Biochimica et Biophysica Acta 1537 (2002; 258 275
`
`Review
`
`Bl(')T‘HlMlCJ\ ET R}(1PH"i'.'\‘t(_A ACTA
`
`
`
`www.bba-direct.con.1
`
`New developments in anti—HIV chemotherapy
`
`Regrr In.sr1'mtefnr 31-i'(.’dr'r.'cz.' Rmtaarrr/1,
`
`Erik De Clercq*
`I\;'crtr‘1r'Jr'i(a/re Ur1iver.s'r'tr».'.r'r .I.(:‘ll1-'(‘P1. Mincirerhr'rJecl'er:s.m'.t1uI‘ IU, B—3l’}Ut'} 1'.reL4ven, Belgiitrpl
`
`Received 24 January 2002; accepted 24 January 2002
`
`
`
`ELSEVIER
`
`Abstract
`
`the treatment of human
`for
`trials,
`the compounds that are currently used, or are subject of advanced clinical
`Virtually all
`immunodeficiency virus (HIV) infections. belong to one of the following classes: (i) nueleoside/nucleotide reverse transcriptase inhibitors
`(NRTIs): i.e. zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), stavudine (d4T). lamivudine (3TC). abacavir (ABC), emtricitahine
`[( — )FTCj,
`tenofovir disoproxil
`fumarate;
`(ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs):
`i.e. nevirapine. delavirdine,
`efavircnz. ernivirine; and (iii) protease inhibitors (Pls): i.e. saquinavir, ritonavir, indinavir. nelfinavir. amprenavir and lopinavir. In addition to
`the reverse transcriptase (RT) and protease reaction, various other events in the HIV rcplicative cycle can be considered as potential targets for
`chemotherapeutic intervention: (i) viral adsorption, through binding to the viral envelope glycoprotein gp120 (polysulfates. polysulfonates,
`polycarboxylates, polyoxometalates, polynueleotides. and negatively charged albumins); (ii) viral entry,
`through blockade of the viral
`coreceptors CXCR4 [bicyclarn (AM1')3l()t)) derivatives] and CCR5 (TAK-779 derivatives); (iii) virus—cell fusion. through binding to the
`viral envelope glycoprotein gp4l
`(T-20. T-1249); (iv) viral assembly and disassembly.
`through NCp7 zinc finger-targeted agents [2.2’ -
`dithiobisbenzamides ('DIBAs). azadicarbonaniidc (ADA')];
`(V) proviral DNA integration. through integrase inhibitors such as 4-aryl-2.4-
`dioxobutanoic acid derivatives; (vi) viral mRNA transcription, through inhibitors of the transcription (transactivation) process (flavopiridol,
`fluoroquinolones). Also. various new NRTIs. NNRTIs and Pls have been developed that possess. respectively: (i) improved metabolic
`characteristics (ie. phosphorarnidate and r,j:c:r'nsaligenyl pronucleotides by-passing the first p}4ospho1"ylation step of the NRTIs), (ii) increased
`activity [“second“ or “third" generation NNRTls (i.e. TMC-I25, DPC-083)] against those lIlV strains that are resistant to the “first"
`generation NNRTls. or (iii) as in the case of Pls. a different, nonpeptidic scaffold [i.e. cyclic urea (mozcnavir). 4-hydroxy-2-pyrone
`(tipranavir}]. Nonpeptidic Pls may be expected to i.nhibit HIV mutant strains that have become resistant to pcptidomimetic Pls. Given the
`multitude of molecular targets with which anti-HIV agents can interact, one should be cautious in extrapolating the mode of action of these
`agents from cell-free enz_vn1atic assays to intact cells. Two examples in point are L-chicoric acid and the nonapeptoid CGP64222. which were
`initially described as an integrase inhibitor or Tat antagonist, respectively, but later shown to primarily act as virus adsorption/entry inl1ibitors.
`the latter through blockade of CXCR4. © 2002 Elsevier Science B.V. All rights reserved.
`
`Ke_vwonIs.' Human irnnrunodeficiency virus (HIV); Reverse transcriptase {HIV}: Protease (HIV); CXCR4 (I-IIV}: (‘CR5 (HIV); Integrase (HIV); Fusion (I-IIVI;
`Transcription (HIV)
`
`Abbre\'t'utz'or1.s'.' HIV, human innnunodeiiciency virus; _\'RTIs. nucleoside/nucleotide reverse transcriptase inhibitors; NNRTIS. non-nucleoside reverse
`transcriptase inhibitors; Pls. protease inhibitors; DIBA, 2.2’-ditlriobisbeiwainide; ADA, azadicarbonannde; AIDS, acquired innnune deficiency syndrome; I-ISV.
`herpes simplex virus; STD. sexually transmitted disease; MII‘-lot and -1|}. macrophage inflannnatory proteins; SDF-l. stronial-cell derived factor; PBMCS.
`peripheral blood mononuelear cells; TM4. transrnenibrane segment; SI. syncytiunzt-inducing; NSI, non-syncytiI.uJ:i—inducing'. NOBA, 3-nitrosobenzainide; AZT.
`zidovudinc; ddl, didanosinc: drl(', zalcitabineg d4T,
`stavudinc; 3T(f,
`Iamivudinc; ABC. ahacavir: bis(P()M}-P_\r1l*I.A. his(pivaloyloxymcthyl)-9—(2-
`phosphonylinetlroxyethyhadenine, adefovir dipivoxyl; bis(POC')-PMPA, bis(isopropyloxycarbonyloxyinethylHR]-9-(2-phosphonylmethoxypiopylladenine.
`te11ofovi1' disoproxil; dOTC, ii I2’-deoxy-3’-oxa-4-thiocytidine;
`(
`lFTC‘, enitricitabine; DAPD. Luntloxovir,
`( VH3»-D-2.6-diauninopurine dioxolane:
`bis(S/\TE)ddAMP, bis(S—acetyl-2—tl1ioethyllphosphotriester of ddA
`Proceedings of the 8th l_nterr1ational Syinpositnn on Molecular Aspects of Chemotherapy. Gdansk. Poland. 5 9 September 2001.
`l Tel; -32—16—33'/341; fax: +32-l6—33734U.
`Email address.‘ erilt.declercq@rega.l€.uleuven.ac.be (E. De Clercq).
`
`0925-4439/02xs — see front matter cc: 2002 Etsevier Science av. All rights reserved.
`*
`Pl]: S(]925—4439(U2 }UU(l89—3
`
`Case No. 2: 1 0-ev-D5954
`
`J°"5*?°” F_''°‘.‘”_°*‘~‘i ‘-9 °‘“'~
`V. Lupin Limited, at al.
`
`PTX79
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`E. De Ck-.'J'r.'q /Bior'h1'im'('u er Bfr1pI1}'sir:a Acta 1587 (20U_’) 258-2 75
`
`259
`
`1. Introduction
`
`three anti-
`least
`Combination therapy, comprising at
`human immunodeficiency virus (HIV) drugs, has become
`the standard treatment of acquired immune deficiency
`syndrome (AIDS) or HIV-infected patients. Virtually all
`drugs that have been licensed for clinical use (or made
`available through expanded access programmes) for the
`treatment of HIV infections fall into one of the following
`three categories: (i) nucleoside/nucleotide reverse transcrip-
`lase inhibitors (NRTIS), that, following two phosphorylation
`steps (tenofovir) or three phosphorylation steps [zidovudine
`("AZ-T). didanosine (ddl), zalcitabine (ddC), stavudine (d4T),
`lamivudine (3TC). abacavir (ABC)]. act, as chain termina-
`tors, at the substrate binding site ofthe reverse transciiptase
`(RT);
`(ii) non-nucleoside reverse transcriptase inhibitors
`(NNRTIS) that interact with the RT at an allosteric. non-
`substrate binding site (nevirapine, delavirdine, efavirenz);
`and (iii) protease inhibitois (PIS) that specifically inhibit, as
`peptidomimetics. the vi1'us—associated protease (saquinaviii,
`ritonavir,
`indinavir, nelfinavir, amprenavir.
`lopinavir).
`Guidelines to the major clinical trials with these compounds
`have been recently published [1].
`Although the long—tenn goal of eradicating the virus from
`latently and chronically infected cells remains forbidding [2],
`the advent of so many new compounds other than those that
`
`have been formally approved. for the treatment of HIV
`infections. will undoubtedly improve the prognosis of
`patients with AIDS and AIDS-associated diseases. Here. I
`will primaiily address those new anti—HlV compounds that (i)
`have emerged as promising anti-HIV drug candidates during
`the last few years, that (ii) are in preclinical or early—clinical
`development. and that (iii) are targeted at well-defined steps
`in the HIV replicative cycle.
`
`2. Virus adsorption (gpl2t)) inhibitors
`
`A great variety of polyanionic compounds have been
`described to block HIV replication through interference with
`virus adsorption (or binding) to the cell surface: i.e. poly-
`sulfates, polysulfonates. polycarboxylates, polyphosphates.
`polyphosphonates, polyoxoinetalates, etc. This class of
`compounds also comprises the cosalane analogues ('1) con-
`taining the polycarboxylate pharmacophore [3]. as well as
`the sulfated polysaccharides extracted from sea algae [4].
`All these compounds, whether synthetic or of natural origin,
`are assumed to exert their anti-HIV activity by shielding off
`the positively charged sites in the V3 loop of the viral
`envelope glycoprotein (gp]20) [5,6]. which is necessary for
`virus attachment
`to the cell surface heparan sulfate, a
`primary binding site. before a more specific binding occurs
`
`
`
`ll
`
`Compound 1
`Cosataue alzatogue
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`260
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`E. De Cleruq / Bfr)cii1'm1'c‘n at Biczpirv.sir.'a Acfu 1.1787 (2002) 258-2 7:?
`
`to the CD4 receptor of the CD4' cells. and to the CXCR4
`coreceptor of the CXCR-4+ cells (the latter in the case of X4
`and dual
`tropic X4/‘R5 HIV strains). Heparan sulfate is
`widely expressed on animal cells and, as it is involved in
`the virus-cell binding of a broad spectrum of enveloped
`viruses, including herpes simplex virus (HSV) [7], dengue
`virus [8] and other flaviviruses (i.e. Japanese encephalitis
`virus) [9]. it also explains why polysulfates have a broad-
`spectrum antiviral activity against HIV, HSV and various
`other enveloped viruses [10].
`
`ligand. namely SDF-1 (“strom-a|—cell derived factor”) has
`been identified. Of these chemokines, the LD78t3 isoform of
`MIP-10: has emerged as the most potent chemokine for
`inhibiting HIV-1 infection in peripheral blood mononuclear
`cells (PBMCS) [14,15] as well as monocytes/‘macrophages
`[16].
`TAK-779. a quaternary ammonium derivative (2) is the
`first nonpeptidic molecule that has been described to block
`the replication of M—tropic R5 HlV—l strains at the CCR5
`level [17].
`
`
`
`Compound 2
`TAK-779
`
`The major role of polysulfates or polyanionic substances
`in general in the management of HIV infections may reside
`in the prevention of sexual transmission of HIV infection,
`these compounds. if applied as a vaginal formulation. may
`successfully block HIV infection through both virus—to-cell
`and cell-to—cell contact. These compounds therefore merit
`being pursued as vaginal microbicides. The fact
`that
`in
`addition to their anti-HIV activity. these polyanjonic sub-
`stances, as demonstrated, for example, for poly(sodium(4-
`styrene)sulfonate). also inhibit other sexually 11'flI‘lSlTllI‘lTBCl
`disease (STD) pathogens,
`i.e. HSV, Neisseria grmorrheae
`and C'l2lmnydr'a tmclzornatis [l I],
`further adds to their
`potential therapeutic. and preventive value.
`
`3. Viral coreccptor antagonists
`
`To enter cells. following binding with the CD4 receptor.
`the HIV-1 particles must interact, again through the viral
`envelope glycoprotein gp120_. with the CXCR4 coreceptor
`[12] or CCR5 coreceptor [13]. CXCR4 is the coreceptor for
`HIV-1 strains that infect T—cells (T—tropic or X4 strains), and
`CCR5 is the coreceptor for HIV-1 strains that infect macro-
`phages (M—tropic or R5 str'ains). CXCR4 and CCR5 have
`not evolved simply to act as coreceptors for HIV entry; they
`normally act
`receptors for chemokines (chemoattractant
`cytokines). The normal
`ligands for CCR5 are RANTES
`(“regulated upon activation, nonnal T-cell expressed and
`secreted”) and MIP—1cx and -16 (“macrophage inflamma-
`tory proteins”), whereas for CXCR4, only one natural
`
`A binding site for TAK-779 has been identified within
`the transmernbrane helices I, 2, 3 and 7 of CCR5 [18].
`TAK-779 has been found to inhibit R5 HIV-1 strains in the
`
`nanomolar concentration range, while not affecting X4
`HIV-1 strains at
`l0,000—tbld higher concentrations [17].
`TAK-7'79 is not a “pure” CCR5 antagonist. as it also
`demonstrates some antagonism towards CCR2b. Unlike
`RANTES, TAK-779 does not
`induce internalization of
`
`CCR5. The clinical potential of TAK-779 and its conge-
`ners [19]
`in the therapy and/or prophylaxis of HIV-1
`infections remains to be further explored. Meanwhile.
`several new CCR5 antagonists have been reported
`[2D.21]_. and a lead clinical candidate (SCH C) for further
`development has been identified.
`i.e.
`three compounds.
`Almost simultaneously [22—24]_.
`the bicyclarn AMD310O [22], [Tyr-5,12.Lys-7]polyphemu-
`sin or T22 [23] and the nonapeptide (D—A1'g)y 01' ALX40—
`4C [24] were announced as CXCR4 antagonists. blocking
`the replication ot"T-tropic X4. but not M-tropic R5, HIV-1
`strains through selective antagonism of CXCR4. The
`bicyclams are the most specific and most potent CXCR4
`antagonists that have been described to date [25,26]. The
`bicyclams had been known as potent and selective HIV
`inhibitors for a number of years
`[27.28]. before their
`target of action was identified as the CXCR4 coreceptor
`[22.29,30]. The bicyclam AMD3l00 (3)
`inhibits the
`replication of X4 HIV-1 strains within the nanomolar
`concentration range [28]. As it
`is not
`toxic to the host
`cells at concentrations up to 500 uM, its selectivity index.
`or ratio of 50% cytotoxic concentration (CC5.3)
`to 50%
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`E. De Ck-.'J'r.'q / 13‘iot'ltt'rm'('u er Bfr1pI1}'s.I'r:a Auto 1587 (20t3_’) 258-2 75
`
`261
`
`antivirally effective concentration (EC_r,c.) can be estimated
`at > 100.000.
`
`.T1“©”Vf“.3.
`l:i\li{ HN:l
`l:1‘\iil
`H1'*«l:l
`l\,/l
`K)
`
`Compound 3
`AMD31fl{i
`
`A close correlation has been found, over a concentration
`range of 01-1000 ng/ml, between the AMD3l00 con-
`centrations required to inhibit (i) HIV-1 NL4-3 replication,
`(ii) monoclonal antibody (mAb 12G5) binding to the
`CXCR4 coreceptor, and (iii) SDF-1-induced signal trans-
`duction (Cazlfltix). suggesting an intimate relationship
`between these three parameters [29.30]_ The inhibitory
`effects of AMD3100 on the T—tropic HIV-1 NL4—3 strain
`have been demonstrated in a wide variety ofcells expressing
`CXCR4,
`including PBMCS; and, vice versa. various T-
`tropic and dual-tropic, but not M-tropic, HIV-1 strains have
`proven sensitive to AMD3100 in PBMC.
`Negatively charged amino acid (i.e. aspartic acid) resi-
`dues in the extracellular regions of CXCR4 must be
`involved in its interaction with both AMD3100 and SDF-
`
`I, and the V3 loop of X4 HIV gp12t), which are all three
`highly basic. Substitutions of a neutral amino acid residue
`for aspaitic acid in the second extracellular loop generated
`resistance to AMD31Cl0 [31].
`in particular,
`the aspartate
`residues at positions 171 and 262,
`located close to the
`extracellular sides of the transmembrane segments TM4
`
`and TM6. may represent crucial sites of interaction with
`the bicyclam AMD3100 [32].
`When the bicyclam AMD3100 was added to PBMC
`infected with clinical HIV isolates displaying the syncy-
`tium-inducing (SI) phenotype, these strains reverted to the
`non—syncytium-inducing (N81) phenotype, and, concomi-
`tantly, these strains switched from CXCR4 to CCR5 cor-
`eceptor use [33]. These findings indicate that selective
`blockade of CXCR4 by AMD3100 may prevent the switch
`from the less pathogenic M-tropic R5 to the more patho-
`genic T—tropic X4 strains of HIV, which in vivo heralds the
`progression to AIDS. AMD310O has proved efficacious,
`alone and in combination with other anti—HlV drugs,
`in
`achieving a marked reduction in viral load in the SCID-hu
`Thy/Liv mouse model [34]. Following a phase I clinical trial
`for safety in normal healthy volunteers [35], AMDBIOO
`recently entered phase II clinical
`trials in HIV-infected
`individuals.
`
`Given their high potency and selectivity as CXCR4
`antagonists, bicyclams, such as AMD3100. may not only
`have great potential for the therapy and/or prophylaxis of
`X4 HIV infections. but also other pathologic processes,
`such as breast cancer metastasis, which are at least partially
`dependent of, or mediated by, signaling through CXCR4
`[36].
`
`4. Viral fusion (gp41) inhibitors
`
`The interaction of the X4 or R5 HIV-1 envelope gly-
`coprotein gp120 with the coreceptor CXCR4 or CCR5,
`respectively.
`is followed by a spring—loaded action of the
`viral glycoprotein gp4l
`[normally covered by the bulkier
`gpl20), which then anchors through its amino terminus
`(the “fusion peptides”) into the target cell membrane. This
`initiates the fusion of the two lipid bilayets,
`that of the
`
`Feséttt)
`
`i_.ettr:.i.oe Zipper
`‘"3-;35l€7’1l
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`:\»le2rti}r'at3e
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`...,.,'»'~'*§<.;:<.)«:;::t—t
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`‘Flt?
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`lf}}‘~ l. (T?
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`.....—r-*“’"M
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`_____,,...,..r---
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`~'_....,,,_
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`“-«--...,__
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`- -M.
`____“_--......__
`[Est.z;as.i...ter:soait;iQa.ss:Naoatmaz.at<was.z..vas'a*it
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`{.1oraptmnti 4
`'1’-2% tgtsrariizifissittii}
`
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`

`
`262
`
`E. De C(E?I‘L‘£f / Bfr)cii1'irtica er Biczpizy.vit.'a Acfu 1.1787 (2002) 258-2 7:?
`
`viral envelope with that of the cellular plasma membrane
`[37]. At
`the onset of the fusion process,
`the hydropho-
`bic grooves on the surface of the N36 coiled coil in the
`gp41 ectodotnain become available for binding with ex-
`traneous inhibitors, such as DP-178 (T—20), a 36-residue
`peptide.
`that binds to the hydrophobic groove of N36
`[37].
`is a synthetic, 36—amino acid
`(4)
`T—20 (pentafuside)
`peptide corresponding to residues 127-162 of the ectodo—
`main of gp4l
`(or residues 643—678 in the gpl60 pre-
`cursor).
`T—20_. previously called DP-178. was modeled
`after a specific domain (within gp41) predictive of ot-
`helical secondary structure: DP—178 consistently afforded
`100% blockade of vims-mediated cell-cell fusion (syncy—
`tium formation) at concentrations ranging from 1 to 10 ng/
`ml,
`i.e. 104- to 105—fold lower than the cytotoxic concen-
`tration [38]. An initial clinical trial has been carried out
`with T—20 at four doses (3. 10. 30 and 100 mg twice daily,
`intravenously. for 14 days) in 16 HIV-infected adults; at
`the highest dose (100 mg, twice daily). T-20 achieved by
`the 15th day a 1.5- to 2.0-fold reduction in plasma HIV
`RNA [39]. These data provide proof-of-concept that HIV
`fusion inhibitors are able to reduce virus replication in
`VIVO.
`
`is the
`The betulinic acid derivative RPR 103611 (5)
`only nonpeptidic low-molecular-weight compound that has
`been reported to block HIV-1 infection through interaction
`with gp4l:
`this liiterpene derivative has been found to
`inhibit the infectivity of a number of HIV-1 strains in the
`10 nM concentration range [41], apparently through inter-
`ference with a post—binding. envelope—dependent step
`involved in the fusion of the virus with the cell plasma
`membrane.
`The exact mode of action of RPR 103611 remains to be
`
`elucidated. Sequence analysis of RPR103611—resistant inu-
`tants indicated that a single amino acid change. 184$.
`in
`HIV-1 gp41 is sufficient
`to confer drug resistance [42].
`However, this I848 mutation did not occur in sotne of the
`
`naturally RPRl036l l -resistant HIV-1 strains such as NDK.
`More recently, the action ofRPR103611 has been thought to
`depend on the accessibility of gp4l
`[43]. and for the
`isomeric betulinic acid derivative IC 9564. HIV-1 gp120_.
`rather than gp41, has been proposed as the prime target
`(based on the mutations G23"/‘R and 1{252K emerging in
`gp120 of drug-resistant mutants) [44]. YK-FH312. a betu-
`linic acid derivative unrelated to RPI-(103611 or IC 9564.
`
`was reported to block the assembly andtor budding of HIV
`particles [45].
`
`
`
`Compound 5
`RPR [0361]
`
`
`
`Meanwhile, T-20 has proceeded to phase 1]:"lII clinical
`trials. and phase 1 clinical trials have been initiated with T-
`1249, a 39-amino acid peptide derived fi‘om DP-107 (which
`is a 38—amino acid peptide corresponding to residues 558-
`595 of gp160); T—1249 would be 10-fold more potent than
`T-20 when evaluated in vitro against HIV under the same
`conditions [40].
`
`5. Nuclcocapsid protein (NCp7) Zn finger-targeted
`agents
`
`The two zinc fingers [Cys-X3-Cys-X4-His-X4-Cys
`(CCHC). whereby X=any amino acid] in the nucleocapsid
`(NCp7) protein [46] comprise the proposed molecular target
`for zinc—ejecting compounds such as 3-nitrosobenzamide
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`E. De Ck-.'J't.'q /13‘ior'l'1irm't'u er Bfr1;>I1}'s1'(:a Auto 1587 (2()tl_’) 258-2 75
`
`263
`
`already equipped with a phosphonate group, and therefore
`only need two phosphorylation steps to be converted to the
`active metabolite [58]. From PMEA and PMPA.
`the oral
`prodrug forms [bis(pivaloyloxymethyl)—9-(2-phosphonyIme-
`thoxyethynadenine (bis(POM)-PMEA) or adefovir dipi—
`voxyl (7). and bistisopropyloxycarbonyloxymethyl)—(R)—9—
`(2-phosphonylmethoxypropyl)adenine (bis(POC)-PMPA) or
`tenofovir disoproxil (8) funiarate. respectively] have been
`prepared. The former is in advanced phase Lll clinical trials
`for the treatment of hepatitis B virus (HBV) infections.
`whereas the latter has completed phase 111 clinical trials for
`the treatment of HIV infections. A new drug application
`(NDA) and market authorization application (MAA) has
`been recently filed for tenofovir disoproxil fumarate with
`the FDA (US) and EMEA (EU). respectively.
`In rhesus
`macaques infected with the highly pathogenic chimeric virus
`SHIV. tenofovir treatment initiated 1 week post-infection. at
`a time when disseminated infection and extensive viral
`
`replication had already been established and CD4 ‘ T-cell
`loss had begun. led to prompt. virtually complete suppres-
`sion of viral replication and long—term stabilization of CD4 ‘
`T-cell levels. which were sustained. ever: after withdrawal of
`
`tenofovir (after 12 weeks of treatment) [59].
`
`i“H—i
`NN
`is >i
`
`i
`K}.
`
`N
`
`W
`
`o
`‘
`P/'
`(c:rr;.)3c—c—o—Crr;—o” “V” \-
`I
`
`I0
`
`Compound 7
`bis(POM)-PMEA
`Adefovir dipivoxyl
`
`ii
`(ca )3CH'-——-0-—~i:~——-t)-—-- CH -—~——o
`3
`2
`
`yo
`:~.: Pg0
`
`CH:
`
`lO
`
`Compound 8
`bis{POC)-PMPA
`'i'enofovi:' disoproxil
`
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`
`(NOBA). 2,2’—dithiobisben7.amide (DIBA). SRR—SB3
`(cyclic DIBA)
`[47]. 1.2—ditl1iane-4,5-diol.l.1-dioxide (di-
`thiane) [48] and azadicarbonamide (ADA) [49,50]. These
`compounds should be able to interfere with both early
`(uncoating. disassernbly) and late phases (packaging, assem-
`bly) of retrovirus replication. Their effect at the early phase
`(disassembly) may also be ascribed to cross-linkage among
`adjacent zinc fingers. The DIBAS are able to enter intact
`virions and the c1'oss—linkage of NCp7 in virions correlates
`with loss of intectivity and decreased proviral DNA syn-
`thesis during acute infection [51]. Electron microscopically,
`the effect bestowed by DIBAs on virus morphology ‘tould
`be described as “core—freezing" [52].
`
`HN
`
`“it
`
`0
`
`ENH
`
`2
`
`)1”
`
`0
`
`Compotmdti
`ADA (azesiicarbonaniide)
`
`Although NOBA, DIBA. dithiane and ADA have been
`shown to dock nicely on the NC137 Zn finger domains [53]
`and are believed to selectively target
`these Zn fingers
`without affecting the cellular Zn finger proteins,
`their
`selectivity indexes [ratio of CC;;{. (50% cytotoxic concen-
`tration) over EC“. (50% effective concentration)] are not
`that
`impressive [53]. Of the NCp7-targeted compounds,
`ADA (6) has been the first to proceed to phase I/II clinical
`trials in advanced AIDS patients. Some preliminary evi-
`dence of efficacy was witnessed with add-on ADA in
`patients failing current antiretroviral
`therapy [54];
`these
`studies should be further extended. Although ADA is an
`HIV NCp7 Zn-finger inhibitor, its action in vivo is likely to
`be multipronged. ADA may well interact with a variety of
`targets and. certainly.
`its inhibitory effects on T-cell
`responses in vitro and in vivo [55] can hardly be attributed
`to a.n action targeted at the HIV NCp7 Zn fingers.
`
`6. RT inhibitors targeted at the substrate binding site
`
`The substrate (dNTP) binding site of the HIV-1 RT is the
`target for a large variety of NRTI analogues, which have for
`several years [56] been recognized as efficacious drugs for
`the treatment of HIV infections: i.e. AZT. ddl. ddC, d4T,
`3TC, ABC. and the yet experimental drug emtricitabine
`[(—)FTC]. Fozivudine tidoxil
`is a thioether lipid AZT
`conjugate that has recently passed phase 11 clinical trials
`[57] and should be as effective as. and potentially better
`tolerated than, AZT. As a rule, all these compounds must be
`phosphorylated to their 5’-triphosphate form, before they can
`act as competitive inhibitors/substrate analogues/chain ter-
`minators at the RT level. In contrast to the nucleoside
`
`analogues. the nucleotide analogues PMEA and PMPA are
`
`

`
`264
`
`E. De C[E?I‘L‘£f / Bfr)cii1'1rtica er Biczpizy.vir.'a Acfu I.1787 (2002) 258-2 7:?
`
`NH2
`
`N
`
`<’
`N
`
`r
`
`I
`
`\
`
`N
`,-/A
`N
`
`NH;
`
`Compound 10
`Amtloxovir
`
`IDAPD, (—}-[5-D-2.6-diantinopu tine dioxolane]
`
`In addition to 3TC and (- )FTC, the stntcturally related
`(i )2’-deoxy-3'—oxa-4'—lhiocylidine (BCI-I-10652.dOTC)
`[60], the dioxolane purine nucleoside analogues [61], the
`methylenecyclopropane nucleoside analogues (and their
`phospl:ioro—L-alaninate diesters) [62.63] and the 4’—ethynyl
`nucleoside analogues [64] have recently been described as
`new anti-I-[IV agents. [( — )FTC] (9) is in phase III trials for
`HIV and phase I/II trials for HBV; it is considered for use
`in the multidrug combination therapy of HIV-1 and HBV
`infections. Amdoxovir [DAPD. (-)-[3-n-2,6-diarninopur-
`ine dioxolane]
`(10), which is converted by adenosine
`deaminase to dioxolane guanine (DXG), has proven active
`against AZ—T- and 3TC—resistant HIV-1 strains and has
`proceeded to phase I/II clinical studies [65.66]. BCH-
`10652 (dOTC)
`(11) has demonstrated activity against
`HIV-1 in the SCID-hu ThyfLiv model. Despite its struc-
`tural similaiity to 3TC. dOTC proved also active against
`3TC—resistant HIV-1 (M184V)_. albeit at a relatively high
`dosage level (400 mg/kg/day) [67]. Also in vitro, dOTC and
`its (+) and (—) enanliomers still retained, albeit reduced.
`activity against 3TC-resistant M]S4V and M1841 HIV-I
`mutants [68].
`
`H0
`
`0
`
`Compound 11
`(:)2*—deoxy—3’—oxa-4’—thiocytidine (dOTC)
`
`Compound 9
`Emtricitabine [(—)F'I‘Cj
`
`The bottleneck in the metabolic pathway leading from
`AZT and the other 2’,3’-dideoxynucleoside (ddN) ana-
`logues to their active 5’-triphosphate form is
`the ['n'st
`phosphorylation step. Therefore. attempts have been made
`at constructing 2’,3"—dideoxynuc1eotide (‘ddNMP) prodrugs
`that, once they have been taken up by the cells, deliver
`the nucleotide (ddNMP) form. This approach has proven
`particularly successful
`for a number of NRTIS such as
`2'3’-dideoxyadenosine (ddA) and d4T. Thus.
`the bis(S-
`acetyI-2-thioethyl)phosphotriester of ddA [bis('SATI:‘]d—
`dAMP] (12) was synthesized and found to be 1000-fold
`more potent against HIV than the parent compound ddA
`[69]. Similarly, aryloxyphosphoramidate derivatives of
`d4T [i.e. S0324, a d4T-MP prodrug containing at
`the
`
`phosphate moiety a phenyl group and the methylesler of
`alanine linked to the phosphate group through a phos-
`phoramidate linkage] have been constructed [70—72].
`After the d4T aryloxyphosphoramidate (13) has been
`taken up by the cells. d4TMP is released intlacellularly
`and then processed onto its active metabolite d4TTP
`[73]. This “thymidine kinase bypass” explains the high
`anti-HIV activity of d4T aryloxyphosphoramidate deriva-
`tives in thymidine kinase deficient cells and resting mono-
`cytes/macrophages [74]. The thymidine kinase (in the
`case of d4T) and the adenosine deaminase (in the case
`of ddA) can also be bypassed by using the cyclic sali-
`genyl approach [75,76]. Cy:-losaljgenyl pronucleotides of
`d4T and ddA deliver exclusively the nucleotides d4TMP
`and ddAMP_. not only under chemical—simulated hydrol-
`ysis conditions but also under
`intracellular conditions
`[77.78]. This has been convincingly shown for the q,vclo—
`saligenyl derivative of d4TMP (14) in a number of cell
`lines [79].
`
`Janssen Ex. 2018
`
`Lupin Ltd. v. Janssen Sciences Ireland UC
`IPRZO15-01030
`
`(Page 7 of 18)
`
`

`
`E. De Clemq /Biot'him1'('u er Bfr1;>I1}'s1'(:a Acta 1587 (20f3_’) ."55’—2 75
`
`265
`
`N
`\
`
`‘>
`
`N
`
`NH;
`
`N/
`
`isN
`
`I
`
`\n-5-:1
`
`0 I
`
`E
`CEi3--—-'C~-"-S"-~-CH2“--Cllzmm-OK /,0
`/ ‘P \
`\ O
`
`cH;,—~— C-~-- s---~ct-12-~-—-Cit,-mi
`I
`
`E0
`
`Conspou ml 1.2
`bis(S—acetyl—2—thioethyhpliosphotriester of ddA [bis(SATE}ddAMP}
`
`[“=:H;‘:v::
`
`H3C0-fi-~ cam as
`0 CH3
`
`x’’0
`
`__...........l
`
`Compound 13
`(HT aryluxyphosphoramidate
`
`O\P’/Ox‘
`\0/ \0 \i/“I0
`
`\ ____
`
`Compound 14
`cyclosaligenyi ai4TMP
`
`7. RT inhibitors targeted at the allusteric, nonsuhstrate
`binding site
`
`More than 30 structurally different classes of com-
`pounds have been identified as NNRTIS, viz. compounds
`that are specifically inhibitory to HIV-1 replication and
`targeted at a nonsubstrate binding site of the RT [80].
`Three NNRTls (nevirapine, delavirdine and efavirenz)
`have so far been formally iicensed fo1' clinical use in the
`
`treatment of HIV-1 infections. ernivirine (MKC-442) (I5)
`is in advanced (phase 111) clinical trials. and others are in
`preclinical or early clinical development. The NNRTIS
`interact with a specific “pocket” site of the HIV-1 RT
`[81], which is closely associated with, but distinct
`fi‘om.
`the substrate binding site. NNRTIS are notorious for
`rapidly eliciting resistance [82], resulting from mutations
`at the amino acid residues that surround the NNRTl-bind-
`
`ing site of HIV-1 RT. However, emergence of NNRTl—
`
`Janssen Ex. 2018
`
`Lupin Ltd. v. Janssen Sciences Ireland UC
`|PR2015-01030
`
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`
`

`
`266
`
`E. De ClE?I‘L‘£f / Bfr)cli1'm1'cr1 at Biczpizy.vit.'a Acfu I.1787 (2002) 258-2 7:?
`
`resistant HTV strains can be prevented it‘ the NNRTIS are
`combined with NRTls and used from the beginning at
`sufficiently high concentrations [80].
`The thiocarboxanilide UC-781 (16) is an exceptionally
`potent inhibitor of HIV-1 replication [80]. It has been found
`to restore the antivi1'al activity of AZT against AZT—resistant
`HIV-1 [83]. UC-781 has been recfignized as a (retro)viru-
`cidal agent, capable of reducing the infectivity of HIV-1
`virions, and. therefore, yielding considerable promise for the
`use in (retro)virucidal formulations to prevent the trans-
`mission of HIV from infected to noninfected individuals
`
`[84]. UC—78l would seem an ideal candidate for application
`as a vaginal microbicide (virucide). i.e. when formulated in
`replens gel [85].
`
`the alkenyldiarylmethane
`urea—PETT derivatives [.97].
`(ADAM) series of compounds [98]. the pyrrolobenzoxaze—
`pinone (PBO) derivatives [99]. the quinoxalinylethylpyridyl
`thioureas (QXPTS) [100] the emivitine (MKC-442) deriv-
`ative SJ-3366 [101] and R165335(TMC125) [102]. As a
`rule, the “new" ("‘second" or “third” generation) NNRTIS
`exhibit higher potency than the “old" (‘‘first’’ generation)
`NNRTIS against wild-type and NNRTI-resistant HIV-l
`[9l_.94—96.99,102]. This is particularly prominent
`for
`DPC 083 (17) and 11165335 (TMC125) (18) that showed
`activity against Ll00l, K.l03N_. Yl8lC, Y188L.
`Kl03N t L100] and KlO3N - Yl8lC RT mutant strains in
`
`the nanomolar concentration range [102]. This makes
`Rl65335 (TMCl25) an excellent candidate for further
`clinical development.
`
`C;
`
`\
`
`.
`
`Compmmd 17
`DPC 083
`
`II
`
`it
`
`t \ t
`/
`
`\\
`
`DY
`Br./lE\[»;«
`
`mg
`
`Compound .18
`R165335 {TMCl25)
`
`Some ofthe new NNRTIS. such as SI-3366 (19). possess
`remarkable features. This compound was reported to inhibit
`HIV-1 replication at a concentration below 1 nM with a
`therapeutic index greater than 4,000,000, and to inhibit HW-
`2 replication (albeit at higher concentrations than those
`required for inhibition of HIV-1) at the viral entry stage
`[10]].
`
`Janssen Ex. 2018
`
`Lupin Ltd. v. Janssen Sciences Ireland UC
`IPRZO15-01030
`
`(Page 9 of 18)
`
`O
`
`(EH3
`
`
`
`Compound 15
`Emivirine (MKC-442}
`
`
`
`ocn (‘H"““ C/
`‘ T 2 ' ‘ T“ \
`
`C113
`
`CH,
`
`s H (
`
`,\N
`H
`
`CH4
`
`|
`
`0
`
`I
`
`\
`
`Compound 36
`Thiaearboxanilide UC—78i
`
`To the new classes of NNRTIS that offer potent anti—HlV—
`1 activity belong the thieno[3,4][l ,2,4]thiadiazine derivative
`QM9652l
`[86],
`the quinoxaline GW420S67X [87],
`the
`irnidazole derivative S-1153 (AGl549, capravirine) [88~
`90],
`( — )-6-chloro-2-[(1-fi1ro[2.3-c]pyridin-5-yl-ethyl)thio]-
`4—pyrimidinamine (PNU—l4272l) [91]. N-[2—(2,5-dimeth0x-
`yphenylethyl]-N’-[2-(5-bromopyridyl]-thiourea (HI-236)
`[92], the pyrido[l,2a]indole derivative BCH—1 [93], the 4-
`cyclopropylalkynyl-4-tritluoromethyl-3.4-dihydro—2( 1H)
`quinaaolinones DPC 961 and DPC 963, the 4—cyclop1'opylal—
`kenyl-4-tritluoromethyl-3,4-dihydro-2(lH)quinazoIin0nes
`DPC 082 and DPC 083 [94]. the thiophene-ethylthiourea
`(TET) derivative HI-443 [95], the cyclohexenylethylthiourea
`derivatives I-ll-346 and HI-445 [96],
`the C115‘-cyclopropyl
`
`

`
`E. De Ck-.'J'r.'q / Bior'hirm'('u er Bfr1pI1}'s1'r:a Acta 1587 (20U_’) ."58—2 75
`
`267
`
`0
`
`‘:33
`
`(*)—Calanolide A (22) is the only naturally occtming
`NNRTI: it was [i1st isolated from a tropical tree (C.‘w'uph_vl'~
`Hum lariigermn) and has already been the subject of a phase
`1 clinical study in healthy, HIV-negative individuals [103]
`
`Compound 19
`SJ—336ti
`
`Capravirine (AGIS49) (20) has a favorable profile of
`resilience to many drug resistance mutations. which has been
`attributed to extensive main chain hydrogen bonding involv-
`ing the main chain ofresidues 101, 103, and 236 ofthe p66
`RT subunit [89]. Capraviiine has proceeded to phase H/IH
`clinical trials [90].
`
`
`
`o \o
`
`on
`
`HIM-I“I
`Compound 22
`{+)-Calanolide A
`
`Recently. an unexpected e[l"ect of NNRTls on HIV-1 RT
`dimerization was documented [I04]: several NNRTIS,
`including efavirenz, were found to enhance the association
`between the RT subunits p66 and p51, apparently due to a
`confonnational change in the p66 subunit that resulted in
`