WO2023242766A1

WO2023242766A1 - Gold nanoconjugates

Info

Publication number: WO2023242766A1
Application number: PCT/IB2023/056148
Authority: WO
Inventors: Raghuraman Kannan; Sai Giridhar Sarma KANDANUR; Abilash Gangula; Agasthya Suresh BABU; Tilak CHHETRI; Dhananjay SURESH; Anandhi UPENDRAN
Original assignee: Alembic Pharmaceuticals Limited
Priority date: 2022-06-15
Filing date: 2023-06-14
Publication date: 2023-12-21

Abstract

The present disclosure provided compounds, nanoconjugates, and compositions thereof for the treatment of cancer.

Description

GOLD NANOCONJUGATES

Cross Reference to Related Applications

[0001] This application claims priority to and benefit of U.S. Appl. No. 63/352,614, filed

June 15, 2022, the entire contents of which are hereby incorporated by reference herein.

Background

[0002] Gemcitabine (Gem) is a well-known broad-spectrum anticancer drug used alone or in combination therapy for treatment of several cancer types including pancreatic, bladder, non-small cell lung cancer, ovarian, breast, head, neck, thyroid, and bone cancers. Despite its high anti-cancer potential, the therapeutic efficacy of Gem is compromised primarily by two of its inherent structural limitations that result in poor delivery of administered drug to the tumor cells. As with other nucleoside analogs with 4-NH2 moiety, Gem is rapidly deaminated into its therapeutically inactive form of 2',2'-difluorodeoxyuridine (dfdu) by the enzyme cytidine deaminase (CD A) which is abundant in blood and liver. Thus, to achieve therapeutically optimal concentrations in the tumor, Gem is administered at high doses (1000 mg/m² for 30 min intravenous infusion) that would cause toxic side effects and development of resistance. In addition, the hydrophilicity of Gem hinders its passive diffusion across the plasma membrane thus necessitating high tumor expression of nucleoside transporters to cause efficient uptake and therapeutic effect. The low tumor expression of nucleoside transporters leads to therapeutic inefficacy and resistance to Gem. In short, poor plasma stability and membrane permeability of Gem are the major roadblocks in realizing its therapeutic potential.

Summary

[0003] The present disclosure provides, among other things, gemcitabine-containing compounds having improved stability and tumor uptake. In some embodiments, such compounds are of Formula I:

I or a pharmaceutically acceptable salt thereof, wherein each of GEM, AA, L, n, y, and x is as defined herein.

[0004] The present disclosure also provides nanoconjugates (e.g., comprising gold nanoparticles) comprising provided compounds, and optionally comprising a thioctic acid terminated peptide.

[0005] In one aspect, the present disclosure provides a gold nanoparticle (AuNP) comprising a compound of the following structure:

— S — I wherein each « is independently a point of attachment of the compound to hydrogen or the AuNP gold surface.

[0006] The present disclosure also provides a pharmaceutical composition comprising a plurality of nanoconjugates, at least one nanoconjugate comprising a compound having the structure:

covalently linked to a gold nanoparticle (AuNP) via at least one Au-S bond.

[0007] The present disclosure further provides pharmaceutical compositions, methods of treating cancer, methods for enhancing the cytotoxicity of gemcitabine, and processes for the preparation of provided compounds, compositions, and nanoconjugates.

Brief Description of the Drawings

[0008] Figure 1 depicts an exemplary synthesis of a Gemcitabine-gold nanoparticle conjugate comprising a targeting peptide.

[0009] Figure 2 depicts hydrodynamic size, zeta potential, and TEM images of (a) Au- [DTGT], (b) P₄BN-Au-[DTGT], and (c) P₄cMET-Au-[DTGT],

[0010] Figure 3 depicts TEM images of Au-[DTGT],

[0011] Figure 4 depicts TEM images of P₄BN-Au-[DTGT],

[0012] Figure 5 depicts TEM images of P₄cMET-Au-[DTGT],

[0013] Figure 6 depicts UV-Visible spectra of Au-[DTGT], P₄BN-Au[DTGT], and

P₄CMET-AU-[DTGT] along with their respective controls.

[0014] Figure 7 depicts HPLC spectra: a) HPLC spectrum of DTGT in 1.5 M NaCN, where the peak at 3.8 min corresponds to Gem that is released on digestion of DTGT with NaCN. b) HPLC-based standard curve of DTGT in 1.5 M NaCN. [0015] Figure 8 depicts standard curves: a) HPLC-based standard curve of P4CMET in 1.5 M NaCN, b) HPLC-based standard curve of P4BN in 1.5 M NaCN.

[0016] Figure 9 depicts standard curves: a) Gemcitabine and b) Theoretical Gemcitabine equivalence in DTGT. Standards of drug in DPBS were processed similar to that of constructs in CDA stability study.

[0017] Figure 10 depicts a stability profile of Gemcitabine and modified Gemcitabine formulations in the presence of cytidine deaminase (CDA).

[0018] Figure 11 depicts MTT assay plots for growth inhibitory analysis of Gemcitabine and its modified analogs in lung cancer (A549 (a), H23 (b)) and pancreatic cancer (PANC-1 (c), BxPC3 (d)) cell lines after 72 hours of treatment.

[0019] Figure 12 depicts SRB assay plots for growth inhibitory analysis of Gemcitabine and its modified analogs in pancreatic cancer cell lines after 72 hours of treatment: MCF-7 (a), MBA-MB-468 (b), MDA-MB-231.

Definitions

[0020] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0021] About: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). [0022] Cancer. The terms "cancer", “malignancy”, "neoplasm", "tumor", and "carcinoma", are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor.

[0023] Excipient: as used herein, refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect.

[0024] Improved, increased, or reduced: As used herein, these terms, or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be “improved” relative to that obtained with a comparable reference agent. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance.

[0025] Natural amino acid. As used herein, the phrase “natural amino acid” refers to any of the 20 amino acids naturally occurring in proteins: glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (He), lysine (Lys), arginine (Arg), histidine (His), proline (Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gin), cysteine (Cys) and methionine (Met). Such natural amino acids include the nonpolar, or hydrophobic amino acids, glycine, alanine, valine, leucine isoleucine, methionine, phenylalanine, tryptophan, and proline. Cysteine is sometimes classified as nonpolar or hydrophobic and other times as polar. Natural amino acids also include polar, or hydrophilic amino acids, such as tyrosine, serine, threonine, aspartic acid (also known as aspartate, when charged), glutamic acid (also known as glutamate, when charged), asparagine, and glutamine. Certain polar, or hydrophilic, amino acids have charged side-chains. Such charged amino acids include lysine, arginine, and histidine. One of ordinary skill in the art would recognize that protection of a polar or hydrophilic amino acid side-chain can render that amino acid nonpolar. For example, a suitably protected tyrosine hydroxyl group can render that tyroine nonpolar and hydrophobic by virtue of protecting the hydroxyl group.

[0026] Patient: As used herein, the term “patient” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals including but not limited to humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disorder or condition. In some embodiments, a patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or presence of one or more tumors. In some embodiments, the patient is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition.

[0027] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applied to the carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0028] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).

[0029] Protecting Group'. The phrase “protecting group,” as used herein, refers to temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, thiols, and acetals and ketals of aldehydes and ketones, respectively. Protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference.

[0030] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[0031] Stable: The term “stable,” when applied to compositions herein, means that the compositions maintain one or more aspects of their physical structure and/or activity over a period of time under a designated set of conditions. In some embodiments, the period of time is at least about one hour; in some embodiments the period of time is about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty-four (24) months, about thirty-six (36) months, or longer. In some embodiments, the period of time is within the range of about one (1) day to about twenty-four (24) months, about two (2) weeks to about twelve (12) months, about two (2) months to about five (5) months, etc. In some embodiments, the designated conditions are ambient conditions (e.g., at room temperature and ambient pressure). In some embodiments, the designated conditions are physiologic conditions (e.g., in vivo or at about 37 °C for example in serum or in phosphate buffered saline). In some embodiments, the designated conditions are under cold storage (e.g., at or below about 4 °C, -20 °C, or -70 °C). In some embodiments, the designated conditions are in the dark.

[0032] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0033] Treatment. As used herein, the term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.

[0034] Unnatural amino acid. As used herein, the phrase “unnatural amino acid” refers to amino acids not included in the list of 20 amino acids naturally occurring in proteins, as described above. Such amino acids include the D-isomer of any of the 20 naturally occurring amino acids. Unnatural amino acids also include homoserine, ornithine, norleucine, and thyroxine. Other unnatural amino acids side-chains are well known to one of ordinary skill in the art and include unnatural aliphatic side chains. Other unnatural amino acids include modified amino acids, including those that are N-alkylated, cyclized, phosphorylated, acetylated, amidated, azidylated, labelled, and the like. This includes, for example, a-, -, ®-, D-, and L- amino acid residues. In some embodiments, an unnatural amino acid is a D-isomer. In some embodiments, an unnatural amino acid is a L-isomer.

Detailed Description of Certain Embodiments

[0035] Prior research on improving the pharmacokinetics or tumor uptake of Gemcitabine (Gem) and other chemotherapeutics includes chemical modifications of the drug structure or the use of nanocarriers as protective delivery vehicles. Amide prodrugs of Gem have been suggested to improve metabolic stability of Gem (Hong, S. et al. Molecules 2018, 23, 2608). In an effort to deliver anticancer drugs to cancer cells, doxorubicin was attached to gold NP through a pH-sensitive linker (US2013/0138032). Other linkers, including aminocarboxylate ligands, have been developed to link radionuclides to gold NP for uses in imaging (Debouttiere et al. Adv. Funct. Mater. 2006, 16, 2330-2339; WO2015/103028), and to link doxorubicin to gold NP for use in chemotherapy (WO2018/129501). Some of these research efforts demonstrated improved in-vitro and in-vivo tumor therapeutic outcome. However, the incorporation of both chemical modification and nanocarrier strategies in the design of new formulations of Gem has not been explored. [0036] The present disclosure encompasses the recognition that a combined strategy that incorporates both chemical modification and nanocarrier delivery of Gem would result in superior overall therapeutic performance in clinical practice. Indeed, the present disclosure validates a synergistic effect by demonstrating that certain chemical modifications of Gem result in improved plasma stability which is further enhanced by conjugating the Gemcitabine modification to a gold nanocarrier.

[0037] Gem is a deoxy cytidine analog and therefore subject to deamination by cytidine deaminase, a process that transforms Gem and other deoxy cytidines (e.g., cytarabine and decitabine) into inactive metabolites. The present disclosure provides insight for improving the enzymatic stability of Gem by synthesizing a novel prodrug of Gem (DTGT) and conjugating it with biocompatible gold nanoparticles to form an Au-DTGT conjugate. For example, the present disclosure modifies Gem at the 4-(N) position with a threonine moiety to generate a metabolically stable threonine derivative of gemcitabine (GT). Then, the resistance to enzymatic degradation is further enhanced by conjugating GT to a gold nanocarrier via a dithiolated (DT) crosslinker to produce Au-DTGT of excellent stability. In some embodiments, nanoparticles are pegylated to further prevent enzymatic degradation, reduce phagocytic clearance, and prolong the circulation time.

[0038] Furthermore, the present disclosure provides superior tumor uptake of Gem by conjugating it to nanoparticles that promote EPR-based tumor accumulation and endocytic internalization, thereby circumventing the need of transporters for membrane permeability. The smaller size (e.g., < 30 nm) of such nanoparticles facilitates efficient penetration of dense tumor extracellular matrix to deliver the drug to the tumor core. In addition, provided nanoparticles can be functionalized with receptor-specific peptides for actively targeting the tumor cells. Overall, the combination of several factors such as 4-(N)-modifi cation of Gem, conjugation with a nanocarrier, the smaller size of the nanocarrier, pegylation of nanocarrier, presence of threonine and targeting peptides together result in a metabolically stable nanoformulation of Gem with a superior tumor uptake potential. The present disclosure therefore provides nanoconjugates of Gem having improved pharmacokinetic, tumor distribution, and therapeutic properties as compared to prior Gem formulations, Gem prodrugs, and Gem alone. Compounds

[0039] In some embodiments, the present disclosure provides compounds of Formula A:

[(Drug)_n(

A or a pharmaceutically acceptable salt thereof, wherein:

L is a multivalent linker moiety having one or more thiol functional groups; each AA is independently a naturally or unnaturally occurring L or D amino acid; each Drug is independently a therapeutic entity capable of being deaminated by cytidine deaminase (CD A); each n is independently 0 or 1 ; x is 1, 2, or 3; and each y is independently 0 or 1 ; wherein n and y cannot both be 0; and wherein all linkages between Drug-AA, Drug-L, and AA- L when present comprise amide bonds.

[0040] In some embodiments, each Drug independently is or comprises a cytidine or deoxy cytidine that is capable of being deaminated by CD A. In some embodiments, each Drug is preferably Gem.

[0041] In some embodiments, the present disclosure provides compounds of Formula I:

I or a pharmaceutically acceptable salt thereof, wherein:

L is a multivalent moiety (e.g., linker) having one or more thiol functional groups; each AA is independently a naturally or unnaturally occurring L or D amino acid; GEM is gemcitabine; each n is independently 0 or 1 ; x is 1, 2, or 3; and each y is independently 0 or 1 ; wherein n and y cannot both be 0; and wherein all linkages between GEM-AA, GEM-L, and AA-L when present comprise amide bonds.

[0042] In some embodiments, the present disclosure provides compounds of Formula I:

I or a pharmaceutically acceptable salt thereof, wherein:

L is a multivalent linker moiety having one or more thiol functional groups; each AA is independently a naturally or unnaturally occurring L or D amino acid;

GEM is gemcitabine; each n is independently 0 or 1 ; x is 1, 2, or 3; and each y is independently 0 or 1 ; wherein n and y cannot both be 0; and wherein all linkages between GEM-AA, GEM-L, and AA-L when present comprise amide bonds.

[0043] It will be appreciated that compounds of Formula A, I, and II are useful for conjugation to a gold nanoparticle (e.g., a thiol functional group of linker moiety can conjugate with the gold surface).

[0044] In some embodiments, L is a multivalent linker moiety having one or more thiol functional groups (e.g., a linker capable of forming covalent Au-S bonds with a gold NP). In some embodiments, L is or comprises a heterofunctional crosslinker containing one or more thiol functional groups and one or more amine reactive groups (e.g., a reactive group capable of forming an amide bond with GEM or AA). In some embodiments, L contains one or more amine reactive groups selected from the group consisting of isothiocyanates, isocyanates, sulfonyl chlorides, aldehydes, carbodiimides, acyl azides, anhydrides, fluorobenzenes, carbonates, NHS esters, imidoesters, epoxides, fluorophenyl esters, and combinations thereof.

[0045] In some embodiments, L is or comprises a polyaminocarboxylate (e.g., aminopolycarboxylic acid). In some embodiments, L is a thiol-functionalized derivative of NTA, EDTA, DTP A, EGTA, BAPTA, NOTA, DOTA, mcotianamine, EDDHA, or EDDS. In some embodiments, L is a thiol-functionalized derivative of DTPA. In some embodiments, L comprises one thiol functional group. In some embodiments, L comprises two thiol functional groups. In some embodiments, L is preferably dithiolated diethylenetriamine pentaacetic acid (DTD TP A):

[0046] In some embodiments, each AA is independently a naturally or unnaturally occurring amino acid. In some embodiments, each AA is independently a naturally occurring amino acid. In some embodiments, each AA is independently an unnaturally occurring amino acid. In some embodiments, AA is preferably threonine (Thr). In some embodiments, AA is preferably L-Thr.

[0047] It will be appreciated that gemcitabine is attached to AA or L via a functional group (e.g., amine) capable of covalently linking gemcitabine directly or indirectly to AA or L, and wherein the linkage comprises an amide bond. In some embodiments, gemcitabine is covalently linked to AA or L via the primary amine group (4-(N)) of gemcitabine. In some embodiments, the primary amine group of Gem is connected to AA via an amide bond. In some embodiments, the primary amine group of Gem is connected to L via an amide bond.

[0048] In some embodiments, x is 3. In some embodiments, x is 2. In some embodiments, x is 1.

[0049] In some embodiments, n is i. In some embodiments, n is 0.

[0050] In some embodiments, x is 3 and each n is i. In some embodiments for a given occurrence of [(GEM)_n(AA)_y], n is 1. In some embodiments for a given occurrence of [(GEM)n(AA)_y], n is 0. In some embodiments where x is 3, n is 1 for two occurrences of [(GEM)n(AA)_y], and n is 0 for the other occurrence of [(GEM)_n(AA)_y]. In some embodiments where x is 3, n is 0 for two occurrences of [(GEM)_n(AA)_y], and n is 1 for the other occurrence of [(GEM)n(AA)_y], In some embodiments where x is 3, n is 0 for each occurrence of [(GEM)n(AA)y],

[0051] In some embodiments where x is 2, n is 0 for one occurrence of [(GEM)_n(AA)_y], and n is 1 for the other occurrence of [(GEM)_n(AA)_y] . In some embodiments where x is 2, n is 0 for each occurrence of [(GEM)_n(AA)_y],

[0052] In some embodiments, x is 1 and n is 1. In some embodiments, x is 1 and n is 0.

[0053] In some embodiments, each y is 0. In some embodiments, each y is 1.

[0054] In some embodiments for a given occurrence of [(GEM)n(AA)_y], y is 1. In some embodiments for a given occurrence of [(GEM)_n(AA)_y], y is 0.

[0055] In some embodiments, x is 3, each n is 1, and each y is 1. In some embodiments where x is 3, two occurrences of [(GEM)_n(AA)_y] have n=l and y=l, and the other occurrence of [(GEM)n(AA)_y] has n=0 and y=l. In some embodiments where x is 3, two occurrences of [(GEM)n(AA)_y] have n=0 and y=l, and the other occurrence of [(GEM)_n(AA)_y] has n=l and y=l.

[0056] In some embodiments, x is 3, each n is 1, and each y is 0. In some embodiments where x is 2, one occurrence of [(GEM)_n(AA)_y] has n=l and y=0, and the other occurrence of [(GEM)n(AA)_y] has n=0 and y=0. In some embodiments where x is 1, n=l and y=0.

[0057] In some embodiments, the present disclosure provides compounds of Formula II:

II or a pharmaceutically acceptable salt thereof, wherein each of GEM, AA, L, n, and y is as defined above and described in classes and subclasses herein, both singly and in combination.

[0058] In some embodiments, a compound of Formula I or II has the structure:

or a pharmaceutically acceptable salt thereof.

Nanoconjugates

[0059] The various types of nanoparticles (NP) that have been explored as delivery vehicles for Gem include polymeric NP, lipid NP, silica NP, magnetic NP, liposomes, and micellar NP. All these formulations involve physical entrapment of Gem within the NP and suffer from two major drawbacks: i) The encapsulation strategies for loading of drug in a nanoparticle are usually inefficient resulting in very low levels of drug loading (<10%). This would result in either low therapeutic response due to insufficient drug concentration in the tumor or elevated systemic toxicity due to significantly high dose of carrier required to be administered to achieve therapeutic levels of drug, ii) Physically entrapped drugs often suffer from drug expulsion during storage and ‘burst release’ resulting in premature drug leakage in the systemic circulation. In contrast, the present disclosure encompasses the recognition that conjugation of a drug on the nanoparticle surface by covalent bonding would allow high drug loading strategies as well as prevent the leakage of the drug. According to one aspect of the present disclosure, a bifunctional crosslinker (DT) enables high loading of Gem by covalent methods. At one end, a single molecule of DT has three carboxylic acid moieties that serve as chemical handles for covalent conjugation of three Gem analogs. On the other end, DT has two sulfhydryl groups that can be readily conjugated to a gold NP. In some embodiments, a single nanoparticle of <10 nm size can accommodate a monolayer containing around 150 molecules of DT, each of which has up to 3 molecules of Gem. Such nanoconjugates can achieve relatively high drug loading of Gem (20-30%) in Au-DTGT bv covalent methods. The metabolic stability of Gem is significantly improved by provided nanoconjugates by i) protection from CDA degradation by chemical modification at 4-(N) position which is susceptible to CDA, and ii) conjugating with pegylated AuNP.

[0060] Another aspect of the present disclosure is the recognition that the use of gold NPs as delivery vehicles offer several synthetic advantages over other NPs being used as delivery vehicles of Gem. Polymeric NPs are susceptible to aggregation and cause toxicity. The practical use of lipid and liposomal NP is limited by low drug loading capacities and poor biodistribution due to high NP uptake by liver and spleen. The inorganic NP such as iron and silica suffer from drawbacks such as low solubility and concerns of toxicity. The synthetic techniques of polymeric, lipid, silica, magnetic and micellar NP involve nanoprecipitation, desolvation, homogenization, ionic gelation, emulsification, sol-gel process, pyrolysis, selfassembly, and co-precipitation. These techniques suffer from limitations such as complexity, lack of reproducibility, use of high temperatures and pressure. The present disclosure provides, among other things, processes for the synthesis of gold nanoparticles which are extremely facile, rapid (15 min), and reproducible. Unlike other inorganic NP vehicles such as silica and iron, the provided gold-based NP are highly water soluble (e.g., up to concentrations of 50 mg/mL). Moreover, gold NP are versatile and adaptable in comparison to other NPs as they can be easily tuned to several sizes, shapes, and surface functionalities. The unique optical properties of gold NP enable them to act as contrast/imaging agent and catalyst for photothermal and photodynamic therapy while simultaneously serving as delivery vehicles. This attribute sets gold NP apart from other NP that just serve as vehicles as it opens opportunities for a clinician to track the drug in- vivo as well as execute multi-modal treatment options, all using a single NP platform. The present disclosure also recognizes, for the first time, that unlike any other NP systems, gold NP possess a unique ability to sensitize tumors to Gem treatment.

[0061] The present disclosure provides nanoconjugates comprising a provided compound covalently linked to a gold nanoparticle (AuNP) via at least one Au-S bond. In some embodiments, a provided compound is compound covalently linked to an AuNP via one Au-S bond. In some embodiments, a provided compound is compound covalently linked to an AuNP via two Au-S bonds. In some embodiments, an AuNP is PEGylated. In some embodiments, an AuNP is PEGylated prior to conjugation with a provided compound. [0062] In some embodiments, a provided nanoconjugate comprises a single layer of compound surrounding the AuNP.

[0063] Targeting peptides can provide for or accentuate accumulation of NP at tumor sites. In some embodiments, a provided nanoconjugate further comprises a targeting peptide. A variety of chemistries are known to the skilled artisan for linking a peptide to an AuNP, by way of nonlimiting example the use of sulfur moieties (e.g., thiols, thioctic acid, disulfides) on the peptide that can form a covalent bond with the gold surface. In some embodiments, a peptide is conjugated to a nanoconjugate via a thioctic acid terminal group on the peptide. In some embodiments, a nanoconjugate comprises a thioctic acid terminated peptide covalently linked to AuNP via at least one Au-S bond. In some embodiments, a thioctic acid terminated peptide is thioctic acid terminated bombesin, thioctic acid terminated cMET or thioctic acid terminated GE11.

[0064] According to one aspect of the present disclosure, nanoconjugates are provided as a plurality of individual nanoconjugates within a composition. Nanoconjugate compositions may be characterized by various parameters, (e.g., average size, drug loading, peptide loading, conjugation efficiency, etc.). In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size of less than about 40 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size of less than about 35 nm, about 30 nm, about 25 nm, about 20 nm, about 15 nm, or about 10 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging from about 5 nm to about 25 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging from about 5 nm to about 35 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging from about 8 nm to about 22 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging from about 5 nm to about 15 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging from about 15 nm to about 25 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging of about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, or about 25 nm.

[0065] In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a zeta potential of about -15 mV or less. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a zeta potential of about -20 mV or less. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a zeta potential of about -10 mV to about -30 mV. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a zeta potential of about -15 mV to about -25 mV. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a zeta potential of about -20 mV to about -25 mV. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a zeta potential of about -15 mV, about -16 mV, about -17 mV, about -18 mV, about -19 mV, about -20 mV, about -21 mV, about -22 mV, about -23 mV, about -24 mV, about -25 mV, about -26 mV, about -27 mV, about -28 mV, about -29 mV, or about -30 mV.

[0066] In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a Drug (e.g., Gem) loading of about 5% to about 60%. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a Drug (e.g., Gem) loading of about 10% to about 50%, about 15% to about 50%, about 25% to about 35%, or about 15% to about 30%.

[0067] In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of at least about 40 mg/mL. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of at least about 45 mg/mL. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of at least about 50 mg/mL. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of about 40 mg/mL to about 75 mg/mL. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of about 40 mg/mL to about 60 mg/mL. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of about 40 mg/mL to about 55 mg/mL.

[0068] In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a targeting peptide (e.g., a thioctic acid terminated peptide) loading of about 5% to about 60%. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a targeting peptide (e.g., a thioctic acid terminated peptide) loading of about 5% to about 10%, about 5% to about 15%, about 15% to about 60%, about 25% to about 60%, about 35% to about 60%, or about 45% to about 60%.

[0069] The present disclosure also provides pharmaceutical compositions of provided nanoconjugates. In some embodiments a pharmaceutical composition comprises a therapeutically effective amount of a nanoconjugate composition and one or more pharmaceutically acceptable excipients. In some embodiments, a pharmaceutical composition is in lyophilized form.

Methods of use

[0070] In some embodiments, the present disclosure provides compositions for use in therapy. In some embodiments, the present disclosure provides a method of treating a cancer in a patient in need of such of treatment, comprising administering to the patient a provided pharmaceutical composition. In some embodiments, a patient exhibits one or more reduced side effects compared to a patient treated with an equivalent amount of Drug (e.g., gemcitabine) alone. In some embodiments the present disclosure provides an improved method of treating cancer in a patient in need of such of treatment, the improvement comprising administering to the patient a provided pharmaceutical composition. In some embodiments, a cancer treated in accordance with the present disclosure is breast, ovarian, non-small cell lung, bladder, testicular, or pancreatic cancer.

[0071] Provided also are methods for killing or inhibiting the growth of a cancer cell, comprising contacting the cell with a provided compound or nanoconjugate composition. The present disclosure further provides methods for enhancing the cytotoxicity or cytostaticity of gemcitabine in a cancer cell, comprising contacting the cancer cell with a provided compound or nanoconjugate composition. In some embodiments, a provided compound or nanoconjugate exhibits an IC50 toward the cancer cell at least 10-fold lower compared to gemcitabine alone.

[0072] The present disclosure also provides compositions and methods for enhancing the stability of a Drug (e.g., Gem) to cytidine deaminase degradation. In some embodiments, such methods comprising providing a compound or nanoconjugate composition prepared by a process described herein. In some embodiments, at least about 25% of the Drug (e.g., Gem) is retained following incubation of a compound or nanoconjugate composition with cytidine deaminase for 72 hours at 37 °C.

[0073] The present disclosure further provides methods for delivering or introducing a Drug (e.g., Gem) into a cancer cell, comprising contacting the cell with a provided compound or nanoconjugate composition. In some embodiments, the present disclosure provides an improved method of delivering gemcitabine into a cancer cell, the improvement comprising contacting the cell with a provided compound or nanoconjugate composition. In some embodiments, gemcitabine is delivered to the cell independent of nucleoside transporters.

General syntheses

[0074] Compounds and nanoconjugate compositions described herein may be made as described in the Exemplification below, as well as by other methods known by one skilled in the art. In some embodiments, the present disclosure provides a process for preparing a compound of Formula I or II, comprising steps of i) covalently linking Gem to AA via an amide bond, and ii) covalently linking DTDTPA to AA via an amide bonds. The exact composition of each AA can be varied using various amino acids and peptide coupling chemistries known in the art (e.g., Hong, S. et al. Molecules 2018, 23, 2608). The present disclosure further provides a process for preparing nanoconjugates comprising covalently linking a compound Formulae A, I, or II to AuNP via at least one Au-S bond. The present disclosure also provides a process for covalently linking a thioctic acid terminated peptide (e.g., thioctic acid terminated bombesin, thioctic acid terminated cMET or thioctic acid terminated GE11) to AuNP via at least one Au-S bond. Such processes may include the use of protecting groups not explicitly exemplified in the Exemplification below, but known to one skilled in the art.

Exemplification

[0075] The present disclosure exemplifies compositions, preparations, formulations, nanoparticles, and/or nanomaterials described herein. The present disclosure also exemplifies methods of preparing, characterizing, and validating compositions, preparations, formulations, nanoparticles, and/or nanomaterials described herein. The ensuing Examples provide exemplary materials and methods of preparing, characterizing, and validating compositions, preparations, nanoparticles, and/or nanomaterials described herein. Other suitable methods are known to the skilled artisan.

Abbreviations:

Preparation of GT intermediate

[0076] An exemplary synthesis of the GT intermediate is described below. The intermediate can also be prepared using methods described by Hong, S. et al. Synthesis of Gemcitabine- Threonine Amide Prodrug Effective on Pancreatic Cancer Cells with Improved Pharmacokinetic Properties. Molecules 2018, 23, 2608.

[0077] Protocol of synthesis of GT: The synthesis of GT is achieved via a two-step procedure. In the first step, the intermediate GT-N-Boc is produced, while in the second step the intermediate is deprotected to obtain GT. The intermediate is synthesized using the following procedure. To a 50 mL two neck round bottom flask fitted with a magnetic stir bar, Gemcitabine HC1 (1 g, 3.33 mmol), l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (0.831 g, 4.33 mmol), and 1 -hydroxybenzotriazole (0.476 g, 3.33 mmol) in DMF/DMSO (10 mL, 3:1) were charged under N2 atmosphere at RT. To this reaction mixture, 4-methylmorpholine (0.336 g, 3.33 mmol) and N-Boc threonine (0.803 g, 3.66 mmol) were charged under N2 atmosphere at RT. The reaction mixture was then stirred in an oil bath at 55 °C for 17 hours, cooled to room temperature, and quenched by adding brine (15 mL). The mixture was then extracted using ethyl acetate (2 50 mL) and the combined organic layer was washed with 100 mL of 20% LiCl solution, 100 mL of saturated NaHCCh aqueous solution, 100 mL of brine solution, dried over MgSO4, and concentrated under reduced pressure to afford the crude intermediate GT-N-Boc. The crude product was purified by silica gel column chromatography (1-2% MeOH/DCM as a solvent system) to afford the desired product GT-Ol(GT-N-Boc) as an off white solid (960 mg, 62%). 1H-NMR (500 MHz, DMSO-d6) 5 11.05 (s, 1H), 8.33 (d, J = 7.5 Hz, 1H), 7.32(d, J = 7 Hz, 1H), 6.55 (d, J = 8.5 Hz, 1H), 6.38 (d, J = 6.5 Hz, 1H), 6.24 (t, J = 7 Hz, 1H), 5.35 (1H), 4.94 (s, 1H), 4.24-4.22 (m, 2H), 4.11 (m, 1H), 3.96 (d, J = 8.5 Hz, 1H), 3.88-3.86 (m, 1H), 3.72 (d, J = 12 Hz, 1H), 1.57 (s, 9H), 1.15 (d, J = 6.5 Hz, 3H).

[0078] The GT-N-Boc obtained above is further deprotected using the following procedure to obtain GT. To a 250 mL one neck round bottom flask fitted with a magnetic stir bar, 960 mg (4.01 mmol) of GT-N-Boc and 40 mL of anhydrous DCM were charged under N2 atmosphere at RT. To the aforementioned solution, 40 mL of 4N HC1 in dioxane was charged and the reaction mixture was stirred overnight (~ 14 hours) under N2 atmosphere at RT. After 14 hours, the solvent was evaporated under reduced pressure, and the residue was triturated with hexane to obtain the desired product GT as a white solid (427 mg, 57%). 1H-NMR (400 MHz, MeOH-d4) 5 8.35 (t, J = 7.5 Hz, 1H), 7.28-7.27 (d, J = 5 Hz, 1H), 6.17 (m, 1H), 4.19-4.17 (m, 2H), 3.98 (m, 1H), 3.89 (dt, 2H), 3.73 (d, J = 10 Hz, 1H), 3.63-3.48 (m, 3H), 1.50 (s, 1H) 1.25 (d, J = 6.5 Hz, 3H); 19F-NMR (564.16 MHz, MeOH-d4) 5 -119.09, 5-119.51, 5-119.98, 5- 120.41; ESLMS: m/z 365.14 [M + H]+, m/z 387.17 [M+Na]+. HPLC purity: 99% [RT: 3 mm; UV detection at 267 nm; Column: C-18, 250 *4.6 mm, 5 pm particle size; Mobile phase A:0.1%TFA in water, Mobile Phase B: 0.1% TFA in acetonitrile, Isocratic run A:B=80:20; Flow rate: 1.0 mL/min; Diluent: Acetonitrile: Water (70:30)].

Example 1

Synthesis of DTGT

[0079] Protocol for the synthesis of S-trityl-DT _T o a 250 mL two neck round bottom flask fitted with a magnetic stir bar, 3 g of DTDTPA (DT) and 45 mL of dry DMF were charged under N2 atmosphere at RT. To this, 3.26 g of trityl chloride was charged under N2 atmosphere at RT. The reaction mixture was stirred for two days under N2 atmosphere at RT. After 2 days, the reaction was quenched by the addition of 240 mL of 10% NaOAc solution to produce a white precipitate. The contents were continued to stir for 30 min and the precipitate was filtered using sintered funnel. The precipitate was washed with 30 mL of water, 60 mL of acetone, and 60 mL of diethyl ether consecutively and dried under high vacuum to obtain the crude S-trityl-DT. 1H- NMR (600 MHz, MeOH-d4) 5 7.40-7.21 (m), 3.33-3.31 (m, 8H), 3.13 (m, 4H), 2.99 (d,4H), 2.40 (4H). DT can be synthesized according to known procedures (see, for example, Debouttiere et al. Adv. Funct. Mater. 2006, 16, 2330-2339).

[0080] Protocol for the conjugation of GT to S-trityl-DT: To a 250 mL two neck round bottom flask fitted with a magnetic stir bar, 1 g of S-trityl-DT and 60 mL of dry DMF were charged under N2 atmosphere at RT. To this, 425 mg of DIPEA was charged under

N2 atmosphere at RT and stirred for 5-10 min. Subsequently, 1.7 g of PyBOP was charged under N2 atmosphere at RT and stirred for 15 min. To this, 1.2 g of GT was charged and the reaction mixture was degassed and stirred for 24 hours under N2 atmosphere at RT. After 24 hours, the reaction was quenched by the addition of 60 mL of water and 20 mL of 10% HCL. The product was extracted from the reaction mixture using 125 mL of ethyl acetate. The organic layer was washed with 50 mL of saturated NaHCOv 50 mL of brine, dried over MgSO-i, and concentrated under reduced pressure to obtain the crude S-trityl-DTGT. The crude S-trityl-DTGT was purified using preparatory HPLC using acetonitrile/water as mobile phase and lyophilized to obtain white powder of S-trityl-DTGT. 1H-NMR (600 MHz, MeOH-d4) 5 8.34 (m,2H), 7.33-7.22 (m,33 H), 6.26 (m, 2H), 4.57 (2H), 3.99-3.83 (8H), 3.45 (3H), 3.10 (7H), 2.37 (3H), 1.25 (d, 9H); 19F- NMR (564.16 MHz, MeOH-d4) 5 -119.09, 5-119.51, 5-119.98, 5-120.41; LC-MS: RT: 17.63, m/z 1017.78 [M/2]+.

[0081] Protocol for the deprotection of sulfur: The S-trityl-DTGT obtained above is further deprotected using the following procedure to obtain DTGT. To a 50 mL two neck round bottom flask fitted with a magnetic stir bar, 60 mg of S-trityl-DTGT, 3 mL of 10% TFA in DCM, and 3 mL of 10% TES in DCM were consecutively charged and the reaction mixture was stirred for 2.5 hours under N2 atmosphere at RT. After 2.5 hours, the reaction was quenched by the addition of 0.3 mL of 10% pyridine in MeOH and the reaction mixture was flushed with N2 to minimize the amount of DCM. To this, 30 mL of diethyl ether was charged to obtain a white precipitate that is filtered by centrifugation. The white solid is dried under vacuum to obtain the final product DTGT. 1H-NMR (600 MHz, MeOH-d4) 5 8.85 (3H), 8.05 (m,3H), 6.16 (3H), 4.46 (m,2H),4.33 (m), 3.99-3.83 (m), 3.47-3.40 (m), 3.23-3.10 (m), 2.64 (m), 1.26 (m); 19F-NMR (564.16 MHz, MeOH-d4) 5 -119.0, 5-119.50, 5-119.98, 5-120, 5-121; LC-MS: RT: 29.20, m/z 1550 [M+H]+.

Example 2

Synthesis of peptide- Au- [DTGT]

[0082] Protocol for the synthesis of Au-[DTGT]: To a 100 mL conical flask fitted with a magnetic stir bar, 45 mL of water, 0.5 mL of IM NaOH, and 1 mL of THPC or Tetrakis (hydroxymethyl) phosphonium chloride (comprised of 1 mL of water + 12 pL of 80% THPC) were charged and stirred for 5 min at 1000 rpm. To this mixture, 2 mL of 25 mM HAuC14.3H2O was quickly added at RT under vigorous stirring. The color of the mixture immediately turned to dark brown indicating the formation of gold nanoparticles. The suspension was allowed to stir for 15 min 1000 rpm. After 15 min, aqueous solution of m-PEG-SH, 2000 daltons (18 mg in 2 mL of H2O) was added to the nanoparticle suspension dropwise at RT under vigorous stirring (1000 rpm). The reaction mixture was continued to stir for 16 hours at 1000 rpm followed by washing with water several times using a 10 kDa (molecular weight cut off) centrifugal filter. The final suspension of gold nanoparticles (AuNP) was concentrated to 1.5 mL. In the next step, to 0.39 mL of concentrated solution of Au NP taken in a 5 mL glass vial fitted with a magnetic stir bar, 0.61 mL of water was charged. To this solution, 0.99 mL of DTGT (5 mg/mL in H2O) was added dropwise while stirring at RT (1000 rpm). The reaction mixture was continued to stir for 16 hours. The unconjugated DTGT was removed by passing the reaction mixture through 10 KD (molecular weight cut off) centrifugal filter. The reaction mixture is further washed with water six times using the 10 KD (molecular weight cut off) centrifugal filter to obtain 0.5 mL concentrated suspension of Au-[DTGT], See also Figure 1.

[0083] Protocol for the synthesis of P4BN: A thioctic-bombesin peptide was synthesized following the traditional solid-phase peptide synthesis (SPPS) procedure employing Fmoc chemistry methodology and the final peptides were purified by HPLC. A 4- hydroxymethylphenoxyacetyl- 4'-methylbenzyhydrylamine resin was used as the solid support for the synthesis. Fmoc-protected amino acids were activated using one equivalent of 0.45 M HBTU/HOBt solutions and two equivalents of N, N-di isopropyl ethylamine. The amino acids were Fmoc deprotected using piperidine and coupled using NMM.HBTU. Following the coupling of all of the amino acids in the appropriate sequence, thioctic acid (lipoic acid) was coupled using DIC.HOBt.

[0084] Cleavage of the peptide from the resin was performed using TFA. This cleavage step also removed the amino acid side chain protecting groups.

[0085] The peptide was purified on a reverse-phase HPLC/C18 column. The purity was confirmed by HPLC reverse-phase chromatography and Electrospray Mass Spectral (ESIMS) analysis. A single peak corresponding to SS-PEG4-BN SS-BN at retention time (tR) of 7.58 min was observed in HPLC validating 98% purity of the peptide. The observed m/z peak correlates well with the calculated molecular ion peak expected for the peptide. The ESI-MS spectrum of pure P4BN shows two peaks, one corresponding to the molecular ion peak at m/z of 1375 (100% abundance) and another at m/2z of 687 corresponding to the doubly charged ion.

(SEQ ID NO: 1)

P4BN

[0086] Protocol for the synthesis of P4BN-Au-[DTGT]: To 200 pL of Au-[DTGT] concentrated suspension taken in a glass vial fitted with a magnetic stir bar, 1.75 mg of P4BN (1 mg/mL in H2O) was added dropwise at RT while stirring (1000 rpm). The reaction mixture was continued to stir for 3 hours. The unconjugated peptide was removed by passing the reaction mixture through a 10 kDa (molecular weight cut off) centrifugal filter. The reaction mixture was further washed with water 4 times using the 10 kDa (molecular weight cut off) centrifugal filter to obtain 0.2 m concentrated suspension of P4BN-Au-[DTGT] (SEQ ID NO: 2).

[0087] Protocol for the synthesis of P4cMET: The peptide was synthesized using standard Fmoc solid-phase peptide chemistry on a multiple peptide synthesizer (Tetras, Advanced ChemTech) on Sieber amide resin. The protected amino acids, as well as the solutions for coupling and deprotecting reactions were separately dissolved and arranged in different bottles of the instrument. The protection groups chosen for the amino acid side chains were: tBu (Tyr and Ser); Trt (His) and Boc (Trp and Lys).

[0088] The peptide chain was assembled by sequential acylation (20 minutes for coupling) with "in situ" activated Fmoc-amino acids. Re-coupling and capping were automatically performed at every cycle.

[0089] The "in situ" activations of Fmoc-amino acids (3 equiv. compared to the resin amount) were carried out using uranium salts (HBTU, 2.7 eq., HOBT 3 equiv.) and DIEA (6 equiv.).

[0090] The Fmoc protecting groups were removed at every subsequent cycle by three treatments with 6% piperazine in 0.1 M HOBt /DMF for 10 min. [0091] The peptidyl-resin was cleaved from the resin and deprotected in a single reaction with TFA, TA, phenol, water, EDT and TIS (87.5:2.5:2.5:2.5:2.5:2.5) for 2 h at room temperature (25 °C). Precipitation and multiple washing with diethyl ether gave the final crudes.

[0092] Crude peptides were dissolved before in a solution of 50 mM TCEP (25% Acetonitrile in Water) in order to keep the sulfhydryl function on the thioctic acid in their reduced form HPLC analysis were then performed on 168-diode array detector, a 507e auto injector and the 32 KARAT software package (Gold System from Beckmann Coulter, Fullerton, CA). The HPLC system was coupled with an ion trap Mass spectrometer (LCQ Fleet from Thermo Fisher, Waltham, MA). For analytical runs a Thermo Scientific BetaBasic Cl 8 analytical column (150 mm x 4.6 mm, 5 pm, 150 A) was used.

[0093] The UV wavelengths used to monitor these runs were 214/280 nm. Eluents used in all runs were water (A), and acetonitrile (B) each containing 0.1% TFA. Gradient used were: linear from 10% to 50% B in 30 min (crude).

[0094] A single peak corresponding to SS-PEG4-cMET was observed in HPLC at retention time (tR) of 29.74 min validating 98% purity of the peptide. The observed m/z peak correlates well with the calculated molecular ion peak expected for the peptide. The ESI-MS spectra of pure P4cMET shows molecular ion peak at m/z of 1893 (10 % abundance) and m/2z of 948 (100% abundance).

(SEQ ID NO: 3)

P4cMET [0095] Protocol for the synthesis of P4cMET-Au-[DTGT] : To 200 pL of Au-[DTGT] concentrated suspension taken in a glass vial fitted with a magnetic stir bar, 2.44 mg of P4cMET (1 mg/mL in H2O) was added dropwise at RT while stirring (1000 rpm). The reaction mixture was continued to stir for 3 hours. The unconjugated peptide was removed by passing the reaction mixture through a 10 kDa (molecular weight cut off) centrifugal filter. The reaction mixture was further washed with water 4 times using the 10 kDa (molecular weight cut off) centrifugal filter to obtain 0.2 mb concentrated suspension of P4cMET-Au-[DTGT] (SEQ ID NO: 4).

[0096] Characterization: The nanoparticle constructs were characterized using hydrodynamic size, zeta potential, and TEM (Figures 2-5), as described in, e.g., WO2018/129501, Silva et al. Bioconjugate Chem. 2016, 27, 1153-1164 (and Supporting Information), and Debouttiere et al. Adv. Funct. Mater. 2006, 16, 2330-2339. The UV-visible spectra of all the DTGT loaded constructs clearly suggested the conjugation of the pro-drug to the NP (Figure 6).

Example 3

Estimation of Gem load in nanoparticles

[0097] The gemcitabine load in the construct is estimated by digesting it in 1.5 M NaCN which dissolves the gold and releases the conjugated gemcitabine. 2 pL of NP suspension is mixed with 18 pL of 1.5 M NaCN and incubated at room temperature for 2.5 hours, 900 RPM. 20 pL of this mixture is then injected into HPLC and analyzed at 272 nm for the presence of gemcitabine. The concentration of gemcitabine is estimated based on a standard curve of gemcitabine in 1.5 M NaCN at 272 nm (Figure 7). The details of HPLC instrumentation and method are as follows. The Agilent 1260 Infinity II HPLC system is connected with a Phenomenex Cl 8 column (Jupiter Cl 8, 250 x 4.6 mm, 5 pm, 300 A), operated at room temperature. The mobile phase consisted of solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile) eluted in gradient at a flow rate of 1 mL/minute. Solvent B starts with 0 % and reaches 10 % at 10 minutes, before returning back to 0 % at 12 minutes followed by reequilibration for 4 minutes. Agilent 1260 Infinity II Diode Array Detector was used for detection at a wavelength of 272 nm. Example 4

Estimation of peptide load in nanoparticles

[0098] The peptide load in the construct is estimated by digesting it in 1.5 M NaCN which dissolves the gold and releases the conjugated peptide. 2 pL of nanoparticle suspension is mixed with 18 pL of 1.5 M NaCN and incubated at room temperature for 2.5 hr. 20 pL of this mixture is then injected into HPLC and analyzed at 280 nm for the presence of gemcitabine. The concentration of peptide is estimated based on a standard curve of peptide in 1.5 M NaCN at 280 nm (Figure 8). The conjugation efficiency of P4BN and P4CMET are 5% and 54 % respectively. The details of HPLC instrumentation and method are as follows. The Agilent 1260 Infinity II HPLC system is connected with a Phenomenex Cl 8 column (Jupiter Cl 8, 250 x 4.6 mm, 5 pm, 300 A), operated at room temperature. The mobile phase consisted of solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile) with the following run program: First 5 minutes, A: B is 95:5, gradually increasing the B to 90% by 15 minutes. For 15 to 17 minutes, the run was maintained at the ratio of A:B = 10:90. Then the system was re-equilibrated back to initial ratio (A: B = 95:5). Peptides were detected at 280 nm with the DAD detector.

Example 5

Metabolic Stability of Gem in DTGT and Au-[DTGT]

[0099] The metabolic stability of Gem in DTGT and Au- [DTGT] was evaluated in-vitro in the presence of pure cytidine deaminase (CD A). The testing with CDA is based on the rationale that CDA is responsible for degradation of Gem into its therapeutically inactive metabolite 2',2'-difluorodeoxyuridine (dFdU) in physiological conditions (primarily in liver/plasma).

[0100] Protocol for the In vitro metabolic stability assay based on CDA: Gem, DTGT and Au- [DTGT] (50 pM Gem equivalent) were incubated in DPBS containing calcium chloride (0.9 mM), magnesium chloride (0.49 mM), potassium chloride (2.66 mM), in the presence of 15 pg/mL of CDA at 37 °C. A sample of each mixture (triplicate; 50 pL) was collected at 2 mins, 10 mins, 1 hour, 24 hours, 72 hours and 2 pL of tetrahydrouridine (10 mg/mL) was added to quench CDA activity. 104 pL of 1 M NaOH was added to each sample and incubated at 45 °C for 2 hours to release gemcitabine. 104 uL of 1 M HC1 was subsequently added to neutralize the mixture. 2 pL of internal standard (1 mg/mL 2' -deoxy cytidine) was then added to all the samples (Gem, DTGT, Au-[DTGT]). 0.2 mb and 0.6 mb of acetonitrile was added to the Gem and DTGT/Au-[DTGT] samples respectively, and vortexed. The mixtures were centrifuged (17,000 g;10 min) and the supernatant was dried under nitrogen flow (40 °C). The residue was resuspended in 50 pL of water, centrifuged and 30 pL of supernatant was injected into HPLC (Agilent). The standard curves for gemcitabine and DTGT were established by spiking DPBS with the drug/construct stocks and processed as described above (Gem-equivalent linear range of 3.1-100 pM). A Phenomenex Jupiter Cl 8 column (5 pm, 250x4.6 mm) was used with a gradient mobile phase of water (0.1%TFA) and acetonitrile (0.1%TFA) (0-10% ACN over 10 minutes followed by re-equilibration). The flow rate was 1.0 mL/min with a column temperature of 50 °C. The detection wavelength for Gemcitabine (RT ~7.7) was 272 nm. The ratio of Gem peak area/IS peak area was applied to the standard curve equations (R²>0.9997) to determine concentration.

[0101] The amount of Gem that remained stable in the constructs in the presence of CDA at various time points of the study was calculated using the standard curves based on HPLC (Figure 9). Gem in its original form completely degraded within 2 min in the presence of CDA, while the modified formulations of Gem, i.e., DTGT and Au-[DTGT] exhibited significant improvement in stability with some/substantial amount of Gem being intact even after 72 hours (Figure 10). More importantly, the design of two-pronged strategy, i.e., 4-(N)- modification of Gem (as in DTGT), and conjugation of the modified derivative to NP (as in Au-[DTGT]) has produced a synergistic effect in strengthening the metabolic stability of Gem. This is validated by the observation that 4-(N)-modification of Gem (DTGT) has resulted in significant improvement in CDA stability which is further enhanced by conjugating the derivative to a gold nanocarrier. At the end of 72 hours, 27% of Gem is intact in DTGT while 69% (2.5-fold) of Gem remained stable in Au-[DTGT], thus highlighting the additional protection rendered by the AuNP (Figure 10).

Example 6

In-vitro cytotoxicity of DTGT and Au-[DTGT] on cancer cell lines [0102] To determine whether AuNP is effective in sensitizing the cancer cells to Gem as well as enhance the uptake and cellular internalization of drug to produce significant inhibition of cell division and growth, two types of cell viability assays (MTT assay and SRB assay) were performed to evaluate the cytotoxicity of the constructs on lung, pancreatic and breast cancer cell lines. The cell lines were selected based on indications approved for Gem by the FDA and expression levels of targeted receptors (cMET and GRPR).

[0103] Protocol for the MTT Assay: To conduct the MTT assay 1x106 cells (at 70% confluency; p+2) were seeded onto 96-well plates overnight (triplicates per dose per construct). Drugs or nanoparticle-constructs at specific concentrations were then prepared in serum-free RPMI media to test the toxicity profile at various concentrations for a period of 72 hours. Drugs or nanoparticle-constructs at specific concentrations were also added to cell-free wells (duplicates) as a background control. At 72 hours, 10 pL of MTT-dye was added per well and incubated for 4 hours as per manufacturer’s protocol, followed by solubilization of formazan crystals (100 pL of solubilization buffer added per well) for an additional two hours in the incubator. The plates were then read at wavelength of 570 nm using a Biotek Synergy Hl plate reader to access the absorption values of the solubilized formazan crystals. Absorption values were then averaged, and background was negated before calculating percent viability. Percent viability values from triplicates were then used in Graphpad Prism software (ver. 8.4.3; [Inhibitor] vs. normalized response — Variable slope) to compute the IC50 of the specific construct (pg/mL dry wt.). Final IC50 values were represented as gemcitabine-equivalent IC50 values shown in Table 1 below.

[0104] Protocol for the SRB Assay: NCI-60 Screening Methodology was followed to evaluate the in vitro efficacy of the constructs. Briefly, cells were seeded in 96-well tissue culture plates at a density appropriate for the cell line. The next day, a control plate was processed as described below to determine the density at TO (zero time). The remaining plates were treated with constructs and controls over a 7 log ug/mL concentration range. The plates were then incubated for 72 hours following which they were fixed with TCA (4 °C; 1 hour; final cone. 10%), dried and stained with sulphorhodamine B (0.4% w/v in 1% acetic acid; 100 uL per well) for 10 minutes. The plates were washed with 1% acetic acid, to remove unbound dye, and dried. Bound dye was extracted with 10 mM Tris Base (200 pL per well). Absorbance readings were obtained at 515 nm using a plate reader. Inhibition of growth was calculated relative to untreated cells and the TO control. GI50 was calculated in Graphpad Prism 8.4.3 using the formula Y=Bottom+(Top-Bottom)/(l+10^A((LogAbsoluteIC50-X)*HillSlope+log((Top- Bottom)/(Fifty-Bottom)-l))). Fifty=(Top+Baseline)/2. All data is represented as Gemcitabineequivalent pg/mL GI50 values.

[0105] Results showed that the gemcitabine-equivalent IC50 of native DTGT-prodrug showed similar or better efficacy than gemcitabine or GT-prodrug in pancreatic cell lines (Figure 11, Table 1). Further, when DTGT was conjugated to gold NP the efficacy was found to be better than DTGT alone (Figure 11, Table 1). When c-MET peptide was attached to the construct the efficacy increased by 20-fold or 7-fold respectively in Panel and BxPC3 Cell lines relative to Au-[DTGT] indicating ability of c-MET to target overexpressed c-MET receptors in the respective cell lines (Figure 11, Table 1). Similarly in the breast cancer cell lines the efficacy of Au-[DTGT] and P4BN-Au-[DTGT] constructs is as good as free Gem or better in some cases (Figure 12, Table 1). The excellent cellular cytotoxicity exhibited by the nanoformulations of Gem validates the competence of the designed gold NP as vehicles for efficient delivery of Gem to the cancer cells.

Table 1: Comparison ofIC-50 (MTT assay, 72 Hours) and GI-50 values (SRB assay, 72 Hours) of free Gemcitabine and its modified formulations in lung, pancreatic and breast cancer cell

[0106] While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

Claims

1. A compound of F ormula I :

I or a pharmaceutically acceptable salt thereof, wherein:

2. The compound of claim 1, wherein the compound is of formula II:

or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1 or 2, wherein L contains two thiol functional groups.

4. The compound of any one of the preceding claims, wherein L is dithiolated di ethylenetriamine pentaacetic acid (DTHTPAt

5. The compound of any one of the preceding claims, wherein AA is L-threonine.

6. The compound of any one of the preceding claims, having the structure:

or a pharmaceutically acceptable salt thereof.

7. A nanoconjugate comprising the compound of any one of claims 1-6 covalently linked to a gold nanoparticle (AuNP) via at least one Au-S bond.

8. The nanoconjugate of claim 7, wherein AuNP is PEGylated.

9. The nanoconjugate of claim 7, wherein the compound is covalently linked to AuNP via two Au-S bonds.

10. The nanoconjugate of claim 7, comprising a single layer of compound surrounding AuNP.

11. The nanoconjugate according to any one of claim 7-10, further comprising a thioctic acid terminated peptide covalently linked to AuNP via at least one Au-S bond.

12. The nanoconjugate of claim 11, wherein the thioctic acid terminated peptide is thioctic acid terminated bombesin, thioctic acid terminated cMET or thioctic acid terminated GE11.

13. A nanoconjugate composition comprising the nanoconjugate of any one of claims 7-10.

14. A nanoconjugate composition comprising the nanoconjugate of any one of claims 11-12.

15. The nanoconjugate composition of claim 13, wherein on average in the composition the nanoconjugates have a hydrodynamic size of less than about 40 nm.

16. The nanoconjugate composition of claim 13 or 15, wherein on average in the composition the nanoconjugates have a zeta potential of about -20 mV or less.

17. The nanoconjugate composition of any one of claims 13-16, wherein on average in the composition the nanoconjugates have a GEM loading between about 15-30% by weight.

18. The nanoconjugate composition of any one of claims 13-17, wherein on average in the composition the nanoconjugates have an aqueous solubility of at least about 40 mg/mL.

19. The nanoconjugate composition of claim 14, wherein on average in the composition the nanoconjugates have a zeta potential of about -15 mV or less.

20. The nanoconjugate composition of claim 14 or 19, wherein on average in the composition the nanoconjugates have a thioctic acid terminated peptide loading of about 5% to about 60%.

21. A gold nanoparticle (AuNP) comprising a compound of any one of claims 1-6.

22. The AuNP of claim 21, wherein the compound is covalently linked to the gold surface via at least one Au-S bond.

23. The AuNP of claim 21 or 22, wherein the AuNP is PEGylated.

24. The AuNP of claim 22 or 23, wherein the compound is covalently linked to the gold surface via two Au-S bonds.

25. The AuNP of any one of claims 21-24, comprising a single layer of compound on the gold surface.

26. A gold nanoparticle (AuNP) comprising a compound of the following structure:

wherein each

is independently a point of attachment of the compound to hydrogen or the AuNP gold surface.

27. The AuNP of claim 26, wherein each -s-l * is a point of attachment of the compound to the AuNP gold surface.

28. The AuNP of any one of claims 21-27, further comprising a thioctic acid terminated peptide covalently linked to the AuNP via at least one Au-S bond.

29. The AuNP of claim 28, wherein the thioctic acid terminated peptide is thioctic acid terminated bombesin, thioctic acid terminated cMET or thioctic acid terminated GE11.

30. An AuNP composition comprising the AuNP of any one of claims 21-29.

31. A pharmaceutical composition comprising a therapeutically effective amount of the nanoconjugate composition of any one of claims 13-20 or the AuNP composition of claim 30 and optionally one or more pharmaceutically acceptable excipients.

32. The pharmaceutical composition of claim 31 or 53 in lyophilized form.

33. A method of treating a cancer in a patient in need of such of treatment, comprising administering to the patient the pharmaceutical composition of claim 32.

34. The method of claim 33, wherein the patient exhibits one or more reduced side effects compared to a patient treated with an equivalent amount of gemcitabine alone.

35. A process for preparing the compound of any one of claims 1-6, comprising covalently linking GEM and DTDTPA to each AA via amide bonds.

36. A process for preparing a nanoconjugate, comprising covalently linking the compound of any one of claims 1-6 to AuNP via at least one Au-S bond.

37. The process of claim 36, further comprising covalently linking a thioctic acid terminated peptide to AuNP via at least one Au-S bond.

38. The process of claim 37, wherein the thioctic acid terminated peptide is thioctic acid terminated bombesin, thioctic acid terminated cMET or thioctic acid terminated GE11.

39. A method for enhancing the stability of gemcitabine to cytidine deaminase degradation, comprising providing a compound of any one of claims 1-6.

40. A method for enhancing the stability of gemcitabine to cytidine deaminase degradation, comprising providing a compound prepared via the process of claim 35.

41. The method of claim 39 or 40, wherein at least about 25% of the gemcitabine is retained following incubation of the compound with cytidine deaminase for 72 hours at 37 °C.

42. A method for enhancing the stability of gemcitabine to cytidine deaminase degradation, comprising providing a nanoconjugate composition of any one of claims 13-20 or the AuNP composition of claim 30.

43. A method for enhancing the stability of gemcitabine to cytidine deaminase degradation, comprising providing a nanoconjugate prepared via the process of claim 36.

44. The method of claim 42 or 43, wherein at least about 50% of the gemcitabine is retained following incubation of the nanoconjugate with cytidine deaminase for 72 hours at 37 °C.

45. A method for introducing gemcitabine into a cancer cell, comprising contacting the cell with the nanoconjugate composition of any one of claims 13-20 or the AuNP composition of claim 30.

46. The method of claim 45, wherein gemcitabine is introduced to the cell independent of nucleoside transporters.

47. A method for killing or inhibiting the growth of a cancer cell, comprising contacting the cell with the nanoconjugate composition of any one of claims 13-20 or the AuNP composition of claim 30.

48. A method for enhancing the cytotoxicity or cytostaticity of gemcitabine in a cancer cell, comprising providing a compound of any one of claims 1-6, and contacting the cancer cell with the compound.

49. A method for enhancing the cytotoxicity or cytostaticity of gemcitabine toward a cancer cell, comprising providing a nanoconjugate composition of any one of claims 13-20 or the AuNP composition of claim 30, and contacting the cancer cell with the nanoconjugate.

50. The method of claim 49, wherein the nanoconjugate exhibits an IC50 toward the cancer cell at least 10-fold lower compared to gemcitabine alone.

51. In a method of delivering gemcitabine into a cancer cell, the improvement comprising contacting the cell with the nanoconjugate composition of any one of claims 13-20 or the AuNP composition of claim 30.

52. In a method of treating cancer in a patient in need of such of treatment, the improvement comprising administering to the patient the pharmaceutical composition of claim 31 or 53.

53. A pharmaceutical composition comprising a plurality of nanoconjugates, at least one nanoconjugate comprising a compound having the structure:

covalently linked to a gold nanoparticle (AuNP) via at least one Au-S bond.