3c protease inhibitors of picorna viruses including enterovirus d68 and human rhino viruses

Abstract

Compounds and treatment methods with dipeptidyl compounds exhibiting antiviral activity and / or inhibition of viral replication against viruses by inhibiting 3CL protease, particularly those belonging to picornavirus, the compounds having a formula:(I) or (I)*.

Description

[0001] 3C PROTEASE INHIBITORS OF PICORNA VIRUSES INCLUDING ENTEROVIRUS D68 AND HUMAN RHINO VIRUSES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 695,205, filed September 16, 2024, entitled 3C PROTEASE INHIBITORS OF PICORNA VIRUSES INCLUDING ENTEROVIRUS D68 AND HUMAN RHINOVIRUSES, incorporated by reference in its entirety herein.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under AH 30092 and AH 61085 awarded by National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Field of the Invention

[0008] The present invention relates to oral antivirals with inhibitory activity against picomaviruses including HRVs and EV-D68.

[0009] Description of Related Art

[0010] Picomaviruses (Picarnaviridae family) are one of the most genetically diverse and contain medically and economically important human and animal pathogens. Enteroviruses belong to the largest genera in the Picomaviridae family and affect millions of people worldwide causing a wide spectrum of diseases from asymptomatic or mild illnesses to severe illnesses. The genus Enterovirus is divided into 10 established and 2 proposed species, and among them, at least 7 species infect humans which includes human rhinovirus (HRV)(A to C) and human enteroviruses (A to D) that include enterovirus D68 (EV-D68), and coxsackievirus A and B (CVA and CVB).

[0011] Although poliovirus (PV) has been nearly eradicated by immunization, non-polio enteroviruses account for more than 85%~95% of aseptic meningitis cases where a specific viral pathogen is identified.

[0012] Human rhinoviruses (HRVs) have more than 100 serotypes and are one of the most commonly implicated viruses in the common cold, being responsible for 30-50% of the cases. HRVs are also important agents for exacerbation of pre-existing respiratory disease such as asthma and chronic obstructive pulmonary disease.

[0013] Enterovirus D68 (EV-D68) is an emerging pathogen responsible for mild to severe respiratory infections that occur mostly in infants, children, and teenagers. EV-D68, originally classified as human rhinovirus (HRV) 87, was first identified in California in 1962, and is biologically more similar to HRV for its acid-lability and the respiratory track as the target tissue than other enteroviruses. EV-D68 can cause mild to severe respiratory illness, which includes fever, runny nose, sneezing, cough, body / muscle aches, wheezing and difficulty breathing at all ages, but infants, children and teenagers are particularly susceptible to severe symptoms. EV-D68 infection causes particularly severe infections in children with asthma or other existing respiratory illnesses. Since its first report in 1962, few outbreaks of EV-D68 had occurred. However, several outbreaks of EV-D68 have been reported in the last 10 years in Europe, Asia and North America. Especially, nationwide outbreak of severe respiratory illness due to EV-D68 infection occurred in the US in 2014 where EV-D68 has been detected in specimens from 12 patients who died. HRVs were recently reclassified into the Enterovirus genus.

[0014] Despite these recognized widespread issues, there are no approved preventive or therapeutic measure against HRVs, EV-D68, or other picornaviruses.

[0015] SUMMARY OF THE INVENTION

[0016] Accordingly, in on aspect, the present disclosure concerns a compound (antiviral, viral inhibitor, protease inhibitor, 3CproL inhibitor) comprising the structure of formula: or a pharmaceutically-acceptable salt thereof, where n is 0-6, each X comprises at least one cyclic moiety; each Y is H or D; Ri is a branched or unbranched Ci-Ce alkyl, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ce-io aryl or arylalkyl, heterocycle, or a natural amino acid side chain selected from the group consisting of leucine, isoleucine, valine, methionine, phenylalanine, fluorophenylalanine, tryptophan, and tyrosine, or an amino acid analog / unnatural side chain such as cyclohexyl alanine, norvaline (n-propyl side chain), norleucine (n-butyl side chain), cyclopropyl methyl alanine, cyclobutyl methyl alanine and gemdimethylcylopropyl proline; and Z is an aldehyde or alpha-ketoamide, provided that when Z is an aldehyde, Ri is not isobutyl (leucine).

[0017] In one aspect, the compound comprises the formula where Y, Z, and Ri are each defined above.

[0018] In a preferred aspect, the compound comprises the formula where n is 0-6, preferably 0-1, R is a halogen, preferably -Cl, and Y, Z, and Ri are each defined above.

[0019] The compound is useful in inhibiting replication of a virus, such as a picornavirus. In one aspect, the picornavirus is human rhinovirus, human enterovirus, including enterovirus D68 (EV- D68), coxsackievirus, or non-polio enterovirus.

[0020] In one aspect, the disclosure concerns an antiviral composition comprising a first protease inhibitor compound according to any of the embodiments and compounds described herein, and a pharmaceutically-acceptable carrier.

[0021] In one aspect, the disclosure concerns a method of inhibiting viral replication in a cell, said method comprising contacting said cell with a compound according to any of the embodiments and compounds described herein, wherein the virus is a picornavirus.

[0022] In one aspect, the disclosure concerns a method of treating or preventing a viral infection in a subject, said method comprising administering to said subject a therapeutically-effective amount of a first compound according to any of the embodiments and compounds described herein, wherein the viral infection is a picomavirus infection. The compound can be administered before or after exposure to the virus and / or before or after observable symptoms of viral infection. The compounds arc useful in reducing viral titers in the subject, such as by inhibiting replication of the vims in the subject.

[0023] DETAILED DESCRIPTION

[0024] The present disclosure is concerned with antiviral compounds that target the 3C or 3C-like protease (3Cpro or 3CLpro) enzyme essential for viral replication in picomaviruses, such as enteroviruses.

[0025] In one aspect, antiviral protease inhibitor compounds are provided which comprise formula

[0026] I, or a pharmaceutically-acceptable salt or prodrug thereof: wherein, each X comprises at least one cyclic moiety, and in particular a substituted (including disubstituted) or unsubstituted C3-C10 cycloalkyl, a substituted or unsubstituted 3-7 membered heterocycle having 1-3 ring heteroatoms selected from N, O, and S, or a substituted or unsubstituted Ce-io aryl group, each of which may be directly attached to the depicted oxygen, or may be connected via a branched or unbranched and substituted or unsubstituted Ci-Ce alkyl linkage (preferably Ci to C2 if linkage present); each Y is H or D;

[0027] Ri is a branched or unbranched Ci-Ce alkyl, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ce-io aryl or arylalkyl, heterocycle, or a natural amino acid side chain selected from the group consisting of leucine, isoleucine, valine, methionine, phenylalanine, fluorophenylalanine, tryptophan, and tyrosine, or an amino acid analog / unnatural side chain such as cyclohexyl alanine, norvaline (n-propyl side chain), norleucine (n-butyl side chain), cyclopropyl methyl alanine, cyclobutyl methyl alanine and gemdimethylcylopropyl proline:

[0028] Z is an aldehyde or alpha-ketoamide (e.g., C(O)C(O)NH-Ra, where Rais a branched or unbranched Ci-Ce alkyl, a C3-C6 cycloalkyl, Ce-io aryl or arylalkyl, acetate esters), provided that when Z is an aldehyde, Ri is not isobutyl / leucine (and preferably Ri comprises a cyclic moiety, more preferably a C3-C10 cycloalkyl or Ce-io aryl or arylalkyl, or heterocycle, such as a cyclic amino acid side chain such as phenylalanine, fluorophenylalanine, or cyclohexyl alanine). Preferably, Rais selected from the group consisting of isobutyl, cyclopropane, benzyl, tert-butyl, cyclohexane, and acetate esters (methyl, ethyl, butyl etc.).

[0029] As noted above, the X group may be connected directly to the depicted oxygen, or may be attached via an alkyl linkage, e.g. the following structure (where the oxygen from formula (I) is depicted for clarity): where n is 0 (no methylene linkage or bridge) or n is 1 to 6, preferably 1 to 2, more preferably where n is 0 or 1, such that a derivative of formula (I) has the structure: , where n is 0-6, preferably 0 or 1.

[0030] When X comprises a substituted aryl group, preferred substituents (i.e., “R”) include halogens (-F, -Cl, -Br, with -Cl particularly preferred) or a sulfoximine substituent, such as: where Rb is an alkyl, preferably and most commonly a methyl group. Sulfoximine can enhance potency, pharmacokinetic properties and oral bioavailability, as the functional group incorporates in its structure a hydrogen bond acceptor (oxygen) and a hydrogen bond donor (NH) which can interact with an enzyme, boosting potency. Substituted aryl groups are preferably phenyl groups, preferably monosubstituted with a halogen substituent (-F, -Cl, -Br, with -Cl particularly preferred). Para- or meta-substitutions are particularly preferred in such embodiments. Other ring substitutions are contemplated herein for the X moiety, including nitrogen, oxygen, and sulfur substitutions.

[0031] In some embodiments, particularly preferred X moieties include the following structures in the brackets below (where the oxygen from formula (I) is depicted for clarity): where R is a halogen or sulfoximine substituent, Y is H or D, Q is CH or N, provided that at least one Q is N subject to a maximum of three nitrogen substitutions per ring, and W is S, NH, or O. The selected structures are all characterized by the presence of one or more hydrogen bond donors and / or acceptors capable of optimally binding to the active site of an enzyme through hydrogen bonding, boosting potency. Similar substituents following these principles arc also cither commercially available or can be readily synthesized (synthetically tractable). Again, X can be directly linked to the depicted oxygen or can be linked via a methylene linkage, preferably Ci to C2 methylene linkage, which, for example, would give the following structure for the first X structure depicted above, where n is 0, 1, or 2, and R is defined as above.

[0032] The present disclosure encompasses deuterated or non-deuterated forms of the foregoing compounds. Deuterium is the naturally-occurring, non-radioactive, stable isotope of hydrogen. Deuterium contains one proton, one electron, and a neutron, effectively doubling the mass as compared to hydrogen, without changing its properties significantly. Because it has reduced electronic polarizability and less hyperconjugative stabilization of adjacent bonds, it potentially can result in weaker van dcr Waals stabilization, and it can produce other changes in properties. Deuteration can improve the potency of compounds and also pharmacokinetic properties via various mechanisms including interfering with Cytochrome P450 enzyme-mediated clearance, lipid peroxidation and / or aldehyde oxidase-driven metabolism.

[0033] As used herein, the term “substituted,” when used to modify a chemical functional group, refers to the replacement of at least one hydrogen radical on the functional group with a substituent. Substituents can include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, oxy, alkoxy, heteroalkoxy, ester, thioester, carboxy, cyano, nitro, amino, amido, acetamide, and halo (e.g., fluoro, chloro, bromo, or iodo). When a chemical functional group includes more than one substituent, the substituents can be bound to the same carbon atom or to two or more different carbon atoms. Thus, the phrase “substituted or unsubstituted” means unsubstituted (i.e., contains the implied hydrogen(s) for that structure or moiety) or substituted with a group e.g., as defined herein. It is understood that substitution at a given atom is limited by valency. In addition, the term “substituted” docs not encompass deuteration or replacement of hydrogen with deuterium. Rather, such structures are referred to as being deuterated (or non-deuterated). For further clarity, the use of a substituent (radical) prefix name such as alkyl without the modifier “unsubstituted” or “substituted” is understood to mean that the particular substituent is unsubstituted. The term “pharmaceutically-acceptable salt” refers to an acid or base salt of a compound of the invention, which salt possesses the desired antiviral activity and is neither biologically nor otherwise undesirable.

[0034] In one aspect, a particularly preferred compound is: where R is a halogen (-F, -Cl, or -Br with -Cl being particularly preferred) or sulfoximine substituent, n is 0, 1, or 2; Y is H or D; Z is an aldehyde or alpha-ketoamide; and Ri is defined above, provided that when Z is an aldehyde, Ri is not isobutyl / leucine (and preferably Ri comprises a cyclic moiety).

[0035] In some embodiments, particularly preferred compounds comprise compounds of the formula:

[0036]

[0037] Prophylactic and / or therapeutic compositions comprising the protease inhibitors with specific or broad-spectrum antiviral activities are also disclosed. Combinations of one or more of the foregoing compounds can also be used in the embodiments herein. The compositions comprise a protease inhibitor compound described herein dispersed in a pharmaceutically-acceptable carrier. The term carrier is used herein to refer to diluents, excipients, vehicles, and the like, in which the protease inhibitor may be dispersed for administration. Suitable carriers will be pharmaceutically acceptable. As used herein, the term “pharmaceutically acceptable” means not biologically or otherwise undesirable, in that it can be administered to a subject without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically-acceptable carrier would be selected to minimize any degradation of the compound or other agents and to minimize any adverse side effects in the subject. Pharmaceutically-acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use, and will depend on the route of administration. For example, compositions suitable for administration via injection are typically solutions in sterile isotonic aqueous buffer. Exemplary carriers include aqueous solutions such as normal (n.) saline (-0.9% NaCl), phosphate buffered saline (PBS), sterile water / distilled autoclaved water (DAW), various oil-in-water or water-in-oil emulsions, as well as dimethyl sulfoxide (DMSO) or other acceptable vehicles, and the like.

[0038] The composition can comprise a therapeutically effective amount of the protease inhibitor compound dispersed in the carrier. As used herein, a “therapeutically effective” amount refers to the amount that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, and in particular elicit some desired therapeutic or prophylactic effect as against the viral infection by slowing and / or inhibiting 3CL protease activity and / or viral replication. Therapeutically effective amounts are those that are effective at reducing viral titers in the subject, such as by inhibiting replication of the virus in the subject. One of skill in the art recognizes that an amount may be considered therapeutically “effective” even if the condition is not totally eradicated or prevented, but it or its symptoms and / or effects are improved or alleviated partially in the subject. In some embodiments, the composition will comprise from about 5% to about 95% by weight of a protease inhibitor compound described herein, and preferably from about 30% to about 90% by weight of the protease inhibitor compound, based upon the total weight of the composition taken as 100% by weight. In some embodiments, combinations of more than one type of the described protease inhibitor compounds can be included in the composition, in which case the total levels of all such compounds will preferably fall within the ranges described above.

[0039] Other ingredients may be included in the composition, such as adjuvants, other active agents, preservatives, buffering agents, salts, other pharmaceutically-acceptable ingredients. The term “adjuvant” is used herein to refer to substances that have immunopotentiating effects and arc added to or co-formulated in a therapeutic composition in order to enhance, elicit, and / or modulate the innate, humoral, and / or cell-mediated immune response against the active ingredients. Other active agents that could be included in the composition include other antiviral compounds (e.g., cathepsins) or any immunogenic active components (e.g., antigens) such as those that resemble a disease-causing microorganism or infectious agent, and / or are made from weakened or killed forms of the same, its toxins, subunits, particles, and / or one of its surface proteins, such that it provokes an immune response to that microorganism or infectious agent. In addition to live, modified, or attenuated vaccine components, active agents using synthetic peptides, carbohydrates, or antigens can also be used. Compositions according to the embodiments disclosed herein are useful in inhibiting protease activity. More specifically, the compositions can be used to inhibit viral infection or viral replication, such as by inhibiting viral infection and replication from picornaviruses in a subject, in particular enteroviruses such as EV-D68, as well as human rhinovirus. Other conditions that can be targets for treatment through inhibition of protease activity include conditions ameliorated by targeting cathepsins, falcipains, and / or calpains, such as malaria, tumor cells, stroke, heart attack, neural degeneration, cataracts, and glaucoma.

[0040] Thus, embodiments described herein have broad-spectrum therapeutic and / or prophylactic uses. The terms “therapeutic” or “treat,” as used herein, refer to processes that are intended to produce a beneficial change in an existing condition (e.g., viral infection, disease, disorder) of a subject, such as by reducing the severity of the clinical symptoms and / or effects of the infection, and / or reducing the duration of the infection / symptoms / effects. The terms “prophylactic” or “prevent,” as used herein, refer to processes that are intended to inhibit or ameliorate the effects of a future viral infection or disease to which a subject may be exposed (but is not currently infected with). Thus, a prophylactic treatment is one given before the subject becomes infected with or has been exposed to the causative agent. Whereas a therapeutic is one administered after the subject has been exposed and / or shows observable signs of infection. In some cases, the composition may inhibit or reduce the development of observable morbidity from viral infection (i.e., near’ 100% prevention of symptoms). In other cases, the composition may only partially prevent and / or lessen the extent of morbidity due to the viral infection (i.e., reduce the severity of the symptoms and / or effects of the infection, and / or reduce the duration of the infection / symptoms / effects, or increase the rate of recovery from the condition).

[0041] In use, a thcrapcutically-cffcctivc amount of a protease inhibitor compound is administered to a subject. In some embodiments, a composition comprising a therapeutically-effective amount of a protease inhibitor compound is administered to a subject. Regardless, the compound or pharmaceutically acceptable salt or prodrug thereof will preferably be administered to the subject in an amount sufficient to provide compound levels (independent of salt, if any) of from about 0.1 mg to about 1,000 mg of compound per kg of body weight of the subject, preferably from about 1 mg / kg to about 100 mg / kg of body weight of the subject, and more preferably from about 10 mg / kg to about 50 mg / kg of body weight of the subject. Thus, it will be appreciated that in the case of compound salts, for example, the formulation may be administered in amounts greater than the above ranges to provide sufficient levels of the active compound. In some embodiments, the protease inhibitor compound or compositions can be provided in unit dosage form in a suitable container. The term “unit dosage form” refers to a physically discrete unit suitable as a unitary dosage for human or animal use. Each unit dosage form may contain a predetermined amount of the protease inhibitor compound (and / or other active agents) in the carrier calculated to produce a desired effect. In other embodiments, the protease inhibitor compound can be provided separate from the carrier (e.g., in its own vial, ampule, sachet, or other suitable container) for on-site mixing before administration to a subject. A kit comprising the protease inhibitor compound(s) is also disclosed herein. The kit further comprises instructions for administering the protease inhibitor compound to a subject. The protease inhibitor compound(s) can be provided as part of a dosage unit, already dispersed in a pharmaceutically-acceptable earner, or it can be provided separately from the carrier. The kit can further comprise instructions for preparing the protease inhibitor compounds for administration to a subject, including for example, instructions for dispersing the protease inhibitor compounds in a suitable carrier.

[0042] In some embodiments, the subject is afflicted with or suffering from a condition (e.g., infection, disease, or disorder) before the protease inhibitor compounds are administered, wherein methods described herein are useful for treating the condition and / or ameliorating the effects of the condition. Preferably, the protease inhibitor compound is administered as soon as possible after infection, preferably within about 7 days from onset of observable symptoms, more preferably within about 5 days from onset of observable symptoms, even more preferably within 3 days from onset of observable symptoms or known contact with virus. It will be appreciated that the sooner the protease inhibitor compound(s) is administered, the increased chance of successfully reducing effects of the viral infection. In other embodiments, the subject is free of a given condition before administering the protease inhibitor compound, wherein the methods described herein are useful for preventing the occurrence or incidence of the condition and / or preventing the effects of the condition, as described above. Thus, the protease inhibitor compounds can be given prophylactically (before exposure or observable infection) or therapeutically (after observable infection).

[0043] The disclosed embodiments are suitable for various routes of administration, depending upon the particular’ carrier and other ingredients used. For example, the prophylactic and / or therapeutic protease inhibitor compounds or compositions can be injected intramuscularly, subcutaneously, intradermally, or intravenously. They can also be administered via mucosa such as intranasally or orally, preferably orally. The protease inhibitor compounds or compositions can also be administered through the skin via a transdermal patch. The protease inhibitor compounds comprising alpha-ketoamide functional groups are particularly suited for oral delivery of the compounds.

[0044] It will be appreciated that therapeutic and prophylactic methods described herein arc applicable to humans as well as any suitable animal, including, without limitation, dogs, cats, and other pets, as well as, rodents, primates, horses, cattle, pigs, etc. The methods can also be applied for clinical research and / or study. Additional advantages of the various embodiments of the disclosure will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and / or integrations of the specific embodiments described and claimed herein.

[0045] As used herein, the phrase "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0046] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).

[0047] EXAMPLES

[0048] The following examples set forth methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration, and nothing therein should be taken as a limitation upon the overall scope of the invention. EXAMPLE 1

[0049] Potent Inhibition of picornaviruses including enterovirus D68 and human rhinoviruses by dipeptidyl a-ketoamide compounds

[0050] In this study, wc evaluated a scries of dipeptidyl compounds against the 3Cpro of picornaviruses, including EV-D68 and HRVs, in enzyme and cell-based assays, and demonstrated that several dipeptidyl compounds are potent inhibitors of HRVs and EV-D68 with high in vitro therapeutic indices (>5000). Our results show that dipeptidyl a-ketoamide compounds, in particular, have potent antiviral activities against EV-D68 as well as multiple strains of HRV with half-maximum concentration (EC50) in the low nanomolar range in cell culture. These a-ketoamide compounds have excellent in vitro safety indices (>10,000). Successful development of a- ketoamide protease inhibitors against hepatitis C virus as oral drugs suggests these compounds may have a high potential as oral antivirals against picornaviruses including EV-D68 and HRVs.

[0051] 2. Materials and methods

[0052] Compounds.

[0053] The genome of enteroviruses is a single- stranded, positive sense RNA molecule that consists of one open reading frame (ORF) that encodes a polyprotein. The ORF is divided into three consecutive regions; processing of the Pl region yields the capsid proteins (VP1-4), while processing of the P2 and P3 regions yields the nonstructural replication proteins 2A-2C and 3A- 3D, respectively. The polyprotein is first cleaved by a virus-encoded 2A protease at the VP1-2A junction, and other junction sites arc cleaved by 3Cpro to generate mature nonstructural and structural proteins. We have previously synthesized peptidyl inhibitors based on the conserved key features of 3Cpro or related 3C-likc protease (3CLpro) encoded by coronaviruses, caliciviruscs or picornaviruses and reported their broad-spectrum antiviral activities against multiple viruses in the enzyme- or cell-based assay systems.

[0054] Libraries of dipeptidyl compounds designed for various 3Cpro or 3CLpro including aldehyde and ketoamide compounds were examined for their activity against EV-D68 and HRV strains (Table 1). Known enterovirus inhibitors, Plecomail, an entry blocker and AG7O88, a 3Cpro inhibitor as well as GC591 (compound 10 in our previous paper, Potent inhibition of enterovirus D68 and human rhinoviruses by dipeptidyl aldehydes and a-ketoamides by Kim et al, Antiviral Research 125, 84-91, 2016) were included in the assays as controls or comparative analysis. Cells, viruses, and reagents.

[0055] WI-38 (human embryonic lung cells) and HeLa cells were maintained in minimal essential medium (MEM) containing 5% fetal bovine serum and antibiotics (chlortetracycline [25 pg / ml], penicillin [250 U / ml], and streptomycin [250 pg / ml]). Viruses used in the study were EV-D68 and HRV (IB and 51 strains) obtained from ATCC.

[0056] Fluorescence resonance energy transfer (FRET) protease assays.

[0057] The FRET substrate (Edans-DFHLQ / GP-Dabcyl) was synthesized by AnaSpec, Inc (Fremont, CA). The FRET protease assay was performed as with EV-D68 3Cpro (prepared from our lab), and HRV14 3Cpro (purchased from Thermofisher). Each enzyme was mixed with serial dilutions of each compound or mock (DMSO) in 25 pl of assay buffer and incubated at 37 °C for 30 min, followed by the addition of 25 pl of substrate (10 mM). Fluorescence readings were obtained at 1 hr following the addition of substrate. The dose-dependent FRET inhibition curves were fitted with variable slope using the GraphPad Prism software (version 6.04, San Diego, CA) in order to determine the 50% inhibitory concentration (IC50) of the compound.

[0058] Cell-based inhibition assays.

[0059] WI-38 cell line was used for the cell-based inhibition assay for EV-D68 and HRVs at 37 °C. The inhibitory effects of each compound against HRV strains were also tested in HeLa cells at 33 °C to confirm the results obtained from WI-38 cells. Briefly, confluent or semi-confluent cells were inoculated with each virus at a multiplicity of infection (MOI) of 0.05 for 1 hr, and the inoculum was replaced with medium containing mock-DMSO (<0.1%) or each compound (up to 100 pM). The virus infected cells were further incubated for up to 168 hr at which time cells show 80-90% cytopathic effects. Viral replication was then determined by the 50% tissue culture infectious dose (TCID50) assay. The EC50 values were determined by using the GraphPad software. Nonspecific cytotoxic effect and enzyme selectivity.

[0060] The 50% cytotoxic concentration (CC50) of each compound was determined in WI-38 cells using a CytoTox 96® non-radioactive cytotoxicity assay kit (Promega) and / or crystal violet staining. The in vitro therapeutic index was calculated by dividing CC50 by EC50. The enzyme selectivity of compounds was evaluated against a panel of representative host proteases including human cathepsin L, B, D, G and neutrophil elastase (HNE), chymotrypsin, trypsin, thrombin and using the enzyme assays (commercially available assays).

[0061] 3. Results

[0062] Table 1. Antiviral compound activity - Structures of representative depeptidyl compounds used in this study and their activity against 3Cpro of EV-D68 or HRV14.

[0063] * The IC50 from enzyme assay and EC50 from cell-based assay are indicated as mean and standarc variation from two or three separate experiments. GC591, AG7088 (Rupintrivir) and Plecornaril are included for controls and comparative analysis. N / T: not tested.

[0064] Inhibitory effects of the compounds against 3Cpro of EV-D68 or HRV14.

[0065] The aldehyde and a-kctomidc compounds in Table 1 were highly effective against both 3Cpro of EV-D68 and HRV14 with IC50 values ranging from 0.004 to 0.054 pM in our assay conditions. Each compound showed comparable activity against 3Cpro of EV-D68 or HRV14, suggesting the similar structural requirement for inhibition in these viral proteases (Table 1). These dipeptidyl compounds have the Gin surrogate at a position corresponding to Pl (Rl) and leucin (Leu), phenylalanine (Phe) or cyclohexyl alanine (Cha) at P2 position (R2) (Table 1). The compounds with Phe at R2 (GC1457 and GC1455) were generally more active than those with Leu or Cha at R2 against 3Cpro of EV-D68 and HRV14 (Table 1). Interestingly, an a-ketoamide [C(O)C(O)NH(Cyc-Prop)] moiety at the Z position increased the activity of the compounds, compared to the corresponding aldehyde (CHO). The most potent compounds for both EV-D68 and HRV 143Cpro were GC1457 and its IC50 values were 0.015 and 0.006 pM, respectively (Table 1).

[0066] The anti-viral effects of the compounds against EV-D68, HRV1B, or HRV51 in cell culture.

[0067] The antiviral effects of the compounds against EV-D68 and HRV strains in cell culture were determined in the human embryonic lung cell line (WI-38 cells) in comparison to AG7088 and Pleconaril (Table 1). The EC50 values of the compounds were generally in line with the IC50 values determined by the protease assay (Tables 1 and 2). Among the tested compounds, a-ketoamide compounds, GC1457 had the most potent activity against the replication of the viruses (EV-D68, HRV1B and HRV51). The EC50 values of GC1457 were 0.006 to 0.03 pM against these viruses (Table 2).

[0068] Cell toxicity and enzyme selectivity.

[0069] Most of listed compounds did not show any cytotoxicity up to 100 pM in proliferating or non-proliferating cells. Since off-target effects for host proteases can be a potential caveat in drug development, the selectivity profile of GC1457 was determined using a representative panel of proteases (Table 2). GC1457 is determined to be a good inhibitor of cathepsin L and B, but it has little inhibitory reactivity toward a panel of other host enzymes up to 50 pM (Table 2).

[0070] Table 2. Inhibitory activity of GC1457 against important host proteases including Cathepsin L, B, D and G, human neutrophil elastase (HNE), chymotrypsin, trypsin, thrombin and carboxypeptidase A.

[0071] 4. Conclusion

[0072] In summary, new derivative compounds of a dipeptidyl compound series that have anti- HRV activity were synthesized to further improve their inhibitory activity against EV-D68 and HRVs. The antiviral potency and the favorable toxicity and enzyme selectivity of GC1457 suggest that this compound has a high potential for picomaviruses including EV-D68 and HRVs.

Claims

CLAIMS:

1. A compound comprising the structure of formula:or a pharmaceutically-acceptable salt thereof, where: n is 0-6, each X comprises at least one cyclic moiety; each Y is H or D;Ri is a branched or unbranched Ci-Ce alkyl, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-10 aryl or arylalkyl, heterocycle, or a natural amino acid side chain selected from the group consisting of leucine, isoleucine, valine, methionine, phenylalanine, fluorophenylalanine, tryptophan, and tyrosine, or an amino acid analog / unnatural side chain such as cyclohexyl alanine, norvaline (n-propyl side chain), norleucine (n-butyl side chain), cyclopropyl methyl alanine, cyclobutyl methyl alanine and gemdimethylcylopropyl proline; andZ is an aldehyde or alpha-kctoamidc, provided that when Z is an aldehyde, Ri is not isobutyl (leucine).

2. The compound of claim 1, where Z is an alpha-ketoamide of the formula C(O)C(O)NH-Ra, where Rais a branched or unbranched Ci-Ce alkyl, a C3-C6 cycloalkyl, Ce-io aryl or arylalkyl, an acetate esters.

3. The compound of claim 1, where X is a substituted (including disubstituted) or unsubstituted C3-C10 cycloalkyl, a substituted or unsubstituted 3-7 membered heterocycle having 1-3 ring heteroatoms selected from N, O, and S, or a substituted or unsubstituted Ce-io aryl group, each of which may be directly attached to the depicted oxygen, or may be connected via a branched or unbranched and substituted or unsubstituted Ci-Ce alkyl linkage.

4. The compound of claim 1, where X is selected from the group consisting of the following structures in the brackets below, where the oxygen from formula (I) is depicted for clarity and where X may be connected directly to the depicted oxygen, or may be attached via an alkyl linkage:where R is a halogen or sulfoximine, Y is H or D, Q is CH or N, provided that at least one Q is N subject to a maximum of three nitrogen substitutions per ring, and W is S, NH, or O.

5. The compound of claim 1 , comprising the formula, where Y, Z, and Ri are each defined as in claim 1.

6. The compound of claim 1, comprising the formula:where n is 0-6, preferably 0-1, R is a halogen (preferably -Cl) or sulfoximine, and Y, Z, and Ri are each defined above.

7. The compound of claim 1, wherein Y is D.

8. The compound of claim 1 , comprising a formula selected from the group consisting9. The compound according to any one of claims 1-8, for use in inhibiting replication of a virus.

10. The compound of claim 9, wherein said virus is a picomavirus.

11. The compound of claim 10, wherein the picornavirus is human rhinovirus, human enterovirus, including enterovirus D68 (EV-D68), coxsackievirus, or non-polio enterovirus.

12. An antiviral composition comprising a first protease inhibitor compound according to any one of claims 1-8 and a pharmaceutically-acceptable earner.

13. The composition of claim 12, wherein said carrier is selected from the group consisting of sterile isotonic aqueous buffer, normal saline, phosphate buffered saline, DMSO, sterile water, oil-in-water emulsion, water-in-oil emulsion, and mixtures thereof.

14. The composition of claim 12, said composition comprising from about 5% to about 95% by weight of said first compound, based upon the total weight of said composition taken as100% by weight.

15. The composition of claim 12, further comprising a second compound.

16. The composition of claim 12, wherein said second compound is a compound according to any one of claims 1 to 7, said first compound being different from said second compound.

17. The composition of claim 12, further comprising one or more of adjuvants, other active agents, preservatives, buffering agents, and salts.

18. A method of inhibiting viral replication in a cell, said method comprising contacting said cell with a compound according to any one of claims 1 to 8, wherein the virus is a picomavirus.

19. A method of treating or preventing a viral infection in a subject, said method comprising administering to said subject a therapeutically-effective amount of a first compound according to any one of claims 1 to 8, wherein the viral infection is a picornavirus infection.

20. The method of claim 19, further comprising administering said first compound repeatedly to said subject over a period of days, weeks, or months.

21. The method of claim 19, wherein said vims is human rhinovirus, human enterovirus, including enterovirus D68 (EV-D68), coxsackievirus, or non-polio enterovirus.

22. The method of claim 19, wherein said subject is suffering from a viral infection prior to said administering.

23. The method of claim 19, wherein said subject is free of observable signs of viral infection prior to said administering.

24. The method of claim 19, wherein said first compound is administered before said subject is exposed to a picornavirus infection.

25. The method of claim 19, wherein said first compound is administered after said subject is exposed to a picornavirus infection.

26. The method of claim 19, wherein said first compound is administered intramuscularly, subcutaneously, intradermally, intranasally, intravenously, orally, or via a transdermal patch, preferably wherein said first compound is administered orally.

27. A kit comprising a compound according to any one of claims 1 to 8; and instructions for administering said compound to a subject in need thereof.

28. The kit of claim 27, wherein the first compound is provided in unit dosage form.

29. The kit of claim 27, wherein the first compound is provided in a first container, said kit further comprising a carrier in a second container; and instructions for preparing said first compound for administration to said subject.

30. Use of a compound according to any one of claims 1 to 8 for the manufacture of a medicament for the treatment or prevention of a viral infection in a subject.

31. The use of claim 30, wherein said viral infection is a picomavirus infection, preferably human enterovirus, human rhinovirus, or non-polio enterovirus.

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