key: cord-1001424-d4qcckh1
authors: Meanwell, Nicholas A.; Serrano-Wu, Michael H.; Snyder, Lawrence B.
title: Chapter 22. Non-HIV antiviral agents
date: 2003-12-31
journal: Annual Reports in Medicinal Chemistry
DOI: 10.1016/s0065-7743(03)38023-6
sha: e9f41ee674c330fe3fb6c9a089af070d6bc6ca8c
doc_id: 1001424
cord_uid: d4qcckh1

Publisher Summary This chapter focuses on non-HIV antiviral agents. The development of antiviral agents to treat non-HIV infections is largely focused on therapies for the treatment of chronic hepatitis infections B and C. Nucleoside analog continue to be the mainstay of Hepatitis B Virus (HBV) therapeutics. The first small molecule inhibitor of Hepatitis C Virus (HCV), the NS3 protease inhibitor BILN-2061, entered phase 2 clinical trials, producing a striking reduction in viral load in treated individuals. The development of the HCV replicon system and its application to screening for antiviral agents provided tangible benefit with the disclosure of mechanistically and structurally diverse HCV inhibitors. Adefovir dipivoxil has been approved in the United States and the European Union for the treatment of HBV, providing a second small molecule antiviral to add to lamivudine (3TC) and the injectable protein IFNα as the only approved agents for treating HBV infection. The chapter also provides details of the inhibitors of hepatitis B and C virus, the inhibitors of simplex virus and human cytomegalovirus, the inhibitors of respiratory viruses and the inhibitors of West Nile virus and Papilloma virus.

Introduction -The development of antiviral agents to treat non-HIV infections is largely focussed on therapies for the treatment of the chronic hepatitis infections B and C (1). Nucleoside analogues continue to be the mainstay of HBV therapeutics and clinical development of several continued during 2002. The last year has seen the first small molecule inhibitor of HCV, the NS3 protease inhibitor BILN-2061, enter phase 2 (P2) clinical trials, producing a striking reduction in viral load in treated individuals.

The development of the first HCV replicon system in 1998 and its application to screening for antiviral agents is beginning to provide tangible benefit with the disclosure of mechanistically and structurally diverse HCV inhibitors. There remains considerable interest in inhibitors of herpes simplex and human cytomegalovirus viruses, particularly non-nucleoside compounds. Developments in the area of respiratory virus inhibitors have focussed more on respiratory syncytial virus with a description of the first antiviral active in animal models following oral administration.

The West Nile virus outbreak in the US, originally confined to the East coast, broadened considerably during the summer of 2002, claiming 230 lives. In the wake of the events of September Ilth, 2001, smallpox was a prominent concern as a potential agent of bioterrorism. Developments in each of these areas will be reviewed.

Inhibitors of Hepatitis B Virus (HBV) -Adefovir dipivoxil iHepsera'") (1) was approved in the US for the treatment of HBV on September 20', 2002 and in the European Union on March 1 lth, 2003, providing a second small molecule antiviral to add to lamivudine (3TC) and the injectable protein IFNa as the only approved agents for treating HBV infection. Adefovir is an effective inhibitor of 3TC-resistant HBV caused by the rtM2041 and rtLl8OM + rtM204V mutations in the reverse transcriptase

(2) and resistance to adefovir has not been seen after 48 weeks of monotherapy (3, 4) .

A clinical study with entecavir (z), currently undergoing P3 trials, compared a dose of 0.1-I mg/day of 2 to 3TC (100 mg/day) for 48 days in 181 patients previously unresponsive to 3TC treatment (5). Compared to 3TC, treatment with 2 resulted in lower overall viral loads, lower ALT levels and a higher proportion of patients with undetectable HBV DNA levels. Moreover, adverse events for 2 were less than those observed with 3TC. A separate P2 trial of 2. in 177 treatment-naive patients for 22 weeks at a dose of 0.5 mglday showed that reduction in viral DNA levels was independent of baseline ALT levels, with treatment resulting in a 4.7-4.8 loglo reduction in HBV DNA (6).

L-nucleosides represent a promising area of antiviral research (7). LdT (telbivudine, 3) is currently in P3 clinical trials designed to evaluate 1200 patients for safety and efficacy compared with standard treatment in HBeAg+ and . Recent P2b data was released from a trial involving 104 adults randomized to receive 3TC plus 2 or 3TC monotherapy once daily for 1 year. Viral load reductions of greater than 6 logto were seen for all patients in the study arms containing 3 and no treatment-limiting or dose-related adverse events were reported. The mechanism of action of L-FMAU (clevudine) (fi), currently in PI/P2 trials, is not firmly understood but recent molecular dynamics simulation experiments have suggested that the triphosphate derivative of 4 may act as a competitive inhibitor rather than as a substrate of HBV polymerase (9,lO). Emtricitabine (CoviracilTM) (5) is also currently in P3 clinical trials for the treatment of HBV. An NDA seeking approval to market 5 for the treatment of HIV was filed in September 2002 (9). Results from a PI/P2 cli%zal trial with ACH-126443 (elvucitabine, 5) have been released (11-13).

In 36 HBV treatment-naive patients receiving single daily doses of l-100 mg of 5, mean declines in plasma HBV DNA of up to 2.5 loglo were observed after 14 days of treatment. Furthermore, plasma levels in excess of the I&O values for wild type and YMDD mutants were achieved in the low dose arms. It is therefore anticipated that S will be efficacious against 3TC resistant infections in an ongoing P2 trial. This mutation, which arises in response to 3TC therapy, is found in the precore region and confers HBeAg negativity.

The major metabolite of 7 formed in rat or human serum is the mono-ester, which is a more potent HBV inhibitor, EC% = 70 nM, than 3TC. It is postulated that the arylthio moiety is responsible for the specificity towards HBV and lower cytotoxicity than PMEA, the active component of adefovir dipivoxil. Additional analogs in this structural class have been prepared with the phenylthio-and 3-methoxyphenylthio ethers showing the most promise whilst other aromatic thioethers exhibited higher cytotoxicity (14, 151) .

Non-nucleoside inhibitors of HBV are beginning to emerge that are anticipated to show reduced cross-resistance with nucleoside analogues. AT130 (B) and its close analog AT61 are active against wild type HBV and the rtLl80M, rtM2041, and rtLl80M+rtL204V mutants (EC50 = 2-5 PM) in HepG2-derived cells (17, 18) . It has been postulated that 8 interferes with the packaging of pregenomic viral RNA resulting in inhibition of viral reverse transcription. Pyridinedicarboxamide S represents the first report of a non-nucleoside inhibitor of HBV reverse transcriptase enantiomer of q is active in cell culture and appears to prevent proper formation of the viral nucleocapsrd.

Inhibitors of Hepatitis C Virus (HCV) -Nearly 170 million individuals are infected with hepatitis C virus (HCV) worldwide and HCV infection is responsible for 8,000-10,000 deaths annually in the United States, a burden expected to increase significantly (22, 23) . A second pegylated interferon-a (IFN), Roche's Pegasys, was approved in 2003, both as mono therapy and in conjunction with ribavirin (11) (CopegusTM) (24). However, safety concerns with combination therapy remain, as the accumulation of 11 in erythrocytes can lead to hemolytic anemia.

This has prompted a search for safer interferon co-therapies which include the active enantiomer of IJ, levovirin, and the prodrug viramidine (12), which improves liver at the expense of erythrocyte exposure (2526).

The development of safe, efficacious, and HCV-specific antiviral agents remains an important goal and the development of subgenomic HCV replicons has dramatically enhanced the potential to identify inhibitors (27, 28) .

A significant advance towards establishing a correlation between replicon inhibition and clinical efficacy was recently accomplished with the disclosure of preliminary clinical data for BILN-2061, a selective inhibitor of the NS3 serine protease of HCV that is structurally related to 13. This highly modified macrocylic tripeptide derivative is extremely potent in vitro, with K, values of 0.3 nM and 0.66 nM towards HCV-la and HCV-1 b NS3 proteases, respectively (29). These figures are similar to the potency observed in cell culture in the cognate HCV replicons, EC50 = 4 nM and 3 nM, respectively (29). Antiviral efficacy was established in a study conducted with 10 patients with chronic HCV and significant liver fibrosis, where all patients treated with BILN-2061 (200 mg p.o. b.i.d.) displayed a decrease in serum HCV RNA levels of at least 2.0 log,, copies/mL after two days of treatment (30). Four of these patients recorded a reduction in viral load of more than 3 orders of magnitude and viral titers returned to baseline following cessation of therapy, with no drug-related safety issues identified (31). The intensity of effort devoted towards the discovery of inhibitors of HCV NS3 has continued, with the focus largely on peptide-based molecules that are required to effectively complement the active site and proximal regions of the protease (32-34). The crystal structure of a macrocyclic inhibitor related to 13 bound to HCV protease has been disclosed and an acyclic tripeptidic inhibitor has been used to generate resistant subgenomic replicons in which mutations mapped to the protease (3836). Amongst several strategies disclosed, the C-terminal carboxylate of peptide inhibitors may be replaced with an N-acyl sulfonamide moiety, as exemplified by 14 (37). Novel approaches to constrain or mimic the peptidic backbone are represented by macrocycle l5, the tetrahydroindolizine B (IC 5~ = 0.12 PM), the imidazolone 17 and the bicyclic proline derivative 18 (K, = 0.042 PM, KS0 = 0.251 pM) (38-46). These inhibitors are constructed around either an a-keto amide or a boronic acid moiety, wellprecedented as serine protease inhibitor motifs that engage the catalytic serine residue in a covalent but reversible interaction.

Ph 1 I However, chemical reactivity is not a prerequisite for potent inhibition in a peptidic background since phenethylamide 19 and the azapeptide 20 inhibit HCV NS3 with KI values of 0.6 and 0.2 PM, respectively (47,48). The more active diastereomer of the ahydroxy amide P3 element explored in the context of 21 was found to possess the (I?)configuration, unanticipated and explained by the presence of an intramolecular hydrogen bond that orients the lipophilic moiety of this isomer into S3, as depicted (49). Non-peptidic inhibitors of NS31NS4A protease are much less common but some progress has been made in this direction.

The bicyclic lactam 22 is a mechanismbased inhibitor of HCV NS3 whilst additional examples of bis-benzimidazole derivatives that rely upon Zr?' to consolidate the enzyme-inhibitor complex have been described (50,51). The NS5B RNA polymerase is another structurally-characterized viral protein that is an attractive target for therapeutic intervention (52-55). Inhibitors of HCV polymerase can be broadly divided into nucleoside and non-nucleoside derivatives. The nucleoside analog ribavirin (II) has been suggested to interfere with both the initiation and elongation steps of HCV RNA replication (52). Several HCV NS5b inhibitors incorporate modified D-ribose elements and include the 2'-Me derivative 23, EC50 = 0.25 PM, the 4'-azido analog 24, EC 50 = 1.2 PM, and the 2'-deoxy-2'-fluoro cytidine derivative 25, EGO = 0.74 PM (56-59).

Several non-nucleoside inhibitors of HCV NS5B have been reported, including a series of phenylalanine derivatives of which compound 28, K, = 2.2 FM, is representative (60,61). This compound has been co-crystallized with NS56 and appears to bind to the inactive, open conformation of the polymerase almost 35 A from the active site (62). Other scaffolds with which HCV polymerase inhibitors have been discovered include an amino thiophene, represented by 27, the enolic rhodanine 28 (I&O = 1 .O PM), and structural variations of previously disclosed benzimidazole derivatives 29 (GO c 0.5 FM) claimed to be active in replicons (61,63-67).

Mechanistic studies with the benzo[l,2,4]thiadiazine polymerase inhibitor 30 suggest interference with the initiation step of viral RNA synthesis, allowing for a potential synergy with existing elongation inhibitors (68).

to HCV therapy include blocking the viral RNA internal ribosomal entry site (IRES), binding of the viral E2 envelope glycoprotein or attachement (69-72). The highly conserved IRES has been targeted by oligonucleotides and artificial ribozymes, but little progress has been made in the development of small molecule inhibitors (70,71). P2 clinical evaluation of the antisense 20-mer oligonucleotide ISIS-14803 revealed a l-2 logto reduction in plasma HCV RNA levels in approximately 30% of the patients after 4 weeks of treatment (73). Another approach, which may prove complementary to virus-specific HCV therapy, is the induction of interferon production in host cells. Small molecules that act via toll-like receptor activation have been identified as activators of an immune response (74). RNA interference (RNAi) is a rapidly emerging technology that has proven to be a powerful means of selectively controlling protein production in cell culture. Inhibition of HCV replication in replicons has been accomplished using this procedure whilst the demonstration of selective targeting of the liver protein Fas in vivo using RNAi holds promise for the treatment of HCV (75-78). Maribavir demonstrated in viva anti-HCMV activity in all of the dosage regimens tested (100, 200, and 400 mg tid, and 600 mg bid), with mean reductions in semen HCMV titers of 2.9 to 3.7 loglo PFUlmL (92).

The HCMV serine protease has been perceived as an attractive antiviral target. Recent crystal structure data demonstrated significant conformational flexibility in the S3 binding pocket of protein complexed with two peptidomimetic inhibtors (94). A series of frans-lactams, represented by 35 and 36, 1% = 0.54 and 0.34 pM, respectively, are derived from the same structural platform as the HCV NS3 inhibitor 22 but inhibit the HCMV serine protease with excellent selectivity (9597).

Mechanistic studies are consistent with acylation of the active site Serlz of HCMV protease in a reversible and time-dependent manner.

Chap. 22

Non-HIV Antiviral Agents Meanwell et al. 219

All but one of the currently licensed drugs available to treat HSV act by inhibiting the viral DNA polymerase, providing a suitable backdrop for the emergence of resistant virus and a rationale for identifying inhibitors of other viral proteins (98,99).

Two groups have reported novel thiazole-containing inhibitors of the HSV helicase-primase. BAY 57-1293 (37) is a leading pre-clinical candidate that is more potent (EC50 = 12 nM) than any anti-herpetic currently used to treat HSV infections (100,101). In a murine lethal challenge model of HSV-1 and HSV-2, 37 was protective with an EDso value of 0.5 mglkg, which compares with the much higher doses of 22 and 16 mglkg for HSV-1 and HSV-2, respectively, required for acyclovir to show efficacy. Additional patent applications that extend this promising chemotype have appeared (102,103).

A second series of HSV helicase-primase inhibitors, of which BILS-179 BS (38) is representative, has been disclosed (104). BILS 179 BS inhibits viral growth with an EC50 of 27 nM, displays an excellent therapeutic index of >2000 and reduces cutaneous HSV-1 and genital HSV-2 disease in a murine model when treatment is initiated 3 hours post-infection.

Interestingly, when treatment was initiated 65 hours after infection, 38 reduced HSV-1 pathology by 75% and HSV-2 mortality by 75% (200 mg/Kglday) when compared to acyclovir or untreated animals (104).

A series of 4-oxo-dihydroquinolone derivatives that are potent and broad spectrum non-nucleoside inhibitors of DNA polymerases of the herpesvirus family, including HCMV, HSV-1, HHV-8 and VZV, have been the subject of a number of recent disclosures (105). These compounds exhibit no significant inhibitory activity towards human CI-, y-, or 6-polymerases.

PNU-183792 (39) is an effective antiviral in cell culture, potently inhibiting HCMV (EC 50 = 0.69 PM), VZV (EC% = 0.37 pM) and HSV (EC50 = 0.58 PM), that is active towards ganciclovir-and cidofovir-resistant HCMV and acyclovir-resistant HSV (106). Excellent oral bioavailability and a protective effect in a murine CMV animal model were also reported.

A series of related analogs have been reported in the recent patent literature (107,108). Pyrazolopyridine derivatives have been reported to possess activity against HSV-1 in Vero 76 cells with 40 having an EC50 of 0.12 FM (109-I 12). Structurally related irnidazopyridine derivatives are active against HSV types l-8 with an E&O of 0.14 PM (113). CMV-423 (41) is a potent inhibitor of HCMV, EC50 = 4-7 nM, that appears to act at a step in viral replrcation preceding DNA polymerization (114).

Overview -Respiratory viruses continue to be a significant source of mortality and morbidity. The annual death rates due to influenza in the US are estimated to have doubled over the last 20 years, attributed to an aging of the population (115, 116) . This study also revealed a greater appreciation of the contribution of respiratory syncytial virus to mortality.

The recently discovered human metapneumovirus (hMPV) was identified as a significant cause of wheezing in infants (117,118). The influenza inhibitor oseltamivir (TamifluTM) was approved for the treatment of influenza in adults and children and for prevention in adults and adolescents by the European Community in June 2002, consolidating its position as the market leading neuraminidase (NA) inhibitor.

However, development of the third neuraminidase inhibitor peramivir was terminated by BioCryst in June after disappointing P3 results in which the orally bioavailable compound failed to meet the key efficacy endpoint of reducing the time to onset of relief of symptoms.

In March, 2003 an outbreak of severe acute respiratory syndrome (SARS) emerged in Southeast Asia for which the culprit was quickly identified as a new coronavirus distinct from any previously identified human coronavirus.

of the 1918 influenza continues and chimeric viruses containing the 1918 NA or Ml ion channel showed susceptibility to oseltamivir and amantadine or rimantadine, respectively, both in vitro an in viva. (119,120).

The role of NA inhibitors in pandemic influenza has been reviewed and the identification of structurally novel NA inhibitors has continued (121). Substitution of the primary amine moiety of oseltamivir with a vinyl group afforded the potent influenza B NA inhibitor 42, Ki = 45 nM. (122) Additional SAR studies around zanamivir have focussed on the C-7 hydroxyl where replacement by F or methylation affords the potent NA inhibitors 43 and 44, ICSO values of 0.8 and 6.1 nM, respectively, which compares favorably with an I&O of 5-10 nM for the prototype (123,124).

Polymeric analogues of zanamivir linked to a polyglutamate backbone via the 7 position showed enhanced influenza inhibitory activity compared to monovalent analogues (125 The O-methyl analogue of zanamivir is claimed to protect mice against a lethal influenza infection following oral administration of the prodrug g whilst the bicyclic ether 48 is the first zanamivir derivative to demonstrate oral efficacy in the mouse model (127, 128) .

The optimization of a screening lead into potent, cyclopentanebased inhibitors of neuraminidase using a combination of structure-based design and combinatorial chemistry has been described in detail (129). Mechanism-based inhibitors of the HRV 3C cysteine protease have been probed using a combination of structure-based design principles and parallel synthesis methods in an effort to find less peptidic inhibitors (132). The chroman a emerged as an inhibitor of HRV-14 replication in cell culture, EC50 = 160 nM; however, the serotype coverage of this compound was poor with much reduced potency against other subtypes, an observation rationalized in the context of structural data (132). The HRV 2A cysteine protease releases itself from the viral P2 polyprotein, cleaves the P2 in both a cis and tram fashion to release the 2B and 2C proteins and also proteolyzes the host cap binding complex in order to compromise host cell transcription.

A series of N-phenylated pyrazole derivatives have been claimed as inhibitors of this essential enzyme with 52 an effective antiviral agent in cell culture, EC50 = 1.8 PM, CC50 = 388 I.IM (133). The HRV RNA-dependent RNA polymerase from HRV-16, a potential drug discovery target, has been cloned, expressed and purified from E. co/i and shown to be enzymatically active (134).

Inhibitors of ResDiratotv Svncvtial Virus (RSV) -The role of RSV in morbidity and mortality continues to be a focus of research designed to provide a more accurate perspective of this virus as a mediator of significant disease burden, (115, 116, 135, 136) .

The enhanced awareness of RSV has stimulated interest in this virus as a therapeutic target with the recent emergence of new structural classes of inhibitor.

However, clinical proof-of-principle remains to be established for RSV antivirals.

Viropharma has suspended development of VP-14637, an RSV fusion inhibitor under examination as a topically administered agent.

The benzimidazole BMS-433771 (53) has been described as the first RSV inhibitor to demonstrate antiviral activity in animal models following oral administration (137, 138) . Mechanism of action studies indicate that g is an inhibitor of the fusion of viral and host cell membranes and targets the RSV F (fusion) protein (137). Analogues of 53 form the basis of proprietary claims (139, 140) whilst Trimeris has disclosed a series of benzimidazole-based inhibitors of RSV fusion (141). Representative of this series is 54, which is active in cell culture, EGO = 10 ng/mL.

-Coronaviruses are enveloped, single-strand, positive-sense RNA viruses most commonly associated with respiratory infections in man (142).

Two coronaviruses are the underlying cause of approximately 30% of upper respiratory tract infections, usually mild to moderate in severity and generally recognized as the common cold, although in the immunocompromised population coronavirus infections can lead to pneumonia.

However, in March, 2003 an outbreak of severe acute respiratory syndrome (SARS) emerged in Southeast Asia that very quickly spread to 27 countries, carried largely by air travelers (143-145). More than 6500 SARS infections were documented worldwide within the following 2 months that were associated with substantial morbidity, including fever, non-productive cough, malaise, chills, headache and dyspnea, and significant mortality, with over 460 deaths reported (146). Whilst SARS appears to have originated in Southern China in November, 2002, reports of major outbreaks in Hong Kong and Toronto, Canada in March 2003 brought the disease to world prominence (143-145). The attributes of modern technology, communication and international teamwork led to the rapid isolation and sequencing of the virus causing SARS, identified as a novel coronavirus with little similarity to previously known human coronaviruses (147-153). Final confirmation came with the demonstration that infection of monkeys with SARS produced a syndrome identical to that seen in man and that virus could subsequently recovered (154). These experiments were rapidly followed by the development of diagnostic assays based on a real-time quantitative PCR method and a TaqMan protocol (148, 155). Transmission of SARS appears to be via person-person contact with infected droplets rather than airborne and, possibly, a fecal-oral route. Inhibitors of coronaviruses are largely unknown although treatment with ribavirin (11) and oseltamivir have been examined in an empirical fashion (143, 156).

scope of the mosquito-borne West Nile virus outbreak in the US broadened considerably in the summer of 2003 extending to almost all states and causing over 2,500 cases of encephalitis and 230 deaths (157,158) .

The virus presents several conventional proteins as targets suitable for intervention including the NS3 protein, which expresses serine protease, helicase and nucleoside triphosphatase activities, and the NS5 RNA-dependent RNA polymerase (159). Moreover, screening using a cell culture assay provides a panoply of less well understood targets. However, few potent and selective WNV inhibitors have been described to date. Ribavirin (II) inhibits WNV RNA production in oligodendroglial cells, a human neural cell line, with an EC50 of -60 FM (160). The nucleoside analogue HMC-HO4 (55) interferes with the helicase activity of WNV NS3 with an I& of 30 pM and is a similarly potent antiviral in cell culture (161). Inhibitors of Vaccinia Virus (Smallpox) -Smallpox has aroused considerable concern with discussion prominently focussed on its potential as an agent of bioterrorism (162) (163) (164) (165) (166) (167) . Many inhibitors of vaccinia virus replication interfere with host cell pathways and include 11 (inosine monophosphate dehydrogenase), the cyclopentenyl nucleoside analogue neplanocin A (S-adenosylhomocysteine hydrolase) and pyrazofurin (OMP decarboxylase) (168, 169) . The @dine and 5-F cytidine analogues of neplanocin A are equally potent inhibitors of several orthopox viruses, including smallpox (170) ORI-1001, which complements the El mRNA start codon of HPV and has demonstrated efficacy in two animal models, is currently undergoing PI12 clinical evaluation.

The fused tetracyclic amide .g is claimed to inhibit the E2-dependent binding of papilloma virus El to DNA, a cntrcal step in viral replication, with an I&J of ~500 nM (175) .

A vaccine derived from an HPV-16 virus-like particle, completely prevented the incidence of cervical neoplasias in HPV-16naite young women (176) . This impressive result provides strong encouragement to the development of a vaccine with a spectrum that, in addition, encompasses the broader range of HPVs (18, 31, 33 and 35) responsible for the majority of cervical cancers (177) . A combined, multivalent vaccine based on self-assembling virus-like particles and directed towards HPV-16 and HPV-18, recognized as MEDI-517, is entering P2 trials (178) .

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September Section IV-Cancer and Infectious Diseases Plattner