Self-assembling amphiphilic polymers as Anti-covid-19 agents

Abstract

To provide methods for treating viral infections including SARS-CoV-2, and to provide polymers for use in such treatments.SOLUTION: Provided are improved amphiphilic comb-type polymers with well-controlled molecular weights, structures, and end groups, and hydrophilic backbones with regularly arranged hydrophobic substituents. The polymers self-assemble in an aqueous environment to form core-corona nanoparticles that are capable of disrupting envelope proteins of a virus and can further encapsulate antiviral drugs or their prodrugs. Regularly arranged targeting groups can also mediate the attachment of the nanoparticles to an envelope of the virus.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to the field of amphiphilic block copolymers, and more particularly to the use of such copolymers in drug delivery. The present invention also relates to the field of targeted antiviral agents. [Background technology]

[0002] Amphiphilic block copolymers, consisting of hydrophobic and hydrophilic blocks, have been widely studied in recent years due to their ability to self-assemble into various nanostructures in response to changes in the surrounding solvent. (See Cameron et al., Can. J. Chem. / Rev. Can. Chim. 77:1311-1326 (1999)). In aqueous solution, the hydrophobic portion of the amphiphilic polymer tends to self-assemble to avoid contact with water and minimize the free surface energy of the system. At the same time, the hydrophilic block forms a hydrated "corona" in the aqueous environment, allowing the aggregates to maintain a thermodynamically stable structure. The result is a stable, latex-like colloidal suspension of polymer aggregate particles with a hydrophobic core and a hydrophilic corona.

[0003] Comb-type amphiphilic copolymers differ from block copolymers in that the main chain is primarily hydrophobic or hydrophilic, and polymer chains of different polarities are attached as side chains to the main chain rather than being incorporated into it. Comb-type copolymers have been created with hydrophobic main chains and hydrophilic side chains (Mayes et al., U.S. Pat. No. 6,399,700) and hydrophilic main chains and hydrophobic side chains (Watterson et al., U.S. Pat. No. 6,521,736). The former have been used for multivalent presentation of ligands to cell surface receptors, while the latter have been used for solubilizing and delivering drugs to cells.

[0004] Amphiphilic polymer aggregates have been investigated as solubilizing carriers for insoluble drugs, targeted drug delivery vehicles, and gene delivery systems. They spontaneously self-assemble into core-corona structures, which are more stable than conventional small molecule micelles, due to chain entanglement or the crystallinity of their internal hydrophobic regions. The polymeric nature of the carriers makes the aggregates relatively immune to the disintegration that occurs when conventional liposomes are diluted below their critical micelle concentration. The absence of a bilayer membrane allows them to easily fuse with cell membranes, delivering needed substances directly to cells. The amphiphilic nature of the aggregates also gives them detergent-like activity, and appropriately targeted aggregates are thought to be capable of fusing with and disrupting viral envelope proteins.

[0005] Due to the excellent biocompatibility of polyethylene glycol (PEG) and the apparent ability of PEG-coated "stealth" particles to evade the reticuloendothelial system, micelles, liposomes, and polymers containing PEG have been widely explored as materials for drug delivery systems. Numerous reports have explored the use of polyethylene glycol (PEG) as the hydrophilic component of PEG-lipids (forming liposomes and micelles). References: Krishnadas et al., Pharm. Res. 20:297-302 (2003). Similarly, self-assembling amphiphilic block copolymers that self-assemble into "polymersomes" with higher stability have also been investigated as carriers for drug solubilization and delivery. (Photos et al., J. Controlled Release, 90:323-334 (2003)). Controlled Release Mater. 20:131 (1993); Kwon et al., Langmuir, 9:945 (1993); Kabanov et al., J. Controlled Release, 22:141 (1992); Allen et al., J. Controlled Release, 63:275 (2000); Inoue et al., J. Controlled Release, 51:221 (1998); Yu and Eisenberg, Macromolecules, 29:6359 (1996); Discher et al., Science, 284:113 (1999); Kim et al., U.S. Patent No. 6,322,805; Seo et al., U.S. Patent No. 6,616,941 and Seo et al., European Patent No. EP 0583955. Luo et al., in Macromolecules 35:3456 (2002), describe PEG-conjugated polyamidoamine ("PAMAM") dendrimers suitable for polynucleotide delivery.

[0006] Comb polymers, produced by random functionalization or copolymerization, are mixtures of thousands of components with different molecular weights and branching patterns. The lack of a single, consistent structure can create problems in characterization and quality control and can be an obstacle when seeking regulatory approval. To overcome this drawback, amphiphilic comb polymers with regular, consistent structures have been introduced (see Diwan et al., U.S. Pat. No. 8,173,764), but the need for strict control of the molecular weight of such polymers remains.

[0007] The recent emergence and global spread of the novel coronavirus (SARS-CoV-2) and the resulting disease, COVID-19, have created an urgent need for effective chemotherapy agents. While some known antiviral agents have shown some effectiveness in reducing the severity and duration of infection, there remains a special need for drugs effective in treating COVID-19 and against future novel coronavirus strains that will almost certainly emerge. Summary of the Invention

[0008] One aspect of the present invention relates to a method for treating viral diseases comprising administering an antiviral agent encapsulated in a polyethylene glycol-based polymeric micelle. The present invention also relates to a polyethylene glycol-based polymeric micelle formulation for treating viral diseases, more specifically, for treating infectious diseases caused by viruses such as SARS-CoV-2. The formulation is composed of polyethylene glycol-based polymeric micelles and contains an encapsulated drug that is effective against viral diseases and has excellent in vitro antiviral activity. Encapsulation according to the present invention significantly improves the pharmacokinetics of the encapsulated drug and also enhances its aqueous solubility.

[0009] The present invention provides improved biocompatible comb polymers having structural formulas (4) and (5) shown below, as well as methods for making the improved polymers. [ka] (4) [ka] (5)

[0010] In the above structural formula, each X is independently OH or NHR, and R is a hydrophobic group of C10 to C18, preferably C14 to C16. The proportion of OH groups among the substituents X is in the range of 10% to 90%, preferably 20 to 65%, and more preferably 25 to 60%. Each L is independently OH or a ligand having specific binding affinity to the virus surface. The average value of M is in the range of 10 to 100, preferably 20 to 50. The value of n is in the range of 5 to 25, and the overall molecular weight of the polymer (structural formula (4)) may be in the range of 2,000 to 25,000 daltons, preferably 5,000 to 15,000 daltons.

[0011] The present invention provides aqueous suspensions of core-corona nanoparticles self-assembled from polymer (4), as well as methods for solubilizing antiviral drugs (and their prodrugs) by incorporating them into the hydrophobic core of the polymer nanoparticles. The present invention also provides hydrophobic prodrugs designed to be soluble in the hydrophobic core of the nanoparticles. In some embodiments of the present invention, polymer (4) can be modified to polymer (5) by covalently attaching a cell-specific, tissue-specific, or virus-specific targeting ligand. The addition of ligands to the repeat units of the polymers of the present invention allows for multivalent display of the ligand on the polymer (5) chain and on the nanoparticles.

[0012] The present invention also provides a method for treating or preventing viral infections in humans or other animals, comprising administering to said animal a suspension of self-assembled nanoparticles comprised of comb polymers having structural formula (4) or (5). The polymer particles preferably have an antiviral agent or prodrug dissolved or dispersed in the hydrophobic nanoparticle core.

[0013] The self-assembled nanoparticles possess inherent antiviral activity, even in the absence of small molecule antiviral agents or prodrugs. This antiviral activity is believed to be due to the detergent-like amphiphilic ability of the amphiphilic polymer to disrupt or denature the viral particle envelope. This activity is enhanced by the binding affinity of the multiple carboxylate groups and / or the ligand L to the target viral surface.

[0014] The present invention further provides methods for making the polymers, nanoparticles, and drug conjugates described herein. The polymers of the present invention self-assemble into polymer aggregates that are efficiently solubilized, distributed, and delivered in vivo, have inherent antiviral activity, and are non-toxic, biocompatible, and stable.

[0015] The present invention also relates to methods for treating viral diseases comprising administering antiviral agents encapsulated in the self-assembled nanoparticles of the present invention. The present invention also relates to pharmaceutical compositions comprising antiviral agents encapsulated in the self-assembled nanoparticles of the present invention, and the use of these compositions in the treatment of viral diseases, more specifically, infections caused by coronaviruses such as SARS-CoV-2. Such formulations significantly improve the pharmacokinetics of the encapsulated drugs and enhance their water solubility. The improved distribution and water solubility allow for the administration of various prodrugs that were previously ineffective. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a graph showing the molecular weight of the π-polymer versus the ratio of DTT to PEG dimaleate.

[0017] [Figure 2] 1 is a synthetic scheme for producing a polymer of the present invention.

[0018] [Figure 3] FIG. 2 is an illustration of FIG. 2, identifying the R groups.

[0019] [Figure 4] 1 is a plot showing cell viability as a function of unencapsulated drug or prodrug concentration.

[0020] [Figure 5] 1 is a plot showing cell viability as a function of encapsulated host concentration.

[0021] [Figure 6] 1 is a plot showing cell viability as a function of encapsulated guest concentration. [Figure 7] A comparison of the efficacy of encapsulated and non-encapsulated compounds plotted as a function of guest compound concentration. [Figure 8] The viability of uninfected cells is plotted against the host concentration of encapsulated compound. DETAILED DESCRIPTION OF THE INVENTION

[0022] Comb polymers having the general structure of the polymers of the invention are described in U.S. Patent Application Nos. 12 / 223,052 and 12 / 518,411 (Publication Nos. 2010 / 0260743 and 2010 / 0008938), which are incorporated herein by reference in their entireties. These materials, referred to as "π-polymers," are characterized in that the side chains R are not randomly or uniformly distributed along the polymer chain, but rather occur in pairs, with each pair being more or less regularly spaced along the polymer chain, depending on the degree of monodispersity of the PEG monomers.

[0023] These materials, referred to as "π-polymers," are characterized by the fact that the R side chains are not randomly or uniformly distributed along the polymer chain, but rather occur in pairs, with each pair being more or less regularly spaced along the polymer chain, depending on the degree of monodispersity of the PEG monomers. The hydrophobic R side chains are preferably C10-C18 alkyl groups, but may incorporate heteroatoms to provide dipole-dipole or hydrogen-bonding interactions with the encapsulated drug or prodrug. For example, ether, ester, amide, sulfoxide, and sulfonyl groups can be incorporated into some or all of the R groups.

[0024] The improved polymers of the present invention are characterized by narrow molecular weight distributions, controlled chain end architecture, low levels of hydrophobic substituents R, and a high density of carboxylate groups that function as affinity ligands for viral coat proteins. Polymers with low levels of hydrophobic substitution (e.g., those in which 10%, 20%, 30%, 40%, or 50% of R are hydrophobic) have been found to be more water-soluble and more suitable for injectable formulations.

[0025] Attaching ligands to the repeating units of the polymers of the present invention allows the ligands to be displayed multivalently on the polymer chain and on the nanoparticle surface, greatly increasing the affinity of the ligand for its target. For example, multivalent antibodies can be much more effective at eliminating their targets than conventional bivalent antibodies. Carbohydrate-binding proteins and sugars are known to be inherently multivalent, and monovalent binding is known to be ineffective. Similarly, multivalent peptides and sugar targeting moieties will be much more effective than single monomers. Increasing molecular weight by conjugation to a polymer reduces the renal clearance rate of peptides and other ligands. Furthermore, the PEG backbone provides peptides with benefits similar to those of PEGylation, including evasion of immune surveillance.

[0026] Furthermore, multivalent targeting moieties decorate and neutralize multivalent targets (e.g., viral particles) much more effectively than monovalent targeting moieties. The ability to display multiple (different) peptides in a multivalent format will lead to improved specificity.

[0027] Broadly, the present invention provides a comb polymer having the structure: [ka] (5) where each X is independently OH or NHR, R is a C10-C18 hydrophobic moiety, and each L is independently OH or a ligand having specific binding affinity for the viral surface. In structure (5), the average value of m ranges from 10 to 100, preferably from 20 to 50, while the value of n ranges from 5 to 25. In view of the synthetic methods described below, it will be understood that structure (5) is an idealized representation, and any of the carboxy groups depicted, including those located at the polymer termini, may be conjugated with a ligand, L.

[0028] Each ligand L may be, for example, any of the following moieties: [ka] In the depicted ligands, each R1 is independently hydrogen or C1-C4 alkyl, and each R2 is independently hydrogen, COR1, or CO2R1.

[0029] The comb polymers described above self-assemble into core-corona nanoparticles when dissolved in water or an aqueous medium. The present invention provides such nanoparticles having an antiviral drug or its prodrug dissolved or dispersed within the hydrophobic core.

[0030] The present invention provides a pharmaceutical composition comprising the above-described self-assembled nanoparticles, in which an antiviral drug or a prodrug thereof may or may not be dissolved, and a pharmaceutically acceptable aqueous carrier.

[0031] The present invention provides methods for treating or preventing viral infections, particularly infections caused by coronaviruses, including SARS-CoV-2, in humans and other animals by administering an effective amount of the comb polymer and the pharmaceutical composition described above.

[0032] A feature of the present invention is the enhanced combined activity of the polymer and the encapsulated drug. The polymeric micelles (host) have the property of degrading viruses by binding to and denaturing the viral envelope glycoprotein, i.e., by blocking the reinfection site of the viral life cycle, i.e., the site of infection of new cells by newly released viral particles. Meanwhile, the encapsulated drug (guest) can block the replication stage of the virus life cycle, where the virus produces progeny viruses within the cell. Sufficient blockage of both life cycle processes results in a synergistic reduction in viral replication and viral load.

[0033] Another feature of the present invention is the improved pharmacokinetics and pharmacodynamics afforded by encapsulation of the drug within polymeric nanoparticles. For example, remdesivir is known to be more effective when injected as a complex with sulfobutylether-β-cyclodextrin (SBECD). An extension of this concept to enhance the pharmacokinetics and pharmacodynamics of remdesivir upon encapsulation in polymeric micelles has been discussed in A. Chakraborty and A. Diwan, "Pharmacodynamics of Remdesivir: An Improved Approach for COVID-19 Treatment," J. Biomed. Res. Environ. Sci. (2020) 1(8):431-438A. In addition, in vivo test results have been reported by Chakraborty et al. in bioRxiv preprints (https: / / doi.org / 10.1101 / 2021.10.22.465399 and https: / / doi.org / 10.1101 / 2021.11.17.468980).

[0034] The polymers of the present invention may be prepared by the process shown in Scheme 1 and FIG. [ka] Scheme 1

[0035] It should be understood that in practice, the illustrated chemical reactions proceed with statistical product distribution and less than perfect efficiency, and that the polymer products shown in the schemes and claims are idealized representations rather than typical or average structures. For example, in the preparation of (3), the amidation reaction is not quantitative, and in practice, quantitativeness is undesirable. Furthermore, treatment of (3) with maleic anhydride (Step D in Figure 2), which the inventors believe is due to competitive Michael addition of the side chain hydroxyl groups to form the lactone ring, results in adduct yields in the range of 30-50%. The subsequent reaction with mercaptosuccinic acid (Step E in Figure 2) can proceed in yields ranging from 20% to 100%, depending on the amount of reagents, time, and temperature; these variables can be manipulated to control the density of carboxyl groups on the final product.

[0036] It is known that the Michael addition of thiols tends to occur at the β-position of maleic acid monoesters. (Yoon, HB, et al., Macromol. Yoon, HB, et al., "Michael Addition of Thiol Compounds to ω-Maleic Polyethylene Oxide: A Model Study for Site-Specific Protein Modification," Macromol. Res. 26:194-203 (2018), doi.org / 10.1007 / s13233-018-6021-4). However, regiochemistry is not 100% specific, and formulas (2), (3), and (4) should be understood to encompass positional isomers in which any sulfur atom is attached to the α- or β-position of any succinic acid carboxylate. Thus, the present invention encompasses compositions that are mixtures of positional isomers at the succinic acid moiety. Due to the asymmetric carbon at the sulfur-bearing carbon, the present invention also encompasses polymers containing mixtures of any or all possible positional and stereoisomeric combinations.

[0037] In structural formulas (3) and (4), the hydrophobic group R is preferably derived from a C8-C18 aliphatic amine RNH2 and is most conveniently attached to the polymer by amidation of the carboxylic acid group of polymer (2), as shown in Scheme 1. The hydrophobic group R is preferably a C8-C20 hydrocarbon group, which may be linear, branched, or contain one or more rings. Examples of groups R include, but are not limited to, n-octyl, 2-ethylhexyl, n-dodecyl, and n-hexadecyl. The solvating power of the hydrophobic core of the self-assembled nanoparticles can be increased by introducing halogen, ether, ester, amide, sulfone, sulfoxide, or nitrile groups into the hydrophobic group R. Here, when the term "hydrophobic" is applied to R, it means that the logP value (octanol / water partition coefficient) of the molecule RH is greater than 2. In a preferred embodiment, the logP value of RH is greater than 2.5.

[0038] In Scheme 1, the end groups of polymer (2) are controlled by using an excess of DTT and capping with maleic acid. As described below, precise control of the amount of DTT allows for the production of a desired molecular weight range. Amidation to produce polymer (3) can be achieved using any of a variety of carboxylic acid activators widely known in the peptide synthesis art. Suitable examples include, but are not limited to, CDI, DCC, DIC, and EDC. N-hydroxysuccinimide or N-hydroxysulfosuccinimide is preferably used in conjunction with a carbodiimide reagent. Carbonyldiimidazole (CDI) is a preferred reagent. Furthermore, esterification with maleic anhydride followed by addition of mercaptosuccinic acid to the resulting maleoyl group can be used to introduce up to six additional carboxyl groups at each branch point, as shown in Structure (4).

[0039] In practice, quantitative incorporation of carboxylate groups has been found to be difficult to achieve, and polymers represented by formula (4) may have an average of 3 to 5 carboxylic acids per branch site, depending on the reagents, reactants, and conditions used in the esterification and Michael addition processes. Therefore, the polymers described herein should be understood to contain some residual (unreacted) and unincorporated functional groups. Accordingly, the scope of the appended claims extends to polymers having from about 20% to 100% of the illustrated functionalization level, unless a specific functionalization level is explicitly recited. [Example]

[0040] General procedure for polymer synthesis The present invention provides a process for producing the comb polymers of the present invention. The primary starting material is polyethylene glycol, which is preferably dried by vacuum stirring at elevated temperatures prior to use. This drying process may require 8 to 12 hours, depending on the quality of the polyethylene glycol. After drying, the polyethylene glycol can be stored indefinitely under a dry, inert gas, such as nitrogen or argon.

[0041] To ensure that the polymers of the present invention have reproducible and consistent properties, it is preferred that the polyethylene glycol be of low dispersity. Most preferred are PEG polymers that are greater than 95% monodisperse, such as those commercially available from Nektar Therapeutics (formerly Shearwater Polymers, Huntsville, Alabama) and Polypure (Oslo, Norway). An example of a monodisperse PEG is Polypure's "PEG-28," which is greater than 95% HO(CH2CHO). 28 H, with a molecular weight of 1252. Also suitable is "Synthetic Grade Polyethylene Glycol 1000" available from MilliporeSigma (Burlington, Massachusetts). Polyethylene glycol from other sources may also be suitable, provided the certificate of analysis indicates a sufficiently narrow molecular weight distribution.

[0042] Controlling the molecular weight is important because polymers of different molecular weights (2) can produce different grades and types of amidated polymers (3) and further derivatized polymers. Regarding the pharmacokinetics of PEG polymers, it is known that smaller polymers are more likely to be excreted via glomerular filtration through the kidney, resulting in a shorter half-life, whereas larger molecular weight polymers circulate longer and may exhibit fecal excretion rather than urinary excretion as the primary route of excretion.

[0043] In theory, when two bifunctional compounds are reacted together, as in the preparation of polymer (2), using equimolar amounts can result in a very high molecular weight product. In practice, molecular weight is controlled by the Carruthers equation, which reflects the fact that the degree of polymerization can be controlled by using an excess of one reactant over the other. In this case, because PEG dimaleate (1) is not purified, it may contain residual maleic acid and / or PEG monomaleic acid, and the results of polymerization with DTT are not entirely predictable. However, the present invention provides a method for obtaining predictable molecular weights.

[0044] First, test reactions were performed on a batch of PEG dimaleate at different molar excesses of DTT using identical conditions, process equipment, and reactor geometry. The resulting polymer (2) was purified, and its molecular weight was measured by size exclusion chromatography and multi-angle light scattering (SEC-MALS). A plot of experimental molecular weight versus DTT / PEG dimaleate ratio was then generated, which is specific to a particular batch of PEG dimaleate.

[0045] A representative plot is shown in Figure 1. From this plot, the DTT / PEG dimaleate ratio required to achieve the desired molecular weight for a particular PEG dimaleate batch can be determined, and this ratio can be used in the manufacturing process to ensure that the desired molecular weight is achieved. This plot is considered to be a specific characteristic associated with that PEG dimaleate batch under specific process conditions. For different PEG dimaleate batches, the process is repeated and new plots generated to obtain the polymerization operating characteristics of that batch.

[0046] All reactions are carried out under an inert atmosphere such as nitrogen or argon with magnetic or mechanical stirring.

[0047] PM2-DTT polymer (2) PEG dimaleate (1) ("P10M2") was prepared from polyethylene glycol 1000 by the method described in U.S. Patent Publication No. 2010 / 0260743. The polymer was melted by heating to 60-80°C under a nitrogen atmosphere, and water was added to a final concentration of 40-50% (w / v). The pH of the solution was adjusted to 6-8.5 by adding DIPEA. Dithiothreitol (DTT) was added as a solution or solid at a ratio of 1.02-1.5 moles per mole of maleic acid double bond. The molar ratio of DTT to P10M2 was set based on the desired molecular weight of the P10M2-DTT polymer (2). The solution pH was monitored with a pH probe, and the viscosity was monitored with an ultrasonic viscometer probe in the reactor. The viscosity became constant after approximately 15 minutes and remained essentially unchanged for the next 30 minutes. Unreacted DTT and terminal sulfhydryl groups were quenched by adding maleic acid until the mixture tested negative with Ellman's reagent. The reaction mixture was acidified to pH 2-4 with 6N hydrochloric acid. The product P10M2-DTT (2) was purified by extraction into dichloromethane (DCM); low-molecular-weight impurities and salts resulting from the reaction of DTT with maleic acid remained in the aqueous layer and were removed. The organic layer was further washed with water, and the dichloromethane was then evaporated under reduced pressure to yield P10M2-DTT polymer (2), suitable for further processing. Alternatively, the P10M2-DTT polymer was precipitated as a low-melting, waxy solid by adding 2-4 volumes of heptane to the dichloromethane. Isolated yield: 70-90% of theoretical.

[0048] The degree of polymerization (DP) of the resulting polymers ranged from 3 to 14 depending on the amount of DTT used, and the molecular weights (by SEC-MALS) ranged from 4 kDa to 18 kDa.

[0049] The solvent extraction process described above is superior to the previously used tangential flow membrane filtration method, which resulted in low yields and very dilute solutions of polymer (2) with insufficient separation from contaminants.

[0050] Amidated PM2-DTT polymer (3) The dried polymer (2) (P10M2-DTT) is dissolved in a solvent such as dichloromethane, and the carboxyl groups are activated by reaction with an activating agent such as diisopropylcarbodiimide (DIC) or N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. N-Hydroxysuccinimide and N-hydroxysulfosuccinimide are used, particularly with carbodiimide-based reagents, to minimize side reactions, such as the conversion of reactive O-acylurea to unreactive N-acylurea. The molar equivalent of activating agent used depends on the desired alkylamine substitution, with the maximum amine substitution being equal to or less than the equivalent of activating agent used. Activation times can range from 15 minutes to 2 hours. For alkylamines such as hexadecylamine (HDA), tetradecylamine, and other C10-C18 alkylamines, a 0.1-0.2 molar excess of activating agent is used. The amidation reaction is performed at temperatures between 20 and 60°C, preferably between 30 and 50°C, depending on the amine. Larger amines have been found to react better at higher temperatures, as lower temperatures can cause micelle formation in the reaction mixture, rendering reactive groups unavailable. The reaction is quenched with water or acidic water to decompose any remaining activated carboxyl groups, and the amidated polymer (3) is extracted into dichloromethane or a suitable water-immiscible solvent and washed with water and dilute acid to remove water-soluble and basic impurities. The amidated polymer is further treated with a strong acid cation exchange resin to remove any remaining alkylamines, and the solvent is removed by vacuum distillation.

[0051] Apparent molecular weight is determined by SEC-MALS. Alkylamine content is quantified by acid hydrolysis of the polymer followed by reaction with an amine-reactive reagent such as fluorescamine. Unreacted carboxyl groups are estimated by determining the acid number of the polymer.

[0052] P10M2-DTT-C16 P10M2-DTT (1 mmol of carboxyl groups) was dissolved in dichloromethane in a reaction vessel equipped with a stirrer, condenser, and thermometer. The pH of the solution was adjusted to 2-4 with a tertiary base such as triethylamine or diisopropylethylamine. Carbonyldiimidazole (CDI, 0.5 mmol) was added under stirring at a temperature of 10-30°C while controlling the evolution of carbon dioxide. The reaction mixture was stirred at room temperature for 15-60 minutes to activate the carboxyl groups on the polymer. Then, n-hexadecylamine (HDA, 0.55-0.65 mmol) was added to the activated polymer. The reaction was stirred at 20-45°C for 2-24 hours, preferably 18-24 hours, or until TLC or mass spectrometry of the reaction mixture indicated the desired degree of reaction. The reaction was terminated by the careful addition of aqueous hydrochloric acid to decompose any remaining activated carboxylates. Further addition of aqueous hydrochloric acid allowed the aqueous layer (containing imidazole, water-soluble salts, and other water-soluble impurities) to be removed. The separated organic phase was washed again with water. Ethanol was added to a final concentration of 30-60%, and the polymer solution was then applied (by column or batch) to a strong acid cation exchange resin (H ) to remove unreacted amines. + The product was isolated by distillation of the dichloromethane-ethanol solvent under reduced pressure to give P10M2-DTT-C16 (3) as a waxy solid.

[0053] Alternatively, the reaction mixture at the end of the reaction can be diluted directly with dichloromethane to a starting polymer content of 4-10% (w / w), and the solution is then loaded (by column or batch) onto a strong acid cation exchange resin (H + The compound was treated with 3 to 10 equivalents of hexadecylamine (1 equivalent of hexadecylamine). The extent of amine removal was followed by TLC and mass spectrometry. The product was isolated by distillation of the dichloromethane solvent under reduced pressure to give P10M2-DTT-C16 (3) as a waxy solid.

[0054] Introduction of multidentate carboxylates: P10M2-DTT-C16-(M-MSA); P10M2-DT-(HDA)x(M-MSA)2 P10M2-DTT-C16(3) was heated to 90-120°C under a nitrogen atmosphere to form a stirrable melt, and then an excess amount of maleic anhydride in methyl isobutyl ketone was added. The amount of maleic anhydride added was at least a 20% stoichiometric excess relative to the calculated amount of hydroxyl groups present, preferably an 80-140% excess, and in some cases even a 300% excess or more. When maleic anhydride is used without a cosolvent such as MEK or MIBK, care must be taken to avoid sublimation. Therefore, maleic anhydride is added when the polymer temperature reaches approximately 50-80°C, preferably approximately 60-70°C. While the polymer is being heated with stirring, maleic anhydride pellets are mixed into the polymer melt. The temperature of the reaction vessel is then further increased to the reaction temperature. The reaction mixture is stirred at 70-140°C, preferably 80-100°C, to generate maleate esters of the DTT hydroxyl groups.

[0055] The reaction mixture is then cooled to approximately 40-70°C, diluted with water, and the pH is raised to 8-9 by the addition of DIPEA (diisopropylethylamine) or TEA (triethylamine). An excess of mercaptosuccinic acid (1-2 equivalents per equivalent of added maleic anhydride, preferably 1.2-1.8 equivalents, more preferably 1.4-1.6 equivalents) is then added to react with the double bond of the maleate ester at a pH of 8-9. The progress of the reaction can be monitored by mass spectrometry. The reaction mixture is then cooled to room temperature and extracted with a 1:1 dichloromethane-isopropyl acetate mixture to remove low-molecular-weight organic wastes. The pH is adjusted to 2-4 with hydrochloric acid, and the polymer is extracted from the aqueous layer into dichloromethane and subsequently precipitated by adding 1-4 volumes of n-heptane. The resulting solid is dissolved in butanol or isoamyl alcohol and reprecipitated by adding n-heptane. The resulting solid is then oven-dried under vacuum or nitrogen atmosphere, resulting in the product P10M2-DTT-C16-(M-MSA), also referred to below as P10M2-DT-(HDA)x(M-MSA)2.

[0056] In an alternative purification procedure, when TEA is used as a basic catalyst for the reaction, the reaction mixture is cooled and then titrated to pH 2-3 with 3-6 N hydrochloric acid, and the resulting free acid form of the polymer is extracted into dichloromethane. The dichloromethane solution is washed with 1 N hydrochloric acid, followed by water, to remove acid and water-soluble residues. The dichloromethane is distilled off under reduced pressure and then under vacuum. Alternatively, the dichloromethane solution is concentrated under reduced pressure, acetone is added, and the mixed solvent is distilled off. This process of replacing dichloromethane with acetone is repeated until dichloromethane is no longer detectable by gas chromatography. The acetone is completely removed by in situ vacuum distillation or, preferably, oven drying under a nitrogen atmosphere, to yield P10M2-DTT-C16-(M-MSA).

[0057] Ligand The representative virus targeting ligand disclosed below has a primary amino group that can be used to link the ligand to the carboxylic acid of the polymer to obtain an active agent.The method described is only a representative example, and other linking means using any of the various linkers and binding reactions known in the field of small molecule-polymer conjugates will be obvious to those skilled in the art.The ligands presented herein are divided into several categories. (a) a ligand containing a nicotinate moiety; (b) a ligand containing a caffeic acid moiety; (c) ligands containing both caffeic acid and nicotinic acid ester moieties, and (d) Ligands belonging to any of the above categories and amidated with L-cysteic acid.

[0058] Nicotinic acid esters [ka] Methyl 6-chloronicotinate is dissolved in tetrahydrofuran (THF) or methyl ethyl ketone (MEK) as a solvent. An equimolar amount of Boc-l-cysteine ​​methyl ester is added, followed by potassium carbonate or a tertiary organic base such as triethylamine or DIPEA (N,N-diisopropylethylamine). Water is added to precipitate the product as a solid, which is isolated by filtration. The filter cake is washed with a methanol-water mixture. The dried product is dissolved in dichloromethane and then treated with hydrochloric acid in dioxane to remove the t-Boc group. The product is isolated as the hydrochloride salt. [ka]

[0059] Synthesis of cysteine ​​amide derivatives (general procedure): Boc-L-cysteic acid and the nicotinic acid ester (a) from above are dissolved in dimethylformamide. The pH is adjusted to 7-8 with triethylamine. A slight molar excess of N-(dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) is added along with an equimolar amount of N-hydroxysuccinimide. After completion of the reaction, the mixture is partitioned between ethyl acetate and aqueous sodium carbonate. Cysteine ​​amide precipitates from the aqueous solution upon neutralization. [ka]

[0060] The nicotinic acid ester is dissolved in a solvent such as acetone. Triethylamine or other base is added to adjust the pH to 7-8.5. Boc-β-alanine-N-hydroxysuccinimide ester (1.05-1.2 equivalents) is added to the solution while maintaining the pH in the range of 7-8.5 by adding a base. After the reaction is complete, water is added to precipitate the product.

[0061] Caffeic acid [ka] Caffeic acid (1 mmol) and H-lysine(Boc)-methyl ester hydrochloride (1 mmol) are mixed in tetrahydrofuran (THF). Triethylamine is added to adjust the pH to the range of 5-8.5, followed by N,N'-diisopropylcarbodiimide, and the reaction mixture is stirred at 30-60°C until amidation is complete. The crude caffeoyl-L-lysine(Boc)-methyl ester is deesterified by treatment with 6N sodium hydroxide at 5-40°C. The deesterified material is extracted into an alkaline phase and acidified to pH 3-5 to precipitate the acid as a solid, which is isolated by filtration to yield caffeoyl-L-lysine(Boc)-OH. The t-Boc group is then removed as described above. [ka]

[0062] The resulting caffeoyllysine is amidated with cysteic acid according to the method described above.

[0063] Ligand L60(OMe)2 [ka] β-Alanine-cys(methylnicotinate)-OMe is suspended in tetrahydrofuran and the pH is adjusted to 8-9.5 using triethylamine. To this suspension, a solution of caffeoyl-lysine (Boc)-OH in THF is added. DIC and NHS (1.2 equivalents each) are added, and the coupling reaction is carried out at 20-40°C. After completion of the reaction, the reaction mixture is acidified to pH 4-4.5 using hydrochloric acid. Water is added to precipitate the product, which is then purified by washing. The t-Boc protecting group is then removed with HCl-dioxane according to the method described above.

[0064] L60(OMe)2L-cysteine ​​acid amide [ka] This compound is prepared from L60(OMe)2 and Boc-L-cysteic acid according to the general procedure previously described. The t-Boc protecting group is removed with HCl-dioxane according to the general procedure previously described.

[0065] Other Ligands The following ligands can be prepared by similar methods: [ka] [ka] [ka] [ka] [ka] [ka]

[0066] Although the above ligands are shown as methyl esters, ethyl, n-propyl, and butyl esters are also contemplated. One or both of the catechol OH groups of caffeic acid may be esterified as carboxymethyl, carboxyethyl, acetate, propionate, etc.

[0067] Ligand attachment to polymers Using standard peptide coupling techniques, the carboxyl groups of polymer (4) are activated with carbonyldiimidazole, N,N-diisopropylcarbodiimide, etc., followed by the addition of the desired ligand. This method allows the conjugation of various virus-specific ligands to the polymer via amide bonds. The amount of ligand can be adjusted as desired. A representative structure (5) is shown below, where L represents the ligand attached by amidating the carboxyl groups of the polymer, and X represents a mixture of the OH and NHR groups described above. While coupling to the least sterically hindered carboxyl group is illustrated, it will be understood that any available carboxyl group, including the end groups of the polymer, can be amidated. [ka] (5)

[0068] Attachment of ligands to polymers The dry polymer P10M2-DT-(HDA)x(M-MSA)2 (4) is dissolved in a suitable inert solvent, such as DMF, at 15-45°C under a nitrogen atmosphere. The carboxylic acid groups of the polymer are activated by adding CDI (5-30% excess relative to the number of carboxylic acid groups intended for amidation). Activation is carried out for 30-60 minutes, after which a solution of the ligand L60(OMe)2 in DMF is added to the activated polymer. The pH is maintained at 7.5-9 using TEA or DIPEA. The coupling reaction is continued for 2-20 hours. Upon completion, the pH is adjusted to 3.5-4.5 with hydrochloric acid, followed by precipitation of the polymer-ligand conjugate with excess water. The polymer-ligand conjugate is then purified by solvent-water extraction, dialysis using an appropriate molecular weight cutoff membrane, or tangential flow filtration.

[0069] Drug Encapsulation: The host polymer and guest drug are dissolved in a common solvent such as dimethyl sulfoxide (DMSO), ethanol, tetrahydrofuran (THF), or dichloromethane (DCM) at a mass ratio of 3:1 to 40:1, preferably 10:1 to 20:1, and mixed to obtain a transparent solution. When combining drugs, the ratio is determined by the degree of the desired effect (e.g., EC 50 (50% effective) and EC 90 It is known that the activity of each compound is balanced if the ratio of physiologically effective concentrations that give the EC (90% effective ratio) is close to the physiologically effective concentration ratio. 50 and E.C. 90 is difficult to measure accurately, so cell culture-based EC 50 and E.C. 90 Therefore, the range of encapsulation ratios for a particular guest and host is determined by their respective EC 50 Value or EC 90The ratio of the guest drug to the total guest drug is preferably in the range of 0.01 to 0.01. Depending on the solvent used, the solution is evaporated in an oven or rotary evaporator, or freeze-dried. The dried mixture is reconstituted in water or a suitable buffer to obtain an emulsion in which the guest drug is distributed within the self-assembled polymer nanoparticles. The encapsulation rate of the guest drug is then determined by an appropriate method, such as HPLC or UV-visible spectroscopy.

[0070] The hydrophobic core of self-assembled nanoparticles allows them to dissolve or suspend hydrophobic drugs and prodrugs, which are typically difficult to formulate into effective formulations. This allows prodrugs to be designed to achieve optimal pharmacokinetics without compromising water solubility and / or bioavailability. Many alkyl- and alkoxycarbonyl-type prodrugs are known to those skilled in the art, and their preparation methods are widely known and, for the most part, routine. Representative examples are provided below, but most known methods are applicable to a variety of substrates. The use of esters of C1-C18 aliphatic and aromatic carboxylic acids, carbonates derived from C1-C18 aliphatic and aromatic alcohols, and carbamates derived from C1-C18 aliphatic and aromatic amines is considered within the scope of the present invention. This invention enables the administration of hydrophobic drugs and prodrugs that may not previously have been considered clinical candidates.

[0071] Antiviral drugs, prodrugs, and drug candidates that can be used in the present invention include, but are not limited to, remdesivir, acyclovir, molnupiravir, PF-00835231, ivermectin, colchicine, mebendazole, CDI-45205, GC-376, and various prodrug esters, amides, and carbamates thereof. The novel and known drug derivatives (i.e., prodrugs) of the present invention are preferably lower alkyl esters or lower alkoxycarbonyl esters (i.e., carbonates) of antiviral drugs known to those skilled in the art, and these are prepared by known acylation methods or modifications thereof. For example, esterification can be carried out using acid anhydrides, acid chlorides, and activated carboxylic acids, as is well known to those skilled in the art. Alkoxycarbonyl esters can also be prepared using alkoxycarbonyl chlorides. Suitable solvents for these reactions are dipolar aprotic solvents such as DMSO, DMF, and NMP.

[0072] Representative drug and drug derivative combinations with polymers (4) and (5) are listed in Tables 1 and 2, respectively, along with the calculated log D values ​​for the guest components. The calculated parameter log D takes into account the overall distribution of both ionized and non-ionized forms of the compound, whereas the log P calculation is based only on the distribution of the non-ionized (neutral) compound. (H. Kubinyi, "Lipophilicity and Drug Activity," Prog. Drug Res. (1979) 23:97-198; doi:10.1007 / 978-3-0348-7105-1_5.(1979) 23:97-198). Table 1 Polymer (4), a compound encapsulated in P10M2-DT-(HDA)x(M-MSA)2 [Table 1] JPEG2025169240000022.jpg7080JPEG2025169240000023.jpg9980JPEG2025169240000024.jpg10881JPEG2025169240000025.jpg11481JPEG2025169240000026.jpg11081JPEG2025169240000027.jpg4580* LogD value is MarvinSketch TM Calculations were performed using software (version 5.4.0.1 or 6.1.2, ChemAxon, Budapest, Hungary). Table 2 Compound P10M2-DT-(HDA)x(M-MSA)2L-60(OMe)2 conjugate encapsulated in polymer (5) [Table 2] *LogD value is MarvinSketch TM The results were determined by ChemAxon software, version 5·4·0·1 or 6.1.2 (ChemAxon, Budapest, Hungary).

[0073] General packaging procedure The host polymer and guest antiviral drug or its derivative are dissolved in a common solvent in a ratio of 3:1 to 20:1, depending on the host and guest, and mixed to obtain a clear solution. Mixing can be achieved by stirring or repeated pumping using a double- or single-barrel syringe. Suitable solvents include, but are not limited to, dimethyl sulfoxide (DMSO), ethanol, tetrahydrofuran (THF), dichloromethane (DCM), and acetone. The solution is evaporated using a vacuum oven, rotary evaporator, or freeze dryer. The dried mixture is reconstituted in water or an appropriate buffer to obtain a nanoemulsion in which the guest component is dispersed within the polymer. The encapsulation rate of the guest drug is determined by an appropriate method, such as HPLC or UV-visible spectroscopy.

[0074] General procedure for esterification The 3-hexanoyloxybutoxymethylphosphonates of adenine and uracil were prepared by reacting 3-hydroxybutoxymethylphosphonate with hexanoic anhydride in dimethyl sulfoxide in the presence of catalytic amounts of 4-dimethylaminopyridine (DMAP). The hydroxyl groups in Boc-protected compounds were also esterified by reaction with various acid anhydrides in the presence of 4-DMAP, followed by removal of the Boc group to give esters with various hydrophobicities suitable for encapsulation. In cases where a heterocyclic nitrogen susceptible to acylation was present, acid chlorides were used instead of acid anhydrides in N,N-dimethylacetamide without a catalyst.

[0075] [4-(6-amino-9H-purin-9-yl)cyclopent-2-enyl]methanol (Example 18) The process described in EP1660498B1 was applied as follows: 5-Amino-4,6-dichloropyrimidine (20 mmol) and (1S,4R)-4-amino-2-cyclopentene-1-methanol hydrochloride (21 mmol) were mixed in 1-butanol (25 mL). Anhydrous sodium bicarbonate (50 mmol) was added, and the reaction mixture was heated to 80–90 °C. The reaction progress was monitored by mass spectrometry. After 2–4 h, the reaction mixture was cooled and filtered to remove inorganic salts and bicarbonate. The residue was washed three times with 5 mL of 1-butanol. The organic layer was washed with water and evaporated to give {4-[(5-amino-6-chloropyrimidin-4-yl)amino]cyclopen-2-en-1-yl}methanol in approximately 90% yield (m / z 241).

[0076] The product (10 mmol) from the above reaction was dissolved in 1-butanol (20 mL). Trimethyl orthoformate (11 mmol) was added, followed by concentrated sulfuric acid (0.5 mmol). The reaction mixture was heated at 80-90 °C for approximately 2 hours until the disappearance of the starting material was determined by mass spectrometry. The reaction mixture was cooled and stirred for 30 minutes to neutralize the acid with sodium bicarbonate. The mixture was filtered to remove salts. Evaporation of the solvent afforded [4-(6-chloro-9H-purin-9-yl)cyclopen-2-en-1-yl]methanol in approximately 80% yield (m / z 251).

[0077] This material (10.7 mmol) was dissolved in a mixture of isopropanol (15 mL) and concentrated aqueous ammonia (15 mL) and heated to 70 °C in a pressure vessel for 24-72 h. The solution was then evaporated to dryness. The solid residue was triturated with hexane-acetone to give [4-(6-amino-9H-purin-9-yl)cyclopen-2-enyl]methanol (m / z 232) in >90% purity by HPLC.

[0078] 2-(6-amino-9H-purin-9-yl)-4-hydroxybutanamide t-Butyl N-[(t-butoxy)carbonyl]-N-(9H-purin-6-yl)carbamate (10 mmol) was dissolved in dry tetrahydrofuran (10 mL). Sodium hydride (20 mmol) was added at room temperature. To form the sodium salt, the suspension was warmed to 60°C and stirred for 5 minutes. 2-Bromo-4-hydroxybutyric acid γ-lactone (20 mmol) was added, and the reaction mixture was refluxed for 3 hours while monitoring the reaction progress by mass spectrometry. The reaction mixture was cooled to room temperature, and methanol was added to quench the reaction. The reaction mixture was filtered to give t-butyl N-[(t-butoxy)carbonyl]-N-[9-(3-methyl-2-oxoxolan-3-yl)-9H-purin-6-yl]carbamate (m / z 420). The product (1.41 g) was dissolved in tetrahydrofuran and concentrated aqueous ammonia (5 mL). The reaction mixture was stirred at room temperature for approximately 2.5 hours and then concentrated to dryness to give the product as a yellow, hygroscopic solid. Mass spectrometry analysis revealed a mixture of the mono-Boc derivative (m / z 337) and the bis-Boc derivative (m / z 437). Silica gel column chromatography using ethyl acetate-methanol as a solvent afforded the mono-Boc derivative as the major product. The N6-Boc protecting group was then removed by treatment with hydrochloric acid in dioxane, followed by treatment with concentrated aqueous ammonia to afford the title compound.

[0079] Alternatively, to remove the t-Boc group, t-butyl N-[(t-butoxy)carbonyl]-N-[9-(2-oxoxolan-3-yl)-9H-purin-6-yl]carbamate was first treated with hydrochloric acid in dioxane. The resulting lactone (148 mg) was suspended in tetrahydrofuran (2 mL) and stirred with concentrated aqueous ammonia (2 mL) at room temperature for 1 hour. After 1 hour, the solvent was removed to give the title compound (160 mg, m / z 237) as a white solid in greater than 90% purity.

[0080] The above procedure was carried out using alkylamines of various chain lengths in place of ammonia to obtain N-alkylbutanamides with a variety of LogD values.

[0081] 2-(6-amino-9H-purin-9-yl)-4-hydroxy-2-methylbutanamide The above procedure was carried out using 2-bromo-2-methyl-4-hydroxybutyric acid γ-lactone to give the title compound.

[0082] [4-(6-amino-9H-purin-9-yl)cyclopent-2-en-1-yl]methanol O-hexanoate (Example 11) [4-(6-amino-9H-purin-9-yl)cyclopent-2-en-1-yl]methanol (0.56 mmol) was dissolved in N,N-dimethylacetamide (1 mL). Hexanoyl chloride (7.15 mmol) was added at room temperature. After approximately 1 hour of reaction at room temperature, the reaction was quenched by adding water to hydrolyze any unreacted hexanoyl chloride. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed with saturated aqueous sodium bicarbonate and then twice with water. The monohexanoate ester obtained after concentration from the ethyl acetate layer was isolated in 55% yield and approximately 95% purity (by HPLC and mass spectrometry).

[0083] 3'-Deoxyadenosine divalerate (Example 10) Following the procedure described above, 3-deoxyadenosine was reacted with valeric acid to give the title compound.

[0084] 3-Hydroxy-4-[(4-methylbenzenesulfonyl)oxy]butoxy}methanephosphonate In a dry round-bottom flask, tetrahydrofuran (50 mL) and sodium hydride (50 mmol, 60% suspension in mineral oil) were combined under ice cooling and nitrogen atmosphere and stirred for 5 minutes. 4-(2-hydroxyethyl)-2,2-dimethyl-1,3-dioxolane (40 mmol) was added, and the temperature was allowed to rise to room temperature. Diethyl p-toluenesulfonyloxymethylphosphonate (44 mmol) dissolved in tetrahydrofuran (20 mL) was added, and the reaction mixture was stirred at room temperature for 2 hours until mass spectrometry indicated the reaction was complete. The product, diethyl {[2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy]methyl}phosphonate (m / z 297), was treated with 1.25 M hydrochloric acid in ethanol to remove the ketal protecting group. The reaction mixture was concentrated to dryness to give the vicinal diol, diethyl [(3,4-dihydroxybutyl)methyl]phosphonate (m / z 257).

[0085] Next, diethyl [(3,4-dihydroxybutyl)methyl]phosphonate (1 mmol) was tosylated at the terminal hydroxyl group with p-toluenesulfonyl chloride (1 mmol) in the presence of triethylamine and dibutyltin oxide (0.02 mmol) in dichloromethane at room temperature. The target compound, diethyl {3-hydroxy-4-[(4-methylbenzenesulfonyl)oxy]butoxy}methanephosphonate (m / z 411), was isolated by silica gel column chromatography.

[0086] Diethyl {[4-(6-amino-9H-purin-9-yl)-3-hydroxybutoxy]methyl}phosphonate diisopropyl {[4-(6-amino-9H-purin-9-yl)cyclopent-3-en-1-yl]methoxy}methylphosphonate The methylphosphonate derivative of adenine was prepared by stirring N6-bis-Boc adenine (1 mmol) and sodium hydride (1.5 mmol, 60% suspension in mineral oil) in tetrahydrofuran (5 mL) for 5 min, adding diethyl {3-hydroxy-4-[(4-methylbenzenesulfonyl)oxy]butoxy}methanephosphonate (10 mmol), and refluxing the reaction mixture overnight to complete the reaction. Water (10 mL) was added, and the reaction mixture was extracted with ethyl acetate. The ethyl acetate layer was concentrated to dryness. Chromatographic purification afforded N6-bis-Boc-protected diethyl {[4-(6-amino-9H-purin-9-yl)-3-hydroxybutoxy]methyl}phosphonate. The purified product (100 mg) was treated with 4N hydrochloric acid in dioxane at room temperature for several hours to remove the Boc protecting group, affording the title product (m / z 374) as a white solid (28.5 mg).

[0087] Diethyl {[4-(6-amino-9H-purin-9-yl)-3-hexanoyloxybutoxy]methyl}phosphonate (Example 13) Using the method used in Example 11, the N6-bis-Boc-protected diethyl {[4-(6-amino-9H-purin-9-yl)-3-hydroxybutoxy]methyl}phosphonate prepared above was reacted with hexanoyl chloride to provide the title compound.

[0088] Diethyl {[4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-3-hydroxybutoxy]methyl}phosphonate A suspension of uracil (1 mmol) and sodium hydride (1.5 mmol) in N,N-dimethylformamide (1 mL) was stirred at room temperature for 5 min. Diethyl {3-hydroxy-4-[(4-methylbenzenesulfonyl)oxy]butoxy}methanephosphonate (1 mmol) dissolved in N,N-dimethylformamide (1 mL) was added, and the mixture was stirred at 70-110 °C for 48 h. The reaction was quenched by adding methanol. After removing the solvent, the residue was purified by column chromatography on silica gel. Elution with 7.5% methanol in ethyl acetate afforded the title compound as a white solid (100 mg) (m / z 351 (M+1), 373 (M+Na), 349 (M-1)).

[0089] Diethyl {[4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-3-hexanoyloxybutoxy]methyl}phosphonate (Example 14) Using the method used in Example 11, diethyl {[4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-3-hydroxybutoxy]methyl}phosphonate, prepared as above, was reacted with hexanoyl chloride to provide the title compound.

[0090] Diisopropyl {[4-(6-amino-9H-purin-9-yl)cyclopent-2-en-1-yl]methoxy}methylphosphonate (Example 12) The compound (1 mmol) was dissolved in N,N-dimethylacetamide (2.5 mL) in a dry round-bottom flask under a nitrogen atmosphere. Sodium t-butoxide (1.2 mmol) was added, and the mixture was stirred at room temperature for 5 minutes. Diisopropyl bromomethylphosphonate (1.1 mmol) was added, and the reaction mixture was stirred at 50 °C. After 4 hours at 50 °C, additional sodium t-butoxide (1.2 mmol) was added, and the reaction mixture was stirred at 50 °C for an additional hour. After a total of 5 hours at 50 °C, when mass spectrometry indicated the reaction was complete, the reaction mixture was cooled to room temperature and quenched by the addition of water (10 mL). The pH was adjusted to 7-7.5 with 1N aqueous hydrochloric acid, and the solution was extracted three times with ethyl acetate. The organic layer was washed once with water. The ethyl acetate layer was acidified with 1N aqueous hydrochloric acid and extracted three times with water to extract the product as the hydrochloride salt. The aqueous layer was adjusted to pH 7-7.5 with sodium bicarbonate and extracted three times with ethyl acetate, followed by one extraction with dichloromethane. The dichloromethane-ethyl acetate mixed phase was concentrated under reduced pressure to give the title compound (65 mg, m / z 410).

[0091] Acyclovir esters Various ester derivatives of acyclovir, ranging from butyrate to palmitate, as well as benzoate and trimethoxybenzoate derived from the primary alcohol, were prepared to investigate their encapsulation efficiency and antiviral properties. Acyclovir was dissolved in dry DMSO to obtain approximately 10–20% solutions. 5–10 mol% 4-dimethylaminopyridine (DMAP) was added to this solution, followed by 1.1–2 mol equivalents of the selected acid anhydride. The solution was stirred until completion of the reaction was confirmed by mass spectrometry. Water was added to precipitate the acyclovir ester as a white solid, which was then washed with water and acetone. Yields were generally greater than 70%.

[0092] Acyclovir octanoate (Example 15) In a 150 mL dry round-bottom flask, acyclovir (2 g, 8.9 mmol) was dissolved in 30 mL of dry DMSO at 20–30°C. To this solution was added DMAP (0.1 g, 0.9 mmol), followed by octanoic anhydride (3.6 g, 13.3 mmol). The reaction mixture was stirred at room temperature for 1–2 h until TLC and mass spectrometry showed that most of the acyclovir had disappeared. Water (300 mL) was added slowly to precipitate the product as a white waxy solid. The product was recrystallized from acetone to give the octanoate ester (2.25 g, 72% of the theoretical yield).

[0093] Remdesivir diacetate In a dry reaction flask equipped with a drying tube and stir bar, remdesivir (1.2 g, 2 mmol) was dissolved in DMSO (5 mL) at room temperature to obtain a clear solution. DMAP (25 mg, approximately 0.2 mmol) was added and stirred until the solution became clear. Acetic anhydride (0.214 g, approximately 0.20 mL, 2.1 mmol) was added, and the reaction was monitored by TQ-MS and TLC. The reaction was complete in less than 1 hour. The reaction mixture was quenched with water, and the resulting white precipitate was washed with water to remove acetic acid.

[0094] Remdesivir dibutanoate (Example 16) In a dry reaction flask equipped with a drying tube and a stir bar, remdesivir (1.2 g, 2 mmol) was dissolved in DMSO (5 mL) at room temperature to obtain a clear solution. DMAP (25 mg, approximately 0.2 mmol) was added and stirred until the solution became clear. Butyric anhydride (332 mg, 2.1 mmol) was added, and the reaction was monitored by TQ-MS and TLC. The reaction was complete in less than 1 hour. Water was added to the reaction mixture to quench the reaction, and the resulting white precipitate was washed with water.

[0095] Encapsulation of Remdesivir (Example 17) A 10–20% (w / w) solution of P10M2-DTT-C16-(M-MSA) polymer (4) was prepared in ethanol. To the polymer solution, 5–20% solid remdesivir was added based on the weight of the polymer used. The mixture was thoroughly stirred to dissolve the remdesivir, then evaporated to dryness under nitrogen at 35–60°C and concentrated to constant weight. The dried material was then dissolved in PBS or water at pH 6–7 and filter-sterilized for further use. The remdesivir concentration was determined by HPLC or UV analysis.

[0096] Effectiveness in cell culture For compounds soluble in ethanol, the compound (guest) was dissolved in ethanol. x (M-MSA) γ The polymer (host) solution was mixed with the guest:host solution at a weight ratio of 1:20. The solution was evaporated under nitrogen in an oven at approximately 50°C. The resulting dried film was redissolved in PBS containing 5% ethanol and subjected to testing. It should be noted that some materials do not dissolve properly in 1x PBS and may precipitate upon refrigeration. Ethanol was added to all samples to ensure uniformity in this study.

[0097] Ethanol can be completely avoided in typical pharmaceutical formulations. Drugs can also be formulated in osmotically balanced or pH-adjusted solutions depending on the route of administration. For example, physiologically balanced solutions containing mannitol, sodium chloride, or other osmolality adjusters can be prepared for injection, infusion, or inhalation. For oral administration, a slightly sour taste is often preferred, and sweeteners, taste masking agents, and flavorings can be added without compromising encapsulation.

[0098] For compounds that were not sufficiently soluble in ethanol, the guest compound was dissolved in DMSO and P10M2DT(HDA) in DMSO. x (M-MSA) γThe DMSO solution was mixed with the host polymer solution at a ratio of 1:20. The resulting DMSO solution was lyophilized. The resulting lyophilized powder was dissolved in PBS containing 5% ethanol as described above.

[0099] The host polymer and remdesivir (RDV) were used as positive controls, and PBS and DMSO were used as solvent controls. Drug and prodrug compounds were in DMSO solution or P10M2DT(HDA). x (M-MSA) γ The nanoparticles were encapsulated in PBS buffer and exposed to MRC5 lung fibroblasts (ATCC CCL-171) infected with hCoV-229E in cell culture plates at different drug concentrations. x (M-MSA) γ Remdesivir, a well-known and approved SARS-CoV-2 treatment with broad anti-coronavirus activity, was also used as a positive control in PBS buffer. The solvent, PBS, was used as a negative control. Remdesivir was dissolved in DMSO due to its poor water solubility, and DMSO was also used as a negative control (results not shown). The improvement in cell viability, which correlated with the inhibition of viral growth, was measured using CellTiter-Glo. TM Assay (Promega, Madison, WI, USA) was used to measure the ATP concentration.

[0100] Cytotoxicity in cell culture Encapsulated and non-encapsulated compounds were added to non-virally infected cell cultures and analyzed using the same CellTiter-Glo TM The assay was used to measure cell viability.

[0101] result Figure 4 plots cell viability versus concentration of unencapsulated compounds, demonstrating the efficacy of selected compounds as antiviral agents. Nearly all compounds exhibited significant concentration-dependent antiviral activity worthy of further investigation.

[0102] Figure 5 plots cell viability versus host polymer concentration, demonstrating the antiviral efficacy of the compounds when encapsulated in P10M2DT(HDA)x(M-MSA)y polymer (4) nanoparticles at a 1:20 ratio. Some of these encapsulated compounds demonstrated superior efficacy to remdesivir (Example 17) in terms of maximal cell viability improvement. In particular, Examples 10, 11, 12, and 13 demonstrated significantly improved efficacy compared to the host polymer (4) itself, confirming their efficacy as comparable to or even superior to remdesivir.

[0103] Figure 6 shows plots of cell viability versus concentration of encapsulated guest compound. These plots show that cell viability improves with increasing amounts of compound, in contrast to remdesivir (Example 17), which exhibits toxicity with increasing concentrations.

[0104] Figure 7 compares the efficacy of encapsulated and unencapsulated compounds when plotted against guest compound concentration. These plots clearly show that encapsulation of the guest compound significantly increases its effective activity.

[0105] Figure 8 shows plots of uninfected cell viability versus host concentration of encapsulated compound. These plots demonstrate the high cytotoxicity of encapsulated remdesivir and the relatively low toxicity of the tested drugs and prodrugs.

[0106] All test compounds, except for remdesivir, showed no toxicity at concentrations up to 200 μg / mL (CC 50 did not reach that level).

[0107] Although remdesivir demonstrated superior maximum viral suppression at lower concentrations than the synthetic and encapsulated drug of the present invention, its efficacy rapidly declined at slightly higher concentrations due to cytotoxicity. Clinical trials of remdesivir have shown that its clinical efficacy was limited, and that higher concentrations were not an option due to toxicity.

[0108] The above examples are not intended to be limiting but merely representative, and the present invention encompasses any combination of known prodrugs, including but not limited to ester, carbonate, ether, and carbamate type prodrugs, in combination with known antiviral drugs amenable to such derivatization, as well as the use of any of these prodrugs in combination with the comb polymers of the present invention.

Claims

1. 1. A method for the treatment or prevention of a viral infection in an animal, comprising administering to said animal a comb polymer having the structure: In the above structure: X is independently OH or NHR, and R is C １０ ~C １８ is a hydrophobic group; 10% to 90% of the X groups are OH; each L is independently OH or a ligand having specific binding affinity for the surface of the virus; The average value of m ranges from 10 to 100; The average value of n ranges from 5 to 25. 【Chemistry 1】

2. 2. The method of claim 1, wherein the comb polymer is in the form of a self-assembled core-corona nanoparticle, and an antiviral drug or a prodrug thereof is encapsulated within the core of the core-corona nanoparticle.

3. Each L group is 【Chemistry 2】 and each L group is R １ are each independently hydrogen or C １ ~C ４ alkyl, and R ２ are each independently hydrogen, COR １ , or CO ２ R １ 2. The method of claim 1, wherein the ligand is selected from the group consisting of compounds

4. The method of claim 3, wherein the comb polymer is in the form of self-assembled core-corona nanoparticles, and further comprises an antiviral drug or a prodrug thereof within the core-corona nanoparticles.

5. The method of any one of claims 1 to 4, wherein the virus is a coronavirus.

6. 5. The method of any one of claims 1 to 4, wherein the virus is SARS-CoV-2.

7. 5. The method of claim 4, wherein the antiviral drug or a prodrug thereof is a compound selected from the group consisting of remdesivir, GS-441524, 3'-deoxyadenosine, acyclovir, molnupiravir, [4-(6-amino-9H-purin-9-yl)cyclopent-2-enyl]methanol, 2-(6-amino-9H-purin-9-yl)-4-hydroxybutanamide, diethyl{[4-(6-amino-9H-purin-9-yl)-3-hydroxybutoxy]methyl}phosphonate, diethyl{[4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-3-hydroxybutoxy]methyl}phosphonate, PF-00835231, nilmatrervir, ritonavir, ivermectin, colchicine, mebendazole, CDI-45205, and GC-376, or a prodrug thereof.

8. A comb polymer having the following structure: In the above structure: Each X group is independently OH or NHR, and R is C １０ ~C １８ a hydrophobic group of 10% to 90% of the X groups are OH; each L group is independently an OH or a ligand having specific binding affinity for the surface of the virus; The average value of m ranges from 10 to 100; The average value of n ranges from 5 to 25. 【Transformation 3】

9. 9. The comb polymer of claim 8, wherein the comb polymer is in the form of self-assembled core-corona nanoparticles, and further comprises an antiviral drug or a prodrug thereof within the core-corona nanoparticles.

10. Each L group is 【Chemistry 4】 and R １ are each independently hydrogen or C １ ~C ４ alkyl, and R ２ are each independently hydrogen, COR １ , or CO ２ R １ 9. The comb polymer of claim 8, wherein the ligand is selected from the group consisting of compounds

11. 11. The comb polymer of claim 10, wherein the comb polymer is in the form of self-assembled core-corona nanoparticles, and further comprises an antiviral drug or a prodrug thereof within the core-corona nanoparticles.

12. 12. The comb polymer of claim 11, wherein the antiviral drug or prodrug thereof is a compound selected from the group consisting of remdesivir, GS-441524, 3'-deoxyadenosine, acyclovir, molnupiravir, [4-(6-amino-9H-purin-9-yl)cyclopent-2-enyl]methanol, 2-(6-amino-9H-purin-9-yl)-4-hydroxybutanamide, diethyl{[4-(6-amino-9H-purin-9-yl)-3-hydroxybutoxy]methyl}phosphonate, diethyl{[4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-3-hydroxybutoxy]methyl}phosphonate, PF-00835231, nilmatrervir, ritonavir, ivermectin, colchicine, mebendazole, CDI-45205, GC-376, and prodrugs thereof.

13. 12. The comb polymer of claim 11, wherein the antiviral drug or prodrug thereof is a compound selected from the group consisting of remdesivir, GS-441524, 3'-deoxyadenosine, acyclovir, molnupiravir, [4-(6-amino-9H-purin-9-yl)cyclopent-2-enyl]methanol, 2-(6-amino-9H-purin-9-yl)-4-hydroxybutanamide, diethyl{[4-(6-amino-9H-purin-9-yl)-3-hydroxybutoxy]methyl}phosphonate, diethyl{[4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-3-hydroxybutoxy]methyl}phosphonate, PF-00835231, nilmatrervir, ritonavir, ivermectin, colchicine, mebendazole, CDI-45205, GC-376, and prodrugs thereof.

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