Remdesivir and remdesivir analogs, solutions, and nanoparticle, liposome, and microparticle compositions for treating viral infections

Abstract

The present invention relates to methods, compounds and compositions for treating viral infections (including COVID-19 viral infections). In certain embodiments, the composition comprises: i) a remdesivir analog, ii) remdesivir or a remdesivir analog, and a surfactant, a cyclodextrin, or a combination thereof, iii) nanoparticles comprising albumin and remdesivir or a remdesivir analog, iv) liposomes comprising lipids and remdesivir or a remdesivir analog; and / or v) microparticles comprising PLA and / or PLGA and remdesivir or a remdesivir analog. In certain embodiments, the composition is aqueous (e.g., for intravenous administration). In other embodiments, the composition is aerosolized or in dry powder form (e.g., for inhalation by an infected subject).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 004,122, filed April 2, 2020, and U.S. Provisional Application No. 63 / 160,407, filed March 12, 2021, the contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to methods, compounds and compositions for treating viral infections, including COVID-19 (SARS-CoV-2) viral infections. In certain embodiments, the composition comprises: i) a remdesivir analog, ii) remdesivir or a remdesivir analog, and a surfactant, a cyclodextrin, or a combination thereof, iii) nanoparticles comprising albumin and remdesivir or a remdesivir analog, iv) liposomes comprising lipids and remdesivir or a remdesivir analog; and / or v) microparticles comprising PLA and / or PLGA, and remdesivir or a remdesivir analog. In certain embodiments, the composition is aqueous (e.g., for intravenous administration). In other embodiments, the composition is aerosolized or in dry powder form (e.g., for inhalation by an infected subject). Background Art

[0004] The COVID-19 pandemic shows no signs of slowing down. Experts predict that the pandemic may continue to be a seasonal disease. Developing treatment options for COVID-19 is critical and urgent. Remdesivir (development code GS-5734, a new antiviral drug in the nucleotide analog class) is considered the most promising drug for the treatment of COVID-19. [1] The expectation for remdesivir to treat COVID-19 is based on the following facts: (a) Remdesivir has broad-spectrum antiviral activity due to its inhibition of viral RNA synthesis [2] (b) Remdesivir has in vitro anti-COVID-19 activity (IC 50 0.77 μM) [1] ; (c) Remdesivir in vitro and in mice [3,4] Monkey [5] (d) Remdesivir was evaluated in human Ebola patients as having adequate plasma exposure and safety profile (although it failed to demonstrate efficacy against Ebola) [6] Remdesivir was introduced in the first COVID-19 case in the United States as a compassionate use medication when the patient developed severe pneumonia. Clinical improvement was observed on the second day of treatment. [7]Clinical trials with a large number of patients are needed to determine the clinical efficacy and safety of the drug. Ten clinical trials are underway. [8] The conclusion is still unclear [9] .

[0005] Remdesivir, a prodrug of nucleoside monophosphate (Nuc-MP), is designed to increase the cell permeability of Nuc-MP and bypass the rate-limiting first phosphorylation step of nucleosides (Nuc) Figure 1 ) [2] After cellular uptake of remdesivir by passive diffusion, remdesivir is converted to Nuc-MP by intracellular hydrolases and then forms the active metabolite nucleoside triphosphate (Nuc-TP), which is expected to interfere with the activity of viral RNA-dependent RNA polymerase (RdRp). [2] Despite the good in vitro antiviral activity of remdesivir, several drawbacks may limit its in vivo efficacy in treating pneumonia caused by COVID-19. Summary of the Invention

[0006] The present invention relates to methods, compositions and compounds for treating viral infections (including COVID-19 viral infections). In certain embodiments, the composition comprises: i) a remdesivir analog, ii) remdesivir or a remdesivir analog, and a surfactant, a cyclodextrin, or a combination thereof, iii) nanoparticles comprising albumin and remdesivir or a remdesivir analog, iv) liposomes comprising lipids and remdesivir or a remdesivir analog; and / or v) microparticles comprising PLA and / or PLGA, and remdesivir or a remdesivir analog. In certain embodiments, the composition is aqueous (e.g., for intravenous administration). In other embodiments, the composition is aerosolized or in dry powder form (e.g., for inhalation by an infected subject).

[0007] In some embodiments, provided herein are methods of treating a subject, comprising administering or providing a composition to a subject infected with a virus, wherein the composition comprises: a) a remdesivir analog, b) remdesivir or a remdesivir analog, and a surfactant, a cyclodextrin, or a combination thereof, c) a plurality of nanoparticles, each of the plurality of nanoparticles comprising albumin and remdesivir or a remdesivir analog, and / or d) a plurality of liposomes, each of the plurality of liposomes comprising a lipid forming a bilayer and remdesivir or a remdesivir analog, and / or e) a plurality of microparticles, each of the plurality of microparticles comprising: i) poly(lactic acid) (PLA) and / or poly(lactic-co-glycolic acid) (PLGA), and remdesivir or a remdesivir analog, and wherein each remdesivir analog is independently a compound of Formula I or Formula II:

[0008]

[0009] wherein X is or comprises an amino acid side chain, R is or comprises a lipid chain or a benzyl group (Formula I), Ar is a substituted or unsubstituted aryl or heteroaryl group, and Y is a C2-C 20 Alkylene or C2-C 20 Alkenylene; and Z is C4-C 40 Alkyl, C4-C 40 Alkenyl, or C4-C 40 Alkynyl (Formula II).

[0010] In some embodiments, the remdesivir analog is a compound of Formula Ia:

[0011] wherein X is or comprises an amino acid side chain, and R is or comprises a lipid chain or a benzyl group.

[0012] In certain embodiments, the virus is SARS-CoV-2, which causes COVID-19. In specific embodiments, the subject is a human. In other embodiments, the virus is severe acute respiratory syndrome-associated coronavirus (SARS-CoV). In other embodiments, the virus is a coronavirus or a virus that causes respiratory illness. In some embodiments, the virus is Ebola virus. In some embodiments, the compound inhibits viral entry into cells and replication.

[0013] In some embodiments, the composition is in the form of an aqueous solution, and wherein administration is via intravenous injection into a subject. In other embodiments, the composition is in aerosolized form, and wherein administration includes releasing the composition into the airways of the subject so that they inhale the composition. In other embodiments, the composition is in aerosolized form, and wherein providing includes distributing a nebulizer device containing the composition to the subject. In other embodiments, the composition is in dry powder form, and wherein administration includes releasing the composition into the airways of the subject so that they inhale the composition. In some embodiments, the composition is in dry powder form, and wherein providing includes distributing a powder dispensing device containing the composition to the subject. In additional embodiments, the composition also includes an excipient and is in micronized powder form, and wherein administration includes releasing the composition into the airways of the subject so that they inhale the composition. In additional embodiments, the composition also includes an excipient and is in micronized powder form, and wherein providing includes distributing a micronized powder dispensing device containing the composition to the subject. In certain embodiments, the excipient is selected from lactose, mannitol, and PVA.

[0014] In some embodiments, provided herein are compositions comprising: remdesivir or a remdesivir analog, and a surfactant, a cyclodextrin, or a combination thereof, wherein each remdesivir analog is independently a compound of Formula I or II:

[0015]

[0016] wherein X is or comprises an amino acid side chain, R is or comprises a lipid chain or a benzyl group, Ar is a substituted or unsubstituted aryl or heteroaryl group, and Y is a C2-C 20 Alkylene or C2-C 20 Alkenylene; and Z is C4-C 40 Alkyl, C4-C 40 Alkenyl, or C4-C 40 Alkynyl.

[0017] In some embodiments, the remdesivir analog is a compound of Formula Ia:

[0018] wherein X is or comprises an amino acid side chain, and R is or comprises a lipid chain or a benzyl group.

[0019] In some embodiments, the composition comprises remdesivir or a remdesivir analog, and a surfactant. In certain embodiments, the surfactant concentration is in the range of 0 to 10%. In certain embodiments, the surfactant comprises Solutol HS 15.

[0020] In some embodiments, the composition comprises remdesivir or a remdesivir analog and a cyclodextrin. In certain embodiments, the concentration of the cyclodextrin is in the range of 5% to 40%. In certain embodiments, the cyclodextrin comprises sulfobutyl ether-β-cyclodextrin or hydroxypropyl-β-cyclodextrin.

[0021] In some embodiments, the composition comprises remdesivir or a remdesivir analog, a surfactant, and a cyclodextrin.

[0022] In certain embodiments, the composition may have Sp isomers, Rp isomers, or mixed Sp / Rp isomers.

[0023] In some embodiments, the pH is between 2 and 5 (e.g., about 2.5, about 3.0, about 3.5, about 4.0, about 4.5). In certain embodiments, the pH is between 3 and 3.5.

[0024] In some embodiments, provided herein is a composition comprising: a plurality of nanoparticles, each of the plurality of nanoparticles comprising albumin and remdesivir or a remdesivir analog, wherein each remdesivir analog is independently a compound of Formula I or II:

[0025]

[0026] wherein X is or comprises an amino acid side chain, R is or comprises a lipid chain or a benzyl group, Ar is a substituted or unsubstituted aryl or heteroaryl group, and Y is a C2-C 20 Alkylene or C2-C 20 Alkenylene; and Z is C4-C 40 Alkyl, C4-C 40 Alkenyl, or C4-C 40 Alkynyl.

[0027] In some embodiments, the remdesivir analog is a compound of Formula Ia:

[0028] wherein X is or comprises an amino acid side chain, and R is or comprises a lipid chain or a benzyl group.

[0029] In certain embodiments, provided herein are compositions comprising: a plurality of liposomes, each of the plurality of liposomes comprising a lipid forming a bilayer and remdesivir or a remdesivir analog, wherein each remdesivir analog is independently a compound of Formula I or II:

[0030]

[0031] wherein X is or comprises an amino acid side chain, R is or comprises a lipid chain or a benzyl group, Ar is a substituted or unsubstituted aryl or heteroaryl group, and Y is a C2-C 20 Alkylene or C2-C 20 Alkenylene, and Z is C4-C 40 Alkyl, C4-C 40 Alkenyl, or C4-C 40 Alkynyl.

[0032] In some embodiments, the remdesivir analog is a compound of Formula Ia:

[0033] wherein X is or comprises an amino acid side chain, and R is or comprises a lipid chain or a benzyl group.

[0034] In certain embodiments, provided herein are compositions comprising: a plurality of microparticles, each of the plurality of microparticles comprising: i) poly(lactic acid) (PLA) and / or poly(lactic-co-glycolic acid) (PLGA), and remdesivir or a remdesivir analog, wherein each remdesivir analog is independently a compound of Formula I or II:

[0035]

[0036] wherein X is or comprises an amino acid side chain, R is or comprises a lipid chain, Ar is a substituted or unsubstituted aryl or heteroaryl group, and Y is a C2-C 20 Alkylene or C2-C 20 Alkenylene, and Z is C4-C 40 Alkyl, C4-C 40 Alkenyl, or C4-C 40 Alkynyl.

[0037] In some embodiments, the remdesivir analog is a compound of Formula Ia:

[0038] wherein X is or comprises an amino acid side chain, and R is or comprises a lipid chain or a benzyl group.

[0039] In other embodiments, provided herein are compositions comprising a compound of Formula I or Formula II:

[0040]

[0041] wherein X is or comprises an amino acid side chain, R is or comprises a lipid chain or a benzyl group, Ar is a substituted or unsubstituted aryl or heteroaryl group, and Y is a C2-C 20 Alkylene or C2-C 20 Alkenylene, and Z is C4-C 40 Alkyl, C4-C 40 Alkenyl, or C4-C 40 Alkynyl.

[0042] In some embodiments, the remdesivir analog is a compound of Formula Ia:

[0043] wherein X is or comprises an amino acid side chain, and R is or comprises a lipid chain or a benzyl group.

[0044] In certain embodiments, each of the plurality of nanoparticles has a diameter of 50 to 200 nm. In other embodiments, in each nanoparticle, albumin forms a shell surrounding Redcievir or Redcievir analogs. In certain embodiments, the composition further comprises water (e.g., sterile saline). In other embodiments, the albumin is human serum albumin. In certain embodiments, the crystalline form of Redcievir or Redcievir analogs in the nanoparticles is an amorphous solid.

[0045] In some embodiments, the molar ratio of redcivir or redcivir analogs to lipids is from about 0.0001: 1 to about 0.5: 1 (e.g., 0.0001: 1 ... 0.01 ... 0.5: 1). In other embodiments, the molar ratio of redcivir or redcivir analogs to lipids is from about 0.01: 1 to about 0.5: 1. In other embodiments, the molar ratio of redcivir or redcivir analogs to albumin is from about 0.05: 1 to about 1: 1. In certain embodiments, each of the plurality of liposomes has a diameter of 50 to 200 nm (e.g., 50 ... 75 ... 125 ... 175 ... 200 nm). In some embodiments, the composition comprises a plurality of nanoparticles. In other embodiments, the composition comprises a plurality of liposomes.

[0046] Provided herein are compounds of Formula I:

[0047]

[0048] wherein X is or comprises an amino acid side chain, R is or comprises a lipid chain or a benzyl group, and Ar is a substituted or unsubstituted aryl or heteroaryl group.

[0049] In some embodiments, the compound is of Formula Ia:

[0050]

[0051] wherein X is or comprises an amino acid side chain, and R is or comprises a lipid chain or a benzyl group.

[0052] Also provided herein are compounds of Formula II:

[0053]

[0054] Where X is or contains an amino acid side chain, and Y is C2-C 20 Alkylene or C2-C 20 Alkenylene; Z is C4-C 40 Alkyl, C4-C 40 Alkenyl, or C4-C 40 alkynyl, and Ar is a substituted or unsubstituted aryl or heteroaryl.

[0055] In additional embodiments, X in Formula I, Ia, or II is or comprises an alanine side chain. In other embodiments, X in Formula I, Ia, or II is or comprises a tryptophan side chain. In additional embodiments, X in Formula I, Ia, or II is or comprises a phenylalanine side chain. In additional embodiments, X in Formula I, Ia, or II is or comprises an amino acid side chain selected from the group consisting of alanine, phenylalanine, valine, leucine, isoleucine, methionine, tryptophan, proline, glycine, cysteine, glutamine, asparagine, serine, tyrosine, and threonine.

[0056] In some embodiments, R in Formula I, Ia, or II is or comprises a lipid chain having 4 to 30 carbons. In additional embodiments, R in Formula I, Ia, or II is derived from a lipid chain lipid, a phospholipid, a sphingolipid, a diglyceride, a dialiphatic glycolipid, a sphingomyelin, a sphingolipid, a steroid lipid, a lipid derived from a hydrophilic polymer, a fatty alcohol, or a mixture thereof. Fatty alcohol includes, for example, 1) normal chain alcohols (saturated): ethanol, 1-propanol, 1-butanol, 1-hexanol, 1-pentanol, 1-heptanol, 1-octanol, 1-nonanol, 1-decanol, 1-undecanol, 1-dodecanol, 1-tridecanol, 1-tetradecanol, 1-pentadecanol, 1-hexadecanol, 1-heptadecanol, 1-octadecanol, 1-nonadecanol, 1-eicosanol, 1-di- Undecanol, 1-docosanol, 1-tricosanol, 1-tetracosanol, 1-hexacosanol, 1-octacosanol, 1-triacontanol; and (unsaturated) 3-butene-1-ol, crotyl alcohol (cis- and trans-), cis-2-penten-1-ol, 4-penten-1-ol, 2,4-hexadien-1-ol, cis-3-hexen-1-ol, trans-2-hexen-1 -ol, 5-hexen-1-ol, cis-2-hexen-1-ol, cis-4-hexen-1-ol, trans-2-hepten-1-ol, cis-4-hepten-1-ol, 6-hepten-1-ol, trans-2-octen-1-ol, cis-3-octen-1-ol, cis-5-octen-1-ol, 7-octen-1-ol, cis-6-nonen-1-ol, cis-2-nonen-1 Other alcohols include: -alcohol, trans-2-nonen-1-ol, cis-3-nonen-1-ol, 8-nonen-1-ol, 9-decen-1-ol, cis-4-decen-1-ol, trans-5-decen-1-ol, 10-undecen-1-ol, trans-2-tridecen-1-ol, linolenyl alcohol, oleyl alcohol, trans-9-octadecenol, trans-2-dodecenol, cis-13-docosenol. Others include: branched alcohols. Classes include tuberculinol; isopranols (such as tetrahydrogeraniol, hexahydrofarnesol, or phytanol); polyprenols (such as geraniol, farnesol, geranylgeraniol, or geranylfarnesol). Others also include phenolic alcohols. The categories include lignin monomers (such as coniferyl alcohol and sinapyl alcohol), phenol phthiocerol, and cyclic alcohols such as 3-(4-methylfuran-3-yl)propan-1-ol.

[0057] In some embodiments, Y is C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene, C9 alkylene, C 10 Alkylene, C 12 Alkylene, C 14 Alkylene, or C 16 Alkylene.

[0058] In some embodiments, Y is C2-C 10 In some embodiments, Y is a C4 alkylene group.

[0059] In some embodiments, Y is C2-C 10 In some embodiments, X is C2, C3, C4, C5, C6, C7, C8, C9, or C 10 Alkenylene.

[0060] In some embodiments, Z is C4-C 20 Alkyl or C4-C 20 In some embodiments, Z is C4, C5, C6, C7, C8, C9, C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19 , or C 20 In selected embodiments, Z is C4-C 10 In some embodiments, X is C4, C5, C6, C7, C8, C9, or C 10 alkyl.

[0061] In some embodiments, Z in Formula II is derived from a fatty acid having 4 to 30 carbon atoms. In additional embodiments, Z in Formula II is derived from a fatty acid moiety selected from the group consisting of: Fatty Acids: 1) Saturated Fatty Acids: Acetic Acid, Propionic Acid, Butyric Acid, Valeric Acid, Hexanoic Acid, Heptanoic Acid, Octanoic Acid, Nonanoic Acid, Decanoic Acid, Undecanoic Acid, Lauric Acid, Tridecanoic Acid, Myristic Acid, Pentadecanoic Acid, Palmitic Acid, Heptadecanoic Acid, Stearic Acid, Nonadecanoic Acid, Arachidic Acid, Heneicosanoic Acid, Behenic Acid, Tricosanoic Acid, Lignoceric Acid, Pentadecanoic Acid, Cerolic Acid, Heptacosanoic Acid, Octacosanoic Acid, Nonacosanoic Acid, and Melisic Acid. 2) Unsaturated fatty acids: crotonic acid, trans-2-pentenoic acid, trans-3-pentenoic acid, trans-2-hexenoic acid, trans-3-hexenoic acid, 2-heptenoic acid (including 3-heptenoic acid), 3-heptenoic acid, trans-2-octenoic acid, 3-octenoic acid, 2-nonenoic acid, 3-nonenoic acid, trans-2-decenoic acid, 3-decenoic acid, 4-decenoic acid, 2-undecenoic acid, 10-undecenoic acid, 2-tridecenoic acid, hexadecenoic acid, palmitic acid Palmitoleic acid, elaidic acid, petroselinic acid, oleic acid, ricinoleic acid, erucic acid, cis-15-tetracosenoic acid, sorbic acid, linoleic acid, linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, linolenic acid, arachidonic acid, EPA, DHA; 3) Fatty dicarboxylic acids: glutaric acid, adipic acid, suberic acid, azelaic acid, sebacic acid, 1,9-nonanedicarboxylic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid and nonadecanedioic acid.

[0062] In some embodiments, Ar is unsubstituted phenyl.

[0063] In some embodiments, Ar is substituted phenyl. In some embodiments, Ar is phenyl substituted with -COOM, wherein M is hydrogen or C1-C6 alkyl. In some embodiments, Ar is substituted with -NL 1 L 2 Substituted phenyl, wherein L 1 and L 2 In some embodiments, Ar is phenyl substituted with -COOM, wherein M is hydrogen or C1-C6 alkyl and -NL 1 L 2 , where L 1 and L 2 Each is independently selected from hydrogen and alkyl. In certain embodiments, Ar is phenyl substituted with -COOH and -NH2.

[0064] In certain embodiments, Ar is wherein M is hydrogen or C1-C6 alkyl. In certain embodiments, Ar is In certain embodiments, Ar is In certain embodiments, Ar is wherein M is hydrogen or C1-C6 alkyl, and L 1 and L 2 Each is independently selected from hydrogen and C1-C6 alkyl. In certain embodiments, Ar is In certain embodiments, Ar is

[0065] In some embodiments, Ar is unsubstituted naphthyl.

[0066] In certain embodiments, the plurality of microparticles each have a diameter of 5 to 30 μm (e.g., 5 ... 10 ... 15 ... 25 ... or 30 μm). In other embodiments, the composition comprises a plurality of microparticles and is in the form of a dry powder. In specific embodiments, the composition comprises a plurality of microparticles, and the microparticles are: solid microparticles, porous microparticles, or microencapsulated nanoparticles. In some embodiments, the composition comprises a plurality of liposomes. In other embodiments, the composition comprises a plurality of microparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Shown is the chemical structure of GS-5734 (Remdesivir) and its metabolic conversion to NTPs.

[0068] Figure 2 Microencapsulated nanoparticles showing remdesivir and new analogs for dry powder inhalation.

[0069] Figure 3 Exemplary remdesivir analogs are shown.

[0070] Figure 4 Shown are exemplary synthetic routes for the remdesivir analogs of Formula Ia disclosed herein.

[0071] Figure 5 Shown are exemplary synthetic routes for the remdesivir analogs of Formula II disclosed herein.

[0072] Figure 6 Shown are exemplary synthetic routes for the remdesivir analogs of Formula I disclosed herein.

[0073] Figure 7 is a graph of the stability of the synthesized compounds in human, hamster, and mouse plasma.

[0074] Figure 8A and 8B is a diagram of the screening and selection of compounds targeting the lung.

[0075] Figures 9A-9H This is a diagram of the tissue targeting of the two agents, MMT5-14 and remdesivir.

[0076] Figures 10A-10FComparison of AUC showing tissue targeting of two formulations, MMT5-14 and remdesivir.

[0077] Figure 11A and 11B Showing a fold increase in tissue targeting for both agents, MMT5-14 and remdesivir.

[0078] Figure 12 Showing the cellular uptake of both formulations, MMT5-14 and remdesivir.

[0079] definition

[0080] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of a conflict, this document (including definitions) shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used for practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are merely exemplary and are not intended to be limiting.

[0081] As used herein, the terms "comprises," "including," "having," "containing," "may," "include," and variations thereof are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates additional embodiments that "comprise, consist of, and consist essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0082] For the recitation of numerical ranges herein, each intervening number with the same degree of precision is expressly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0083] As used herein, the terms "host," "subject," and "patient" refer to any animal that is studied, analyzed, tested, diagnosed, or treated, including but not limited to humans and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, etc.). Unless otherwise indicated, as used herein, the terms "host," "subject," and "patient" are used interchangeably. In certain embodiments, the subject is a human (e.g., a human with a viral infection such as a COVID-19 infection).

[0084] Definitions of specific functional groups and chemical terms are described in more detail below.For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, on the inside cover of the Handbook of Chemistry and Physics, 75th edition, and specific functional groups are generally defined as described therein.

[0085] Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th ed., John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd ed., Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0086] As used herein, the term "aliphatic" or "aliphatic group" refers to a hydrocarbon moiety that can be straight (i.e., non-branched), branched, or cyclic (including fused, bridged, and spirofused polycyclics), and can be fully saturated, or can contain one or more unsaturated units but is not aromatic. Unless otherwise indicated, an aliphatic group contains 1 to 30 carbon atoms. In certain embodiments, an aliphatic group contains 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Suitable aliphatic groups include, but are not limited to, straight or branched alkyl, alkenyl, and alkynyl groups, and their hybrids, such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl, or (cycloalkyl) alkenyl.

[0087] As used herein, the term "alkyl" refers to a straight or branched saturated hydrocarbon chain. For example, C4-C 40Alkyl means a straight or branched saturated hydrocarbon chain containing 4 to 40 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 4,4-dimethylpent-2-yl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0088] As used herein, the term "alkenyl" means a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond.

[0089] As used herein, the term "alkynyl" means a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond.

[0090] As used herein, the term "alkenylene" refers to a divalent group derived from a straight or branched chain hydrocarbon having at least one carbon-carbon double bond.

[0091] As used herein, the term "alkylene" refers to a divalent group derived from a straight-chain or branched saturated hydrocarbon of 1 to 20 carbon atoms.

[0092] As used herein, the term "amino acid side chain" refers to a moiety attached to the α-carbon (or another backbone atom) of an amino acid or amino acid residue. For example, the amino acid side chain of alanine is a methyl group, the amino acid side chain of phenylalanine is a benzyl group, the amino acid side chain of cysteine ​​is a thiomethyl group, the amino acid side chain of aspartic acid is a carboxymethyl group, and the amino acid side chain of tyrosine is a 4-hydroxyphenylmethyl group.

[0093] As used herein, the term "aryl" refers to an aromatic carbocyclic ring system having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic) (including fused ring systems) and zero heteroatoms. As used herein, the term "aryl" contains 6-20 carbon atoms (C6-C 20 aryl), 6 to 14 ring carbon atoms (C6-C 14 aryl), 6 to 12 ring carbon atoms (C6-C 12 aryl), or 6 to 10 ring carbon atoms (C6-C 10 Representative examples of aryl include, but are not limited to, phenyl, naphthyl, anthracenyl, and phenanthrenyl.

[0094] As used herein, the term "benzyl" refers to a group having the structure C6H5CH2-.

[0095] As used herein, the term "fatty acid chain" refers to a molecule, ester or residue derived from a triglyceride or phospholipid and consisting of a carboxylic acid with a saturated or unsaturated long aliphatic tail (chain). The fatty acid chain can have any degree of unsaturation and be in any position within the tail. Most naturally occurring fatty acids have chains with an even number of carbon atoms (generally greater than 4 carbon atoms). Short-chain fatty acids (SCFA) are fatty acids with an aliphatic tail less than six carbons. Medium-chain fatty acids (MCFA) are fatty acids with an aliphatic tail of 6-12 carbons, which can form medium-chain triglycerides. Long-chain fatty acids (LCFA) are fatty acids with an aliphatic tail of 13 to 21 carbons. Very long-chain fatty acids (VLCFA) are fatty acids with an aliphatic tail longer than 22 carbons. In some embodiments, the fatty acid chain, or ester or residue thereof, may contain 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 carbon atoms, where any of the stated values ​​may form upper or lower endpoints as appropriate.

[0096] As used herein, the term "heteroaryl" refers to an aromatic monocyclic or aromatic bicyclic ring system. An aromatic monocyclic ring is a five- or six-membered ring containing at least one heteroatom independently selected from N, O, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). A five-membered aromatic monocyclic ring has two double bonds, and a six-membered aromatic monocyclic ring has three double bonds. An example of a bicyclic heteroaryl is a monocyclic heteroaryl ring attached to a parent molecular moiety and fused to a monocyclic cycloalkyl as defined herein, a monocyclic aryl as defined herein, a monocyclic heteroaryl as defined herein, or a monocyclic heterocycle as defined herein. Representative examples of heteroaryl include, but are not limited to, indolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothiophenyl, benzofuranyl, isobenzofuranyl, furyl, oxazolyl, isoxazolyl, purinyl, isoindolyl, quinoxalinyl, indazolyl, quinazolinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, isoquinolyl, quinolyl, 6,7-dihydro-1,3-benzothiazolyl, imidazo[1,2-a]pyridinyl and naphthyridinyl.

[0097] As used herein, the term "lipid chain" refers to a saturated or unsaturated hydrocarbon chain derived from the hydrophobic tail of a lipid, such as an alkyl, alkenyl or alkynyl chain, as described elsewhere herein. The lipid chain can be derived from a lipid derived from a dialiphatic chain lipid, a phospholipid, a diglyceride, a dialiphatic glycolipid, a sphingomyelin, a sphingolipid, a steroidal lipid or a hydrophilic polymer. In some embodiments, the lipid chain comprises 4 to 40 carbons, such as 4 to 20 carbons, 10 to 40 carbons, 10 to 30 carbons, 10 to 20 carbons or 20 to 30 carbons.

[0098] The term "substituted" refers to a group that can be further substituted by one or more substituents. Substituents include, but are not limited to, halogen, =O (oxo), =S (thio), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl. For example, if a group is described as "optionally substituted" (such as alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heteroalkyl, heterocycle, or other groups such as R groups), it can have 0, 1, 2, 3, 4, or 5 substituents independently selected from halogen, =0 (oxo), =S (thio), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0099] In some cases, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl and alkenyl) is indicated by the prefix "C x -C y ” indicates an alkyl group containing from 1 to 3 carbon atoms, where x is the minimum number of carbon atoms in the substituent and y is the maximum number. Thus, for example, “C1-C3 alkyl” refers to an alkyl substituent containing from 1 to 3 carbon atoms.

[0100] For the compounds described herein, radicals and substituents may be chosen based on the allowed valences of the atoms and substituents such that selection and substitution results in stable compounds, eg, which do not spontaneously undergo transformations such as by rearrangement, cyclization, elimination, or the like.

[0101] When substituents are specified by their conventional chemical formula written from left to right, such formula also encompasses the same substituents resulting from writing the structure from right to left. For example, -CH2NH- is also intended to encompass -NHCH2-. DETAILED DESCRIPTION

[0102] The present invention relates to methods and compositions for treating viral infections (including COVID-19 viral infections). In certain embodiments, the composition comprises: i) a remdesivir analogue with tissue targeting and antiviral activity (including inhibition of viral entry and viral replication), ii) remdesivir or a remdesivir analogue, and a surfactant, cyclodextrin, or a combination thereof, iii) nanoparticles comprising albumin and remdesivir or a remdesivir analogue, iv) liposomes comprising lipids and remdesivir or a remdesivir analogue; and / or v) microparticles comprising PLA and / or PLGA, and remdesivir or a remdesivir analogue. In certain embodiments, the composition is aqueous (e.g., for intravenous administration (iv)). In other embodiments, the composition is atomized or in dry powder form (e.g., for inhalation by an infected subject).

[0103] The prodrug remdesivir is unstable and rapidly hydrolyzes into nucleoside monophosphate (Nuc-MP) [10,11] Only the prodrug, remdesivir, has the potential to distribute and penetrate tissues. However, nucleoside monophosphates (Nuc-MPs) are charged and polar in plasma and therefore cannot penetrate membranes into cells to demonstrate efficacy (unless they are taken up by an unknown transporter, which is unlikely as Nuc-MPs have not shown activity).

[0104] When remdesivir was administered intravenously to monkeys (10 mg / kg, equivalent to 100-200 mg in humans), the plasma concentration of remdesivir was a few micromolar and rapidly decreased to below 0.5 μM within 30 minutes. [2] Plasma nucleoside monophosphate (Nuc-MP) concentrations are approximately 0.5-1 μM over 12-24 hours. The plasma exposure of both the prodrug remdesivir and nucleoside monophosphate (Nuc-MP) misleadingly predicts their efficacy.

[0105] The active metabolite, nucleoside triphosphate (Nuc-TP), can only be formed intracellularly by nucleoside phosphokinase and is not detectable in plasma but can only be detected at high concentrations (30-40 μM) in blood cells (such as PBMCs). [2] This could indeed help suppress the COVID-19 virus in PBMCs (or perhaps even in lymphocytes) since COVID-19 patients do have lower lymphocyte counts. [4]However, the high intracellular concentration of Nuc-TP in PBMCs could not be translated into high intracellular concentration in lung cells.

[0106] Treatment of COVID-19-induced pneumonia requires drug accumulation in the lungs, especially in the alveoli where the virus primarily attacks, so low lung accumulation of intravenously administered remdesivir will limit its efficacy. [2,10] . However, any polar molecule will have high plasma exposure (high concentrations and AUC), but based on the mass balance principles of pharmacokinetics, it may have very limited tissue exposure. This may be the reason why many polar molecules have high plasma exposure and good safety properties, because they do accumulate in organs. In this case, high plasma exposure (low tissue exposure) may lead to poor efficacy. This phenomenon is often overlooked during drug discovery and development, where high plasma exposure is often used as a good surrogate for tissue exposure and they are selected as good drug candidates for efficacy studies. Although plasma exposure is a good predictor for many drugs, this is not the case for many other cases (perhaps more than 30% of cases, as seen for remdesivir).

[0107] Preclinical data in mice (25-50 mg / kg) and monkeys (10 mg / kg), which is equivalent to a 100-200 mg dose in humans, did not achieve measurable concentrations of remdesivir in the lungs. [2,3] , while the active metabolite nucleoside triphosphate (Nuc-TP) was only detected in the lung at low concentrations (0.8-1.5 μM in monkey lungs and ~3 μM in mouse lungs) [2,3] These low concentrations in the lung are expected based on the physiochemical properties of remdesivir and its stability in plasma, raising questions about the efficacy of remdesivir in treating COVID-19 patients with mild / severe / critical pneumonia.

[0108] The inactive prodrug Remdesivir needs to enter the intracellular lung cells and form the active metabolite nucleoside triphosphate (Nuc-TP) to obtain its antiviral activity. Therefore, the newly formed Nuc-TP is negatively charged and accumulates inside the cell, with a concentration >25-100 times higher than the extracellular concentration of Remdesivir. This phenomenon has been confirmed in a variety of human and rhesus monkey cells (PBMC, monocytes, Hela, microphages and HUVEC). [2] In another study using B24 cells, intracellular Nuc-TP accumulated more than three times as much as extracellular remdesivir.

[10] On average, it is reasonable to assume that intracellular Nuc-TP accumulates more than 10-fold more than extracellular remdesivir. Therefore, the in vitro EC of remdesivir against COVID-19 virus cell lines 50 (0.77 μM) and EC 90 (1.76μM) [1]At least equivalent to the EC of intracellular Nuc-TP against COVID-19 virus 50 (>7.7 μM) and EC 90 (>17.6μM).

[0109] However, after intravenous injection of remdesivir (10 mg / kg) in marmosets (equivalent to 100-200 mg in humans), remdesivir was undetectable in the lungs, and the concentration of Nuc-TP in the lungs was 0.8-1.5 μM. [3] If the lung Nuc-TP / plasma remdesivir concentration ratio is assumed to be the same between monkeys and humans, a single intravenous dose of 200 mg remdesivir would only achieve a concentration of Nuc-TP in the lung (2-4 μM), where the intracellular concentration of Nuc-TP in the lung may be only 4-8 μM, which is lower than the estimated EC50 of Nuc-TP for inhibition of COVID-19. 50 and EC 90 Unfortunately, systemic side effects such as hepatotoxicity have prevented intravenous dose escalation of remdesivir to >200 mg / day.

[0110] In addition to suppressing the virus in the lungs of COVID-19, it may also be necessary to consider suppressing the virus in lymphocytes, lymph nodes, and spleens. In fact, most viruses typically attack these lymphoid organs as well. In fact, COVID-19 patients often have low lymphocyte counts and significantly smaller spleens. [4] In certain embodiments, remdesivir is delivered to these lymphoid organs (in addition to the lungs) to achieve its efficacy or better efficacy against COVID-19. Based on the physiochemical properties of remdesivir, low drug exposure in these lymphoid organs can be expected. It is also unclear whether high concentrations of nucleoside triphosphates (Nuc-TP) in PBMCs [2] Whether it can be transformed into high concentrations in lymphocytes, lymph nodes or spleen.

[0111] Provided herein are methods, compounds, compositions, and systems for improving the efficacy of remdesivir. In certain embodiments, remdesivir nanoformulations are generated for intravenous infusion to improve drug delivery to the lungs, spleen, and lymph nodes. Specific nanoformulations, such as albumin nanoparticles and liposomes, can be used to enhance drug exposure in the lungs, lymph nodes, and spleen to improve efficacy in patients with COVID-19 or other respiratory viral diseases.

[0112] Nanoparticles (such as liposomes, albumin nanoparticles, PLGA) form a stable shell around the drug core, which can protect the drug from degradation by enzymes in water or in the body

[16] Different nanoformulations will have different abilities to improve drug exposure in the lungs, spleen, and lymph nodes [12,13]The spleen and lungs are part of the reticuloendothelial system, where nanoparticles naturally accumulate.

[17] Previous studies have also demonstrated that nanomedicine can enhance lymphatic drug delivery to treat metastatic tumors.

[18] .

[0113] Compared with intravenous infusion, pulmonary delivery of remdesivir provides higher lung concentrations of remdesivir and Nuc-TP and reduces systemic toxicity. The total volume of human lung tissue is 1.17 L, and the total intracellular fluid volume in the lung is 0.54 L (46%)

[19] In vitro metabolism data showed that approximately 55% of remdesivir was converted into Nuc-TP in human cells, and other metabolites accounted for 45%. [2] To achieve an intracellular Nuc-TP concentration of 17.6 μM in human lung (in vitro EC 90 ), the total amount of remdesivir to be delivered to lung cells is about 17.6 μM * 0.54 L ÷ 55% * 603 g / mole = 10.5 mg. If it is assumed that 50% of remdesivir is swallowed during inhalation and the remaining 50% of the dose is delivered to the lungs, a daily inhaled dose of about 21 mg of remdesivir is expected to achieve an EC of Nuc-TP intracellularly in human lungs. 90 This dose can be achieved by inhalation.

[0114] Pulmonary drug delivery using devices such as metered-dose inhalers, nebulizers, and dry powder inhalers provides higher lung concentrations and reduced systemic toxicity compared with intravenous infusion, thus facilitating targeted treatment of respiratory diseases.

[20] Some clinicians have proposed nebulized therapy to deliver drugs directly to the lungs, but due to the instability of remdesivir in its aqueous phase, aerosol inhalation of aqueous solutions is not feasible because once remdesivir is hydrolyzed into nucleoside monophosphate (Nuc-MP), it is more difficult for it to enter cells to maximize its efficacy.

[0115] This article provides at least two methods for solving this problem. In certain embodiments, dry powder inhalation is used, wherein about 2.5um redciwei crystalline form can be mixed with lactose of different sizes (15um and 200um) for preparation and manufacture. Another solution is to prepare nanoformulations to protect redciwei from degradation in aqueous phase (such as liposomes, albumin nanoparticles, or PLGA nanoparticles). Nanoformulations can be used as a nebulized inhalation of an aqueous solution or as 2.5um nanoparticle aggregates in dry powder inhalation.

[0116] Previous results showed that nanomedicine can protect drugs from degradation in aqueous phase

[21] In addition, the nanoformulation method has unique advantages over the traditional microparticle form of drugs: (1) Increased solubility and stability of drugs in lung secretions

[22] ; (2) leading to uniform distribution of drugs in the lungs and longer retention time

[22] (3) Promotes drug penetration through the lung surfactant (PS) layer, which is a strong barrier that prevents drugs or particles from entering the alveoli

[23] Amikacin Liposomal Inhalation Suspension In 2018, it was approved by the FDA for the treatment of Mycobacterium avium complex (MAC) lung disease.

[24] .

[0117] I. Albumin Nanoformulation of Remdesivir for Intravenous Injection

[0118] In certain embodiments, in order to improve the tissue distribution of remdesivir or its analogs (especially in the lungs, spleen and lymph nodes), injectable albumin nanoformulations of remdesivir or remdesivir analogs are used to treat COVID-19 infection and other viral infections. The preparation contains albumin and drugs in different ratios. An exemplary manufacturing method is shown in Example 1 below. In some embodiments, the size of the nanoparticles is adjustable in the range of 50 to 200 nm with a narrow particle size distribution (PDI <0.15). In certain embodiments, the nanoformulation achieves a higher encapsulation efficiency (>90%) and good stability. The product is usually in the form of a lyophilized powder.

[0119] Nanoformulations of albumin and remdesivir (or its analogs) may offer improved properties. First, albumin nanoparticles exhibit extremely rapid tissue distribution and high volume distribution.

[26] More importantly, albumin nanoparticles can increase lung accumulation and permeation of encapsulated drugs

[12] . Secondly, the stable albumin nanoparticles of the drug increase the accumulation in the spleen and lymph nodes. Third, the albumin nanoformulation can form a stable albumin shell on the outside of the drug, which can prevent the drug from being hydrolyzed by various enzymes in the blood. As the stability of the drug increases, the total amount of drug distributed to the tissue (especially the lungs) increases. Fourth, compared with free drugs, albumin nanoparticles reduce blood drug concentrations, thereby reducing the uptake of drugs by blood cells to further increase the amount of drugs distributed to the tissues.

[0120] II. Liposomal Nanoformulation of Remdesivir for Intravenous Injection

[0121] In certain embodiments, injectable liposomal nanoformulations of remdesivir or its analogs are used to treat COVID-19 infection and other viral infections. In certain embodiments, such formulations are composed of one or more vesicle-forming lipids selected from the following: di-aliphatic chain lipids, such as phospholipids; diglycerides; di-aliphatic glycolipids; single lipids, such as sphingomyelin or glycosphingolipids; steroidal lipids; lipids derived from hydrophilic polymers, or mixtures thereof. In some embodiments, the one or more vesicle-forming lipids used in the liposomes prepared according to the methods of the present invention are present in an amount such that the molar ratio of ionizable drug to vesicle-forming lipid is in the range of about 0.0001:1 to about 0.5:1, preferably about 0.01:1 to about 0.5:1. In certain embodiments, liposomal nanoformulations for remdesivir (or its analogs) are prepared by various methods, including: 1) hydrating the lipid film using ultrasonic treatment or extrusion to optimize the particle size distribution; 2) solvent evaporation, such as ethanol injection, ether injection, reverse phase evaporation; or 3) detergent removal methods, by combining the drug and lipid before forming the vesicle (passive drug loading technology), or by "loading" the drug into the lipid vesicle after the lipid vesicle is formed (active drug loading technology), Remdesivir or the like can be encapsulated into the liposome. In certain embodiments, the liposomes prepared by the method of the present invention are unilamellar liposomes with a size of 50nm to about 200nm. In some embodiments, after intravenous injection, the liposomal formulation prolongs the circulation time. The liposomal formulation increases the stability of the drug and prevents it from degrading in the blood. In certain embodiments, the liposomal formulation changes the mass balance of the drug biodistribution and increases the drug distribution in the lungs and spleen

[27] .

[0122] III. Albumin and Liposomal Nanoformulations of Remdesivir for Nebulized Inhalation

[0123] Compared with other routes of administration, aerosol inhalation has multiple advantages in the treatment of respiratory diseases. However, due to its instability in water, Redcievir cannot be used for aerosol treatment. In certain embodiments, albumin or liposome nanoformulations are used to form a stable shell outside the Redcievir (or analog) drug core, thereby preventing Redcievir or analogs from hydrolyzing in aqueous media. In some embodiments, the albumin nanoformulation encapsulates Redcievir or analog drug amorphous form inside and stabilizes the form so that the preparation does not require a crystalline form of the drug. In certain embodiments, for aerosol delivery, the size of the albumin or liposome nanoparticles can be adjusted from 20-1000nm.

[0124] In certain embodiments, in order to prepare a liquid aerosol for use in a nebulizer, freeze-dried albumin or liposome preparation powder is resuspended with medical saline, and the concentration of the compound should be adjusted based on the inhalable dose. In certain embodiments, the drug (remdesivir or the like) is evenly distributed in the aerosol droplets. In some embodiments, aerosol inhalation allows drug particles to disperse in the lungs, and then releases nanoparticles from the microparticles in the lungs for better alveolar accumulation and longer lung retention. In specific embodiments, aerosol inhalation of nanoformulations reduces premature mucociliary clearance of hydrophobic drugs and increases absorption, and / or aerosol inhalation increases cellular uptake by lung cells. In some embodiments, aerosol inhalation prolongs the retention of the drug in the lungs. [28,29] Muralidharan, P. et al. (Nanomedicine, 2015.11(5): p. 1189-99) provide guidance for other drugs made into nanoformulations for dry powder inhalers. In certain embodiments, aerosol inhalation directly delivers the drug to the lungs to achieve high local concentrations with lower doses, but reduces systemic drug exposure to reduce liver toxicity. In some embodiments, aerosol inhalation using liposomes that encapsulate the drug in a lipid bilayer or inside a liposome increases stability and solubility. In other embodiments, aerosol inhalation using a liposomal formulation provides sustained drug release characteristics and prolongs the retention of the drug in the lungs.

[0125] IV. Remdesivir for Dry Powder Inhalation

[0126] Generally speaking, the preferred size of particles for inhalation is about 2.5 μm (e.g. 1-5 μm). Particles smaller than 0.5 μm cannot be deposited in the lungs at all because they can be easily exhaled.

[30] . The preparation of remdesivir or its analogs for dry powder inhalation can be performed as follows. First, a crystalline form of remdesivir (or an analog) is selected and ground to generate microparticles (e.g., 0.5-5um). The drug microparticles (0.5-5um) are mixed with excipients (such as lactose, mannitol, and PVA) of different sizes (~200um, <15um) to form micronized particles. Secondly, dry powder inhalation delivers the drug directly to the lungs to achieve high local concentrations with lower doses, but reduces systemic drug exposure to reduce liver toxicity.

[0127] V. Albumin and Liposomal Nanoformulations of Remdesivir for Dry Powder Inhalation

[0128] It is difficult to use dry powder inhalation directly because nanoparticles have a tendency to agglomerate due to the huge increase in Gibbs free energy caused by the large surface area. Moreover, the preferred size of particles for inhalation is about 2.5 μm; particles smaller than 0.5 μm cannot be deposited in the lungs at all because they can be easily exhaled.

[30] Therefore, in some embodiments, albumin or liposome microencapsulated nanopowder of Remdesivir (or its analogs) is constructed into a nanopowder for inhalation (e.g. Figure 2 As shown). First, albumin or liposome nanoparticles of remdesivir (or the like) are prepared as described herein. Any method can be used to convert nanoparticles into microparticles. One is to use a freeze-dried powder cake to generate microencapsulated nanoparticles (0.5-5um). Another method is to convert a nanosuspension in water into a respirable microaggregate via techniques such as spray drying and spray freeze drying (0.5-5um). The microencapsulated nanoparticles (0.5-5um) are then mixed with excipients (such as lactose, mannitol, and PVA) of different sizes (~200um, <15um) to form micronized particles. Secondly, the micronized powder allows the drug particles to disperse in the lungs, and then releases the nanoparticles from the microparticles in the lungs for better alveolar accumulation and longer lung retention. In some embodiments, nanoformulations for dry powder inhalation reduce premature mucociliary clearance of hydrophobic drugs and increase absorption. In specific embodiments, nanoformulations for dry powder inhalation increase cellular uptake of lung cells. In other embodiments, nanoformulations for dry powder inhalation prolong drug retention in the lungs [28,29] A variety of drugs have been formulated into nanoformulations for use as dry powder inhalers.

[28] . In some embodiments, nano drug powder inhalation delivers the drug directly to the lungs to achieve high local concentrations with lower doses, but reduces systemic drug exposure to reduce liver toxicity. In specific embodiments, albumin nanoparticle inhalation encapsulates the amorphous form of the drug inside, which increases the solubility and stability of the drug. This also eliminates the need for a crystalline form of the drug. In certain embodiments, liposome formulation inhalation encapsulates the drug in or inside a lipid bilayer to increase stability and solubility. In some embodiments, liposome formulation inhalation provides sustained drug release characteristics to prolong the retention of the drug in the lungs.

[0129] VI. PLA / PLGA Microparticles of Remdesivir for Dry Powder Formulation

[0130] Due to their unique biocompatibility and versatility, poly(lactic acid) (PLA) or poly(lactic-co-glycolic acid) (PLGA) particles have been shown to be excellent carriers for drugs, and the number of commercial products for their use in drug delivery systems is increasing. Therefore, in certain embodiments, respirable PLA / PLGA microparticles (MPs) incorporating microparticles, larger porous microparticles (LMPs), and microencapsulated nanoparticles are prepared by various methods such as single / double emulsion-solvent evaporation technology, spray drying, spray freeze drying, supercritical fluid drying, and nanoprecipitation [31, which is incorporated herein by reference in its entirety, particularly with respect to the microparticle form]. The larger porous microparticles (LMPs) exhibit a larger geometric diameter (5-30 μm), a low density (<0.4 g / cm3 ) and an acceptable aerodynamic diameter (1-3 μm), which is important for improving deep lung localization and avoiding macrophage clearance

[31] In certain embodiments, the microencapsulated nanoparticles (nanocomposites) are designed to release the primary NPs from the inert microcarriers into the lung lining fluid after reaching the alveolar surface, which prolongs the retention of the drug (remdesivir or an analogue) in the lung.

[0131] VII. Modification of Remdesivir to Alter Tissue Targeting and Achieve Higher Encapsulation in Nanoformulations

[0132] Provided herein are remdesivir analogs of compounds of Formula I or II:

[0133]

[0134] wherein X is or comprises an amino acid side chain, R is or comprises a lipid chain or a benzyl group, Ar is a substituted or unsubstituted aryl or heteroaryl group, and Y is a C2-C 20 Alkylene or C2-C 20 Alkenylene; and Z is C4-C 40 Alkyl, C4-C 40 Alkenyl, or C4-C 40 Alkynyl.

[0135] In some embodiments, Ar is substituted or unsubstituted phenyl or naphthyl.

[0136] In some embodiments, Ar is unsubstituted phenyl. In some embodiments, Ar is substituted phenyl. In some embodiments, Ar is phenyl substituted with -COOM, where M is hydrogen or C1-C6 alkyl. In some embodiments, Ar is substituted with -NL 1 L 2 Substituted phenyl, wherein L 1 and L 2 In some embodiments, Ar is phenyl substituted with -COOM, wherein M is hydrogen or C1-C6 alkyl and -NL 1 L 2 , where L 1 and L 2 Each is independently selected from hydrogen and alkyl. In certain embodiments, Ar is phenyl substituted with -COOH and -NH2.

[0137] In certain embodiments, Ar is wherein M is hydrogen or C1-C6 alkyl. In certain embodiments, Ar is In certain embodiments, Ar is In certain embodiments, Ar is wherein M is hydrogen or C1-C6 alkyl, and L 1 and L 2 Each is independently selected from hydrogen and C1-C6 alkyl. In certain embodiments, Ar is In certain embodiments, Ar is

[0138] In some embodiments, Ar is unsubstituted naphthyl.

[0139] In some embodiments, the remdesivir analog is a compound of Formula Ia:

[0140] wherein X is or comprises an amino acid side chain, and R is or comprises a lipid chain or a benzyl group.

[0141] In some embodiments, X is or comprises an amino acid side chain selected from the group consisting of alanine, phenylalanine, valine, leucine, isoleucine, methionine, tryptophan, proline, glycine, cysteine, glutamine, asparagine, serine, tyrosine, and threonine. In some embodiments, X is or comprises an alanine side chain. In some embodiments, X is or comprises a tryptophan side chain. In some embodiments, X is or comprises a phenylalanine side chain.

[0142] In some embodiments, R comprises a lipid chain having 4 to 30 carbons. In some embodiments, the lipid chain comprises 10 to 20 carbons. In specific embodiments, the lipid chain comprises 12 carbons. In exemplary embodiments, the lipid chain comprises n-dodecane.

[0143] In some embodiments, R comprises a lipid chain having 18 carbons. In some embodiments, the lipid chain is

[0144] In some embodiments, R is benzyl.

[0145] In some embodiments, Y is C2-C 10 In some embodiments, Y is C4 alkylene. In some embodiments, Y is C2-C 10 Alkenylene.

[0146] In some embodiments, Z is C4-C 20 Alkyl or C4-C 20 In selected embodiments, Z is C4-C 10 alkyl.

[0147] The following describes the synthesis of remdesivir analogs by adjusting their proteinaceous portion to achieve higher encapsulation in albumin and liposome nanoparticles. Because the proteinaceous portion is cleaved intracellularly to release nucleoside monophosphates, which are subsequently activated after phosphorylation to nucleoside triphosphates, modifications to the proteinaceous portion should not alter the activity of remdesivir.

[0148] The following formula Ia shows the general analog structure:

[0149] wherein R is a lipid chain and X is an amino acid side chain.

[0150] A more specific structure is shown in e.g. Figure 3 are shown in Series 1-3 and described further below.

[0151] Series 1. The first series of analogs were designed by retaining L-alanine and replacing only the 2-ethylbutyl group with lipid chains of various lengths (see Figure 3 ). First, it was found that Remdesivir (Sp isomer) and its Rp isomer were similar in potency, and the Sp isomer was chosen based on its crystalline properties that allowed for rapid scalability in synthesis. Therefore, this stereocenter was not taken into consideration in the design, and the analogs presented herein are mixtures of two diastereomers (Sp, Rp)

[25] Second, the 2-ethylbutyl group is replaced with a lipid chain for at least one of the following reasons: i) to increase lipophilicity for better permeability, thereby enhancing cellular uptake; ii) to enhance drug tissue distribution, particularly in the lungs, spleen, and lymph nodes; iii) to enhance encapsulation efficiency by albumin nanoparticles; iv) to introduce the drug into the liposome bilayer; or v) so that the lipid chain can form micelles to protect the drug from nonspecific hydrolysis in the bloodstream. However, once the drug enters the cell, it can be readily activated by intracellular hydrolases.

[0152] Series 2. A second series of analogs was designed by replacing L-alanine with N-methyl-D-tryptophan and replacing the 2-ethylbutyl group with various lipid chains (see Figure 3 ). First, it was found that Remdesivir (Sp isomer) and its Rp isomer were similar in potency, and the Sp isomer was chosen based on its crystalline properties that allowed for rapid scalability in synthesis. Therefore, this stereocenter was not taken into consideration in the design, and the analogs presented herein are mixtures of two diastereomers (Sp, Rp)

[25] In certain embodiments, the 2-ethylbutyl group is replaced with a lipid chain: i) to increase lipophilicity for better permeability, thereby enhancing cellular uptake; ii) to enhance drug tissue distribution, particularly in the lungs, spleen, and lymph nodes; iii) to enhance encapsulation efficiency by albumin nanoparticles; iv) to introduce the drug into the liposome bilayer; or v) to allow the lipid chain to form micelles to protect the drug from nonspecific hydrolysis in the bloodstream. In certain embodiments, alanine is replaced with N-methyl-D (or L) -tryptophan: i) to enhance encapsulation efficiency by albumin nanoparticles; ii) to increase lipophilicity for better permeability, thereby enhancing cellular uptake, to enhance drug tissue distribution, particularly in the lungs, spleen, and lymph nodes; iii) to introduce the drug into the liposome bilayer; or iv) to enhance immune cell function to improve the antiviral efficacy of the drug.

[0153] Series 3. A third series of analogs was designed by replacing L-alanine with L(or D)-phenylalanine and replacing the 2-ethylbutyl group with various lipid chains (see Figure 3 ). First, it was found that Remdesivir (Sp isomer) and its Rp isomer were similar in potency, and the Sp isomer was chosen based on its crystalline properties that allowed for rapid scalability in synthesis. Therefore, this stereocenter was not taken into consideration in the design, and the analogs presented herein are mixtures of two diastereomers (Sp, Rp)

[25] In certain embodiments, the 2-ethylbutyl group is replaced with a lipid chain: i) to increase lipophilicity for better permeability, thereby enhancing cellular uptake; ii) to enhance drug tissue distribution, particularly in the lungs, spleen, and lymph nodes; iii) to enhance encapsulation efficiency by albumin nanoparticles; iv) to introduce the drug into the liposomal bilayer; or v) to allow the lipid chain to form micelles to protect the drug from nonspecific hydrolysis in the bloodstream. In certain embodiments, alanine is replaced with L (or D) -phenylalanine: i) to enhance encapsulation efficiency by albumin nanoparticles; ii) to increase lipophilicity for better permeability, thereby enhancing cellular uptake; iii) to enhance drug tissue distribution, particularly in the lungs, spleen, and lymph nodes; iv) to introduce the drug into the liposomal bilayer.

[0154] The following formula II shows the general analog structure:

[0155]

[0156] Where X is an amino acid side chain and Y is a C2-C 20 Alkylene or C2-C 20 Alkenylene; Ar is substituted or unsubstituted aryl or heteroaryl; and Z is C4-C 40 Alkyl, C4-C 40 Alkenyl, or C4-C 40A more specific structure is shown in Figure 3 are shown in Series 4-6 and described further below.

[0157] Series 4. Using aliphatic diols as the linker between the fatty acid and alanine, a fourth series of analogs was designed by retaining L-alanine and replacing the 2-ethylbutyl group with fatty acid chains of various lengths (see Figure 3 ). First, it was found that Remdesivir (Sp isomer) and its Rp isomer were similar in potency, and the Sp isomer was chosen based on its crystalline properties that allowed for rapid scalability in synthesis. Therefore, this stereocenter was not taken into consideration in the design, and the analogs presented herein are mixtures of two diastereomers (Sp, Rp)

[25] Second, the 2-ethylbutyl group is replaced with a fatty acid chain for at least one of the following reasons: i) to increase lipophilicity for better permeability, thereby enhancing cellular uptake; ii) to enhance drug tissue distribution, particularly in the lungs, spleen, and lymph nodes; iii) to enhance encapsulation efficiency by albumin nanoparticles; iv) to introduce the drug into the liposome bilayer; or v) so that the fatty acid chain can form micelles to protect the drug from nonspecific hydrolysis in the bloodstream. However, once the drug enters the cell, it can be readily activated by intracellular hydrolases.

[0158] Series 5. Using aliphatic diols as linkers between fatty acids and N-methyl-D-tryptophan, a fifth series of analogs was designed by replacing L-alanine with N-methyl-D-tryptophan and replacing the 2-ethylbutyl group with fatty acid chains of varying lengths (see Figure 3 ). First, it was found that Remdesivir (Sp isomer) and its Rp isomer were similar in potency, and the Sp isomer was chosen based on its crystalline properties that allowed for rapid scalability in synthesis. Therefore, this stereocenter was not taken into consideration in the design, and the analogs presented herein are mixtures of two diastereomers (Sp, Rp)

[25] In certain embodiments, the 2-ethylbutyl group is replaced with a fatty acid chain: i) to increase lipophilicity for better permeability, thereby enhancing cellular uptake; ii) to enhance drug tissue distribution, especially in the lungs, spleen, and lymph nodes; iii) to enhance encapsulation efficiency by albumin nanoparticles; iv) to introduce the drug into the liposome bilayer; or v) to allow the fatty acid chain to form micelles to protect the drug from nonspecific hydrolysis in the bloodstream. In certain embodiments, alanine is replaced with N-methyl-D (or L) -tryptophan: i) to enhance encapsulation efficiency by albumin nanoparticles; ii) to increase lipophilicity for better permeability, thereby enhancing cellular uptake, iii) to enhance drug tissue distribution, especially in the lungs, spleen, and lymph nodes; iv) to introduce the drug into the liposome bilayer; or v) to enhance immune cell function to improve the antiviral efficacy of the drug.

[0159] Series 6. Using aliphatic diols as linkers between fatty acids and L (or D) -phenylalanine, the sixth series of analogs were designed by replacing L-phenylalanine with L (or D) -phenylalanine and replacing the 2-ethylbutyl group with fatty acid chains of varying lengths (see Figure 3 ). First, it was found that Remdesivir (Sp isomer) and its Rp isomer were similar in potency, and the Sp isomer was chosen based on its crystalline properties that allowed for rapid scalability in synthesis. Therefore, this stereocenter was not taken into consideration in the design, and the analogs presented herein are mixtures of two diastereomers (Sp, Rp)

[25] In certain embodiments, the 2-ethylbutyl group is replaced with a fatty acid chain: i) to increase lipophilicity for better permeability, thereby enhancing cellular uptake; ii) to enhance drug tissue distribution, particularly in the lungs, spleen, and lymph nodes; iii) to enhance encapsulation efficiency by albumin nanoparticles; iv) to introduce the drug into the liposome bilayer; or v) to allow the fatty acid chain to form micelles to protect the drug from nonspecific hydrolysis in the bloodstream. In certain embodiments, alanine is replaced with L (or D) -phenylalanine: i) to enhance encapsulation efficiency by albumin nanoparticles; ii) to increase lipophilicity for better permeability, thereby enhancing cellular uptake; iii) to enhance drug tissue distribution, particularly in the lungs, spleen, and lymph nodes; iv) to introduce the drug into the liposome bilayer.

[0160] The following formula I shows the general structure of remdesivir analogs:

[0161] wherein R is a lipid chain or a benzyl group, X is an amino acid side chain and Ar is a substituted or unsubstituted aryl group. Specific compounds are contemplated in Series 7 and Examples 10 and 11.

[0162] Series 7. The seventh series of analogs was designed by replacing the phenyl group with naphthalene, replacing L-alanine with N-methyl-D-tryptophan, and replacing the 2-ethylbutyl group with various lipid chains and benzyl groups. First, it was found that remdesivir (Sp isomer) and its Rp isomer were similar in potency, and the choice of the Sp isomer was based on its crystalline properties that can be rapidly scaled in synthesis. Therefore, this stereocenter was not taken into consideration in the design, and the analogs presented herein are mixtures of two diastereomers (Sp, Rp). In certain embodiments, the phenyl group is replaced with a naphthyl group: i) to enhance encapsulation efficiency by albumin nanoparticles; ii) to increase lipophilicity to obtain better permeability, thereby enhancing cellular uptake, iii) to enhance drug tissue distribution, especially in the lungs, spleen, and lymph nodes; iv) to introduce the drug into the liposome bilayer. In certain embodiments, 2-ethylbutyl is replaced with a lipid chain and a benzyl group: i) to increase lipophilicity for better permeability, thereby enhancing cellular uptake; ii) to enhance drug tissue distribution, particularly in the lungs, spleen, and lymph nodes; iii) to enhance encapsulation efficiency by albumin nanoparticles; iv) to introduce the drug into the liposome bilayer; or v) to allow the lipid chain to form micelles to protect the drug from nonspecific hydrolysis in the bloodstream. In certain embodiments, alanine is replaced with N-methyl-D (or L) -tryptophan: i) to enhance encapsulation efficiency by albumin nanoparticles; ii) to increase lipophilicity for better permeability, thereby enhancing cellular uptake, iii) to enhance drug tissue distribution, particularly in the lungs, spleen, and lymph nodes; iv) to introduce the drug into the liposome bilayer; or v) to enhance immune cell function to improve the antiviral efficacy of the drug.

[0163] Additionally disclosed are compounds selected from the group consisting of:

[0164]

[0165]

[0166] Compounds can exist as stereoisomers having asymmetric or chiral centers. Depending on the configuration of the substituents around the chiral carbon atom, the stereoisomer is "R" or "S". The terms "R" and "S" as used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The present disclosure contemplates various stereoisomers and mixtures thereof, and these are specifically included within the scope of the present disclosure. Stereoisomers include enantiomers and diastereomers, as well as mixtures of enantiomers or diastereomers. Individual stereoisomers of a compound can be synthesized from commercially available raw materials containing asymmetric or chiral centers, or by preparing a racemic mixture followed by resolution methods well known to those of ordinary skill in the art. Examples of such resolution methods are: (1) attaching a mixture of diastereomers to a chiral auxiliary, separating the resulting mixture of diastereomers by recrystallization or chromatography, and optionally liberating the optically pure product from the auxiliary, as described in Furniss, Hannaford, Smith and Tatchell, "Vogel's Textbook of Practical Organic Chemistry," 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England (or a more recent edition thereof), or (2) direct separation of the mixture of optical enantiomers on a chiral chromatographic column, or (3) fractional recrystallization methods.

[0167] It is understood that compounds may have tautomeric forms as well as geometric isomers and these also constitute embodiments of the present disclosure.

[0168] The present disclosure also includes isotopically labeled compounds that are identical to those described in Formula I, Ia, or II, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from that normally found in nature. Examples of suitable isotopes for inclusion in the compounds of the present disclosure are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, respectively, such as, but not limited to, 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P. 35 S. 18 F and 36Cl. With heavier isotopes such as deuterium (e.g. 2 H) substitution may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in some circumstances. Compounds may incorporate positron emitting isotopes for use in medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron emitting isotopes that may be incorporated into compounds of formula (I) are 11 C. 13 N. 15 O and 18 F. Isotopically labeled compounds of Formula I, Ia or II can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples, using an appropriate isotopically labeled reagent in place of a non-isotopically labeled reagent.

[0169] The disclosed compounds can be introduced into pharmaceutically acceptable compositions. Pharmaceutical compositions may include a "therapeutically effective amount" or a "prophylactic effective amount" of one or more compounds. A "therapeutically effective amount" refers to an amount that effectively achieves the desired therapeutic outcome at the necessary dosage and time period. The therapeutically effective amount of a composition can be determined by one skilled in the art and can vary according to factors such as the individual's disease state, age, sex, and weight, as well as the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the beneficial effects of the treatment exceed any toxic or deleterious effects of the compounds of the present invention (e.g., compounds of formula (I)). A "prophylactic effective amount" refers to an amount that effectively achieves the desired prophylactic outcome at the necessary dosage and time period. Typically, since prophylactic doses are used in subjects before disease or in the early stages of disease, the prophylactic effective amount will be less than the therapeutically effective amount.

[0170] Pharmaceutical compositions and formulations may include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose, and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffers such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffers, and other nontoxic, compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0171] The route of administration of the disclosed compound and the form of the composition will determine the type of carrier used. The composition can be in a variety of forms suitable for, for example, systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implant, or parenteral) or local administration (e.g., skin, lung, nose, ear, eye, liposome delivery system, or iontophoresis).

[0172] Carriers for systemic administration typically include at least one of a diluent, lubricant, binder, disintegrant, colorant, flavoring, sweetener, antioxidant, preservative, glidant, solvent, suspending agent, wetting agent, surfactant, combinations thereof, etc. All carriers in the composition are optional.

[0173] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; glycols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols such as glycerol; mannitol; and sorbitol. The amount of one or more diluents in a systemic or topical composition is typically about 50 to about 90%.

[0174] Suitable lubricants include silicon dioxide, talc, stearic acid and its magnesium and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter. The amount of one or more lubricants in the systemic or local composition is typically from about 5 to about 10%.

[0175] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; gum tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of one or more binders in the systemic composition is typically from about 5 to about 50%.

[0176] Suitable disintegrants include agar, alginic acid and its sodium salt, effervescent mixtures, cross-linked carboxymethylcellulose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays and ion exchange resins. The amount of one or more disintegrants in the systemic or topical composition is typically from about 0.1 to about 10%.

[0177] Suitable coloring agents include colorants such as FD&C dyes.When used, the amount of coloring agent in the systemic or topical compositions is typically from about 0.005 to about 0.1%.

[0178] Suitable flavoring agents include menthol, peppermint, and fruit flavors.When used, the amount of one or more flavoring agents in the systemic or topical compositions is typically from about 0.1 to about 1.0%.

[0179] Suitable sweeteners include aspartame and saccharin.The amount of one or more sweeteners in the systemic or topical compositions is typically from about 0.001 to about 1%.

[0180] Suitable antioxidants include butylated hydroxyanisole ("BHA"), butylated hydroxytoluene ("BHT"), and vitamin E. The amount of one or more antioxidants in the systemic or topical compositions is typically from about 0.1 to about 5%.

[0181] Suitable preservatives include benzalkonium chloride, methylparaben, and sodium benzoate.The amount of one or more preservatives in systemic or topical compositions is typically from about 0.01 to about 5%.

[0182] Suitable glidants include silicon dioxide.The amount of one or more glidants in the systemic or topical compositions is typically from about 1 to about 5%.

[0183] Suitable solvents include water, isotonic saline, ethyl oleate, glycerol, hydroxylated castor oil, alcohols such as ethanol, dimethyl sulfoxide, N-methyl-2-pyrrolidone, dimethylacetamide, and phosphates (or other suitable buffers). The amount of one or more solvents in a systemic or topical composition is typically from about 0 to about 100%.

[0184] Suitable suspending agents include AVICEL RC-591 (available from FMC Corporation of Philadelphia, Pa.) and sodium alginate.The amount of one or more suspending agents in the systemic or topical compositions is typically from about 1 to about 8%.

[0185] Suitable surfactants include lecithin, polysorbate 80 and sodium lauryl sulfate, and TWEENS (available from Atlas Powder Company of Wilmington, Del.). Suitable surfactants include those disclosed in CTFA Cosmetic Ingredient Handbook, 1992, pp. 587-592; Remington's Pharmaceutical Sciences, 15th edition, 1975, pp. 335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of one or more surfactants in a systemic or topical composition is typically from about 0.1% to about 5%.

[0186] Although the amounts of the components of the systemic composition may vary depending on the type of systemic composition being prepared, in general, systemic compositions include 0.01% to 50% of the active compound (e.g., a compound of Formula (I)) and 50% to 99.99% of one or more carriers. Compositions for parenteral administration typically include 0.1% to 10% of the active substance and 90% to 99.9% of a carrier, which includes a diluent and a solvent.

[0187] Compositions for oral administration can have various dosage forms. For example, solid forms include tablets, capsules, granules and bulk powders. These oral dosage forms include a safe and effective amount (usually at least about 5%, and more particularly about 25% to about 50%) of the active substance. Oral dosage compositions include about 50% to about 95% carrier, and more particularly about 50% to about 75%.

[0188] Tablets can be compressed, tablet-milled, enteric-coated, sugar-coated, film-coated, or repeatedly compressed. Tablets typically include an active ingredient and a carrier, which comprises a composition selected from a diluent, a lubricant, a binder, a disintegrant, a colorant, a flavoring, a sweetener, a glidant, and a combination thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and cross-linked carboxymethyl cellulose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific coloring agents are FD&C dyes, which can be added for appearance. Chewable tablets preferably contain sweeteners, such as aspartame and saccharin, or flavorings such as menthol, peppermint, fruit flavors, or a combination thereof.

[0189] Capsules (including implants, timed-release and sustained-release formulations) typically include an active compound (e.g., a compound of formula (I)) and a carrier comprising one or more of the above-disclosed diluents in a capsule comprising gelatin. Granules typically include the disclosed compound and preferably a glidant (e.g., silicon dioxide) to improve flow characteristics. Implants may be biodegradable or non-biodegradable.

[0190] The choice of ingredients in the carrier for oral compositions depends on secondary considerations such as taste, cost, and storage stability, which are not critical for the purposes of the present invention.

[0191] Solid compositions can be coated by conventional methods, typically with a pH or time-dependent coating, so that the disclosed compound is released in the gastrointestinal tract proximal to the desired site of administration or at different points and times to prolong the desired effect. The coating typically comprises one or more selected from cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, ethyl cellulose esters, Components of the coating (obtained from Evonik Industries of Essen, Germany), wax, and shellac.

[0192] Compositions for oral administration can have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted by non-effervescent granules, suspensions reconstituted by non-effervescent granules, effervescent preparations reconstituted by effervescent granules, elixirs, tinctures, syrups, etc. Liquid oral compositions typically include the disclosed compounds and a carrier, that is, a carrier selected from diluents, colorants, flavorings, sweeteners, preservatives, solvents, suspending agents, and surfactants. Oral liquid compositions preferably include one or more ingredients selected from colorants, flavorings, and sweeteners.

[0193] Other compositions that can be used to achieve systemic delivery of the compounds of the invention include sublingual, buccal, and nasal dosage forms. Such compositions typically include one or more soluble filler substances such as diluents, including sucrose, sorbitol, and mannitol; and binders, such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include lubricants, colorants, flavorings, sweeteners, antioxidants, and glidants.

[0194] The disclosed compounds can be administered topically. Topical compositions for topical application to the skin can be in any form, including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-in and rinse-off hair conditioners, emulsions, cleansers, moisturizers, sprays, skin patches, and the like. Topical compositions comprise: a disclosed compound (e.g., a compound of formula (I)) and a carrier. The carrier of the topical composition preferably facilitates penetration of the compound into the skin. The carrier may further include one or more optional components.

[0195] The amount of carrier employed in conjunction with the disclosed compounds is sufficient to provide a practical amount of the composition administered per unit dose of the compound. Techniques and compositions for preparing dosage forms useful in the methods of the present invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, edited by Banker & Rhodes (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd edition, (1976).

[0196] The carrier may comprise a single component or a combination of two or more components. In topical compositions, the carrier comprises a topical carrier. Suitable topical carriers include one or more components selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, and combinations thereof. More particularly, carriers for skin application include propylene glycol, dimethyl isosorbide and water, and even more specifically phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols and symmetrical alcohols.

[0197] The carrier of the topical composition may further comprise one or more ingredients selected from the group consisting of emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.

[0198] Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadec-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, peanut oil, castor oil, acetylated lanolin alcohol, naphtha, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. Specific emollients for use on the skin include stearyl alcohol and dimethicone.The amount of one or more emollients in the skin-based topical composition is typically from about 5% to about 95%.

[0199] Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof.The amount of one or more propellants in the topical composition is typically from about 0% to about 95%.

[0200] Suitable solvents include water, ethanol, methylene chloride, isopropyl alcohol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethanol and isopropyl alcohol. The amount of one or more solvents in the topical composition is typically from about 0% to about 95%.

[0201] Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of one or more humectants in the topical composition is typically 0% to 95%.

[0202] The amount of thickening agent(s) in the topical composition is typically from about 0% to about 95%.

[0203] Suitable powders include beta-cyclodextrin, hydroxypropyl cyclodextrin, chalk, talc, Fuller's earth, kaolin, starch, gum, colloidal silica, sodium polyacrylate, tetraalkylammonium smectite, trialkylaryl ammonium smectite, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethylcellulose, ethylene glycol monostearate, and combinations thereof. The amount of one or more powders in the topical composition is typically from 0% to 95%.

[0204] The amount of fragrance in topical compositions is typically from about 0% to about 0.5%, especially from about 0.001% to about 0.1%.

[0205] Suitable pH adjusting additives include HCl or NaOH in an amount sufficient to adjust the pH of the topical pharmaceutical composition.

[0206] VIII. Treatment Methods

[0207] Disclosed herein are methods of treating viral infections using the compounds and compositions described herein (e.g., a compound of Formula (I) or a compound of Formula (II), or a composition comprising a compound of Formula (I) or a compound of Formula (II)).

[0208] Viruses that cause infections that can be treated by the methods, compounds, and compositions disclosed herein include, but are not limited to, herpes simplex virus type 1; herpes simplex virus type 2; encephalitis virus; papillomavirus; varicella zoster virus; herpes simplex virus; cytomegalovirus; human herpes virus type 8; BK virus; JC virus; smallpox (herpes zoster virus); adenovirus; hepatitis A virus; hepatitis B virus; hepatitis D virus; hepatitis E virus; human bocavirus; parvovirus B19; human astrovirus; norovirus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; foot-and-mouth disease virus, severe acute Respiratory syndrome (SARS); SARS-CoV-2; coronavirus; flavivirus; hepatitis C virus; yellow fever virus; dengue virus; West Nile virus; rubella virus; hepatitis E virus; human immunodeficiency virus (HIV); vaccinia virus; influenza virus; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabiá virus; Crimean-Congo hemorrhagic fever virus; Ebola virus (e.g., Ebola-Zaire, Ebola-Sudan, and Ebola-Ivory) Coast and Ebola-Reston); Marburg virus; measles virus; mumps virus; parainfluenza virus; respiratory syncytial virus; human metapneumovirus; Hendra virus; Nipah virus; rabies virus; hepatitis D; rotavirus; orbivirus; Koro virus; Banna virus; human enterovirus; Hantavirus; West Nile virus; Middle East respiratory syndrome (MERS) coronavirus; Japanese encephalitis virus; arboviral encephalitis virus; vesicular herpes virus; and Eastern equine encephalitis. In some embodiments, the virus is SARS-CoV-2. In some embodiments, the virus is Ebola virus.

[0209] A non-limiting list of viral diseases includes AIDS (HIV), AIDS-related cytomegalovirus infection, HIV-associated nephropathy, lipodystrophy, hepatitis A, B, C, D or E, herpes, herpes zoster (fowl pox), monkeypox, cowpox, German measles (rubella virus), yellow fever, dengue fever, etc. (arbovirus), influenza (influenza virus), hemorrhagic infectious diseases such as Ebola virus disease (Marburg virus or Ebola virus), Ross River virus infection, West Nile virus (WNV) disease, human papillomavirus (HPV) infection, respiratory tract infection such as adenovirus infection, avian (H5N1) influenza, influenza, RSV infection, severe acute respiratory syndrome (SARS), and swine (H1N1) influenza, viral skin diseases such as B19 parvovirus infection, epidemics such as Ebola hemorrhagic fever, Marburg hemorrhagic fever, Nipah virus disease, SARS, MERS and COVID-19. In some embodiments, the viral disease is COVID-19. In some embodiments, the viral disease is Ebola virus disease.

[0210] A therapeutically effective amount of a compound disclosed herein, or a composition thereof, can be administered alone or in combination with a therapeutically effective amount of at least one additional therapeutic agent. In some embodiments, effective combination therapy is achieved with a single composition or pharmacological formulation comprising both agents, or with two different compositions or formulations administered simultaneously, one comprising a compound of the invention and the other comprising a second agent. Alternatively, in other embodiments, therapy precedes or follows treatment with the other agent by an interval ranging from minutes to months.

[0211] A variety of secondary therapies can be used in conjunction with the compounds and compositions of the present disclosure. The secondary therapy can be a combination of a second therapeutic agent, or it can be a second therapy that is not associated with the administration of another agent. Such secondary therapies include, but are not limited to, steam inhalation, supplemental oxygen and / or mechanical ventilatory support, administration of a second therapeutic agent, such as a decongestant, a steroid, an analgesic, and a second antiviral agent.

[0212] In some embodiments of the methods disclosed herein, the compound or composition can be co-administered with an additional antiviral agent. In some embodiments, the additional antiviral agent is selected from abacavir, acyclovir, adefovir, amantadine, amprenavir, atazanavir, baloxavir maposiate, bictegravir, boceprevir, bulevirtide, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docosin, dolutegravir, doravirine, edoxuridine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, foscarnet, ganciclovir, ibacitabine, ibacitabine, sore throat, imiquimod, isoprolin (imunovir), indinavir, lamivudine, levofloxacin, lopinavir, lovir

[00135] The invention further comprises the following: selegiline, acetaminophen, fenvalerate, fenvalerate, morphine, nelfinavir, nevirapine, sorafenib, nexavir, nitazoxanide, oseltamivir, penciclovir, peramivir, penciclovir, peramivir, puraconazole, podophyllotoxin, raltegravir, remdesivir, ribavirin, rilpivirine, rilpivirine, rimantadine, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, tarivirine, telaprevir, telbivudine, tenofovir (e.g., tenofovir alafenamide or tenofovir disoproxil fumarate), tipranavir, trifluridine, tromantanide, arbidol, valacyclovir, valganciclovir, viraviroc, vidarabine, zalcitabine, zanamivir, and zidovudine, and any combination thereof.

[0213] Example

[0214] Example 1

[0215] Preparation of albumin nanoparticles containing remdesivir for injection or aerosol therapy

[0216] This example describes the preparation of albumin nanoparticles of remdesivir for injection or aerosolization, which will be used for subsequent lung distribution and anti-COVID 19 viral efficacy testing.

[0217] To prepare albumin nanoparticles, the organic phase was prepared by dissolving Remdesivir or modified Remdesivir (100 mg) in chloroform (2 ml). 20 ml of commercially available HSA solution (5%) was then mixed with the organic phase and vigorously dispersed by a stator-rotor homogenizer (Ultra-Turrax T25, 8K rpm–12K rpm, 5 minutes) to form a milky emulsion. The emulsion was then treated with a high-pressure homogenizer (Nano DeBEE) with parameters set to pressure = 15,000 psi-20,000 psi, condensation temperature = 50 ° C, and number of cycles = 6. The remaining organic solvent in the product was removed by a rotary evaporator. The final nanosuspension was filtered with a 0.22 μm membrane and then placed directly into a 10 ml vial (2 ml / vial) for lyophilization (primary drying temperature = -5 ° C for 30 hours and secondary drying temperature = 30 ° C for 6 hours). The vial was filled with nitrogen, then sealed and stored at -20 ° C.

[0218] To prepare the injection solution of the remdesivir nanoformulation, the lyophilized powder was resuspended with 2 ml of medical saline, which should suspend 5 mg / ml of the compound in a 5% HSA solution. To prepare the liquid aerosol used in the nebulizer, the powder was resuspended with medical saline, and the concentration of the compound should be adjusted based on the inhalable dose. A normal external power supply can be used to generate the liquid aerosol.

[0219] Example 2

[0220] Preparation of liposomes of remdesivir for injection and aerosol therapy

[0221] This example describes the preparation of liposomes of remdesivir for injection or aerosolization, which will be used for subsequent lung distribution and anti-COVID 19 viral efficacy testing.

[0222] In order to prepare liposome formulations of Remdesivir and its modified compounds, 100 mg of compound was dissolved in an organic solution such as chloroform together with other lipids (the concentration of lipid was 10-20 mg). The solvent was removed with a rotary evaporator, and a lipid / compound film was formed on the side of the round-bottom flask. The lipid film was thoroughly dried to remove residual organic solvent by placing the flask on a vacuum pump overnight. 5 ml of ddH2O was added to the flask to hydrate the film, the temperature was set to 60 ° C, and the time was set to 1 hour. Vigorous shaking, mixing, or stirring was used to completely hydrate the lipid film. Larger multilamellar vesicles will be generated after the hydration step and can be broken by a bath sonicator to form unilamellar vesicles (SUVs) and sonicated for 5-10 minutes at a temperature higher than the Tc of the lipid. The final formulation was filtered with a Sephadex column to remove free compounds and then placed directly into a 10 ml vial (2 ml / vial) for lyophilization (primary drying temperature = -5 ° C for 30 hours, and secondary drying temperature = 30 ° C for 6 hours). The vial was filled with nitrogen, then sealed and stored at -20°C.

[0223] To prepare the injection solution of the remdesivir nanoformulation, the lyophilized powder was resuspended with 2 ml of medical saline, which should suspend 5 mg / ml of the compound in a 5% HSA solution. To prepare the liquid aerosol used in the nebulizer, the powder was resuspended with medical saline, and the concentration of the compound should be adjusted based on the inhalable dose. A normal external power supply can be used to generate the liquid aerosol.

[0224] Example 3

[0225] Preparation of albumin or liposome nanoparticle dry powder inhalation formulation of remdesivir

[0226] This example describes the preparation of a dry powder inhalation formulation of albumin or liposome nanoparticles of remdesivir.

[0227] In order to prepare albumin or liposome nanoparticles of redcivir and its modified compounds, a similar procedure is performed and a nanosuspension in water is produced. Dry powder for inhalation is prepared using spray freeze drying. Non-inhalable carriers such as lactose with different sizes (~200 μm, <15 μm) are blended with the dry powder and inhalation-grade capsules are prepared. Upon actuation, the capsule ruptures or is pinhole-shaped by the DPI device, and the powder is released for aerosolization.

[0228] Example 4

[0229] Drug modification for higher encapsulation of albumin and liposomal nanoparticles

[0230] The production route of remdesivir analogs is shown in Figure 4 middle.

[0231] Step 1: The alcohol is dissolved in dichloromethane and cooled to 0°C. A catalytic amount of 4-(dimethylamino)pyridine (DMAP, 10%) is added and the reaction mixture is stirred at 0°C under argon for 20 minutes. At 0°C, N-Boc-amino acid (1 equivalent) and N-(3-diaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl, 1 equivalent) are added to the reaction. The mixture is stirred for 1 hour, warmed to room temperature and stirred overnight. The mixture is diluted with dichloromethane (CH2Cl2), washed with saturated NaHCO3 solution, then with brine, and dried over sodium sulfate. The crude ester obtained after evaporation is purified by column chromatography on silica gel.

[0232] Step 2: Under argon, intermediate I is added once to a HCl / dioxane solution (4 ml, 4 M) cooled to 0 ° C. The ice bath is removed, and the mixture is kept stirred at room temperature for 4 hours. After completion, the reaction mixture is concentrated by rotary evaporation under high vacuum. The residue is then placed in anhydrous ether and collected by filtration to give a deprotected alanine ester as a salt. The crude product and phenol dichlorophosphate (1 equivalent) are dissolved in anhydrous dichloromethane and cooled to -78 ° C under argon. Anhydrous trimethylamine (2 equivalents) is added dropwise, and the reaction is allowed to slowly warm to room temperature and stirred for 2 hours. After completion, the solvent is evaporated under reduced pressure, and the resulting residue is dissolved in anhydrous ether and filtered. The filtrate is reduced to dryness to give intermediate II, which is used in the next step without further purification.

[0233] Step 3: Under argon, to a stirred anhydrous THF solution of Redcivir metabolite GS-441524 (DCCHEMICALS, 1 equivalent) was added intermediate II (3 equivalents) dissolved in anhydrous THF. Methylimidazole (5 equivalents) was added dropwise to the reaction mixture within 5 minutes at -78 ° C. After 15 minutes, the reaction was allowed to slowly warm to room temperature and stirred overnight. After completion, the solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane and washed with 0.5M HCl. The organic layer was dried over sodium sulfate, filtered, evaporated, and purified on silica gel by column chromatography.

[0234] Example 5

[0235] Additional modifications for higher encapsulation of albumin and liposomal nanoparticles

[0236] The production route of remdesivir analogs is shown in Figure 5 middle.

[0237] Step 1: Dissolve the mono-TBDMS protected diol in dichloromethane and cool to 0°C. Add a catalytic amount of 4-(dimethylamino)pyridine (DMAP, 10%) and stir the reaction mixture at 0°C under argon for 20 minutes. At 0°C, N-Boc-amino acid (1 equivalent) and N-(3-diaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl, 1 equivalent) are added to the reaction. The mixture is stirred for 1 hour, warmed to room temperature and stirred overnight. The mixture is diluted with dichloromethane (CH2Cl2), washed with saturated NaHCO3 solution, then washed with brine, and dried over sodium sulfate. The crude ester obtained after evaporation is purified on silica gel by column chromatography.

[0238] Step 2: General procedure for deprotection of TBDMS from the parent compound.

[0239] Use 1.1 equivalents of TBAF, add alcohol (1.0 equivalent) and anhydrous tetrahydrofuran of TBDMS protection in the 15mL polypropylene tube equipped with a magnetic stirrer. Buffered tetrabutylammonium fluoride solution (1.1 equivalents of fluoride) is added to the THF solution, screw cap is added to the polypropylene tube, and the reaction mixture is stirred at 23 ° C. The reaction was completed after 3 hours, and now the crude material is directly loaded onto the silica gel quick column. Chromatography (30: 70 ethyl acetate-hexane, gradually increased to 50: 50 ethyl acetate-hexane) provides intermediate II.

[0240] Step 3: The purified intermediate II was dissolved in dichloromethane and cooled to 0°C. A catalytic amount of 4-(dimethylamino)pyridine (DMAP, 10%) was added and the reaction mixture was stirred at 0°C under argon for 20 minutes. At 0°C, fatty acid (1 equivalent) and N-(3-diaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC HCl, 1 equivalent) were added to the reaction. The mixture was stirred for 1 hour, warmed to room temperature and stirred overnight. The mixture was diluted with dichloromethane (CH Cl), washed with saturated NaHCO solution, then washed with brine, and dried over sodium sulfate. The evaporated crude intermediate III was purified on silica gel by column chromatography.

[0241] Step 4: Under argon, intermediate III is added once to HCl / dioxane solution (4ml, 4M) cooled to 0°C. The ice bath is removed, and the mixture is kept stirred at room temperature for 4 hours. After completion, the reaction mixture is concentrated by rotary evaporation under high vacuum. Then, the residue is placed in anhydrous ether and collected by filtration to provide the deprotected ester as a salt. The crude product and phenol dichlorophosphate (1 equivalent) are dissolved in anhydrous dichloromethane and cooled to -78°C under argon. Anhydrous trimethylamine (2 equivalents) is added dropwise, and the reaction is allowed to slowly warm to room temperature and stirred for 2 hours. After completion, the solvent is evaporated under reduced pressure, and the residue obtained is dissolved in anhydrous ether and filtered. The filtrate is reduced to dryness to provide intermediate IV, which is used in the next step without further purification.

[0242] Step 5: Under argon, to a stirred anhydrous THF solution of Redcivir metabolite GS-441524 (DCCHEMICALS, 1 equivalent) was added intermediate IV (3 equivalents) dissolved in anhydrous THF. Methylimidazole (5 equivalents) was added dropwise to the reaction mixture within 5 minutes at -78 ° C. After 15 minutes, the reaction was allowed to slowly warm to room temperature and stirred overnight. After completion, the solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane and washed with 0.5M HCl. The organic layer was dried over sodium sulfate, filtered, evaporated, and purified on silica gel by column chromatography.

[0243] Example 6

[0244] Improving the stability of remdesivir in water through nanoformulation

[0245] In order to verify the stability of the redoxivir nanoformulation for aerosol treatment in saline in certain embodiments, the nanoformulation freeze-dried powder was resuspended with saline to prepare the final medical solution used in the nebulizer. The control group was simply prepared by dissolving redoxivir in saline. The two groups were incubated at 4 ° C, 20 ° C and 37 ° C for 0, 2, 5, 10, 15 and 20 minutes, respectively. A small drop of sample was taken from each group and mixed with a methanol / acetonitrile mixture (1: 1, v / v) and a 10% aqueous solution containing 5-(2-aminopropyl) indole (5-IT, 20nM) (volume ratio of 1: 4). The concentrations of the parent drug (redoxivir) and metabolites (alanine metabolites, nucleoside monophosphate, nuc, nucleoside triphosphate) were detected using LC-MS.

[0246] To verify the plasma stability of remdesivir nanoformulations for intravenous therapy, nanoformulations and control solutions were prepared as previously described. The two groups were incubated with plasma collected from wild-type (WT) C57BL / 6 mice and / or esterase-deficient (Ces1c− / −) C57BL / 6 mice for 0, 5, 10, 30, 60 minutes, and 2 hours at 37°C. Samples were then prepared as previously described and filtered sequentially (Agilent Captiva 96 0.2μm) and dried under a stream of nitrogen. After reconstitution in a mixture of acetonitrile / formic acid solution (100:1:10,000, v / v / v), 10μL aliquots were analyzed by LC-MS. This method was used to determine the concentrations of the parent drug (remdesivir) and metabolites (alanine metabolites, nucleoside monophosphates, nucleoside triphosphates).

[0247] Example 7

[0248] Improved lung (spleen, lymph node) distribution and in vivo stability through nanoformulation

[0249] The nanoformulation was dissolved in 0.9% sterile saline, and redcivir was dissolved in 100% DMSO for in vitro studies and in a vehicle containing 12% sulfobutyl ether-β-cyclodextrin in an aqueous solution (with HCl / NaOH, pH 5) for in vivo studies. Hamsters were randomly assigned to 6 groups (n=24) and administered redcivir, redcivir albumin nanoparticles, and redcivir liposomes at a dose of 5 mg / kg by intravenous injection. And the atomized albumin-based nanoparticles, atomized liposomes, and atomized PLA / PLGA NP / MP were administered in equal doses by inhalation, and then plasma was isolated from three parallel mice at 0.25, 0.5, 1, 2, 4, 6, 8, and 12 hours after administration. Using the same dosing regimen, three male rhesus monkeys in each group (n=8) were intravenously administered a 10 mg / kg dose of remdesivir, and then plasma was separated at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration. The plasma concentrations of remdesivir, nucleotide monophosphate (Nuc-MP), diphosphate (Nuc-DP) and triphosphate (Nuc) were measured to determine the in vivo stability of the prodrug and the pharmacokinetic characteristics of the nanoformulation.

[0250] For both mice and rhesus monkeys, lung, spleen, and lymph node tissues were separated and snap-frozen at different time points after administration. The frozen lung samples were crushed and weighed. Dry ice cooled extraction buffer, which contained potassium hydroxide (0.1%) and EDTA (67mM) in 70% methanol and adenosine chloride (0.5mM). After clarifying and centrifuging for 20 minutes at 20,000g, the supernatant was dried in a centrifugal evaporator. Then, the dried sample was reconstructed with 60ml of mobile phase A, which contained an aqueous solution of 3mM ammonium formate (pH 5) and 10mM dimethylhexylamine, and centrifuged for 20 minutes at 20,000g. The final supernatant was transferred to an LC / MS injection vial to obtain the concentration of lung nucleotide monophosphate (Nuc-MP), diphosphate (Nuc-DP), and triphosphate (Nuc-TP). In addition, 25ml of plasma separated from both mice and rhesus monkeys was processed and the in vitro improvement of stability of the nanoformulation was analyzed as described above.

[0251] Example 8

[0252] Improving antiviral efficacy through nanoformulation

[0253] The nanoformulations were dissolved in 0.9% sterile saline, and remdesivir was dissolved in 100% DMSO for in vitro studies and in a vehicle containing 12% sulfobutyl ether-β-cyclodextrin in water (with HCl / NaOH, pH 5) for in vivo studies.

[0254] 1) In vitro efficacy and cytotoxicity in different types of human lung cell lines.

[0255] To test whether the nanoparticle-based formulation has similar efficacy to remdesivir in inhibiting viral replication in vitro, a comparative antiviral assay was performed using Calu-3 2B4 cells (human lung epithelial cell line), NHBE cells (human bronchial epithelial cell line), and HAE cells (primary human airway epithelial cell line). Briefly, cells were seeded into plates and plated at 5×10 4 24 hours after the cells were plated, fresh culture medium was added. In triplicate, the cells were infected with SARS-CoV-2 (COVID 19) diluted in growth medium (MOI of 0.08) at 37 ° C for 1 hour, after which the virus was removed, the culture was rinsed once, and fresh culture medium containing dilutions of redcivir or nanoformulations was added. At 48 hours post-infection (hpi), viral replication was measured by nLUC assay (Promega), and cytotoxicity was measured via CellTiter-Glo (Promega) assay and then read on a SpectraMax plate reader (Molecular Devices). IC 50Values ​​were defined in GraphPad Prism 7 (GraphPad) as the concentration that reduced viral replication by 50% using UV-treated MERS-nLUC (100% inhibition) and vehicle alone (0% inhibition) as controls. CC was determined by comparing data with data from cell-free (100% cytotoxicity) and vehicle-only (0% cytotoxicity) samples. 50 At 5 days post infection (dpi), supernatants from the plate wells were collected and the amount of virus was quantified by endpoint dilution assay, which was performed by preparing serial dilutions of the assay medium and adding these dilutions to fresh cell monolayers in the plate to determine the tissue culture infectious dose (TCID) that causes 50% cytopathic effect by the Spearman-Karber method. 50 ). In addition, to measure the levels of viral RNA from infected cells, total RNA was extracted using an RNA isolation kit and quantified using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay using primers and probes specific for the SARS-Covid 19 nucleoprotein gene.

[0256] 2) In vivo efficacy studies:

[0257] Male and female hamsters were randomly divided into 14 groups (n=8) with 10 5 TCID 50The SARS-CoV-2 original virus was inoculated intranasally at a dose of 500 mg / kg. The treatment in the four groups before virus inoculation included vehicle (1 ml / kg), redoxivir (5 mg / kg, IV), redoxivir's albumin nanoparticles (5 mg / kg, IV), redoxivir's liposomes (5 mg / kg), aerosolized albumin-based nanoparticles (5 mg / kg, inhalation) and aerosolized PLA / PLGA NP / MP, and continued once a day until 7 dpi. The other four groups began to be administered with the same regimen 12 hours after inoculation. Body weight and respiratory rate were then recorded every day. At 7 dpi, mice were dissected to collect different tissues to screen for viral replication and histopathological changes. The main organs were grossly examined and then fixed in 10% buffered formalin solution, and paraffin sections (thickness 3-4 μm) were routinely prepared. Histopathological changes in all organs were identified using hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, and modified Masson's trichrome staining. After different preparations were processed, blood was collected in EDTA tubes for ELISA analysis of specific IgG for SARS-CoV. Then, lung tissue sections (thickness 3-4 μm) were routinely prepared, and IHC immunohistochemical staining (microscope) was combined with immunofluorescence staining (confocal) to evaluate the viral antigens in different groups. In addition, total RNA was extracted from the lungs and reverse transcribed using PrimerScript RT Reagent test kit, and then qRT-PCR reactions were performed using PowerUp SYBG Green Master Mix test kit to determine the viral replication in different groups.

[0258] Next, a second experiment was performed to evaluate their therapeutic efficacy in monkeys (macaques). Rhesus monkeys (3–6 years old) were randomly assigned to 12 experimental treatment groups (n=4), stratified by sex (equal number of males and females in each group) and balanced by weight using SAS statistical software. Treatment with remdesivir and remdesivir-loaded nanoformulations as described above (remdesivir dose of 10 mg / kg and vehicle of 2 ml / kg) was started on the day before / after SARS-CoV-2 (COVID 19) virus infection. Animals were observed at least twice a day to monitor signs of disease and clinical scores were assigned based on a scoring table approved by the Institutional Animal Care and Use Committee (IACUC), including impaired walking that prevented access to food or water, severe respiratory distress (open mouth breathing, lack of activity or cyanosis), lack of physical and mental alertness, or body temperature ≤35°C.

[0259] Example 9

[0260] Synthesis of Remdesivir analogs

[0261] The production route of remdesivir analogs is shown in Figure 6 middle.

[0262] Step 1: The alcohol is dissolved in dichloromethane and cooled to 0°C. A catalytic amount of 4-(dimethylamino)pyridine (DMAP, 10%) is added and the reaction mixture is stirred at 0°C under argon for 20 minutes. At 0°C, N-Boc-amino acid (1 equivalent) and N-(3-diaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl, 1 equivalent) are added to the reaction. The mixture is stirred for 1 hour, warmed to room temperature and stirred overnight. The mixture is diluted with dichloromethane (CH2Cl2), washed with saturated NaHCO3 solution, then with brine, and dried over sodium sulfate. The crude ester obtained after evaporation is purified by column chromatography on silica gel.

[0263] Step 2: Under argon, intermediate I was added in one portion to a solution of HCl / dioxane (4 ml, 4 M) cooled to 0°C. The ice bath was removed and the mixture was stirred at room temperature for 4 hours. After completion, the reaction mixture was concentrated by rotary evaporation under high vacuum. The residue was then taken up in anhydrous ether and collected by filtration to give the deprotected alanine ester as a salt.

[0264] Step 3: Intermediate II (1.1 equivalents) and aryloxy phosphate dichloride (1 equivalent) are dissolved in anhydrous dichloromethane and cooled to -78 ° C under argon. Triethylamine (5 equivalents) is added dropwise, and the reaction is allowed to slowly warm to room temperature and stir for 2 hours. The mixture is then cooled to 0 ° C, and 4-nitrophenol (1 equivalent) is added, followed by the slow addition of triethylamine (1.1 equivalents). The mixture is allowed to slowly warm to room temperature and stir for 3 hours. After completion, the solvent is evaporated under reduced pressure, and the residue of the gained is dissolved in anhydrous ether and filtered. The filtrate is reduced to dryness, to provide intermediate III, which is purified on silica gel by column chromatography.

[0265] Step 4: Under argon and room temperature, sulfuric acid (18M, 1.3 equivalents) was added dropwise to a stirred acetone solution of Remdesivir metabolite GS-441524 (DCCHEMICALS, 1 equivalent) and 2,2-dimethoxypropane (4.8 equivalents). The reaction mixture was stirred for 30 minutes and warmed to 45 ° C. After 30 minutes, the reaction was allowed to slowly cool to room temperature, and solid sodium bicarbonate and water were added sequentially. The mixture was stirred for 20 minutes and the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate and water. The aqueous layer was extracted with ethyl acetate, and the organic layer was dried over sodium sulfate, filtered, evaporated, and purified by column chromatography on silica gel.

[0266] Step 5: To a solution of intermediate III (1.2 equivalents) in acetonitrile was added intermediate IV (1 equivalent) and magnesium chloride (1 equivalent) at room temperature. The solution was heated to 50°C for 15 minutes and N,N-diisopropylethylamine (2.5 equivalents) was added. After 1 hour, the reaction mixture was allowed to cool to room temperature and then diluted with ethyl acetate. The organic layer was washed with 5% aqueous citric acid solution, saturated aqueous ammonium chloride solution, 5% aqueous potassium carbonate solution and brine. The organic layer was dried over anhydrous sodium sulfate and reduced to dryness to give intermediate V, which was purified by column chromatography on silica gel.

[0267] Step 6: To a stirred solution of intermediate V (1 equivalent) in tetrahydrofuran was slowly added 37% aqueous hydrochloric acid at 0°C. The reaction mixture was allowed to warm to room temperature. After 6 hours, the reaction mixture was diluted with water and adjusted to pH = 8 by adding saturated aqueous sodium bicarbonate solution. The resulting mixture was extracted with ethyl acetate, and the organic layer was then washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product obtained after evaporation was purified by column chromatography on silica gel to give a redcivir analog.

[0268] Compound MMT5-14:

[0269]

[0270] 1 H NMR (500 MHz, methanol-d4) δ7.85 (s, 1H), 7.29 (dt, J = 9.1, 7.4 Hz, 2H), 7.21–7.10 (m, 3H), 6.93–6.85 (m, 2H), 5.43–5.23 (m, 4H), 4.79 (d, J = 5.4 Hz, 1H), 4.47–4.33 (m, 2H), 4.33–4.23 (m, 1H),4.19(dt,J=17.5,5.5Hz,1H),4.10–3.94(m,2H),3.92–3.76(m,1H),2.76(t,J=6.3H z,2H),2.11–1.98(m,4H),1.60–1.52(m,2H),1.41–1.19(m,23H),0.89(t,J=6.8Hz,3H); 13CNMR (126 MHz, methanol-d4) δ 173.49, 155.83, 150.44, 146.93, 146.89, 129.51, 129.49, 129.29, 129.26, 127.64, 124.64, 124.14, 120.01, 119.97, 119.96, 119.92, 116.53, 116.17, 110.97, 110.91, 101.1 8,82.92,82.85,79.89,79.70,74.21,70.28,70.12,65.00,64.93,50.04,48.09,31.24,29.30,29.06,29.04,28.89,28.88,28.85,28.28,28.22,26.74,25.49,25.48,25.12,22.20,19.03,13.01; 31 P NMR (202 MHz, methanol-d4) δ 3.52 (s), 3.50 (s); MS (m / z) = 767.2 [M+1]

[0271] Compound MMT5-15:

[0272]

[0273] 1 H NMR (500 MHz, methanol-d4) δ8.09 (ddd, J = 24.8, 7.6, 2.2 Hz, 1H), 7.88–7.78 (m, 2H), 7.66 (t, J = 7.9 Hz, 1H), 7.52–7.46 (m, 2H), 7.46–7.40 (m, 1H), 7.38–7.28 (m, 1H), 6.87–6.77 (m, 2H), 5.43–5.18 (m, 4H), 4.69 (t, J = 5.5 Hz, 1H), 4 .49–4.41(m,1H),4.42–4.37(m,1H),4.34(td,J=10.6,10.0,4.4Hz,1H),4.19(t,J=5.5Hz,1H),4.03–3.91(m ,3H),2.75(t,J=6.3Hz,2H),2.09–1.91(m,4H),1.55–1.42(m,2H),1.39–1.07(m,23H),0.89(t,J=6.8Hz,3H); 13C NMR (126 MHz, methanol-d4) δ 173.46, 155.62, 146.86, 134.68, 129.51, 129.49, 127.64, 127.63, 127.41, 126.30, 125.99, 125.94, 125.02, 124.52, 124.47, 124.09, 121.31, 121.24, 116.17, 114.74, 110.92, 11 0.79,101.22,83.01,79.82,74.28,74.21,70.28,69.99,65.36,65.02,64.91,48.09,31.23,29.27,29.04,29.02,28.86,28.83,28.23,28.19,26.74,26.72,25.46,25.44,25.12,22.19,18.93,13.01; 31 P NMR (202 MHz, methanol-d4) δ 3.93 (s), 3.87 (s); MS (m / z) = 817.1 [M+1]

[0274] Compound MM5-1:

[0275]

[0276] 1 H NMR (500 MHz, methanol-d4) δ 8.12–8.01 (m, 1H), 7.88–7.78 (m, 2H), 7.65 (dd, J = 10.7, 8.2 Hz, 1H), 7.52–7.36 (m, 3H), 7.35–7.20 (m, 6H), 6.91–6.71 (m, 2H), 5.09–4.95 (m, 2H), 4.67 (dd, J = 9.2, 5.5 Hz, 1H), 4.47–4.25 (m, 3H), 4.20–4.13 (m, 1H), 4.02–3.89 (m, 1H), 1.31–1.24 (m, 3H); 13 C NMR (126 MHz, methanol-d4) δ 172.32, 156.25, 146.86, 146.44, 135.73, 128.10, 128.08, 127.84, 127.83, 127.81, 127.37, 126.29, 125.00, 124.46, 121.19, 116.45, 101.20, 74.21, 70.23, 70.16, 66.51, 66.42, 48.08, 38.46, 18.90; 31P NMR (202 MHz, methanol-d4) δ 3.95 (s), 3.82 (s); MS (m / z) = 659.4 [M+1].

[0277] Compound MMT4-70:

[0278]

[0279] 1 H NMR(500MHz, methanol-d4)δ7.85(d,J=7.5Hz,1H),7.29(dt,J=9.2,7.2Hz,2H),7 .22–7.07(m,3H),6.97–6.73(m,2H),4.79(d,J=5.4Hz,1H),4.45–4.33(m,2 H),4.33–4.24(m,1H),4.18(dt,J=17.3,5.5Hz,1H),4.10–3.96(m,3H),1.6 4–1.48(m,2H),1.26(tdd,J=13.7,5.7,2.2Hz,25H),0.89(t,J=6.8Hz,3H); 13 C NMR (126 MHz, methanol-d4) δ 173.49, 155.83, 150.78, 150.72, 146.91, 146.87, 129.28, 129.25, 124.68, 124.64, 124.15, 124.12, 120.01, 116.52, 116.17, 110.97, 110.91, 101.19, 83.08, 82.92, 82.85, 79. 88,79.70,74.23,74.22,71.49,70.28,70.15,70.12,65.78,65.74,65.01,64.94,50.05,48.09,31.64,29.32,29.31,29.25,29.19,29.03,28.90,28.28,28.21,27.56,25.49,25.48,22.30,13.01; 31 P NMR (202 MHz, methanol-d4) δ 3.53 (s), 3.49 (s); MS (m / z) = 687.0 [M+1]. Compound MMT4-98:

[0280]

[0281] 1H NMR (500 MHz, methanol-d4) δ 8.16–8.02 (m, 1H), 7.87–7.79 (m, 2H), 7.66 (t, J = 8.0 Hz, 1H), 7.53–7.40 (m, 3H), 7.34 (dt, J = 9.5, 7.9 Hz, 1H), 6.88–6.76 (m, 2H), 4.69 (t, J = 5.6 Hz, 1H),4.46(dddd,J=11.4,8.9,6.0,2.8Hz,1H),4.41–4.30(m,2H),4.19(t,J=5.5Hz, 1H),4.04–3.90(m,3H),1.58–1.43(m,2H),1.37–1.09(m,25H),0.89(t,J=6.8,3H); 13 C NMR (126 MHz, methanol-d4) δ 173.42, 155.83, 146.94, 146.87, 134.83, 127.40, 126.30, 125.98, 125.93, 125.01, 124.46, 124.10, 124.02, 121.31, 121.23, 116.46, 116.17, 114.74, 110. 91,110.78,101.21,79.82,74.28,74.21,70.28,70.16,69.99,65.03,64.92,48.08,31.63,29.29,29.22,29.15,29.13,29.03,28.88,28.86,28.23,28.19,25.44,22.30,13.01; 31 P NMR (202 MHz, methanol-d4) δ 3.92 (s), 3.87 (s); MS (m / z) = 737.1 [M+1].

[0282] Example 10

[0283] Bioanalysis of remdesivir analogs and related metabolites

[0284] For nucleoside triphosphate analysis, LC-MS / MS was performed by ion pairing chromatography using an LC20AD UFLC (Shimadzu, Japan) coupled to an API 5500A (AB Sciex, USA). Separation was achieved on a 50×2.1 mm Agilent 3.5 μm C18 column. The mobile phase consisted of 3 mM ammonium formate in water containing 10 mM dimethylhexylamine (mobile phase A) and ACN containing 0.1% formic acid (mobile phase B). A multi-step linear gradient started at 2% B for 1 minute, increased to 95% B over 2 minutes, and maintained at 95% B for 1 minute at a flow rate of 0.4 mL / min. Detection was performed in negative ion mode and multiple reaction monitoring mode.

[0285] Remdesivir, analogs, and their metabolites, alanine metabolites, and Nuc were analyzed on an X500R QTOF system (AB Sciex, USA). Chromatographic separation was obtained on a 50×2.1 mm Agilent 3.5 μm C18 column using a constant flow rate of 0.6 mL / min. The gradient mobile phase consisted of an aqueous solution containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B), running from 5% B to 95% B in 3 minutes, and then maintaining 95% B for 1 minute. The eluent was introduced into a quadrupole time-of-flight mass spectrometer by electrospray ionization (ESI) operated in positive mode.

[0286] A 10-point standard curve ranging from 5 to 5000 ng / mL was used to quantify all analytes.

[0287] Example 11

[0288] Plasma stability of remdesivir analogs

[0289] To test in vitro plasma stability, 5ul of 100μM test compounds (Redcivir, MMT4-70, MMT4-98, MMT5-1) were added to 495ul of human, hamster and mouse plasma, respectively. The structures of MMT4-70, MMT4-98 and MMT5-1 are shown below. 40μL aliquots were taken from the reaction solution and terminated by adding 160μL of cold acetonitrile at different sampling times (0, 10 minutes, 30 minutes, 60 minutes and 120 minutes). The incubation solution was centrifuged at 3500rpm for 10 minutes to precipitate the protein. The supernatant was used for LC / MS / MS analysis.

[0290] like Figure 7As shown, all compounds were metabolized to ALA at different rates, and no other metabolites such as NUC and NTP were observed. In mouse plasma, all compounds were metabolized within 10 minutes, and the synthetic compounds (MMT4-70, MMT4-98 and MMT5-1) had similar / better stability when ALA production was taken into account. The stability of the compounds in human and hamster plasma was much better, with at least 20% of the parent compound observed after 2 hours. MMT5-1 performed best in human plasma, while no significant differences were observed between the synthetic compounds in hamster plasma. Overall, MMT4-70, MMT4-98 and MMT5-1 were more stable than remdesivir in different plasmas.

[0291] Example 12

[0292] Pharmacokinetics and initial tissue targeting

[0293] Different compounds were compared using a short-term PK test. Five remdesivir analogs (MMT4-70, MMT4-98, MMT5-1, MMT5-14, and MMT5-15) were compared with remdesivir. The structures of MMT5-14 and MMT5-15 are shown below. The albumin nanocomplexes of remdesivir analogs were prepared according to the following procedure: the organic phase was prepared by dissolving the synthesized compound (20 mg) in chloroform (1 ml). Then, 20 ml of commercially available HSA solution (5%) was mixed with the organic phase and vigorously dispersed by a stator-rotor homogenizer (Ultra-Turrax T25, 8K rpm–12K rpm, 5 minutes) to produce a milky emulsion. The emulsion was then treated with a high-pressure homogenizer (Nano DeBEE) with parameters set to pressure = 15,000 psi-20,000 psi, condensation temperature = 50°C, and number of cycles = 6. The remaining organic solvent in the product was removed by a rotary evaporator. The final nanosuspension was filtered with a 0.22 μm membrane and freeze-dried (primary drying temperature = -5°C for 30 hours and secondary drying temperature = 30°C for 6 hours).The vial was filled with nitrogen, then sealed and stored at -20°C.

[0294] The formulation and remdesivir (commercially available formulation) were administered intravenously to intubated hamsters at a dose of 10 mg / kg. Two hamsters were included in each group. At different time points (0.25 hours, 1 hour, 2 hours, and 4 hours), plasma was collected for future LC-MS analysis. At the 4-hour time point, the hamsters were euthanized and blood and lungs were collected for future LC-MS analysis.

[0295] like Figure 8AAs shown, changes in plasma concentrations were observed in different groups. Nano-MMT5-15 and nano-MMT5-14 had the highest stability in plasma, followed by nano-MMT4-98 and nano-MMT4-70. Nano-MMT5-1 and Rem were both rapidly metabolized in plasma. In the Rem group, high concentrations of ALA and NUC were observed at short time points. No NTPs were tested in plasma for any of the groups, meaning that NTPs were generated intracellularly according to known pathways. This was also confirmed by blood samples taken at the 4-hour time point ( Figure 8B ), where the NTP concentration of the synthesized compound was 10-15 times higher than that of Remdesivir. The highest concentration of the parent compound in the blood was observed in nano MMT5-15, which demonstrates the best stability of the compound. Lung tissue is the most important viral target. Higher concentrations of the parent compound (2-10 times) were observed for all synthetic compounds except MMT5-1. This higher stability also resulted in higher active metabolite NTPs in lung tissue, an increase of 2-5 times. For similar structures (MMT4-70 vs MMT4-98, MMT5-14 vs MMT5-15), more stable compounds tended to have higher parent compounds and ALA, but lower late metabolites such as NUC and NTP.

[0296] Example 13

[0297] Tissue targeting of the formulation

[0298] MMT5-14 was selected for further PK analysis due to its balanced stability and higher concentrations in blood and lung tissue. In addition to the nanoformulation, a cyclodextrin-based formulation of MMT5-14 was also prepared. In short, 2% hydrogenated castor oil and 2% ethanol were added to dissolve MMT5-14 and then mixed with 20% SBE-CD. The two formulated MMT5-14 compounds were intravenously administered to tube-placed hamsters at a dose of 10 mg / Kg together with Redcivir (commercially available formulation). There were two hamsters in each group. At different time points (0.5 hours, 1 hour, 4 hours, 7 hours and 24 hours), the hamsters were euthanized and samples including plasma, blood, blood vessels, heart, intestines, kidneys, lungs, lymph nodes, spleen, trachea and others were collected for future LC-MS analysis.

[0299] The concentration-time curves of different dosing groups and tissues are shown in Figure 9. The parent compound of Redcievir is rapidly metabolized by all tissues, while the other two groups (MMT5-14, nano-MMT5-14) have much higher parent concentrations at short time points. For most tissues except blood vessels, kidneys and trachea, no significance was observed between the MMT5-14 and nano-MMT5-14 groups, and higher parent compounds were observed in the MMT5-14 group. Similar trends in changes in ALA metabolites were also found in both the MMT5-14 and nano-MMT5-14 groups, with higher ALA metabolites observed. However, for the late metabolite NUC, higher concentrations were observed in several tissues of the Redcievir group, such as the heart and trachea. For the active metabolite NTP, the Redcievir group accumulated better in blood vessels, heart and lymph nodes, while the MMT5-14 and nano-MMT5-14 groups performed better in blood, lungs and spleen.

[0300] After calculating the AUC for these concentration-time plots, a clear comparison between the different tissues can be observed in Figure 10. For the parent compound and ALA, MMT5-14 and nano-MMT5-14 had higher AUCs in all tissues analyzed. For NUC and NTP, higher metabolite accumulation was observed in the kidney, which can be explained by higher specific metabolizing enzymes in this tissue. To further compare the formulated MMT5-14 group with remdesivir, the AUC fold is shown in Figure 11. For NUC and NTP metabolites, the highest increase was observed in lung tissue, with a 4-5 fold change. Tissues such as blood and spleen also showed a 2-3 fold increase in NTP metabolites.

[0301] Example 14

[0302] Cellular uptake of the formulation

[0303] THP-1, Calu-3, HUVEC, and AMJ2-C11 cells (0.5-1×10 6 ) and incubated for 12 hours before any treatment. 10 μM Remdesivir, MMT5-14, and nano-MMT5-14 were added to the cells and incubated for 2 hours, 6 hours, 12 hours, and 24 hours. At different time points, the cells were collected by adding trypsin, and the medium containing the drug was removed by washing the cell suspension three times with ice-cold PBS. After the last wash, ice-cold 70% methanol (200 μl) was added to the isolated cells, and the cell solution was stored at -20 ° C overnight. The extract was centrifuged at 15,000g for 15 minutes, and the supernatant was tested by LC-MS.

[0304] At different time points, the parent compound and the relevant active metabolites ( Figure 12). The parent compound of MMT5-14 and nano-MMT5-14 showed much higher concentrations in all cells and slowly decreased to lower levels with increasing incubation time. In contrast, the alanine metabolite showed higher concentrations of remdesivir in AMJ2-C11, Calu-3 and THP-1 cells. As seen in AMJ2-C11 and Calu-3 cells, the instability of remdesivir also partially leads to higher production of NTPs. However, similar amounts of NTPs were also observed for MMT5-14 and remdesivir in HUVEC and THP-1 cells. MMT5-14 and nano-MMT5-14 showed similar cellular uptake behavior, while the overall production of NUC and NTPs was lower in the nano-MMT5-14 group, which may be due to the higher stability of the compound in the nanoformulation.

[0305] References

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[0341] All publications and patents mentioned in the above description are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the compositions and methods of the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various modifications of the pattern for implementing the present invention that are apparent to those skilled in the relevant art are within the scope of the present invention.

Claims

1. A compound of formula I, in: X is the alanine side chain; R is or comprises a lipid chain comprising 10 to 20 carbons; and Ar is unsubstituted phenyl or naphthyl.

2. The compound according to claim 1, wherein the compound is a compound of formula Ia: in: X is an alanine side chain; and R is or comprises a lipid chain comprising 10 to 20 carbons.

3. The compound of claim 1, wherein the compound is:

4. A composition comprising an effective amount of the compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. The composition of claim 4, wherein the composition comprises a plurality of nanoparticles comprising albumin.

6. The composition of claim 5, wherein each of the plurality of nanoparticles has a diameter of 50 to 200 nm.

7. The composition of claim 5 or 6, wherein in each nanoparticle, albumin forms a shell surrounding the compound.

8. The composition of any one of claims 5-7, wherein the albumin is human serum albumin.

9. The composition of claim 4, wherein the composition comprises a plurality of liposomes, each liposome comprising lipids forming a bilayer.

10. The composition of claim 9, wherein the molar ratio of the compound to the lipid is from 0.0001:1 to 0.5:

1.

11. The composition of claim 9 or 10, wherein the molar ratio of the compound to the lipid is from 0.01:1 to 0.5:

1.

12. The composition of any one of claims 9-11, wherein each of the plurality of liposomes has a diameter of 50 to 200 nm.

13. The composition of claim 4, wherein the composition comprises a plurality of microparticles, each of the plurality of microparticles comprising: i) poly(lactic acid) (PLA) and / or poly(lactic-co-glycolic acid) (PLGA), and ii) the compound.

14. The composition of claim 13, wherein each of the plurality of microparticles has a diameter of 5 to 30 μm.

15. The composition of claim 13 or 14, wherein the microparticles are solid microparticles, porous microparticles or micro-encapsulated nanoparticles.

16. The composition of any one of claims 4-15, wherein the composition further comprises water.

17. The composition of any one of claims 4-16, wherein the composition is in aerosolized form.

18. The composition of any one of claims 4-17, wherein the composition is in the form of a dry powder.

19. The composition of any one of claims 4-18, wherein the composition further comprises an excipient and is in the form of a micronized powder.

20. The composition of claim 19, wherein the excipient is selected from the group consisting of lactose, mannitol, and PVA.

21. Use of a compound according to any one of claims 1 to 3 or a composition thereof in the manufacture / preparation of a medicament for treating SARS-CoV-2.

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