Dancing with chemical formulae of antivirals: A panoramic view (Part 2)

Biochemical Pharmacology 86 (2013) 1397–1410

Contents lists available at ScienceDirect

Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Commentary

Dancing with chemical formulae of antivirals: A panoramic view (Part 2) Erik De Clercq * Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 August 2013 Accepted 12 September 2013 Available online 23 September 2013

In this second part of ‘‘Dancing with antivirals as chemical formulae’’ I will focus on a number of chemical compounds that in the last few years have elicited more than common attraction from a commercial viewpoint: (i) favipiravir (T-705), as it is active against influenza, but also several other RNA viruses; (ii) neuraminidase inhibitors such as zanamivir and oseltamivir; (iii) peramivir and laninamivir octanoate, which might be effective against influenza virus following a single (intravenous or inhalation) administration; (iv) sofosbuvir, the (anticipated) cornerstone for the interferon-free therapy of HCV infections; (v) combinations of DAAs (direct antiviral agents) to achieve, in no time, a sustained virus response (SVR) against HCV infection; (vi) HIV protease inhibitors, the latest and most promising being darunavir; (vii) the integrase inhibitors (INIs) (raltegravir, elvitegravir, dolutegravir), representing a new dimension in the anti-HIV armamentarium; (viii), a new class of helicase primase inhibitors (HPIs) that may exceed acyclovir and the other anti-herpes compounds in both potency and safety; (ix) CMX-001, as the latest of Dr. Antonı´n Holy´’s legacy for its activity against poxviruses and CMV infections, and (x) noroviruses for which the ideal antiviral compounds are still awaited for. ß 2013 Elsevier Inc. All rights reserved.

Keywords: Favipiravir Neuraminidase inhibitors Sofosbuvir Direct acting antivirals (DAAs) HIV protease inhibitors (PIs) Integrase inhibitors (INIs) Helicase primase inhibitors (HPIs) CMX-001 Norovirus Chemical compounds studied in this article: Favipiravir (PubChem CID: 492405) Zanamivir (PubChem CID: 60855) Oseltamivir (PubChem CID: 65028) Peramivir (PubChem CID: 154234) Laninamivir octanoate (PubChem CID: 9847629) Sofosbuvir (PubChem CID: 45375808) Daclatasvir (PubChem CID: 25154714) Ledipasvir (PubChem CID: 67505836) Raltegravir (PubChem CID: 54671008) Elvitegravir (PubChem CID: 5277135) Dolutegravir (PubChem CID: 54726191) CMX-001 (PubChem CID: 483477)

1. Introduction Chemical formulas have continuously attracted my spirit in life, perpetually inspiring me how to develop new avenues on how to (try to) develop new antiviral compounds. The basis for scientific progress in the development of antiviral compounds could greatly profit from the chemical advances made in this area of research. Personally I have been fascinated by the chemical structure entities that have determined the enormous progress made in the antiviral area. I have previously reviewed [1] the compounds I have been personally acquainted with in my crusade on the development of

* Corresponding author. Tel.: +32 26 337367; fax: +32 16 337340. E-mail address: [email protected] 0006-2952/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2013.09.010

new antivirals. Here, I want to describe a more detailed account on those compounds I have not been personally involved with, but which I considered particularly fascinating, just because of their chemical beauty, or as John Keats once said, a ‘‘piece of beauty (=chemistry) is a joy for ever’’.

2. Favipiravir Although favipiravir (T-705) (Toyama Chemical Co., Ltd, Tokyo, Japan) is a pyrazine derivative (i.e. 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) and ribavirin (virazole, 1-b-D-ribofuranosyl1,2,4-triazole-3-carboxamide) is a nucleoside analogue, they share a common structural feature, that is a carboxamide entity (Fig. 1A). This carboxamide group may be considered as the pharmacophore

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The efficacy of T-705 (that in the meantime was referred to as favipiravir) was also found efficacious against lethal H5N1 influenza A virus [7], even if resistant to oseltamivir [8]. In fact, favipiravir has been found active in vitro againt various oseltamivir-resistant variants carrying the H274Y mutation in their neuraminidase [9]. Combinations of favipiravir and peramivir would perform better than suboptimal doses of each compound alone for the treatment of influenza virus infections in mice [10]. Besides orthomyxoviruses, various other ()RNA viruses have been found sensitive to T-705 (favipiravir): arenaviruses (Junin, Pichinde and Tacaribe viruses) and bunyaviruses (La Crosse, Punta Toro, Rift Valley fever, and sandfly viruses) [11]. This list has been extended to yet other arenaviruses (Junin, Machupo and Guanarito), where, again, the RNA-dependent RNA polymerase, was thought to be the target of action [12]. Favipiravir offered significant protection in a model of lethal arenavirus (Pichinde´) in hamsters [13]. Favipiravir would also be effective against hantaviruses, i.e. Maporalvirus (MPRLV), a surrogate for Andes

explaining the interaction of T-705 and ribavirin with the metabolic pathway guanosine (GMP-GTP) that contains a similar carboxamide (HN1–OC6–) in its heterocyclic part. Although ribavirin was originally described as active against both DNA and RNA viruses, it is mainly active against RNA viruses [2]. It would owe its major antiviral activity to interference with the IMP dehydrogenase, a key enzyme in the biosynthesis of GMP and GTP [3]. Yet, Eriksson et al. [4] reported inhibition of influenza virus RNA polymerase by ribavirin triphosphate. Furuta et al. [5] were the first to report the in vitro and in vivo activities of T-705, and attributed the mode of action of T-705 mainly to a specific inhibition of the influenza virus RNA polymerase by the T-705 RTP [6]. To this end, T-705 should be first converted to its ribofuranosylmonophosphate (RMP) by a phosphoribosyl transferase [analogous to the hypoxanthine guanine phosphoribosyl transferase (HGPT)], and then converted to the diphosphate (RDP) and triphosphate (RTP) (Fig. 1B), before the latter would interact in a GTP-competitive manner with the viral RNA synthesis (Fig. 1C).

A

B O F O

F

N

N

NH2

HO

O

N

F

OH

T-705 Favipiravir

N

NH2

NH2

T-705

N

HO

Phosphoribosyl transferase HO

O

Nucleosidase N

N

NH2 OH

N

O

OH

F

Nucleodase

N

O

Ribavirin HO

P

O

N

O

O

HO

N

O

OH

T-705 ribofuranose

O

OH OH

HO

T-705 RMP Nucleode kinase O F O HO

P

N

NH2

O O

P

OH

O

N

O

O

OH OH

HO

T-705 RDP Nucleoside diphosphate kinase

O F

O HO

P OH

P OH

NH2

O

O O

N

O

P

O

N

O

O

OH HO

OH

T-705 RTP Fig. 1. (A) Structural formulae of T-705 (favipiravir) and ribavirin; (B) metabolic pathways of T-705 (favipiravir); (C) mode of action of T-705 (favipiravir) compared to ribavirin.

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C

T-705

IMP IMP dehydrogenase (minor target)

T-705 RMP XMP

T-705 RDP

GMP GDP GTP

T-705 RTP

RNA polymerase (major target) RNA synthesis

Ribavirin

IMP dehydrogenase (major target) XMP

Ribavirin DP

GMP GDP GTP

Ribavirin TP

reduced susceptibility (I117V, K150N, I292V/T/K, and S246N) were found in 0.8% of human and 2.9% of avian isolates [20]. Zanamivir is normally given by inhalation, but under an emergency investigational new drug program in the United States, it can be given intravenously [21]. The use of intravenous zanamivir seems warranted in critically ill patients with pandemic influenza A (H1N1) infection [22]. The antiviral efficacy of zanamivir can be significantly enhanced when attached via a flexible linker to biodegradable polymers such as poly-L-glutamine (PGN) [23]. Attaching zanamivir to a polymeric chain such as PGN may be potentially useful for minimizing drug resistance [24]. A Q136K mutation in the neuraminidase gene of pandemic 2009 H1N1 influenza A virus confers resistance to zanamivir but exhibits reduced fitness in the guinea pig transmission model [25]. The Q136K mutation conferred resistance to zanamivir in seasonal H1N1 virus [26], whereas the I223R mutation conferred resistance to both oseltamivir and zanamivir [27]. The unexpected dominance (98%) of oseltamivir-resistant H1N1 viruses from 2007 to 2009 demonstrated that NA inhibitor resistance could enhance fitness and transmissibility [28]. NA inhibitor-resistant recombinant H5N1 viruses retain their replication efficiency and pathogenicity in vitro and in vivo [29], including ferrets. 4. Neuraminidase inhibitors: Peramivir and laninamivir octanoate

IMP Ribavirin MP

1399

RNA polymerase (minor target) RNA synthesis Fig. 1. (Continued ).

virus (ANDV), the principal cause of the hantavirus cardiopulmonary syndrome (HCPS) in Argentina [14]. Ribavirin may lead to lethal mutagenesis (‘‘error catastrophe’’, ‘‘error-prone’’ replication) of (+)RNA viruses [15]. Now that favipiravir has also been postulated to induce lethal mutagenesis in influenza A (H1N1) virus (an ()RNA virus) [16], it could mean that T-705 RTP (see Fig. 1B), like the triphosphate of ribavirin, is also incorporated into the viral RNA. 3. Neuraminidase inhibitors: Zanamivir and oseltamivir Foremost among the antiviral agents active against influenza A virus [17] are the neuraminidase (NA) inhibitors zanamivir (GlaxoSmithKline, London, UK) and oseltamivir (Hoffmann-La Roche, Basel, Switzerland). Therapy with (oral) oseltamivir or (inhaled) zanamivir may provide a net benefit over no treatment of influenza [18]. Both zanamivir and oseltamivir can be considered as transition state analogues of the sialic acid (Fig. 2) involved in the trapping of the release of influenza virions from the infected cells. This has also led to the design and development of new inhibitors, i.e. DFSAs (difluorosialic acids (e.g. compound 5 (Fig. 2)) as new mechanism-based covalent NA inhibitors with broadspectrum influenza antiviral activity [19]. Established markers of NA inhibitor resistance (E119A, H274Y and N294S) were found in 2.4% of human and 0.8% of avian isolates, and the markers of

Whereas oseltamivir and zanamivir are traditionally administered for 5 days, at 75 mg BID orally for oseltamivir (Tamiflu1) and 10 mg BID by inhalation for zanamivir (Relenza1), peramivir (Rapiacta1, Peramiflu1) (BioCryst Pharmaceuticals, Birmingham, AL) is administered parenterally (intravenously or intramuscularly) at 600 mg QD for 5–10 days, and laninamivir octanoate (Inavir1) (Daiichi Sankyo, Tokyo, Japan) as a single dose, by inhalation [30]. Influenza virus resistance to NA inhibitors has been the subject of comprehensive review articles [i.e. [31]]. High resistance (>50-fold) change compared with wild-type has been reported with amino acid substitution H274Y (H1N1 and H5N1) for oseltamivir and peramivir, E119A/D (H3N2, B) for oseltamivir, zanamivir and peramivir [32]. In vitro passaging of pandemic H1N1/09 virus selects for high-level resistance to oseltamivir and peramivir, but not zanamivir [33]. The single I223R mutant was associated with reduced susceptibility to oseltamivir (53-fold), zanamivir (7-fold) and peramivir (10-fold) [34]. Intramuscular peramivir could constitute an alternative treatment of oseltamivir-resistant influenza H1N1 infections [35]. Repeated intravenous injection of peramivir against influenza A (H1N1) 2009 infection was found efficacious in immunosuppressed mice [36] and intravenous peramivir has been found efficacious in children with 2009 pandemic H1N1 influenza virus infection [37]. Through the Emergency Use Authorization (EUA), intravenous peramivir has been advocated for use in hospitalized patients during the pandemic H1N1 influenza virus infection in 2009–2010. The clinical effectiveness of peramivir in critically ill patients has not yet been fully assessed [38]. Laninamivir octanoate (LO) (Fig. 3) has been licensed in Japan (Inavir1) for the treatment of influenza virus infection in both adult and pediatric patients. LO is the octanoyl ester prodrug (the 3-acyl form being the major and the 2-acyl form being the minor form) (Fig. 3). LO generated a prolonged high retention of laninamivir in the mouse respiratory tissues [39], due to three consecutive series of steps: (i) uptake of LO into the airway epithelial cells, (ii) hydrolysis of LO to laninamivir by intracellular esterases, and (iii) limited efflux of the generated laninamivir by its poor membrane permeability [39]. Concentration profiles of laninamivir in epithelial lining fluid (ELF) support its long-lasting efficacy for treatment of patients with influenza by single inhaled

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O

OH

HO

O

AcHN HO

O O

HO

OH

O O

HO

state

Η3C

O

O

H3C

O

Ο

OEt

AcHN

ΗΝ Η2Ν

δ+

Neuraminidase

O

AcHN HO

O

AcHN HO

Sialic acid at cell surface HO

OH

HO

Η3Ν

ΝΗ2

Zanamivir

Oseltamivir HO

O

OH

OH F

O

AcHN HO

F HN H2N

NH

Difluorosialic acid (DFSA) Compound 5 Fig. 2. Chemical structures of cell surface sialic acid, neuraminidase transition site, zanamivir (Relenza1), oseltamivir (Tamiflu1), and DFSA (difluorosialic acid) (compound 5 [19]).

administration of LO [40]. For treatment of children with influenza, LO may be more convenient than zanamivir as it requires only a single inhalation [41]. 5. Sofosbuvir The triphosphate of 20 -a-F-20 -b-C-methyl uridine is a potent inhibitor of the NS5B polymerase and exhibits a long intracellular half-life of 36 h, and a phosphoramidate produg approach was used to address the enzymatic blockade at the monophosphorylation step and to see if the long intracellular triphosphate half-life would translate into once a day dosing in the clinic [42,43]. PSI7851, a 1:1 mixture of diastereomers (Fig. 4) was selected as a development candidate. When this compound was combined with PEG-IFN and/or ribavirin or other DAAs that included NS3/4 protease inhibitors, synergistic or additive effects were observed in the replicon assay [44]. As noted for other 20 -methylnucleosides, the S282T mutant replicon was resistant (16-fold) to PSI-7851: in a phase 1 clinical trial the compound was shown to be generally safe and well tolerated at doses up to 800 mg q.d. In this clinical trial no virus–drug resistance emerged. Further development of 20 -a-F-20 b-C-methyl uridine phosphoramidate then proceeded with the single Sp diastereomer PSI-7977 (Fig. 4) which was obtained by selective crystallization from the mixture and was shown to be 10fold more active than the Rp isomer [42]. In a 14-day monotherapy study in genotype 1 patients, PSI-7977 (now termed GS-7977 or sofosbuvir) achieved a 5.0 log10 IU/ml reduction in viral load with 85% of patients achieving undetectability. An unprecedentedly fast progress has been made in the annals of drug development with sofosbuvir (alias PSI-7977 and GS-7977) (Gilead Sciences, Foster City, CA), which is now intended to become a cornerstone of interferon-free, all-oral treatment for HCV. In

patients with HCV genotype 2 or 3 infection, sofosbuvir plus ribavirin for 12 weeks without interferon, or with interferon for 4, 8 or 12 weeks, achieved a sustained virologic response at 24 weeks [45]. Lawitz et al. [46] then reported that 90% of the genotype 1/4/ 5/6 patients, sofosbuvir plus ribavirin plus pegylated interferon for 12 weeks achieved a sustained virologic response at 12 weeks (SVR12) (NEUTRINO Study); in genotype 2/3 patients sofosbuvir plus ribavirin was shown to provide a SVR12 in 67% of the patients (FISSION Study). The S282T mutation (the only variant known to be associated with resistance to sofosbuvir) was not detected [46]. The response rates among patients with genotype 3 infection were lower than for genotype 2 infection: these could have been improved by adding pegylated interferon and/or extending the duration of treatment with sofosbuvir and ribavirin. In the third paper, the results of the POSITRON and FUSION Studies were reported [47]. The POSITRON Study in genotype 2/3 patients which were interferon–intolerant, –ineligible or –unwilling, sofosbuvir plus ribavirin achieved a SVR12 rate of 78%; in the FUSION Study, the SVR16 rate was 73%. These findings suggest that 12 weeks of treatment with sofosbuvir and ribavirin may be an effective option for patients with HCV genotype 2 infection, but that for patients with genotype 3 infection, particularly those who have cirrhosis or who have not had a response to prior treatment with interferon, extending the duration of treatment to 16 weeks may provide additional benefit [47]. 6. Daclatasvir and ledipasvir Daclatasvir (BMS-790052) (Bristol-Myers Squibb, New York, NY) is the first-in-class HCV NS5A inhibitor [48]; HCV NS5A is a 447 amino acid (aa) phosphoprotein which does not possess any enzymatic activity, but which, according to the two modes of

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1401

NH HN

NH O

NH2

H3C H N

H3C

OH

O OH CH3

HN

H3C

O

NH2

H N O

OH

O O

OH

CH3 OH

Peramivir

Lan inamivir

Laninamivir octanoate (LO) NH O H3C H3C

NH2

HN H N

O

OH

O O

OH

CH3

O O

3 -acy l form (major) And NH O H3C H3C

HN

NH2

H N O

O

O HO

OH

O

CH3

O

2-acyl form (minor) Fig. 3. Structural formulae of peramivir, laninamivir and laninamivir octanoate (LO). LO is 9:1 mixture of the 3-acyl form and 2-acyl form.

action delineated for daclatasvir may be involved in viral RNA synthesis and virion assembly [49]: these functions would correspond to NS5A domain I (aa 37-213) and domain III (aa 356-447), respectively, whereas domain II (aa 250-342) would be involved with binding to cyclophilin A [50]. Resistance to daclatasvir has been mapped to several residues within domain I (the N-terminal region of NS5A), most notably L31 and Y93 in genotype 1b and M28, Q30, L31 and Y93 in genotype 1a [51,52]. NS5A dimerization is correlated with RNA binding and HCV replication [53], and the dimeric structure of NS5A seems to be related to the symmetric structure of daclatasvir, built around a central biphenyl linkage, which could be readily extended to a straight triphenyl-linked compound (such as 11a [54]) (Fig. 5).

However, a bidentate structure, as in daclatasvir, is not absolutely necessary for the anti-HCV activity of NS5A inhibitors as monodentate molecules have been found to inhibit HCV replication in the picomolar activity range (i.e. 5a [55]) (Fig. 5). The combination of an oral HCV NS3/4A protease inhibitor (asunaprevir) (Bristol-Myers Squibb) with a NS5A inhibitor (daclatasvir) has been hailed as a watershed moment by some [56]. Chayama et al. [57] demonstrated that the combination of daclatasvir (60 mg q.d.) and asunaprevir (600 mg bid) in null responders (HCV genotype 1b) caused a sustained viral response (SVR) after 24 weeks of therapy. Combination treatment of daclatasvir with asunaprevir is effective against recombinant genotype 1a, 2a and 3a HCV [58]. Daclatasvir (at a dose of 60 mg

E. De Clercq / Biochemical Pharmacology 86 (2013) 1397–1410

1402

O

O

N H O

P

O

O

O

N

NH

O O HO

F

PSI-7851 2’-α-F-2’-β-C-methyluridine 5’-phosphoramidate 1:1 mixture of diastereomers

O

O

N H O

P

O

O

O

N

NH

O O HO

F

PSI-7977 GS-7977 Sofosbuvir 2’-α-F-2’-β-C-methyluridine 5’-phosphoramidate sp diastereomer Fig. 4. Chemical structure of sofosbuvir.

daily) also increased the potency of pegylated interferon and ribavirin [59]. A triple-drug combination containing asunaprevir, daclatasvir and BMS-791325 (a NS5B RNA polymerase inhibitor) [60] should allow to remove interferon and ribavirin from future combination cocktails. Lok and colleagues [61] have shown that the combination of daclatasvir with asunaprevir given for 24 weeks resulted in a sustained virologic response in 36% of patients who had not had response to prior therapy with peginterferon and ribavirin. Could we do better? According to Chung [56], certainly, i.e. if a nucleotide polymerase inhibitor such as sofosbuvir is added to the treatment regimen of patients with HCV genotype 2 or 3. GS-5885 (ledipasvir) (Gilead Sciences) is a new, symmetric bidentate molecule that has demonstrated significant antiviral activity in genotype 1a and 1b HCV-infected patients over a oncedaily dose range of 1, 3, 10 and 30 mg [62], a 3 log10 reduction in HCV RNA was observed after 3, once-daily doses of 1, 3, 10 or 30 mg [63]. Ledipasvir has been found safe and well-tolerated in over 1000 patients treated in phase 2 studies [64]. 7. Protease inhibitors (PIs) There are presently 10 protease inhibitors (PIs) approved for clinical use in the treatment of HIV infections: four belong to the first-generation [saquinavir (Hoffmann-La Roche), ritonavir

(Abbott, Chicago, IL), indinavir (Merck, Summit, NJ), nelfinavir (Pfizer, New York, NY)], the other six belong to the second generation protease inhibitors (PIs): amprenavir (GlaxoSmithKline), fosamprenavir (GlaxoSmithKline), lopinavir (Abbott), atazanavir (Bristol-Myers Squibb), tipranavir (Boehringer-Ingelheim, Ingelheim am Rhein, Germany) and darunavir (Johnson & Johnson, New Brunswick, NJ) [65]. With the exception of tipranavir, which contains a dihydropyrone (coumarin) core, all other nine PIs can be considered as peptidomimetic in that the peptide linkage is replaced centrally by a hydroxyethylene, –CH2–CHOH–, whereby the OH in fosamprenavir is esterified by a phosphate group (Fig. 6A and B). Cobicistat (Gilead Sciences) can be considered as a derivative of ritonavir, where the hydroxyethylene is replaced by an ethylene group (–CH2–CH2–). Cobicistat is a pharmacoenhancer (‘‘booster’’) which by itself has no anti-HIV activity and thus cannot elicit anti-HIV resistance. Except for nelfinavir, all other PIs are ‘‘boosted’’ by ritonavir [81]. Daily pill burden varies from 1  2 (or 1  3) to 2  2(or 2  3, 2  4, or 2  5) for both therapy-naı¨ve and -experienced patients [65]. Darunavir has outperformed most other approved PIs in inhibiting HIV infectivity (IC50: 3–6 nM), exhibiting a selectivity index (CC50/EC50) of >20,000 [66]. Only amprenavir displays crossresistance to darunavir, which may be related to the fact that both compounds share a sulfonamide group [67]. What sets darunavir apart from the other PIs is its high genetic barrier to resistance development: resistance develops very slowly after multiple passages, only at concentrations of less than 200 nM [68]. Moreover, darunavir also has the unique ability to act as a dual inhibitor, blocking not only the cleavage of the natural peptide substrate but also inhibiting dimerization of the HIV-1 protease (at a concentration as low as 0.01 mM) [66]. Tipranavir is the only other PI sharing this property with darunavir [67]. High-level resistance to darunavir usually requires a minimum of 3 to 4 mutations [69]. Ritonavir is currently exclusively used as a pharmacokinetic enhancer of other PIs, predominantly due to ritonavir’s potent inhibition of the cytochrome P450 3A4 isoenzyme [70]. Atazanavir boosted with ritonavir (atazanavir/r) has less potential for longterm complications of hyperlipidemia and insulin resistance compared with other PIs, and its high genetic barrier to resistance makes atazanavir/r an excellent option for initial HIV treatment [71]. If simplification to monotherapy is selected to treat some patients, twice-daily lopinavir/ritonavir or preferably once-daily darunavir/ritonavir should be chosen [72]. Except for the combination with elvitegravir in Stribild1, cobicistat has so far not been routinely used as a pharmacoenhancer to boost in vivo efficacy of PIs. It would be worth evaluating whether for this indication cobicistat could replace ritonavir. 8. HIV Integrase inhibitors (INIs) Two integrase inhibitors (INIs), raltegravir (Merck) and elvitegravir (Gilead Sciences) (Fig. 7) have now been approved by regulatory agencies for use in the treatment of HIV infections, and a third such drug, dolutegravir (GlaxoSmithKline), has in the mean time been approved (12 August 2013) on the basis of several phase three clinical trials [73]. Several resistance mutations toward INIs have been identified, the most prominent being mutations at position Q148 [74]. From the REALMRK Study, raltegravir has been identified as generally safe and well tolerated with potent efficacy regardless of gender or race [75]. From a 96week, double-blind, active-controlled, phase 3 study once-daily elvitegravir appeared non-inferior to twice-daily raltegravir [76]. The fact that elvitegravir can be given once a day as compared to twice a day for raltegravir, might improve the patients’ adherence [77]. Primary INI resistance-associated mutations occurring in

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1403

O O

NH

O

H N

N

N

N N

N H

O

O

HN O

Daclatasvir (BMS-790052)

O O

NH

O N

H N

N

N N

N H

O

O

HN O

11a (Shi et al. [69])

H N N3

N N

O

NH

O

Br OCH3

5a (Amblard et al. [70]) Fig. 5. Daclatasvir, a symmetrical bidentate NS5A inhibitor, another symmetrical bidentate NS5A inhibitor (compound 11a [54]) and a monodentate derivative thereof (compound 5a [55]).

HIV-1-infected patients failing elvitegravir-containing regimens in clinical studies were T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H [78]. In combination with cobicistat (COBI) tenofovir disoproxil fumarate (TDF) and emtricitabine (()FTC), elvitegravir (EVG) has proven non-inferior to analogous combination regimes containing ritonavir-boosted atazanavir [79], or efavirenz [80], and regulator approval has been granted to the single-tablet, oncedaily, elvitegravir-based regimen (EVG/COBI/()FTC/TDF), Stribild1, for the initial treatment of HIV infection. The durable efficacy and safety of EVG/COBI/()FTC/TDF has been confirmed [81]. Dolutegravir boasts good tolerability, once-daily dosing with no need for a pharmacological enhancer, and a relative resilience to resistance development [82]. Dolutegravir has good activity against raltegravir- and elvitegravir-resistant virus but reduced susceptibility has been reported for virus with the Q148 or G140 signature mutations [83]. Dolutegravir would possess (as compared to raltegravir and elvitegravir) a higher genetic barrier to drug-resistance development [84]. The non-inferior efficacy and similar safety profile of dolutegravir compared with raltegravir means that combination treatment of once-daily dolutegravir and fixed-dose nucleoside reverse transcriptase inhibitors would be an effective new option for treatment of HIV-1 infection in treatmentnaı¨ve patients [85]. Meanwhile, new INIs have been developed: i.e. 2-hydroxyisoquinoline-1,3(2H,4H)-diones [86], and dihydro-1H-isoindole

derivatives [87], which may have higher potency and/or a higher barrier to resistance than raltegravir. A prototypic example of the second class of these new INIs is XZ-259 (Fig. 7). 9. HPIs (helicase–primase inhibitors) The helicase–primase complex consists of the viral proteins UL5 (helicase), UL8 (an ancillary protein) and UL52 (primase): it unwinds HSV DNA at the replication fork and primes both the lagging strand and leading strand during DNA synthesis [88]. The first two HPIs reported to be effective HSV inhibitors were BAY 571293, reported by Kleymann et al. [89] and BILS 22 BS, described by Crute et al. [90] (Fig. 8). In fact, Crute et al. [91] had already described how the HSV helicase–primase could be considered as a complex of three virus-encoded gene products that could be targeted for inhibition of HSV replication, and Spector et al. described inhibition of HSV replication by a 2-aminothiazole via interaction with the helicase compound (UL5) of the UL5–UL8– UL52 complex [92] (Fig. 8). From the start, BAY 57-1293 was announced as ‘‘superior’’ in efficacy to acyclovir in its efficacy against HSV in animal models [89], and the activity of BAY 57-1293 was not only found superior to that of acyclovir [93] but also to that of famciclovir in BALB/c mice infected with HSV-1 [94]. The enthusiasm generated by BAY 57-1293 was dampened, however, by the detection of HSV-1 mutants that were highly resistant to BAY 57-1293 [95]. HPI-resistance mutations may have pre-existed

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1404

A (Leu) Asn

Gln

(Phe) Tyr

(Ile) Val

Pro

Substrate

(Ser) Val

peptidic bond

N N H O

O

Tipranavir

hydroxyethylene bond

Inhibitor N H

N OH O

Ritonavir

Saquinavir

Indinavir

Atazanavir

Nelfinavir

Amprenavir

Lopinavir

Darunavir

Fosamprenavir

Cobicistat

B

Ritonavir Norvir® O

H N

N

O

N N H NH2

O

N

O

OH O

O

NH

H3C

CH3SO2 OH

N

CH3

S

N N

H3C CH3

CH3

N H

N

O

OH

N H

O

S N

Cobicistat

O

H N

N H

O

CH3

CH3

O

N H

CH3

H3C

Saquinavir hard gel capsules, Invirase® so gelan capsules, Fortovase®

H3C

N

S CH3

O H N

O

N

S N

OH

H N

N

O H3C

O

NH

HO CH3

CH3

Indinavir Crixivan® Fig. 6. (A) HIV protease inhibitors. Peptidic bond replaced by hydroxyethylene bond as in saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, fosamprenavir, lopinavir, atazanavir and darunavir, or dihydropyrone ring as in tipranavir; (B) chemical structures of saquinavir, ritonavir, cobicistat, indinavir, nelfinavir, amprenavir/fosamprenavir, lopinavir, atazanavir, darunavir and tipranavir.

E. De Clercq / Biochemical Pharmacology 86 (2013) 1397–1410

1405

CH3 H3C CH3

O

HO

CH3

N

NH

O

S

CH3SO2 OH

N H

OH

O

N OH

O

H N

N H

O N

H N

N H

O

O O

Nelfinavir Viracept® Atazanavir Reyataz® O

O

O O

N H

S

O

N

H CH3

OH

NH2

O H

CH3

NH2

OH H N

O H O

N O

S

O

Ph

O

Amprenavir Agenerase®, Prozei®

O

O

O O

N H O HO

S

Darunavir (TMC-114) Prezista® O

N P

CH3

O OH

O

NH2

O O

CH3

OH

N H

Fosamprenavir Lexiva®, Telzir®

S

O

F

N F

F

Tipranavir (U-140690) Apvus® OH

CH3 O O CH3

H N

O N H H3C

N

NH

O CH3

Lopinavir combined with ritonavir at 4/1 rao Kaletra® Fig. 6. (Continued ).

[96]. A mutation conferring resistance to BAY 57-1293 was found in the HSV-1 UL52 primer (A899T) which did not confer resistance to BILS 22 B5 [97]. Resistance of HSV to HPI BAY 57-1293 [which has been renamed as AIC 316 (pritelivir) (AiCuris, Wuppertal,

Germany)] is not as important a problem as originally thought [98]. No resistant HSV was observed in a phase II clinical trial with AIC 316 [99]. In a phase I/II clinical trial AIC 316 showed excellent activity against genital herpes [100].

E. De Clercq / Biochemical Pharmacology 86 (2013) 1397–1410

1406

O N

H3C

N

H3C

N H2N

H N

N

O H3C

F

OH

N

H N

O

Cl

CH3

S

O

T157602

Raltegravir

O

CH3

OH

H N

N

CH3

CH3

N

N

O

O

N

S

OH

Cl F

O H3C

H2N

O

BILS 179 BS

Elvitegravir CH3

O

F

F

O

N O

O

OH N

N

H N

N H

O

Dolutegravir

BILS 45 BS F O

OH

O

Cl N

HO N O O

S N

BILS 22 BS

CH3

H3C

XZ-259

H3C O

Fig. 7. Chemical formulae of raltegravir, elvitegravir, dolutegravir and XZ-259 [87].

10. Hexadecyloxypropyl-cidofovir (CMX-001)

H2N

O Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, (S)-HPMPC] (Gilead Sciences) has since 1996 been licensed for clinical use for the treatment of human cytomegalovirus (HCMV) infections, particularly (H)CMV retinitis in AIDS patients [101]. Off-label the compound has been used in the systemic treatment (intravenously at a dose not exceeding 5 mg/kg every other week) or topical (1% gel) treatment of pox-, adeno-, or papilloma virus infections (i.e. orf) [102]. However, cidofovir suffers from two major shortcomings: nephrotoxicity and lack of oral bioavailability. Therefore, alkoxyalkyl [hexadecyloxypropyl (HDP) and octadecyloxyethyl (ODE)] derivatives have been developed for both cidofovir and its predecessor, (S)-9-(3hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] [103]. HDP- and ODE-(S)-HPMPA markedly increased the oral

N

S S

O

N

N CH3

BAY 57-1293 (AIC 316, pritelivir) Fig. 8. Chemical formulae of HPIs [T157602, BILS 179 BS, BILS 45 BS, BILS 22 BS, and BAY 57-1293 (AIC 316, pritelivir)].

efficacy of the parent compound against both pox- and herpesvirus infections in mice. In particular, the hexadecyloxypropyl derivative of cidofovir (HDP-cidofovir) (Fig. 9) has received most attention as a potential drug (CMX-001) (Chimerix, Durham, NC) for the treatment of

E. De Clercq / Biochemical Pharmacology 86 (2013) 1397–1410

NH2 N O

O

N

P

O

OH

OH HO

(S)-HPMPC Cidofovir Visde® CDV NH2 N

herpes simplex virus (HSV) infections [108]. CMX-001 has been found to potentiate the efficacy of acyclovir in HSV infections [109]. In pregnant guinea pigs CMX-001 proved effective in a congenital model of CMV infection [110]. It also proved effective in the therapy of a lethal monkeypox virus infection in a mouse stat 11 model [111] and respiratory mousepox, an animal model of smallpox [112]. In the treatment of CMV infections, CMX-001 leads to the development of the same resistance development mutation as its parent compound, cidofovir, namely the D542E mutation in the CMV DNA polymerase (UL54) gene [113]. As has been shown for cidofovir in several anecdotal reports (see, for example Erickson et al. [114]), CMX-001 should be effective in the treatment of molluscum contagiosum caused by the molluscipoxvirus MCV (molluscum contagiosum virus) [115]. The cyclic analogue, hexadecyloxypropyl-cyclic cidofovir could prevent viral retinitis for up to 68 days in rabbits following a single intravitreal injection [116]. CMX-001 has since several years been considered as a candidate drug for the treatment of selected DNA virus infections [117]. CMX-001 should be entertained for the treatment of all DNA virus infections that are amenable to cidofovir, that is human cytomegalovirus (HCMV) infections, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections, adenovirus infections, polyomavirus infections (BK, JC), orthopoxvirus (smallpox, vaccinia, cowpox, monkeypox), molluscipox (molluscum contagiosum) and parapox (orf) virus infections. CMX-001 may be a therapeutic option for the treatment of severe adenovirus infections in immunocompromised patients [118].

O

N

O

O

P

O(CH2)3O(CH2)15CH3

OH HO

Hexadecyloxypropyl-cidofovir HDP-CDV CMX-001 NH2

11. Norovirus inhibitors Norovirus is responsible for infectious gastroenteritis, a common illness that is characteristically self-limiting but can become debilitating and life-threatening in immunocompromised patients [119]. Noroviruses are nonenveloped viruses with a single-stranded (ss) RNA genome that make up the genus norovirus of the family Caliciviridae and are (+) ssRNA viruses, just as are the Picornaviridae. The selective inhibitors of picornavirus replication [120] may thus serve as a paradigm for norovirus inhibitors. Ribavirin was shown to inhibit the replication of Norwalk virus, a norovirus strain [121], but this was not confirmed in a case report of van de Ven et al. [122]. Styrylchromones would have potential as anti-norovirus agents [123], and even favipiravir (T-705, see Section 2) has been reported to inhibit murine norovirus

N

O

CH3 H N

O

1407

O

N O

P

N H

CH3

O

HO

O

O

O(CH2)2O(CH2)17CH3

OH

H N

N H

S O H N

O

O

O H O S N

O

O O

O S

S O

O

S

O

O

O

poxvirus and CMV infections [104]. The conversion of cidofovir to CMX-001 increased its oral bioavailability and diminished its accumulation in the kidney [105]. The inhibitory potential of CMX001 has been well described against human cytomegalovirus infection [106], murine cytomegalovirus infection [107], and

S

O

O

O

Octadecyloxyethyl-cidofovir ODE-CDV Fig. 9. Alkoxyalkyl esters of cidofovir: hexadecyloxypropyl (HDP)- and octadecyloxyethyl (ODE)-cidofovir.

O

Suramin

O

O

O

S

S

O

O O

O H N

O

O O

N H

O S

O

O

O

O O

O H N

N H

O

S

O S

S O

NF023 Fig. 10. Chemical formulae of suramin and NF023.

O

O

1408

E. De Clercq / Biochemical Pharmacology 86 (2013) 1397–1410

replication in vitro [124]. Also, 20 -C-methylcytidine has been shown to inhibit (murine) norovirus replication [125], and so was its closely related analogue 20 -F-20 -C-methylcytidine [126]. Ribavirin antagonized the anti-norovirus activity of 20 -C-methylcytidine, just as it had been found to antagonize the anti-HCV activity of 20 -Cmethylcytidine [127]. Remarkably, suramin and NF023, another polysulfonate analogue (Fig. 10), have been found to be structurebased inhibitors of the RNA-dependent RNA polymerase (RdRp) of norovirus [128]. Suramin and NF023 were shown to occupy the same RdRp site between the fingers and thumb domain. This does not mean that suramin and NF023 would have a meaningful effect on norovirus replication, but the anti-RdRp effect of suramin is reminiscent of its inhibitory effect on the reverse transcriptase of murine retroviruses which, once upon a time, led to the initiation of the avalance of anti-HIV agents, active against AIDS.

12. Conclusions The 10 compounds I have discussed here are all representatives of the most recent directions antiviral drug development has currently taken: thereby ecompassing all important viral pathogens: influenza, hepatitis C virus (HCV), HIV, HSV, CMV and norovirus. The compounds addressed are (i) favipiravir (T-705), (ii) zanamivir, oseltamivir, (iii) peramivir, laninamivir, (iv) sofosbuvir, (v) daclatasvir, ledipasvir; (vi) HIV protease inhibitors, (vii) HIV integrase inhibitors (INIs), (viii) helicase-primase inhibitors (HPIs), (ix) CMX-001, and (x) norovirus inhibitors. There are some fascinating aspects linked to these 10 compounds or families. (i) How can the mode of action of favipiravir be rationalized, and to which extent shares it common features with ribavirin from both a structural and mechanistic viewpoint? (ii) Are neuraminidase inhibitors such as oseltamivir and zanamivir poised to see their beneficial effects curtailed by drug resistance development? (iii) Can the practical usefulness of the neuraminidase inhibitors peramivir and laninamivir be ascertained with single (intravenous) injection, or single oral inhalation, respectively? (iv) Is sofosbuvir likely to be the cornerstone for the future interferonfree all-oral treatment of HCV infection? (v) May sofosbuvir, in combination with oher direct antiviral agents (DAAs) such as NS5A inhibitors, be expected to fulfil an old dream, that is to eradicate a virus (i.e. HCV), whereas for other viruses (i.e. HIV, HBV), this goal has proven unrealistic? (vi) What is the magic underlying the success of the central hydroxyethylene peptidomimetic core present in nine of the 10 licensed HIV protease inhibitors (PIs), including darunavir, the latest PI to be approved for clinical use? (vii) The INIs represent the last class added to the armamentarium of drugs for the treatment of AIDS: how do dolutegravir and elvitegravir compare with raltegravir in terms of potency, safety and resistance development. (viii) For the treatment of HSV infections, a new drug, the helicase primase inhibitor (HPI) AIC 316 (pritelivir), is forthcoming. How will its clinical efficacy and safety compare with that of the gold standard for the treatment of HSV infections (acyclovir/valaciclovir)? (ix) CMX-001 as prodrug of cidofovir should be effective against all DNA virus infections, that are amenable to cidofovir therapy, including particularly, pox-, adeno-, papilloma-, herpes- and CMV-infections. (x) Norovirus has emerged as a new chemotherapeutic target: it is curious that those inhibitors that have so far been described as norovirus inhibitors have been known to inhibit various other (unrelated) viruses such as influenza (i.e. favipiravir) and HIV (i.e. suramin). Acknowledgments I thank Christiane Callebaut for her proficient and never waning editorial assistance.

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