Methods for inhibition of viral infection

Abstract

ABSTRACT Compounds, compositions, and methods for treating viral infection or inhibiting viral main proteases. The methods may include administering to a subject in need thereof an effective amount of main protease inhibitor, a salt thereof, an analog thereof, or a derivative thereof, to treat viral infection or inhibit viral main protease.

Description

SD2023-315-2PCT Attorney Docket No.24978-0928 METHODS FOR INHIBITION OF VIRAL INFECTION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 509,230, filed June 20, 2023, which is incorporated by reference herein. TECHNICAL FIELD

[0002] The present disclosure relates to the prevention and treatment of viral infection. BACKGROUND

[0003] Between the declaration of the coronavirus disease 2019 (COVID-19) pandemic in March 2020 and March 2024, approximately 774.9 million severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections were reported worldwide, with over 7.04 million deaths1. Non-pharmacological protocols and the development and emergency approval of vaccines2were pivotal to tackling the pandemic3.

[0004] However, access to vaccines has been uneven worldwide4, and the vaccines do not prevent infections and transmission5. In addition, vaccine hesitancy poses a significant public health risk6because it contributes to the viral spread7. Vaccine hesitancy also demonstrates a need for non-pharmacological interventions8. Regarding drugs, initial strategies focused on drug repurposing, such as remdesivir9, as well as the design, discovery, and development of new antivirals10,11that have provided therapeutic options to decelerate the COVID-19 pandemic and associated deaths.

[0005] A number of antiviral drugs are approved to treat COVID-19, such as the RNA polymerase inhibitor, molnupiravir12, and the SARS-CoV-2 main protease (Mpro) inhibitors ensitrelvir13and ritonavir-boosted nirmatrelvir14. Nirmatrelvir has limitations, including being recommended only for patients with mild-to-moderate COVID-19 symptoms15to prevent progression to severe disease16. Also, it exhibits detrimental drug- drug interactions (due to CYP3A4 inhibition)17,18with many antiarrhythmics, and some anticonvulsants, anxiolytics, and cancer drugs19. Last, there are reports of Mpromutations associated with drug resistance20in vitro and in vivo for both ensitrelvir21and nirmatrelvir22,23, including those that occur naturally for nirmatrelvir24, which raises theSD2023-315-2PCT Attorney Docket No.24978-0928 concern of drug resistant strains25that may undermine the utility of the current treatment options25,23.

[0006] Ligand- and structure-based drug design (LBDD and SBDD) strategies26have enhanced the discovery and development of bioactive compounds targeting viral proteases27, such as the SARS-CoV-2 papain-like protease (PLpro) and Mpro, which are believed to be essential for viral replication28. The SARS-CoV-2 Mpro, which shares 90- 96% genome identity with SARS-CoV and other bat coronaviruses29–31, is a potential multi-coronavirus target32–34, including for mutated strains35,36. Targeting Mprofor therapeutics, therefore, may provide a bulwark against future outbreaks, epidemics, and pandemic scenarios37,38.

[0007] SARS-CoV-2 Mprois a structurally well-described drug target, and there has been a strong focus on computational approaches to derive noncovalent and covalent inhibitors39,40. A recent case of success was the development of a noncovalent reversible SARS-CoV-2 single Mproinhibitor drug during the COVID-19 Moonshot initiative41, ensitrelvir. This Mproinhibitor was discovered by a SBDD docking-based pharmacophore virtual screening of an in-house corporate library (hundreds of thousands of compounds) and evaluated for its biological activity13. Additional efforts have also discovered inhibitors of both Mproand PLpro42, and of both these and human cathepsin L43.

[0008] Improvements in methods for preventing and treating viral infection are needed. In particular, inhibitors of viral proteases, such as the SARS-CoV-2 main protease (SARS- CoV-2 Mpro) would be desirable. SUMMARY OF THE INVENTION

[0009] In one aspect, methods of preventing or treating viral infections are provided. In some embodiments, the methods include administering to a subject in need thereof a therapeutically effective amount of a main protease inhibitor to prevent or treat the viral infection of the subject. The main protease inhibitor may include any one or more of the compounds described herein, or a salt, an analog, or a derivative thereof. In some embodiments, the main protease inhibitor is selected from the group consisting of (i) Compound 5, a salt thereof, an analog thereof, or a derivative thereof -SD2023-315-2PCT Attorney Docket No.24978-0928

[0010] ,

[0011] (ii) Compound 9, a salt thereof, an analog thereof, or a derivative thereof – –

[0015] (iv) Compound 5’, a salt thereof, an analog thereof, or a derivative thereof –SD2023-315-2PCT Attorney Docket No.24978-0928 O

[0016] .

[0017] In another aspect, methods of inhibiting a viral main protease are provided. In some embodiments, the methods include administering to a subject in need thereof a therapeutically effective amount of a main protease inhibitor to inhibit the viral main protease. The main protease inhibitor may be selected from any one or more of the compounds described herein, a salt, an analog, or a derivative thereof. In some embodiments, the main protease inhibitor is selected from Compound 5, Compound 5’, Compound 9, Compound 10, a salt thereof, an analog thereof, a derivative thereof, or a combination thereof.

[0018] In yet another aspect, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions include a pharmaceutically active agent, and optionally a pharmaceutically acceptable carrier. The pharmaceutically active agent may be selected from any one or more of the compounds described herein, a salt thereof, an analog thereof, or a derivative thereof. In some embodiments, the pharmaceutically active agent is selected from Compound 5, Compound 5’, Compound 9, Compound 10, a salt thereof, an analog thereof, a derivative thereof, or a combination thereof. The pharmaceutical compositions generally may be formulated for any route of administration, such as intravenous administration, oral administration, or topical administration.

[0019] Additional aspects will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the aspects described herein. The advantages described herein may be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is toSD2023-315-2PCT Attorney Docket No.24978-0928 be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG.1 depicts Mproalignment of different coronaviruses sequences.

[0021] FIG.2 depicts concentration-response curves of compounds against SARS-CoV- 2 Mpro.

[0022] FIG.3 depicts concentration-response curves of resynthesized compounds against SARS-CoV Mpro.

[0023] FIG.4A depicts concentration-response curves for inhibition of SARS-CoV-2 Mproactivity by an embodiment of an active compound.

[0024] FIG.4B depicts concentration-response curves for inhibition of SARS-CoV-2 Mproactivity by an embodiment of an active compound.

[0025] FIG.4C depicts concentration-response curves for inhibition of SARS-CoV-2 Mproactivity by an embodiment of an active compound.

[0026] FIG.5 depicts the results of an assay configured to test whether the inhibition of SARS-CoV-2 Mproby embodiments of active compounds was reversible.

[0027] FIG.6A depicts a predicted binding site residue in an Mproenzyme.

[0028] FIG.6B depicts a predicted binding site residue in an Mproenzyme.

[0029] FIG.6C depicts a predicted binding site residue in an Mproenzyme.

[0030] FIG.6D depicts a predicted binding site residue in an Mproenzyme.

[0031] FIG.7A, FIG.7B, and FIG.7C depict Redocking of Mproco-crystallized ligands using GOLD.

[0032] FIG.8A depicts the docking of an embodiment of an active compound with an Mproenzyme.SD2023-315-2PCT Attorney Docket No.24978-0928

[0033] FIG.8B depicts the docking of an embodiment of an active compound with an Mproenzyme.

[0034] FIG.8C depicts the docking of an embodiment of an active compound with an Mproenzyme.

[0035] FIG.8D depicts the docking of an embodiment of an active compound with an Mproenzyme.

[0036] FIG.8E depicts the docking of an embodiment of an active compound with an Mproenzyme.

[0037] FIG.8F depicts the docking of an embodiment of an active compound with an Mproenzyme.

[0038] FIG.9A and FIG.9B depict MD simulations of the co-crystallized ligand GC376 against SARS-CoV-2 Mpro.

[0039] FIG.10A—FIG.10D depict RMSD and RMSF values for compounds 9 (FIG. 10A and FIG.10B, respectively) and 10 (FIG.10C and FIG.10D, respectively).

[0040] FIG.11A and FIG.11B depict proposed binding modes of compounds 9 and 10 in the active site of SARS-CoV-2 Mpro.

[0041] FIG.11C is a violin plot displaying a variation of calculated binding energy, using MM / GBSA along the entire MD trajectory, and normalized to the natural log number of heavy atoms.

[0042] FIG.11D depicts distances of a reactive ethynyl group of compound 9 to the catalytic cysteine.

[0043] FIG.11E depicts the average distance of reactive atoms from the reactive catalytic cysteine’s sulfur.

[0044] FIG.12A and FIG.12B depict concentration-response of compounds 9 and 10 against SARS-CoV-2.SD2023-315-2PCT Attorney Docket No.24978-0928

[0045] FIG.12A and FIG.12B depict concentration-response of compounds 9 and 10 against SARS-CoV-2. DETAILED DESCRIPTION

[0046] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0047] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.

[0048] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, 2nded. (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Remington, The Science and Practice of Pharmacy, 20thed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22thed., (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012).

[0049] Compounds

[0050] Provided herein are compounds, which may include any one or more compounds disclosed herein, including salts thereof, analogs thereof, and / or derivatives thereof.SD2023-315-2PCT Attorney Docket No.24978-0928

[0051] In some embodiments, the compound is Compound 5 (methyl (R,E)-5-((S)-2- (((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-4-oxohex-2-enoate), a salt thereof, an analog thereof, or a derivative thereof:

[0052] .

[0053] In some embodiments, the compound is Compound 5’ (methyl (R,Z)-5-((S)-2- (((benzyloxy(carbonyl)amino)-3-phenylpropanamido)-4-oxohex-2-enoate), a salt thereof, an analog thereof, or a derivative thereof:

[0054] .

[0055] In some embodiments, the compound is Compound 9 (benzyl ((R)-1-oxo-1-(((R)- 3-oxopent-4-en-2-yl)amino)-3-phenylpropan-2-yl)carbamate), a salt thereof, an analog thereof, or a derivative thereof:SD2023-315-2PCT Attorney Docket No.24978-0928

[0056] .

[0057] In some embodiments, theCompound 10 (benzyl ((R)-1-oxo-1- (((S)-3-oxopent-4-en-2-yl)amino)-3-phenylpropan-2-yl)carbamate), a salt thereof, an analog thereof, or a derivative thereof:

[0058] .

[0059] In some embodiments, the compound selected from the group consisting of Compound 6, Compound 7, Compound 8, a salt thereof, an analog thereof, and a derivative thereof.

[0060] In some embodiments, the compound is Compound A, a salt thereof, an analog thereof, or a derivative thereof:SD2023-315-2PCT Attorney Docket No.24978-0928

[0061] In some embodiments, the compound is Compound B, a salt thereof, an analog thereof, or a derivative thereof:

[0062] Pharmaceutical Compositions

[0063] Also provided herein are pharmaceutical compositions. The pharmaceutical compositions may include a pharmaceutically active agent and optionally a pharmaceutically acceptable carrier.

[0064] The pharmaceutically active agent may include any one or more of the compounds disclosed herein, including salts thereof, analogs thereof, and / or derivatives thereof.

[0065] The pharmaceutical compositions may be formulated as a solution, emulsion, suspension, or dispersion. The pharmaceutical compositions may be formulated for any route of administration, such as intravenous administration, oral administration, or topical administration.

[0066] MethodsSD2023-315-2PCT Attorney Docket No.24978-0928

[0067] Also provided herein are methods of preventing or treating viral infection, such as a viral infection caused by a coronavirus.

[0068] In some embodiments, the methods include administering to a subject in need thereof a therapeutically effective amount of a main protease inhibitor to prevent or treat viral infection of the subject. The main protease inhibitor may include any one or more compounds disclosed herein, including salts thereof, analogs thereof, and / or derivatives thereof.

[0069] Also provided are methods of inhibiting a viral main protease. In some embodiments, the methods include administering to a subject in need thereof a therapeutically effective amount of a main protease inhibitor to inhibit the viral main protease. The main protease inhibitor may include any one or more compounds disclosed herein, including salts thereof, analogs thereof, and / or derivatives thereof. The viral main protease may include SARS-CoV-2 cysteine proteases, main protease (Mpro) and papain- like protease (PLpro).

[0070] The viral infection generally may be any infection caused by a virus, such as a coronavirus. The coronavirus may be SARS-CoV, such as CoV-2, which causes COVID- 19. The coronavirus may be MERS-CoV.

[0071] The subject may be a mammal, such as an animal or human.

[0072] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by,” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a fusion protein, a pharmaceutical composition, and / or a method that “comprises” a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the fusion protein, pharmaceutical composition and / or method.

[0073] As used herein, the transitional phrases “consists of” and “consisting of” exclude any element, step, or component not specified. For example, “consists of” or “consistingSD2023-315-2PCT Attorney Docket No.24978-0928 of” used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of” or “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of” or “consisting of” limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0074] As used herein, the transitional phrases “consists essentially of” and “consisting essentially of” are used to define a fusion protein, pharmaceutical composition, and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.

[0075] When introducing elements of the present invention or the preferred embodiment(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0076] The term “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 in combination with any one or more of the listed items. For example, the expression “A and / or B” is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination. The expression “A, B and / or C” is intended to mean 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.

[0077] It is understood that aspects and embodiments of the invention described herein include “consisting” and / or “consisting essentially of” aspects and embodiments.

[0078] It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered toSD2023-315-2PCT Attorney Docket No.24978-0928 have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Values or ranges may also be expressed herein as “about,” from “about” one particular value, and / or to “about” another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0079] As used herein, “patient” or “subject” means a human or animal subject to be treated.

[0080] As used herein the term “pharmaceutical composition” refers to pharmaceutically acceptable compositions, wherein the composition comprises a pharmaceutically active agent, and in some embodiments further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination of pharmaceutically active agents and carriers

[0081] The term “combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where one or more active compounds and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals. In some circumstances, the combination partners show a cooperative, e.g., synergistic effect. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarilySD2023-315-2PCT Attorney Docket No.24978-0928 administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0082] As used herein the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and / or non- human mammals.

[0083] As used herein the term “pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle with which demethylation compound(s), is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compositions in combination with carriers are known to those of skill in the art. In some embodiments, the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g.,SD2023-315-2PCT Attorney Docket No.24978-0928 Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

[0084] Sterile compositions are within the present disclosure, including compositions that are in accord with national and local regulations governing such compositions. Pharmaceutical compositions, alone or in combination with other active ingredient(s), described herein may be formulated as solutions, emulsions, suspensions, or dispersions in suitable pharmaceutical solvents or carriers, or as pills, tablets, lozenges, suppositories, sachets, dragees, granules, powders, powders for reconstitution, or capsules along with solid carriers according to conventional methods known in the art for preparation of various dosage forms. A glycosidase, alone or in combination with other active ingredient(s), described herein, and preferably in the form of a pharmaceutical composition, may be administered by a suitable route of delivery, such as oral, parenteral, rectal, nasal, topical, or ocular routes, or by inhalation. In some embodiments, the compositions are formulated for intravenous or oral administration.

[0085] For oral administration, Main protease inhibitor may be provided in a solid form. In embodiments, Main protease inhibitor or derivatives thereof, alone or in combination with another active ingredient, may be provided in a tablet or capsule, or as a solution, emulsion, or suspension. To prepare the oral compositions, Main protease inhibitor or derivatives thereof, alone or in combination with another active ingredient, may be formulated to yield an effective dosage. Oral tablets may include the active ingredient(s) mixed with compatible pharmaceutically acceptable excipients such as diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservative agents. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Starch, polyvinyl-pyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid are exemplary disintegrating agents. Binding agents may include starch and gelatin. The lubricating agent, if present, may be magnesium stearate, stearic acid, or talc. If desired, the tabletsSD2023-315-2PCT Attorney Docket No.24978-0928 may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract, or may be coated with an enteric coating.

[0086] Capsules for oral administration include hard and soft gelatin capsules. To prepare hard gelatin capsules, active ingredient(s) may be mixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsules may be prepared by mixing the active ingredient with water, an oil, such as peanut oil or olive oil, liquid paraffin, a mixture of mono and di-glycerides of short chain fatty acids, polyethylene glycol 400, or propylene glycol.

[0087] Liquids for oral administration may be in the form of suspensions, solutions, emulsions, or syrups, or may be lyophilized or presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically-acceptable excipients such as suspending agents (for example, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and the like); non-aqueous vehicles, e.g., oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol, or water; preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents such as lecithin; and, if desired, flavoring or coloring agents.

[0088] For parenteral use, including, intravenous, intramuscular, intraperitoneal, intranasal, or subcutaneous routes, Main protease inhibitor, alone or in combination with other active ingredient(s), may be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity or in parenterally acceptable oil. Suitable aqueous vehicles can include Ringer's solution and isotonic sodium chloride. Such forms may be presented in unit-dose form such as ampoules or disposable injection devices, in multi-dose forms such as vials from which the appropriate dose may be withdrawn, or in a solid form or pre-concentrate that can be used to prepare an injectable formulation. Illustrative infusion doses range from about 1 to 1000 μg / kg / minute of agent admixed with a pharmaceutical carrier over a period ranging from several minutes to several days.

[0089] For nasal, inhaled, or oral administration, main protease inhibitor, alone or in combination with other active ingredient(s), may be administered using, for example, a spray formulation also containing a suitable carrier.SD2023-315-2PCT Attorney Docket No.24978-0928

[0090] For topical applications, main protease inhibitor, alone or in combination with other active ingredient(s), are preferably formulated as creams or ointments or a similar vehicle suitable for topical administration. For topical administration, main protease inhibitor, alone or in combination with other active ingredient(s), may be mixed with a pharmaceutical carrier at a concentration of about 0.1% to about 10% of drug to vehicle. Another mode of administering a main protease inhibitor, alone or in combination with other active ingredient(s), may utilize a patch formulation to effect transdermal delivery.

[0091] One of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration. In particular, a main protease inhibitor, alone or in combination with other active ingredient(s), may be modified to render them more soluble in water or other vehicle. It is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of the compositions, alone or in combination with other active ingredient(s), in order to manage the pharmacokinetics of the present compositions for maximum beneficial effect in a patient.

[0092] As used herein, “therapeutically effective amount” refers to an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions. When used with reference to a method, the method is sufficiently effective to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions. For example, an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses.

[0093] As used herein, the terms “treat,” “treatment,” or “treating” embraces at least an amelioration of the symptoms associated with diseases in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. a symptom associated with the disease or condition being treated. As such, “treatment” also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g. prevented from happening)SD2023-315-2PCT Attorney Docket No.24978-0928 or stopped (e.g. terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition.

[0094] As used herein, and unless otherwise specified, the terms "prevent," "preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be interchangeably used with the term "prophylactic treatment."

[0095] As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a prophylactic benefit in the prevention of the disease. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[0096] A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

[0097] EXAMPLES

[0098] The following examples describe the screening of 141 cysteine protease inhibitors for inhibition of SARS-CoV-2 Mprocatalysis. These inhibitors were the dry powder remnants of a larger compound collection assembled between 1997 and 2012 to target the major cathepsin L-like cysteine protease in Trypanosoma cruzi, the causative agent of Chagas disease44–48.SD2023-315-2PCT Attorney Docket No.24978-0928

[0099] Five compounds showed first-pass inhibition of SARS-CoV-2 Mproand the two most potent entities were resynthesized, in addition to the synthesis of a new isomer. Concentration-response assays determined that the three newly synthesized compounds were nanomolar Mproinhibitors of both SARS-CoV and SARS-CoV-2 (i.e., SARS- related).

[0100] Rapid dilution enzyme assays supported by density functional theory (DFT) calculations identified the compounds as reversible inhibitors. Further, a combination of Mprostructural characterization, docking and molecular dynamics (MD) simulations supported the three compounds as SARS-related Mproinhibitors. Finally, one of the three inhibitors decreased SARS-CoV-2 viral titers in Vero-infected cells at low nanomolar concentration.

[0101] Example 1 - SARS-CoV-2, SARS-CoV and MERS-CoV Mproinhibition assays

[0102] The 141 compounds tested in the examples provided herein were the dry powder remnants from a ~500 compound collection of protease inhibitors that had been synthesized between 1997 and 2012 to target the major cysteine protease, cruzain, of Trypanosoma cruzi, which causes Chagas disease44–47,49. The remnant collection contained mainly dipeptidyl molecules with various reactive groups, such as vinyl sulfones, nitriles, ketones, epoxyketones, and acrylamides with molecular weights ranging between 285.3 and 677.8 Da. The compounds’ nomenclature incorporated the acronym “WRR” after William R. Roush (of the former Scripps Research Institute, Jupiter, FL), the principal chemist involved in the legacy project44,45.

[0103] Compound powders were dissolved as 10 mM stocks in DMSO in 96-well polypropylene microplates, which were stored at -80 °C.Recombinant SARS-CoV-2 Mprowas expressed and purified as described50. The inhibitor collection was initially screened at 10 and 5 µM using a described assay51.

[0104] Briefly, assays were performed in 384-well black microplates following a 15 minute pre-incubation of the compounds with 100 nM enzyme diluted in assay buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.01% Tween 20). The reaction was started by addition of the fluorogenic substrate Ac-Abu-Tle-Leu-Gln-MCA (Biosynth,SD2023-315-2PCT Attorney Docket No.24978-0928 FA178674; 20 μM) diluted in the same buffer. Catalysis was measured for up to 2 hours at 37 °C in a Synergy HTX (Biotek, USA) microplate reader with excitation / emission wavelengths of 360 / 460 nm. The final enzyme and substrate concentrations were 50 nM and 10 μM, respectively, and the final DMSO concentration was up to 0.2%. The protease inhibitor nirmatrelvir (100 nM) was used as a positive control. Enzymatic activity was normalized to DMSO controls, and the percent inhibition was calculated (mean and standard error). Compounds that inhibited enzyme activity by ≥ 50% were also assessed under the same assay conditions against recombinant SARS-CoV and MERS-CoV Mpro(R&D Systems, E720 and E719, respectively), as reported40. One or two independent assays, each in triplicate, were performed (n ≥ 3 data points).

[0105] Compounds meeting the ≥ 50% inhibition criterion progressed to eight-point (0.001 - 40 μM) concentration-response assays against SARS-CoV-2 and SARS-CoV Mpro. The half-maximal inhibitory concentration (IC50) was calculated by nonlinear regression (r2> 0.9), and the deviation of each data point from the calculated nonlinear regression was < 10%. To evaluate time-dependent covalent inhibition, the same assay was performed without the 15-minute pre-incubation period52. For both experiments, two independent assays were performed, each in triplicate (n = 6 data points). To evaluate reversible inhibition, a rapid dilution enzyme assay was performed.

[0106] Briefly, 100-fold of the final assay concentration of Mprowas incubated for 30 minutes with the active inhibitors at 10-times their respective IC50values. A total of 0.5 μL of this mixture was transferred to 384-well black microplates and diluted 100-fold with assay buffer containing 10 μM Ac-Abu-Tle-Leu-Gln-MCA in a final volume of 50 μL. Thus, the final enzyme and compound concentrations were 50 nM and 0.1-times the compounds’ IC50values, respectively. Residual catalysis was measured in the Synergy HTX as described above, for up to 2 hours at 37 °C. Two independent assays in triplicate were performed (n = 6 data points). Data were analyzed using GraphPad Prism 9.0 (GraphPad Software, San Diego, California, USA).

[0107] Example 2 - Discovering new SARS-related Mproinhibitors from a small legacy collectionSD2023-315-2PCT Attorney Docket No.24978-0928

[0108] Using the remnants of a legacy small molecule library designed to target cruzain, the major cysteine protease of T. cruzi, five (sub)micromolar inhibitors of the Mproenzyme of SARS-CoV-2 were discovered. All five inhibitors shared an amino-3-phenylpropan moiety. The two most potent Mproinhibitors, 5 and 9, contained an ethene-ester and an ethynyl electrophilic warhead, respectively. Given the age of both compounds (stored for at least 10 years), they were resynthesized along with the R,S version of 9, termed 10. Low nanomolar inhibition of SARS-CoV-2 Mprowas confirmed for all three inhibitors and shown for the related enzyme of SARS-CoV, but not MERS-CoV. This was consistent with the greater identity between the two SARS-related Mproenzymes29(96%) compared to the Mproof MERS-CoV (~51%; FIG.1). Of the three inhibitors, 10 was the most potent with an IC50value of 9.3 nM, i.e., almost half the value obtained for the current oral COVID-19 drug, nirmatrelvir, in the tested assay conditions (FIG.2).

[0109] FIG.2 depicts concentration-response curves of compounds against SARS-CoV- 2 Mpro. The IC50values from two independent assays in triplicate (n = 6 data points), were calculated by nonlinear regression (r2> 0.9). Analysis considered the standard deviation of each point in the curve, and deviations were less than 10%. At least eight different compound concentrations (0.001 - 40 µM) were assessed. The data were analyzed using GraphPad Prism 9.0.

[0110] FIG.1 depicts Mproalignment of different coronaviruses sequences (SARS-CoV- 2, SARS-CoV, MERS-CoV, and MHV-A59). Sequences were retrieved from UniProt after being selected in PDB. Accession numbers: P0DTD1 (SARS-CoV-2; PDB ID 7D1M), P0C6U8 (SARS-CoV; PDB ID 6W2A), T2B9U0 (MERS-CoV; PDB ID 6VH3), and P0C6X9 (MHV-A59; PDB ID 6JIJ). Sequences were aligned with MUSCLE from EMBL-EBI (available at https: / / www.ebi.ac.uk / jdispatcher / msa / muscle). The identity (%) between each sequence is displayed as a matrix (bottom right).

[0111] In terms of the structure-activity relationship (SAR), the presence of the ester group after the ethene substituent in 5, compared to 10, decreased the IC50 value potency by between 2.8- and 172-times depending on the Mprotarget (Table 2). Compounds (N- to C-terminus) 6 (morpholine, hydroxyethyl-cyclopropenone, and butylbenzene substituents), 7 (morpholine-ethynylester, isopentane, and methylsulfonyl-benzene substituents) and 8 (morpholine, butylbenzene, and methylsulfonyl-benzene substituents), respectively, wereSD2023-315-2PCT Attorney Docket No.24978-0928 902 – 1,812-times less potent than 10 against the SARS-CoV-2 Mproand were also inactive against SARS-CoV Mpro.

[0112] In addition, 10 was twice as potent against SARS-CoV-2 Mprocompared to its enantiomer 9, both of which did not inhibit the orthologous enzyme of MERS-CoV. Other “WRR” analogs were previously reported against SARS-CoV Mpro97, two of which, WRR- 182 (30) and 183 (31), were available in a legacy collection. Compound 31, the S,R version of 30 (S,R), was 10-times more potent than its enantiomer against SARS-CoV Mpro97. On the other hand, 30 was more active than 31 against another cysteine protease, cruzain44. These epoxyketones, 30 and 31, did not inhibit the SARS-CoV-2 Mproactivity by more than 50% at 10 µM, despite preserving the amino-3-phenylpropan moiety of hits described herein (Table A) and having an ethylene oxide substituent instead of the ethene in 5, or ethynyl in 9 and 10.

[0113] Table A. Compounds “30” and “31” against SARS-CoV-2 MproμMa

[0114]

[0115] Example 3 - SARS-CoV-2 PLproinhibition assays

[0116] To understand whether the second major SARS-CoV-2 cysteine protease, PLpro, could be inhibited, each of the 141 compounds was pre-incubated at 10 μM for 15 minutesSD2023-315-2PCT Attorney Docket No.24978-0928 with 100 nM of recombinant enzyme (Acro Biosystems, PAE-C5184) in an assay buffer comprising 50 mM HEPES at pH 6.5, 150 mM NaCl, 0.01% Tween 20 and 0.1 mM DTT.

[0117] Assays were performed in 384-well black microplates and the reaction was started by the addition of the fluorogenic substrate Z-Arg-Leu-Arg-Gly-Gly-AMC (Bachem, 369 I1690), diluted with an equal volume of the same buffer (100 µM final). Catalysis was measured in the Synergy HTX as described above for 1 hour at 37 °C. The final enzyme and substrate concentrations were 50 nM and 50 μM, respectively. The protease inhibitor GRL-0617, (10 μM) was used as a positive control53. Enzymatic activity was normalized to DMSO controls, and the percent inhibition was calculated. The data were analyzed using GraphPad Prism 9.0. One independent assay in triplicate was performed (n = 3 data points).

[0118] Example 4 - Preparation of Compounds

[0119] The (re)synthesis was achieved by processes depicted at the following scheme.

[0120] Scheme 1. (Re)synthesis of Compound 5

[0121] Reactions and conditions of Scheme 1. (a) N-Cbz-L-phenylalanine 1 (1.00 eq), NHS (1.00 eq), DCC (1.10 eq), CHCl3 (0.05 M), rt, 1 hour. (b) D-alanine (1.00 eq), NaHCO3(2.00 eq.), H2O / acetone (1:2, 0.05 M), rt, 16 h (89% over two steps). (c) N-Cbz- L-phenylalanyl-D-alanine 2 (1.00 eq), N,O-dimethyl-hydroxylamine hydrochloride (1.10 eq), ethyl chloroformate (1.00 eq), N-methylmorpholine (1.00 eq), freshly prepared diazomethane (6.00 eq), THF (0.1 M), -78 °C to rt, 16 hours (37%). (d) Diazoketone 3 (1.00 eq), freshly prepared DMDO (3.00 eq), acetone (0.02 M), -78 °C to rt, 16 hours (99%). (e) Glyoxal 4 (1.00 eq), methyl 2-(dimethoxyphosphoryl)acetate (1.10 eq), NaHSD2023-315-2PCT Attorney Docket No.24978-0928 (1.10 eq), CH3CN (0.05 M), 0 °C to rt, 16 hours (12%). This procedure, or a similar procedure, may be used to produce Compound 5’.

[0122] N-Cbz-L-phenylalanyl-D-alanine (2). To a solution of N-Cbz-L-phenylalanine (1.00 g, 3.34 mmol, 1.00 eq) and NHS (0.384 g, 3.34 mmol, 1.00 eq) in CHCl3(60 ml), DCC (0.758 g, 3.67 mmol, 1.10 eq) was added at room temperature. The resulting mixture was stirred at room temperature for 1 hour. The precipitate was filtered off, and the filtrate was concentrated en vacuo. The resulting crude was used in the subsequent reaction without further purification. To a solution of the crude intermediate in acetone (80 ml), a solution of D-alanine (0.298 g, 3.34 mmol, 1.00 eq) and NaHCO3 (0.561 g, 6.68 mmol, 2.00 eq) in water (40 ml) was added at room temperature. The reaction mixture was stirred at room temperature. After 16 hours, the mixture was concentrated en vacuo to remove the acetone. The resulting mixture was diluted with 60 mL water, and the remaining precipitate was removed by filtration. The filtrate was acidified to pH 3, and the resulting precipitate and crude product was collected by filtration (1.10 g, 3.00 mmol, 89%).1H NMR (600 MHz, CDCl3) δ 7.37 – 7.27 (m, 4H), 7.27 – 7.09 (m, 7H), 6.74 (d, J = 7.1 Hz, 1H), 5.75 (d, J = 8.6 Hz, 1H), 5.06 (d, J = 12.2 Hz, 1H), 5.01 (d, J = 12.4 Hz, 1H), 4.61 (s, 1H), 4.46 (s, 1H), 3.03 (d, J = 7.5 Hz, 2H), 1.22 (d, J = 7.2 Hz, 3H) ppm. MS (ESI) [M+H]+371.1.

[0123] Benzyl ((S)-1-(((R)-4-diazo-3-oxobutan-2-yl)amino)-1-oxo-3-phenylpropan- 2-yl)carbamate (Diazoketone 3). To a solution of N-Cbz-L-phenylalanyl-D-alanine (0.500 g, 1.35 mmol, 1.00 eq) in anh. THF (13 ml), methylmorpholine (0.137 g, 1.35 mmol, 1.00 eq) was added, followed by ethyl chloroformate (0.146 g, 1.35 mmol, 1.00 eq). Freshly distilled solution of diazomethane (prepared using diazaled and KOH in ethanol and water) was slowly added (4.0 ml) at -78 °C. The reaction mixture was slowly warmed to room temperature and stirred overnight. After 16 hours, the reaction mixture was diluted with cold saturated NaHCO3and extracted with EtOAc (3x). The organic extracts were combined and dried over Na2SO4. The crude mixture was concentrated in vacuo and purified via column chromatography (up to 50% EtOAc in hexanes) to yield to product as a white solid (0.490 g, 1.19 mmol, 88%).1H NMR (600 MHz, DMSO-d6) δ 8.43 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.39 – 7.15 (m, 10H), 6.08 (s, 1H), 4.98SD2023-315-2PCT Attorney Docket No.24978-0928 (d, J = 2.6 Hz, 2H), 4.32 – 4.17 (m, 2H), 2.93 (dd, J = 13.6, 5.2 Hz, 1H), 2.80 (dd, J = 13.4, 9.7 Hz, 1H), 2.51 (s, 2H), 1.13 (d, J = 7.3 Hz, 3H) ppm.

[0124] Benzyl ((S)-1-(((R)-3,4-dioxobutan-2-yl)amino)-1-oxo-3-phenylpropan-2- yl)carbamate (Glyoxal 4). To a solution of benzyl ((S)-1-(((R)-4-diazo-3-oxobutan-2- yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate 3 (0.200 g, 0.507 mmol, 1.00 eq) in acetone (1 ml), freshly prepared DMDO (25 ml; prepared from 50 g Oxone and 48 g NaHCO3 in H2O / acetone) was added slowly at -78 °C. The reaction mixture was slowly warmed to room temperature and stirred overnight. After 16 hours, the resulting mixture was concentrated in vacuo and used in the subsequent step without further purification (0.193 g, 0.505 mmol, 99%). MS (ESI) [M+H]+383.1

[0125] Methyl (R,E)-5-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)- 4-oxohex-2-enoate (5). The desired ylide was first prepared. To a solution of methyl 2- (dimethoxyphosphoryl)acetate (0.033 mg, 0.180 mmol, 1.00 eq) in anhydrous CH3CN (1.0 ml), NaH (0.0044 g, 0.180 mmol, 1.00 eq) was added. The resulting suspension was added to a solution of glyoxal 4 (0.070 g, 0.180 mmol, 1.00 eq) in anh. CH3CN (1.0 ml) at 0 °C. ,SD2023-315-2PCT Attorney Docket No.24978-0928

[0126] Scheme 2. Preparation of Benzyl ((R)-1-oxo-1-(((S)-3-oxopent-4-en-2-yl)amino)- 3-phenylpropan-2-yl)carbamate (10)

[0127] Reactions and conditions of Scheme 2. (a) N-Cbz-D-phenylalanine 6 (1.00 eq), NHS (1.00 eq), DCC (1.10 eq), CHCl3(0.05 M), rt, 1 h. (b) D-alanine (1.00 eq), NaHCO3(2.00 eq.), H2O / acetone (1:2, 0.05 M), rt, 16 h (91% over two steps). (c) N-Cbz-D- phenylalanyl-D-alanine 7 (1.00 eq), N,O-dimethyl-hydroxylamine hydrochloride (1.10 eq), HATU (1.10 eq), DIPEA (2.50 eq), DMF (0.1 M), rt, 16 h (88%). (d) Weinreb amide 8 (1.00 eq), vinyl magnesium bromide (3.00 eq), THF (0.1 M), 0 °C to rt, 3 h (58%).

[0128] N-Cbz-D-phenylalanyl-D-alanine (7). The synthesis of 2 was closely followed using N-Cbz-D-phenylalanine (1.00 g, 3.34 mmol, 1.00 eq), (0.384 g, 3.34 mmol, 1.00 eq), DCC (0.758 g, 3.67 mmol, 1.10 eq), D-alanine (0.298 g, 3.34 mmol, 1.00 eq) and NaHCO3 (0.561 g, 6.68 mmol, 2.00 eq). MS (ESI) [M+H]+371.1.

[0129] Benzyl ((R)-1-(((R)-1-(methoxy(methyl)amino)-1-oxopropan-2-yl)amino)-1- oxo-3-phenylpropan-2-yl)carbamate (Weinreb amine 8). To a solution of 7 (0.500 g, 1.35 mmol, 1.00 eq) in anh. DMF (13 ml), N,O-dimethyl-hydroxylamine hydrochloride (0.145 g, 1.48 mmol, 1.10 eq) was added at room temperature, followed by HBTU (0.563 g, 1.48 mmol, 1.10 eq) and DIPEA (0.436 g, 3.37 mmol, 2.50 eq). The reaction mixture was stirred at room temperature for 16 hours. The resulting mixture was diluted with water (20 mL) and extracted with EtOAc (3x). The organic extracts were combined and washed with water (5x), washed with brine, and dried over Na2SO4. The crude mixture was concentrated en vacuo and purified via column chromatography (up to 40% EtOAc / Hexanes) as a white solid (0.490 g, 1.19 mmol, 88%).1H NMR (600 MHz, CDCl3) δ 7.82 – 7.66 (m, 10H), 5.55 – 5.50 (m, 2H), 5.36 – 5.30 (m, 1H), 4.92 (dd, J = 13.1, 7.1 Hz, 1H), 4.58 (q, J = 7.1 Hz, 2H), 4.20 (s, 3H), 3.63 (s, 3H), 3.53 (p, J = 5.8 Hz, 2H), 1.61 (d, J = 7.0 Hz, 3H) ppm. MS (ESI) [M+H]+413.2.

[0130] Benzyl ((R)-1-oxo-1-(((R)-3-oxopent-4-en-2-yl)amino)-3-phenylpropan-2- yl)carbamate (9) and benzyl ((R)-1-oxo-1-(((S)-3-oxopent-4-en-2-yl)amino)-3- phenylpropan-2-yl)carbamate (10). To a solution of Weinreb amide 8 (0.080 g, 0.190 mmol, 1.00 eq) in THF (2 ml), vinyl magnesium bromide (0.7 M in hexanes, 0.83 ml, 3.00 eq) was added at 0 °C. The reaction was slowly warmed to room temperature and stirredSD2023-315-2PCT Attorney Docket No.24978-0928 for 3 hours. The resulting mixture was quenched with ice (20 mL) and extracted with EtOAc (3x). The combined organic extracts were combined and dried over Na2SO4. The crude mixture was concentrated in vacuo and purified via column chromatography (up to 30% EtOAc / Hexanes) as a racemic mixture of two diastereomers (0.043 g, 0.110 mmol, 58%). The diastereomers were separated via chiral preparative HPLC.

[0131] Benzyl ((R)-1-oxo-1-(((R)-3-oxopent-4-en-2-yl)amino)-3-phenylpropan-2- yl)carbamate (9)1H NMR (600 MHz, DMSO-d6) δ 8.46 (d, J = 7.1 Hz, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.35 – 7.16 (m, 9H), 6.63 (s, 0H), 6.48 (dd, J = 17.4, 10.6 Hz, 1H), 6.26 (dd, J = 17.4, 1.6 Hz, 1H), 5.83 (dd, J = 10.5, 1.5 Hz, 1H), 4.94 (s, 2H), 4.56 (t, J = 7.1 Hz, 1H), 4.28 (ddd, J = 10.8, 8.6, 4.3 Hz, 1H), 2.99 (dd, J = 13.8, 4.3 Hz, 1H), 2.73 (dd, J = 13.8, 10.6 Hz, 1H), 1.22 (d, J = 7.2 Hz, 3H) ppm. MS (ESI) [M+H]+381.2.

[0132] Benzyl ((R)-1-oxo-1-(((S)-3-oxopent-4-en-2-yl)amino)-3-phenylpropan-2- yl)carbamate (10).1H NMR (600 MHz, DMSO-d6) δ 8.42 (d, J = 7.3 Hz, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.37 – 7.18 (m, 9H), 6.70 (s, 0H), 6.57 (dd, J = 17.5, 10.6 Hz, 1H), 6.27 (dd, J = 17.5, 1.6 Hz, 1H), 5.83 (dd, J = 10.5, 1.6 Hz, 1H), 4.95 (s, 2H), 4.53 (t, J = 7.2 Hz, 1H), 4.33 – 4.25 (m, 1H), 2.94 (dd, J = 13.7, 4.9 Hz, 1H), 2.77 (dd, J = 13.7, 10.2 Hz, 1H), 1.13 (d, J = 7.2 Hz, 3H) ppm. MS (ESI) [M+H]+381.2.

[0133] Example 5 - Cytotoxicity evaluation

[0134] The effective concentration at which the viability of Vero (ATCC® CCL-81™) and L929 (mouse adipose fibroblasts; ATCC® CCL-1™) cells was reduced by 50% (CC50), was calculated employing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; Thermo Fisher Scientific, USA) assay54. Cells were cultured at 37 °C and 5% CO2in Dulbecco’s Modified Essential Medium (MEM; Cultilab, Brazil) supplemented with 1% heat inactivated FBS (Cultilab, Brazil), 100 IU / mL penicillin (Cellofarm, Brazil), 100 μg / mL streptomycin (Merck, Germany), and 0.25 μg / mL amphotericin B (Cultilab, Brazil), in a humidified incubator at 37 °C and a 5% CO2 atmosphere.

[0135] Compounds were assessed in a serial dilution over eight concentrations (200 to 1.56 μM) performed in 96-well microplates containing 4 x 104cells per well, which wereSD2023-315-2PCT Attorney Docket No.24978-0928 incubated for 48 h, as reported55. Linear regression was used to calculate CC50 values, considering only those data for which r2> 0.85. Inhibition of cell viability was normalized to DMSO controls. Two independent assays in triplicate were performed (n = 6 data points).

[0136] Example 6 - Anti-SARS-CoV-2 in Vero cells

[0137] The SARS-CoV-2 isolate, SP02 / BRA (SARS.CoV2 / SP02.2020.HIAE.Br), from the Wuhan original strain was propagated in Vero cells and titrated as described56,57. Antiviral activity assays were performed in a BSL-3 laboratory at the Federal University of Minas Gerais (UFMG). This work was registered the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen), Brazil, under the registration number A9AC252. Cells were seeded into 24-well microplates containing 1 x 105cells / well and incubated for 24 h at 37 °C in a 5% CO2 atmosphere. Compounds diluted in MEM without FBS were added (700 μL) to wells at different concentrations (0.01, 0.1, 1.0, 10, 20, 40, 60, 80 and 100 μM) together with 100 μL of SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1, that is, one viral particle per 10 cells, and incubated for 1 h.

[0138] After 1 hour of viral adsorption, the medium was removed, and 700 μL of MEM containing 1% FBS and the same concentrations of each compound were added to each well. After 48 hours, the supernatants were removed and centrifuged for 5 minutes at 1,850 g and 25 °C to separate cell debris. Supernatants were stored at -80 °C.

[0139] Viral titration was performed in 24-well microplates (1 x 105cells / well) containing 100 μL supernatant in a 1:10 ratio (diluted 10-1to 10-5) and incubated for 1 hour. Subsequently, 1 mL of medium 199 supplemented with 2% FBS and 1% carboxymethylcellulose (CMC) was added to each well and incubated for 72 hours. The contents of each well were fixed overnight with 10% formalin (3.7% v / v), washed with water, and stained with violet crystal (1% v / v) for 20 minutes. Microplates were washed with water and allowed to dry at room temperature. All wells containing between 10 and 100 plaque-forming units (PFU) were counted by visual inspection. The reduction in viral titer (PFU / mL) was measured by calculating a decrease in infection (PFU per well) of at least 50% at each compound’s dilution compared to the controls (infected cells in MEMSD2023-315-2PCT Attorney Docket No.24978-0928 containing DMSO)57,58. This plaque plaque-forming neutralization effect of the compounds was compared to amantadine (AMA)59as an inhibition control. Selectivity indices (SI) were calculated as CC50 / IC50. The data were analyzed using GraphPad Prism 9.0. One or two independent assays in triplicate were performed (n ≥ 3 data points).

[0140] Example 7 - Anti-MHV-3 activity in L929 cells

[0141] To understand the potential selectivity of the identified inhibitors for SARS- related Mpro, a murine coronavirus, mouse hepatitis virus 3 (MHV-3) was also tested. Despite the fact that this mouse model is used to mimic the debilitating conditions associated with SARS-CoV-260, no activity should be expected from selective SARS-CoV or SARS-CoV-2 Mproinhibitors. Antiviral activity was measured using the MTT assay, as reported55.

[0142] Briefly, viral suspensions at a MOI of 0.1 in DMEM (no FBS) were incubated with a serial dilution of compounds (100 to 1.56 μM) for 48 h under the same conditions as in the cytotoxicity assays. The effective concentration at which compounds were effective in protecting cells from viral infection by 50% (EC50) were calculated relative to the DMSO controls (infected cells in DMEM containing DMSO). The hepatitis C drug, ribavirin, was used as a positive control. SI values were calculated as CC50 / EC50. One or two independent assays in triplicate were performed (n ≥ 3 data points).

[0143] Example 8 - Binding site characterization

[0144] The SARS-CoV-2 Mprostructure co-crystallized with GC376 (bisulphite adduct of GC373), a reversible covalent cysteine protease inhibitor of feline infectious peritonitis virus (FIPV) and other coronaviruses61, was selected from the Protein Data Bank62(PDB ID: 7D1M63, 1.35 Å resolution), due to its high resolution and the relevance of the binding ligand64. In addition, SARS-CoV (PDB ID: 6W2A65, 1.65 Å resolution) and MERS-CoV (PDB ID: 6VH365, 2.2 Å resolution) Mpro, both co-crystallized with the inhibitor 7j65(IUPAC name (1S,2S)-2-[[(2S)-2-[(4,4-difluorocyclohexyl)methoxycarbonylamino]-4- methylpentanoyl]amino]-1-hydroxy-3-[(3S)-2-oxopyrrolidin-3-yl]propane-1-sulfonic acid)), were also included for comparison.SD2023-315-2PCT Attorney Docket No.24978-0928

[0145] The structures were submitted to the web servers FTSite66, FTMap67and PrankWeb68. FTSite predicts binding sites and amino acid residues, while FTMap uses 16 different molecules as probes (e.g., ethanol) to predict interactions with these binding sites and residues. In addition, PrankWeb predicts and ranks the potential binding points clustered on the protease surfaces, by calculating the physicochemical and geometric properties of residues. Thus, residues that would be more relevant to a potential inhibitor were selected in a consensus prediction. The PyMOL69software (v2.5.7 Schrödinger Inc., New York, NY, USA) was used to generate images.

[0146] Example 9 - Preparation of the compounds for docking and MD simulations

[0147] Three-dimensional (3D) structural models of the compounds were generated using Discovery Studio (BIOVIA, USA, 2017). These compounds were submitted to OMEGA 3.1.1.2 (OpenEye Scientific Software, USA, 2019) to create a conformer set70. The lowest energy of each compound was generated using the command line “–maxconfs 1”. In other words, the most stable conformational unit of a compound was generated among a maximum number of possible conformations. All structures had their ionization states corrected to pH 7.4 employing the FixpKa application in the QUACPAC 2.0.1.2 software (OpenEye Scientific Software, USA, 2019) to allow for a single favorable ionization state.

[0148] Example 10 - Molecular docking of compounds with SARS-CoV-2 Mpro

[0149] Molecular docking with the SARS-CoV-2, SARS-CoV and MERS-CoV Mprostructures was performed with the GOLD 5.171software. Also, docking with SARS-CoV- 2 Mprowas performed with GLIDE72to the prepare for MD simulations input. The intrinsic parameters of the programs, such as scoring function, the flexibility of residues and atoms and the number of poses (binding modes), were standardized to better reproduce (lowest RMSD values) the experimental binding mode of co-crystallized ligands, as previously described73.

[0150] Here, the redocking of the co-crystallized ligands considered root-mean-square deviation (RMSD) values ≤ 2.5 Å. The redocking poses provided binding mode references for comparison purposes and validate the docking protocol. Target structures wereSD2023-315-2PCT Attorney Docket No.24978-0928 prepared by fixing missing sidechains, adding hydrogen atoms, removing water molecules and calculating atomic partial charges with default parameters74. The searching parameters in GLIDE employed the program’s default, that is, considering tautomer binding to the target, ionized states and water molecules with three or more hydrogen bonds to residues. The searching parameters in GOLD were: (i) all protein rotatable bonds were fixed; (ii) binding site defined with the co-crystallized ligand with all atoms within 6Å; (iii) chemscore_kinase as the template; (iv) 200 genetic algorithm (GA) runs; (v) CHEMPLP as the scoring function, with no early termination allowed; and (vi) the GA search option set as slow. The PyMOL software (v2.5.7) was used to generate images.

[0151] Example 11 - Molecular dynamics simulations and trajectory analyses

[0152] MD simulations were performed according to established protocol39,40. Briefly, simulations used the Desmond software75and OPLS4 force field76, the TIP3P explicit solvent model77and counterions to neutralize the total charge of the system (Na+or Cl−depending on the overall system charge). The systems were treated as a cubic box with periodic boundary conditions with a size equal to 13 Å. Short-range coulombic interactions were calculated using 1 fs time steps and a 9.0 Å cut-off value, whereas long- range coulombic interactions were estimated using the Smooth Particle Mesh Ewald method, which is sufficient to approximate long-range interactions on large timescales78,79. A constant temperature of 310 K was kept throughout the simulation using the Nose- Hoover thermostat algorithm80and the Martyna-Tobias-Klein Barostat algorithm81,82was used to maintain 1 atmosphere of pressure. After minimization and relaxation of the system, a multiple production step of at least 200 ns was continued, with frames being recorded and saved every 1,000 ps.

[0153] The production simulations were carried out in ten independent replicates of 200 ns with random seeds (yielding a total of 2 µs per ligand). Trajectories and interaction data are available on the Zenodo repository under the code, 10.5281 / zenodo.7841336. Representative structures were selected by hierarchical clustering analysis based on changes in the RMSD, that is, a representative frame was selected at random at points along the trajectory where the RMSD was not fluctuating after equilibration. Protein- ligand interactions were determined using the Simulation Interaction Diagram pipeline implemented in Maestro (Maestro v2023.1).SD2023-315-2PCT Attorney Docket No.24978-0928

[0154] Lastly, the ligands’ binding energies were calculated by the Born and surface area continuum solvation (MM / GBSA) models using Prime83and the implemented thermal MM / GBSA script. Calculated free-binding energies are represented by the MM / GBSA and normalized by the number of heavy atoms (HAC), according to the following formula: ligand efficiency = ln (docking score) / (1 + ln (HAC)).

[0155] Example 12 - Density functional theory calculations

[0156] Compounds 5, 9 and 10, as well as the inhibitor controls nirmatrelvir and GC373, a peptide aldehyde irreversible cysteine protease inhibitor of coronaviruses Mpro61, were submitted to a density functional theory (DFT) calculation. This method aimed to calculate the energy of adduct formation with a given cysteine residue, for example, Cys145, as previously reported84. Briefly, used in this example was the B3LYP functional85,86with 6– 31g* and 6–311++g** basis set87,88to an implicit solvation model with water, and considering a dielectric of 78.30. These were assessed by the C-PCM method89in Spartan ’20 (v.1.1.4, Wavefunction Inc., CA, USA). Calculations were performed using the 3D structure of compounds building the cysteine and cysteine-compound adducts as previously described90. Last, the energy of adduct formation, that is, the energy of binding, was calculated as the energy of the adduct minus the sum of the ligand and the cysteine energy.

[0157] Example 13 - Potential Mproinhibitors identified in a legacy cysteine protease inhibitor collection

[0158] From 141 compounds screened at 5 and 10 µM (Table B), five hits (5 – 9), inhibited at least 50% of the SARS-CoV-2 Mproactivity at 10 µM.SD2023-315-2PCT Attorney Docket No.24978-0928

[0159] Table B-1. Inhibitory activity of 141 compounds against SARS-CoV-2 MproCompound% inhibition% inhibition % inhibition % inhibition (10 μMa) (5 μMa)Compound(10 μMa) (5 μMa) 1 RR 12 21 1 4 RR 241 4 1 227 52 84 88 19 92 58 61 96 83 35 63 79 .4 07 38 18 00 47 89 32 14 46 02 76 51 34 74 66 11 62 49 82 22 57 97 22 12 .6 03 .2 16 22 .3 35calculated from one independent assay in triplicate. Errors are given by the ratio of the standard deviation to the square root of the number of measurements (n = 3). Initial hits are highlighted in bold and italics. WRR: William R. Roush.SD2023-315-2PCT Attorney Docket No.24978-0928

[0161] Table B-2. Inhibitory activity of 141 compounds against SARS-CoV-2 MproCompound% inhibition% inhibition % inhibition % inhibition (10 μMa) (5 μMa)Compound(10 μMa) (5 μMa) 1 RR 2 4 2 11 RR 4 2 1 4 53 28 76 08 13 48 .8 18 17 66 29 33 05 85 74 31 08 17 07 97 97 53 46 02 84 07 )calculated from one independent assay in triplicate. Errors are given by the ratio of the standard deviation to the square root of the number of measurements (n = 3). Initial hits are highlighted in bold and italics. WRR: William R. Roush.

[0163] Of these, only 5 and 9 maintained ≥50% inhibition at 5 µM (Table 1). Despite having a common amino-3-phenylpropan moiety with the analogs 6, 7 and 8, compounds 5 and 9 were the most similar in structure, differing by an electrophilic ethene-ester warhead at 5’s P1 position, while 9 has only an ethynyl group.

[0164] Table 1. Hits that inhibited SARS-CoV-2 Mproactivity at 10 and 5 μM Compound (MW) 10 μMa5 μMaCompound (MW) 10 μMa5 μMaSD2023-315-2PCT Attorney Docket No.24978-0928two independent assays in triplicate (n = 6 data points). Errors are given by the ratio of the standard deviation to the square root of the number of measurements (n ≥ 6). Molecular weights are in parenthesis. *The protease inhibitor nirmatrelvir was tested at 100 nM (0.1 μM).

[0166] Because the compounds had been stored at room temperature for over 10 years, the two most potent compounds, 5 and 9, were resynthesized, as well as the latter’s (R,S) enantiomer, termed 10. The resynthesized compounds yielded low nanomolar IC50 values with the newly synthesized 10 being the most potent with a value of 9.8 nM (Table 2 and FIG.2). Compounds 6 – 8, as well as the resynthesized 5, 9 and 10 were also evaluated for inhibition of SARS-CoV Mproand MERS-CoV Mpro(Table 2 and FIG.3). Only 5, 9 and 10 inhibited SARS-CoV Mprowhereas no compound inhibited MERS-CoV Mpro.

[0167] FIG.3 depicts concentration-response curves of resynthesized compounds against SARS-CoV Mpro. The IC50 values from two independent assays in triplicate (n = 6 data points) were calculated by nonlinear regression (r2> 0.9). Analysis considered theSD2023-315-2PCT Attorney Docket No.24978-0928 standard deviation of each point in each curve, and the deviations were less than 10%. At least eight different compound concentrations (0.02 - 10 µM) were assessed. The data were analyzed using GraphPad Prism 9.0.

[0168] With both the SARS enzymes, the IC50values of 5, 9 and 10 were comparable to or better than those generated by nirmatrelvir in the tested assay conditions, although the latter was also active against MERS-CoV. Finally, none of the 141 compounds inhibited enzymatic activity of against SARS-CoV-2 PLproby ≥ 50% at 10 µM (Table C).

[0169] Table 2. IC50 values of hit compounds against Mproof SARS-related and MERS- CoV Compound (μM) SARS-CoV-2 SARS-CoV MERS-CoV [017 atapoints) were calculated by nonlinear regression (r2> 0.9). Analysis considered the standard deviation of each point in each curve, and the deviations were less than 10%. Errors are given by the ratio of the standard deviation to the square root of the number of measurements (n ≥ 6). *(Re)Synthesized compound.SD2023-315-2PCT Attorney Docket No.24978-0928

[0171] Table C. Inhibitory activity of 141 compounds against SARS-CoV-2 PLproCompound% inhibition% inhibition % inhibition (10 μMa) Compound (10 μMa)Compound(10 μMa) 1 RR 12 4 RR 2 1 RR 2 2 157 51 85 18 65 18 02 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ± 6 0.0 19 89 68 0.0 0.0 0.0 73 0.0 0.0 0.0 47 0.0 0.0 61 0.0 0.0 0.0 0.0 0.0 0.0 77 0.0 86 0.0 0.0 37 0.0 03 - -calculated from one independent assay in triplicate. Errors are given by the ratio of theSD2023-315-2PCT Attorney Docket No.24978-0928 standard deviation to the square root of the number of measurements (n = 3). WRR: William R. Roush.

[0173] Example 14 - Compounds 5, 9 and 10 reversibly inhibited SARS-CoV-2 Mpro

[0174] Also considered was whether the hit compounds, 5, 9 and 10, showed time- dependent inhibition of SARS-CoV-2 Mpro, which might indicate covalent binding52. To do this, a comparison was made of the inhibition-response curves obtained with and without a 15 minute preincubation period before addition of substrate (FIG.4A, FIG.4B, FIG.4C). Each of the hit compounds exhibited distinct behaviors. The curves for 5 and 9 were shifted to the left resulting in a two- and five-fold decrease in the respective IC50values, whereas for 10, the curves were essentially unaltered.

[0175] FIG.4A, FIG.4B, and FIG.4C depict concentration-response curves for inhibition of SARS-CoV-2 Mproactivity by compounds 5 (FIG.4A), 9 (FIG.4B) and 10 (FIG.4C). The IC50values from two independent assays in triplicate (n = 6 data points), were calculated by nonlinear regression (r2> 0.9). Analysis considered the standard deviation of each point in each curve, and the deviations were less than 10%. The black and green curves represented assays performed with and without a 15-minute pre- incubation period prior to addition of substrate, respectively. At least eight different compound concentrations were assessed, ranging from 0.01 up to 40 µM. The data were analyzed using GraphPad Prism 9.0.

[0176] Rapid dilution assays then were conducted to investigate whether inhibitor binding was reversible or irreversible. Inhibitors were pre-incubated at 10-times the IC50value with 100x of the standard enzyme concentration (50 nM). The mixture was diluted to achieve the pre-determined proportions of substrate and enzyme of 10 μM and 50 nM, respectively, and 0.1-times the compounds IC50 value. Nirmatrelvir and GC373 were used as reversible and irreversible control inhibitors, respectively.

[0177] Upon dilution, none of the three inhibitors (5, 9 and 10) and nirmatrelvir maintained inhibition of the enzyme, suggesting reversible inhibition, whereas inhibition by GC373 was not affected by dilution (FIG.5).SD2023-315-2PCT Attorney Docket No.24978-0928

[0178] As depicted at FIG.5, the inhibition of SARS-CoV-2 Mproby 5, 9 and 10 was reversible. Following pre-incubation of the enzyme with excess inhibitor, the mixture was diluted, and the formation of the cleavage product was monitored for 2 hours. Two independent assays in triplicate were performed (n = 6 data points). Image displays a single data point of product formation (i.e., cleaved substrate) represented by the relative fluorescence units (RFU) over time (s).

[0179] Example 15 - Docking of 9 and 10 and characterized Mprosites support compounds’ SARS-related selectivity

[0180] To understand the target selectivity of these reversible Mproinhibitors, the testing also focused on characterizing the conserved residues between the Mproenzymes of SARS-CoV-2, SARS-CoV and MERS-CoV. Characterization of the binding sites was first conducted using FTSite and FTMap, and 13 residues of interest were concomitantly identified for SARS-CoV-2 (FIG.6A), SARS-CoV (FIG.6B), and MERS-CoV (FIG. 6C) Mpro. Among these residues, namely Thr26, Leu27, His41, Phe140, Leu141, Gly143, Ser144, Cys145, His163, Met165 and Glu166, two important substitutions were evidenced between the two SARS-related and MERS-CoV Mpro, namely Asn142Cys and His164Gln (FIG.6D), which could influence potential interactions. In addition, His172 was uniquely predicted for MERS-CoV, whereas Thr25 was only predicted for the SARS-related enzymes. For the latter, Met49 was only predicted in SARS-CoV-2 Mpro. All these residues were also predicted by PrankWeb to be favorable for ligand interaction (Table D).

[0181] FIG.6A, FIG.6B, FIG.6C, and FIG.6D depict predicted binding site residues in different Mproenzymes. Predictions using FTSite and FTMap resulted in potential residues for interaction with ligands in (FIG.6A) SARS-CoV-2 (white), (FIG.6B) SARS-CoV (aquamarine) and (FIG.6C) MERS-CoV (slate purple). In this analysis, the catalytic site residues, His41 and Cys145, were predicted, in addition to Thr26, Leu27, Phe140, Leu141, Asn142 / Cys142, Gly143, Ser144, His163, His164 / Gln164, Met165 and Glu166. (FIG.6D) Superimposed structures display the different conformations due to residue substitutions Asn142 / Cys142 and His164 / Gln164. Images were generated with PyMOL (v2.5.7).SD2023-315-2PCT Attorney Docket No.24978-0928

[0182] Table D. SARS-CoV-2, SARS-CoV, and MERS-CoV Mproresidues predicted with PrankWeb arget SiSite #2 Site #3 Tte #1Score (accuracy, %)Score (accuracy, %) Score (accuracy, %) , 8, , , , , , , , , , , , ,

[0184] To understand whether the differences in the three enzymes’ residues noted above would impact the binding of inhibitors, a docking of the most potent inhibitors, 9 and 10, was performed with the three coronavirus Mproenzymes using GOLD. The docking method was validated (RMSD values ≤2.5 Å91) with redocking of each co- crystallized ligand (FIG.7A, FIG.7B, and FIG.7C) resulting in RMSD values of 1.49, 0.73 and 2.19 Å for MERS-CoV, SARS-CoV and SARS-CoV-2, respectively.SD2023-315-2PCT Attorney Docket No.24978-0928

[0185] FIG.7A, FIG.7B, and FIG.7C depict Redocking of Mproco-crystallized ligands using GOLD. Redocking poses of the co-crystallized ligands GC376 (RMSD of 2.19 Å) in SARS-CoV-2 Mpro(PDB ID: 7D1M), (FIG.7B) 7j (RMSD of 0.73 Å) in SARS-CoV (aquamarine) Mpro(PDB ID: 6W2A), and (FIG.7C) 7j (RMSD of 1.49 Å) in MERS-CoV (slate purple) Mpro(PDB ID: 6VH3). Catalytic residues His41 and Cys145 are highlighted. Images were generated with PyMOL (v2.5.7).

[0186] The redocking results of this example matched those found in the original docking study with GC376, which used the GLIDE software92. This validation was supported with additional redocking to other coronaviruses Mproco-crystallized ligands (Table E). This cross-study and software validation was believed to be important for accurately predicting residues that interact with a ligand and its overall pose. This was the case in the design of GC376 against SARS-CoV-2 Mpro64,92, SARS-CoV and MERS-CoV Mpro93, and the Michael acceptor inhibitor N3 for the same enzymes94.

[0187] Table E. SARS-CoV-2, SARS-CoV, and MERS-CoV Mproresidues interacting with co-crystallized ligands predicted from GOLD redocking PDB Inhibitor PDB Inhibitor PDB Inhibitor PDB Inhibitor 7D1M GC376 7C6U GC376 7C6S boceprevir 7BRP boceprevir rSD2023-315-2PCT Attorney Docket No.24978-0928 His163 His164 His163 His163 His164 His166 Gln164 Gln164 Glu166 Pro168 Glu166 Met165 -CoV.

[0189] Docking of 9 and 10 to SARS-CoV-2 Mpro(FIG.8A and FIG.8B) predicted similar poses for both compounds, with 10 displaying a potential interaction of the P4 benzyl-esther with Gln189, which would correlate with the activities of 9 and 10 (FIG.4). Similar poses were also predicted for both compounds to SARS-CoV Mpro(FIG.8C and FIG.8D), with a different P3 benzyl accommodation, which correlates to their similar enzyme activity (Table 2). In contrast, for MERS-CoV, both compound poses were predicted with the P1 group facing the opposite direction from the catalytic cysteine (FIG. 8E and FIG.8F), potentially supporting weak or an absence of binding. In support of this notion, both 9 and 10 did not inhibit the catalytic activity of MERS-CoV Mpro(Table 2), possibly due to interference by the Asn142Cys and His164Gln substitutions (FIG.8E and FIG.8F, and FIG.6D).

[0190] FIG.8A—FIG.8F depict the docking of 9 and 10 with different Mproenzymes. Docking was performed using GOLD for SARS-CoV-2 (white), which resulted in a predicted binding pose for (FIG.8A) 9 (orange) and (FIG.8B) 10 (blue), similar to the respective poses in (FIG.8C) and (FIG.8D) for SARS-CoV (aquamarine). In contrast, the potential interactions of (FIG.8E) 9 and (FIG.8F) 10 with MERS-CoV (stale purple) showed that the electrophilic ethynyl warhead faces away from the catalytic cysteine. The catalytic site residues, His41 and Cys145, are highlighted as are some of the predicted binding site residues, such as Asn142 / Cys142, Gly143, Ser144, His163, His164 / Gln164, Glu166 and Gln189. Images were generated with PyMOL (v2.5.7).

[0191] Example 16 - A reversible inhibitor binding mode predicted by MD simulations

[0192] Also performed were MD simulations to assess the binding stability of 9 and 10 in SARS-CoV-2 Mpro, as well as predict the frequency of interaction with each residue. Initially, the system was validated by simulating with the co-crystallized cysteine protease inhibitor, GC376 (PDB ID: 7D1M). Five replicates of 200 ns (1 µs) were simulated toSD2023-315-2PCT Attorney Docket No.24978-0928 predict the residues that bind to GC376 and their frequencies of interactions: Phe140 (48%), Cys145 (93%), His163 (90%), His164 (75%), Glu166 (96%) and Gln189 (74%), as well as with Thr26 (30%) and Gly143 (40%) that are mediated by water contacts (FIG. 9A). Here, Cys145 was predicted to interact with a hydroxyl group, whereas Phe140, His163, His164, Glu166 and Gln189, were predicted to interact with the amide in GC376. Also performed were simulations with GC376 co-crystallized with a monomer of SARS- CoV-2 Mpro(PDB ID: 7C6U) to compare the conservation of interactions and the ligand’s stability between two different experimentally determined binding modes (FIG.9B). Residues Gly143, Cys145, Glu166 and Gln189 were consistent in their interactions across all replicates, also corroborating previous predictions (FIG.6A—FIG.6D, and FIG. 7A—FIG.7C).

[0193] FIG.9A and FIG.9B depict MD simulations of the co-crystallized ligand GC376 against SARS-CoV-2 Mpro. Five replicates of 200 ns simulations were performed for the inhibitor GC376 in two experimentally determined binding modes (FIG.9A) PDB ID: 7D1M and (FIG.9B) PDB ID: 7C6U. Similar interaction frequencies with amide groups are observed for Phe140, His163, His164, Glu166 and Gln189, in addition to interactions of Thr26, Gly143 and Cys145 with a hydroxyl group. Interaction types and trend lines: hydrogen bonding or polar, ionic or negatively charged, hydrophobic, and water-mediated. Images were generated with Schrodinger.

[0194] Also performed was a combination of multiple short MD simulations, that is, 10 replicas of 200 ns for each ligand, to identify any differences between 9 and 10. These calculations were followed by clustering, geometry, and binding energy analysis along the calculated trajectories (FIG.10A, FIG.10B, FIG.10C, FIG.10D). The proposed binding modes of both compounds are represented by the most populated cluster conformation from the MD simulations for 9 (FIG.11A) and 10 (FIG.11B).

[0195] FIG.10A—FIG.10D depict RMSD and RMSF values for compounds 9 (FIG. 10A and FIG.10B, respectively) and 10 (FIG.10C and FIG.10D, respectively). Variation of the RMSD values along with the simulation, for both template crystal structures and simulations with docking pose. Additionally, changes in RMSF are normalized by residue for the protein backbone.SD2023-315-2PCT Attorney Docket No.24978-0928

[0196] Both configurations rely on hydrogen interactions between the amide linker of the inhibitor and the backbone of Glu166, as well as pi-mediated interactions between the phenyl ring of the inhibitor and the His41 sidechain. In addition, compound 10 does not stably occupy the S1 pocket (Cys145 and Gly143) but rather interacts with the S3 and S4 pockets with additional polar contacts with the Gln189 and Thr190. These additional interactions are reflected in the predicted binding energy (FIG.11C), which shows that 10 has a lower binding energy compared to 9 and consistent with its lower IC50 values measured in the enzymatic assays (Table 2). These simulations also suggested that 9 and 10 are unlikely to establish a stable covalent bond, in spite of the short distance (3.6 – 4 Å) between the reactive ethynyl group of the inhibitor 9 and the catalytic cysteine in the most populated clusters (FIG.11D). The distance between the ethynyl warhead’s reactive atoms and the sulfur of the active site’s cysteine also remains unstable along the simulation trajectory (FIG.11E), which are consistent with the reversible inhibition profile of both compounds measured in the enzymatic assays (FIG.4 and FIG.5).

[0197] FIG.11A and FIG.11B depict proposed binding modes of 9 and 10 in the active site of SARS-CoV-2 Mpro, represented by the most populated cluster conformation from the MD simulations for (FIG.11A) 9 (orange) and (FIG.11B) 10 (blue). Residues performing polar contacts are labeled in bold and their interaction frequency in the analyzed trajectory time (10 x 200 ns per ligand) are shown as percentage values. FIG. 11C is a violin plot displaying the variation of the calculated binding energy, using MM / GBSA along the entire MD trajectory, and normalized to the natural log number of heavy atoms. Median energy values are highlighted. Differences between distributions were analyzed by a one-sample Mann-Whitney multiple comparison test (****p < 0.0001). As shown at FIG.11D, most populated clusters depict a short distance (3.6 – 4 Å) of the reactive ethynyl group of 9 to the catalytic cysteine (orange box). However, 9 and 10 were unlikely to form stable covalent bonds, because, as depicted at FIG.11E, the average distance of the warhead’s reactive atoms from the reactive catalytic cysteine’s sulfur remains unstable along the simulation’s trajectory.

[0198] Finally, as different electrophilic functional groups40may still be predicted with similar or the same interactions in MD simulations, additional methods can be employed to further describe their effects, such as DFT calculations95. Herein, DFT values (Table 3)SD2023-315-2PCT Attorney Docket No.24978-0928 using B3LYP / 6-311++g** for inhibitors 9 and 10 (-15.59 to -15.97 kcal / mol) were closer to those for nirmatrelvir (-18.89 kcal / mol) than the irreversible inhibitor GC373 (-20.14 kcal / mol). A similar behavior was calculated using B3LYP / 6-311+g with an exception to nirmatrelvir’s values (-8.14 kcal / mol). These results were consistent with the unstable covalent behavior predicted in the binding of the ethynyl electrophilic group of 9 and 10 to the catalytic cysteine residue (FIG.8 and FIG.11), in spite of the distance (3.6 – 4 Å) for a potential reversible covalent bond of 9 (FIG.11D). Also, a more thermodynamically favorable behavior observed for GC373 would correspond to its irreversibility, as opposed to the reversibility for 9, 10 and nirmatrelvir (FIG.5).

[0199] Table 3. Calculated total energies for ligands, the active site cysteine residue and adducts (atomic unity): adduct energy formation (kcal / mol) with B3LYP / 6–31g* and B3LYP / 6-311++g** methods B3LYP Compound Eligand ECys145 Eadduct Ebinding 6-31g* 9 -792885.67 -453084.25 -1245986.78 -16.86 10Nirmatrelvir -1110945.38 -453084.25 -1564037.77 -8.14 GC373 -852466.23 -453084.25 -1305571.91 -21.43 B3LYP Compound EligandECys145EadductEbinding-311++g** 9 -792924.2 -453103.91 -1246043.7 -15.59 10 -Nirmatrelvir -1110996.27 -453103.91 -1564119.07 -18.89 GC373 -852511.12 -453103.91 -1305635.17 -20.14

[0200] Example 17 - Compound 10 is a nanomolar inhibitor of SARS-CoV-2 replication in Vero host cells

[0201] For compounds 5, 9 and 10, the CC50values were between 17 and 49 µM for Vero cells and 74 and 102 µM for L929 cells (Table 4), which suggested that the compounds are relatively non-toxic. Also considered was whether 9 and 10 could decrease viral titers of the SARS-CoV-2 Wuhan strain in Vero cells. For comparison, and to verify the selectivity of the inhibitors for SARS-related Mproenzymes, also evaluated was their ability to inhibit the replication of MHV-3 in L929 cells. MHV-3 is a mouse coronavirus from the same genus of SARS-related coronaviruses96, like MERS-CoV, but shares a lower Mproidentity than the SARS-related coronaviruses (FIG.4).SD2023-315-2PCT Attorney Docket No.24978-0928

[0202] Compounds 9 and 10 decreased the viral titers (PFU / mL) with IC50 values of 10.45 and 0.018 µM, respectively (FIG.12A and FIG.12B).

[0203] FIG.12A and FIG.12B depict concentration-response of 9 and 10 against SARS-CoV-2. Vero cells CCL-81 were infected with SARS-CoV-2 at multiplicity of infection (MOI) of 0.1. After the adsorption, inhibitors were incubated for 48 h and the supernatant was collected and titrated by plaque assay to count the plaque forming-units (PFU). IC50 values calculated for (FIG.12A) 10 (two assays) aside to (FIG.12B) 9 (single assay). The tested concentration at 100 μM impairs the cell monolayer due to its toxicity, which prevents the calculation of PFU reduction, resulting in the dropping points at (B) and (C). The data were analyzed with GraphPad Prism 9.0. All conditions were tested in triplicate in one (n = 3 data points) or two (n = 6 data points) independent assays.

[0204] These values, when factored into the respective CC50 values of 48.98 and 37.53, generated SI values of 4.69, and an impressive 2,085, respectively. Last, 5 did not show significant antiviral activity up to 100 μM, and none of the three analogs showed antiviral activity in the MHV-3 / L929 system up to 100 µM (Table 4).

[0205] Table 4. Cytotoxicity and antiviral activity of inhibitors against SARS-CoV-2 and MHV-3. Vero SARS-CoV-2 L929 MHV-3not tested.

[0207] Example 17 - Compounds 9 and 10 interact selectivity as SARS-related reversible MproinhibitorsSD2023-315-2PCT Attorney Docket No.24978-0928

[0208] Given the differences in IC50 values of the hit compounds against the three Mprotargets, also characterized were their potential binding sites using FTSite, FTMap and PrankWeb. The predictions corroborated residues that usually engage inhibitors against different SARS-CoV-2 Mprostructures in such simulations98–100. The predictions highlighted MERS-CoV Mprosubstitutions (Asn142Cys and His164Gln) that would not favor interactions with an inhibitor101. The docking simulations using GOLD then predicted 9 and 10 P1 orientation facing the opposite direction of the catalytic cysteine of MERS-CoV Mpro(FIG.8E and FIG.8F), while no changes were predicted for the two SARS-related proteases. These supported the compounds lack of biochemical inhibition of MERS-CoV Mpro(Table 2), in addition to MERS-CoV lower identity compared to SARS- related coronaviruses (FIG.4). This behavior was demonstrated for ensitrelvir, which form hydrogen bonds with Asn142 in crystal structures of SARS-CoV-2 and SARS-CoV Mpro, but no bonds are observed to the substituted residue in MERS-CoV Mpro102.

[0209] Additionally, the 1 µs MD simulations of the examples herein predicted a lower binding energy for 10 compared to 9, consistent with the lower IC50 values for the former compound. Also, it suggested that both compounds are unlikely to establish a stable covalent bond, supporting the data from time-dependent and rapid dilution assays as noncovalent reversible inhibitors (FIG.4, FIG.5, and FIG.11). This was also the case for MD simulations assessing nirmatrelvir’s reversible binding to the SARS-CoV-2 Mpro103. Last, the DFT calculations herein predicted similar binding formation (kcal / mol) energies for 9 and 10, and their values are closer to that for nirmatrelvir than the irreversible inhibitor GC373. In this sense, it may be likely that the ethenyl substituents in 9 and 10 could favor reversible bonds104, as was observed for nirmatrelvir’s reversible binding to SARS-CoV-2 Mpro, ultimately being confirmed in vitro105.

[0210] Example 18 - Compound 10 is a nanomolar inhibitor comparable to current SARS-CoV-2 drugs

[0211] Compounds 9 and 10 reduced the viral titers of the SARS-CoV-2 Wuhan strain in Vero cells at (sub)micromolar concentrations. Compound 10 was especially potent with an IC50 value of 18 nM and an associated SI value of 2,085, i.e., essentially non-toxic to Vero cells. It is known that stable and conserved interactions of inhibitors with SARS- CoV-2 Mproresidues usually support their potency and correlate equivalence inSD2023-315-2PCT Attorney Docket No.24978-0928 biochemical and antiviral activities105, which would correspond to predictions for the enantiomer (R,S) 10 with Gln189 (FIG.8 and FIG.11).

[0212] Also, inhibitors interactions with Gln189, Glu166, His163, Gly143 and His41 in simulations are postulated to impact their inhibitory activity significantly39, which was observed for compound 10. The current IC50 and SI values of 10 are competitive with data for other antiviral agents that covalently or noncovalently bind to SARS-CoV-2 Mpro, as shown in Table 5. Accordingly, compound 10 is a promising hit for an expanded SAR program that would include further in vitro and in vivo efficacy studies58,59including other SARS-related strains36or inhibitor-induced mutations21,105.

[0213] Table 5. Noteworthy activity of 10 compared to other SARS-CoV-2 Mproinhibitors. Inhibitor CC50(µM) IC50or EC50(µM) SI 5

[0214] 107(Vero E6 cells), and e108(Calu-3 cells).SD2023-315-2PCT Attorney Docket No.24978-0928

[0215] The foregoing example describe the discovery of three nanomolar inhibitors that are selective for SARS-related Mproenzymes from a legacy library of protease inhibitors. The foregoing enzyme and cell assays against the SARS-CoV-2 Wuhan strain demonstrated compound 10 with nanomolar potency comparable to or better than ensitrelvir and nirmatrelvir. This discovery successfully combined computer-aided simulations with experimental validation to characterize this inhibitor activity, similar to the discovery of the current Mprodrugs. Therefore, 10 could be a feasible alternative as a lead candidate for the current and future challenges of COVID-19, opening possibilities for further structural modifications and the synthesis of new analogs while carrying out complementary in vitro and in vivo studies.

[0216] Listing of Embodiments.

[0217] The following is a non-limiting listing of embodiments of the present disclosure.

[0218] Embodiment 1. A compound selected from (i) Compound 5 (methyl (R,E)-5- ((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-4-oxohex-2-enoate), a salt thereof, an analog thereof, or a derivative thereof:

[0219] ; and / or

[0220] (ii) Compound 5’ (methyl (R,Z)-5-((S)-2-(((benzyloxy(carbonyl)amino)-3- phenylpropanamido)-4-oxohex-2-enoate), a salt thereof, an analog thereof, or a derivative thereof:SD2023-315-2PCT Attorney Docket No.24978-0928

[0221] .

[0222] Embodiment 2. A compound selected from Compound 9 (benzyl ((R)-1-oxo-1- (((R)-3-oxopent-4-en-2-yl)amino)-3-phenylpropan-2-yl)carbamate), a salt thereof, an analog thereof, or a derivative thereof:

[0223] .

[0224] Embodiment 3. A compound selected from Compound 10 (benzyl ((R)-1-oxo-1- (((S)-3-oxopent-4-en-2-yl)amino)-3-phenylpropan-2-yl)carbamate), a salt thereof, an analog thereof, or a derivative thereof:SD2023-315-2PCT Attorney Docket No.24978-0928

[0225] .

[0226] Embodiment 4. A compound selected from the group consisting of Compound 6, Compound 7, Compound 8, a salt thereof, an analog thereof, and a derivative thereof.

[0227] Embodiment 5. A pharmaceutical composition comprising, consisting essentially of, or consisting of a pharmaceutically active agent and optionally a pharmaceutically acceptable carrier, wherein the pharmaceutically active agent comprises, consists essentially of, or consists of the compound of any one or more of Embodiments 1 to 4.

[0228] Embodiment 6. The pharmaceutical composition of Embodiment 5, wherein the pharmaceutical composition is formulated as a solution, emulsion, suspension, or dispersion.

[0229] Embodiment 7. The pharmaceutical composition of Embodiment 5 or 6, wherein the pharmaceutical composition is formulated for intravenous administration, oral administration, or topical administration.

[0230] Embodiment 8. A method of preventing or treating viral infection, the method comprising, consisting essentially of, or consisting of administering to a subject in need thereof a therapeutically effective amount of a main protease inhibitor to prevent or treat viral infection of the subject; wherein the main protease inhibitor comprises, consists essentially of, or consists of any one or more of Embodiments 1 to 4, or is present in a pharmaceutical composition of any one of Embodiments 5 to 7.SD2023-315-2PCT Attorney Docket No.24978-0928

[0231] Embodiment 9. The method of Embodiment 8, wherein the viral infection is caused by a coronavirus.

[0232] Embodiment 10. The method of Embodiment 9, wherein the coronavirus is SARS-CoV, such as CoV-2, which causes COVID-19.

[0233] Embodiment 11. The method of Embodiment 9, wherein the coronavirus is MERS-CoV.

[0234] Embodiment 12. A method of inhibiting a viral main protease, the method comprising, consisting essentially of, or consisting of administering to a subject in need thereof a therapeutically effective amount of a main protease inhibitor to inhibit the viral main protease; wherein the main protease inhibitor comprises, consists essentially of, or consists of any one or more of Embodiments 1 to 4, or is present in a pharmaceutical composition of any one of Embodiments 5 to 7.

[0235] Embodiment 13. The method of Embodiment 12, wherein the viral main protease is selected from SARS-CoV-2 cysteine proteases, main protease (Mpro) and papain-like protease (PLpro).

[0236] Embodiment 14. The method of any one of Embodiments 8 to 13, wherein the subject is a mammal.

[0237] Embodiment 15. The method of Embodiment 14, wherein the mammal is a human.

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Claims

SD2023-315-2PCT Attorney Docket No.24978-0928 What is claimed is:

1. A method of preventing or treating a viral infection, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a main protease inhibitor to prevent or treat the viral infection of the subject; wherein the main protease inhibitor is selected from the group consisting of- (i) Compound 5, a salt thereof, an analog thereof, or a derivative thereof -, (ii) Compound 9, a salt thereof, an analog thereof, or a derivative thereof - ,(iii) Compound 10, a salt thereof, an analog thereof, or a derivative thereof -SD2023-315-2PCT Attorney Docket No.24978-0928.

2. The method of claim 1, wherein the main protease inhibitor is Compound 5, the salt thereof, the analog thereof, or the derivative thereof.

3. The method of claim 1, wherein the main protease inhibitor is Compound 5’, the salt thereof, the analog thereof, or the derivative thereof.

4. The method of claim 1, wherein the main protease inhibitor is Compound 9, the salt thereof, the analog thereof, or the derivative thereof.

5. The method of claim 1, wherein the main protease inhibitor is Compound 10, the salt thereof, the analog thereof, or the derivative thereof.SD2023-315-2PCT Attorney Docket No.24978-0928 6. The method of claim 1, wherein the viral infection is a coronavirus infection.

7. The method of claim 6, wherein the coronavirus infection is a SARS-CoV infection.

8. The method of claim 6, wherein the coronavirus infection is a MERS-CoV infection.

9. The method of claim 1, wherein the subject is a human.

10. The method of claim 1, wherein the main protease inhibitor is a component of a pharmaceutical composition.

11. The method of claim 10, wherein the pharmaceutical composition is formulated for oral or intravenous administration.

12. A method of inhibiting a viral main protease, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a main protease inhibitor to inhibit the viral main protease; wherein the main protease inhibitor is selected from the group consisting of- (i) Compound 5, a salt thereof, an analog thereof, or a derivative thereof -, (ii) Compound 9, a salt thereof, an analog thereof, or a derivative thereof -SD2023-315-2PCT Attorney Docket No.24978-0928 ,-a an or aSD2023-315-2PCT Attorney Docket No.24978-0928.

13. The method of claim 12, wherein the subject is a human.

14. The method of claim 12, wherein the main protease inhibitor is a component of a pharmaceutical composition.

15. The method of claim 14, wherein the pharmaceutical composition is formulated for oral or intravenous administration.

16. A pharmaceutical composition comprising: a pharmaceutically active agent; and a pharmaceutically acceptable carrier; wherein the pharmaceutically active agent is selected from the group consisting of- (i) Compound 5, a salt thereof, an analog thereof, or a derivative thereof -SD2023-315-2PCT Attorney Docket No.24978-0928, (ii) Compound 9, a salt thereof, an analog thereof, or a derivative thereof - ,(iii) Compound 10, a salt thereof, an analog thereof, or a derivative thereof -SD2023-315-2PCT Attorney Docket No.24978-0928 (iv) Compound 5’, a salt thereof, an analog thereof, or a derivative thereof-.

17. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition is formulated for intravenous administration, oral administration, or topical administration.

18. The pharmaceutical composition of claim 16, wherein the pharmaceutically active agent is Compound 5, the salt thereof, the analog thereof, or the derivative thereof.

19. The pharmaceutical composition of claim 16, wherein the main protease inhibitor is Compound 5’, the salt thereof, the analog thereof, or the derivative thereof.

20. The pharmaceutical composition of claim 16, wherein the pharmaceutically active agent is Compound 9, the salt thereof, the analog thereof, or the derivative thereof.

21. The pharmaceutical composition of claim 16, wherein the pharmaceutically active agent is Compound 10, the salt thereof, the analog thereof, or the derivative thereof.

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