WO2019089734A1

WO2019089734A1 - Methods and compositions for the treatment of influenza

Info

Publication number: WO2019089734A1
Application number: PCT/US2018/058417
Authority: WO
Inventors: Jun Wang; Chunlong MA; Christopher Hulme
Original assignee: University of Arizona
Current assignee: University of Arizona
Priority date: 2017-10-31
Filing date: 2018-10-31
Publication date: 2019-05-09
Anticipated expiration: 2020-04-30

Abstract

This invention is in the field of medicinal chemistry. In particular, the invention relates to a new class of small-molecules having a tetrazolyl-piperidinyl-benzoimidazole structure: Structure (I) which function as inhibitors influenza polymerase activity, and their use as therapeutics for the treatment of influenza.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF INFLUENZA

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of U.S. Provisional Application No. 62/579,590, filed October 31, 2017, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention is in the field of medicinal chemistry. In particular, the invention relates to ng a tetrazolyl-piperidinyl-benzoimidazole structure

which function as inhibitors influenza polymerase activity, and their use as therapeutics for the treatment of influenza.

INTRODUCTION

Influenza virus infection is responsible for both seasonal influenza as well as sporadic influenza pandemics (see, e.g., Palese, P.; Shaw, M. L. Orthomyxoviridae: The Viruses and Their Replication. In: Knipe DM, Howley PM, eds. Fields Virology. 5th ed. Philadelphia:

Lippincott Williams & Wilkins. pp 1647-1690. 2007). In the annual influenza season, an estimated 10-20% of the human population is infected with the influenza virus. Despite the availability of influenza vaccines and small molecule antiviral drugs, the death toll of influenza virus-related illness surpasses that of breast cancer, which places the influenza virus among the top ten leading causes of death in the United States. Moreover, the convenient transmission through airways, coupled with the high mortality rates associated with pandemic influenza viruses and highly pathogenic avian influenza (HPAI) viruses, renders the influenza virus a major public health concern (see, e.g., Zhang, W. Q.; Webster, R. G. Science 2017, 357, 111- 111). For example, the CDC estimated the 2009 HlNl influenza pandemic led to 284,000 deaths globally in the first 12 months of outbreak (see, e.g., Dawood, F. S.; et al, Lancet Infect Dis 2012, 12, 687-95). Over 400 cases of human infection by HPAI H7N9 were reported in the recent 2017 outbreak in China and the mortality rate was -40% (see, e.g., Zhu, H.; et al, Curr Opin Virol 2016, 16, 106-113). Therefore, next-generation vaccines and antiviral drugs with improved efficacy and antiviral spectrum are clearly needed to combat influenza virus infection.

Influenza vaccines remain the mainstay for the prophylaxis of influenza infection. They are generally effective in preventing seasonal influenza virus infection with an overall effectiveness of -60% (see, e.g., Jackson, M. L.; et al, N Engl J Med 2017, 377, 534-543). However, there is generally a six-month delay from strain identification to batch production, which impedes its use at the beginning of an influenza outbreak (see, e.g., Koszalka, P.; et al, Influenza Other Respir Viruses 2017, 11, 240-246). Thus, small molecule antivirals are highly desired. They are not alternatives, but essential complements of influenza vaccines.

There are currently two classes of FDA-approved small molecule influenza antivirals: M2 channel blockers such as amantadine and rimantadine that inhibit the early stage of viral uncoating (see, e.g., Wang, I; Li, F.; Ma, C. Biopolymers 2015, 104, 291-309), and

neuraminidase inhibitors, such as oseltamivir, peramivir, and zanamivir that inhibit the last stage of viral egress (see, e.g., Loregian, A.; et al, Cell Mol Life Sci 2014, 71, 3659-83). The increasing incidences of drug-resistant viruses now call for the development of the next generation of influenza antivirals (see, e.g., Webster, R. G.; Govorkova, E. A. Ann N Y Acad Sci 2014, 1323, 115-39). Indeed, amantadine and rimantadine are no longer recommended due to the widespread M2-S31N mutant (see, e.g., Li, F.; et al, J Med Chem 2017, 60, 1580-1590; Li, F.; et al, ACS Infect Dis 2016, 2, 726-733). The 2008-2009 seasonal HlNl strain circulating in the United States and Japan is completely resistant to the only orally bioavailable drug, oseltamivir, due to the H275Y mutation in the neuraminidase (see, e.g., Hurt, A. C. Curr Opin Virol 2014, 8, 22-9; Matsuzaki, Y.; et al, Virol J 2010, 7, 53). The emergence of drug-resistant viruses with acquired fitness of transmission is a timely reminder of the urgent need for the next generation of antivirals with a novel mechanism of action and a high genetic barrier to drug resistance.

Accordingly, there is an urgent need for novel influenza antiviral agents.

The current invention addresses this need.

SUMMARY OF THE INVENTION

In the search for novel influenza antivirals, experiments were conducted to establish a fast-track drug discovery program by exploring multi-component reaction (MCR) products for inhibitors that target the influenza polymerase subunit PAC-PBIN interactions. As drug discovery involves iterative cycles of design, synthesis, and pharmacological characterization, it was envisioned that MCRs would greatly accelerate lead optimization during the drug discovery process, as final products can be conveniently synthesized in a one-pot one-step reaction, often containing 3 or more point of diversification (see, e.g., Domling, A.; et al, Chemical Reviews 2012, 112, 3083-3135).

The influenza virus RNA-dependent RNA polymerase, composed of PBl, PB2 and PA subunits, is responsible for viral RNA transcription and replication (FIG. 1 A) (see, e.g., Pflug, A.; et al, Virus Res 2017; Stevaert, A.; et al, Med Res Rev 2016, 36, 1127-1173). X-ray crystal structures show that the N-terminal tail of PBl (PBIN) interacts extensively with the C-terminal domain of PA (PAc) (FIG. IB), and as such the concave shape of the PBlN-binding site in PAc presents an attractive target for rational drug design (see, e.g., Stevaert, A.; et al, Med Res Rev 2016, 36, 1127-1173; Massari, S.; et al., J Med Chem 2016, 59, 7699-718). Moreover, the PAc domain is highly conserved among different types and subtypes of influenza viruses, and compounds that inhibit PAc-PB IN interactions have shown to have broad-spectrum antiviral activity (see, e.g., Yuan, S.; et al., Antiviral Res 2016, 125, 34-42; Massari, S.; et al., J Med Chem 2015, 58, 3830-42; Muratore, G; et al, Proc Natl Acad Sci U S A 2012, 109, 6247-52).

Using the crystal structure of PAc bound to the PBIN peptide (PDB: 3CM8) as a template, experiments conducted during the course of developing embodiments for the present invention screened two thousand compounds from an in-house library using the Schrodinger Glide standard precision docking program. The in-house library comprises a diverse set of compounds prepared by multicomponent reaction methodologies (see, e.g., Hulme, C; Ayaz, M.; Martinez- Ariza, G; Medda, F.; Shaw, A. Recent Advances in Multicomponent Reaction Chemistry. In Small Molecule Medicinal Chemistry, John Wiley & Sons, Inc: 2015; pp 145- 187). Top hits prioritized by in silico docking were tested in a PAC-PBIN ELISA assay.

Compound 5 ( ⁵ ) was found to inhibit PAC-PBIN interaction in a dose- dependent manner with an IC50 of 4.3 ± 0.1 μΜ. The antiviral activity of compound 5 was confirmed by the plaque assay and it had single to submicromolar EC50 values against several influenza A and B viruses, including both oseltamivir-sen ant strains.

Subsequent lead optimization led to the discovery of 12a ( ^12a ) with an improved selectivity index. Similarly, compound 12a had potent and broad-spectrum influenza antiviral activity against several human clinical isolates of influenza A and B viruses. More importantly, no resistant virus was selected under the drug selection pressure of compound 12a.

Accordingly, the invention rel small-molecules having a tetrazolyl-

piperidinyl-benzoimidazole structure

) which function as inhibitors influenza polymerase activity, and their use as therapeutics for the treatment of influenza.

Indeed, the present invention generally relates to small-molecules having a tetrazolyl- piperidinyl-benzoimidazole structure or pharmaceutically acceptable salts thereof, and to uses of such small molecules for inhibiting the replication of influenza viruses, for reducing the amount of influenza viruses, for inhibiting influenza polymerase activity (e.g., through inhibiting interaction between the PA and PBl submits of influenza polymerase), for preventing influenza, and for treating influenza.

In a particular embodiment, small-molecules having a tetrazolyl

benzoimidazole structure encompassed within Formula I are provided:

including pharmaceutically acceptable salts, solvates, and/or prodrugs provided that if R2 is then Rl cannot be

provided that if R2 is then Rl cannot be

provided that if R2 is Rl cannot and

provided that if R2 is

, then Rl cannot be

In some embodiments, the resulting compound is able to inhibit influenza polymerase activity. In some embodiments, the resulting compound is able to inhibit interaction between PA and PB1 (PAC-PB IN) of an influenza polymerase. In some embodiments, the resulting compound is able to inhibit and/or prevent influenza virus activity. In some embodiments, the resulting compound is able to inhibit the replication of drug-resistant influenza viruses.

In some embodiments, the following compounds are contemplated for Formula I:



pharmaceutically acceptable salt, solvate, or prodrug thereof.

Tables 1 , 2, 3, 4 and 5 (see, Examples) show inhibition against influenza strains for various compounds encompassed within Formula I.

In certain embodiments, the invention is directed to a method of reducing the amount of influenza viruses in a biological in vitro sample or in a subject (e.g., a mammalian subject) (e.g., a human subject), comprising administering to the sample or subject an effective amount of a small-molecule having a tetrazolyl-piperidinyl-benzoimidazole structure disclosed herein (e.g., one or more of the compounds described herein).

In certain embodiments, the invention is directed to a method of inhibiting the replication of influenza viruses in a biological in vitro sample or in a subject (e.g., a mammalian subject) (e.g., a human subject), comprising administering to the sample or subject an effective amount of a small-molecule having a tetrazolyl-piperidinyl-benzoimidazole structure disclosed herein (e.g., one or more of the compounds described herein).

In certain embodiments, the invention is directed to a method of inhibiting influenza polymerase activity in a biological in vitro sample or in a subject (e.g., a mammalian subject) (e.g., a human subject), comprising administering to the sample or subject an effective amount of a small-molecule having a tetrazolyl-piperidinyl-benzoimidazole structure disclosed herein (e.g., one or more of the compounds described herein). In some embodiments, inhibiting influenza polymerase activity comprises inhibiting interaction between the PA and PB1 submits (e.g., PAC-PBIN) of the influenza polymerase.

In certain embodiments, the invention is directed to a method of preventing or treating influenza in a subject (e.g., a mammalian subject) (e.g., a human subject), comprising administering to the subject a therapeutically effective amount of a small-molecule having a tetrazolyl-piperidinyl-benzoimidazole structure disclosed herein (e.g., one or more of the compounds described herein). In some embodiments, the method further comprises coadministration of one or more small molecule influenza antivirals (e.g., M2 channel blockers such as amantadine and rimantadine) (e.g., neuraminidase inhibitors such as oseltamivir, peramivir, and zanamivir). BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 : Structures of the influenza polymerase. (A) X-ray crystal structures of the viral polymerase complexes from the bat influenza A/H17N10 virus (PDB: 4WSB) (see, e.g., Reich, S.; et al., Nature 2014, 516, 361). PA: green; PB1 : yellow; PB2: magenta. (B) X-ray crystal structure of PAC-PBIN (PDB: 3CM8) (see, e.g., He, X.; et al, Nature 2008, 454, 1123-1126). PA: green; PB1 : yellow.

FIG. 2: Inhibition of PA-PB1 by compound 5 in ELISA assay. (A) Cartoon

representation of the PAC-PB IN ELISA assay. (B) IC50 curve of compound 5 in the PAC-PB IN ELISA assay.

FIG. 3: Structure-activity relationship studies of compound 5. Oseltamivir carboxylate and nucleozin were tested at 200 nM and 1 μΜ, respectively. All other compounds were tested at 5 μΜ. The results are the mean ± S.D. from two independent experiments.

FIG. 4: Compound 12a has a high in vitro genetic barrier to drug resistance as shown by the serial viral pasage experiment. (A) Cartoon representation of the serial drug passage experiment. (B) Comparsion of the in vitro genetic barrier of drug resistance between oseltamivir carboxylate and compound 12a.

FIG. 5: Docking model of compound 12a in the PB1 -binding pocket in PA. (A) Surface view of the docking model of compound 12a in PAc. (B) Ligand interaction diagram of compound 12a with residues in the binding site.

DETAILED DESCRIPTION OF THE INVENTION

Influenza viruses are RNA viruses of the family Orthomyxoviridae, which comprises five genera: Influenza virus A, Influenza virus B, Influenza virus C, ISA virus and Thogoto virus.

The Influenza virus A genus has one species, influenza A virus. Wild aquatic birds are the natural hosts for a large variety of influenza A. Occasionally, viruses are transmitted to other species and may then cause devastating outbreaks in domestic poultry or give rise to human influenza pandemics. The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease. The influenza A virus can be subdivided into different serotypes based on the antibody response to these viruses. The serotypes that have been confirmed in humans, ordered by the number of known human pandemic deaths, are: H1N1 (which caused Spanish influenza in 1918), H2N2 (which caused Asian Influenza in 1957), H3N2 (which caused Hong Kong Flu in 1968), H5N1 (a pandemic threat in the 2007-08 influenza season), H7N7 (which has unusual zoonotic potential), H1N2 (endemic in humans and pigs),

H9N2, H7N2, H7N3 and H10N7.

The Influenza virus B genus has one species, influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza A. The only other animal known to be susceptible to influenza B infection is the seal. This type of influenza mutates at a rate 2-3 times slower than type A and consequently is less genetically diverse, with only two lineages,

Yamagata and Victoria. As a result of this lack of antigenic diversity, a degree of immunity to influenza B is usually acquired at an early age. However, influenza B mutates enough that lasting immunity is not possible. This reduced rate of antigenic change, combined with its limited host range (inhibiting cross species antigenic shift), ensures that pandemics of influenza

B do not occur. Nevertheless, influenza B viruses are found to be the predominant circulating strains in certain influenza seasons and human infection with influenza B viruses have been shown to led to morbidity and mortality.

The Influenza virus C genus has one species, influenza C virus, which infects humans and pigs and can cause severe illness and local epidemics. However, influenza C is less common than the other types and usually seems to cause mild disease in children.

Influenza A, B and C viruses are very similar in structure. The virus particle is 80-120 nanometers in diameter and usually roughly spherical, although filamentous forms can occur.

Unusual for a virus, its genome is not a single piece of nucleic acid; instead, it contains seven or eight pieces of segmented negative-sense RNA. The Influenza A genome encodes 1 1 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), Ml , M2, NS 1, NS2(NEP), PA,

PB 1, PB1 -F2 and PB2.

HA and NA are large glycoproteins on the outside of the viral particles. HA is a lectin that mediates binding of the virus to target cells and entry of the viral genome into the target cell, while NA is involved in the release of progeny virus from infected cells, by cleaving sugars that bind the mature viral particles. Thus, these proteins have been targets for antiviral drugs.

Furthermore, they are antigens to which antibodies can be raised. Influenza A viruses are classified into subtypes based on antibody responses to HA and NA, forming the basis of the H and N distinctions (vide supra) in, for example, H5N1.

Current treatment options for influenza include vaccination, and chemotherapy or chemoprophylaxis with antiviral medications. Vaccination against influenza with an influenza vaccine is often recommended for anyone aged 6 months old and older, especially for high-risk groups, such as children and the elderly, or in people that have asthma, diabetes, or heart disease.

However, it is possible to get vaccinated and still get influenza. The vaccine is reformulated each season for a few specific influenza strains but cannot possibly include all the strains actively infecting people in the world for that season. It may take six months for the manufacturers to formulate and produce the millions of doses required to deal with the seasonal epidemics;

occasionally, a new or overlooked strain becomes prominent during that time and infects people although they have been vaccinated (as by the H3N2 Fujian flu in the 2003-2004 influenza season). It is also possible to get infected just before vaccination and get sick with the very strain that the vaccine is supposed to prevent, as the vaccine may require several weeks to become effective.

Further, the effectiveness of these influenza vaccines is variable. Due to the high mutation rate of the virus, a particular influenza vaccine usually confers protection for no more than a few years. A vaccine formulated for one year may be ineffective in the following year, since the influenza virus changes rapidly over time, and different strains become dominant. Antiviral drugs can also be used to treat influenza, with neuraminidase inhibitors being particularly effective, but viruses can develop resistance to the standard antiviral drugs.

In response to an urgent need for the next generation of influenza antivirals with broad- spectrum antiviral activity and a high genetic barrier to drug resistance, experiments conducted during the course of developing embodiments for the present invention established a fast-track drug discovery platform by exploring multi-component reaction products for antiviral drug candidates. Specifically, molecular docking was applied to screen a small molecule library derived from the Ugi-azide four-component reaction methodology for inhibitors that target the influenza polymerase PAC-PB IN interactions.

One hit compound 5 ( ⁵ ) was confirmed to inhibit PAC-PBIN interactions in an ELISA assay and had potent antiviral activity in an antiviral plaque assay. Subsequent lead optimization led to the discovery of compound 12a

( ^2a ), which had broad-spectrum antiviral activity against a panel of human clinical isolates of influenza A and B viruses, including both oseltamivir-sensitive and oseltamivir-resistant influenza viruses. Moreover, compound 12a had a higher in vitro genetic barrier to drug resistance than oseltamivir, and no resistant virus emerged under drug selection pressure. Overall, the discovery of compound 12a as a broad-spectrum influenza antiviral with a high in vitro genetic barrier to drug resistance is significant, as it offers a second line of defense to combat the next influenza epidemics and pandemics if vaccines and oseltamivir fail to confine the disease outbreak.

Accordingly, the invention relates to a new class of small-molecules having a tetrazolyl-

piperidinyl-benzoimidazole structure (

Indeed, the present invention generally relates to small-molecules having a tetrazolyl- piperidinyl-benzoimidazole structure or pharmaceutically acceptable salts thereof, and to uses of such small molecules for inhibiting the replication of influenza viruses, for reducing the amount of influenza viruses, for inhibiting influenza polymerase activity (e.g., through inhibiting interaction between the PA and PB1 submits of influenza polymerase), for preventing influenza, and for treating influenza. In a articular embodiment, benzoic acid compounds encompassed within Formula I are

provided:

including pharmaceutically acceptable salts, solvates, and/or prodrugs thereof;

provided that if R2 is

, then Rl cannot and

provided that if R2 is

then Rl cannot be

In some embodiments, the resulting compound is able to inhibit influenza polymerase activity. In some embodiments, the resulting compound is able to inhibit interaction between PA and PBl (PAC-PBIN) of an influenza polymerase. In some embodiments, the resulting compound is able to inhibit and/or prevent influenza virus activity. In some embodiments, the resulting compound is able to inhibit drug resistant influenza virus activity.





pharmaceutically acceptable salt, solvate, or prodrug thereof.

Tables 1, 2, 3, 4 and 5 (see, Examples) show inhibition activity against influenza strains for small-molecules having a tetrazolyl-piperidinyl-benzoimidazole structure encompassed within Formula I.

Such small-molecules having a tetrazolyl-piperidinyl-benzoimidazole structure encompassed within Formula I can exist in or form different polymorphic forms. As known in the art, polymorphism is an ability of a compound to crystallize as more than one distinct crystalline or "polymorphic" species. A polymorph is a solid crystalline phase of a compound with at least two different arrangements or polymorphic forms of that compound molecule in the solid state. Polymorphic forms of any given compound are defined by the same chemical formula or composition and are as distinct in chemical structure as crystalline structures of two different chemical compounds. Generally, different polymorphs can be characterized by analytical methods such as X-ray powder diffraction (XRPD) pattern, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), or by its melting point, or other techniques known in the art. As used herein, the term "polymorphic form" includes solvates and neat polymorphic form that does not have any solvates.

Unless otherwise indicated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, cis-trans, conformational, and rotational) forms of the structure. For example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in this invention, unless only one of the isomers is drawn specifically. As would be understood to one skilled in the art, a substituent can freely rotate around any rotatable bonds. Therefore, single

stereochemical isomers as well as enantiomeric, diastereomeric, cis/trans, conformational, and rotational mixtures of the present compounds are within the scope of the invention. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

One aspect of the present invention is generally related to the use of the small-molecules having a tetrazolyl-piperidinyl-benzoimidazole structure disclosed herein (e.g., one or more of the compounds described herein) for inhibiting the replication of influenza viruses in a biological sample or in a patient, for reducing the amount of influenza viruses (reducing viral titer) in a biological sample or in a patient, and for treating influenza in a patient.

In certain embodiments, the invention is directed to a method of reducing the amount of influenza viruses in a biological in vitro sample or in a subject (e.g., a mammalian subject) (e.g., a human subject), comprising administering to the sample or subject an effective amount of a small-molecule having a tetrazolyl-piperidinyl-benzoimidazole structure disclosed herein.

In certain embodiments, the invention is directed to a method of preventing or treating influenza in a subject (e.g., a mammalian subject) (e.g., a human subject), comprising administering to the subject a therapeutically effective amount of a small-molecule having a tetrazolyl-piperidinyl-benzoimidazole structure disclosed herein (e.g., one or more of the compounds described herein).

In one embodiment, the present invention is generally related to the use of the compounds disclosed herein (e.g., in pharmaceutically acceptable compositions) for any of the uses specified above.

In yet another embodiment, the compounds disclosed herein (e.g., one or more of the compounds described herein) can be used to reduce viral titre in a biological sample (e.g. an infected cell culture) or in humans (e.g. lung viral titre in a patient).

The terms "influenza virus mediated condition", "influenza infection", or "Influenza", as used herein, are used interchangeably to mean the disease caused by an infection with an influenza virus. Influenza is an infectious disease that affects birds and mammals caused by influenza viruses. Influenza viruses are RNA viruses of the family Orthomyxoviridae, which comprises five genera: Influenza virus A, Influenza virus B, Influenza virus C, ISA virus and Thogoto virus. Influenza virus A genus has one species, influenza A virus which can be subdivided into different serotypes based on the antibody response to these viruses: H1N1 , H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3 and H10N7. Additional examples of influenza A virus include H3N8 and H7N9. Influenza virus B genus has one species, influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza A. Influenza virus C genus has one species, Influenza virus C virus, which infects humans and pigs and can cause severe illness and local epidemics. However, Influenza virus C is less common than the other types and usually seems to cause mild disease in children.

In some embodiments of the invention, influenza or influenza viruses are associated with Influenza virus A or B. In some embodiments of the invention, influenza or influenza viruses are associated with Influenza virus A. In some specific embodiments of the invention, Influenza virus A is H1N1 , H2N2, H3N2 or H5N1. In some specific embodiments of the invention,

Influenza virus A is H1N1, H3N2, H3N8, H5N1 , and H7N9. In some specific embodiments of the invention, Influenza virus A is H1N1, H3N2, H3N8, and H5N1.

In humans, common symptoms of influenza are chills, fever, pharyngitis, muscle pains, severe headache, coughing, weakness, and general discomfort. In more serious cases, influenza causes pneumonia, which can be fatal, particularly in young children and the elderly. Although it is often confused with the common cold, influenza is a much more severe disease and is caused by a different type of virus. Influenza can produce nausea and vomiting, especially in children, but these symptoms are more characteristic of the unrelated gastroenteritis, which is sometimes called "stomach flu" or "24-hour flu".

Symptoms of influenza can start quite suddenly one to two days after infection. Usually the first symptoms are chills or a chilly sensation, but fever is also common early in the infection, with body temperatures ranging from 38° C. to 39° C. (approximately 100° F. to 103° F.). Many people are so ill that they are confined to bed for several days, with aches and pains throughout their bodies, which are worse in their backs and legs. Symptoms of influenza may include: body aches, especially joints and throat, extreme coldness and fever, fatigue, headache, irritated watering eyes, reddened eyes, skin (especially face), mouth, throat and nose, abdominal pain (in children with influenza B). Symptoms of influenza are non-specific, overlapping with many pathogens ("influenza-like illness"). Usually, laboratory data is needed in order to confirm the diagnosis. The terms, "disease", "disorder", and "condition" may be used interchangeably here to refer to an influenza virus mediated medical or pathological condition.

As used herein, the terms "subj ect" and "patient" are used interchangeably. The terms "subject" and "patient" refer to an animal (e.g., a bird such as a chicken, quail or turkey, or a mammal), specifically a "mammal" including a non-primate (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog, and mouse) and a primate (e.g., a monkey, chimpanzee and a human), and more specifically a human. In one embodiment, the subject is a non-human animal such as a farm animal (e.g., a horse, cow, pig or sheep), or a pet (e.g., a dog, cat, guinea pig or rabbit). In a preferred embodiment, the subject is a "human".

The term "biological sample", as used herein, includes, without limitation, cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof; blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

As used herein, the terms "treat", "treatment" and "treating" refer to both therapeutic and prophylactic treatments. For example, therapeutic treatments includes the reduction or amelioration of the progression, severity and/or duration of influenza viruses mediated conditions, or the amelioration of one or more symptoms (specifically, one or more discernible symptoms) of influenza viruses mediated conditions, resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as a compound or composition of the invention). In specific embodiments, the therapeutic treatment includes the amelioration of at least one measurable physical parameter of an influenza virus mediated condition. In other embodiments the therapeutic treatment includes the inhibition of the progression of an influenza virus mediated condition, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the therapeutic treatment includes the reduction or stabilization of influenza viruses mediated infections. Antiviral drugs can be used in the community setting to treat people who already have influenza to reduce the severity of symptoms and reduce the number of days that they are sick.

The term "chemotherapy" refers to the use of medications, e.g. small molecule drugs (rather than "vaccines") for treating a disorder or disease.

The terms "prophylaxis" or "prophylactic use" and "prophylactic treatment" as used herein, refer to any medical or public health procedure whose purpose is to prevent, rather than treat or cure a disease. As used herein, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk of acquiring or developing a given condition, or the reduction or inhibition of the recurrence or said condition in a subject who is not ill, but who has been or may be near a person with the disease. The term "chemoprophylaxis" refers to the use of medications, e.g., small molecule drugs (rather than "vaccines") for the prevention of a disorder or disease.

As used herein, prophylactic use includes the use in situations in which an outbreak has been detected, to prevent contagion or spread of the infection in places where a lot of people that are at high risk of serious influenza complications live in close contact with each other (e.g. in a hospital ward, daycare center, prison, nursing home, or the like). It also includes the use among populations who require protection from the influenza but who either do not get protection after vaccination (e.g., due to weak immune system), or when the vaccine is unavailable to them, or when they cannot get the vaccine because of side effects. It also includes use during the two weeks following vaccination, since during that time the vaccine is still ineffective. Prophylactic use may also include treating a person who is not ill with the influenza or not considered at high risk for complications, in order to reduce the chances of getting infected with the influenza and passing it on to a high-risk person in close contact with him (for instance, healthcare workers, nursing home workers, or the like).

According to the US CDC, an influenza "outbreak" is defined as a sudden increase of acute febrile respiratory illness (AFRI) occurring within a 48 to 72 hour period, in a group of people who are in close proximity to each other (e.g. in the same area of an assisted living facility, in the same household, etc.) over the normal background rate or when any subject in the population being analyzed tests positive for influenza. One case of confirmed influenza by any testing method is considered an outbreak.

A "cluster" is defined as a group of three or more cases of AFRI occurring within a 48 to 72 hour period, in a group of people who are in close proximity to each other (e.g. in the same area of an assisted living facility, in the same household, etc.).

As used herein, the "index case", "primary case" or "patient zero" is the initial patient in the population sample of an epidemiological investigation. When used in general to refer to such patients in epidemiological investigations, the term is not capitalized. When the term is used to refer to a specific person in place of that person's name within a report on a specific

investigation, the term is capitalized as Patient Zero. Often, scientists search for the index case to determine how the disease spread and what reservoir holds the disease in between outbreaks. Note that the index case is the first patient that indicates the existence of an outbreak. Earlier cases may be found and are labeled primary, secondary, tertiary, and the like.

In one embodiment, the methods of the invention are a preventative or "pre-emptive" measure to a patient, specifically a human, having a predisposition to complications resulting from infection by an influenza virus. The term "pre-emptive" or "pre-emptively", as used herein, for example, in 'pre-emptive' use, is the prophylactic use in situations in which an "index case" or an "outbreak" has been confirmed, in order to prevent the spread of infection in the rest of the community or population group.

In another embodiment, the methods of the invention are applied as a "pre-emptive" measure to members of a community or population group, specifically humans, in order to prevent the spread of infection.

As used herein, an "effective amount" refers to an amount sufficient to elicit the desired biological response. In the present invention the desired biological response is to inhibit the replication of influenza virus, to reduce the amount of influenza viruses or to reduce or ameliorate the severity, duration, progression, or onset of an influenza virus infection, prevent the advancement of an influenza viruses infection, prevent the recurrence, development, onset or progression of a symptom associated with an influenza virus infection, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy used against influenza infections. The precise amount of compound administered to a subject will depend on the mode of

administration, the type and severity of the infection and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. When coadministered with other antiviral agents, e.g., when co-administered with an anti -influenza medication, an "effective amount" of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of a compound described herein being used. In cases where no amount is expressly noted, an effective amount should be assumed. For example, the compounds disclosed herein can be administered to a subject in a dosage range from between approximately 0.01 to 100 mg/kg body weight/day for therapeutic or prophylactic treatment.

Generally, dosage regimens can be selected in accordance with a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the renal and hepatic function of the subject; and the particular compound or salt thereof employed, the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts. The skilled artisan can readily determine and prescribe the effective amount of the compounds described herein required to treat, to prevent, inhibit (fully or partially) or arrest the progress of the disease.

Dosages of the compounds described herein can range from 0.01 to 100 mg/kg body weight/day, 0.01 to 50 mg/kg body weight/day, 0.1 to 50 mg/kg body weight/day, or 1 to 25 mg/kg body weight/day. It is understood that the total amount per day can be administered in a single dose or can be administered in multiple dosing, such as twice a day (e.g., every 12 hours), three times a day (e.g., every 8 hours), or four times a day (e.g., every 6 hours).

In some embodiments, dosages of the small-molecules having a tetrazolyl-piperidinyl- benzoimidazole structure encompassed within Formula I and pharmaceutically acceptable salts thereof are in a range of 100 mg to 1,600 mg, such as 400 mg to 1 ,600 mg or 400 mg to 1 ,200 mg. Each dose can be taken once a day (QD), twice per day (e.g., every 12 hours (BID)), or three times per day (e.g., q8h (TID)). It is noted that any combinations of QD, BID, and TID can be employed, as desired, such as BID on day 1, followed by QD thereafter.

In some embodiments, dosages of the small-molecules having a tetrazolyl-piperidinyl- benzoimidazole structure disclosed herein and pharmaceutically acceptable salts thereof are in a range of 100 mg to 1,600 mg, such as 400 mg to 1,600 mg or 400 mg to 1 ,200 mg. Each dose can be taken once a day (QD), twice per day (e.g., every 12 hours (BID)), or three times per day (e.g., q8h (TID)). It is noted that any combinations of QD, BID, and TID can be employed, as desired, such as BID on day 1, followed by QD thereafter, or, when a loading dosage is employed on day 1 , BID on day 2, followed by QD thereafter.

In one specific embodiment, dosages of the compounds described herein are 400 mg to 1,600 mg, 400 mg to 1,200 mg, or 600 mg to 1,200 mg once a day. In another specific embodiment, dosages of the compounds described herein are 400 mg to 1 ,600 mg, 400 mg to 1 ,200 mg, or 300 mg to 900 mg twice a day. In yet another specific embodiment, dosages of the compounds described herein are 400 mg to 1 ,000 mg once a day. In yet another specific embodiment, dosages of the compounds described herein are 600 mg to 1 ,000 mg once a day. In yet another specific embodiment, dosages of the compounds described herein are 600 mg to 800 mg once a day. In yet another specific embodiment, dosages of the compounds described herein are 400 mg to 800 mg twice a day (e.g., 400 mg to 800 mg every 12 hours). In yet another specific embodiment, dosages of the compounds described herein are 400 mg to 600 mg twice a day.

In some embodiments, a loading dosage regimen is employed. In one specific embodiment, a loading dose of 400 mg to 1 ,600 mg is employed on day 1 of treatment. In another specific embodiment, a loading dose of 600 mg to 1 ,600 mg is employed on day 1 of treatment. In another specific embodiment, a loading dose of 800 mg to 1 ,600 mg is employed on day 1 of treatment. In yet another specific embodiment, a loading dose of 900 mg to 1,600 mg is employed on day 1 of treatment. In yet another specific embodiment, a loading dose of 900 mg to 1 ,200 mg is employed on day 1 of treatment. In yet another specific embodiment, a loading dose of 900 mg is employed on day 1 of treatment. In yet another specific embodiment, a loading dose of 1,000 mg is employed on day 1 of treatment. In yet another specific embodiment, a loading dose of 1 ,200 mg is employed on day 1 of treatment.

In one specific embodiment, the dosage regimen of the compounds described herein employs a loading dosage of 600 mg to 1,600 mg on day 1 and with a regular dosage of 300 mg to 1 ,200 mg for the rest of the treatment duration. Each regular dose can be taken once a day, twice a day, or three times a day, or any combination thereof. In a further specific embodiment, a loading dosage of 900 mg to 1 ,600 mg, such as 900 mg, 1 ,200 mg, or 1,600 mg, is employed. In another further specific embodiment, a loading dosage of 900 mg to 1 ,200 mg, such as 900 mg or 1 ,200 mg, is employed. In yet another further specific embodiment, a regular dosage of 400 mg to 1 ,200 mg, such as 400 mg, 600 mg, or 800 mg, is employed for the rest of the treatment duration. In yet another further specific embodiment, a regular dosage of 400 mg to 1 ,000 mg for the rest of the treatment duration. In yet another further specific embodiment, a regular dosage of 400 mg to 800 mg is employed for the rest of the treatment duration. In yet another further specific embodiment, a regular dosage of 300 mg to 900 mg twice a day is employed. In yet another further specific embodiment, a regular dosage of 600 mg to 1 ,200 mg once a day is employed. In yet another further specific embodiment, a regular dosage of 600 mg twice a day on day 2, followed by 600 mg once a day for the rest of the treatment duration.

For therapeutic treatment, the compounds described herein can be administered to a patient within, for example, 48 hours (or within 40 hours, or less than 2 days, or less than 1.5 days, or within 24 hours) of onset of symptoms (e.g., nasal congestion, sore throat, cough, aches, fatigue, headaches, and chills/sweats). Alternatively, for therapeutic treatment, the compounds described herein can be administered to a patient within, for example, 96 hours of onset of symptoms. The therapeutic treatment can last for any suitable duration, for example, for 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, etc. For prophylactic treatment during a community outbreak, the compounds described herein can be administered to a patient within, for example, 2 days of onset of symptoms in the index case, and can be continued for any suitable duration, for example, for 7 days, 10 days, 14 days, 20 days, 28 days, 35 days, 42 days, etc., up to the entire flu season. A flu season is an annually -recurring time period characterized by the prevalence of outbreaks of influenza. Influenza activity can sometimes be predicted and even tracked geographically. While the beginning of major flu activity in each season varies by location, in any specific location these minor epidemics usually take 3-4 weeks to peak and another 3-4 weeks to significantly diminish. Typically, Centers for Disease Control (CDC) collects, compiles and analyzes information on influenza activity year round in the United States and produces a weekly report from October through mid-May.

In one embodiment, the therapeutic treatment lasts for 1 day to an entire flu season. In one specific embodiment, the therapeutic treatment lasts for 3 days to 14 days. In another specific embodiment, the therapeutic treatment lasts for 5 days to 14 days. In another specific embodiment, the therapeutic treatment lasts for 3 days to 10 days. In yet another specific embodiment, the therapeutic treatment lasts for 4 days to 10 days. In yet another specific embodiment, the therapeutic treatment lasts for 5 days to 10 days. In yet another specific embodiment, the therapeutic treatment lasts for 4 days to 7 days (e.g., 4 days, 5 days, 6 days, or 7 days). In yet another specific embodiment, the therapeutic treatment lasts for 5 days to 7 days (e.g., 5 days, 6 days, or 7 days). In one specific embodiment, the prophylactic treatment lasts up to the entire flu season.

In one specific embodiment, the compounds described herein are administered to a patient for 3 days to 14 days (e.g., 5 days to 14 days) with a loading dosage of 900 mg to 1,600 mg on day 1 and with a regular dosage of 300 mg to 1 ,200 mg for the rest of the treatment duration. In another specific embodiment, the compounds described herein are administered to a patient for 3 days to 14 days (e.g., 5 days to 14 days) with a loading dosage of 900 mg to 1,200 mg on day 1 and with a regular dosage of 400 mg to 1 ,000 mg for the rest of the treatment duration. In yet another specific embodiment, the compounds described herein are administered to a patient for 3 days to 14 days (e.g., 5 days to 14 days) with a loading dosage of 900 mg to 1,200 mg on day 1 and with a regular dosage of 400 mg to 800 mg for the rest of the treatment duration. In yet another specific embodiment, the compounds described herein are administered to a patient for 3 days to 14 days (e.g., 5 days to 14 days) with a loading dosage of 900 mg to 1,200 mg on day 1 and with a regular dosage of 400 mg to 800 mg for the rest of the treatment duration. Each dose can be taken once a day, twice a day, or three times a day, or any combination thereof.

In one specific embodiment, the compounds described herein are administered to a patient for 3 days to 14 days with a loading dosage of 900 mg to 1 ,600 mg on day 1 and with a regular dosage of 600 mg to 1,000 mg once a day for the rest of the treatment duration. In another specific embodiment, the compounds described herein are administered to a patient for 3 days to 14 days with a loading dosage of 900 mg to 1,200 mg on day 1 and with a regular dosage of 600 mg to 800 mg (e.g., 600 mg, 650 mg, 700 mg, 750 mg, or 800 mg) once a day for the rest of the treatment duration. In some embodiments, the treatment duration is for 4 days to 10 days, 5 days to 10 days, or 5 days to 7 days.

In one specific embodiment, the compounds described herein are administered to a patient for 3 days to 14 days with a loading dosage of 900 mg to 1 ,600 mg on day 1 and with a regular dosage of 400 mg to 800 mg twice a day for the rest of the treatment duration. In another specific embodiment, the compounds described herein are administered to a patient for 3 days to 14 days with a loading dosage of 900 mg to 1 ,200 mg on day 1 and with a regular dosage of 400 mg to 600 mg (e.g., 400 mg, 450 mg, 500 mg, 550 mg, or 600 mg) twice a day for the rest of the treatment duration. In some embodiments, the duration is for 4 days to 10 days, 5 days to 10 days, or 5 days to 7 days.

In one specific embodiment, the compounds described herein are administered to a patient for 4 days or 5 days with a loading dosage of 900 mg to 1 ,200 mg (e.g., 900 mg or 1 ,200 mg) on day 1 and with a regular dosage of 400 mg to 600 mg (e.g., 400 mg or 600 mg) twice a day for the rest of the treatment duration (e.g., days 2 through 4, or days 2 through 5). In another specific embodiment, the compounds described herein are administered to a patient for 4 days or 5 days with a loading dosage of 900 mg to 1,200 mg (e.g., 900 mg or 1,200 mg) on day 1 and with a regular dosage of 600 mg to 800 mg (e.g., 600 mg or 800 mg) once a day for the rest of the treatment duration.

An effective amount can be achieved in the method or pharmaceutical composition of the invention employing a compound of the invention (including a pharmaceutically acceptable salt or solvate (e.g., hydrate)) alone or in combination with an additional suitable therapeutic agent, for example, an antiviral agent or a vaccine. When "combination therapy" is employed, an effective amount can be achieved using a first amount of a compound of the invention and a second amount of an additional suitable therapeutic agent (e.g. an antiviral agent or vaccine).

In another embodiment of this invention, a compound of the invention and the additional therapeutic agent, are each administered in an effective amount (i.e., each in an amount which would be therapeutically effective if administered alone). In another embodiment, a compound of the invention and the additional therapeutic agent, are each administered in an amount which alone does not provide a therapeutic effect (a sub-therapeutic dose). In yet another embodiment, a compound of the invention can be administered in an effective amount, while the additional therapeutic agent is administered in a sub-therapeutic dose. In still another embodiment, a compound of the invention can be administered in a sub-therapeutic dose, while the additional therapeutic agent, for example, a suitable cancer-therapeutic agent is administered in an effective amount.

As used herein, the terms "in combination" or "co-administration" can be used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and/or therapeutic agents). The use of the terms does not restrict the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to a subject.

Co-administration encompasses administration of the first and second amounts of the compounds of the coadministration in an essentially simultaneous manner, such as in a single pharmaceutical composition, for example, capsule or tablet having a fixed ratio of first and second amounts, or in multiple, separate capsules or tablets for each. In addition, such coadministration also encompasses use of each compound in a sequential manner in either order.

In one embodiment, the present invention is directed to methods of combination therapy for inhibiting Influenza viruses replication in biological samples or patients, or for treating or preventing Influenza virus infections in patients using the compounds described herein.

Accordingly, pharmaceutical compositions of the invention also include those comprising an inhibitor of Influenza virus replication of this invention in combination with an anti-viral compound exhibiting anti-Influenza virus activity.

Methods of use of the compounds described herein and compositions of the invention also include combination of chemotherapy with a compound or composition of the invention, or with a combination of a compound or composition of this invention with another anti-viral agent and vaccination with an Influenza vaccine.

When co-administration involves the separate administration of the first amount of a compound of the invention and a second amount of an additional therapeutic agent, the compounds are administered sufficiently close in time to have the desired therapeutic effect. For example, the period of time between each administration which can result in the desired therapeutic effect, can range from minutes to hours and can be determined taking into account the properties of each compound such as potency, solubility, bioavailability, plasma half-life and kinetic profile. For example, a compound of the invention and the second therapeutic agent can be administered in any order within 24 hours of each other, within 16 hours of each other, within 8 hours of each other, within 4 hours of each other, within 1 hour of each other or within 30 minutes of each other.

More, specifically, a first therapy (e.g., a prophylactic or therapeutic agent such as a compound of the invention) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a prophylactic or therapeutic agent such as an anti-cancer agent) to a subject.

It is understood that the method of co-administration of a first amount of a compound of the invention and a second amount of an additional therapeutic agent can result in an enhanced or synergistic therapeutic effect, wherein the combined effect is greater than the additive effect that would result from separate administration of the first amount of a compound of the invention and the second amount of an additional therapeutic agent.

As used herein, the term "synergistic" refers to a combination of a compound of the invention and another therapy (e.g., a prophylactic or therapeutic agent), which is more effective than the additive effects of the therapies. A synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) can permit the use of lower dosages of one or more of the therapies and/or less frequent administration of said therapies to a subject. The ability to utilize lower dosages of a therapy (e.g., a prophylactic or therapeutic agent) and/or to administer said therapy less frequently can reduce the toxicity associated with the administration of said therapy to a subject without reducing the efficacy of said therapy in the prevention, management or treatment of a disorder. In addition, a synergistic effect can result in improved efficacy of agents in the prevention, management or treatment of a disorder. Finally, a synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) may avoid or reduce adverse or unwanted side effects associated with the use of either therapy alone.

When the combination therapy using the compounds of the present invention is in combination with an Influenza vaccine, both therapeutic agents can be administered so that the period of time between each administration can be longer (e.g. days, weeks, or months).

The presence of a synergistic effect can be determined using suitable methods for assessing drug interaction. Suitable methods include, for example, the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied with experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration- effect curve, isobologram curve and combination index curve, respectively.

Specific examples that can be co-administered with a compound described herein include neuraminidase inhibitors, such as oseltamivir (Tamiflu®) and Zanamivir (Rlenza®), viral ion channel (M2 protein) blockers, such as amantadine (Symmetrel®) and rimantadine

(Flumadine®), and antiviral drugs described in WO 2003/015798, including T-705 under development by Toyama Chemical of Japan. (See also Ruruta et al, Antiviral Research, 82: 95- 102 (2009), "T-705 (flavipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections"). In some embodiments, the compounds described herein can be co- administered with a traditional influenza vaccine.

In some embodiments, the compounds described herein can be co-administered with zanamivir. In some embodiments, the compounds described herein can be co-administered with flavipiravir (T-705). In some embodiments, the compounds described herein can be coadministered with oseltamivir. In some embodiments, the compounds described herein can be co-administered with amantadine or rimantadine. Oseltamivir can be administered in a dosage regimen specified in its label. In some specific embodiments, it is administered 75 mg twice a day, or 150 mg once a day.

The compounds described herein can be formulated into pharmaceutical compositions that further comprise a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound of the invention described above, and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present invention is a pharmaceutical composition comprising an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices.

An "effective amount" includes a "therapeutically effective amount" and a

"prophylactically effective amount". The term "therapeutically effective amount" refers to an amount effective in treating and/or ameliorating an influenza virus infection in a patient infected with influenza. The term "prophylactically effective amount" refers to an amount effective in preventing and/or substantially lessening the chances or the size of influenza virus infection outbreak. A pharmaceutically acceptable carrier may contain inert ingredients which do not unduly inhibit the biological activity of the compounds. The pharmaceutically acceptable carriers should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic or devoid of other undesired reactions or side-effects upon the administration to a subject. Standard pharmaceutical formulation techniques can be employed.

The pharmaceutically acceptable carrier, adjuvant, or vehicle, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's

Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds described herein, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention. As used herein, the phrase "side effects" encompasses unwanted and adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Side effects are always unwanted, but unwanted effects are not necessarily adverse. An adverse effect from a therapy (e.g., prophylactic or therapeutic agent) might be harmful or uncomfortable or risky. Side effects include, but are not limited to fever, chills, lethargy, gastrointestinal toxicities (including gastric and intestinal ulcerations and erosions), nausea, vomiting, neurotoxicities, nephrotoxicities, renal toxicities (including such conditions as papillary necrosis and chronic interstitial nephritis), hepatic toxicities (including elevated serum liver enzyme levels), myelotoxicities (including leukopenia, myelosuppression, thrombocytopenia and anemia), dry mouth, metallic taste, prolongation of gestation, weakness, somnolence, pain (including muscle pain, bone pain and headache), hair loss, asthenia, dizziness, extra-pyramidal symptoms, akathisia, cardiovascular disturbances and sexual dysfunction.

Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as twin 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil;

glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

The compounds and pharmaceutically acceptable compositions described above can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated.

Liquid dosage forms for oral administration include, but are not limited to,

pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile inj ectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U. S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a compound described herein, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Inj ectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide- polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are specifically suppositories which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin,

polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, 0 absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this invention.

Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

The compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Specifically, the compositions are administered orally, intraperitoneally or intravenously.

Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include, but are not limited to, lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the pharmaceutical compositions described herein may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions described herein may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically -transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol,

polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, specifically, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

The pharmaceutical compositions may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

The compounds for use in the methods of the invention can be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

One of ordinary skill in the art will readily recognize that the foregoing represents merely a detailed description of certain preferred embodiments of the present invention. Various modifications and alterations of the compositions and methods described above can readily be achieved using expertise available in the art and are within the scope of the invention. EXAMPLES

The following examples are illustrative, but not limiting, of the compounds,

compositions, and methods of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the invention.

Example I.

Compound 5 was synthesized by the Ugi-azide 4-CR in methanol at room temperature in a 72% yield (Scheme 1 A). A focused library of compounds 9a-9u with diversity elements at the amine (Ri), aldehyde (R2), and isocyanide (R3) was synthesized according to the general procedure employed to afford compound 5 (Scheme IB). Compounds 5 and 9a-9u were synthesized and tested as enantiomeric mixtures, whilst compounds 12a and 12b were prepared as diastereomeric mixtures (Scheme 1C), and separated by silica gel flash chromatography. The absolute stereochemistry of compound 12b was determined by X-ray crystallography (Scheme 1C).

Scheme 1. Synthesis routes and structures for compounds 5, 9a-9u and 12a-12b.

5 (yield: 72%)

B. General synthesis route of compound 9 by Ugi-azide 4CR

9e 9f 9g

41 C. Synthesis of diastereomers 12a and 12b by Ugi-Azide 4CR

Example II.

This example describes PAC-PBIN inhibition and antiviral activity of the initial hit compound 5.

The initial hit compound 5 was confirmed as a potent inhibitor of the PAC-PB IN polymerase subunit interactions in the ELISA assay with an IC50 of 4.3 ± 0.1 μΜ (FIG. 2). The antiviral activity of compound 5 was tested in the antiviral plaque assay. It was found that compound 5 inhibits multiple strains of influenza A and B viruses with single to submicromolar EC50 values (Table 1). The cellular cytotoxicity of compound 5 in MDCK cells with a 48 h incubation time was 17.4 ± 1.2 μΜ; therefore, the antiviral activity of compound 5 was not due to its cellular cytotoxicity.

Table 1. Broad-spectrum antiviral activity and cytotoxicity of compound 5.

The values are the mean ± S.D. from two independent experiments. Example III.

This example describes structure-activity relationship studies of compound 5.

Encouraged by the broad-spectrum antiviral activity of 5, the inventors were thus interested in pursuing SAR studies further to optimize the antiviral potency and selectivity index of compound 5. To this end, experiments were conducted that synthesized a focused library of 21 compounds (9a-9u) using the expeditious Ugi-azide 4-CR. The library included compounds that have points of diversity at the amine component Ri (9a-9c), the aldehyde component R2 (9d-9o), and the aldehyde and isocyanide components combined (R2 & R3) (9p-9u, and 12a- 12b). All compounds were initially tested at 5 μΜ against the A/WSN/33 (H1N1) virus in the plaque assay to rule out compounds that have no cellular antiviral activity (FIG. 3). Nucleozin and oseltamivir carboxylate, two known influenza antivirals, were included as positive controls. It was found that the amine component, 4-(2-keto-l-benzimidazolinyl)piperidine, is essential for the antiviral activity, as compounds with amine modifications 9a-9c had no antiviral activity

(FIG. 3 and Table 2). Amongst compounds with modifications of the aldehyde input R2 (9d-9o), compounds 9i and 9k-9o had significantly improved antiviral activity compared with compound 5, and infected cells had less than 30% plaque formation when treated with 5 μΜ of compound. Compounds 9d, 9e, 9g, and 9h had moderate antiviral activity, while compounds 9f and 9j were not active. These results suggest that hydrophobic aromatic groups such as benzene (9g, 9i-91), thiophene (9m, 9o), and furan (9n) are preferred at the R2 position. When R2 substitution was benzene, a methoxyl group was tolerated at the ortho-, meta-, and /¾zra-positions (9k and 91). A small alkyl group such as ethyl (9g) was also tolerated at the para- position; however, branched alkyl group such as isopropyl (9j) and a bulky substitution such as benzene (9f) were not tolerated. For compounds simultaneously modified at both the aldehyde component R2 and the isocyanide component R3 (9p-9u), it was found that benzyl is preferred at R3 (9p, 9u versus 9q, 9r, 9s and 9t), and compound 9p had similar antiviral activity as 9o (12.3% versus 19.5% plaque formation at 5 μΜ). Compound 9t was not active (73.0% plaque formation at 5 μΜ), possibly due to the presence of the bulky naphthalene substitution at the R2 position. Compound 9u had antiviral activity similar to compound 5 (22.7% vs 16.5% plaque formation at 5 μΜ) as it combines the favorable substitutions from both the aldehyde component ((o- trifluoromethoxy)phenyl) and the isocyanide component (benzyl).

One feature of Ugi-azide 4-CR is that it produces a new chiral center during the reaction, and hence the product is a mixture of enantiomers. Although the enantiomers could be separated by chiral HPLC or other methods, chiral separation is generally time consuming and expensive, which presents a challenge for future development. Indeed, this somewhat compromises the advantages of exploring MCR products in the drug discovery arena. With grams of material required for possible downstream pharmacokinetic and in vivo animal studies, experiments therefore sought to develop a convenient synthesis and separation strategy to bypass chiral separation. Hence, experiments employed a chiral isocyanide, (<S)-(-)-a-methylbenzyl isocyanide in Ugi-azide 4-CR, which we felt should be well-tolerated based on the SAR results of 9a-9u . With this chiral isocyanide strategy, a mixture of diastereomers was produced (Scheme 1C) that were conveniently separated by silica gel flash column chromatography. The absolute stereochemistry of compound 12b was determined by X-ray crystallography as (R, S). It was found that the (S, S) diastereomer 12a had potent antiviral activity (1.9% plaque formation at 5 μΜ), while the (R, S) diastereomer 12b was not active (FIG. 3).

Selected compounds were also tested for their inhibition of PAC-PBIN interactions in the ELISA assay. For compounds 9i, 9k, 9m, 9o, and 12a, which had potent antiviral activity (less than 20% plaque formation at 5 μΜ), the IC50 values in the ELISA assay ranged from 7.6 μΜ to 15.1 μΜ. For compounds 9c, 9f, and 12b that had no antiviral activity (greater than 90% plaque formation at 5 μΜ), the IC50 values were above 26.9 μΜ. Overall, there is in general a positive correlation between the compounds' PAC-PBIN inhibition and their antiviral efficacy. The lack of a straight linear correlation is expected, as the antiviral efficacy is a combined effect of PAC- PB IN inhibition, cellular permeability, and potential off-target interactions.

Table 2. Antiviral activity and PA-PB1 inhibition of tetrazole analogs.

The results are the mean ± S.D. from two independent experiments. N.D. = not determined.

Next, the cellular cytotoxicity and broad-spectrum antiviral activity of one of the most potent compounds 12a were further profiled (Table 3). Compound 12a was not cytotoxic to either MDCK or A549 cells and the CC50 values were greater than 150 μΜ and 98.1 ± 2.5 μΜ, respectively. When tested against a panel of human clinical isolates of influenza A and B viruses, compound 12a showed broad-spectrum antiviral activity, with EC50 values ranging from 0.6 μΜ to 2.7 μΜ. It is noteworthy that compound 12a had no cross-resistance with the FDA- approved influenza antivirals amantadine and oseltamivir, as shown by the results that compound 12a had potent antiviral activity against viruses that are resistant to amantadine, oseltamivir, or both.

Table 3. Broad-spectrum antiviral activity and cytotoxicity of compound 12a.

B/Memphis/20/1996

2.7 ± 0.2 >55.6/36.3 (Yamagata)

Amantadine

B/Utah/09/2014 resistant

1.5 ± 0.3 > 100/65.4

(Yamagata) Oseltamivir

sensitive

B/Phuket/3073/2013

1.2 ± 0.2 >125/81.8

(Yamagata)

B/Brisbane/60/2008

0.6 ± 0.3 >250/163.5 (Victoria)

aThe values are the mean ± S.D. from two independent experiments. ^bSelectivity index is expressed as MDCK/A549.

Example IV.

This example demonstrates that compound 12a has a higher in vitro genetic barrier to drug resistance than oseltamivir.

Drug-induced resistance is one of the major obstacles facing antiviral drugs (see, e.g., Loregian, A.; et al, Cell Mol Life Sci 2014, 71, 3659-83). Experiments therefore designed serial viral passage experiments to characterize the genetic barrier to drug resistance of compound 12a (FIG. 4) (see, e.g., White, K. M.; et al, ACS Infect Dis 2015, 1, 98-109; Ma, C; et al, Antiviral Res 2016, 133, 62-72; Ma, C; et al, Mol Pharmacol 2016, 90, 188-98; Hu, Y.; et al., Antiviral Res 2017, 140, 45-54). In this experiment, the A/WSN/33 (H1N1) virus was amplified in the presence of increasing concentrations of compound 12a and the drug sensitivity of the resulting viruses at different passages was assayed against compound 12a using a plaque assay.

Oseltamivir carboxylate was included as a control. Gratifyingly, viruses at passage 10 remained sensitive to compound 12a, and no increase in EC50 value was observed (FIG. 4B and Table 4). In contrast, the EC50 for oseltamivir carboxylate increased 10-fold at passage six and onwards, which is consistent with previous reports (see, e.g., Ehrhardt, C; et al, Cell Microbiol 2013, 15, 1198-211; Shih, S. R.; et al, J Antimicrob Chemother 2010, 65, 63-71). These results indicate that compound 12a targets a vital viral replication component such as the viral polymerase that is less prone to mutate. Overall, compound 12a demonstrated a high in vitro genetic barrier to drug resistance, rendering it a desired drug candidate for further development.

Table 4. In vitro serial drug passage experiment with compound 12a.

4 80 N.D. 8 N.D.

5 160 N.D. 16 N.D.

6 320 60.1 ± 14.7 32 0.5 ± 0.3

7 640 N.D. 64 N.D.

8 640 N.D. 64 N.D.

9 640 N.D. 64 N.D.

10 640 70.2 ± 18.0 64 0.7 ± 0.4

Passage was performed using the A/WSN/33 (HlNl) virus by following our reported procedure (see, e.g., Ma, C; et al., Antiviral Res 2016, 133, 62-72; Ma, C; et al, Mol Pharmacol 2016, 90, 188-98; Hu, Y.; et al., Antiviral Res 2017, 140, 45-54). Oseltamivir carboxylate was included as a control. The EC50 values at selected passages were determined by plaque assay. The values are the mean ± S.D. from two independent experiments. N.D. = not determined.

Example V.

This example shows the % plaque formation, EC50, and CC50 values for specific compounds of the present invention against the A/California/07/2009 (HlNl) virus (see, Table 5).

Table 5.



Example VI.

This example describes a docking model of compound 12a in PAc.

To gain insights on how compound 12a binds to the PB1 -binding pocket in PAc, experiments performed molecular docking using Schrodinger Glide software. In the docking model of 12a in PA, 12a snuggly fits in the PB1 -binding pocket in PA (FIG. 5 A), forming extensive hydrophobic interactions and multiple π-π interactions (FIG. 5B). For example, the phenyl ring from the isocyanide input (R3) in 12a interacts with F707 and K643 through π-π and cation-π interactions, respectively. The thiophene ring from 12a forms π-π interactions with F710. In addition, the carbonyl from the benzimidazol-2-one in 12a forms a hydrogen bond with the E623 backbone amide NH, while the benzene ring from benzimidazol-2-one fits in the hydrophobic pocket formed by F41 1 and 1621.

Example VII.

This example describes the materials and methods for Examples I-VI.

Chemistry. All chemicals that were commercially available were used without further purification. All final compounds were purified by flash column chromatography. Compound 5 and 9a-9u were synthesized and tested as a mixture of enantiomers. Compounds 12a and 12b were separated as pure diastereomers and tested individually. ¾ and ¹ C NMR spectra were recorded on a Bruker-400 NMR spectrometer. Chemical shifts are reported in parts per million referenced with respect to residual solvent (DMSO-d6) 2.50 ppm and (Chloroform-d) 7.26 ppm or from internal standard tetramethylsilane (TMS) 0.00 ppm. The following abbreviations were used in reporting spectra: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublets. HPLC-grade solvents were used for all reactions. Flash column chromatography was performed using silica gel (230-400 mesh, Merck). Low- resolution mass spectra were obtained using an ESI technique on a 3200 Q Trap LC/MS/MS system (Applied Biosystems). High-resolution mass spectra were obtained using the positive ESI method for all the compounds, obtained in an Ion Cyclotron Resonance (ICR) spectrometer. The purity was assessed by using a Shimadzu LC-MS with a Waters XTerra MS C-18 column (part #186000538), 50 ^χ 2.1 mm, at a flow rate of 0.3 mL/min; λ = 250 and 220 nm; mobile phase A, 0.1% formic acid in H2O, and mobile phase B', 0.1% formic in 60% isopropanol, 30% CH3CN and 9.9% H2O. All compounds submitted for testing in the ELISA and plaque assays were confirmed to be > 95.0% purity by LC-MS traces. All compounds were characterized by proton and carbon NMR and MS.

Synthesis Procedures. Amine (1.0 equiv) and aldehyde (1.0 equiv) were mixed in methanol (5 ml) and stirred at room temperature for 15 minutes. Then TMS-azide (1.0 equiv) and isocyanide (1.0 equiv) were added sequentially and the resulting mixture was stirred at room temperature overnight. After that, the solvent was removed under reduced pressure and the crude product was purified with flash silica gel chromatography (Ethyl acetate in hexane 20-70%). l -(l -{l -[l -(2,3 -dihydro -1, 4 -benzodioxin -6 -yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ] -phenylpropyljpi peridin-4-yl) -2, 3-dihydro-lH-l, 3-benzodiazol-2-one (5). White solid. Yield: 65%. ¾ NMR (400 MHz, CDCh) δ 10.31 (s, 1H), 7.27-6.89 (m, 12H), 4.24-4.22 (m, 5H), 3.95-3.85 (m, 1H), 2.97- 2.95 (m, 1H), 2.86-2.19 (m, 9H), 1.84-1.70 (m, 2H); ¹³C NMR (101 MHz, CDCh) δ 144.8,

143.5, 134.8, 133.6, 130.2, 118.6, 118.1, 117.9, 117.7, 116.7, 115.8, 110.9, 110.6, 107.8, 107.6, 104.3, 99.5, 99.0, 54.0, 53.9, 46.7, 40.3, 38.6, 37.5, 22.1, 19.2, 19.1, 18.6; C30H31N7O3 HRMS (ESI) m/z calculated for [M + H] ⁺ = 538.25611, found 538.25612. l -(l -{l -[l -(2,3 -dihydro -1, 4 -benzodioxin -6 -yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ] -3 -phenylpropylj piperidin-4-yl) -3-methyl-2,3-dihydro-lH-l, 3-benzodiazol-2-one (9a). Beige oil. Yield: 89%. ¾ NMR (400 MHz, DMSO-de) δ 7.28-7.24 (m, 3H), 7.18-7.10 (m, 5H), 7.07-7.06 (m, 2H), 7.04- 7.02 (m, 2H), 4.33 (2, 4H), 4.14-3.96 (m, 2H), 3.28 (s, 3H), 2.87 (d, J= 12.1 Hz, 1H), 2.66-2.59 (m, 2H), 2.46 (d, J = 9.4 Hz, 1H), 2.31-2.05 (m, 5H), 1.63 (d, J= 13.8 Hz, 1H), 1.52 (d, J= 11.5 Hz, 1H), 1.33-1.17 (m, 1H); ¹ C NMR (101 MHz, DMSO-de) δ 154.1, 152.9, 144.9, 143.6, 141.1, 129.7, 128.3, 128.2, 127.8, 126.7, 125.9, 120.7, 120.6, 118.5, 117.6, 114.7, 108.6, 107.8, 64.22, 64.15, 56.3, 50.5, 47.9, 47.7, 31.8, 29.03, 28.97, 26.7; C31H33N7O3 HRMS (ESI) m/z calculated for [M + H]⁺ = 552.27245, found 552.27176.

8-{l -[1 -(2, 3 -dihydro -1, 4 -benzodioxin -6 -yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ] -3 -phenylpropyl} -1 - phenyl-l, 3,8-triazaspiro[4.5]decan-4-one (9b). White solid. Yield: 66%. ¾ NMR (400 MHz, DMSO-de) δ 8.62 (s, 1H), 7.31 (d, J = 2.3 Hz, 1H), 7.28-7.13 (m, 7H), 7.12-7.01 (m, 2H), 6.81- 6.68 (m, 3H), 4.53 (s, 2H), 4.33-4.30(m, 4H), 3.95-3.92 (m, 1H), 3.08-3.02 (m, 1H), 2.89-2.78 (m, 2H), 2.62-2.57 (m, 3H), 2.49-2.40 (m, 1H), 2.38-2.22 (m, 3H), 1.56 (d, J = 13.2 Hz, 1H), 1.50-1.41 (m, 1H); ¹ C NMR (101 MHz, DMSO-de) δ 176.0, 154.1, 144.9, 143.7, 143.2, 141.1, 128.9, 128.3, 128.2, 126.7, 125.9, 118.3, 117.6, 117.5, 114.4, 113.9, 64.2, 64.1, 58.6, 58.1, 56.4, 45.4, 44.2, 31.8, 28.7, 28.6; C31H33N7O3 HRMS (ESI) m/z calculated for [M + H]⁺ = 552.27176, found 552.27210.

3-(l -{l -[l -(2,3 -dihydro -1, 4 -benzodioxin -6 -yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ] -3 -phenylpropyl} piper idin -4 -yl) -lH -indole (9c). White solid. Yield: 75%. ¾ NMR (400 MHz, CDCb) δ 8.02 (s, 1H), 7.66-7.57 (m, 1H), 7.41-7.34 (m, 1H), 7.30-7.23 (m, 3H), 7.23-7.08 (m, 6H), 7.00-6.92 (m, 3H), 4.46-4.21 (m, 4H), 3.92 (dd, J = 8.6, 5.4, 1H), 2.97 (d, J= 11.4, 1H), 2.84-2.72 (m, 2H), 2.74-2.62 (m, 2H), 2.60-2.46 (m, 2H), 2.45-2.26 (m, 2H), 2.11 (d, J= 13.1, 1H), 1.97 (d, J = 12.8, 1H), 1.83-1.56 (m, 2H). ¹ C NMR (101 MHz, CDCb) δ 154.10, 145.11, 143.93, 140.88, 136.41, 128.47, 128.37, 127.18, 126.55, 126.09, 121.94, 121.23, 119.64, 119.14, 119.11, 118.35, 117.87, 114.81, 111.21, 64.40, 64.29, 57.41, 49.81, 49.09, 33.58, 33.42, 33.06, 32.61, 29.12; C31H32N6O2 EI-MS: m/z (M+H⁺): 521.6 (calculated), 521.0 (found). l -(l -{l -[l -(2,3 -dihydro -1, 4 -benzodioxin -6 -yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl Jpropyljpiperidin - 4-yl) -2, 3-dihydro-lH-l, 3-benzodiazol-2-one (9d). Beige solid. Yield: 34%. ¾ NMR (400 MHz, DMSO-de) δ 10.79 (s, 1H), 7.35-7.34 (m, 1H), 7.17-7.11 (m, 2H), 7.08-7.05 (m, 1H), 6.99- 6.94 (m, 3H), 4.34 (s, 4H), 4.13-3.93 (m, 2H), 2.90 (d, J= 10.4 Hz, 1H), 2.51-2.46 (m, 3H), 2.23-1.99 (m, 4H), 1.62 (d, J = 13.7 Hz, 1H), 1.51 (d, J = 11.8 Hz, 1H), 0.89 (t, J = 7.3 Hz, 3H); ¹ C NMR (100 MHz, DMSO-de) δ 154.3, 153.6, 144.9, 143.7, 129.1, 128.3, 127.0, 120.5, 120.3, 118.6, 117.7, 114.7, 108.8, 108.6, 64.2, 58.9, 49.9, 48.4, 47.5, 29.0, 28.9, 20.7, 11.2;

C24H27N7O3 HRMS (ESI) m/z calculated for [M + H]⁺ = 462.22481, found 462.22522. 1-(1-{1-[1-(2,3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl J -2 -phenylethyljpi peridin-4-yl)-2,3-dihydro-lH-l,3-benzodiazol-2-one (9e). White solid. Yield: 75%. ¾NMR (400 MHz, DMSO-ce) δ 10.81 (s, 1H), 7.30-7.13 (m, 5H), 7.08-6.92 (m, 5H), 6.83 (d, J= 2.5 Hz, 1H), 6.71 (dd,J= 8.6, 2.5 Hz, 1H), 4.35-4.23 (m, 5H), 4.07-3.96 (m, 1H), 3.44-3.27 (m, 2H), 3.07 (d,J= 11.4 Hz, 1H), 2.65 (d, J= 11.3 Hz, 1H), 2.50-2.42 (m, 1H), 2.30 (dd, J= 12.2, 9.8 Hz, 1H), 2.22-2.01 (m, 2H), 1.64 (d,J= 11.9 Hz, 1H), 1.54 (d,J= 11.7 Hz, 1H).¹ C NMR (101 MHz, DMSO-ce) δ 154.36, 154.05, 145.46, 144.08, 138.50, 129.77, 129.53, 128.80, 128.76, 126.96, 126.88, 120.97, 120.76, 118.85, 118.10, 114.93, 114.26, 109.28, 109.07, 64.67, 64.61, 60.14, 50.27, 49.07, 48.45, 35.35, 29.42, 29.30; C29H29N7O3 EI-MS: m/z (M+H+): 524.6 (calculated), 525.0 (found).

1-[1-({[1,1 '-biphenyl ] -4-yl}[l -(2,3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 - yl]methyl)piperidin-4-yl] -2,3-dihydro-lH-l,3-benzodiazol-2-one (9f). White solid. Yield: 62%. ¾ NMR (400 MHz, DMSO-ce) δ 10.81 (s, 1H), 7.74-7.65 (m, 4H), 7.56-7.43 (m, 4H), 7.42- 7.33 (m, 1H), 7.24 (d, J= 2.5 Hz, 1H), 7.17-6.92 (m, 6H), 5.27 (s, 1H), 4.41-4.22 (m, 4H),

4.06-3.84 (m, 1H), 2.89 (dd, J= 30.1, 10.5 Hz, 2H), 2.35-2.03 (m, 4H), 1.66-1.53 (m, 2H).¹³C NMR (101 MHz, DMSO-ce) δ 154.93, 154.07, 145.64, 144.15, 140.44, 140.06, 134.07, 130.30, 129.62, 129.43, 128.74, 128.07, 127.17, 126.98, 120.98, 120.77, 119.35, 118.15, 115.46, 109.25, 109.01, 64.74, 64.63, 62.55, 50.28, 50.16, 49.28, 29.21, 29.11; C34H31N7O3 EI-MS: m/z

(M+H+): 586.7 (calculated), 587.0 (found). l-(l-{[l-(2,3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ]( 4-ethylphenyl)met hyl}piperidin-4-yl)-2,3-dihydro-lH-l,3-benzodiazol-2-one (9g). White solid. Yield: 69%. ¾ NMR (400 MHz, DMSO-ce) δ 10.80 (s, 1H), 7.32 (d, J= 8.2 Hz, 2H), 7.29-7.15 (m, 3H), 7.15- 7.06 (m, 2H), 7.04-6.92 (m, 4H), 5.14 (s, 1H), 4.44-3.78 (m, 5H), 2.84 (dd, J= 26.0, 10.1 Hz, 2H), 2.62 (q,J=7.6Hz, 2H), 2.31-2.13 (m, 3H), 2.02 (t,J= 11.4 Hz, 1H), 1.57 (t,J= 14.4 Hz, 2H), 1.20(t,J=7.6Hz, 3H).¹³CNMR(101 MHz, DMSO-ce) δ 155.13, 154.07, 145.62, 144.19, 144.14, 132.29, 129.60, 128.73, 128.10, 127.12, 120.97, 120.77, 119.32, 118.14, 115.43, 114.26, 109.24, 109.00, 64.73, 64.62, 62.69, 50.31, 50.13, 49.34, 29.16, 29.07, 28.28, 15.88;

C30H31N7O3 EI-MS: m/z (M+H+): 538.6 (calculated), 539.0 (found). l-(l-{[l-(2,3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl Jfpyridin -4 -yl)methy ljpiperidin-4 -yl) -2, 3 -dihydro -lH-1 , 3 -benzodiazol -2 -one (9h). Whites solid. Yield: 43%. ¾ NMR (400 MHz, DMSO-de) δ 10.08 (s, 1H), 8.60 (d, J= 6.0 Hz, 2H), 7.61-7.37 (d, J= 6.0 Hz, 2H), 7.30 (d, J= 2.1 Hz, 1H), 7.19-7.03 (m, 3H), 6.97 (m, 3H), 5.39 (s, 1H), 4.34 (t, J= 3.3 Hz, 4H), 4.18-3.81 (m, 1H), 2.88 (d, J= 9.3 Hz, 1H), 2.74 (d, J = 10.0 Hz, 1H), 2.35-1.91 (m, 4H), 1.72 - 1.40 (m, 2H). ¹ C NMR (100 MHz, DMSO-de) δ 153.58, 153.23, 149.56, 145.17, 143.66, 142.55, 129.09, 128.27, 126.75, 124.25, 120.52, 120.28, 118.83, 117.65, 114.93, 108.80, 108.55, 79.16, 64.28, 64.16, 61.14, 49.60, 49.32, 48.45, 28.73, 28.66; C27H26N8O3 EI-MS: m/z (M+H⁺): 511.6 (calculated), 512.0 (found). l -(l -{[l -(2, 3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ] (phenyl)methyl}pip eridin-4-yl) -2, 3-dihydro-lH-l,3-benzodiazol-2-one (9i). White solid. Yield: 88%. ¾ NMR (400 MHz, DMSO-de) δ 10.79 (s, 1H), 7.41-7.33 (m, 5H), 7.19-7.18 (m, 1H), 7.11-7.07 (m, 2H), 7.01-6.94 (m, 4H), 5.19 (s, 1H), 4.36-4.33 (m, 4H), 4.02-3.94 (m, 1H), 2.87 (d, J = 9.7 Hz, 1H), 2.80 (d, J = 11.9 Hz, 1H), 2.51-2.50 (m, 1H), 2.28-2.12 (m, 2H), 1.61-1.52 (m, 1H); ¹ C NMR (101 MHz, DMSO-de) δ 154.5, 153.6, 145.1, 143.7, 134.5, 129.2, 129.1, 128.3, 128.2, 126.7, 120.5, 120.3, 118.8, 117.7, 115.0, 108.8, 108.5, 64.3, 64.1, 62.4, 59.7, 49.8, 49.6, 48.8, 28.6, 20.7, 14.1, 10.8; C28H27N7O3 HRMS (ESI) m/z calculated for [M + H]⁺ = 510.22481, found 510.22513. l -(l -{[l -(2, 3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ][ 4 -(propan -2 -yl)ph enyl]methyl}piperidin-4-yl) -2, 3-dihydro-lH-l, 3-benzodiazol-2-one (9j). White solid. Yield: 65%. ¾ NMR (400 MHz, DMSO-c e) δ 10.80 (s, 1H), 7.33 (d, J = 8.3 Hz, 2H), 7.26 (d, J= 8.3 Hz, 2H), 7.21-7.07 (m, 3H), 7.05-6.92 (m, 4H), 5.14 (s, 1H), 4.48-4.24 (m, 4H), 4.07-3.89 (m, 1H), 2.94-2.77 (m, 3H), 2.30-2.10 (m, 3H), 2.03 (t, J= 11.5 Hz, 1H), 1.64-1.52 (m, 2H), 1.22 (d, J= 6.9 Hz, 6H). ¹ C NMR (101 MHz, DMSO-c e) δ 155.15, 154.07, 148.77, 145.62, 144.13, 132.43, 129.60, 128.73, 127.13, 126.63, 120.97, 120.77, 119.35, 118.13, 115.46, 109.24, 109.01, 64.73, 64.62, 62.67, 50.30, 50.16, 49.35, 33.58, 29.16, 29.06, 24.29, 24.24; C31H33N7O3 EI-MS: m/z (M+H⁺): 552.7 (calculated), 553.0 (found). l -(l -{[l -(2, 3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ]( 2, 3 -dimethoxyphe nyl)methyl}piperidin-4-yl) -2, 3-dihydro-lH-l, 3-benzodiazol-2-one (9k). White solid. Yield: 56%. ¾ NMR (400 MHz, CDCh) <H0.00 (s, 1H), 7.32-7.29 (m, 1H), 7.18-7.16 (m, 1H), 7.10-7.06 (m, 2H), 7.04-6.98 (m, 4H), 6.92-6.85 (m, 2H), 5.56 (s, 1H), 4.35-4.30 (m, 4H), 4.26-4.21 (m, 1H), 3.87 (s, 3H), 3.64 (s, 3H), 3.10-2.98 (m, 2H), 2.54-2.34 (m, 3H), 2.32-2.16 (m, 1H), 1.84- 1.71 (m, 2H); ¹ C NMR (101 MHz, CDCh) δ 155.2, 154.8, 152.7, 147.3, 145.5, 144.1, 129.2, 129.0, 128.2, 127.0, 123.9, 122.1, 121.3, 121.1, 119.1, 118.1, 115.4, 112.7, 109.8, 109.7, 64.5, 64.4, 60.7, 56.0, 55.9, 50.8, 50.5, 50.1, 29.6; C30H31N7O5 HRMS (ESI) m/z calculated for [M + H]⁺ = 570.24594, found 570.24716. l -(l -{[l -(2, 3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ]( 4 -methoxyphenyl) methyl}piperidin-4-yl) -2, 3-dihydro-lH-l,3-benzodiazol-2-one (91). White solid. Yield: 76%. ¾ NMR (400 MHz, CDCh) δ 10.16 (s, 1H), 7.35 (d, J = 8.3 Hz, 2H), 7.26-7.15 (m, 1H), 7.15- 6.96 (m, 4H), 6.96-6.84 (m, 3H), 6.77 (dd, J= 8.6, 2.5 Hz, 1H), 4.89 (s, 1H), 4.34 (t, J = 3.8 Hz, 4H), 4.31 (s, 1H), 3.81 (s, 3H), 3.07 (d, J= 11.1 Hz, 1H), 2.96 (d, J= 7.0 Hz, 1H), 2.58-2.30 (m, 3H), 2.11 (m, 1H), 1.75 (m, 2H). ¹³C NMR (101 MHz, CDCh) δ 159.85, 155.25, 154.95, 145.63, 144.16, 130.59, 129.15, 128.19, 126.89, 126.79, 121.30, 121.11, 118.95, 118.15, 115.27, 114.10, 109.88, 109.73, 64.53, 64.43, 63.19, 55.44, 50.74, 50.50, 49.99, 29.47, 29.35;

C29H29N7O4 EI-MS: m/z (M+H⁺): 540.6 (calculated), 541.0 (found). l -(l -{[l -(2, 3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ]( thiophen -2 -yl)met hyl}piperidin-4-yl) -2, 3-dihydro-lH-l, 3-benzodiazol-2-one (9m). White solid. Yield: 61%. ¾ NMR (400 MHz, CDCh) δ 10.31 (s, 1H), 7.37-7.35 (m, 1H), 7.18-6.92 (m, 8H), 5.35 (s, 1H), 4.35-4.21 (m, 5H), 3.13-3.02 (m, 2H), 2.64-2.56 (m, 1H), 2.49-2.37 (m, 2H), 2.26-2.19 (m, 1H), 1.84-1.76 (m, 2H); ¹³C NMR (100 MHz, CDCh) δ 155.3, 153.6, 145.7, 144.3, 136.8, 129.1, 128.6, 128.2, 126.9, 126.8, 126.7, 121.4, 121.2, 118.6, 118.3, 115.0, 110.0, 109.6, 64.5, 64.4, 60.5, 58.4, 50.6, 50.0, 48.8, 29.5, 29.3, 21.2, 14.3, 13.8; C26H25N7O3S HRMS (ESI) m/z calculated for [M + H]⁺ = 516.18123, found 516.18154. l -(l -{[l -(2, 3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ] (fur an -3 -yl)methylj piperidin-4-yl) -2, 3-dihydro-lH-l,3-benzodiazol-2-one (9n). White solid. Yield: 68%. 'H NMR (400 MHz, CDCh) δ 9.69 (s, 1H), 7.66-7.39 (m, 2H), 7.25-7.15 (m, 2H), 7.14-7.00 (m, 5H),

6.67 (dd, J= 1.9, 0.9 Hz, 1H), 5.05 (s, 1H), 4.41-4.30 (m, 4H), 4.30-4.18 (m, 1H), 3.04 (dd, J = 40.0, 11.3 Hz, 2H), 2.71-2.04 (m, 4H), 1.92-1.72 (m, 2H). ¹³C NMR (101 MHz, CDCh) δ 154.90, 153.74, 145.45, 144.08, 143.33, 142.36, 129.11, 127.97, 127.00, 121.21, 121.05, 118.44, 118.12, 118.10, 114.81, 111.53, 109.68, 109.47, 64.44, 64.37, 54.67, 50.54, 50.33, 47.81, 29.50, 29.16; C26H25N7O4 EI-MS: m/z (M+H⁺): 500.5 (calculated), 500.0 (found). l -(l -{[l -(2, 3 -dihydro -1, 4 -benzodioxin -6-yl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ]( thiophen -3 -yl)met hyl}piperidin-4-yl) -2, 3-dihydro-lH-l, 3-benzodiazol-2-one (9o). White solid. Yield: 76%. ¾ NMR (400 MHz, CDCh) δ 9.82 (s, 1H), 7.42-7.26 (m, 3H), 7.25-7.15 (m, 1H), 7.16-7.00 (m, 5H), 6.93 (dd, J= 8.6, 2.5 Hz, 1H), 5.17 (d, J= 0.5 Hz, 1H), 4.42-4.32 (m, 4H), 4.25 (tt, J = 12.2, 4.2 Hz, 1H), 3.52 (d,J=4.9Hz, 1H), 3.18-3.10 (m, 1H), 3.04-2.96 (m, 1H), 2.62-2.34 (m, 3H), 2.23-2.12 (m, 1H), 1.86-1.78 (m, 2H).¹ C NMR (101 MHz, CDCh) δ 154.97, 154.08, 145.49, 144.08, 134.78, 129.09, 128.44, 128.00, 126.83, 126.10, 125.46, 121.21, 121.04, 118.60, 118.10, 114.95, 109.70, 109.52, 64.43, 64.35, 58.57, 50.57, 50.34, 48.79, 29.48, 29.23;

C26H25N7O3S EI-MS: m/z (M+H+): 516.6 (calculated), 517.0 (found).

1 -{1-[(1 -benzyl -lH-1, 2, 3, 4 -tetrazol -5 -yl) ( (hiophen -3 -yljmethyl Jpiperidin -4 -ylj -2, 3 -dihyd ro-lH-l,3-benzodiazol-2-one (9p). White solid. Yield: 82%. ¾ NMR (400 MHz, DMSO-ce) δ 10.81 (s, 1H), 7.55 (d, J= 4.5 Hz, 2H), 7.46-7.16 (m, 6H), 7.16-6.79 (m, 4H), 6.01-5.76 (m, 2H), 5.63 (s, 1H), 3.97 (m, 1H), 3.00 (d, J= 6.9 Hz, 1H), 2.89 (d, J= 10.8 Hz, 1H), 2.37-1.93 (m, 4H), 1.68-1.45 (m, 2H).¹ CNMR(101 MHz, DMSO-ce) δ 154.22, 153.64, 134.86, 134.37, 129.10, 128.80, 128.74, 128.25, 128.15, 127.72, 125.88, 125.66, 120.48, 120.25, 108.76, 108.62, 79.16, 57.27, 54.88, 50.29, 49.84, 48.62, 47.89, 28.79, 28.52; C25H25N7OS EI-MS: m/z (M+H+): 472.6 (calculated), 473.0 (found).

1 -{1-[(1 -cyclohexyl -lH-1, 2, 3, 4 -tetrazol -5 -yl) ( (hiophen -3 -yljmethyl Jpiperidin -4 -yl} -2, 3 -d ihydro-lH-l,3-benzodiazol-2-one (9q). White solid. Yield: 65%. ¾ NMR (400 MHz, DMSO- de)510.79 (s, 1H), 7.71-7.44 (m, 2H), 7.27 (dd,J=4.8, 1.4 Hz, 1H), 7.22-7.06 (m, 1H), 7.06- 6.79 (m, 3H), 5.66 (s, 1H), 4.03 (m, 1H), 3.04 (d,J= 10.8 Hz, 1H), 2.87 (d,J= 10.8 Hz, 1H), 2.46-2.15 (m, 3H), 2.16-2.00 (m, 2H), 2.00-1.77 (m, 5H), 1.77-1.38 (m, 5H), 1.38-1.20 (m, 1H).¹ CNMR(101 MHz, DMSO-ce) δ 153.63, 153.24, 134.77, 129.21, 128.67, 128.32, 125.92, 125.41, 120.48, 120.20, 108.81, 108.34, 79.16, 57.51, 57.12, 50.29, 49.76, 47.93, 32.87, 32.60, 28.91, 28.63, 24.85, 24.82, 24.63; C24H29N7OS EI-MS: m/z (M+H+): 464.6 (calculated), 465.0 (found). l-[l-({l-[(4 -tnethylbenzenesulfonyljmethyl ] -lH-1, 2, 3, 4 -tetrazol -5 -yl}( (hiophen -3 -yl)met hyl)piperidin-4-yl] -2,3-dihydro-lH-l,3-benzodiazol-2-one (9r). White solid. Yield: 53%. ¾ NMR (400 MHz, DMSO-ce) δ 10.79 (s, 1H), 7.73-7.62 (m, 2H), 7.58 (dd, J= 5.0, 2.9 Hz, 1H), 7.55-7.42 (m, 3H), 7.25-7.14 (m, 2H), 7.04-6.86 (m, 3H), 6.72-6.50 (m, 2H), 5.47 (s, 1H),

4.04-3.87 (m, 1H), 2.92 (dd,J= 53.6, 11.2 Hz, 2H), 2.42 (s, 3H), 2.39-2.14 (m, 3H), 2.03-1.84 (m, 1H), 1.62 (dd,J=31.7, 11.8 Hz, 2H).¹³CNMR(101 MHz, DMSO) δ 154.84, 153.64, 146.08, 133.76, 133.12, 130.24, 129.19, 128.72, 128.27, 125.80, 125.77, 120.50, 120.28, 108.74, 108.61, 64.73, 57.23, 49.82, 49.71, 47.81, 28.86, 28.56, 21.22; C26H27N7O3S2 EI-MS: m/z (M+H⁺): 550.7 (calculated), 551.0 (found). l -(l -{[l -(2, 6-dimethylphenyl) -lH-1, 2, 3, 4 -tetrazol -5 -yl ]( thiophen -3 -yl)methyl}piperidin - 4-yl) -2, 3-dihydro-lH-l, 3-benzodiazol-2-one (9s). White solid. Yield: 72%. ¾ NMR (400 MHz, DMSO-c¾) 5 10.78 (s, 1H), 7.55 (dd, J = 5.0, 3.0 Hz, 1H), 7.50 (t, J= 7.6 Hz, 1H), 7.44 (dd, J = 3.0, 1.3 Hz, 1H), 7.41 (m, 1H), 7.29 (m, 1H), 7.21 (dd, J= 5.0, 1.3 Hz, 1H), 7.08 (m, 1H), 7.02- 6.92 (m, 3H), 4.87 (s, 1H), 3.98 (m, 1H), 3.10-2.72 (m, 2H), 2.39-2.11 (m, 3H), 2.06 (s, 3H), 2.04-1.90 (m, 1H), 1.70-1.53 (m, 2H), 1.48 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 155.34, 153.61, 135.41, 135.35, 134.17, 131.42, 131.05, 129.23, 128.90, 128.79, 128.52, 128.25, 126.45, 126.10, 120.46, 120.28, 108.76, 108.34, 79.16, 57.98, 51.00, 49.84, 48.14, 28.67, 28.41, 17.15, 16.28; C26H27N7OS EI-MS: m/z (M+H⁺): 486.6 (calculated), 486.0 (found).

1 -{1 -[(1 -cyclopentyl -lH-1, 2, 3, 4 -tetrazol -5 -yl)(naphthalen -1 -yljmethyl Jpiperidin -4 -yl} -2, 3-dihydro-lH-l, 3-benzodiazol-2-one (9t). White solid. Yield: 62%. ¾ NMR (400 MHz, CDCh) δ 9.66 (s, 1H), 8.53-8.50 (m, 1H), 7.91-7.85 (m, 2H), 7.65-7.61 (m, 1H), 7.58-7.53 (m, 2H), 7.49-7.45 (m, 1H), 7.22-7.20 (m, 1H), 7.12-7.05 (m, 3H), 5.93 (s, 1H), 4.91-4.83 (m, 1H), 4.40-4.33 (m, 1H), 3.27-3.23 (m, 1H), 2.92-2.88 (m, 1H), 2.81-2.76 (m, 1H), 2.72-2.59 (m,M), 2.53-2.44 (m, 1H), 1.92-1.78 (m, 8H), 1.64-1.59 (m, 2H); ¹³C NMR (101 MHz, CDCh) δ 155.1, 153.5, 134.3, 131.7, 131.5, 129.7, 129.4, 129.1, 128.1, 127.0, 126.6, 126.3,

125.1, 123.7, 121.4, 121.2, 109.8, 109.4, 61.3, 59.3, 52.0, 51.2, 50.0, 33.5, 33.3, 29.6, 24.9, 24.7; C29H31N7O HRMS (ESI) m/z calculated for [M + H] ⁺ = 494.26629, found 494.26652.

1 -{1 -[(1 -benzyl -lH-1, 2, 3, 4 -tetrazol -5 -yl)[ 2 -( (rifluoromethoxyjphenyl ] methyl Jpiperidin -4 -yl}-2, 3-dihydro-lH-l, 3-benzodiazol-2-one (9u). White solid. Yield: 79%. ¾ NMR (400 MHz, CDCh) δ 10.15 (s, 1H), 7.89-7.86 (m, 1H), 7.40-7.31 (m, 5H), 7.28-7.26 (m, 1H), 7.22-7.19 (m, 2H), 7.12-7.01 (m, 4H), 5.78 (d, J= 15.3 Hz, 1H), 5.56 (d, J= 15.3 Hz, 1H), 5.36 (s, 1H), 4.19-4.12 (m, 1H), 2.88-2.85 (m, 1H), 2.76-2.75 (m, 1H), 2.46-2.29 (m, 2H), 2.19-2.12 (m, 2H), 1.73-1.67 (m, 2H); ¹³C NMR (101 MHz, CDCh) δ 155.3, 154.0, 147.6, 133.3, 131.9, 130.2, 129.3, 129.2, 129.1, 128.2, 127.6, 126.9, 126.2, 121.8, 121.4, 121.2, 119.8, 109.9, 109.5, 55.8, 51.5, 50.6, 50.5, 49.3, 29.4, 29.3; C28H26F3N7O2 HRMS (ESI) m/z calculated for [M + H] ⁺ = 550.21728, found 550.21728. IS) -1 -p enylethyl J -lH-1, 2, 3, 4 -tetrazol -5 -yl}(thiophen -3 -yljmethyl Jpiperid in-4-yl} -2, 3-dihydro-lH-l,3-benzodiazol-2-one (12a). C26H27N7OS EI-MS: m/z (M+H⁺): 486.6 (calculated), 487.0 (found). l -{l -[(R) -{l -[( IS) -1 phenylethyl J -lH-1, 2, 3, 4 -tetrazol -5 -yl}( thiophen -3 -yljmethyl Jpiperid in-4-yl} -2, 3-dihydro-lH-l,3-benzodiazol-2-one (12b). C26H27N7OS EI-MS: m/z (M+H⁺): 486.6 (calculated), 487.0 (found).

Plaque assay. The plaque reduction assay was performed as previously reported (see, e.g., Wang, J.; et al, PNAS 2013, 110, 1315-1320), except MDCK cells expressing ST6Gal I were used instead of regular MDCK cells (see, e.g., Hatakeyama, S.; et al, Journal of Clinical Microbiology 2005, 43, 4139-4146). Briefly, the confluent monolayers of ST6Gal MDCK cells were incubated with -100 pfu virus samples in DMEM with 0.5% BSA for 1 h at 4 °C, then 37 °C for 1 h. The inoculums were removed, and the cells were washed with phosphate buffered saline (PBS). The cells were then overlaid with DMEM containing 1.2% Avicel microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA) and NAT (2.0 μg/mL). To examine the effect of the compounds on plaque formation, the overlay media was supplemented with compounds at testing concentrations. At two days after infection, the monolayers were fixed and stained with crystal violet dye solution (0.2% crystal violet, 20% methanol). Influenza A virus A/WSN/33 (H1N1) was obtained from Dr. Robert Lamb at the Northwestern University. The influenza viruses A/Texas/04/2009 (H1N1), B/Wisconsin/1/2010, and B/Brisbane/60/2008 were obtained from Dr. James Noah at the Southern Research Institute. Influenza A and B viruses

A/Switzerland/9715293/2013 X-247 (H3N2), FR-1366; A/Washington/29/2009 (H1N1), FR- 460; A/California/07/2009 (H1N1), FR-201; A/Washington/29/2009 (H1N1), FR-460;

B/Memphis/20/1996, FR-486; B/Utah/9/2014, FR-1372; and B/Phuket 3073/2013, FR-1364; were obtained through the Influenza Reagent Resource, Influenza Division, WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, Atlanta, GA, USA. The influenza viruses A/Denmark/524/2009 (H1N1) and A/Denmark/528/2009 (H1N1) was obtained from Dr. Elena Govorkova at St. Jude Children's Research Hospital.

Cytotoxicity assay. Evaluation of the cytotoxicity of compounds was carried out using the neutral red uptake assay (see, e.g., Repetto, G; del Peso, A.; Zurita, J. L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 2008, 3, 1125-31). Briefly, 80,000 cells/mL of MDCK or A549 cells in DMEM medium supplemented with 10% FBS and 100 U/mL Penicillin-Streptomycin were dispensed into 96-well cell culture plates at 100 μίΛνβΙΙ. Twenty-four hours later, the growth medium was removed and washed with 100 PBS buffer; then for the cytotoxicity assay, 200 fresh DMEM (no FBS) medium containing serial diluted compounds was added to each well. After incubating for 48 h at 37 °C with 5% CO2 in a CO2 incubator, the medium was removed and replaced with 100 μΐ. DMEM medium containing 40 μg/mL neutral red for four hours at 37 °C. The amount of neutral red uptake was determined at absorbance 540 nm using a Multiskan FC Microplate Photometer (Fisher Scientific). The CC50 values were calculated from best-fit dose response curves with variable slope in GraphPad Prism version 5.

ELISA assay. To test the inhibitory activity of compound on PAC-PB IN interaction, ELISA was performed (see, e.g., Yuan, S.; et al, Antiviral Res 2016, 125, 34-42). Briefly, microliter plates were coated with 400 ng of His-tagged PA239-716 (PAc ) for 3 h at 37 °C, followed by blocking with 2% (wt/vol) BSA in phosphate buffer saline (PBS) for 1 h. After washing with PBS containing 0.3% Tween 20, plates were incubated with 200 ng of GST- tagged PB11-25 (PB IN ) protein and compounds overnight at room temperature. Then the PAC- PB IN interaction was detected using a horseradish peroxidase (HRP)- conjugated anti-GST monoclonal antibody and its chromogenic substrate TMB. The absorbance at 450 nm was read on a plate reader. The IC50 values were calculated from best-fit dose response curves with variable slope in GraphPad Prism version 5.

Serial drug passage experiments. Serial drug passage experiments were performed accordingly to previously published protocol (see, e.g., Ma, C; et al., Antiviral Res 2016, 133, 62-72; Ma, C; et al, Mol Pharmacol 2016, 90, 188-98; Hu, Y.; et al., Antiviral Res 2017, 145, 103-113). Briefly, MDCK cells were infected with the A/WSN/33 (H1N1) virus at MOI 0.001 for 1 h. Then the inoculum was removed and MDCK cells were incubated with 1 μΜ compound 12a in the first passage and the concentration of 12a was gradually increased 2-fold in passages 2-7 and kept constant at 64 μΜ in passages 7-10. In each passage, the viruses were harvested when a significant cytopathic effect was observed, which usually takes 2-3 days after virus infection. The titers of harvested viruses were determined by plaque assay. The drug sensitivity after passages 3, 6, and 10 was determined via plaque assay as described previously (see, e.g., Hu, Y.; et al., Eur J Med Chem 2017, 135, 70-76). Oseltamivir carboxylate was included as a control and similar fold of drug selection pressure was applied. The drug sensitivity of oseltamivir at passages 3, 6, and 10 was determined via plaque assay.

Having now fully described the invention, it will be understood by those of skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety. INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

What Is Claimed Is:

1. A compound having Formula I:

including pharmaceutically acceptable

provided that if R2 is

provided that if R2 is , t en Rl cannot be

provided that if R2 is

, then Rl cannot be and provided that if R2 is

, then Rl cannot be

wherein the resulting compound is able to:

inhibit influenza polymerase activity; and/or

inhibit interaction between PA and PBl (PAC-PBIN) of an influenza polymerase; and/or inhibit and/or prevent influenza virus activity; and/or

inhibit drug resistant influenza virus activity.

The compound of claim 1 , wherein Rl is selected from the group consisting of Rl is

64

65

66

5. A pharmaceutical composition comprising a compound according to claim 1 and at least one pharmaceutically acceptable carrier or excipient.

6. A method of reducing the amount of influenza viruses in a biological in vitro sample or in a subject, comprising administering to the sample an effective amount of one or more of the compounds described herein.

7. A method of inhibiting the replication of influenza viruses in a biological in vitro sample or in a subject, comprising administering to the sample an effective amount of one or more of the compounds described herein.

8. A method of treating influenza in a subject, comprising administering to the subject a therapeutically effective amount of one or more of the compounds described herein.

9. A method of treating influenza through inhibiting influenza polymerase activity in a biological in vitro sample or in a subject, comprising administering to the sample an effective amount of one or more of the compounds described herein.

10. The method of claim 9, wherein inhibiting influenza polymerase activity comprises inhibiting interaction between the PA subunit and PBl subunit (PAC-PBIN) of an influenza polymerase.

11. The method of any one of claims 6-10, further comprising co-administering one or more additional therapeutic agents to the subject.

12. The method of claim 11, wherein the additional therapeutic agents include an anti-virus drug.

13. The method of claim 12, wherein the anti-virus drug is a neuraminidase inhibitor.

14. The method of claim 13, wherein the neuraminidase inhibitor is oseltamivir or zanamivir.

15. The method of claim 12, wherein the anti-virus drug is a polymerase inhibitor.

16. The method of claim 15, wherein the polymerase inhibitor is flavipiravir.

17. The method of any one of claims 6-10, wherein the influenza viruses are influenza A viruses.

18. The method of claim 17, wherein the influenza A viruses are H1N1 viruses.

19. The method of claims 6-10, wherein the subject is a human subject.

20. A kit comprising a compound of claim 1 and instructions for administering said compound to a patient suffering from an influenza virus.

21. The kit of claim 20 further comprising one or more one or more additional therapeutic agents

22. The kit of claim 21, wherein the additional therapeutic agents include an anti -virus drug. The kit of claim 22, wherein the anti-virus drug is a neuraminidase inhibitor.

The kit of claim 23, wherein the neuraminidase inhibitor is oseltamivir or zanamivir.

The kit of claim 21, wherein the anti-virus drug is a polymerase inhibitor.

The kit of claim 25, wherein the polymerase inhibitor is fiavipiravir.

The kit of claim 21, wherein the influenza viruses are influenza A viruses.