Prodrugs of Peptide Epoxy Ketone Protease Inhibitors

TECHNICAL FIELD

This disclosure features compounds that are useful as pro-drugs of epoxy ketone protease inhibitors.

BACKGROUND

The proteasome has been validated as a therapeutic target, as demonstrated by the FDA approval of bortezomib, a boronic acid proteasome inhibitor, for the treatment of various cancer indications, including multiple myeloma. However, other more highly proteasome-specific inhibitors that could have fewer toxic side effects have recently been described. These compounds include peptide epoxy ketones such as epoxomicin, described in U.S. Pat. No. 6,831,099, the contents of which are hereby incorporated by reference, and those described in U.S. Pat. Nos. 7,687,456; 7,737,112; 7,232,818; 7,417,042; 8,080,576; 8,088,741; and 8,357,683, the contents of each is hereby incorporated by reference in its entirety.

SUMMARY

In some embodiments, the compounds described herein themselves exhibit lower therapeutic activity when compared with said corresponding epoxy ketone protease inhibitors.

In some embodiments, the compounds described herein themselves exhibit lower therapeutic activity when compared with said corresponding epoxy ketone protease inhibitors and exhibit enhanced solubility, permeability, pharmacokinetics and/or pharmacodynamics properties in vivo when compared with said corresponding epoxy ketone protease inhibitors.

In some embodiments, the epoxy moiety of the epoxy ketone protease inhibitors is replaced with an activated diol. In some embodiments, the activated diol contains a PEG moiety. In some embodiments, the epoxy moiety of the epoxy ketone protease inhibitors is replaced with an activated halohydrin (e.g., iodohydrin). In some embodiments, the ketone moiety of the epoxy ketone protease inhibitors is replaced with a masked ketone moiety, e.g., acylhydrazones, oximes, oxazolidine, or thiazolidine. In some embodiments, the masked ketone moiety contains a PEG moiety. In some embodiments, the backbone amide of the epoxy ketone protease inhibitors is replaced with N-acyloxymethyl, N-acyloxymethylcarbamate or sulfonamide. In some embodiments, the N-acyloxymethyl, N-acyloxymethylcarbamate, or sulfonamide moiety contains PEG. In some embodiments, the N-terminal “cap” of the epoxy ketone protease inhibitors (e.g., the morpholino and 2-methylthiazole cap) is converted to a quaternary salts (e.g., by the addition of a N-acyloxymethyl group). In some embodiments, the quaternary salt contains a PEG moiety. In some embodiments, the prodrugs are cleavable by pH change and/or enzymes such as, but not limited to, esterases, cytochrome P450, phosphodiesterase, phosphoamidase, phosphatase, and DT-diaphorase, or any combination thereof. In some embodiments, the PEG is a linear PEG. In some embodiments, the PEG is a bifunctional PEG, that can conjugate 1-2 compounds per PEG. In some embodiments, the PEG is a four-arm PEG that can conjugate 1-4 compounds per PEG. In some embodiments, the PEG is an eight-arm PEG with a hexaglycerin core, that can conjugate 1-8 compounds per PEG. In some embodiments, the PEG is an eight-arm PEG with a tripentaerythritol core, that can conjugate 1-8 compounds per PEG. In some embodiments, the PEG is a branched two-arm PEG. In some embodiments, the PEG is a branched four-arm PEG. In addition, the compounds can further include solubilizers, permeability enhancers, masking agents, macromolecular carriers, targeting moieties, and biologics to improve half-life and disease specificity that are attached directly to the compound or indirectly attached via a spacer moiety.

While not wishing to be bound by theory, it is believed that having the protease inhibitory activity temporarily masked until the compounds described herein have reached and released the active into system circulation can reduce side effects associated with routes of administration. This can also facilitate subcutaneous administration and extend half-life, e.g., to beyond four hours. In some embodiments, the human plasma half-life of the prodrug is longer than 0.5 hr. In some embodiments, the human plasma half-life of the prodrug is between 0.5 and 5 hr. In some embodiments, the human plasma half-life of the prodrug is longer than 5 hr. In some embodiments, the human plasma half-life of the prodrug is between 5 and 100 hr. In some embodiments, the human plasma half-life of the prodrug is longer than 100 hr. In some embodiments, the human plasma half-life of the prodrug is between 100 and 836 hr. In some embodiments, the human plasma half-life of the prodrug is between 200 and 300 hr. In some embodiments, the human plasma half-life of the prodrug is about 267 hr. In some embodiments, the human plasma half-life of the prodrug is up to 836 hr. In some embodiments, this will improve both dosing and patient convenience and compliance.

In one aspect, compounds of formula (I) are featured:

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or a pharmaceutically acceptable salt thereof, in which:

R¹is defined according to (A) or (B) below:

(A) R¹has formula (II):

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wherein Het is heterocyclyl that includes from 5-6 ring atoms, wherein at least one ring atom is a nitrogen atom selected from —N(H)—, —N(M)-, or —N(C₁-C₃alkyl)-, wherein said ring nitrogen atom is further optionally substituted with a group R¹¹, thereby forming a quaternary nitrogen atom and wherein the positive charge associated with the quaternary nitrogen atom is balanced by a pharmaceutically acceptable anion; and M is C₁-C₁₂alkyl; or

(B) R¹is heteroaryl that includes from 5-6 ring atoms, wherein at least one ring atom is a nitrogen atom selected from ═N— and —N(C₁-C₃alkyl)-, wherein said ring nitrogen atom is further optionally substituted with a group R¹¹, thereby forming a quaternary nitrogen atom and wherein the positive charge associated with the quaternary nitrogen atom is balanced by a pharmaceutically acceptable anion;

R²and R³, together with the carbon atoms to which each is attached, form an epoxide ring; or

R²is R¹², and R³is hydroxyl or activated hydroxyl;

R⁴and R⁵, together with the carbon atom to which both are attached, form a carbonyl group; or

R⁴and R⁵, together with the carbon atom to which both are attached, form R¹³,

R^S1, R^S2, R^S3, and R^S4are each independently selected from C_1-6alkyl, C_1-6hydroxyalkyl,

C_1-6alkoxyC_1-6alkyl, C_6-10aryl, and C_7-12aralkyl, each of which is optionally substituted with a group selected from amide, amine, carboxylic acid or a pharmaceutically acceptable salt thereof, carboxyl ester, thiol, and thioether;

R^N1, R^N2, R^N3, and R^N4are each independently selected from H and R¹⁴;

R¹¹is a moiety that is removable at a pH>7 or in the presence of an esterase;

R¹²is a leaving group;

R¹³is a masked carbonyl group that is removable at a pH<6.5 or in the presence of an enzyme selected from esterase, amidase, phosphodiesterase or phosphoamidase and CYP P450, or any combination thereof;

each R¹⁴is, independently, a moiety that is removable at a pH>7 or in the presence of an esterase;

provided that at least one group selected from R¹¹, R¹², R¹³, and R¹⁴is present.

In another aspect, compounds having the formula (SM)_m-PEG, or a pharmaceutically acceptable salt thereof, are featured, in which each SM is an independently selected compound of formula (I) as defined in above and anywhere herein and is attached to PEG (e.g. covalently attached to PEG); and m is 2-10. Examples of such compounds are provided below:

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(VIII), in which each of the attendant variables in each of said formula can be independently as defined anywhere herein.

In one aspect, a therapeutic agent is featured, which is prepared by a process that includes reacting a compound as described anywhere herein under conditions sufficient to remove any one or more of R¹¹, R¹², R¹³, and R¹⁴.

In one aspect, a pharmaceutical composition is featured, which includes a compound as described anywhere herein and a pharmaceutically acceptable carrier.

In one aspect, methods are featured for treating a disease or condition selected from the group consisting of cancer, autoimmune disease, graft or transplant-related condition, neurodegenerative disease, fibrotic-associated condition, ischemic-related conditions, infection (viral, parasitic or prokaryotic) and diseases associated with bone loss, the method includes administering to a patient a therapeutically effective amount of a compound as described anywhere herein.

In one aspect, methods for treating cancer (e.g., multiple myeloma, e.g., multiple myeloma that is relapsed and/or refractory) in a patient are featured, which include administering to a patient a therapeutically effective amount of a compound as described anywhere herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the disclosure will be apparent from the following detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme showing cleavage of an embodiment of an epoxy ketone protease inhibitor by peptidases and epoxide hydrolase to inactive metabolites.

FIG. 2 is a scheme showing embodiments of modifications of peptide epoxy ketone protease inhibitors.

FIG. 3 is a line graph showing that more intensive dosing of epoxy ketone protease inhibitors can yield greater efficacy in both solid and hematologic tumor models.

FIG. 4 is a scheme showing cyclization rates of embodiments of epoxy ketone protease inhibitor prodrugs in pH 7.4 buffer.

FIG. 5 is a line graph showing rates of re-formation of an active epoxy ketone protease inhibitor from embodiments of epoxy ketone protease inhibitor prodrugs in human plasma, where the re-formation rate is adjustable by tuning a linker group's electron density.

FIG. 6 is a line graph showing proteolytic cleavage assessed via non-cleavable conjugates (liver homogenate), where the remaining active epoxy ketone protease inhibitor is estimated from metabolite loss. A 2 kDa conjugate had a proteolytic t_1/2of <1 hr; while a 20 kDa-4 Arm conjugate had a proteolytic t_1/2of greater than 6 hrs.

FIG. 7 is a graph showing the effect of PEG size on renal clearance.

FIG. 8 is a line graph showing the plasma levels of embodiments of epoxy ketone protease inhibitor and inhibitor PEG-conjugates. High plasma concentrations were observed for PEG conjugates with significantly extended half life for PEG conjugate compound 41.

FIG. 9 is a bar graph showing in vivo mouse PD results for embodiments of epoxy ketone protease inhibitor PEG-conjugates.

FIG. 10 is a scheme showing that proteolytic cleavage is reduced by increasing PEG size and/or branching and that cleavage of the epoxide moiety was observed for aliphatic sulfonate linkers in embodiments of epoxy ketone protease inhibitor PEG-conjugates.

FIG. 11 is a scheme showing embodiments of epoxy ketone proteasome inhibitor prodrug undergoing pH driven reconversion to the active inhibitor.

FIG. 12 is a scheme showing embodiments of epoxy ketone proteasome inhibitor prodrug being released by hydroxide-driven reconversion to the active inhibitor.

FIG. 13 is a scheme showing an embodiment of an epoxy ketone proteasome inhibitor prodrug containing a sulfonate-linking group that is conjugated to a PEG, which is then released.

FIG. 14 is a scheme showing an embodiment of an epoxy ketone proteasome inhibitor prodrug conjugated to a sulfonate-linking group containing a PEG moiety by way of the PEG-containing sulfonyl chloride.

FIG. 15 is a scheme showing the conjugation of a PEG of molecular weight between 1-40 kDa onto a sulfonate-containing epoxy ketone proteasome inhibitor prodrug.

FIG. 16 is a scheme showing the general synthesis of an alkyl sulfonate-containing epoxy ketone proteasome inhibitor prodrug.

FIG. 17 is a scheme showing the general synthesis of a phenoxysulfonate-containing epoxy ketone proteasome inhibitor prodrug.

FIG. 18 is a scheme showing the general synthesis of an aryl sulfonate-containing epoxy ketone proteasome inhibitor prodrug.

FIG. 19 is a scheme showing an an embodiment of epoxy ketone proteasome inhibitor prodrug containing a masked ketone, which undergoes pH driven reconversion to the active inhibitor.

FIG. 20 is a scheme showing an an embodiment of epoxy ketone proteasome inhibitor prodrug containing a masked ketone, which undergoes reconversion to the active inhibitor through an esterase, amidase, phosphodiesterase, or phosphoamidase.

FIG. 21 is a scheme showing an an embodiment of epoxy ketone proteasome inhibitor prodrug containing a masked ketone, which undergoes hydrolysis to the active inhibitor.

FIG. 22 is a scheme showing an an embodiment of epoxy ketone proteasome inhibitor prodrug containing a masked ketone, which undergoes hydrolysis by an esterase then oxidation to the active inhibitor.

FIG. 23 is a scheme showing embodiments of N-acyloxymethyl-linked epoxy ketone proteasome inhibitor prodrugs containing a morpholine moiety.

FIG. 24 is a scheme showing embodiments of N-acyloxymethyl-linked epoxy ketone proteasome inhibitor prodrugs containing a thiazole moiety.

FIG. 25 is a scheme showing an approach to regioselective syntheses of N-acyloxymethyl-linked epoxy ketone proteasome inhibitor prodrugs.

FIG. 26 is a scheme showing an alternate approach to regioselective syntheses of N-acyloxymethyl-linked epoxy ketone proteasome inhibitor prodrugs.

FIG. 27 is a scheme showing embodiments of N-acyloxymethyl-linked quaternary epoxy ketone proteasome inhibitor prodrugs.

FIG. 28 is a scheme showing embodiments of N-acyloxymethyl-linked quaternary epoxy ketone proteasome inhibitor prodrugs, with the quaternary ion forming with an aryl ring nitrogen.

FIG. 29 is a scheme showing embodiments of N-benzyloxy-linked quaternary epoxy ketone proteasome inhibitor prodrugs.

FIG. 30 is a scheme showing embodiments of iodohydrin ester or carbonate epoxy ketone proteasome inhibitor prodrugs.

FIG. 31 is a scheme showing the putative mechanism of iodohydrin phenoxy ester or carbonate epoxy ketone proteasome inhibitor prodrug release.

FIG. 32 is a scheme showing the putative mechanism of iodohydrin succinamide ester or carbonate epoxy ketone proteasome inhibitor prodrug release.

FIG. 33 is a scheme showing embodiments of iodohydrin succinamide ester or carbonate epoxy ketone proteasome inhibitor prodrugs.

FIG. 34 is a scheme showing embodiments of N-acylmethyloxy ester and iodohydrin succinamide ester/carbonate linked PEG prodrugs of epoxy ketone proteasome inhibitors.

FIG. 35 is a scheme showing embodiments of copper-free [3+2] cycloaddition conditions to form PEG prodrugs of epoxy ketone proteasome inhibitors.

FIG. 36 is a scheme showing a general approach to embodiments of amide-linked PEG prodrugs of epoxy ketone proteasome inhibitors.

FIG. 37 is a scheme showing the synthesis of an embodiment of a precursor to a sulfonate-linked PEG prodrug of epoxy ketone proteasome inhibitors.

FIG. 38 is a scheme showing the synthesis of an embodiment of a thiazole carboxamide prodrug of epoxy ketone proteasome inhibitors.

FIG. 39 is a scheme showing the synthesis of an embodiment of an alkynyl sulfonate precursor to a prodrug of epoxy ketone proteasome inhibitors.

FIG. 40 is a scheme showing the synthesis of an embodiment of an alkyl sulfonate precursor to a prodrug of epoxy ketone proteasome inhibitors.

FIG. 41 is a scheme showing the synthesis of an embodiment of a quaternary ammonium precursor to prodrugs of epoxy ketone proteasome inhibitors.

FIG. 42 is a scheme showing the synthesis of an alternative embodiment of a quaternary ammonium precursor to prodrugs of epoxy ketone proteasome inhibitors.

FIG. 43 is a scheme showing the synthesis of an embodiment of an iodohydrin ester prodrug of an epoxy ketone proteasome inhibitor conjugated to a multi-arm PEG.

FIG. 44 is a scheme showing the synthesis of an embodiment of a precursor to an acyl hydrazone prodrug of an epoxy ketone proteasome inhibitor.

FIG. 45 is a scheme showing the synthesis of an embodiment of N-acyloxymethyl prodrug of an epoxy ketone proteasome inhibitor.

DETAILED DESCRIPTION

This disclosure features compounds that are useful as pro-drugs of epoxy ketone protease inhibitors (e.g., such as those described in U.S. Pat. Nos. 7,687,456; 7,737,112; 7,232,818; 7,417,042; 8,080,576; 8,088,741; and 8,357,683). In some embodiments, the compounds described herein include one or more moieties that (i) can confer enhanced solubility, permeability, pharmacokinetics and/or pharmacodynamics properties relative to compounds that do not contain such moieties; and (ii) can be removed in vivo. In some embodiments, the epoxy moiety of the epoxy ketone protease inhibitors is replaced with an activated diol. In some embodiments, the epoxy moiety of the epoxy ketone protease inhibitors is replaced with an activated halohydrin. In some embodiments, the epoxy moiety of the epoxy ketone protease inhibitor is replaced an activated diol that is conjugated to polyethylene glycol (PEG). In some embodiments, the ketone moiety of the epoxy ketone protease inhibitors is replaced with a masked ketone moiety, e.g., acylhydrazones, oximes, oxazolidine, or thiazolidine. In some embodiments, the masked ketone moiety contains a PEG moiety. In some embodiments, the backbone amide of the epoxy ketone protease inhibitors is replaced with N-acyloxymethyl, N-acyloxymethylcarbamate or sulfonamide. In some embodiments, the N-acyloxymethyl, N-acyloxymethylcarbamate, or sulfonamide moiety contains PEG. In some embodiments, the N-terminal “cap” of the epoxy ketone protease inhibitors (e.g., the morpholino and 2-methylthiazole cap) is converted to a quaternary salts (e.g., by the addition of a N-acyloxymethyl group). In some embodiments, the quaternary salt contains a PEG moiety. In some embodiments, the prodrugs are cleavable by pH change and/or enzymes such as, but not limited to, esterases, cytochrome P450, phosphodiesterase, phosphoamidase, phosphatase, and DT-diaphorase, or any combination thereof. In some embodiments, the PEG is a linear PEG. In some embodiments, the PEG is a bifunctional PEG, that can conjugate 1-2 compounds per PEG. In some embodiments, the PEG is a four-arm PEG that can conjugate 1-4 compounds per PEG. In some embodiments, the PEG is an eight-arm PEG with a hexaglycerin core, that can conjugate 1-8 compounds per PEG. In some embodiments, the PEG is an eight-arm PEG with a tripentaerythritol core, that can conjugate 1-8 compounds per PEG. In some embodiments, the PEG is a branched two-arm PEG. In some embodiments, the PEG is a branched four-arm PEG. In addition, the compounds can further include solubilizers, permeability enhancers, masking agents, macromolecular carriers, targeting moieties, and biologics to improve half-life and disease specificity that are attached directly to the compound or indirectly attached via a spacer moiety.

Compound Scaffolds

In some embodiments, the compounds (or a pharmaceutically acceptable salt thereof) can have Formula I, in which R¹, R², R³, R⁴, R⁵, R^N2, R^N3, R^N4, R^S1, R^S2, R^S3, and R^S4can be as defined anywhere herein.

In other embodiments, the compounds can have the formula (SM)_m-PEG, or a pharmaceutically acceptable salt thereof, in which each SM is an independently selected compound of formula (I) as defined in above and anywhere herein and is attached to PEG; and m is 2-10 compounds having any one of formulas III-VIII).

Variables R¹and R¹¹

In some embodiments, has formula (II):

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In some embodiments, Het is:

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In some embodiments, M is CH₂.

In some embodiments, the ring nitrogen atom is further substituted with a group R¹¹, thereby forming a quaternary nitrogen atom and wherein the positive charge associated with the quaternary nitrogen atom is balanced by a pharmaceutically acceptable anion.

In some embodiments, when R¹has formula (II). R^S1and R^S3are each independently C_1-6alkyl, and R^S2and R^S4are each independently C_7-12aralkyl.

In some embodiments, when R¹has formula (II), R^S1and R^S3are both isobutyl, R^S4is phenylethyl, and R^S2is phenylmethyl.

In other embodiments, R¹is heteroaryl that includes from 5-6 ring atoms, wherein at least one ring atom is a nitrogen atom selected from ═N— and —N(C₁-C₃alkyl)-, wherein said ring nitrogen atom is further optionally substituted with a group R¹¹, thereby forming a quaternary nitrogen atom and wherein the positive charge associated with the quaternary nitrogen atom is balanced by a pharmaceutically acceptable anion.

In some embodiments, R¹is:

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In some embodiments, when R¹is heteroaryl that includes from 5-6 ring atoms, R^S2and R^S3are each independently C_1-6alkoxyC_1-6alkyl, and R^S1is C_1-6aralkyl.

In some embodiments, when R¹is heteroaryl that includes from 5-6 ring atoms, R^S2and R^S3are both methoxymethyl, and R^S1is phenylmethyl.

In some embodiments, the R¹Het or heteroaryl ring nitrogen atom is substituted with a group R¹¹, thereby forming a quaternary nitrogen atom and wherein the positive charge associated with the quaternary nitrogen atom is balanced by a pharmaceutically acceptable anion.

In some embodiments, R¹¹is:

(i) —CH₂OC(═O)R¹⁵;
(ii) —C(═O)OCH₂C(═O)R¹⁵;

(iii) —SR¹⁵; or

(iv) —CH₂Ar—R¹⁵;

wherein:

Ar is C_6-10aryl, optionally substituted with from 1-3 substituents independently selected from C_1-6alkyl, C_1-6alkoxy, and halo;

R¹⁵is C_1-6alkyl, C_1-6haloalkyl, C_1-6alkoxyC_1-6alkyl, C_6-10aryl, C_7-12aralkyl, C_3-7cycloalkyl, heteroaryl including from 5-10 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), or heterocyclyl including from 3-7 ring atoms (wherein from 1-2 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, and O), each of which is optionally substituted with from 1-3 R^a;

wherein: R^aat each occurrence is, independently selected from halo, cyano, nitro, hydroxyl, C_1-6alkoxy, heteroalkyl, C_6-10aryloxy, C_7-12aralkoxy, CF₃, quaternary ammonium ion, sugar, —Y′_p—Z′, C_1-6alkyl, —C(═O)(C_1-6alkyl), —SO₂(C_1-6alkyl), —C(═O)O(C_1-6alkyl), —C(═O)O(heteroalkyl), —C(═O)NH(C_1-6alkyl), —C(═O)NH(heteroalkyl), —C(═O)(phenyl), —SO₂(phenyl), phosphate (or a salt thereof), —C(═O)—Y_n—Z, and —SO₂—Y_n—Z;

or

R¹⁵is Y_n—Z; wherein:

Y is a divalent spacer comprising one or more of the following moieties (e.g., comprising 1, 2, 3, 4, or 5 of the following moieties; e.g., comprising 1, 2, or 3 of the following moieties; e.g., consisting of 1, 2, 3, 4, or 5 of the following moieties; e.g., consisting of 1, 2, or 3 of the following moieties): heteroatom (e.g. N, O, or S), alkylene chain, C_6-10arylene, heteroarylene from 5-10 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), heteroalkylene chain, polyheteroalkylene chain, alkenylene chain, —OC(═O)—, —C(═O)O—, —NHC(═O)—, —C(═O)NH—, —C(═O)—, cyclodextrin, human serum albumin, amino acid, amino acid mimetic, or hydrazine;

Z is conjugate for enhancing solubility, stability, half-life, permeability, volume of distribution, and/or target specificity, which comprises one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g., comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): alkylene chain, heteroalkylene chain, polyheteroalkylene chain, alkenylene chain, cyclodextrin, alkylated cyclodextrin, human serum albumin, —OC(═O)—, —C(═O)O—, —NHC(═O)—, —C(═O)NH—, —C(═O)—, monoclonal anti-body, quaternary ammonium ion, NH₂, docosahexenoic acid, hyaluronic acid, poly(L-glutamic acid), N-(2-hydroxypropyl)methacrylamide copolymer, or dendrimers (e.g., nitro-substituted dendrimers); and

n is 0 or 1.

In some embodiments. R¹¹is: (i) —CH₂C(═O)R¹⁵; (ii) —C(═O)OCH₂C(═O)R¹⁵; or (iii) —SR¹⁵.

In certain embodiments, R¹¹is —CH₂OC(═O)R¹⁵.

In certain embodiments, R¹¹is —CH₂C(═O)R¹⁵, and R¹⁵is C_1-6alkyl, C_1-6haloalkyl, C_1-6alkoxyC_1-6alkyl, C_6-10aryl, C_7-12aralkyl, C_3-7cycloalkyl, heteroaryl including from 5-10 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), or heterocyclyl including from 3-7 ring atoms (wherein from 1-2 of the ring atoms are independently selected from N. NH, N—C₁-C₆alkyl, and O), each of which is optionally substituted.

In some embodiments, R¹¹is —CH₂C(═O)R¹⁵, and R¹⁵is C_1-6alkyl. In certain embodiments, R¹⁵is tert-butyl.

In some embodiments, the pharmaceutically acceptable anion is selected from chloride, iodide, acetate, mesylate, tosylate, and citrate.

In some embodiments, R¹¹is —CH₂C(═O)R²¹—R²⁰-PEG or —CH₂OSO₂R²¹—R²⁰-PEG.

In some embodiments, R¹¹is —CH₂C(═O)R²¹—R²⁰-PEG.

In some embodiments, R¹¹is —CH₂OSO₂R²¹—R²⁰-PEG

For ease of exposition, the term “PEG” refers to a polyethylene glycol (PEG) chain and includes oligomers and polymer of ethylene oxide polyethylene glycol chain (linear or branched) having a molecular weight of from 300 g/mol to 10,000,000 g/mol. The PEG can have any one of the architectures described herein (e.g., formulas 2A-2H; table AA).

Further, it is understood that one or more compound can be conjugated to a polyethylene glycol (PEG) polymer chain.

In some embodiments, the prodrug has Formula 1B:

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wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the prodrug has Formula 1B′:

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wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the prodrug has Formula 1C:

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wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, m is 4.

In some embodiments, m is 2.

In some embodiment, m is 1.

In some embodiments, the prodrug has Formula (VIII):

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in which Ar is C_6-10aryl (e.g., phenyl), optionally substituted with from 1-3 substituents independently selected from C_1-6alkyl, C_1-6alkoxy, and halo; and wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The following definitions apply to formulas 1B, 1W, 1C, and VIII as well as —CH₂C(═O)R²¹—R²⁰-PEG and —CH₂OSO₂R²¹—R²⁰-PEG. For purposes of clarification, it is understood that the R²⁰/R²¹-containing substituent in each of these formulas 1B, 1W, 1C, and VIII is attached to the Het or heteroaryl ring nitrogen atom, thereby forming a quaternary nitrogen atom and wherein the positive charge associated with the quaternary nitrogen atom is balanced by a pharmaceutically acceptable anion.

R²⁰is a heteroarylene including 5-6 ring atoms ((wherein from 1-4 of the ring atoms are independently selected from N. NH, N—C₁-C₆alkyl, O, and S; e.g., containing one or more, e.g., from 1-4, 1-3, 1-2 nitrogen atoms), optionally substituted with 1, 2, or 3 substituents independently selected from C_1-6alkyl, C_1-6hydroxyalkyl, C_1-6alkoxyC_1-6alkyl, C_6-10aryl, or C_7-12aralkyl, each of which is optionally substituted with a group selected from amide, amine, carboxylic acid or a pharmaceutically acceptable salt thereof; or R²⁰is absent (e.g., R²⁰is present and is as defined above).

In some embodiments, R²⁰is

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In some embodiments, R^S5is H, C_1-6alkyl, C_1-6hydroxyalkyl, C_1-6alkoxyC_1-6alkyl, C_6-10aryl, or C_7-12aralkyl, each of which is optionally substituted with a group selected from amide, amine, carboxylic acid or a pharmaceutically acceptable salt thereof, carboxyl ester, thiol, and thioether.

In certain embodiments, R^S5is H or C_1-6alkyl.

In certain embodiments, R^S5is H.

In other embodiments, R²⁰is absent.

R²¹is a divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g., comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g. N. O, or S), alkylene chain, heteroalkylene chain, polyheteroalkylene chain, alkenylene chain, arylene including 6-10 ring atoms, heteroarylene including from 5-10 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH. N—C₁-C₆alkyl, O, and S), heterocycloalkylene including 3-9 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), —OC(═O)—, —C(═O)O—, —NHC(═O)—, —C(═O)NH—, —N(C_1-6alkyl)C(═O), —C(═O)N(C_1-6alkyl)-, —C(═O)—. NHC(═O)NH—, cyclodextrin, human serum albumin, amino acid, amino acid mimetic, or hydrazine, wherein arylene including 6-10 ring atoms, heteroarylene including from 5-10 ring atoms, and heterocycloalkylene including 3-9 ring atoms are each optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, cyano, nitro, hydroxyl, C_1-6alkoxy, heteroalkyl, C_6-10aryloxy, C_7-12aralkoxy. CF₃, quaternary ammonium ion, sugar. C_1-6alkyl, —C(═O)(C_1-6alkyl), —SO₂(C_1-6alkyl), —C(═O)O(C_1-6alkyl), —C(═O)O(heteroalkyl), —C(═O)NH(C_1-6alkyl), —C(═O)NH(heteroalkyl), —C(═O)(phenyl), —SO₂(phenyl), and phosphate (or a salt thereof);

In some embodiments, R²¹is divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g. comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g. N, O, or S), alkylene chain, heteroalkylene chain, arylene including 6-10 ring atoms, heteroarylene including from 5-10 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), heterocycloalkylene including 3-9 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), —OC(═O)—, —C(═O)O—, —NHC(═O)—, —C(═O)NH—, —N(C_1-6alkyl)C(═O), —C(═O)N(C_1-6alkyl)-, —C(═O)—, or —NHC(═O)NH—, wherein 6-10 membered arylene, 5-10 membered heteroarylene, and 6-10 membered cycloalkylene are each optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, cyano, C_1-6alkoxy, heteroalkyl, CF₃, and C_1-6alkyl.

In some embodiments, R²¹is divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g., comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g., N, O, or S), alkylene chain, heteroalkylene chain, arylene including 6-10 ring atoms, heteroarylene including from 5-10 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), heterocycloalkylene including 3-9 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), —NHC(═O)—, —C(═O)NH—, —N(C_1-6alkyl)C(═O), —C(═O)N(C_1-6alkyl)-, —C(═O)—, or —NHC(═C)NH—, wherein 6-10 membered arylene, 5-10 membered heteroarylene, and 6-10 membered cycloalkylene are each optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo. C_1-6alkoxy, heteroalkyl, CF₃, and C_1-6alkyl.

In some embodiments, R²¹is a divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g., comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g., N, O, or S), alkylene chain, heteroalkylene chain, phenylene, indolylene, indolinylene, thiophenylene, or furanylene, wherein phenylene, indolylene, indolinylene, thiophenylene, and furanylene are each optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, C_1-6alkoxy, heteroalkyl, CF₃, and C_1-6alkyl.

In some embodiments, R²¹is a divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g., comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g., N, O, or S), alkylene chain, heteroalkylene chain, or phenylene, wherein phenylene is optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, C_1-6alkoxy, heteroalkyl. CF₃, and C_1-6alkyl.

In some embodiments, R²¹is a divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3.4, or 5 of the following moieties; e.g., comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g., N, O, or S), alkylene chain, heteroalkylene chain, —C(═O)—, or phenylene, wherein phenylene is optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, C_1-6alkoxy, heteroalkyl, CF₃, and C_1-6alkyl.

In some embodiments, R²¹is a phenylene optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, C_1-6alkoxy, heteroalkyl, CF₃, and C_1-6alkyl.

The PEG can have any one of the architectures described herein (e.g., formulas 2A-2H; table AA).

In some embodiments, PEG has a molecular weight of greater than 1 kDa.

In some embodiments, PEG has a molecular weight of about 1 kDa.

In some embodiments, PEG has a molecular weight of greater than 2 kDa.

In some embodiments, PEG has a molecular weight of about 2 kDa.

In some embodiments, PEG has a molecular weight of greater than 5 kDa.

In some embodiments, PEG has a molecular weight of about 5 kDa.

In some embodiments, PEG has a molecular weight of greater than 10 kDa.

In some embodiments, PEG has a molecular weight of about 10 kDa.

In some embodiments, PEG has a molecular weight of greater than 20 kDa.

In some embodiments, PEG has a molecular weight of about 20 kDa.

In some embodiments, PEG has a molecular weight of greater than 30 kDa.

In some embodiments, PEG has a molecular weight of about 30 kDa.

In some embodiments, PEG has a molecular weight of greater than 40 kDa.

In some embodiments, PEG has a molecular weight of about 40 kDa.

In some embodiments, PEG has a molecular weight of greater than 50 kDa.

In some embodiments, prior to conjugation to epoxy ketone protease inhibitors, PEG has a plurality of reactive functional groups (e.g., azide groups).

In some embodiments, prior to conjugation to epoxy ketone protease inhibitors, PEG has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 reactive functional groups (e.g., azide groups).

Variables R², R¹², and R³

In some embodiments, R¹²is present (i.e., R²is R¹²).

In some embodiments, when R¹²is present, R³is activated hydroxyl as defined anywhere herein.

In some embodiments, when R¹²is present, R³is hydroxyl.

In some embodiments, R²and R³, together with the carbon atoms to which each is attached, form an epoxide ring.

In some embodiments, R¹²is:

(i) halo;

(ii) —OSO₂—R¹⁶;

(iii) —OC(═O)R¹⁶;

(iv) —OP(═O)(R¹⁶)₂,
(v) —OP(═O)(OR¹⁶)₂;
(vi) —OP(═O)(R¹⁶)(OR¹⁶); or

(vii) —O—(N-linked heteroaryl);

wherein:

R¹⁶is C_1-6alkyl, C_1-6haloalkyl, heteroalkyl, C_6-10aryl, C_7-12aralkyl, C_3-7cycloalkyl, heteroaryl including from 5-10 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), or heterocyclyl including from 3-7 ring atoms (wherein from 1-2 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, and O), each of which is optionally substituted with from 1-3 R^a;

or

R¹⁶is —Y′_p—Z′; wherein:

Y′ is a divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g., comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g., N, O, or S), alkylene chain, heteroalkylene chain, polyheteroalkylene chain, alkenylene chain, —OC(═O)—, —C(═O)O—, —NHC(═O)—, C(═O)NH—, —C(═O)—, cyclodextrin, human serum albumin, amino acid, amino acid mimetic, or hydrazine;

Z′ is conjugate for enhancing solubility, stability, half-life, permeability, volume of distribution, and/or target specificity, which comprises one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g. comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): alkylene chain, heteroalkylene chain, polyheteroalkylene chain, alkenylene chain, cyclodextrin, alkylatal cyclodextrin, human serum albumin, —OC(═O)—, —C(═O)O—, —NHC(═O)—, —C(═O)NH—, —C(═O)—, monoclonal anti-body, quaternary ammonium ion, NH₂, docosahexenoic acid, hyaluronic acid, poly(L-glutamic acid), N-(2-hydroxypropyl)methacrylamide copolymer, or dendrimers (e.g., nitro-substituted dendrimers);

p is 0 or 1; and

R^aat each occurrence is, independently selected from halo, cyano, nitro, hydroxyl, C_1-6alkoxy, heteroalkyl, C_6-10aryloxy, C_7-12aralkoxy, CF₃, quaternary ammonium ion, sugar, —Y′_p—Z″. C_1-6alkyl, —C(═O)(C_1-6alkyl), —SO₂(C_1-6alkyl), —C(═O)O(C_1-6alkyl), —C(═O)O(hetero alkyl), —C(═O)NH(C_1-6alkyl), —C(═O)NH(heteroalkyl), —C(═O)(phenyl). —SO₂(phenyl), phosphate (or a salt thereof), —C(═O)—Y′_p—Z′, and SO₂—Y′_p—Z′.

In some embodiments, R¹²is −OSO₂—R¹⁶. In some embodiments, R¹⁶is C_1-6alkyl (e.g., CH₃) or C_6-10aryl. (e.g., phenyl).

In some embodiments, R¹²is halo (e.g., iodo),.

In some embodiments. R¹²is halo (e.g., iodo), and R³is activated hydroxyl.

In some embodiments, R¹²is halo (e.g., iodo), and R³is hydroxyl.

In some embodiments, R³is hydroxyl.

In some embodiments, R³is activated hydroxyl, e.g., —O—C(═O)—R²²—R²⁰-PEG or —O—SO₂—R²²—R²⁰-PEG.

In certain embodiments, R³is —O—C(═O)—R²²—R²⁰-PEG.

In certain embodiments, R³is —O—SO₂—R²²—R²⁰-PEG.

It is understood that one or more prodrugs can be conjugated to a polyethylene glycol (PEG) polymer chain. In some embodiments, the prodrug has Formula ID:

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In some embodiments, the prodrug has Formula 1E:

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In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, m is 4.

In some embodiments, m is 2.

In some embodiment, m is 1.

R²⁰is a heteroarylene including 5-6 ring atoms ((wherein from 1-4 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S; e.g., containing one or more, e.g., from 1-4, 1-3, 1-2 nitrogen atoms), optionally substituted with 1, 2, or 3 substituents independently selected from C_1-6alkyl, C_1-6hydroxyalkyl, C_1-6alkoxyC₁-6alkyl, C_6-10aryl, or C_7-12aralkyl, each of which is optionally substituted with a group selected from amide, amine, carboxylic acid or a pharmaceutically acceptable salt thereof; or R²⁰is absent (e.g., R²⁰is present and is as defined above).

In some embodiments, R²⁰is

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In certain embodiments, R^S5is H or C_1-6alkyl.

In certain embodiments, R^S5is H.

In other embodiments, R²⁰is absent.

R²²is a divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties: e.g., comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom N, O, or S), alkylene chain, heteroalkylene chain, polyheteroalkylene chain, alkenylene chain, arylene including 6-10 ring atoms, heteroarylene including from 5-10 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), heterocycloalkylene including 3-9 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), —OC(═O)—, —C(═O)O—, —NHC(═O)—, —C(═O)NH—, —N(C_1-6alkyl)C(═O), —C(═O)N(C_1-6alkyl)-, —C(═O)—, —NHC(═O)NH—, cyclodextrin, human serum albumin, amino acid, amino acid mimetic, or hydrazine, wherein arylene including 6-10 ring atoms, heteroarylene including from 5-10 ring atoms, and heterocycloalkylene including 3-9 ring atoms are each optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, cyano, nitro, hydroxyl, C_1-6alkoxy, heteroalkyl, C_6-10aryloxy, C_7-12aralkoxy, CF₃, quaternary ammonium ion, sugar, C_1-6alkyl, —C(═O)(C_1-6alkyl), —SO₂(C_1-6alkyl), —C(═O)O(C_1-6alkyl), —C(═O)O(heteroalkyl), —C(═O)NH(C_1-6alkyl), —C(═O)NH(heteroalkyl), —C(═O)(phenyl), —SO₂(phenyl), and phosphate (or a salt thereof).

In some embodiments, R²²is divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties: e.g., comprise 1, 2, or 3 of the following moieties; e.g. consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g. N. O, or S), alkylene chain, heteroalkylene chain, arylene including 6-10 ring atoms, heteroarylene including from 5-10 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), heterocycloalkylene including 3-9 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N. NH, N—C₁-C₆alkyl, O, and S), —OC(═O)—, —C(═O)O—, —NHC(═O)—, —C(═O)NH—, —N(C_1-6alkyl)C(═O), —C(═O)N(C_1-6—C(═O)—, or —NHC(═O)NH—, wherein 6-10 membered arylene, 5-9 membered heteroarylene, and 6-10 membered cycloalkylene are each optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, cyano, C_1-6alkoxy, heteroalkyl, CF₃, and C_1-6alkyl.

In some embodiments, R²²is divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g., comprise 1, 2, or 3 of the following moieties: e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g., N, O, or S), alkylene chain, heteroalkylene chain, arylene including 6-10 ring atoms, heteroarylene including from 5-10 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), heterocycloalkylene including 3-9 ring atoms (wherein from 1-3 of the ring atoms are independently selected from N, NH, N—C₁-C₆alkyl, O, and S), —NHC(═O)—, —C(═O)NH—, —N(C_1-6alkyl)C(═O), —C(═O)N(C_1-6alkyl)-, —C(═O)—, or —NHC(═O)NH—, wherein 6-10 membered arylene, 5-10 membered heteroarylene, and 6-10 membered cycloalkylene are each optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, C_1-6alkoxy, heteroalkyl, CF₃, and C_1-6alkyl.

In some embodiments, R²²is a divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g., comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g., N, O, or S), alkylene chain, heteroalkylene chain, phenylene, indolylene, indolinylene, thiophenylene, or furanylene, wherein phenylene, indolylene, indolinylene, thiophenylene, and furanylene are each optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, C_1-6alkoxy, heteroalkyl, CF₃, and C_1-6alkyl.

In some embodiments, R²²is a divalent spacer comprising one or more of the following moieties (e.g., comprise 1, 2, 3, 4, or 5 of the following moieties; e.g., comprise 1, 2, or 3 of the following moieties; e.g., consist of 1, 2, 3, 4, or 5 of the following moieties; e.g., consist of 1, 2, or 3 of the following moieties): heteroatom (e.g., N, O, or S), alkylene chain, heteroalkylene chain, or phenylene, wherein phenylene is optionally substituted with 1, 2, or 3 substituents each independently selected from the group consisting of halo, C_1-6alkoxy, heteroalkyl. CF₃, and C_1-6alkyl.

In some embodiments, R²²is a heteroalkylene chain.