A Journey around the Medicinal Chemistry of Hepatitis C Virus

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A Journey around the Medicinal Chemistry of Hepatitis C Virus Inhibitors Targeting NS4B: From Target to Preclinical Drug Candidates Rolando Cannalire, Maria Letizia Barreca, Giuseppe Manfroni,* and Violetta Cecchetti Department of Pharmaceutical Sciences, Università degli Studi di Perugia, Via A. Fabretti, 48-06123 Perugia, Italy

ABSTRACT: Hepatitis C virus (HCV) infection is a global health burden with an estimated 130−170 million chronically infected individuals and is the cause of serious liver diseases such as cirrhosis and hepatocellular carcinoma. HCV NS4B protein represents a validated target for the identification of new drugs to be added to the combination regimen recently approved. During the last years, NS4B has thus been the object of impressive medicinal chemistry efforts, which led to the identification of promising preclinical candidates. In this context, the present review aims to discuss research published on NS4B functional inhibitors focusing the attention on hit identification, hit-to-lead optimization, ADME profile evaluation, and the structure− activity relationship data raised for each compound family taken into account. The information delivered in this review will be a useful and valuable tool for those medicinal chemists dealing with research programs focused on NS4B and aimed at the identification of innovative anti-HCV compounds. and Europe,7 while gt 3 is common in Central Asia and Middle East.8,9 Finally, HCV gt 4 and gt 5 are found almost exclusively in Africa,10 and HCV gt 6 is endemic in East and Southeast Asia.11,12 Gt 1 and gt 4 are the hardest to treat and are associated with a particularly aggressive course of the disease.3,13 HCV is a small, enveloped RNA virus belonging to the Flaviviridae family and is the only member of the genus Hepacivirus. HCV genome is a 9.6-kb single-stranded plus RNA (ss-(+)-RNA) composed of a long open reading frame (ORF) flanked by 5′- and 3′-nontranslated regions (Figure 1A). The 3′-nontranslated region contains a poly U/UC tract and a highly conserved 98-nucleotide element that is required for viral RNA synthesis.14,15 The 5′-nontranslated region is the most conserved among the different gts and contains the internal ribosomal entry site (IRES) element, which is essential for a direct Cap-independent translation of the ORF region.16−18 Translation affords a precursor polyprotein of approximately 3000 amino acids (aa) successively processed by host cell and viral proteases into both structural (S) and nonstructural (NS) proteins, respectively, whose main

1. AN OVERVIEW ON THE HEPATITIS C VIRUS The infection caused by hepatitis C virus (HCV) is an escalating global health issue. With an estimated ∼130−170 million chronically infected individuals worldwide, HCV is responsible for ∼27% of all cases of liver cirrhosis and ∼25% of hepatocellular carcinoma.1,2 These data explain why chronic hepatitis C represents the main indication for liver transplantation in industrialized countries.3 The prevalence of HCV infection is highly variable with the highest prevalence in Africa and in the Middle East, and with the lowest in the Americas, Australia, and Northern and Western Europe.1−3 HCV infection occurs through exposure to blood products, especially in injected drug users and with less extent by sexual and vertical way.3,4 An acute and often asymptomatic phase develops after the virus−host interaction, with a high percentage (nearly 80%) of patients that become chronically infected.3,5 During their life course patients can incur in heavy hepatic damages such as liver-destroying cirrhosis up to hepatocellular carcinoma and death, if liver transplantation is not possible.3,5,6 At least six HCV genotypes (gts) have been identified until now and they can be divided in multiple subtypes. The global distribution of HCV gts varies depending on the geographical area with HCV gt 1 being the most common in North and South America, Europe, and Australia.7,8 HCV gt 2 is widespread in America © 2015 American Chemical Society

Received: May 29, 2015 Published: August 4, 2015 16

DOI: 10.1021/acs.jmedchem.5b00825 J. Med. Chem. 2016, 59, 16−41

Journal of Medicinal Chemistry

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Figure 1. (a) Genomic organization of HCV and proteins’ processing; (b) membrane topology and main functions of cleaved viral proteins. (Reprinted with permission from ref 36. Copyright 2013 Macmillan Publishers Limited.)

and virus replication.32 NS5B is a RNA-dependent RNA polymerase responsible for replication of the viral genome within the replication complex.33 The crystal structure of the protein reveals a typical right-hand architecture usually observed in this class of enzyme with palm, thumb, and finger domains.34,35 NS5B proceeds via the synthesis of a complementary ss-(−)-RNA used as a template for the generation of numerous copies of ss-(+)-RNA. Initiation of RNA synthesis has been found to proceed as a de novo mechanism in the absence of primer.33 When the discovery of HCV was published in 1989 by Houghton and co-workers at Chiron,37 the only available treatment was an aspecific antiviral therapy based on the combination of pegylated interferon-α (pegIFN-α) and ribavirin (RBV).38 Peg-IFN-α/RBV treatment is moderately successful in treating infected patients and shows marked differences depending on the HCV gt. In fact, only 40−50% of patients infected with gt 1 and gt 4 achieves a sustained virological response, indicative of a cure, which increases up to 75% for those infected by gt 2 and gt 3.38,39 In addition, this regimen is difficult to tolerate and is associated with many toxicities, especially due to the use of IFN-α.38 Anyway, this treatment has remained the standard-of-care (SOC) until 2011 for gt 1, and until 2014 for the other gts. Over the past 20 years, the development of new models and tools to reveal the different steps of the HCV life cycle and tremendous efforts made by industrial companies and some academic groups have allowed the development of direct-acting antivirals (DAAs) that specifically target HCV proteins.36,40 In 2011 the first DAAs, which are the NS3/4A HCV protease inhibitors (PIs) telaprevir (Incivek; Vertex)41 and boceprevir (Victrelis; Merck),42 were approved by the US Food and Drug Administration (FDA), each in combination with pegIFN-α plus RBV (Figure 2).43 Although the cure rates have improved significantly (SVR =

functions are schematized in Figure 1B and briefly discussed below.19−21 The S proteins include the nucleocapsid core (C),22 the highly glycosylated transmembrane proteins E1 and E2 involved in the HCV adhesion to host cell receptors,23 and the viroporin p7 that likely forms ion channels essential for assembly and release of infectious virions.24 The remainder of the precursor protein contains the NS proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B.19 These proteins are released by protease activity of the two viral enzymes NS2 and NS3 that in turn are autocatalytically produced from the polyprotein precursor.19 In particular, a first cleavage occurs at the NS2/ NS3 junction by the zinc-dependent autoproteolytic NS2 protease and the N-terminal one-third of NS3.25 NS3 is a bifunctional protein endowed with a serine protease domain, that cleaves at the NS3/4A, 4A/4B, 4B/5A, and 5A/5B junctions, and a nucleoside triphosphatase (NTPase)/helicase domain with RNA binding, unwinding, and NTP hydrolysis functions.26 The NS3 requires the NS4A protein as essential cofactor for an efficient enzymatic function.26 As all ss(+)-RNA viruses, also HCV replicates its genome in intimate association with host intracellular membranes.19,27 In particular, HCV induces the formation of novel membrane structures, termed membranous web (MW), that facilitate RNA replication.19,28,29 The main protagonist in this process is the highly hydrophobic transmembrane protein NS4B.30 Through aggregation of membranous vesicles derived from the endoplasmic reticulum (ER), NS4B leads to the formation of the MW, the platform where the NS viral proteins and host factors associate to replicate the HCV RNA.31 The NS5A is a phosphoprotein with no apparent enzymatic activity, which exists in two phosphorylated forms. It is organized into three domains and acts as a regulator of cellular pathways as host cell growth, immunity, escape from immune-mediated response, 17



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70%), the new SOC provides limited clinical benefit against HCV gts 2−6, and many patients cannot tolerate the severe side effects due to the combination therapy thus remaining untreated.44 From the end of 2013 to date, six new additional HCV DAAs have been approved: the second generation NS3/4A PIs simeprevir45,46 and paritaprevir;47,48 the NS5B polymerase inhibitors sofosbuvir49−52 and dasabuvir;48,53 and the HCV inhibitors targeting NS5A ledipasvir54,55 and ombitasvir48,56 (Figure 3). Experimental data from literature strongly demonstrate that the IFN era is coming to its end, and IFNfree strategies are proving the ability of achieving SVR up to 95−96% for combinations of DAAs.40 Several other combinations of DAAs, targeting NS3 protease, NS5B polymerase, and NS5A, are in the late stages of clinical development and/or are close to the approval by FDA and/or European Medicines Agency (EMA). More detailed information about the clinical trial panorama for the treatment of HCV can be retrieved from some literature reviews40,57 and trusted Web sites.58,59 Until a few years ago, little or nothing was present in literature regarding DAAs acting on NS4B. The last couple of years have strongly highlighted that also NS4B represents a druggable and appealing antiviral target, thus making the NS4B

Figure 2. First-generation of HCV NS3/4A protease inhibitors.

Figure 3. Latest approved drugs for the treatment of HCV infection. 18



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the last entry target in HCV drug discovery process.31,60−63 To date, no drugs acting against NS4B protein have been approved by FDA, but we recognize that all main pharma companies are working on the discovery and development of clinical candidates. On this context, the present review aims to report: (i) the description of the in vitro testing models currently available for the identification of NS4B ligands; (ii) the discussion of all the published chemical classes and miscellaneous compounds targeting NS4B along with the available data of their activity, cytotoxicity, pharmacokinetic (PK) properties, drug-resistance profile, and structure−activity relationship (SAR); and (iii) our critical point of view on the discussed topic and perspectives for future development.

most documented functions of NS4B.28,29 Hence, the NS4B has been proposed to play a structural role in HCV RNA replication due to the ability to reorganize the intracellular membranes into new membranous (e.g., MW) structures hosting the HCV replication complex.66,69,71 The NS4B central region harbors a nucleotide-binding motif (NBM) Walker A located between TM2 and TM3 domains (aa 129−135) with the typical “GXXXXGK” motif of a NTPase (Figure 4).65,72 The NBM Walker A is found in almost all HCV gts, permitting binding and hydrolysis of GTP and ATP and the synthesis of ATP and AMP from two ADP molecules.73 The NMB Walker A is essential for HCV life cycle as shown by the dramatic loss in viral replication effectiveness after introduction of point mutations at NBM sequence.72 Although the precise role of the NBM mediated NTPase activity remains still unclear, it has been proposed that the NS4B GTPase activity plays an important role in cells transformation and tumor formation.74 The analysis of the secondary structure of NS4B C-term reveals two α-helices, named H1 and H2 (Figure 4).66 The first helix extends from residue 200 to 213 and is highly conserved among HCV gts,75 while the 3D structure of H2 determined by NMR (PDB code 2KDR) reveals a twisted amphipathic α-helix composed by aa 229−253, with a more variable degree.71,75,76 H2 mediates membrane association and is also involved in the formation of functional HCV replication complex.66,77,78 In addition, researchers at Stanford University reported that the Cterm domain includes arginine-rich motifs at residues 192−193 and 247−248 able to bind the 3′ end of the HCV ss-(−)-RNA, an essential property for an efficient in vitro viral replication.79 NS4B C-term has also two palmitoylation sites at two ending cysteine residues (aa 257 and 261)80 probably involved in NS4B oligomerization, but the role of C-terminal palmitoylation of NS4B in the HCV life cycle remains still unclear. Interestingly, HCV NS4B significantly differs from the same protein present in the HCV-related flavivirus sharing a negligible resemblance in the amino acid sequence.81 Although HCV and flavivirus NS4B are considered orthologue but not homologue proteins,82 they show the same hydrophobic and transmembrane nature with some shared functional features (e.g., induction of MW formation, dimerization/oligomerization functionality).81 2.2. In Vitro Testing Models for Identifying NS4B Ligands. Most of NS4B ligands have been identified thanks to HCV replicon based high-throughput-screening (HTS) campaigns aimed at the identification of novel anti-HCV compounds whose mode of action has been determined by subsequent genetic validation. In particular, the identification of mutations in the NS4B sequence conferring drug viral resistance confirmed this protein as target of the tested antiHCV compounds. However, the transmembrane nature of NS4B made difficult the expression, the biochemical and structural characterization of the protein thus hampering the development of quick screenings. Nonetheless, some in vitro assays based on biophysical and/ or biochemical methods that enabled HTS for potential HCV inhibitors targeting NS4B have been developed over the years. These in vitro assays include: (i) microfluidic RNA-binding inhibition assay,79 (ii) AH2-mediated lipid vesicle aggregation inhibition assay,83 (iii) quenching fluorescence binding assay,84 and (iv) nontraditional approach based on encoded library technology (ELT).85

2. NS4B AS A PROMISING TARGET FOR HCV THERAPY: FROM STRUCTURE AND FUNCTION TO POTENTIAL DRUG CANDIDATES 2.1. Structure and Function. NS4B is very tricky to study due to its integral membrane association, and therefore, it is the less characterized HCV NS protein.64 NS4B is a predominantly hydrophobic protein of 27 kDa, which is released from the precursor polyprotein by the NS3−4A protease cleavage.31 Localized at ER, NS4B is formed by (i) a N-terminal part (aa 1−69), (ii) a central core with at least four predicted transmembrane (TM) domains (aa 70−190), and (iii) a Cterminal part (aa 191−261) (Figure 4).64−66 The NS4B N-term

Figure 4. Membrane topology of NS4B: two N-terminal amphipathic α-helices (AH1 and AH2), transmembrane segments (TM1−4), and two C-terminal α-helices (HI and H2). The N-terminus of NS4B can translocate into the ER, thereby creating a fifth transmembrane segment (TMX). Nucleotide-binding motif (NBM) Walker A is located at the cytoplasmic loop between TM2 and TM3. The C-term contains the RNA binding motif, which is located between TM3 and H1.

consists of two amphipathic α-helices, AH1 (aa 6−29) and AH2 (aa 42−66), the last being well conserved in all HCV genotypes and critical for HCV replication.67−69 The N-term is believed to be oriented on the cytosolic face of ER, but the NS4B AH2 seems to cross the membrane yielding the fifth additional TM domain (TMX) and promote AH1 translocation into the ER lumen (Figure 4).65 This post-translational event allows to hypothesize two topologies of NS4B exerting two distinct functions of the protein during virus life cycle.65,70 In particular, the N-term can translocate upon NS4B dimerization/multimerization promoting lipid vesicle aggregation that in turn seems to play an important role in MW formation for the recruitment of the replication complex thus allowing HCV RNA replication.66,68−71 The formation of ER membranesderived MW during HCV genome replication is one of the 19



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no one compound is known yet to specifically block this function and achieve an anti-HCV effect. To date, different chemical classes targeting NS4B have been published and are herein reviewed. 2.3.1. Clemizole and Structurally Related Compounds. The hydrochloride salt of clemizole, an old drug approved as H1 histamine receptor antagonist in the late 1950s,91 was reported in 2008 by Glenn et al. as one of the earliest NS4B inhibitors (Figure 5).79,92 In particular, scientists screened a

The microfluidic affinity assay has been developed by Glenn et al. at Stanford University to evaluate the capability of small molecules to hamper NS4B-RNA binding.79 In this assay the IC50 is measured through a method based on mechanical trapping of molecular interactions.86−88 In particular, a flow layer containing fluorescently labeled HCV RNA is delivered through a chamber where a static layer constituted by immobilized NS4B protein can bind the nucleic acid. The increase of unbound RNA is related to the reduced affinity of NS4B for the RNA induced by the inhibitor, and its ability to interfere with the NS4B-RNA binding is expressed as IC50. More recently, Glenn et al. have also developed a NS4B AH2-mediated lipid vesicle aggregation inhibition assay to evaluate the ability of small molecules to inhibit the NS4B mediated MW formation.83 In particular, this assay consists of two consecutive experiments based on different biophysical methods, which are the fluorescence microscopy and the dynamic light scattering. At first, fluorescence microscopy monitors the aggregation of fluorescently labeled synthetic lipid vesicles upon addition of a synthetic AH2 peptide in the absence and in the presence of compounds. The molecules endowed with antiaggregation activity are further evaluated in a secondary screen in which dynamic light scattering measurements of lipid vesicle size are performed in the presence of a compound. The quenching fluorescence binding assay has been described in a patent by Chunduru et al. and led to the identification of several classes of anti-HCV compounds targeting NS4B.84 In particular, this assay is based on monitoring changes in intrinsic protein fluorescence of a recombinant NS4B as an indirect measure of candidate ligand binding, allowing the evaluation of KD. Very recently, an innovative approach has been pursued by Thompson et al. at GlaxoSmithKline who have advantageously exploited the ELT to screen unprecedented size collections of small molecules as N4SB binders.85 The ELT approach is a technology platform that bridges the fields of combinatorial chemistry and molecular biology.89,90 Different combinatorial libraries are built by conjugating drug-like building blocks with coding short double strand DNA tags, used as markers of each chemical library. Using combinatorial split/mix methods, a broad chemical diversity can be easily achieved. The DNA tagged derived libraries are screened by affinity selection on the immobilized NS4B target protein. After multirounds of capture to the target, binding molecules are separated from the nonbinding molecules and removed from NS4B by heat elution. The chemical libraries containing the NS4B protein binders are indirectly identified translating the amplified DNA tagging sequences into reporter proteins. After off-DNA resynthesis of the chemical libraries, the KD of each compound is separately determined with a displacement assay using a radiolabeled known NS4B ligand. 2.3. Targeting NS4B. Unlike other HCV NS proteins, NS4B has remained for a long time an undisclosed target within the HCV drug discovery programs. The first report on NS4B binders is from a patent published ten years ago by Chunduru et al. at Viropharma.84 However, impressive drug discovery efforts have been made during the past few years, and several promising molecules from literature have provided evidence that small molecules targeting NS4B could suppress HCV replication by impairing AH2-mediated MW formation or RNA binding property. To our knowledge, although the NTPase activity of NS4B has been described from more than ten years,

Figure 5. Structures and activities of clemizole and its analogues.

library of 1280 pharmacologically active compounds against a purified NS4B-GFP fusion protein through the microfluidic RNA binding assay (see section 2.2).79 Clemizole inhibited NS4B RNA binding (IC50 = 24 nM) and HCV gt 2a replication (EC50 = 8 μM) (Figure 5). Moreover, clemizole was 3-fold less active against gt 1b (EC50 = 23 μM).93 The evident low correlation between the potency in the biochemical assay (RNA binding inhibition) and the anti-HCV activity in the replicon assays was explained by the authors in terms of poor compound cellular permeability.79 In order to confirm NS4B as target, in vitro treatment of Huh-7 cells carrying HCV gt 1b with clemizole revealed two significant aa alterations: W55R mutation within the AH2 and R214Q mutation within the cytoplasmic C-terminal segment of NS4B.79 When clemizole was re-evaluated in two engineered HCV gt 2a (J6/JFH) clones individually carrying the W55R or R214Q mutations, the drug resulted 2-fold (W55R) and 5-fold (R214Q) less potent than in the wild type gt 2a clone.79 Compared to the NS4B wild type, the W55R and the R214Q 20



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(KD = 0.662 μM), and it was also able to reduce HCV protein expression as determined by an ELISA-based HCV replication assay without showing significant toxicity.84,99 Later on, Glenn et al. have deeply studied anguizole demonstrating that it was able to hamper the interaction of the NS4B AH2 with lipid vesicles thus inhibiting the lipid vesicle aggregation promoted by NS4B.83 These results suggested a direct binding of the compound to the AH2 region.83,100 When tested in the HCV replicon luciferase reporter assay, anguizole resulted as the first NS4B ligand having sub-μM antiviral activity (EC50 against gts 1a and 1b of 0.56 and 0.31 μM, respectively); the compound was instead inactive against HCV gt 2a (Figure 6).100 In addition, resistant mutants selection assay using ET cells carrying HCV gt 1b replicon gave the genetic evidence that NS4B was the target of anguizole. In particular, the H94R mutation in NS4B was found as the most common one conferring anguizole resistance; the F98L and the V105M mutations were also observed in some HCV colonies.100 Interestingly, these mutations occurred in the TM1 segment of NS4B and not within the AH2 helix identified as the binding region for anguizole by the AH2 mediated vesicle aggregation inhibition assay. In order to explain these contrasting results, Glenn et al. argued that mutations in the AH2 region are very poorly tolerated by the virus, while mutations at position 94 are well tolerated with the H94R alteration counteracting anguizole’s ability to inhibit HCV replication. The H94R alteration might be responsible for a conformational change in the NS4B protein able to partially reduce the anguizole binding.100 More recently, Lee et al. have provided novel insights into the anguizole mechanism of action,101 demonstrating the compound’s ability to impair NS4B dimerization/multimerization process and thus to alter the protein subcellular localization. The anguizole-mediated inhibition process is involved in the disruption of MW formation thereby reducing HCV replication. In addition, anguizole seemed to be also able to inhibit the interaction between NS4B and NS5A.101 The racemic mixture of tetrahydropyrazole[1,5-a]pyrimidine derivative rac-5, a partly saturated analogue of anguizole, was identified by researchers at Apath through a HTS using cellbased HCV gt 1b subgenomic replicon luciferase reporter assay (Figure 7).102 More recently, it has been found that the

mutants allowed the protein to possess a greater affinity for HCV RNA as demonstrated by the microfluidic assay. The authors explained this experimental evidence by hypothesizing a postmutational NS4B conformational change that simultaneously reduced the affinity for clemizole and increased the affinity for viral RNA.79 In a different study, the same group reported a very promising antiviral synergistic effect of clemizole in combination with a first generation HCV PI (i.e., boceprevir or telaprevir). These associations also granted a non-gt dependent effect together with no increase of cytotoxicity and reduction of drug resistance outbreak.93 Successive research programs aimed at developing clemizole analogues with improved anti-HCV activity were undertaken at Stanford University leading to compounds 1−4 (Figure 5).94−97 In particular, several clemizole analogues exemplified by compounds 1 and 2 were reported as HCV replication inhibitors with EC50 ≤ 5 μM against HCV gt 1b or 2a.94,95 These data indicated that the pyrrolidinomethylene substituent at C-2 of benzimidazole core can be removed without affecting the antiviral activity (e.g., compound 1). Conversely, the N-1(4-chloro)benzyl moiety was present into almost all the active clemizole analogues thus appearing as an essential chemical feature. When clemizole analogues were tested for their ability to block hEGR channel, they showed a significant inhibition in the μM range.94,95 This undesired effect might represent an issue for further developments of these potential drug candidates. Compounds 3 and 4 are representative analogues of a compound series obtained through bioisosteric replacement of the benzimidazole core of clemizole with an indazole ring bearing a basic substituent at the C-3 position.96,97 These derivatives were able to inhibit the NS4B RNA binding, generally showing EC50 (gt 1b) below 5 μM in cell assay (e.g., compounds 3 and 4).96,97 Among all the derivatives, only clemizole has entered into Phase 1B clinical trials in HCV-infected patients (gt 1 and gt 2), also thanks to its wide and well-known safety profile. The results of the study have not yet been published and are currently under evaluation by the FDA.98 2.3.2. Anguizole and Structurally Related Compounds. Employing a quenching fluorescence binding assay (see section 2.2), the pyrazolo[1,5-a]pyrimidine anguizole was first identified in 2005 by Chunduru et al. at Viropharma (Figure 6).84,99Anguizole showed a valuable NS4B binding property

Figure 7. Structure and activities of compound rac-5 and its active synenantiomer 5.

selective HCV inhibitory activity of rac-5 was due to its synenantiomer 5 (AP80978) having 5S,7R configuration;103 this molecule was able to inhibit HCV replication in the sub-μM range with an EC50 (gt 1b) = 0.63 μM. Compound 5 showed a comparable activity against HCV gt 1a but was inactive against gt 2a, and its potency, gt specificity, and resistance profile were very similar to those of anguizole.103 Furthermore, compound 5 was endowed with a great virus specificity showing no activity

Figure 6. Structure and activities of anguizole. aELISA-based HCV replication assay; bHCV replicon luciferase reporter assay. cCrystal violet staining based assay. dCell proliferation reagent WST-1 based assay. 21



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Figure 8. Structures and activities of representative imidazo[1,2-a]pyridines summarizing the chemical optimization process from hit compound 6 to 11.

Figure 9. Structures and activities of representative piperazinone/piperazine imidazo[1,2-a]pyridines derived from compound 11.

However, before changing the thiophene ring of 6, the optimization strategy started with the replacement of the C-3 bromine with a chlorine and of the C-5 1H-4-pyrazolyl with a 3-furyl, in analogy to anguizole, leading to a new derivative 7 with valuable biological profile (Figure 8).104 These chemical modifications were well tolerated, and the authors focused on the optimization of the amide side chain in the context of C-3Cl and C-5-furyl substituents (compounds 8−11, Figure 8).104 The chiral substituted pyrrolidine was replaced by the 6-terms piperidine, yielding to achiral derivatives represented by compound 8, an almost equipotent HCV replication inhibitor of 7. Replacement of the 2-thiophenyl ring with the 1pyrrolidinyl one (e.g., 9) caused a total loss of activity while the insertion of a carbonyl moiety at C-2 position of the pyrrolidine ring restored the NS4B binding ability and anti-HCV activity (e.g., 10). Interstingly, the replacement of pyrrolidinone ring with an oxazolidinone moiety (e.g., 11) gave the best antiviral

against a panel of HCV unrelated and related viruses such as flavivirus. Lead optimization studies based on derivatives structurally related to anguizole resulted in a novel imidazo[1,2-a]pyridine series with sub-nM anti-HCV gt 1b activity.104−108 In particular, Shotwell et al. reported the development of this new class of NS4B binders starting from hit compound 6 (EC50 in both gt 1a and gt 1b < 55 nM), which was identified at GlaxoSmithKline through a cell-based HTS using a HCV gt 1b replicon luciferase reporter assay (Figure 8).104 Derivative 6 showed a nM binding constant to NS4B measured by a isothermal tritation calorimetry. Resistance passaging of 6 in HCV replicon gt 1b revealed that key mutants in NS4B (i.e., H94N, F98L, and V105M) were the same observed for anguizole. It was also found that hit 6 underwent to thiophene-related quick in vitro/ in vivo metabolism, prompting optimization efforts aimed at mainly improving the PK properties of this chemotype. 22



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Figure 10. Structures and activities of representative imidazo[1,2-a]pyridines summarizing the chemical optimization process from hit compound 16 to 22.

potency. The high activity of these compounds was explained by the authors due to the presence of an H-bond acceptor able to improve the binding to the viral protein. Despite its favorable activity profile, derivative 11 still exhibited rapid in vivo clearance in rat due to metabolism of oxazolidinone ring.104 After several chemical modifications, Shotwell et al. identified the functionalized piperazinone nucleus as suitable replacer of the piperidinyloxazolidinone, as exemplified by compounds 12−14 (Figure 9).104 Although Nmethylpiperazinone 12 was both a weak NS4B binder and HCV replication inhibitor (12 vs 8, 10, and 11), the replacement of the methyl with an aromatic moiety gave the more promising derivative 13 showing 50-fold improvement in NS4B affinity and potent replicon inhibition in the sub-μM range. Furthermore, the saturation of N-4-phenyl ring of 13 to the corresponding cyclohexyl group led to the highly potent HCV replication inhibitor 14. Hence, the presence of the hydrophobic pendant ring resulted in a key feature for obtaining a strong NS4B binding, leading to hypothesize the filling of a pocket in the NS4B binding site. While the presence of a carbonyl group in the amide side chain was favored in the

piperidine subset (9 vs 10), the removal of the carbonyl function of the piperazinone moiety did not significantly affect the activity (13 vs 15). Since derivative 14 was still far from optimal PK properties (it suffered from quick clearance in rat due to direct metabolic oxidation of cyclohexyl), further chemical optimizations were carried out at GlaxoSmithKline.104 In particular, the insertion of a 4-hydroxyl substituent (i.e., 16, Figure 10) conferred a significant improvement in PK properties (experimental data not reported by the authors) while maintaining comparable NS4B affinity and HCV replicon activity with the anti configuration preferred over the corresponding syn arrangement (compound 17, Figure 10). Since the furyl ring of 16 underwent extensive metabolism (experimental data not reported by the authors),104 a further optimization entailed the replacement of the C-5-furyl functionality with small alkyl and/or cycloalkyl groups.105−107 The outcome of these efforts is represented by C-5-cyclopropyl derivatives 18−21 (Figure 10) that retained the same tight binding to NS4B and comparable anti-HCV activity with respect to parent 16.104−108 Compound 18 was characterized by a greater 23



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Figure 11. Structures and activities of representative pyrazolopyridines.

Table 2. In Vitro Predicted Clearance (mL/min/kg)a of 26 and 27b

metabolic stability, a better in vivo clearance, and more favorable oral bioavailability in rats and dogs (Table 1) than the direct analogue 16. 104 Due to the advantageous combination of an excellent antiviral potency (EC50 < 10 nM) with a very promising PK profile (Table 1), derivative 18 became the lead compound within this series. As expected, in vitro resistance passaging experiments employing wild-type HCV replicons revealed that single-point mutations within the NS4B protein (H94N or V105M) rendered the virus partially resistant to compound 18 (Figure 10).104 Very recently, Thompson et al. at GlaxoSmithKline have reported the anti-HCV activity of compound 18 against a panel of different gts.85 In particular, this compound showed EC50 in the nM range against gts 3a (42.6 nM), 4a (6.98 nM), and 5a (2.53 nM), while it was only a weak inhibitor of gts 2b (2.4 μM) and 6a (4.47 μM) and resulted inactive against gts 2a and 6o. In order to collect additional data to support preclinical development, Peat et al. at GlaxoSmithKline explored the in vivo antiviral activity of lead compound 18.108 Despite the improved PK properties, imidazopyridine 18 failed to achieve in rats the required high plasma drug concentration for preclinical safety studies due to reprecipitation phenomena. Thus, by adopting a prodrug strategy, the synthesis of the imidazopyridine phosphate 22 allowed to reach higher bioavailability respect to active drug 18.108 In particular, prodrug 22 achieved an EC90 of 29 nM in PXB mice (chimeric humanized mice sensible to infection by human liver pathogens such as HCV and HBV)109 infected by HCV gt 1a leading to a viral load reduction of 4 log units in a 7-day study.108 To date, this study represents the only in vivo proof-of-concept of the usefulness of HCV inhibitors targeting NS4B. Unfortunately, an adverse cardiovascular effect observed for 18 during the 7-day safety study led to the termination of further development.108

a

Cl (mL/min/kg) Vdss (L/kg)d PO T1/2 (h)e AUC0−24 (ng·h/mL)f f (%)g

ratb

dogb

20 4.1 3 3800 98%

1.1 0.9 10 52000 65%

human

monkey

dog

rat

mouse

26 27

18 70-fold). Interestingly, this mutation is localized in the TM1 domain of NS4B, the same region involved in the

Figure 17. Structures and activities of representative N-(4′-(indol-2yl)phenyl)sulfonamides.

derivative 42 resulted significantly less active than the hit 41, (ii) polar substituents (such as hydroxyl, morpholinomethyl, hydroxymethyl, methoxymethyl, carboxamido, and carboxyl) were not well tolerated with the C-6 carboxyl derivative 43 being completely inactive, (iii) small lipophilic fluorinated substituents at C-6 (44−46) gave compounds endowed with good potency (EC50 ranging from 0.17 to 0.36 μM), and (iv) compounds having C-5 substitution as well as C-5/C-6 27



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Figure 18. Structures and activities of selected N-(4′-(indol-2-yl)phenyl)sulfonamides.

Figure 19. Structures and activities of 4-(indol-2-yl)benzensulfonamides 51−55: (A) favorable chemical modifications and (B) unfavorable chemical modifications.

Toward this end, the contemporary presence of two adjacent substituents at C-5 and C-6 positions would block arene oxide formation.114 Fluorine atom was chosen as the sole C-5 substituent retaining good activity in the parent indolphenyl sulfonamide series (compound 47 in Figure 17).114 As expected, the insertion of a fluorine atom at C-5 position of the indole ring gave the equipotent compound 63 (Figure 22A).114 Afterward, keeping constant the fluorine atom at C-5 position of the indole ring, the insertion of small alkyls such as a methyl (64) and an ethyl (65) group at C-6 position resulted in compounds retaining the single-digit nM activity, good PK properties, and greater metabolic stability (Figure 22A).114 A second set of modifications entailed the replacement of the N-1 cyclobutyl substituent of compound 62 with a pyrimidine ring, leading to the analogue 66 endowed with high potency and improved oral bioavailability in rats (Figure 22B).114 Moreover, when the 6-difluoromethoxy group of compound 66 was replaced by a methyl (i.e., 67) excellent potency was retained (EC50 = 3 nM) (Figure 22B).114 Then, the synthesis of analogues containing both 5-F/6-alkyl disubstitution and N-1 pyrimidinyl moiety was pursued leading to derivatives 68, 69, and 70 (PTC275) (Figure 22C).114 Compound 70 emerged as the most promising lead among several synthesized N-heteroaryl analogues, due to its excellent potency and very favorable balance in PK properties (Table 4).114,115 When assayed against gt 1a, compound 70 showed impressive potency (EC50 = 7 nM) coupled with a high degree of selectivity; however, it resulted significantly less active against HCV gt 2a (EC50 = 2.2 μM). The low potency against this latter gt could be explained by a difference in the aa NS4B sequence within the two gts, given that at position 98 there is a leucine (gt 2a) or a phenylalanine (gt 1b). This hypothesis is supported by the evidence that F98L mutation is responsible for drug resistance in gt 1b. The inhibitory activity of

Figure 20. Structures and activities of representative 6-(indol-2yl)pyridine-3-sulfonamides.

generation of escape mutants identified for anguizole and related compounds (see section 2.3.2). More recently, medicinal chemistry efforts involving a further progression of this chemical class have been reported by Zhang et al.114 In this regard, the aforementioned compound 62 was chosen as a suitable starting point for a hit-to-lead optimization strategy especially focused on the improvement of the physicochemical/PK properties, while retaining a comparable anti-HCV activity. In this project, two distinct sets of chemical modifications were carried out involving the benzene ring of the indole core and the N-1 substituent.114 The first set of modifications were planned in order to reduce the oxidative metabolism observed at the expense of the indole benzene ring. 28



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Figure 21. Structures and activities of representative 6-(indol-2-yl)pyridine-3-sulfonamides modified at aminosulfonyl alkyl chain.

Figure 22. Structures and activities of representative 6-(indol-2-yl)pyridine-3-sulfonamides summarizing the chemical optimization process leading to preclinical candidate 70. (A) Representative C-5/C-6 disubstituted indoles. (B) Representative N-1-pyrimidinyl indoles. (C) Compounds derived merging C-5/C-6 and N-1 modifications.

evidence needs further in-depth investigation in order to elucidate the exact mode of action of compound 70.115 After oral administration, lead compound 70 highlighted good PK properties in rats and dogs, while a poor oral exposure was observed in monkeys (F = 18%) (Table 4).115 Compound 70 also showed an excellent safety profile being well tolerated at doses up to 2000 mg/kg/day in a 14-day pilot rat safety study.115 No information has been reported to date neither in literature nor in specialized Web sites regarding the efficacy in animal models. As a continuation of their efforts, the teams at PTC Therapeutics and Merck have recently reported a series of

compound 70 in a combination with other DAAs was also studied in Huh7 cells carrying HCV gt 1b replicon, obtaining an additive effect with boceprevir and a synergistic effect (i.e., enhancement of antiviral activity and reduction of viral drugresistance outbreak) with the non-nucleoside NS5B inhibitor 5(3,3-dimethyl-1-butyn-1-yl)-3-{(trans-4-hydroxycyclohexyl)[(trans-4-methylcyclohexyl)carbonyl]amino}-2-thiophenecarboxylic acid (VX-222).115,116 Although 70 shared the same resistance profile (H94R, F98L, and V105M)113 with anguizole, it did not alter the subcellular distribution of NS4B protein as anguizole when cells were microscopically analyzed. This 29



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Table 4. In Vivo PK Profiles of Lead Compound 70 Following Single Dose Administrationa PK parameters

rat

dog

monkey

AUC0‑∞b (PO,c nM·h) Cmaxd (PO,c nM) Cle (IV,f mL/min/kg) T1/2g (IV,f h) Vdssh (L/kg) f (%)i plasma/(8 h)/EC50 liver/plasma/brain ratio

20000 2400 10.1 7.5 3.9 62 325 25/1/0.22

29000 4000 4.6 8.9 2.1 78 460 NDj

3100 540 11.0 8.0 3.8 18 50 NDj

compounds 71−74 (Figure 23).117 In comparison with the 6(indol-2-yl)pyridine-3-sulfonamide series, a slight decrease in potency was observed for the 4- or 5-azaindoles (e.g., 71 and 72, respectively), while the 6-azaindole subseries (e.g., 73) showed a drop in potency. Conversely, potent derivatives exemplified by compound 74 were obtained by placing the nitrogen atom at 7-position. In the context of 7-azaindole derivatives, small lipophilic substituents at C-5 gave the best compounds (75 and 76), while a decrease in the activity was observed for the unsubstituted analogue 77 or moving the C-5 substituent to the C-6 position of the indole core (78 vs 74) (Figure 24).117 Increasing the size of the substituent also resulted detrimental causing a significant potency loss in C-5-propoxy derivatives 79 and 80.117 Hit 76, the most potent compound within this series, showed also promising PK properties comparable to that of compound 70. In summary, a SAR can be extrapolated for the potent NS4B targeting N-(4′-(indol-2-yl)phenyl)sulfonamides and related compounds (Figure 25): (i) the essential pharmacophore for the anti-HCV activity is constituted by an indole central core bearing a cyano group at the C-3 position, a para-substituted aryl group at the C-2 position and an alkylated nitrogen at N-1 position; (ii) core bioisosteric replacement of the indole ring with a 7-azaindole is also tolerated; (iii) the replacement of the C-2 phenyl ring with a pyridinyl one grants improvement both on the anti-HCV activity and on the physicochemical properties; (iv) small cycloalkyls are the best substituents at the N-1 position, even though heteroaryl are also well tolerated, with 2-pyrimidyl conferring favorable PK properties; (v) the presence of a monosubstituted sulfonamide moiety in paraposition of the C-2 aryl ring furnishes the most potent compounds; (vi) the best substituents for the sulfonamide side chain have to be small and lipophilic in nature, as hydrophilic substituents are very detrimental for the activity; (vii) small lipophilic substituents, such as alkyl and fluorinated groups are preferred at C-6 position; (viii) a fluorine atom at C-5 is not only well tolerated for the activity but also contributes to improve the PK profile. 2.3.4. Piperazinone Derivatives. Kakarla et al. at Pharmasset have recently reported the identification and optimization of a piperazinone class as novel anti-HCV chemotype targeting NS4B.118 Initially, a cell-based HTS using HCV 1b replicon luciferase-reporter assay led to the identification of the hit compound 81 showing promising and selective anti-HCV

a

Data from ref 115. bAUC0‑∞ = area under the curve infinity. cDoses: 10 mg/kg in rat, 5 mg/kg in dog, 5 mg/kg in monkey (PO). dCmax = maximal plasma concentration. eCl = clearance. fDoses: 6 mg/kg in rat, 2.5 mg/kg in dog, 2.5 mg/kg in monkey (IV). gT1/2 = elimination half-life. hVdss = steady-state volume of distribution. if(%) = oral bioavailability. jND = not determined.

Figure 23. Structures and activities of 6-(azaindol-2-yl)pyridine-3sulfonamides.

compounds having an endocyclic nitrogen atom in different positions of the indole core.117 The anti-HCV activity of different azanidoles was explored by walking the endocyclic nitrogen around the six-membered ring as in representative

Figure 24. Structures and activities of 6-(6-azaindol-2-yl)pyridine-3-sulfonamides. 30



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Figure 25. SAR summary of NS4B targeting N-(4′-(indol-2-yl)phenyl)sulfonamides and related compounds. Difference in potency: ≈ equipotent, > from 2- to 10-fold, ≫ more than 10-fold, ≫ more than 100-fold. The substituents depicted in red granted the best PK properties.

activity (Figure 26).118 The mode of action of this new antiHCV chemotype was revealed through the generation of

Figure 26. Structures and activities of piperazinone hit 81 and its isomer 82. aRange of values from four different cell lines (Huh7, HepG2, BxPC3, and CEM).

mutant replicons characterized by mutations at aa 90 and 98 of NS4B (the exact pattern of aa substitution is not reported by the authors). Piperazinone 81 was characterized by a diisobutylpiperazin-2-one core with a S,S configuration at C-3 and C-6 stereocenters, and a (2-phenylcyclopropyl)carbonyl side chain having a trans-R,R configuration. The stereochemistry of compound 81 was indeed very relevant for the activity as different stereochemistry combinations resulted detrimental. For instance, compound 82 with the (S,S) configuration of the cyclopropyl group was completely inactive (Figure 26).118 Researchers at Pharmasset continued their studies with distinct chemical modifications either on the piperazinone core or on the amide side chain, as exemplified by compounds 83− 112 (Figures 27−30).118−120 Thus, keeping constant the stereochemistry of hit 81, alkylation of N-1 amide nitrogen of piperazinone (83) as well as removal of the C-2 carbonyl group (84) resulted in 10-fold loss in anti-HCV activity (Figure 27). These results clearly indicated the endocyclic amide of the piperazinone scaffold as a key pharmacophoric element. Further investigations of the C-3 and C-6 core substituents showed that changes in the alkyls generally led to potency loss (85 and 86 in Figure 27). The replacement of the C-6-isobutyl in compound 81 with a n-propyl (87) was the only permitted modification (Figure 27). Regarding the amide side chain, different replacers of the cyclopropyl bridge were investigated showing that a trans double bond (88), an oxazole ring (89), and an oxadiazole ring (90) were well tolerated (Figure 28A). Removal of the side-

Figure 27. Structures and activities of representative piperazinone derivatives.

chain carbonyl group of derivative 88 did not affect significantly the anti-HCV activity (91 in Figure 28A). However, the replacement of the cyclopropyl group with a cis double bond (92) as well as with a phenyl (93) or other heterocycles (94 and 95) caused a loss of antiviral activity (Figure 28B). Moreover, the presence of a phenyl group in the amide side chain resulted essential as shown by the inactivity of derivative 96 (Figure 28B). Based on the encouraging results obtained for cinnamide 88 and 5-phenyloxazolylamide 89 derivatives, the authors explored the influence on biological activity of selected para substituents at the phenyl ring. Different results were obtained modifying cinnamide 88 to compounds 97−100 and 5-phenyloxazolylamide 89 to derivatives 101−104 (Figure 29). In general, both electron-donating (97 vs 88) and electron-withdrawing (99 vs 88) groups resulted detrimental for the activity in cinnamide 31



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Figure 28. Structures and activities of representative piperazinones modified on the amide side chain. (A) Favorable chemical modifications. (B) Unfavorable chemical modifications.

Figure 29. Structures and activities of representative (A) cinnamate and (B) oxazole piperazinones modified at the para-position of the phenyl ring.

Figure 30. Structures and activities of representative oxazole piperazinones modified at the C-6 position of piperazinone core.

32



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Figure 31. SAR summary of NS4B targeting piperazinone class. Difference in potency: ≈ equipotent, > from 2- to 10-fold, ≫ more than 10-fold, ≫ more than 100-fold.

Figure 32. Structures and activities of representative 2-oxadiazoloquinolines summarizing the chemical optimization process from the initial hit 113 to 120.

series (Figure 29A). Only, p-chloro 101 and the p-fluoro 102 phenyl-substituted derivatives showed improvements in potency over their parent 88 (Figure 29A). Conversely, substituted 5-phenyloxazolylamide analogues 101−104 retained reasonable antiviral activity with the p-chloro 103 and the p-fluoro 104 piperazinones having sub-μM potency (Figure 29B). Finally, the authors came back on the C-6 position of the piperazinone core by replacing the isobutyl group with either

different hydrophobic moieties (acyclic, branched, cyclic, and saturated/unsaturated alkyls) or alkyl and aryl substituents having H-bond acceptor or donor properties.118−120 In general, acyclic and cyclic alkyls (e.g., 105 and 106) were tolerated with the exception of the totally inactive C-6 sec-butyl derivative 107 (Figure 30). Adding polar functionalities such as a hydroxyl (108) or a methoxyl (109) group at the C-6 alkyl substituents also caused a significant decrease in antiviral potency (Figure 30). On the contrary, the insertion of unsaturated systems gave 33



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recombinant HCV NS4B gt 1b), showing a KD of 31 nM (Figure 32). The classical bioisoteric replacement of the oxygen with a nitrogen in the C-6 substituent (116) further enhanced the anti-HCV activity (Figure 32).121 Starting from derivative 115, the methylene-1,3-dioxolane group was replaced by substituted cyclobutyl groups, furnishing compounds (117− 120, Figure 32) potent against both HCV gt 1b and 2a.121 Stereochemistry of the cyclobutyl moderately influenced the activity especially against HCV gt 2a, with the 1,3-cissubstitution pattern being the best (117 vs 119). The combination of the hydroxycyclobutyl substituent with the C6-trifuoloethylamino group as in compound 120, resulted in an impressive increase in the anti-HCV gt 2a activity (EC50 = 25 nM, Figure 32). After a systematic exploration of cycloalkyl alchols, the (1S,3R)-cis-aminocyclohexanol side chain proved to be the most efficacious, with compound 121 endowed with pM antiHCV gt 1b activity and a single-monodigit nM anti-HCV gt 2a activity (Figure 33).121 Given its impressive potency, this new

a slight increase in potency as in the propenyl derivative 110 and phenyl analogue 111 (Figure 30). Notably, the introduction of heterocyclic rings at C-6 position resulted very advantageous, with the C-6-(2-thiophene) analogue 112 displaying nM inhibitory potency in the replicon assay (EC90 (gt 1b) = 0.006 μM) (Figure 30). In addition, derivative 112 showed good activity against HCV gt 1a (EC90 = 0.022 μM) but resulted inactive against HCV gt 2. Due to their lack of broad genotype coverage, the anti-HCV piperazinones targeting NS4B were abandoned by Pharmasset and did not progress into further preclinical development.118 To sum up, a partial SAR on anti-HCV piperazinone derivatives targeting NS4B can be delineated (Figure 31): (i) the N-unsubstituted piperazinone is a key pharmacophore element as well as the S configuration at both C-3 and C-6 stereocenters; (ii) the only information on the C-3 position of piperazinone core is about the replacement of the isobutyl group with the ethyl moiety, which leads to a complete loss of activity; (iii) a 2-thiophene at C-6 of piperazinone core grants the highest potency (in the monodigit nM range) but a phenyl, 1-propenyl, and n-propyl or isobutyl group is also tolerated, while smaller as well as bulkier alkyls are instead detrimental; (iv) the carbonyl group of the N-4 side chain is important but not essential for the activity; (v) an oxazole as well as a trans(R,R)-cyclopropyl group or oxadiazole result in the best linkers in the N-4 side chain; (vi) the phenyl group of the side chain is fundamental since its removal leads to loss of potency and its replacement with heteroarenes is not favorable; (vii) the paraposition of the phenyl was extensively explored, and a fluorine turned out to be the best substituent. 2.3.5. 2-Oxadiazoloquinoline Derivatives. Very recently, Phillips et al. at Gilead Sciences reported the development of 2oxadiazoloquinoline derivatives as NS4B functional inhibitors endowed with potent pan-genotypic anti-HCV activity, although the authors did not clearly indicate how this class of compounds was first identified.121 The initial hit 113 had excellent and selective activity against HCV gt 1b (EC50 = 5.9 nM, CC50 = 86.3 μM), but it was inactive against HCV gt 2a (EC50 > 20 μM) and characterized by high lipophilicity (LogD = 5.6) (Figure 32).121 Impressive chemical modifications were realized with the double aim of (i) improving the physicochemical properties (i.e., reduction of lipophilicity) and (ii) obtaining anti-HCV gt 2a activity. The optimization strategy adopted by the authors was oriented toward decreasing the molecular planarity of hit 113 by modifying the two phenyl groups at each end of the molecule. Thus, the phenyl ring at the aminoxadiazole moiety was replaced by several cycloalkyl ethers with the methylene-1,3-dioxolane moiety emerging as the best one. In addition, alkyl ethers (e.g., trifluoroethyloxy) at C-6 position of quinoline core proved to be effective replacers of the C-6-phenyl group. As a consequence of these medicinal chemistry efforts, derivative 114 was identified (Figure 32).121 Compared to 113, compound 114 showed (i) reduced lipophilicity (LogD = 3.6); (ii) increased anti-HCV gt 2a activity (EC50 = 15.7 μM); (iii) while maintaining equal potency against HCV gt 1b (Figure 32). A 4-fold increase of the potency in gt 1b replicon (EC50 = 1.3 nM) as well as significant improvement against gt 2a (EC50 = 3.6 μM) was instead observed by replacing the C-8-trifluoromethyl on the quinoline nucleus with a tert-butyl group (115) (Figure 32).121 In order to clarify the mode of action of this new anti-HCV class, representative compound 115 was then submitted to a NS4B binding assay (scintillation proximity assay using a

Figure 33. Structure and activities of pan-gt HCV inhibitor 121.

derivative was also evaluated in a panel of HCV replicons to assess the activity across a broad range of gts as well as against 1b-resistant mutants. Of note, derivative 121 displayed high potency (EC50 < 4 nM) against all the replicons included in the study. Due to its excellent biological profile, the lead compound 121 was selected for PK studies in rats, and the results indicated it is a very promising drug candidate (Table 5). Anyway, to date no information about progression in clinical trials has been reported for compound 121. Although no chemical modifications on both the quinoline core and the aminoxadiazole moiety have been described so far, it is possible to extrapolate a partial SAR on the basis of the substituents placed around the quinolineoxadiazole scaffold (Figure 34): (i) at C-6 position of quinoline core, small fluorinated alkylamino or alkyloxy chains are superior Table 5. Physicochemical Properties and PK Profile of Compound 121a PK profile in rat LogD

HLM Clb (L/h/kg)

Clc (L/h/kg)

T1/2d (h)

f (%)e

3.6

0.22

0.65

11

88

a Data from ref 121. bHLM Cl = predicted metabolic drug clearance in human. cCl = clearance. dT1/2 = elimination half-life. ef (%) = oral bioavailability.

34



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Figure 34. SAR summary of NS4B targeting 2-oxadiazoloquinoline class. Difference in potency: > from 2- to 10-fold, ≫ more than 10-fold.

Figure 35. Structures and activities of miscellaneous compounds.

NS4B sequence (K52R, G120V, A210S).84 To our knowledge none of these compounds has been subjected to further development with the exception of derivatives 126 and 128 for which related molecules have been reported in a patent as antivirals active against several viruses including HCV.122 In 2010, Gleen et al. claimed in a patent a series of amiloride analogues identified through AH2-mediated lipid vesicle aggregation inhibition assay.123 These derivatives, exemplified by compound 129, showed interesting anti-HCV activity against both gt 1b and gt 2a coupled with no toxicity in cell line (Figure 35).83,123 Very recently, Thompson et al. at GlaxoSmithKline pursued an ELT screening approach89,90 (see section 2.2) using immobilized NS4B and 28 libraries containing 1 million to 8 billion compounds with diverse chemical structures.85 After the screening, two families of NS4B binders have been identified, one dominated by the bipiperidyl-triazine scaffold and the other by the spyro-diazaundecane pyrimidine core. Within the latter

compared to phenyl and methoxyl groups; (ii) at C-8 position a tert-butyl is favored over the trifluoromethyl, even though the SAR at this position has not been fully explored; (iii) four- to six-membered cycloalkyl alcohols with syn-arrangement at the aminoxadiazole moiety are more favorable for the activity than simple cycloalkyls, dioxolane, and aryl substituents. 2.3.6. Miscellaneous. In this section NS4B ligands are reported for which, despite their interesting biological profile, no information regarding a systematic chemical optimization aimed at developing preclinical drug candidates is available in the literature. In particular, Chunduru et al. identified compounds 122− 128 by employing a quenching fluorescence binding assay (Figure 35).84 These compounds were shown to bind NS4B with an affinity in the low μM range and to inhibit HCV protein expression, as determined by an ELISA-based HCV replication assay. Only for the triazinoindole derivative 127 genetic validation was provided with mutations located in 35



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Figure 36. Schematic representation of NS4B structure showing the mutation sites that confer drug resistance to representative ligands. Amino acid changes associated with resistance are depicted in color-coded circles according to the different ligand classes.

family, compound 130 (GSK2189, Figure 35) was the best ELT-identified hit.85 It was endowed with high affinity to NS4B and good potency against HCV gt 1b, but its activity decreased from moderate to weak in gt 1a and 2a, respectively (data not reported by the authors). An early optimization aimed at enlarging the gts coverage allowed the synthesis of derivative 131 (GSK0109, Figure 35) endowed with a better biological profile against gts 1a and 2a.85 After in-depth biological evaluation, compound 131 was found to be a potent HCV replicon inhibitor of gts 3a and 5a, a good inhibitor of gts 4a and 6a and a modest inhibitor of gts 2b and 6o. Mutational studies highlighted that the activity of the tested compound was sensitive to F98L alteration, while it was not affected by H94R and V105M mutations. Overall, the mutational profile is partially different from those observed for other classes of NS4B binders such as anguizole, anguizole-related compounds, and indole-based compounds, while a mutation at aa 98 was similarly observed in the case of piperazinone derivatives.

the MW formation (AH2-mediated lipid vesicle aggregation inhibition assay). More recently, an innovative approach has entailed the use of nontraditional ELT screening to detect specific NS4B binders. Despite the availability of such biochemical assays, most of the promising NS4B binders reported so far derive from optimization process of hits identified by HTS campaigns using Huh cells containing HCV replicons. Successive mutational studies revealed NS4B as target and allowed the identification of the protein region involved in the ligand binding. Unfortunately, this combined approach is mainly suitable to big pharma companies, and thus, it is very desirable that the aforementioned in vitro assays could become widely available, reliable, and fast to help researchers in testing large compound libraries. The information available about mutation sites was collected and schematized in Figure 36 with the aim to provide an easy tool that could assist medicinal chemists to associate a NS4B binder with its interaction site within the protein. Briefly (i) H94R, F98L, and V105M mutations at the TM1 segment confer resistance to anguizole and related compounds as well as to other structurally unrelated chemical families (i.e., indolopyridine solfonamides and piperazinones); (ii) the single mutation F98L was observed for the spiro-diazaundecane pyrimidines; (iii) W55R mutation within the AH2 amphipathic α helix and R214Q within the cytoplasmic C-terminal segment of NS4B confer resistance to clemizole and analogues; (iv) the K52R replacement at the AH2 region, G120V in the TM2 segment, and A210S within the C-terminal segment are responsible of resistance for triazinoindole derivatives. The mode of action of some of the aforementioned NS4B binders has been deeply characterized. Anguizole and some

3. SUM-UP AND DISCUSSION During the past years, significant improvements have been made in understanding the NS4B function and structure as well as in the identification of new anti-HCV agents targeting this viral protein. Structural data regarding NS4B protein are not yet available thus hampering structure-based drug design programs. Indeed, NS4B purification and crystallization are, and probably will be in the near future, a challenge due to its membranous nature. Toward the discovery of new NS4B ligands, some biochemical assays have been developed (see section 2.2) to identify compounds that specifically bind NS4B preventing either the RNA binding activity (microfluidic affinity assay) or 36



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leads targeting NS4B until now reported, no final inference can be drawn. In conclusion, the information herein reported can provide a helpful instrument for those medicinal chemists dealing with research programs focused on NS4B and aimed at the identification of innovative anti-HCV compounds.

analogues proved to affect MW formation by inhibiting the NS4B AH2 vesicle aggregation-promoting activity, while clemizole and derivatives inhibited NS4B RNA binding activity. It has been also demonstrated that indolopyridine 70 did not alter the subcellular distribution of NS4B; this behavior is worth noting considering that 70 shares the same mutation pattern with anguizole. We believe that the different chemical classes acting on the NS4B TM1 domain (Figure 36) might have a common ligandbinding site, as suggested by the similar chemical features and resistance profile shared by these compounds. Our hypothesis is supported by a work published by Thompson et al. in the early 2015, where different NS4B TM1 ligands were shown to have similar 3D geometry by using a computational molecular operating environment method.85 In this review particular attention was dedicated to the SAR for each class of compounds highlighting the efforts made to improve physicochemical/PK properties of the identified hits. Our analysis has revealed that all known NS4B inhibitors are heterocyclic compounds with suitable physicochemical features, and chemical characteristics in agreement with the criteria identified for most drugs (e.g., Lipinski’s rule). The comprehensive work has been conceived after an in-depth study of the available literature, but we have realized that in the past few years several patents on NS4B inhibitors have been published. This means that a possible new drug candidate may be already available among the very numerous chemical entities reported in these patents. To our knowledge, the most advanced NS4B binder is the antihistamine drug clemizole hydrochloride, which is the only one in phase Ib clinical trials to test its safety and tolerability when administered to treatmentnaive patients chronically infected with HCV. Moreover, imidazopyridine 18 is the only compound for which in vivo proof-of-concept for anti-HCV agents targeting NS4B has been reported. Other promising preclinical candidates are the pyrazolopyridine 27, the indole 70, and the 2-oxadiazoloquinoline 121. The activity of derivative 27 and 70 is gt dependent and limited to HCV gt 1 as for most of the NS4B targeting compounds. Conversely, 2-oxadiazoloquinoline 121 has broad anti-HCV gt coverage and may represent a starting point for the development of a pan-gt anti-HCV agent. The recent approval of DAAs combination treatments has opened a new era for the therapy of patients with chronic HCV. Given that DAAs aim to eradicate the infection or to reach at least a SVR > 90%, these new therapies may have damped the interest in new HCV inhibitors. Sooner or later, however, these therapeutic regimens will have to deal with drug resistance development thus making necessary the use of new DAAs. In this context, the development of drugs targeting NS4B is certainly of great interest and may increase the barrier to resistance. Apart from any therapeutic use of NS4B binders for the treatment of chronic hepatitis C, the efforts made for setting specific in vitro testing models, especially the NS4B AH2mediated lipid vesicle aggregation inhibition assay, and the inactivity demonstrated by compound 5 on flavivirus suggest that (i) a similar assay might be developed to identify compounds targeting flavivirus NS4B since the MW formation property is shared by HCV and flavivirus NS4B proteins and (ii) it is not obvious that anti-HCV compounds able to interact with the HCV NS4B can also prove the efficacy against flavivirus. However, due to the absence of strong experimental data regarding the antiflavivirus activity of all the anti-HCV

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +39-0755855126. Fax: +39-075-5855115. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. Biographies Rolando Cannalire received his master degree in Pharmacy summa cum laude at the University of Perugia (Italy) in 2011. He is currently a student of the Ph.D. course “Medicinal Chemistry” at the University of Perugia under the supervision of Prof. Cecchetti. In 2013 he spent a period at the Instituto de Higiene e Medicina Tropical of Universidade Nova de Lisboa (Portugal) under the supervision of Prof. Viveiros, working on antimicrobial resistance. In 2014 he was also a visiting Ph.D. student in the group headed by Prof. Otto at Stratingh Institute for Chemistry at University of Groningen (Netherlands), focusing on dynamic combinatorial chemistry. He is the author of two papers in the HCV research field, and his main research interests concern the design and synthesis of antiviral small molecules. Maria Letizia Barreca obtained her MSc cum laude in Pharmaceutical Chemistry and Technology (1992), received her Ph.D. in Pharmaceutical Sciences (1996), and was appointed as Assistant Professor (1999) at the University of Messina. Between 1993 and 1999 she spent long research periods at the University of Bari, and in 2000−2001 was Visiting Scientist at the University of Houston. In 2007, she was Visiting Scientist at the Department of Chemistry, University of Perugia, and in 2008 she moved to the Department of Pharmaceutical Sciences of the same university. Her research focuses on computational modeling and drug discovery, and the main topics include HIV, HCV, neurodegenerative, and inflammatory diseases. She is the author of 64 papers and one patent. In 2008, she was awarded the “Farmindustria Prize for Pharmaceutical Research”. Giuseppe Manfroni graduated with full marks in Pharmaceutical Chemistry and Technology (March 2001) and received his PhD in Medicinal Chemistry (February 2006) from the University of Perugia (Italy). From 2006 to 2008 he worked as a postdoctoral researcher at the University of Perugia. From 2008 to date he is Assistant Professor at the Department of Pharmaceutical Sciences and he is a lecturer in Pharmaceutical Analysis. He spent short periods as a visiting PhD student at Rega Institute for Medical Research (Leuven, Belgium) and at the Molecular Modelling Laboratory (University of Perugia) under the supervision of Prof. Johan Neyts and Prof. Gabriele Cruciani, respectively. He is the author of 32 papers, and his research is mainly focused on Medicinal Chemistry of antiviral (HIV, HCV, and flavivirus), antitumor, and anti-inflammatory (p38 inhibitors) agents. Violetta Cecchetti graduated summa cum laude in Pharmacy (1979) and received her PhD in Medicinal Chemistry (1987) at the University of Perugia. Since 2000 she has been Full Professor at the University of Perugia. She spent working periods as Visiting Researcher at the Laboratory of Molecular Biophysics, Oxford University (UK) and at 37



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(14) Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.; Rice, C. M. Hepatitis C Virus-encoded Enzymatic Activities and Conserved RNA Elements in the 3′ non-Translated Region are Essential for Virus Replication in vivo. J. Virol. 2000, 74, 2046−2051. (15) Yanagi, M.; St Claire, M.; Emerson, S. U.; Purcell, R. H.; Bukh, J. In vivo Analysis of the 3′ Untranslated Region of the Hepatitis C Virus After in vitro Mutagenesis of an Infectious cDNA Clone. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 2291−2295. (16) Tsukiyama-Kohara, K.; Iizuka, N.; Kohara, M.; Nomoto, A. Internal Ribosome Entry Site within Hepatitis C Virus RNA. J. Virol. 1992, 66, 1476−1483. (17) Wang, C.; Sarnow, P.; Siddiqui, A. Translation of Human Hepatitis C Virus RNA in Cultured Cells is Mediated by an Internal Ribosome-binding Mechanism. J. Virol. 1993, 67, 3338−3344. (18) Otto, G. A.; Puglisi, J. D. The Pathway of HCV IRES-mediated Translation Initiation. Cell 2004, 119, 369−380. (19) Ashfaq, U. A.; Javed, T.; Rehman, S.; Nawaz, Z.; Riazuddin, S. An Overview of HCV Molecular Biology, Replication and Immune Responses. Virol. J. 2011, 8, 161−171. (20) Eckart, M. R.; Selby, M.; Masiarz, F.; Lee, C.; Berger, K.; Crawford, K.; Kuo, C.; Kuo, G.; Houghton, M.; Choo, Q. L. The Hepatitis C Virus Encodes a Serine Protease Involved in Processing of the Putative non-Structural Proteins from the Viral Polyprotein Precursor. Biochem. Biophys. Res. Commun. 1993, 192, 399−406. (21) Hijikata, M.; Mizushima, H.; Akagi, T.; Mori, S.; Kakiuchi, N.; Kato, N.; Tanaka, T.; Kimura, K.; Shimotohno, K. Two Distinct Proteinase Activities Required for the Processing of a Putative nonStructural Precursor Protein of Hepatitis C Virus. J. Virol. 1993, 67, 4665−4675. (22) Gawlik, K.; Gallay, P. A. HCV Core Protein and Virus Assembly: What We Know without Structures. Immunol. Res. 2014, 60, 1−10. (23) Bartosch, B.; Dubuisson, J.; Cosset, F. L. Infectious Hepatitis C Virus Pseudo-Particles Containing Functional E1-E2 Envelope Protein Complexes. J. Exp. Med. 2003, 197, 633−642. (24) Khaliq, S.; Jahan, S.; Hassan, S. Hepatitis C Virus p7: Molecular Function and Importance in Hepatitis C Virus Life Cycle and Potential Antiviral Target. Liver Int. 2011, 31, 606−617. (25) Dentzer, T. G.; Lorenz, I. C.; Evans, M. J.; Rice, E. C. Determinants of the Hepatitis C Virus non-Structural Protein 2 Protease Domain Required for Production of Infectious Virus. J. Virol. 2009, 83, 12702−12713. (26) Raney, K. D.; Sharma, S. D.; Moustafa, I. M.; Cameron, C. E. Hepatitis C Virus non-Structural Protein 3 (HCV NS3): a Multifunctional Antiviral Target. J. Biol. Chem. 2010, 285, 22725− 22731. (27) Suhy, D. A.; Giddings, T. H., Jr.; Kirkegaard, K. Remodeling the Endoplasmic Reticulum by Poliovirus Infection and by Individual Viral Proteins: an Autophagy-like Origin for Virus Induced Vesicles. J. Virol. 2000, 74, 8953−8965. (28) Egger, D.; Wölk, B.; Gosert, R.; Bianchi, L.; Blum, H. E.; Moradpour, D.; Bienz, K. Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex. J. Virol. 2002, 76, 5974−5984. (29) Gosert, R.; Egger, D.; Lohmann, D.; Bartenschlager, R.; Blum, H. E.; Bienz, K.; Moradpour, D. Identification of the Hepatitis C Virus RNA Replication Complex in Huh-7 Cells Harbouring Subgenomic Replicons. J. Virol. 2003, 77, 5487−5492. (30) Moradpour, D.; Gosert, R.; Egger, D.; Penin, F.; Blum, H. E.; Bienz, K. Membrane Association of Hepatitis C Virus non-Structural Proteins and Identification of the Membrane Alteration that Harbors the Viral Replication Complex. Antiviral Res. 2003, 60, 103−109. (31) Gouttenoire, J.; Penin, F.; Moradpour, D. Hepatitis C Virus non-Structural Protein 4B: a Journey into Unexplored Territory. Rev. Med. Virol. 2010, 20, 117−129. (32) Schmitz, U.; Tan, S. L. NS5A − From Obscurity to New Target for HCV Therapy. Recent Pat. Anti-Infect. Drug Discovery 2008, 3, 77− 92.

the Institut für Lasermedizin, Heinrich-Heine-Universität (Germany). She collaborated in the development and introduction of therapy for the first once-daily antibacterial fluoroquinolone, rufloxacin. Currently she is the director of the five-year degree in Pharmacy at the University of Perugia. Her scientific activity is documented by 90 publications and six patents. Prof. Cecchetti’s group is mainly involved in the design/ synthesis of bioactive heterocycles and molecular modelling studies with particular regard to anti-infective drug discovery.

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ABBREVIATIONS USED aa, amino acid; DAA, direct-acting antiviral; ELT, encode library technology; EMA, European Medicinal Agency; ER, endoplasmic reticulum; FDA, Food and Drug Administration; HCV, hepatitis C virus; gts, genotypes; HLM, human liver microsomal; HTS, high-throughput screening; IFN, interferon; IRES, internal ribosome entry site; MW, membranous web; NMB, nucleotide binding motif; NS, nonstructural; NTP, nucleotide triphosphate; ORF, open riding frame; PI, protease inhibitors; PK, pharmacokinetic; RBV, ribavirin; S, structural; SAR, structure−activity relationship; SOC, standard-of-care; ss(+)-RNA, single strand plus RNA; TM, transmembrane

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A Journey around the Medicinal Chemistry of Hepatitis C Virus

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