`proline also providc.:d compounds with antivirul potencies in a
`low micromolar range. It therefore appears that the amino acid
`core could br: eonsidcrnbly varied to give ::!.~lt! 1.'!ra! ~~g~nts '<'.'!!~:
`potencies within a I 0-fold range in replieon cells. Importantly.
`potency l'ptimization r~quircs consideration 0f both amino and
`and ester moieties a~ most clearly shown t\lr the ethyl and benzyl
`esters of the L-phcnylalanine analogues. Moro::ovcr, quite di;tinct
`SARs c·mcrgcd frc>1n this l'runily versus ![(.'\' as co1nparcd t(l
`our prioc studies in olhc1· tilmili~s.
`We also explored the possibility to replace the phenyl
`substilllent un the phosphate with a more hydrophobic moiety.
`]-naphthyl. Previously. we noted an increase of in uitm pot·~ncy
`,,f 1-naphthyl-phosphommidates comparee! to the cc1rrcsponding
`phenyl phosphoramidaks whe11 investigating FlVdU phospho(cid:173)
`n:unidaLc:::
`in an anticancer assay. 2(1 \Vc synth..:sizcd 33_ the
`!--naphthyl analogue of 17 (t.-alaninc benzyl e,.rcr). As shown
`in Table 3, compound 33 inhibited IK'V replication with an
`ECso Df 0.22 p.M. leading to a further increase in antiviral
`activity (>450-lold) in comparison to 4'-az!duuridine ('fable
`3). One of the two phosplwrus diastcrcoisomers Cl>ttld be
`pu l'ified using a C-1 ~ rcv~rsc phase scmiprcpe~rativc II PL C OnL'
`of the two main rractions obtained sltowccl only llnc :'1 r Nl\1 R
`peak. The second li<tction was less pur~. although tltc second
`lhc major .:omponent ol· the
`diustcrcoisomer appeared as
`mixture. We have previously reported a method
`tor the
`pr.:dicti@ or the phosphoms configuration of such diastcreo(cid:173)
`isomers based on a different 1 II NMR prolilc or the mctltyknc
`protons of the benzyl ester2 r' Applying this concept to cum(cid:173)
`pounds 34 and 35, we noticed thaL in tJne case (more polar. 35)
`a clear AB-system was l'bsecved whik, fur the <'thee diastcrc,J(cid:173)
`isomer (less polar, 34), the two protons displayed an appar.ont
`doubkt. Confonnational studies were rerllmncd using the Sybyl
`7.0 softwa1·e package. The lowest energy conlormation tound
`fur ea~h diastcreoisomer is shown in Figure 2. These di ffercnccs
`in proton profiles can be explained by tlte ability of one. but
`not the other, cliastereoisom.cr to fonn ::r-~7. interactions between
`the naphthyl and the phenyl group of the benzyl ester resulting
`in a constrained conformation. This interacliun can l'lily l'ccur
`with tlte S phosrhorus conllgtu~ttion (35) with the lwo methyknc
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`1 H-44 Journul t~( ,o\ledidual Chemistry, 20n7, Vol_ 50. No. 8
`
`Perrone et of.
`
`Figu re 2. L('l\vcst energy contOrmaUons <)f comp<.)und" 34 and JS.
`
`protons becoming no nmugnctically equivalent (AB syste m). For
`the tliastercoisomcr w ith R phosphorus configuration (34), th is
`interaction doc<s not occur a nd the higher degree of ncxibility
`around the mcthyknc renders its protons more magn~tically
`similar (apparent do ublet). T he bio logica l activities or the
`scpurutcd diastcreoisorncrs (34 and 35) were comparable to each
`other and to the mixture (33) (Table 3 ).
`Interestingly. application of similar ProTide methods to the
`active 4'-azidocytidinc gave little or no boost in anli-HC'V
`activity (claw m>t shown). implying a rather cf11cie nt phospho(cid:173)
`rylation of this nucleoside a nalogue, with which ProTide
`methods presumably cannot compete.
`
`C onclusion
`A series of phosphoram idatc Pro Tides of 4'-azidouridine were
`prepared and evaluated as inhibitors or HCV replication in vitro.
`Th~ phosphoramidate ilpproach provided nove l compounds with
`high ly increased potency in the rep Iicon assay when compared
`to the inactive parent ~ompo u nd, com:sponcling to boosts in
`anti-I-ICV potency of > 450-fo ld. All phosphoramidmes tested
`were nontox ic in the rep Iicon assay (CC5o > lOO p M). The most
`active compo und p repa red in the series was the !-naphthyl
`L-alanine benzyl ester p hosphoramidate wi th an ECso or 0.22
`11M in the replicon assay. The cliastercoisomers of this corn(cid:173)
`pound were separated by HPLC and their absolu te phosphonts
`wnfigurations predicted by mode ling and NMR. However. they
`did not show any differe nces in biological activity. 1l1is report
`demonstrates the ability of' the ProTide approach to success!ttlly
`bypass the ra te limiting initial phosphorylation o r a ri bonucleo-.
`s ide analog ue and thu s confer signiti cant a ntiviral activity on
`an inactiw parent nuc leoside.
`·
`
`Expe rimenh1l Section
`Biology. HC'V rcplicon assay wo> rcrlonncd in the stable
`rcpl icon cell line 2209-23 derived from Huh-7 cells stubly trans(cid:173)
`li:ctcd with o bicistronic HCY rqllicon (genotype ! b) expressing
`the rcni lla lucifcrnsc reporter gene. as described' T he RNA
`synthesis activity of n.:combinant HCV pulymerasc proteins was
`measured as incotpormion oJ'mJ iolabclcd UMI' intu ucid-insolublc
`RNA products using HCV genome derived c!RES RNA os o
`template in a prinwr-indcpcndc nt RNA synthesis assay.' Rccom(cid:173)
`bin:mt proteins used were truncated at amino acid position 570 and
`deri ved from genotype tb strain BK (NS5B570n-BK) or Con i
`(NSS 8570-Conl ).
`Chemistr y. General Procedures. All experiments involving
`water-sensitive compounds were conducted tmdcr scr upulously dry
`conditions. Anhydrou ~ tctrahydrofuran and dichlororncthanc w.;rc
`purchased from Aldrich. Proton. carbon, and phosphortts Nuckar
`Magnetic Rcsomt11cc C'H. "C. 31 P NMR) spectra were recorded
`on a Brukcr Avance spectrometer operating at 500, 125, and 202
`Mrl z, rc spc~t ivcly . All "C and >tp spectra were rc~ordcd protun(cid:173)
`dccouplctl . All N1VIR spectra were 1·c~o rdcd in CD30D at room
`tcmpcratun; (20 "C ± 3 "C I. Che mica l shilts for 1H and 1•1C spectra
`arc quoted in parts per million duwnti.:td !'rum tctramcthybi lanc.
`Coupling t:onstants nrc rcfctTCU to as .I values. Signal splitting
`p;ntcrns '""' dc>.;ribcd d $ single t (s) , doubkt (d). rtipkt (t), quart,·t
`
`' .....__/
`
`(q), broad signal (br), doublet o f doublet (dd), doublet of tripl et
`(dt). or multiplet (m). Chemical shifts for 31 P spectra arc quoted in
`parts per mi ll ion relative 10 <Jn external phosphatic acid standard.
`Many proton and carbon NMR signals were spl it due to the presence
`of (phosphate) diastcrcoisomcrs in tltc samples. T he mode of
`ionization for mass spectroscopy was fast atom bombardment (FAB)
`using MNOBA as matrix. Column chromatography refers to flash
`column chrontotogrnphy carried out using Merck silica gcl60 (40-
`60 11M) as stationary phase.
`For convenience. standard procedures have been given, as similar
`procedures were employed for reactions concerning the synthesis
`of precursors and derivatives of ProTides. Variations from these
`procedures and individua l purification methods arc given in the
`main text. Preparative and spectroscopic data on individual precur(cid:173)
`sor, blue ked nuclcosidcs arc given as Suppot1ing fnformation only
`(s~e below), excluding only the tirst example.
`St3ndard Procedure l: Prcpar3tion ~,~r 2',3'-0,0-Cyclopen(cid:173)
`tyli!lcne-4'-azidouridine Phosp horamidates. 'BuMgCl (2.0 mol
`cquiv) and 2',3'-0,0-cyclopcntylidene-4'-azidouridinc (1.0 mol
`cquiv) were dissolved in dry THF (31 mot cqui v) a nd stirred for
`15 min. Then a I M solution o f the appropriate phosphorochloridatc
`(2.0 mol equiv) in dry THF was acld~d dropwisc and then stirred
`ovcmight. A saturdteJ solution of NH4CI was added, and the solvent
`wa~ removed under red need pressure to give a yellow solid, which
`was consequently purified by chromatography.
`Standard Procedure 2: Deprotection or 2' ,3'-Protected 4'(cid:173)
`Azidouridinc Phospho ramidates. The appropriate 2',3'-0 ,0-
`cyclopcntylidcnc-4'-azidoutidim: phosphoramidatc was added to a
`solution 80% of fom1ic acid in water. The reaction was stilTed at
`room temperature lor 4 h. 1l1e solvent w<Js removed under reduced
`pressure, and the obtained oil was purified by chromatography.
`Standard Procedure 3: Preparation of 4' -Azidouridine Phos(cid:173)
`phoramida tes vin Free Nucleoside. 'BuMgCI (2.0 mol cquiv) and
`4'-azidouridinc ( 1.0 mo l cquiv) were dissolved in dry THF (31 mol
`cquiv) and stirred to r IS min. Then a I M solution of the appropriate
`phosphorochloridatc (2.0 mol cquiv) in dry THF was added
`drop wise and then stirred ovcmight. A saturated sollltion of NH~­
`C I was added, and the solvent was removed under reduced pressure
`to give " yellow solid, which was purifi ed by chromatography.
`HPLC Ml•thod Used for the Separation or Compound 34 a nd
`35. Varian ProS tar instrument usinu: a Polaris C 18-A 1 Ott column;
`elution was pcrfom1cd using tl mobi le pha~c consisting of wntcr/
`acctonitrik 70% f-120 /30% CHJC'N, 17 min dution time with o flow
`o f 20 mlfmin. Optimal loading on column: 8 mg of phosphora(cid:173)
`m idate per run.
`Syn thesis of 2',3' -0,0-Cyclopcntylidcne-4'-azidouridine 5'-
`0-[Phcnyt(mclhylox)'· l.-alaninyi)J Phosphate (Methyl N-l{l(cid:173)
`{(3n R,4R.6R,6uS)-4-Azido-tctrahyclro-4-(hydroxymethyt)-2,2-
`cyclopcn tyl furu I J .4-d] 1 •~d io xo 1-6-yiJ p y rim id in e-2,4 ( 1H,3 H)(cid:173)
`di onc} (Phenoxy)-phosphoryiJ-L-alaninate). Prepared according
`to the standard proccdttt·e I, from 2',3'-0, 0-cyclop entyl id~nc-4'­
`aziclouridinc (150 mg. 0.427 mmol), 'Bul'v!gCI (0.85 mL, I M
`so lution in THF, 0.854 mmoi), and phcnyl(methyloxy-L-alaninyl)
`phosphoroc hloridatc (0.85 mL of solution I M in TH F', 0.854
`mtnol ). The crude product was puri lied by column chromatography,
`using o~ eluent CHCI;/McOH (95i5 J. The pure product was a white
`sol id ( 156 mg, 0.263 mmol, 6 1%). <lr (d.-CHi) H): 3. 14, 3.04; <\1
`(d4-CH 30H): 7.66 (IH, t, H6-uridinc), 7.35 (2 H, t. 2 CH-phcnyl),
`7.28-7. 19 (3H, m. 3 CH-phcnyl), 5.97 ( I H, dd, H 1'-uridine), 5.70
`( t H, dd, H5-urid ine). 5. 12-5.04 (2 H, m, H2' -uridine, H3'-uridine),
`4.31-4.27 (2 H. m, H5'-uridinc), 4.01 ( I H. m, CHu), 3.70 (3H, d,
`CH3-methyl), 2.2 1- 2.1 1 (2H, m, CH2-cyclopcntyl). 1.79- 1.73 (6H,
`m, 3 CH2-cyclopcntyl), 1.37 (3 H. t. CH3-alanine, J = 9. 5 Hz).
`Synthesis or 4'-Azidouridine 5' -0-{Phenyl(methyloxy-L-alani(cid:173)
`nyl)J Phosphate (Methyl N-1 {1-{2R,3S,4R,5R)-5-Aziclo-tetrahy(cid:173)
`d.-o-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)pyrimidine-2,4-
`(1H,3fl)-d ione} (Phenoxy)-phosphoryi{-L-alaninatc) (11). Prepared
`accordi ng to the standnrd procedure 2, from 2~ ,3'- 0, 0-cyclopcn­
`tyl idene-4' -azid,,twicti nc 5' -0-[phcnyl(mcthyloxy-L-alaninyl)] pho5-
`phate ( 135 mg, 0.222 mmol), and a sol ution 80% o f HCOOH in
`water (t O mLl. The crttdc was purified by colunm chromatography,
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`Phosplzoramidate PmTide Appmuch 10 4'-;l=id<lill'idine
`
`Joumul ~l Akdidnal Chemi.l'lf)'. 2UU7. Vol. 50. No. 8 18~ 5
`
`us ing as eluent C'HCI;/McOH (8!2). The obtained pur~ fll't'dnct wa s
`a white solid (65 mg, 0. 116 mmol. 54%). <)r (d~-C HtO H) : ~ .50,
`3.3 .1; ou (d,-C'H,OH): 7 .65 (lH, dd. H6-Uiidincl. 7.Jg (2H. m , 2
`C H-phenyl), 7.28-7.20 OH. nt, 3 CH -phcnyl). 6.15 (I H, m. HI '(cid:173)
`Ulidinc), 5.72 ( l H, m, H5-uridinc), 4.42 -4.3n (2 H. m. H2' -uridinc,
`H3'-uridinc), 4 .26-4. 18 (2H, m, 1-15'-uriclinc), 4.00 ( I H, q, C Hll ),
`3.70 (JH, d , CH,-m cthyl), 1.35 (3 H, dd, CH.;-:IIHninc ). M S (FJ !):
`549.1124 (MNa''J; C 1oH"NoOwNaP requires 549. 11 11 Anal.
`(Ct9H2JN60toP) C, H. N.
`Synthesis of 2',3'-0,0-Cyclopcntylidcnc-4'-nzidouridinc 5' -
`(Ethyl N-1 ( l(cid:173)
`0-[Phenyl(ethyloxy-L.-alsninyi)J Phosphate
`[(3nR,4R,6R,6aS)-4- Azido-tetrnhydro-4-(hydroxymethyl)-2.2-cy(cid:173)
`clopcn tylru ro [ 3,4-d l '"'d io xo 1-6-yl J pyri mid i n e-2,4( I f/,31-J)-di(cid:173)
`one} (Pbenoxy)-phosphoryi}-L·alaninntc). Sec Supporting lnl'or(cid:173)
`mation for pre parative and spectroscopic data.
`Synthesis of 4'-Azidouridine 5'-0-]Pitc nyl(rthylox~·-l.-al au i­
`nyi)JPilosphatc (Ethyl N-1 ( i-(2il,JS,'IR,5R)-5-Az ido'tctrohyriro-
`3,4-dihydroxy-S-(IIydroxymcth yl) fu ro n-2-yl) py ri midi ne-2,4-
`(IH,3H)-dione} (Phenoxy)-pbospltoryii·L-alaninatc) (12). Pre(cid:173)
`pared according to thQ standard pro~cdurc 2, (i·om 2',3'-0, 0 -
`QyQ!opcntylidcnc-4' -azidouridi11e 5' -0 -[j>hcny I( Qthy loxy-L-ahulinyl ) 1-
`phosphate ( 135 mg, 0.222 mmol), Jnd