Dendrimer-glutamine antagonist conjugates and methods of using same

Abstract

Compositions of dendrimers conjugated with one or more glutamine antagonists that inhibit glutamine metabolism, preferably in activated microglia, and methods of using them to treat, alleviate, and / or prevent one or more neurological, oncological, and / or immune disorders associated with pathogenic or dysregulated glutamine-dependent pathways and / or glutamate transmission have been developed. The dendrimers conjugated with one or more glutamine antagonists effectively inhibit glutaminase activity without any systemic toxicity. The dendrimers conjugated with one or more glutamine antagonists accumulate in activated microglia associated with damaged or diseased cells and tissues.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 174,878, filed April 14, 2021, which is hereby incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention is generally in the field of drug delivery, and in particular glutamine antagonists conjugated via dendrimers for selective uptake into the site or region of diseased and damaged cells and tissues in need thereof. [Background technology]

[0003] 2. Background of the Invention The broadly active glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) has been investigated for 60 years as a potential anti-cancer therapeutic. Studies have shown that DON has robust anti-cancer potential, including the treatment of brain cancer. DON has also been demonstrated to block glutamate release from activated microglia and normalize glutamate production in activated immune cells, providing broad therapeutic utility in neurological, neuropsychiatric, and immune disorders involving neuroinflammation and excitotoxicity.

[0004] Despite the therapeutic potential of DON, clinical development has not been carried out due to its systemic toxicity, which is mostly gastrointestinal (GI) related. To obtain adequate therapeutic concentrations of DON in target tissues, peripheral administration doses must be large, causing intolerable GI side effects. Therefore, there is a need for novel glutamine antagonists that have similar biological activity and therapeutic potential as DON, but with reduced or minimal systemic toxicity.

[0005] It is therefore an object of the present invention to provide compositions, and methods of making and using same, for reducing and preventing pathological processes associated with dysregulated glutamine-dependent pathways and / or glutamate transmission.

[0006] It is also an object of the present invention to provide compositions for treating or preventing a variety of CNS, oncological, and immune disorders in which dysregulated glutamine-dependent pathways and / or glutamate transmission are presumed to be pathogenic. Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention 6-Diazo-5-oxo-L-norleucine (DON) analog compositions have been developed.

[0008] In some other examples, the DON analogs include the following, where "AA" refers to an amino acid: [ka] wherein R1 is selected from -OCH2CH3, -OCH(CH3)2-OC(CH3)3, -NH2, NHCH3, -NH-AA-COOCH(CH3)2, and -NH-AA-COOC(CH3)3; R2 is selected from -H, -CH3, -CH2phenyl, -CH2CH(CH3)2, -CH2(indol-3-yl), and -CH2CH2C(=O)CH=N≡N; R3 is selected from -NH2 and -OH; n is an integer value from 1 to 32. [ka] wherein R1 is selected from -OCH2CH3, -OCH(CH3)2-OC(CH3)3, -NH2, NHCH3, -NH-AA-COOCH(CH3)2, and -NH-AA-COOC(CH3)3; R3 is selected from -NH2 and -OH; n is an integer value from 1 to 16. [ka] wherein R2 is selected from -H, -CH3, -CH2phenyl, -CH2CH(CH3)2, -CH2(indol-3-yl), or -CH2CH2C(=O)CH=N≡N; R4 is selected from -H, -CH3, -CH2phenyl, or -CH2CH(CH3)2; R5 is -H, -Ac, -Fmoc, dimethylglycine, Ac-AA-C(=O)-, dimethylglycine-NH-AA-C(=O)-, dimethylglycine-NH-DON-C(=O)-, or CH 17 OC(=O)CH(NH2)CH2CH2C(=O)-NH-6-diazo-5-oxo-L-norleucine-C(=O)-; n is an integer value from 1 to 32. These have one of three general chemical formulas:

[0009] DON analogs include molecules having any one of structures I-X: [ka] [ka] [ka] [ka] [ka]

[0010] Compositions of dendrimers conjugated to one or more DON analogs are also described. Compositions are provided that include dendrimers complexed, covalently conjugated, or intramolecularly dispersed or encapsulated with one or more 6-diazo-5-oxo-L-norleucine (DON) analogs having any of structures I-X or general formulas A-C.

[0011] Exemplary dendrimers are fourth, fifth, sixth, seventh or eighth generation dendrimers. In some embodiments, the dendrimer is a poly(amidoamine) (PAMAM) dendrimer, such as a hydroxyl-terminated PAMAM dendrimer. In preferred embodiments, the dendrimer is a fourth, fifth or sixth generation hydroxyl-terminated PAMAM dendrimer.

[0012] In some embodiments, the dendrimer is covalently conjugated to one or more DON analogs, optionally via a linker or spacer moiety.In some embodiments, the dendrimer is further complexed or conjugated to one or more therapeutic, preventive or diagnostic agents.Exemplary therapeutic agents include neuroprotective agents, anti-inflammatory agents, and / or chemotherapeutic agents.

[0013] Also provided are pharmaceutical compositions comprising a dendrimer complexed, covalently conjugated, intramolecularly dispersed or encapsulated with one or more 6-diazo-5-oxo-L-norleucine (DON) analogs having any of structures I-XII or formulas A-C, and one or more pharma- ceutically acceptable excipients.

[0014] The pharmaceutical compositions are formulated for parenteral or oral administration, for example, in the form of hydrogels, nanoparticles or microparticles, suspensions, powders, tablets, capsules, and solutions.

[0015] A method for treating one or more of neurological, oncological, and / or immune disorders in a subject in need of such treatment includes administering to the subject an effective amount of a dendrimer complexed, covalently conjugated, intramolecularly dispersed, or encapsulated with one or more 6-diazo-5-oxo-L-norleucine (DON) analogs having any of structures I-XII or general formulas A-C to treat, alleviate, and / or prevent one or more symptoms associated with one or more of the neurological, oncological, and immune disorders. Preferably, the composition is administered in an amount effective to reduce glutaminase activity in diseased or damaged tissues of one or more of the neurological, oncological, and immune disorders. For example, in some embodiments, the composition is administered in an amount effective to reduce glutaminase activity in activated microglia associated with diseased and / or damaged tissues of one or more of the neurological, oncological, and immune disorders. Exemplary neurological disorders that can be treated with these DON analogs include stress-induced mood disorders, cognitive deficits, ischemia, neuroinflammation, Alzheimer's disease, and multiple sclerosis. In one embodiment, the disease or disorder is a stress-induced mood disorder, such as depression. In some embodiments, the composition is administered in an amount effective to reduce neuroinflammation, improve cognition, or a combination thereof. Exemplary oncological disorders that can be treated by the method include breast cancer, ovarian cancer, uterine cancer, prostate cancer, testicular germ cell tumor, brain cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, renal cell carcinoma, colorectal cancer, and pancreatic cancer. In some embodiments, the composition is administered in an amount effective to reduce tumor size. Exemplary immune disorders that can be treated by the method include rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), type 1 diabetes, inflammatory bowel disease, and thyroid disease. In further embodiments, the composition is administered to the subject in combination with one or more of an immune checkpoint modulator, a chemotherapeutic agent, an anti-infective agent, an adoptive T cell therapy, a cancer vaccine, surgery, and radiation therapy.Exemplary immune checkpoint modulators include PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists.

[0016] Methods for making and conjugating to dendrimers one or more 6-diazo-5-oxo-L-norleucine (DON) analogs having any of structures IX or formulas AC are also described. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram of the synthesis of compound I.

[0018] [Diagram 2] FIG. 2 is a schematic diagram of the synthesis of compound II.

[0019] [Diagram 3] FIG. 3 is a schematic diagram of the synthesis of compound III.

[0020] [Figure 4] FIG. 4 is a schematic diagram of the synthesis of compound IV.

[0021] [Diagram 5] FIG. 5 is a schematic diagram of the synthesis of compound V.

[0022] [Figure 6] FIG. 6 is a schematic diagram of the synthesis of compound VI.

[0023] [Figure 7] FIG. 7 is a schematic diagram of the synthesis of compound VII.

[0024] [Figure 8] FIG. 8 is a schematic diagram of the synthesis of compound VIII.

[0025] [Figure 9]FIG. 9 is a schematic diagram of the synthesis of compounds IX and X.

[0026] [Figure 10] FIG. 10 is a schematic diagram of the synthesis of compound XI.

[0027] [Figure 11] FIG. 11 is a scheme showing a synthetic scheme for compound XII.

[0028] [Figure 12] FIG. 12 is a schematic diagram of a general synthetic route for dendrimer-glutamine antagonist conjugates.

[0029] [Figure 13] Figure 13 is a schematic diagram showing the experimental setup using mice. Mice were intracranially injected with LPS (2.5 mg / kg) into their right striatum. Three hours later, dendrimer-TTM020 or empty dendrimer was administered IP at a dose of 2 mg / kg DON equivalent. After 24 and 72 hours, mice were sacrificed, the right hemisphere was removed, and CD11b cells were extracted for glutaminase activity measurement.

[0030] [Figure 14] FIG. 14 is a schematic showing the chemical structure of the dendrimer conjugated to TTM020.

[0031] [Figure 15] FIG. 15 is a bar graph showing glutaminase activity measured in fmol / mg / hr in control mice and mice treated with unconjugated dendrimer (D-EMPTY) or DON analog TTM020-conjugated dendrimer (D-DON) at 24 and 72 hours after drug treatment.

[0032] [Figure 16]Figure 16 is a bar graph showing uptake of dendrimer-Cy5(AUC) in the contralateral, peritumoral and tumor regions of the brain in a rodent tumor-inoculated 9L gliosarcoma model, n=6.

[0033] [Figure 17] FIG. 17 is a schematic diagram showing the chemical structure of a dendrimer conjugated to a DON analog via a linker.

[0034] [Figure 18] Figures 18A and 18B are bar graphs showing tumor mass (Figure 18A) and tumor burden as a percentage of brain mass (Figure 18B) in untreated controls, mice treated with unconjugated DON (free DON), and mice treated with dendrimer-KMN-045 conjugate (D-045), respectively, in the GL261 murine model of GBM in C57BL / 6 mice.

[0035] [Figure 19] FIG. 19 is a bar graph showing % DON release over time for the in vitro stability of dendrimer-KMN-045 conjugates in brain homogenates.

[0036] [Figure 20A] FIG. 20A is a line graph showing the release of DON (μM) in tumor homogenates over 60 minutes. [Figure 20B] FIG. 20B is a line graph showing inhibition of tumor volume in mm 3 over 14 days in a syngeneic EL4 lymphoma model upon treatment with D-DON (TTM020) or vehicle. [Figure 20C] FIG. 20C is a line graph showing the percent body weight change over 14 days upon treatment with D-DON (TTM020) or vehicle.

[0037] [Figure 21]Figure 21 is a bar graph showing glutaminase activity (fmol / mg / hr) in three experimental groups including control mice treated with vehicle (Ctr+veh), CSDS mice treated with vehicle (CSDS+veh), and CSDS mice treated with D-TTM020 (CSDS+D-TTM020). **p<0.01, comparison between Ctr+veh and CSDS+veh. ##p<0.01, comparison between CSDS+veh and CSDS+D-TTM020. Analyzed by one-way ANOVA, Tukey's post-hoc test. n=5 per condition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Detailed Description of the Invention I. Definition The term "active agent" or "biological agent" refers to a therapeutic, prophylactic or diagnostic chemical or biological compound that induces a desired pharmacological and / or physiological effect, which may be prophylactic, therapeutic or diagnostic. These may be nucleic acids, nucleic acid analogs, small molecules having a molecular weight of less than 2 kD, more typically less than 1 kD, peptidomimetics, proteins or peptides, carbohydrates or sugars, lipids, or combinations thereof. These terms also encompass pharma- ceutically acceptable and pharmacologically active derivatives of the agents, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, and analogs.

[0039] The term "therapeutic agent" refers to an agent that can be administered to treat one or more symptoms of a disease or disorder.

[0040] The term "diagnostic agent" generally refers to an agent that can be administered to localize, identify, and reveal a pathological process. A diagnostic agent can label target cells and allow for subsequent detection or imaging of these labeled target cells. In some embodiments, a diagnostic agent can target / bind to activated microglia in the central nervous system (CNS) via a dendrimer or suitable delivery vehicle.

[0041] The term "prophylactic agent" refers generally to an agent, such as a vaccine, that can be administered to prevent a disease or its symptoms or to prevent a particular condition.

[0042] The term "analog" refers to a chemical compound that has a similar structure to that of another (reference compound) but differs therefrom with respect to certain constituents, functional groups, atoms, etc. The term "6-diazo-5-oxo-L-norleucine (DON) analog" or "DON analog" refers to a chemical compound that has a similar but slightly different structure to that of 6-diazo-5-oxo-L-norleucine. Exemplary DON analogs include any of the compounds encompassed by structures I-X or general formulas A-C.

[0043] The term "derivative" refers to a compound formed from a parent compound by one or more chemical reactions.

[0044] The term "alkyl" as used herein means a linear or branched saturated hydrocarbon chain containing from 1 to 10 carbon atoms, including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms. The term "lower alkyl" or "C1-C6-alkyl" means a linear or branched hydrocarbon chain containing from 1 to 6 carbon atoms, including 1, 2, 3, 4, 5, and 6 carbon atoms. The term "C1-C3-alkyl" means a linear or branched hydrocarbon chain containing from 1 to 3 carbon atoms, including 1, 2, and 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0045] The term "alkylenyl," as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 5 carbon atoms, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, for example, 1, 2, 3, 4, and 5 carbon atoms. Representative examples of alkylenyl include, but are not limited to, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, and -CH2-CH2-CH2-CH2-CH2-CH2-CH2-.

[0046] The term "alkenylenyl," as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon of 2 to 50 carbon atoms, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 carbon atoms, and in which at least one carbon-carbon bond is a double bond. Representative examples of alkenylenyl include, but are not limited to, -CH=CH-, -CH=CH-CH2-, -CH2-CH=CH-CH2-, and -CH2CH2-CH=CH-CH2-.

[0047] The term "alkynylenyl," as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon of 2 to 50 carbon atoms, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 carbon atoms, where at least one carbon-carbon bond is a triple bond. Representative examples of alkynylenyl include, but are not limited to, -C≡C-, -C≡C-CH2-, -C≡C-CH2-CH2-, and -CH2-C≡C-CH2-.

[0048] The terms "alkoxyl" or "alkoxy", "aroxy" or "aryloxy" generally refer to a group of the formula -OR v The present invention describes a compound represented by the formula: vExamples include, but are not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkylheteroaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted carbonyl, sugar group, phosphonium, phosphanyl, phosphonyl, sulfinyl, silyl, thiol, amido, and amino. Exemplary alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, and the like. A lower alkoxy group is an alkoxy group containing 1 to 6 carbon atoms. An ether is two functional groups covalently linked by an oxygen as defined below. Thus, the substituent of an alkyl that renders the alkyl an ether is or resembles an alkoxyl and can be represented, for example, by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aryl, -O-heteroaryl, -O-polyaryl, -O-polyheteroaryl, -O-heterocyclyl, and the like.

[0049] The term "carbonyl" as used herein refers to a group having the general formula: [ka] is represented by wherein X is a bond or represents oxygen or sulfur; R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, hydroxyl, alkoxy, phosphonyl, amido, amino, or -(CH) m E" is absent or E" is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl; R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, hydroxyl, alkoxy, phosphonyl, amido, amino, or -(CH) m-R"; R" represents hydroxyl, substituted or unsubstituted carbonyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, alkoxy, phosphonyl, amido, or amino; and m is 0 or an integer ranging from 1 to 8. When X is an oxygen and R is defined as above, the moiety is also referred to as a carboxyl group. When X is an oxygen and R is hydrogen, the formula represents a "carboxylic acid". When X is an oxygen and R' is hydrogen, the formula represents a "formate". When X is an oxygen and R or R' is not hydrogen, the formula represents an "ester". When X is a bond and R is not hydrogen, the above formula represents a "ketone". When X is a bond and R is hydrogen, the above formula represents an "aldehyde".

[0050] The term "phosphoryl" as used herein refers to a group having the general formula: [ka] is represented by wherein E is absent, oxygen, substituted or unsubstituted alkoxy, or substituted or unsubstituted aroxy; and independently of E, R vi and R vii are independently hydroxyl, substituted or unsubstituted alkoxy, or substituted or unsubstituted aroxy, as defined above. vi and R viiWhen is substituted, the substituents include, but are not limited to, halogen, azido, azo, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), silyl, ether, ester, thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, amino (e.g., quaternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl, and combinations thereof.

[0051] "Substituted," as used herein, refers to all permissible substituents of the compounds or functional groups described herein. In the broadest sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic grouping containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally containing one or more heteroatoms, e.g., oxygen, sulfur, or nitrogen groupings, in linear, branched, or cyclic structural formats. Representative substituents include substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted aralkyl, halogen, hydroxyl, alkoxy, phenoxy, aroxy, silyl, thiol, alkylthio, substituted alkylthio, phenylthio, arylthio, cyano, isocyano, nitro, substituted or unsubstituted carbonyl, carboxyl, amino, amido, azo, oxo, sulfinyl, sulfonyl, sulfonic acid, phosphonium, phosphanyl, phosphoryl, phosphonyl, amino acid, polymer, peptide, and sugar groups. Such substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted aralkyl, halogen, hydroxyl, alkoxy, phenoxy, aroxy, silyl, thiol, alkylthio, substituted alkylthio, phenylthio, arylthio, cyano, isocyano, nitro, substituted or unsubstituted carbonyl, carboxyl, amino, amido, oxo, sulfinyl, sulfonyl, sulfonic acid, phosphonium, phosphanyl, phosphoryl, phosphonyl, amino acid, polymer, peptide, and sugar groups may be further substituted.

[0052] When the term "independently selected" is used, the referenced substituents (e.g., R groups, e.g., R1 groups, R2 groups, etc., or variables, e.g., "m" and "n") can be the same or different. For example, both R1 and R2 can be substituted alkyls, or R1 can be hydrogen and R2 can be substituted alkyl, and the like.

[0053] The description of the compounds of the present disclosure is limited by the principles of chemical bonding known to those skilled in the art.Therefore, when a group can be substituted with one or more of several substituents, such substitutions are likely to be unstable under ambient conditions, such as aqueous, neutral, and some known physiological conditions, and are selected to obtain compounds that are not inherently unstable and / or would be known to those skilled in the art according to the principles of chemical bonding.For example, heterocycloalkyl or heteroaryl is bonded to the rest of the molecule through ring heteroatoms according to the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0054] The term "pharmacologically acceptable" or "biocompatible" refers to compositions, polymers, and other materials and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase "pharmacologically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material involved in carrying or transporting any subject composition from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not harmful to the patient.

[0055] The term "pharmaceutically acceptable salt" is recognized in the art and includes relatively non-toxic inorganic and organic acid addition salts of a compound. Examples of pharmaceutically acceptable salts include those derived from inorganic acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of inorganic bases suitable for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. By way of example, classes of such organic bases can include mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines, such as mono-, di-, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; and N-benzylphenethylamine.

[0056] The term "biodegradable" generally refers to a material that will break down or erode under physiological conditions into smaller units or chemical species that can be metabolized, eliminated, or excreted by a subject. Degradation time is a function of composition and morphology.

[0057] The term "therapeutically effective amount" refers to an amount of a therapeutic agent that, when incorporated into and / or onto a dendrimer, produces some desired effect at a reasonable benefit / risk ratio applicable to any medical procedure. The effective amount may vary depending on factors such as the disease or condition being treated, the particular targeting construct being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without undue experimentation. In some embodiments, the term "effective amount" refers to an amount of a therapeutic or prophylactic agent to reduce or reduce one or more symptoms of a disease or disorder, for example, to reduce tumor size (e.g., tumor volume) or to reduce or lessen pain and swelling in the small joints of the wrist and hands and feet in patients with one or more symptoms of an autoimmune disease, such as rheumatoid arthritis. In the case of cancer or tumors, an effective amount of a drug may have the effect of reducing the number of cancer cells; reducing tumor size; inhibiting the invasion of cancer cells into peripheral organs; inhibiting tumor metastasis; inhibiting tumor growth; and / or alleviating one or more of the symptoms associated with the disorder. An effective amount may be administered in one or more administrations.

[0058] The term "inhibit" or "reduce" in the context of inhibition means a decrease or reduction in activity and amount. This may be a complete inhibition or reduction or a partial inhibition or reduction in activity or amount. The inhibition or reduction may be compared to a control or standard level. The inhibition may be 5, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%. For example, a dendrimer composition comprising one or more DON analogs may inhibit or reduce the activity and / or amount of activated microglia associated with glutaminase by about 5%, 10%, 20%, 30%, 40%, 50% or more than 50% from the activity and / or amount of the same cells in a comparable tissue of a subject that was not given or treated with the dendrimer composition. In some embodiments, the inhibition and reduction are compared at the mRNA, protein, cell, tissue, and organ levels. For example, the inhibition and reduction in the rate of neuronal loss, the rate of brain weight loss, or the rate of hippocampal volume loss when compared to a non-treated control subject.

[0059] "Treating" or "preventing" a disease, disorder, or condition from occurring in an animal that is susceptible to the disease, disorder, or condition but has not yet been diagnosed as having the disease, disorder, or condition; inhibiting the disease, disorder, or condition, e.g., preventing its progression; and / or alleviating the disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating a disease or condition includes reversing at least one symptom of a particular disease or condition, even if the underlying pathophysiology is not affected, for example, treating pain in a subject by administering a painkiller, even if such an agent does not treat the cause of the pain. Desirable effects of treatment include slowing the rate of disease progression, reversing or alleviating the disease state, and remission or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated, including, but not limited to, a slowing of the rate of neuronal loss, a decrease in symptoms attributable to the disease, an improvement in the quality of life of those afflicted with the disease, a reduction in the dosage of other medications required to treat the disease, a delay in the progression of the disease, and / or an increase in the survival of the individual.

[0060] The term "dendrimer" includes, but is not limited to, molecular structures with an inner core, inner layers (or "generations") of repeating units regularly attached to this initial core, and an outer surface of terminal groups attached to the outermost generation.

[0061] The term "functionalization" means modifying a compound or molecule in a manner that results in the attachment of a functional group or moiety. For example, a molecule may be functionalized by introducing a molecule that makes it a strong nucleophile or a strong electrophile.

[0062] The term "targeting moiety" refers to a moiety that is localized or remote from a specific location. The moiety may be, for example, a protein, a nucleic acid, a nucleic acid analog, a carbohydrate, or a small molecule. The entity may be, for example, a therapeutic compound, for example, a small molecule, or a diagnostic entity, for example, a detectable label. The location may be a tissue, a specific cell type or ligand, or a subcellular compartment. In one embodiment, the targeting moiety localizes the agent. In a preferred embodiment, the dendrimer composition selectively targets one or more types of immune cells, such as activated microglia, in the absence of additional targeting moieties.

[0063] The term "extended residence time" refers to an increase in the time required for an agent to clear from a patient's body or from an organ or tissue of the patient. In certain embodiments, "extended residence time" refers to an agent that clears with a half-life that is 10%, 20%, 50% or 75% longer than a comparison standard, e.g., a comparison agent that is not conjugated to a delivery vehicle such as a dendrimer. In certain embodiments, "extended residence time" refers to an agent that clears with a half-life that is 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 or 10000 times longer than a comparison standard, e.g., a comparison agent that does not contain a dendrimer that specifically targets a particular cell type.

[0064] The terms "incorporated" and "encapsulated" refer to incorporating, formulating, or otherwise including such an agent in and / or on a composition that permits release, e.g., sustained release, of the agent in the desired application. Agents or other materials may be incorporated into such dendrimers by binding to one or more surface functional groups of the dendrimer (by covalent, ionic, or other binding interactions), by physical mixing, by enveloping the agent within the dendritic structure, and / or by encapsulating the agent within the dendritic structure.

[0065] II. Composition Analogues of glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) have been developed.It has been established that systemically administered hydroxyl-terminated PAMAM dendrimers can deliver one or more DON analogues to damaged / activated target cells and / or tissues, while minimizing peripheral exposure and thus minimizing associated systemic toxicity, including GI toxicity.Therefore, DON analogues are described that are conjugated to dendrimer nanoparticles suitable for delivery to one or more target immune cells in subjects.

[0066] Compositions are provided of dendrimers complexed, covalently conjugated, or intramolecularly dispersed or encapsulated with one or more 6-diazo-5-oxo-L-norleucine (DON) analogs having any of structures I-X or general formulas A-C. Exemplary dendrimers include fourth, fifth, sixth, seventh, or eighth generation dendrimers. In some embodiments, the dendrimer is a poly(amidoamine) (PAMAM) dendrimer, such as a hydroxyl-terminated PAMAM dendrimer. In preferred embodiments, the dendrimer is a fourth, fifth, or sixth generation hydroxyl-terminated PAMAM dendrimer. In some embodiments, the dendrimer is covalently conjugated to one or more DON analogs, optionally via a linker or spacer moiety.

[0067] In certain embodiments, the dendrimer compositions disclosed herein have the structure of formula (I): [ka] have where D is a dendrimer, preferably a G2 to G10 poly(amidoamine) (PAMAM) dendrimer; L 1 and L 2 are each independently absent or C1 to C 12 Alkylenyl, C2-C12 Alkenyl, C2-C 12 Alkynylenyl, or (-CH2CH2O-) q where q is 1 to 10; Y1 and Y2 are each independently absent or a linker selected from secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamate (-OCONH-;-NHCOO-), tertiary carbamate (-OCONR-;-NRCOO-), carbonate (-OC(O)-O-), urea (-NHCONH-;-NRCONH-;-NHCONR-,-NRCONR-), carbinol (-CHOH-,-CROH-), a disulfide group, hydrazone, hydrazide, and ether (-O-), where R is an alkyl group, an aryl group, or a heterocyclic group; Z is a DON analog of formula (IIIa) or formula (IIIb) or selected from structures I-X or general formulas A-C; n is from 1 to 100; m is from 1 to 4000).

[0068] In some embodiments, the presently disclosed dendrimer compositions have the structure of formula (Ia): [ka] have where D is a dendrimer, preferably a G2 to G10 poly(amidoamine) (PAMAM) dendrimer; L 1 does not exist or C1~C 12 Alkylenyl, C2-C 12 Alkenyl, C2-C 12 Alkynylenyl, or (-CH2CH2O-) q where q is 1 to 10; Y1 and Y2 are each independently absent or a linker selected from secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamate (-OCONH-;-NHCOO-), tertiary carbamate (-OCONR-;-NRCOO-), carbonate (-OC(O)-O-), urea (-NHCONH-;-NRCONH-;-NHCONR-,-NRCONR-), carbinol (-CHOH-,-CROH-), a disulfide group, hydrazone, hydrazide, and ether (-O-), where R is an alkyl group, an aryl group, or a heterocyclic group; Z is a DON analog of formula (IIIa) or formula (IIIb) or selected from structures I-X or general formulas A-C; p is 1 to 10; n is 1 to 100; m is 1 to 4000).

[0069] In certain embodiments, the dendrimer compositions disclosed herein have the structure of formula (II): [ka] have where D is a dendrimer, preferably a G2 to G10 poly(amidoamine) (PAMAM) dendrimer; L 1 and L 2 are each independently absent or C1 to C 12 Alkylenyl, C2-C 12 Alkenyl, C2-C 12 Alkynylenyl, or (-CH2CH2O-) q where q is 1 to 10; Y1 and Y2 are each independently absent or a linker selected from secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamate (-OCONH-;-NHCOO-), tertiary carbamate (-OCONR-;-NRCOO-), carbonate (-OC(O)-O-), urea (-NHCONH-;-NRCONH-;-NHCONR-,-NRCONR-), carbinol (-CHOH-,-CROH-), a disulfide group, hydrazone, hydrazide, and ether (-O-), where R is an alkyl group, an aryl group, or a heterocyclic group; Z is a DON analog of formula (IIIa) or formula (IIIb) or selected from structures I-X or general formulas A-C; n is from 1 to 100; m is from 1 to 4000).

[0070] In some embodiments, the dendrimer compositions disclosed herein have the structure of formula (IIa): [ka] have where D is a dendrimer, preferably a G2 to G10 poly(amidoamine) (PAMAM) dendrimer; L 1 does not exist or C1~C 12 Alkylenyl, C2-C 12 Alkenyl, C2-C 12 Alkynylenyl, or (-CH2CH2O-) q where q is 1 to 10; Y1 and Y2 are each independently absent or a linker selected from secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamate (-OCONH-;-NHCOO-), tertiary carbamate (-OCONR-;-NRCOO-), carbonate (-OC(O)-O-), urea (-NHCONH-;-NRCONH-;-NHCONR-,-NRCONR-), carbinol (-CHOH-,-CROH-), a disulfide group, hydrazone, hydrazide, and ether (-O-), where R is an alkyl group, an aryl group, or a heterocyclic group; Z is a DON analog of formula (IIIa) or formula (IIIb) or selected from structures I-X or general formulas A-C; p is 1 to 10; n is 1 to 100; m is 1 to 4000).

[0071] In certain embodiments of Formula (I), (Ia), (II), and / or (IIa), Z is a DON analog of Formula (IIIa) or Formula (IIIb): [ka] (wherein X′ and Y′ are independently oxygen or NH; R1 and R2 are independently absent, substituted or unsubstituted alkyl, carbonyl, or phosphoryl.

[0072] DON analog dendrimer conjugates are particularly suitable for treating or preventing one or more symptoms associated with various neurological, oncological, and immune disorders in which dysregulated glutamine-dependent pathways and / or glutamate transmission are presumed to be pathogenic.Generally, one or more DON analogs are encapsulated, associated, and / or conjugated in dendrimer complexes at a concentration of about 0.01% to about 30% by weight, preferably about 1% to about 20% by weight, and more preferably about 5% to about 20% by weight.Preferably, DON analogs are covalently conjugated to dendrimers via one or more linkages, such as disulfide, ester, ether, thioester, carbamate, carbonate, hydrazine, and amide linkages, and optionally via one or more spacers.

[0073] Dendrimers have the advantage that multiple therapeutic, prophylactic, and / or diagnostic agents can be delivered using the same dendrimer. One or more types of agents may be encapsulated, complexed, or conjugated to the dendrimer. In one embodiment, the dendrimer is complexed or conjugated to two or more different classes of agents to provide for simultaneous delivery with different or independent release kinetics at the target site. In another embodiment, the dendrimer is covalently linked to at least one detectable moiety and at least one class of agent. In a further embodiment, dendrimer complexes each carrying a different class of agent are administered simultaneously for combination treatment. The additional agent contained in the particle to be delivered may be a protein or peptide, a sugar or carbohydrate, a nucleic acid or oligonucleotide, a lipid, a small molecule (e.g., molecular weight less than 2000 Daltons, preferably less than 1500 Daltons, more preferably 300-700 Daltons), or a combination thereof. The nucleic acid may be an oligonucleotide encoding a protein, such as a DNA expression cassette or an mRNA. Exemplary oligonucleotides include siRNA, microRNA, DNA, and RNA. In some embodiments, the agent is a therapeutic antibody.

[0074] Exemplary additional active agents include anti-inflammatory drugs, chemotherapeutic agents, anticonvulsants, vasodilators, and anti-infective agents.

[0075] The presence of additional agents may affect the zeta potential or surface charge of the particle. In one embodiment, the zeta potential of the dendrimer is between -100mV and 100mV, between -50mV and 50mV, between -25mV and 25mV, between -20mV and 20mV, between -10mV and 10mV, between -10mV and 5mV, between -5mV and 5mV, or between -2mV and 2mV. In a preferred embodiment, the surface charge is neutral or near neutral. The above ranges include all values ​​from -100mV to 100mV.

[0076] A. Glutamine antagonists Glutamine antagonists, which preferably inhibit glutamine metabolism in activated microglia, are useful for treating one or more neurological, oncological, and / or immune disorders in which dysregulated glutamine-dependent pathways and / or glutamate transmission are suspected to be pathogenic or contribute to the pathogenesis.

[0077] It has been discovered that systemically administered hydroxyl-terminated PAMAM dendrimers can selectively deliver glutamine antagonists to damaged / activated tissues, where they are retained for at least 2 weeks, but are rapidly cleared from the periphery (plasma t1 / 2 is approximately 6-24 hours). Thus, to enhance delivery of glutamine antagonists to activated target tissues while simultaneously minimizing peripheral exposure and GI toxicity, one or more glutamine antagonists are preferably conjugated to a dendrimer, preferably a PAMAM dendrimer, for selective delivery to damaged / activated tissues. In a preferred embodiment, the glutamine antagonist is an analog of 6-diazo-5-oxo-L-norleucine.

[0078] 1.6-Diazo-5-oxo-L-norleucine (DON) analogue Analogs of 6-diazo-5-oxo-L-norleucine (DON) have been developed as glutamine antagonists to inhibit glutamine metabolism in subjects in need thereof, preferably with less toxicity than DON.

[0079] Many tumors become highly dependent on glutamine to obtain the carbon and nitrogen building blocks required for growth. Tumor glutamine dependency has been targeted with selective glutaminase inhibitors with some success. Several allosteric inhibitors, including BPTES (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide), compound 968, and CB-839 (Calithera), have shown robust activity in cell culture experiments and promising single-agent preclinical activity (Elgogary A, et al., Proc Natl Acad Sci USA 2016;113(36):E5328-3611;Wang JB, et al., Cancer Cell 2010;18(3):207-19;Gao M, et al., Mol Cell 2015;59(2):298-308). CB-839 is progressing into clinical studies. Although target engagement was clearly observed (Harding JJ, et al., Journal of Clinical Oncology 2015;33(15_suppl):2512), single agent antitumor activity was minimal. Combination trials are currently ongoing with promising early results (Meric-Bernstam F, et al., Journal of Clinical Oncology 2016;34(15_suppl):4568; DeMichele A, et al., Journal of Clinical Oncology 2016;34(15_suppl):1011).

[0080] 6-Diazo-5-oxo-norleucine (DON) is the best-studied broadly active glutamine antagonist with multiple supporting biochemical, preclinical and clinical evaluations. DON was originally isolated from the fermentation broth of Streptomyces in the 1950s. DON inhibits glutamine-utilizing enzymes, including glutaminase, at low micromolar levels, and also inhibits multiple glutamine amidotransferases involved in de novo purine and pyrimidine synthesis, coenzyme synthesis, amino acid synthesis, and hexosamine production. Clinical studies of DON in the 1950s using low daily doses suggested antitumor activity, but subsequent phase I and II clinical trials of DON given intermittently at high doses were hampered by dose-limiting nausea and vomiting. Further clinical development of DON was discontinued.

[0081] Given the enormous therapeutic potential of glutamine antagonism, several DON analogs have been synthesized.

[0082] In some other examples, the DON analogs include the following, where "AA" refers to an amino acid: [ka] wherein R1 is selected from -OCH2CH3, -OCH(CH3)2-OC(CH3)3, -NH2, NHCH3, -NH-AA-COOCH(CH3)2, and -NH-AA-COOC(CH3)3; R2 is selected from -H, -CH3, -CH2phenyl, -CH2CH(CH3)2, -CH2(indol-3-yl), and -CH2CH2C(=O)CH=N≡N; R3 is selected from -NH2 and -OH; n is an integer value from 1 to 32. [ka] wherein R1 is selected from -OCH2CH3, -OCH(CH3)2-OC(CH3)3, -NH2, NHCH3, -NH-AA-COOCH(CH3)2, and -NH-AA-COOC(CH3)3; R3 is selected from -NH2 and -OH; n is an integer value from 1 to 16. [ka] wherein R2 is selected from -H, -CH3, -CH2phenyl, -CH2CH(CH3)2, -CH2(indol-3-yl), or -CH2CH2C(=O)CH=N≡N; R4 is selected from -H, -CH3, -CH2phenyl, or -CH2CH(CH3)2; R5 is -H, -Ac, -Fmoc, dimethylglycine, Ac-AA-C(=O)-, dimethylglycine-NH-AA-C(=O)-, dimethylglycine-NH-DON-C(=O)-, or CH 17 OC(=O)CH(NH2)CH2CH2C(=O)-NH-6-diazo-5-oxo-L-norleucine-C(=O)-; n is an integer value from 1 to 32. These have one of three general chemical formulas:

[0083] Preferred DON analogs are shown below in structures I-X: [ka] [ka] [ka] [ka] [ka]

[0084] B. Dendrimer Dendrimer-glutamine antagonists include dendrimers complexed, covalently conjugated, intramolecularly dispersed or encapsulated with one or more 6-diazo-5-oxo-L-norleucine (DON) analogs having any of the structures I-X or general formulas A-C.

[0085] Dendrimers are three-dimensional hyperbranched, monodisperse, spherical, multivalent macromolecules that contain a high density of surface end groups (Tomalia, DA, et al., Biochemical Society Transactions, 35, 61 (2007); and Sharma, A., et al., ACS Macro Letters, 3, 1079 (2014)). Due to their unique structural and physical features, dendrimers are useful as nanocarriers for various biomedical applications including targeted drug / gene delivery, imaging and diagnostics (Sharma, A., et al., RSC Advances, 4, 19242 (2014); Caminade, A.-M., et al., Journal of Materials Chemistry B, 2, 4055 (2014); Esfand, R., et al., Drug Discovery Today, 6, 427 (2001); and Kannan, RM, et al., Journal of Internal Medicine, 276, 579 (2014)).

[0086] Dendrimer surface groups significantly affect its biodistribution (Nance, E., et al., Biomaterials, 101, 96 (2016)). Hydroxyl-terminated fourth generation PAMAM dendrimers (approximately 4 nm size), without any targeting ligand, cross the impaired BBB significantly more (>20-fold) when administered systemically in a rabbit model of cerebral palsy (CP) compared to healthy controls, and selectively target activated microglia and astrocytes (Lesniak, WG, et al., Mol Pharm, 10 (2013)).

[0087] The term "dendrimer" includes, but is not limited to, a molecular configuration having an inner core and layers (or "generations") of repeating units attached to and extending from the inner core, each layer having one or more branch points and with an exterior surface of terminal groups attached to the outermost generation. In some embodiments, the dendrimer has a standard dendrimer or a "starburst" molecular structure.

[0088] Typically, dendrimers have a diameter between about 1 nm and about 50 nm, more preferably between about 1 nm and about 20 nm, between about 1 nm and about 10 nm, or between about 1 nm and about 5 nm. Conjugates generally fall within the same size range, but larger proteins, such as antibodies, may be as large as 5-15 nm in size. Typically, drugs are encapsulated in larger generation dendrimers (i.e., 4th or higher) at a drug to dendrimer ratio of between 1:1 and 4:1. In a preferred embodiment, the dendrimers have a diameter effective to pass through brain tissue and be retained in target cells for extended periods of time.

[0089] In some embodiments, the dendrimers have a molecular weight between about 500 and about 100,000 daltons, preferably between about 500 and about 50,000 daltons, and most preferably between about 1,000 and about 20,000 daltons.

[0090] Suitable dendrimer scaffolds that may be used include poly(amidoamine), also known as PAMAM, or STARBURST™ dendrimers, polypropylamine (POPAM), polyethyleneimine, polylysine, polyester, iptycene, aliphatic poly(ether), and / or aromatic polyether dendrimers. The dendrimers may be carboxyl, amine, and / or hydroxyl terminated. In a preferred embodiment, the dendrimers are hydroxyl terminated. Each dendrimer of the dendrimer complex may be the same or of similar or different chemical nature to the other dendrimers (e.g., a first dendrimer may comprise a PAMAM dendrimer, while a second dendrimer may be a POPAM dendrimer).

[0091] The term "PAMAM dendrimer" refers to a poly(amidoamine) dendrimer of any generation, including but not limited to a first generation PAMAM dendrimer, a second generation PAMAM dendrimer, a third generation PAMAM dendrimer, a fourth generation PAMAM dendrimer, a fifth generation PAMAM dendrimer, a sixth generation PAMAM dendrimer, a seventh generation PAMAM dendrimer, an eighth generation PAMAM dendrimer, a ninth generation PAMAM dendrimer, or a tenth generation PAMAM dendrimer, which may contain different cores and have amidoamine building blocks and may have carboxyl, amine, and hydroxyl termini. In a preferred embodiment, the dendrimer is soluble in the formulation and is a fourth, fifth, or sixth generation ("G") dendrimer. The dendrimer may have hydroxyl groups attached to its functional surface groups.

[0092] Methods for making dendrimers are known to those skilled in the art and generally involve a two-step repeating reaction sequence that produces concentric shells (generations) of dendritic β-alanine units around a central starting core (e.g., an ethylenediamine core). Each subsequent growth step represents a new "generation" of polymer with a larger molecular diameter, twice the number of reactive surface sites, and approximately twice the molecular weight of the previous generation. Dendrimer scaffolds suitable for use are commercially available in various generations. Preferably, the dendrimer composition is based on a 0th, 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th generation dendrimer scaffold. Such scaffolds have 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096 reactive sites, respectively. Thus, dendrimer compounds based on these scaffolds may have up to a corresponding number of combined targeting moieties, if any, and agents.

[0093] In some embodiments, the dendrimers contain multiple hydroxyl groups. Some exemplary high density hydroxyl group-containing dendrimers include commercially available polyester dendritic polymers, such as hyperbranched 2,2-bis(hydroxyl-methyl)propionic acid polyester polymers (e.g., hyperbranched bis-MPA polyester-64-hydroxyl, 4th generation), dendritic polyglycerols.

[0094] In some embodiments, the high-density hydroxyl-containing dendrimer is an oligoethylene glycol (OEG)-like dendrimer. For example, second-generation OEG dendrimers (D2-OH-60) can be synthesized using highly efficient, robust, and atom-economical chemical reactions, such as Cu(I)-catalyzed alkyne-azide click and photocatalyzed thiol-ene click chemistry. By using orthogonal hypermonomer and hypercore strategies, very low-generation high-density polyol dendrimers can be achieved with minimal reaction steps, as described, for example, in WO2019094952. In some embodiments, the dendrimer backbone has non-cleavable polyether bonds throughout the structure, avoiding the in vivo disintegration of the dendrimer and allowing such dendrimers to disappear from the body as a single entity (non-biodegradable).

[0095] In some embodiments, the dendrimers specifically target a particular tissue region and / or cell type, preferably activated microglia in the CNS, hi preferred embodiments, the dendrimers specifically target a particular tissue region and / or cell type that does not have a targeting moiety.

[0096] In a preferred embodiment, the dendrimer has multiple hydroxyl (-OH) groups on the periphery of the dendrimer. The preferred hydroxyl (-OH) group surface density is at least 1 OH group / nm 2 (number of hydroxyl surface groups / nm surface area 2 For example, in some embodiments, the hydroxyl group surface density is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10; preferably at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or greater than 50. In further embodiments, the hydroxyl (-OH) group surface density is between about 1 and about 50, preferably 5-20 OH groups / nm 2 (number of hydroxyl surface groups / nm surface area 2 ) and at the same time have a molecular weight between about 500 Da and about 10 kDa.

[0097] In some embodiments, dendrimers may have a fraction of hydroxyl groups exposed on the exterior surface, with another fraction in the interior core of the dendrimer. In preferred embodiments, dendrimers have at least 1 OH group / nm 3 (number of hydroxyl groups / volume nm 3 For example, in some embodiments, the hydroxyl group volume density is 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, 15, 20, 25, 30, 35, 40, 45, and 50. In some embodiments, the hydroxyl group volume density is from about 4 to about 50 groups / nm 3 , preferably about 5 to about 30 groups / nm 3 , and more preferably about 10 to about 20 groups / nm 3 It is.

[0098] C. Coupling Agents and Spacers Dendrimer complexes may be formed from glutamine antagonists with one or more additional therapeutic agents or compounds conjugated or attached to the dendrimer, dendritic polymer, or hyperbranched polymer, if desired. Optionally, the agent is conjugated to the dendrimer via one or more spacers / linkers through different linkages, such as disulfide, ester, carbonate, carbamate, thioester, hydrazine, hydrazide, and amide linkages. The one or more spacers / linkers between the dendrimer and the agent may be designed to provide an in vivo releasable or non-releasable form of the dendrimer-active complex. In some embodiments, the conjugation occurs through a suitable spacer that provides an ester bond between the agent and the dendrimer. In some embodiments, the conjugation occurs through a suitable spacer that provides an amide bond between the agent and the dendrimer. In preferred embodiments, the one or more spacers / linkers between the dendrimer and the agent are added to achieve the desired and effective release kinetics in vivo.

[0099] The term "spacer" includes compositions used to link therapeutic agents to dendrimers. Spacers can be either a single chemical entity or two or more chemical entities that are linked together to bridge the polymer and the therapeutic or imaging agent. Spacers can include sulfhydryls, thiopyridines, succinimidyls, maleimides, vinyl sulfones, and any small chemical entity, peptide, or polymer with a carbonate terminus.

[0100] The spacer may be selected from the classes of sulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone and carbonate terminated compounds. The spacer may be a thiopyridine terminated compound such as dithiodipyridine, N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDP or sulfo-LC-SPDP. Spacers can also include linear or cyclic peptides substantially having sulfhydryl groups, such as glutathione, homocysteine, cysteine ​​and derivatives thereof, arg-gly-asp-cys (RGDC), cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)), cyclo(Arg-Gly-Asp-d-Tyr-Cys), cyclo(Arg-Ala-Asp-d-Tyr-Cys). Spacers can be mercapto acid derivatives, such as 3-mercaptopropionic acid, mercaptoacetic acid, 4-mercaptobutyric acid, thiolan-2-one, 6-mercaptohexanoic acid, 5-mercaptovaleric acid, and other mercapto derivatives, such as 2-mercaptoethanol and 2-mercaptoethylamine. The spacer can be thiosalicylic acid and its derivatives, (4-succinimidyloxycarbonyl-methyl-alpha-2-pyridylthio)toluene, (3-[2-pyridylthio]propionylhydrazide. The spacer can be maleimide terminated, and the spacer can be a polymer or a small chemical entity, such as bis-maleimidodiethylene glycol and bis-maleimidotriethylene glycol, bis-maleimidoethane, bismaleimidohexane. The spacer can be vinyl sulfone, such as 1,6-hexane-bis-vinyl sulfone. The spacer can be a thioglycoside, such as thioglucose. The spacer can be a reduced protein, such as bovine serum albumin and human serum albumin, any thiol-terminated compound capable of forming a disulfide bond. The spacer can be maleimide, succinimidyl, and thiol-terminated polyethylene glycol.

[0101] The glutamine antagonist or additional drug and / or targeting moiety may be covalently bonded, dispersed intramolecularly or encapsulated.The dendrimer is preferably a PAMAM dendrimer of up to 10th generation with carboxyl, hydroxyl or amine end.In a preferred embodiment, the dendrimer is linked to the drug via a spacer that ends with disulfide, ester or amide bond.

[0102] D. Additional medication to be delivered The dendrimer-DON analog complex can be used to deliver one or more additional active agents, in particular one or more agents for preventing or treating one or more symptoms of a disease or disorder. Suitable therapeutic, diagnostic, and / or prophylactic agents can be biomolecules, such as peptides, proteins, carbohydrates, nucleotides or oligonucleotides, or small molecule agents (e.g., molecular weight less than 2000 amu, preferably less than 1500 amu), including organic, inorganic, and organometallic agents. The agents can be encapsulated within the dendrimer, dispersed within the dendrimer, and / or covalently or non-covalently associated with the dendrimer surface.

[0103] 1. Therapeutic and preventive drugs In some embodiments, the dendrimer comprises one or more therapeutic, prophylactic, or prognostic agents complexed or conjugated to the dendrimer. Exemplary therapeutic agents include, but are not limited to, neuroprotective agents, anti-inflammatory agents, antioxidants, anti-infective agents, and combinations thereof.

[0104] In one embodiment, the additional therapeutic agent is a steroid.Suitable steroids include biologically active forms of vitamins D3 and D2, such as those described in U.S. Patent Nos. 4,897,388 and 5,939,407.Steroids may be co-administered to further aid in neurogenesis stimulation or induction and / or prevention of neuronal loss, particularly in the treatment of Alzheimer's disease.Estrogens and estrogen-related molecules, such as allopregnanolone, may be co-administered with neuroenhancement agents to enhance neuroprotection, as described in Brinton (2001) Learning and Memory 8(3): 121-133.

[0105] Other neuroactive steroids, such as various forms of dehydroepiandrosterone (DHEA), as described in U.S. Patent No. 6,552,010, may also be co-administered to further aid in neurogenic stimulation or induction and / or prevention of neuronal loss. Other agents that induce neuronal growth and neuronal network extension, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), may be administered simultaneously with, before, or after administration of THP. In addition, inhibitors of neuronal apoptosis, such as calpain and caspase, and other cell death mechanisms, such as necrosis, may be co-administered with the neuroenhancement agent to further prevent neuronal loss associated with certain neurological diseases and deficits.

[0106] Representative small molecules include steroids such as methylprednisone, dexamethasone, nonsteroidal anti-inflammatory agents including COX-2 inhibitors, corticosteroidal anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressants, anti-inflammatory agents and anti-angiogenic agents, anti-excitotoxic agents such as valproic acid, D-aminophosphonovalerate, D-aminophosphonoheptanoate, glutamate formation / release inhibitors, baclofen, NMDA receptor antagonists, salicylate anti-inflammatory agents, ranibizumab, anti-VEGF agents including aflibercept, and rapamycin.Other anti-inflammatory agents include nonsteroidal drugs such as indomethacin, aspirin, acetaminophen, diclofenac sodium and ibuprofen.The corticosteroid may be fluocinolone acetonide and methylprednisolone.

[0107] Representative oligonucleotides include siRNA, microRNA, DNA, and RNA.

[0108] 2. Diagnostic Agents In some cases, the additional agent is a diagnostic agent. Examples of diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, X-ray contrast agents, and imaging media. Examples of other suitable imaging agents include radiopaque gases or gas-emitting compounds. The dendrimer may further comprise an agent that is useful for determining the location of the administered composition. Useful agents for this purpose include fluorescent tags, radionuclides, and imaging agents.

[0109] Exemplary diagnostic agents include dyes, fluorescent dyes, near-infrared dyes, SPECT contrast agents, PET contrast agents, and radioisotopes. Representative dyes include carbocyanine, indocarbocyanine, oxacarbocyanine, thueicarbocyanine, and merocyanine, polymethine, coumarin, rhodamine, xanthene, fluorescein, boron-dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor647, HiLyte Fluor680, HiLyte Fluor750, IRDye800CW, IRDye800RS, IRDye700DX, ADS780WS, ADS830WS, and ADS832WS.

[0110] Exemplary SPECT or PET imaging agents include chelating agents, such as diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diaminedithiols, activated mercaptoacetyl-glycyl-glycyl-glycine (MAG3), and hydrazidonicotinamide (HYNIC).

[0111] Exemplary isotopes include Tc-94m, Tc-99m, In-111, Ga-67, Ga-68, Gd3+, Y-86, Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57, F-18, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, and Dy-166.

[0112] In a preferred embodiment, the dendrimer conjugate comprises one or more radioisotopes suitable for positron emission tomography (PET) imaging. Exemplary positron-emitting radioisotopes include carbon-11 ( 11 C), copper-64( 64 Cu), Nitrogen-13( 13 N), oxygen-15( 15 O), Gallium-68( 68 Ga), and Fluorine-18( 18 F), for example, 2-deoxy-2- 18 F-fluoro-β-D-glucose ( 18 F-FDG).

[0113] In a further embodiment, a single dendrimer complex composition is capable of simultaneously treating and / or diagnosing a disease or condition at one or more locations in the body.

[0114] III. Pharmaceutical Preparations A pharmaceutical composition comprising a dendrimer and one or more 6-diazo-5-oxo-L-norleucine (DON) analogues is described. The pharmaceutical composition comprising the dendrimer and the DON analogue may be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries, that facilitate processing of the active compound into a medicament that can be used pharma- ceutical. The appropriate formulation depends on the route of administration selected. In a preferred embodiment, the composition is formulated for parenteral delivery. In some embodiments, the composition is formulated for intravenous injection. Typically, the composition will be formulated in a sterile saline or buffer solution for injection into the tissue or cells to be treated. The composition may be stored lyophilized in a single-use vial for rehydration immediately prior to use. Other means for rehydration and administration are known to those skilled in the art.

[0115] In some embodiments, the pharmaceutical formulation contains one or more dendrimer conjugates in combination with one or more pharma- ceutically acceptable excipients. Representative excipients include solvents, diluents, pH modifiers, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizers, and combinations thereof. Suitable pharma-ceutically acceptable excipients are preferably selected from materials that are generally recognized as safe (GRAS) and can be administered to an individual without causing undesired biological side effects or undesirable interactions.

[0116] In general, pharmaceutically acceptable salts may be prepared by reacting the free acid or free base form of the drug with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include salts of drugs derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts, as well as salts formed by reacting drugs with suitable organic ligands (e.g., quaternary ammonium salts). Lists of suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704. Examples of ophthalmic drugs that are sometimes administered in the form of pharmaceutically acceptable salts include timolol maleate, brimonidine tartrate, and diclofenac sodium.

[0117] The composition is preferably formulated into a dosage unit form for ease of administration and uniformity of dosage. The phrase "dosage unit form" refers to a physically discrete unit of the conjugate appropriate for the patient to be treated. However, it will be understood that the total single dose of the composition will be determined by the attending physician within the scope of sound medical judgment. The therapeutically effective dose may be estimated initially in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may be used to achieve a desired concentration range and route of administration. Such information should then be useful in determining useful doses and routes of administration in humans. The therapeutic efficacy and toxicity of the conjugate, e.g., ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal to 50% of the population), may be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of the toxic to therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating different dosages for use in humans.

[0118] In certain embodiments, the composition is administered locally, for example, by direct injection into the site to be treated.In some embodiments, the composition is injected, locally applied, or otherwise directly administered into the vasculature on vascular tissue at or near the site of injury, surgery, or transplantation.For example, in some embodiments, the composition is locally applied to vascular tissue exposed during surgery.Typically, local administration results in an increased local concentration of the composition, which is higher than can be achieved by systemic administration.

[0119] Pharmaceutical compositions formulated for parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection) and enteral routes of administration are described.

[0120] A. Parenteral Administration In some embodiments, the pharmaceutical composition comprising the dendrimer and the DON analogue is formulated for parenteral administration. The phrases "parenteral administration" and "administered parenterally" are art-recognized terms and include modes of administration other than enteral and topical administration, such as intravenous (iv), intramuscular (im), intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intradennal, intraperitoneal (ip), transtracheal, subcutaneous (sc), subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The dendrimer can be parenterally administered by subdural, intravenous, intrathecal, intraventricular, intraarterial, intraamniotic, intraperitoneal, or subcutaneous routes.

[0121] For liquid formulations, the pharma- ceutically acceptable carrier may be, for example, an aqueous or non-aqueous solution, suspension, emulsion, or oil. Parenteral vehicles (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oil. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters, such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions, or suspensions, including saline and buffered media. Dendrimers can also be administered as emulsions, for example, water-in-oil. Examples of oils are those of animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish liver oil, sesame oil, cottonseed oil, corn oil, olive, and mineral oil. Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0122] Formulations suitable for parenteral administration may include aqueous and non-aqueous sterile suspensions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

[0123] Injectable pharmaceutical carriers for injectable compositions are well known to those of skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)).

[0124] B. Enteral Administration In some embodiments, the pharmaceutical composition comprising dendrimer and DON analogue is formulated for enteral administration.The carrier or diluent useful for the composition that can be enterally administered can be the solid carrier or diluent such as capsule or tablet for solid preparation, the liquid carrier or diluent for liquid preparation, or their mixture, and can also comprise food mixture and liquid feeding preparation.

[0125] For liquid preparations, pharma- ceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters, such as ethyl oleate.Aqueous carriers include, for example, water, alcohol / aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.

[0126] Examples of oil are petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish-liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral.The fatty acid suitable for use in parenteral formulations includes, for example, oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0127] Vehicles may include, for example, liquid nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose. In general, water, saline, aqueous dextrose and related sugar solutions are preferred liquid carriers. These may also be formulated with proteins, lipids, sugars and other components of artificial feeding.

[0128] In a preferred embodiment, the composition is formulated for oral administration. Oral formulations may be in the form of liquid or suspension, chewing gum, gel strips, tablets, capsules or lozenges. Encapsulating materials for preparing enteric coated oral formulations include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, and methacrylic acid ester copolymers. Solid oral formulations, such as capsules or tablets, are preferred. Elixirs and syrups are also well-known oral formulations.

[0129] IV. METHODS OF MAKING DENDRIMERS AND CONJUGATS OR COMPLEXES THEREOF Methods are provided for making one or more 6-diazo-5-oxo-L-norleucine (DON) analogs having any of structures I-X and preparing dendrimers conjugated with one or more of these DON analogs. Methods for making one or more 6-diazo-5-oxo-L-norleucine (DON) analogs having structures encompassed by general formulas A-C as discussed above and preparing dendrimers conjugated with one or more of these DON analogs may be accomplished by the same or similar synthetic methods used to make structures I-X and these dendrimers, as described below, which synthetic methods are well within the skill of one of ordinary skill in the art.

[0130] A. Methods for making analogs of 6-diazo-5-oxo-L-norleucine Exemplary syntheses of one or more 6-diazo-5-oxo-L-norleucine (DON) analogs having any of structures IX are described in Example 1.

[0131] In some embodiments, the synthetic scheme for structure I is as shown in FIG. 1, and exemplary reaction conditions include: (i) Fmoc-Cl, NaHCO3, dioxane, 0° C., 2 hours, 67%; (ii) isopropyl (S)-2-amino-6-diazo-5-oxohexanoate, phenyl dichlorophosphate, 2,4,6-trimethylpyridine, THF, 0° C. to room temperature, 5 hours, 41%; (iii) piperidine, DCM, room temperature, 2.5 hours, 73%.

[0132] In some embodiments, the synthetic scheme for structure II is as shown in FIG. 2, and exemplary reaction conditions include: (i) HATU, DIEA, Fmoc-L-Leu-OH, DCM, 0° C. to room temperature, 20 hours, 76%; (ii) piperidine, DCM, room temperature, 2 hours, 93%; (iii) 5-(Fmoc-amino)-3-oxapentanoic acid, HATU, DIEA, DMF, 0° C. to room temperature, 1 hour, 70%; (iv) piperidine, DCM, room temperature, 2 hours, 93%.

[0133] In some embodiments, the synthetic scheme for structure III is as shown in FIG. 3, and exemplary reaction conditions include: (i) Fmoc-9-amino-4,7-dioxanonanoic acid, HATU, DIEA, DMF, 0° C. to room temperature, 1 hour, 67%; (ii) piperidine, DCM, room temperature, 3 hours, 89%.

[0134] In some embodiments, the synthetic scheme for structure IV is as shown in FIG. 4, and exemplary reaction conditions include: (i) HATU, DIEA, Fmoc-L-Leu-OH, DCM, 0° C. to room temperature, 20 hours, 96%; (ii) diethylamine, DCM, room temperature, 1.5 hours, 88%; (iii) Fmoc-9-amino-4,7-dioxanonanoic acid, HATU, DIEA, DMF, 0° C. to room temperature, 1 hour, 86%; (iv) piperidine, DCM, room temperature, 1 hour, 87%.

[0135] In some embodiments, the synthetic scheme for structure V is as shown in FIG. 5, with exemplary reaction conditions including: (i) 5-(Fmoc-amino)-3-oxapentanoic acid, HATU, DIEA, DMF, 0° C. to room temperature, 1 hour, 89%; (ii) piperidine, DCM, room temperature, 1 hour, 74%.

[0136] In some embodiments, the synthetic scheme for structure VI is as shown in FIG. 6, and exemplary reaction conditions include: (i) allyl bromide, Cs2CO3, MeCN, rt, 1 hr, 96%; (ii) TFA, DCM; (iii) Boc-L-Phe-OH, HATU, DIEA, DMF, rt, 3 hr, 88%; (iv) TFA, DCM; (v) a DIEA, DCM; b (S)-2-(Fmoc-amino)-6-diazo-5-oxohexanoic acid, EDC, HOBt, DCM, rt, 2 hr, 71%; (vi) diethylamine, DCM, 75%; (vii) Fmoc-L-Leu-OH, DIEA, HBTU, DCM, rt, 2 hr, 47%; (viii) Pd(PPh3)4, dimedone, THF, 63%.

[0137] In some embodiments, a synthetic scheme for structure VII is as shown in FIG. 7, with exemplary reaction conditions including: (i) allyl bromide, Cs2CO3, MeCN, room temperature, 1 hour, 96%; (ii) TFA, DCM; (iii) Boc-L-Phe-OH, HATU, DIEA, DMF, room temperature, 3 hours, 88%; (iv) TFA, DCM; (v) a DIEA, DCM; b (S)-2-(Fmoc-amino)-6-diazo-5-oxohexanoic acid, EDC, HOBt, DCM, room temperature, 2 hours, 71%; (vi) diethylamine, DCM, 75%; (vii) Fmoc-L-Leu-OH, DIEA, HBTU, DCM, room temperature, 2 hours, 47%; (viii) Pd(PPh3)4, dimedone, THF, 63%.

[0138] In some embodiments, a synthetic scheme for structure VIII is as shown in FIG. 8, with exemplary reaction conditions including: (i) HATU, DIEA, H-Gly-OAll*HCl, DCM, 0° C. to rt, 2.5 hr, 77%; (ii) diethylamine, DCM, rt, 2.5 hr; (iii) (((4-nitrophenoxy)carbonyl)oxy)(phenyl)methyl pivalate, DMF, rt, 4 hr, 54% over two steps; (iv) Pd(PPh3)4, phenylsilane, DCM, rt, 0.5 hr; (v) HATU, DIEA, H2N-PEG5-COOAll*TFA salt, DMF, 0° C. to rt, 4 hr; (vi) Pd(PPh3)4, phenylsilane, DCM, rt, 0.5 hr.

[0139] In some embodiments, the synthetic scheme for structures IX and X is as shown in FIG. 9, and exemplary reaction conditions are: (i) LiHMDS, Fmoc-Cl, THF, −78° C.; (ii) n-BuLi, TMSCHN2, THF, −116 to −78° C.; (iii) piperidine, DMF, room temperature; (iv) Pd(PPh3)4, PhSiH3, DCM, 0° C.; (v) HATU, DCM, DIEA, 0° C.; (vi) piperidine, DMF, room temperature; (vii) CH 17OH, DCC, DMAP, DCM, room temperature; (viii) Pd(PPh3)4, PhSiH3, DCM, room temperature; (ix) HATU, DIEA, DCM, 0°C; (x) piperidine, DMF, room temperature; (xi) HOBt, EDC, DIEA, THF, room temperature.

[0140] B. Methods for Making Dendrimers Dendrimers conjugated with one or more DON analogues may be prepared via various chemical reaction steps. Dendrimers are usually synthesized according to methods that allow control of their structure at all stages of construction. Dendritic structures are mainly synthesized by two major different approaches: divergent or convergent.

[0141] In some embodiments, dendrimers are prepared using a divergent method, in which dendrimers are assembled from a multifunctional core, which is extended outward by a series of reactions, usually Michael reactions. The strategy involves coupling monomer molecules bearing reactive and protecting groups to the multifunctional core moiety, which leads to the stepwise addition of generations around the core, followed by removal of the protecting groups. For example, PAMAM-NH2 dendrimers are first synthesized by coupling N-(2-aminoethyl)acrylamide monomers to the ammonia core.

[0142] In other embodiments, dendrimers are prepared using convergent methods, where the dendrimer is built up from small molecules that are ultimately located on the surface of a sphere, and the reaction proceeds inwards, building inwards and ultimately attaching to the core.

[0143] Many other synthetic routes exist for preparing dendrimers, e.g., the orthogonal approach, the accelerated approach, the two-step convergent or hypercore approach, the hypermonomer or branched monomer approach, the double exponential approach; the orthogonal coupling approach or the two-step approach, the two-monomer approach, the AB2-CD2 approach.

[0144] In some embodiments, the dendrimer core, one or more branching units, one or more linkers / spacers, and / or one or more surface groups may be modified to allow conjugation to additional functional groups (branching units, linkers / spacers, surface groups, etc.), monomers, and / or drugs via click chemistry using one or more of Copper-Assisted Azide-Alkyne Cycloaddition (CuAAC), Diels-Alder reactions, thiol-ene and thiol-yne reactions, and azide-alkyne reactions (Arseneault M et al., Molecules. 2015 May 20;20(5):9263-94). In some embodiments, prefabricated dendrons are clicked into high density hydroxyl polymers. "Click chemistry" involves the coupling of two different moieties (e.g., a core group and a branching unit; or a branching unit and a surface group) via a 1,3-dipolar cycloaddition reaction between, for example, an alkyne moiety (or equivalent) on the surface of a first moiety and an azide moiety on a second moiety (e.g., a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc., present on a triazine composition or equivalent).

[0145] In some embodiments, the dendrimer synthesis is amenable to one or more reactions, such as thiol-ene click reaction, thiol-yne click reaction, CuAAC, Diels-Alder click reaction, azide-alkyne click reaction, Michael addition, epoxy ring opening, esterification, silane chemistry, and combinations thereof.

[0146] Any existing dendritic platform may be used to create dendrimers of desired functionality, i.e., high density of surface hydroxyl groups, by conjugating high hydroxyl-containing moieties, such as 1-thio-glycerol or pentaerythritol. Exemplary dendritic platforms may be synthesized and studied, such as polyamidoamine (PAMAM), poly(propyleneimine) (PPI), poly-L-lysine, melamine, poly(ether hydroxylamine) (PEHAM), poly(esteramine) (PEA), and polyglycerol.

[0147] Dendrimers may also be prepared by combining two or more dendrons. A dendron is a wedge-shaped section of a dendrimer that has a reactive focal functional group. Many dendron scaffolds are commercially available. They are sold as 1st, 2nd, 3rd, 4th, 5th, and 6th generation, with 2, 4, 8, 16, 32, and 64 reactive groups, respectively. In certain embodiments, one type of drug is linked to one type of dendron and a different type of drug is linked to another type of dendron. The two dendrons are then connected to form a dendrimer. The two dendrons may be linked via click chemistry, i.e., a 1,3-dipolar cycloaddition reaction between an azide moiety on one dendron and an alkyne moiety on the other dendron to form a triazole linker.

[0148] Exemplary methods for making dendrimers are detailed in International Patent Applications WO2009 / 046446, WO2015168347, WO2016025745, WO2016025741, WO2019094952, and U.S. Patent No. 8,889,101.

[0149] C. Dendrimer-DON Analogue Conjugate Dendrimer conjugates may be formed from DON analogs conjugated or complexed to dendrimers, dendritic polymers, or hyperbranched polymers. Exemplary dendrimer-DON analog conjugates are shown in Table 1 below.

[0150] The conjugates may also include additional therapeutic, prophylactic, or diagnostic agents. Conjugation of one or more agents to dendrimers is known in the art and is described in detail in U.S. Patent Application Publications US2011 / 0034422, US2012 / 0003155, and US2013 / 0136697.

[0151] In some embodiments, one or more drugs are covalently attached to the dendrimer. In some embodiments, the drugs are attached to the dendrimer via a linking moiety designed to be cleaved in vivo. The linking moiety may be designed to be cleaved hydrolytically, enzymatically, or a combination thereof, thereby providing sustained release of the drug in vivo. Both the composition of the linking moiety and its attachment point to the drug are selected such that cleavage of the linking moiety releases either the drug or a suitable prodrug thereof. The composition of the linking moiety may also be selected in consideration of the desired release rate of the drug.

[0152] In some embodiments, the bond is formed through one or more of disulfide, ester, ether, thioester, carbamate, carbonate, hydrazine, or amide bond. In preferred embodiments, the bond is formed through a suitable spacer that provides an ester bond or an amide bond between the drug and the dendrimer, depending on the desired release kinetics of the drug. In some cases, the ester bond is introduced for the releasable form of the drug. In other cases, the amide bond is introduced for the non-releasable form of the drug.

[0153] The linking moiety generally comprises one or more organic functional groups. Examples of suitable organic functional groups include secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamate (-OCONH-;-NHCOO-), tertiary carbamate (-OCONR-;-NRCOO-), carbonate (-OC(O)-O-), urea (-NHCONH-;-NRCONH-;-NHCONR-,-NRCONR-), carbinol (-CHOH-,-CROH-), disulfide group, hydrazone, hydrazide, ether (-O-), and ester (-COO-,-CH2O2C-,CHRO2C-), where R is an alkyl group, an aryl group, or a heterocyclic group. In general, the identity of one or more organic functional groups in the linking moiety is selected in consideration of the desired release rate of the drug. Furthermore, one or more organic functional groups may be selected to facilitate covalent attachment of the drug to the dendrimer. In a preferred embodiment, attachment may occur via a suitable spacer that provides a disulfide bridge between the drug and the dendrimer. The dendrimer complex allows for rapid release of the drug in vivo by thiol exchange reaction under reducing conditions found in the body.

[0154] In certain embodiments, the linking moiety comprises one or more of the organic functional groups described above in combination with a spacer group. The spacer group may be composed of any assembly of atoms, including oligomeric and polymeric chains, but the total number of atoms in the spacer group is preferably between 3 and 200 atoms, more preferably between 3 and 150 atoms, more preferably between 3 and 100 atoms, and most preferably between 3 and 50 atoms. Examples of suitable spacer groups include alkyl groups, heteroalkyl groups, alkylaryl groups, oligo- and polyethylene glycol chains, and oligo- and poly(amino acid) chains. Variations in the spacer group further control the release of the drug in vivo. In embodiments in which the linking moiety comprises a spacer group, one or more organic functional groups will generally be used to connect the spacer group to both the anti-inflammatory drug and the dendrimer.

[0155] Reactions and strategies that are useful for covalently attaching drugs to dendrimers are known in the art. See, e.g., March, “Advanced Organic Chemistry,” 5 th Edition, 2001, Wiley-Interscience Publication, New York) and Hermanson, "Bioconjugate Techniques," 1996, Elsevier Academic Press, USA. An appropriate method for the covalent attachment of a given agent may be selected taking into consideration the desired linking moiety and the structures of the agent and dendrimer, and generally relates to functional group compatibility, protecting group strategies, and the presence of labile linkages.

[0156] Optimal drug loading will necessarily depend on many factors, including the choice of drug, the structure and size of the dendrimer, and the tissue to be treated. In some embodiments, one or more drugs are encapsulated, associated, and / or conjugated to the dendrimer at a concentration of about 0.01% to about 45% by weight, preferably about 0.1% to about 30% by weight, about 0.1% to about 20% by weight, about 0.1% to about 10% by weight, about 1% to about 10% by weight, about 1% to about 5% by weight, about 3% to about 20% by weight, and about 3% to about 10% by weight. However, the optimal drug loading for any given drug, dendrimer, and target site can be identified by routine methods, such as those described above.

[0157] In some embodiments, drug and / or linker conjugation occurs via one or more surface and / or internal groups. Thus, in some embodiments, drug / linker conjugation occurs via about 1%, 2%, 3%, 4%, or 5% of all available surface functional groups of the dendrimer prior to conjugation, preferably hydroxyl groups. In other embodiments, drug / linker conjugation occurs with less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% of all available surface functional groups of the dendrimer prior to conjugation. In preferred embodiments, the dendrimer complex retains an effective amount of surface functional groups to target specific cell types and simultaneously conjugates to an effective amount of drug to treat, prevent, and / or image a disease or disorder.

[0158] V. Usage Methods of using dendrimer and DON analog conjugate compositions are described. In some embodiments, the dendrimer and DON analog conjugates are used to treat cancer. In other embodiments, the dendrimer and DON analog conjugates are used to treat CNS and immune disorders. The methods typically include administering to a subject in need thereof an effective amount of a composition comprising a dendrimer and one or more active agents to modulate glutamine metabolism, e.g., to enhance anti-tumor response. The methods can be used to treat one or more conditions and / or diseases associated with dysregulated glutamine-dependent pathways and / or glutamate transmission. In some embodiments, the methods are used to effectively reduce tumor growth. The methods include administering to a subject in need thereof an effective amount of a composition comprising a dendrimer complexed, conjugated, or encapsulated with one or more analogs of 6-diazo-5-oxo-L-norleucine (DON). In a preferred embodiment, the method comprises administering to the subject an effective amount of a composition comprising a dendrimer complexed or conjugated to one or more DON analogs shown in structures I-XII or analogs of general formulas A-C, or pharma- ceutically acceptable salts thereof.

[0159] A. Treatment The dendrimer and DON analogue compositions and their formulations may be administered to treat disorders associated with infection, inflammation, and / or cancer, particularly those associated with dysregulated glutamine-dependent pathways and / or glutamate transmission in the CNS.The compositions may also be used to treat other diseases, disorders and injuries, including proliferative diseases, and to treat other tissues in which nerves play a role in disease or disorder.The compositions and methods are also suitable for prophylactic use.

[0160] Typically, an effective amount of a dendrimer complex comprising a combination of a dendrimer and one or more analogs of 6-diazo-5-oxo-L-norleucine (DON) is administered to an individual in need thereof. The dendrimer may also contain targeting agents, although as demonstrated in the examples, these are not required for delivery to damaged or diseased tissues in the brain.

[0161] The amount of dendrimer and DON analog conjugate administered to a subject is selected to deliver an effective amount to reduce, prevent, or otherwise ameliorate one or more clinical or molecular symptoms of the disease or disorder being treated, as compared to a control, e.g., a subject treated with an active agent without the dendrimer.

[0162] In some embodiments, the dendrimer and DON analog complex is administered to a subject in an amount effective to reduce glutaminase activity and / or reduce glutamate release in one or more target cells associated with diseased or damaged tissue of one or more of neurological, oncological, and immune disorders. In a preferred embodiment, the composition is administered in an amount effective to reduce glutaminase activity and / or reduce glutamate release in activated microglia, particularly those associated with diseased or damaged tissue of one or more of neurological, oncological, and immune disorders.

[0163] B. The condition to be treated The composition of dendrimer and DON analogue complex is suitable for treating one or more of neurological, oncological and / or immune diseases, conditions and disorders in which dysregulated glutamine-dependent pathways and / or glutamate transmission are presumed to be pathogenic.In a preferred embodiment, one or more of neurological, oncological and immune diseases, conditions and disorders are further associated with the pathological activation of microglia and astrocytes.These include, but are not limited to, lung, colorectal, brain and pancreatic cancer, depression, cognitive deficits, ischemia, neuroinflammation, Alzheimer's disease, multiple sclerosis, tuberculosis, lupus, rheumatoid arthritis and viral infections.

[0164] Microglia are a type of neuroglial cell found throughout the brain and spinal cord. They represent 10-15% of all cells found in the brain. They are involved in the primary response to microbes, neuroinflammation, homeostasis, and tissue regeneration, as well as contributing to the pathogenesis of neurodegenerative diseases. Studies have shown that microglial diversity, multifunctionality, and their association with glutamate are important for determining their role in these diseases.

[0165] As resident macrophage cells, they act as the first and major form of active immune defense in the central nervous system (CNS). Microglia play an important role after CNS injury and can have both protective and detrimental effects based on the timing and type of injury (Kreutzberg, GW Trends in Neurosciences, 19, 312 (1996);Watanabe, H., et al., Neuroscience Letters, 289, 53 (2000);Polazzi, E., et al., Glia, 36, 271 (2001);Mallard, C., et al., Pediatric Research, 75, 234 (2014);Faustino, JV, et al., The Journal of Neuroscience : The Official Journal Of The Society For Neuroscience, 31, 12992 (2011);Tabas, I., et al., Science, 339, 166 (2013);and Aguzzi, A., et al., Science, 339, 156 (2013)).Alterations in microglial function also affect normal neuronal development and synaptic pruning (Lawson, LJ, et al., Neuroscience, 39, 151 (1990); Giulian, D., et al., The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience, 13, 29 (1993); Cunningham, TJ, et al., The Journal of Neuroscience : The Official Journal Of The Society For Neuroscience, 18, 7047 (1998); Zietlow, R., et al., The European Journal Of Neuroscience, 11, 1657 (1999); and Paolicelli, RC, et al., Science, 333, 1456 (2011)). Microglia undergo a dramatic change in morphology from branched to amoeboid structures and proliferate after injury. The resulting neuroinflammation disrupts the blood-brain barrier at the site of injury, leading to acute and chronic neuronal and oligodendrocyte death. Therefore, targeting pro-inflammatory microglia should be a powerful and effective therapeutic strategy. The damaged BBB in neuroinflammatory diseases can be exploited to transport drug-loaded nanoparticles to the brain.

[0166] In some embodiments, the dendrimer and DON analog complex composition is administered in an amount effective to treat cells / tissues with pathogenic dysregulated glutamine-dependent pathways and / or glutamate transmission without any associated toxicity. In a preferred embodiment, the dendrimer complex composition is administered in an amount effective to treat microglia-mediated pathology in a subject in need of such treatment without any associated toxicity. In a further preferred embodiment, the dendrimer complex composition is administered in an amount effective to treat pathogenic dysregulated glutamine-dependent pathways and / or glutamate transmission in activated microglial cells without any associated toxicity.

[0167] In some embodiments, the subject to be treated is a human. In some embodiments, the subject to be treated is a child or infant. All methods may include a step of identifying and selecting a subject in need of treatment or who would benefit from administration of the composition.

[0168] 1. Cancer In some embodiments, the dendrimer and DON analog compositions and formulations thereof are used in a method for treating cancer in a subject in need thereof.A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dendrimer and a DON analog to treat cancer, for example, to reduce or prevent one or more symptoms of cancer.

[0169] Cancer in a patient refers to the presence of cells with typical characteristics of cells that lead to cancer, such as uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cell markers. In some circumstances, cancer cells will be in the form of a tumor; such cells may be present locally in an animal body or circulate in the bloodstream as independent cells, e.g., leukemia cells. Tumor refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all precancerous and cancerous cells and tissues. Solid tumors are generally abnormal masses of tissue that do not contain cysts or liquid areas. Solid tumors may be present in the brain, colon, breast, prostate, liver, lung, esophagus, head and neck, ovaries, cervix, stomach, colon, rectum, bladder, uterus, testes, and pancreas, as non-limiting examples. In some embodiments, after a solid tumor is treated with the methods disclosed herein, the solid tumor regresses or its growth slows or stops. In other embodiments, the solid tumor is malignant. In some embodiments, the cancer comprises stage 0 cancer. In some embodiments, the cancer comprises stage I cancer. In some embodiments, the cancer comprises stage II cancer. In some embodiments, the cancer comprises stage III cancer. In some embodiments, the cancer comprises stage IV cancer. In some embodiments, the cancer is refractory and / or metastatic. For example, the cancer may be resistant to radiation therapy, treatment with chemotherapy, or monotherapy with immunotherapy.Cancers include newly diagnosed or recurrent cancers including, but not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, progressive soft tissue sarcoma, brain cancer, metastatic or invasive breast cancer, breast cancer, bronchogenic carcinoma, choriocarcinoma, chronic myeloid leukemia, colon cancer, colorectal cancer, Ewing's sarcoma, gastrointestinal cancer, glioma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, Hodgkin's disease, intracranial ependymoblastoma, colorectal cancer, leukemia, liver cancer, lung cancer, Lewis lung carcinoma, lymphoma, malignant fibrous histiocytoma, breast tumor, melanoma, mesothelioma, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, pontine tumor, premenopausal breast cancer, prostate cancer, rhabdomyosarcoma, reticulum cell sarcoma, sarcoma, small cell lung cancer, solid tumors, gastric cancer, testicular cancer, and uterine cancer. In some embodiments, the cancer is acute leukemia. In some embodiments, the cancer is acute lymphoblastic leukemia. In some embodiments, the cancer is acute myeloid leukemia. In some embodiments, the cancer is progressive soft tissue sarcoma. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is breast cancer (e.g., metastatic or invasive breast cancer). In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is bronchogenic carcinoma. In some embodiments, the cancer is choriocarcinoma. In some embodiments, the cancer is chronic myeloid leukemia. In some embodiments, the cancer is colon cancer (e.g., adenocarcinoma). In some embodiments, the cancer is colorectal cancer (e.g., colorectal carcinoma). In some embodiments, the cancer is Ewing's sarcoma. In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is glioma. In some embodiments, the cancer is glioblastoma multiforme. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is Hodgkin's disease. In some embodiments, the cancer is intracranial ependymoblastoma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is lung cancer (e.g., lung cancer). In some embodiments, the cancer is Lewis lung carcinoma. In some embodiments, the cancer is lymphoma. In some embodiments, the cancer is malignant fibrous histiocytoma. In some embodiments, the cancer includes breast tumors.In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is a mesothelioma. In some embodiments, the cancer is a neuroblastoma. In some embodiments, the cancer is an osteosarcoma. In some embodiments, the cancer is an ovarian cancer. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer includes pontine tumors. In some embodiments, the cancer is premenopausal breast cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is a rhabdomyosarcoma. In some embodiments, the cancer is a reticulum cell sarcoma. In some embodiments, the cancer is a sarcoma. In some embodiments, the cancer is a small cell lung cancer. In some embodiments, the cancer includes solid tumors. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is testicular cancer. In some embodiments, the cancer is uterine cancer.

[0170] Cancers that may be prevented, treated, or otherwise reduced by the compositions include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovium, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, and gastric carcinoma (for reviews of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., JB Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America).

[0171] The methods and compositions as described are useful for both prophylactic and therapeutic treatments.

[0172] Therapeutic treatment involves administering to a subject, after cancer has been diagnosed, a therapeutically effective amount of a composition as described, or a pharma- ceutically acceptable salt thereof.

[0173] In further embodiments, the compositions are used for prophylactic use, i.e., to prevent, delay onset, reduce, eradicate, or delay the progression of signs or symptoms after onset, and prevent recurrence.For prophylactic use, a therapeutically effective amount of the compounds and compositions as described or their pharmacologic acceptable salts is administered to a subject before onset (e.g., before obvious signs of cancer), during early onset (e.g., at the first signs and symptoms of cancer), or after the established occurrence of cancer.Prophylactic administration may be performed from days to years before symptoms appear.Prophylactic administration may be used, for example, for chemopreventive treatment of subjects who show precancerous lesions, those diagnosed with early malignant disease, and subgroups that have a susceptibility to certain cancers (e.g., familial, racial, and / or occupational).

[0174] In some embodiments, the subject to be treated has one or more solid tumors. A solid tumor is an abnormal mass of tissue that does not usually contain cysts or liquid areas. A solid tumor can be benign (not cancerous) or malignant (cancer). Examples of solid tumors are sarcoma, carcinoma, and lymphoma. In preferred embodiments, the compositions and methods are effective in treating one or more symptoms of cancer, such as skin, lung, liver, pancreas, brain, kidney, breast, prostate, colon and rectum, bladder, etc. In further embodiments, the tumor is localized lymphoma or follicular lymphoma.

[0175] 2. Neurological and neurodegenerative diseases The dendrimer-DON analogue compositions and their formulations can be used to diagnose and / or treat one or more neurological and neurodegenerative diseases. The compositions and methods are particularly suitable for treating one or more neurological or neurodegenerative diseases associated with defective metabolism and function of sphingolipids, including sphingomyelin. In some embodiments, the disease or disorder is selected from, but is not limited to, some psychiatric disorders (e.g., depression, schizophrenia (SZ), alcohol use disorder, and morphine antinociceptive tolerance) and neurological disorders (e.g., Alzheimer's disease (AD), Parkinson's disease (PD)). In one embodiment, the dendrimer complex is used to treat Alzheimer's disease (AD) or dementia.

[0176] Neurodegenerative diseases are chronic progressive disorders of the nervous system that affect neurological and behavioral functions, with biochemical changes resulting in distinct histopathological and clinical syndromes (Hardy H, et al., Science. 1998;282:1075-9). Abnormal proteins that are resistant to cellular degradative mechanisms accumulate within cells. The pattern of neuronal loss is selective, meaning that one group is affected while the other remains intact. There is often no obvious precipitating event for the disease. Diseases classically described as neurodegenerative are Alzheimer's disease, Huntington's disease, and Parkinson's disease.

[0177] Neuroinflammation mediated by activated microglia and astrocytes is a key hallmark of various neurological disorders and represents a potential therapeutic target (Hagberg, H et al., Annals of Neurology 2012, 71, 444; Vargas, DL et al., Annals of Neurology 2005, 57, 67; and Pardo, CA et al., International Review of Psychiatry 2005, 17, 485). Several scientific reports suggest that mitigating neuroinflammation at an early stage by targeting these cells can delay the onset of the disease and thus provide a longer therapeutic window for treatment (Dommergues, MA et al., Neuroscience 2003, 121, 619; Perry, VH et al., Nat Rev Neurol 2010, 6, 193; Kannan, S et al., Sci. Transl. Med. 2012, 4, 130ra46; and Block, ML et al., Nat Rev Neurosci 2007, 8, 57). Delivering therapeutics across the blood-brain barrier is a difficult challenge. Neuroinflammation leads to the breakdown of the blood-brain barrier (BBB). The damaged BBB in neuroinflammatory disorders can be exploited to transport drug-loaded nanoparticles throughout the brain (Stolp, HB et al., Cardiovascular Psychiatry and Neurology 2011, 2011, 10; and Ahishali, B et al., International Journal of Neuroscience 2005, 115, 151).

[0178] The composition and method may also be used to deliver active agents for treating neurological or neurodegenerative diseases or disorders, or central nervous system disorders.In a preferred embodiment, the composition and method are effective in treating and / or alleviating the neuroinflammation associated with neurological or neurodegenerative diseases or disorders, or central nervous system disorders.The method typically comprises administering to a subject an effective amount of the composition for enhancing cognition or reducing cognitive decline, enhancing cognitive function or reducing cognitive decline, enhancing memory or reducing memory decline, enhancing learning capacity or capacity or reducing learning capacity or capacity decline, or a combination thereof.

[0179] Neurodegeneration refers to the progressive loss of neuronal structure or function, including neuronal death. For example, the compositions and methods can be used to treat diseases or disorders such as Parkinson's disease (PD) and PD-related diseases, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and other dementias, prion diseases such as Creutzfeldt-Jakob disease, corticobasal degeneration, frontotemporal dementia, HIV-associated cognitive impairment, mild cognitive impairment, motor neuron disease (MND), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), Friedreich's ataxia, Lewy body disease, Alpers' disease, Batten disease, cerebro-oculo-facial-skeletal syndrome, corticobasal degeneration, Gerstmann-Sträussler-Scheinker disease, kuru, Leigh's disease, monomelic muscular atrophy (Monomelic Amyotrophy, multiple system atrophy, multiple system atrophy with orthostatic hypotension (Shy-Drager syndrome), multiple sclerosis (MS), neurodegeneration with brain iron accumulation, opsoclonus myoclonus, posterior cortical atrophy, primary progressive aphasia, progressive supranuclear palsy, vascular dementia, progressive multifocal leukoencephalopathy, dementia with Lewy bodies (DLB), lacunar syndrome, hydrocephalus, Wernicke-Korsakoff syndrome, postencephalitic dementia, cancer and chemotherapy-related cognitive impairment and dementia, and depression-induced dementia and pseudodementia may be used to treat subjects. In one embodiment, the neurological disease or disorder is stress-induced mood disorder, particularly depression.

[0180] In further embodiments, the disease or disorder is selected from, but not limited to, injection-limited amyloidosis, cerebral amyloid angiopathy, myopathy, neuropathy, brain trauma, frontotemporal dementia, Pick's disease, multiple sclerosis, prion disorder, type 2 diabetes, fatal familial insomnia, cardiac arrhythmia, isolated atrial amyloidosis, atherosclerosis, rheumatoid arthritis, familial amyloid polyneuropathy, hereditary non-neuropathic systemic amyloidosis, Finnish amyloidosis, lattice corneal dystrophy, systemic AL amyloidosis, and Down's syndrome. In a preferred embodiment, the disease or disorder is Alzheimer's disease or dementia.

[0181] Criteria for assessing improvement in a particular neurological factor include methods for assessing cognitive skills, motor skills, memory capacity, etc., as well as methods for assessing physical changes in selected areas of the central nervous system, such as magnetic resonance imaging (MRI) and computed tomography scans (CT) or other imaging methods. Such assessment methods are well known in the fields of medicine, neurology, psychology, etc., and can be appropriately selected to diagnose a particular neurological dysfunction state. A selected assessment or evaluation test, or test group, for assessing changes in Alzheimer's disease or associated neurological changes is performed prior to initiating administration of the dendrimer composition. After this initial assessment, a treatment regimen for administering the dendrimer composition is initiated and continued for various time intervals. At a selected time interval following the initial assessment of neurological deficit dysfunction, the same assessment or evaluation test is used again to reassess changes or improvements in the selected neurological criteria.

[0182] 3. Immune disorders In some embodiments, the composition of dendrimers conjugated or complexed with one or more DON analogs is administered to a subject with an immune disorder. Exemplary immune disorders include autoimmune or inflammatory diseases or disorders. Autoimmune diseases occur when the body's natural defense system cannot effectively distinguish between the body's own cells and foreign cells, leading the body to mistakenly attack normal cells. There are more than 80 types of autoimmune diseases that affect a wide range of parts of the body. Common autoimmune diseases include rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), type 1 diabetes, inflammatory bowel disease, and thyroid disease.

[0183] In some embodiments, the composition is intended to treat an autoimmune or inflammatory disease or disorder, such as, for example, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiac myopathy, celiac sprue dermatitis, chronic fatigue syndrome immune deficiency, chronic inflammatory demyelinating polyneuropathy (CFIDS), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), Iga nephropathy, insulin-dependent diabetes mellitus (type I), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome syndrome), polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff man syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0184] In some embodiments, the dendrimer composition is administered in an amount and dosage regimen effective to induce a desired immunological outcome in a subject in need thereof. Administration of the composition leads to improving or rebalancing the immune environment in an individual with an immunological disorder.

[0185] 4. Viral infection In some embodiments, the dendrimer compositions and formulations thereof are used in methods for treating one or more viral infections. In some embodiments, the dendrimer compositions and formulations thereof are used to reduce or inhibit viral replication, viral load, and / or viral release, particularly when activated microglia and astrocytes are targeted / infected by the virus.

[0186] In some embodiments, dendrimer-DON analogue compositions are effective in reducing inflammatory response to viral infections in which virus-specific immune response is involved as a mediator of neuronal damage.For example, the compositions are effective in reducing lymphocyte proliferation in draining lymph nodes, reducing leukocyte infiltration into CNS, and reducing inflammatory cytokine levels, which reduces one or more symptoms associated with elevated inflammatory response, such as cytokine storm.

[0187] C. Dosage and Effective Amount Dosage and dosing regimen depend on the severity and location of the injury or damage and / or the method of administration and are known to those skilled in the art. The therapeutically effective amount of the dendrimer composition used in the treatment of neurological disease is typically sufficient to reduce or alleviate one or more symptoms of the neurological disease. Preferably, the agent does not target or modulate the activity or amount of healthy cells that are not present in or associated with the diseased or target tissue, or targets or modulates at a low level compared to the target cells, including activated microglial cells in the CNS. In this way, the by-products and other side effects associated with the composition are reduced. Administration of the composition results in improved or enhanced neurological function in individuals with neurological disease, neurological injury, or age-related neuronal decline or dysfunction. In some in vivo approaches, the dendrimer complex is administered to a subject in a therapeutically effective amount to stimulate or induce neuronal mitosis leading to the generation of new neurons and provide a neurogenic effect. Also provided are compositions in an effective amount to prevent, reduce, or end the deterioration, dysfunction, or death of neurons, neurites, and neural networks in an individual, and to provide a neuroprotective effect.

[0188] A therapeutically effective amount of the dendrimer composition used in the treatment of cancer is typically sufficient to reduce or alleviate one or more symptoms of cancer. The symptoms of cancer may be physical, e.g., tumor burden, or biological, e.g., proliferation of cancer cells. Thus, the amount of dendrimer complex may be effective, for example, to kill tumor cells or inhibit tumor cell proliferation or metastasis. Preferably, the dendrimer composition including one or more active agents, e.g., immune modulating agents, is preferentially delivered to cells in and around the tumor tissue, e.g., cancerous cells or immune cells (e.g., M2 macrophages) associated with the tumor tissue. Preferably, the active agent does not target or modulate the activity or amount of healthy cells not present in or associated with the cancer tissue, or targets or modulates at a lower level compared to cancer or cancer-associated cells. In this way, by-products and other side effects associated with the composition are reduced, preferably leading directly or indirectly to cancer cell death. In some embodiments, the active agent directly or indirectly reduces cancer cell migration, angiogenesis, immune evasion, radioresistance, or a combination thereof. In some embodiments, the active agent directly or indirectly induces changes in the cancer cells themselves or their microenvironment that reduce the suppression or induce activation of the immune response against the cancer cells. In some in vivo approaches, the dendrimer complex is administered to a subject in a therapeutically effective amount to reduce tumor size. In some embodiments, an effective amount of the composition is used to cause cancer remission and / or maintain cancer remission. An effective amount of the composition is also provided to reduce or stop the proliferation of cancer stem cells.

[0189] The actual effective amount of the dendrimer conjugate may vary according to factors including the particular agent administered, the particular composition formulated, the mode of administration, and the age, weight, condition, and route of administration of the subject to be treated, and the disease or disorder. The dosage of the composition may be about 0.01 to about 100 mg / kg body weight; about 0.1 mg / kg body weight to about 10 mg / kg body weight; about 0.5 mg to about 5 mg / kg body weight. Generally, for intravenous injection or infusion, the dosage may be lower than that for oral administration.

[0190] Generally, the timing and frequency of administration will be adjusted to balance the effectiveness of a given treatment or diagnostic schedule and the side effects of a given delivery system. Exemplary dosing frequencies include continuous infusion, single and multiple doses, e.g., hourly, daily, weekly, monthly or yearly dosing.

[0191] The composition may be administered daily, twice weekly, weekly, biweekly, or less frequently, in an amount to provide a therapeutically effective increase in blood levels of the therapeutic agent. If administration is by other than an oral route, the composition may be delivered over more than an hour, for example, 3-10 hours, to produce a therapeutically effective dose within 24 hours. Alternatively, the composition may be formulated for controlled release, and the composition is administered as a single dose that is repeated on a weekly or less frequent regimen.

[0192] Dosage may vary and may be administered daily in single or multiple dose administrations for one or several days. Guidance can be found in the literature regarding appropriate dosages for a given class of pharmaceutical agent. Optimal dosing schedules can be calculated from measurements of drug accumulation in the subject's or patient's body. Those skilled in the art can easily determine optimal dosages, dosing methodologies, and repetition rates. Optimal dosages may vary depending on the relative potency of individual pharmaceutical compositions, and are generally determined by the EC50- ... 50 can be inferred based on

[0193] In some embodiments, the regimen includes one or more cycles of a treatment round followed by a rest period (e.g., no drug). The rest period may be 1, 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, 4 weeks, or 1, 2, 3, 4, 5, or 6 months.

[0194] D. Combination Therapies and Procedures The dendrimer and DON analog compositions may be administered alone or in combination with one or more conventional therapies. Examples of preferred additional therapeutic agents include other conventional therapies known in the art for treating the desired disease, disorder or condition.

[0195] In the context of Alzheimer's disease, other therapeutic agents can include one or more of acetylcholinesterase inhibitors (e.g., tacrine, rivastigmine, galantamine or donepezil), beta-secretase inhibitors, e.g., JNJ-54861911, antibodies, e.g., aducanumab, agonists for the 5-HT2A receptor, e.g., pimavanserin, sargramostim, AADvac1, CAD106, CNP520, gantenerumab, solanezumab, and memantine.

[0196] In the context of Lewy body dementia, other therapeutic agents can include one or more of: an acetylcholinesterase inhibitor, such as tacrine, rivastigmine, galantamine, or donepezil; the N-methyl d-aspartate receptor antagonist memantine; a dopamine-based treatment, such as levodopa or selegiline; an antipsychotic, such as olanzapine or clozapine; a REM disorder treatment, such as clonazepam, melatonin, or quetiapine; an antidepressant and anxiolytic, such as a selective serotonin reuptake inhibitor (such as citalopram, escitalopram, sertraline, paroxetine), or a serotonin and noradrenaline reuptake inhibitor (venlafaxine, mirtazapine, and bupropion) (see, e.g., Macijauskiene, et al., Medicina (Kaunas), 48(1):1-8 (2012)).

[0197] Exemplary neuroprotective agents are also known in the art and include, for example, glutamate antagonists, antioxidants, and NMDA receptor agonists. Other neuroprotective agents and treatments include caspase inhibitors, trophic factors, anti-protein aggregation agents, therapeutic hypothermia, and erythropoietin.

[0198] Other common active agents for treating neurological dysfunction include amantadine and anticholinergics for treating motor symptoms, clozapine for treating psychosis, cholinesterase inhibitors for treating dementia, and modafinil for treating daytime sleepiness.

[0199] 1. Cancer treatment In some embodiments, the composition of dendrimers conjugated or complexed with one or more 6-diazo-5-oxo-L-norleucine (DON) analogs is administered in combination with one or more conventional therapies, e.g., conventional cancer therapies. In some embodiments, the conventional therapies include administration of one or more of the compositions in combination with one or more additional active agents. Combination therapy may include administering the active agents together in the same addition mixture or in separate addition mixtures. Thus, in some embodiments, the pharmaceutical composition includes two, three or more active agents. Such formulations typically include an effective amount of an immunomodulatory agent that targets the tumor microenvironment. The additional active agent(s) may have the same or different mechanisms of action. In some embodiments, the combination provides an additive effect on the treatment of cancer. In some embodiments, the combination provides a more than additive effect on the treatment of a disease or disorder.

[0200] In some embodiments, the formulation is formulated for intravenous, subcutaneous or intramuscular administration to subjects, or for enteral administration.In some embodiments, the formulation is administered before, together with, after, or alternating with one or more additional therapies or procedures.In some embodiments, the additional therapies are administered during or off drug cycles, and are part of the dosing regimen of the composition.For example, in some embodiments, the additional therapies or procedures are surgery, radiation therapy, or chemotherapy.

[0201] Additional therapeutic agents include traditional cancer treatments, such as chemotherapy agents, cytokines, chemokines, and radiation therapy. The majority of chemotherapy drugs can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. These drugs affect and function in some way on cell division or DNA synthesis. Additional therapeutic agents include monoclonal antibodies and tyrosine kinase inhibitors, such as imatinib mesylate (GLEEVEC® or GLIVEC®), which directly target molecular abnormalities in certain types of cancer (chronic myeloid leukemia, gastrointestinal stromal tumors).

[0202] Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, mechlorethamine, melphalan, mexanthine, melphalan ... These include lecaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and its derivatives, trastuzumab (HERCEPTIN®), cetuximab, and rituximab (RITUXAN® or MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine taurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2)5, and combinations thereof.

[0203] In some embodiments, the compositions and methods are used prior to or in conjunction with immunotherapy, such as inhibiting checkpoint proteins, e.g., components of the PD-1 / PD-L1 axis or the CD28-CTLA-4 axis, using one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists), adoptive T cell therapy, and / or cancer vaccines. Exemplary immune checkpoint modulators used in immunotherapy include pembrolizumab (anti-PD1 mAb), durvalumab (anti-PDL1 mAb), PDR001 (anti-PD1 mAb), atezolizumab (anti-PDL1 mAb), nivolumab (anti-PD1 mAb), tremelimumab (anti-CTLA4 mAb), avelumab (anti-PDL1 mAb), and RG7876 (CD40 agonist mAb).

[0204] Methods of adoptive T cell therapy are known in the art and are used in clinical practice. In general, adoptive T cell therapy involves isolating tumor-specific T cells and expanding them ex vivo to obtain a larger number of T cells than can be obtained by vaccination alone. The tumor-specific T cells are then infused into the body of a patient with cancer to try to endow the immune system with the ability to suppress residual tumors via T cells that can attack and kill cancer. Several forms of adoptive T cell therapy can be used to treat cancer, including, but not limited to, culturing tumor-infiltrating lymphocytes or TILs; isolating and expanding one specific T cell or clone; and using T cells engineered to recognize and attack tumors. In some embodiments, T cells are taken directly from the patient's blood. Methods of priming and activating T cells in vitro for adaptive T cell cancer therapy are known in the art. See, e.g., Wang, et al, Blood, 109(11):4865-4872 (2007) and Hervas-Stubbs, et al, J. Immunol., 189(7):3299-310 (2012).

[0205] Historically, adoptive T cell therapy strategies have mostly focused on the infusion of tumor antigen-specific cytotoxic T cells (CTLs) that can directly kill tumor cells. However, CD4+ T helper (Th) cells, such as Th1, Th2, Tfh, Treg, and Th17, can also be used. Th can activate antigen-specific effector cells and recruit cells of the innate immune system, such as macrophages and dendritic cells, to help antigen-presenting cells (APCs), and antigen-primed Th cells can directly activate tumor antigen-specific CTLs. As a result of activating APCs, antigen-specific Th1 has been implicated as an initiator of epitope or determinant spreading, which broadens immunity to other antigens in tumors. The ability to induce epitope spreading may broaden the immune response to many potential antigens in tumors, resulting in more efficient tumor cell killing due to the ability to initiate heterologous genotypic responses. In this way, adoptive T cell therapy can be used to stimulate endogenous immunity.

[0206] In some embodiments, the T cells express chimeric antigen receptors (CARs, CAR T cells, or CARTs). Artificial T cell receptors are engineered receptors that graft specific specificities onto immune effector cells. Typically, these receptors are used to graft the specificity of monoclonal antibodies onto T cells and may be engineered to target virtually any tumor-associated antigen. First generation CARs typically had the intracellular domain from the CD3ζ-chain, which is the main mediator of signals from endogenous TCRs. Second generation CARs add intracellular signaling domains from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to T cells, and third generation CARs combine multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to further enhance efficacy.

[0207] In some embodiments, the compositions and methods are used prior to or together with cancer vaccines, such as dendritic cell cancer vaccines. Vaccination typically involves administering an antigen (e.g., a cancer antigen) to a subject with an adjuvant to induce therapeutic T cells in vivo. In some embodiments, the cancer vaccine is a dendritic cell cancer vaccine in which the antigen delivered by dendritic cells is primed ex vivo to present the cancer antigen. An example is PROVENGE® (sipuleucel-T), a dendritic cell-based vaccine for treating prostate cancer (Ledford, et al., Nature, 519, 17-18 (05 March 2015)). Such vaccines as well as other compositions and methods for immunotherapy are reviewed in Palucka, et al., Nature Reviews Cancer, 12, 265-277 (April 2012).

[0208] In some embodiments, the compositions and methods are used prior to or in conjunction with surgical removal of a tumor, for example, in preventing primary tumor metastasis, hi some embodiments, the compositions and methods are used to enhance the body's own anti-tumor immune function.

[0209] E. Control The therapeutic outcome of a dendrimer conjugate composition comprising one or more 6-diazo-5-oxo-L-norleucine (DON) analogs may be compared to a control. Suitable controls are known in the art and include, for example, untreated subjects, or untreated cells. A typical control is a comparison of the condition or symptom of a subject before and after administration of a targeted agent. The condition or symptom may be a biochemical, molecular, physiological, or pathological readout. For example, the effect of the composition on a particular symptom, pharmacological, or physiological indicator may be compared to an untreated subject, or to the condition of the subject before treatment. In some embodiments, the symptom, pharmacological, or physiological indicator is measured in the subject before treatment and again one or more times after treatment begins. In some embodiments, the control is a reference level or average determined based on the measurement of the symptom, pharmacological, or physiological indicator in one or more subjects (e.g., healthy subjects) that do not have the disease or condition to be treated. In some embodiments, the effect of the treatment is compared to a conventional treatment known in the art.

[0210] VI. Kit The composition may be packaged as a kit. The kit may include a single or multiple doses of the composition comprising one or more 6-diazo-5-oxo-L-norleucine (DON) analogs encapsulated in, associated with, or conjugated with dendrimers, and instructions for administering the composition. In particular, the instructions indicate that an effective amount of the composition is administered to an individual with a particular disease, disorder, or disorder indicated. The composition may be formulated as described above with reference to a particular treatment method, and may be packaged in any convenient manner.

[0211] The invention will be further understood by reference to the following non-limiting examples. EXAMPLES

[0212] Example 1 Synthetic methods for glutamine antagonists Synthetic methods for glutamine antagonists FIG. 1 is a schematic showing the synthesis scheme for compound I: (i) Fmoc-Cl, NaHCO3, dioxane, 0° C., 2 h, 67%; (ii) isopropyl (S)-2-amino-6-diazo-5-oxohexanoate, phenyl dichlorophosphate, 2,4,6-trimethylpyridine, THF, 0° C. to room temperature, 5 h, 41%; (iii) piperidine, DCM, room temperature, 2.5 h, 73%.

[0213] Figure 2 is a scheme showing the synthesis scheme for compound II: (i) HATU, DIEA, Fmoc-L-Leu-OH, DCM, 0°C to rt, 20 h, 76%; (ii) piperidine, DCM, rt, 2 h, 93%; (iii) 5-(Fmoc-amino)-3-oxapentanoic acid, HATU, DIEA, DMF, 0°C to rt, 1 h, 70%; (iv) piperidine, DCM, rt, 2 h, 93%.

[0214] Figure 3 is a scheme showing the synthesis scheme for compound III: (i) Fmoc-9-amino-4,7-dioxanonanoic acid, HATU, DIEA, DMF, 0°C to room temperature, 1 hour, 67%; (ii) piperidine, DCM, room temperature, 3 hours, 89%.

[0215] Figure 4 is a scheme showing the synthesis scheme for compound IV: (i) HATU, DIEA, Fmoc-L-Leu-OH, DCM, 0°C to rt, 20 h, 96%; (ii) diethylamine, DCM, rt, 1.5 h, 88%; (iii) Fmoc-9-amino-4,7-dioxanoic acid, HATU, DIEA, DMF, 0°C to rt, 1 h, 86%; (iv) piperidine, DCM, rt, 1 h, 87%.

[0216] 5 is a scheme showing the synthetic scheme for compound V. (i) 5-(Fmoc-amino)-3-oxapentanoic acid, HATU, DIEA, DMF, 0° C. to room temperature, 1 h, 89%; (ii) piperidine, DCM, room temperature, 1 h, 74%.

[0217] Figure 6 is a schematic showing the synthesis scheme for compound VI: (i) allyl bromide, Cs2CO3, MeCN, rt, 1 h, 96%; (ii) TFA, DCM; (iii) Boc-L-Phe-OH, HATU, DIEA, DMF, rt, 3 h, 88%; (iv) TFA, DCM; (v) a DIEA, DCM; b (S)-2-(Fmoc-amino)-6-diazo-5-oxohexanoic acid, EDC, HOBt, DCM, rt, 2 h, 71%; (vi) diethylamine, DCM, 75%; (vii) Fmoc-L-Leu-OH, DIEA, HBTU, DCM, rt, 2 h, 47%; (viii) Pd(PPh3)4, dimedone, THF, 63%.

[0218] Figure 7 is a schematic showing the synthesis scheme for compound VII: (i) allyl bromide, Cs2CO3, MeCN, 50°C, 1 h, 96%; (ii) TFA, DCM; (iii) Boc-L-Phe-OH, HATU, DIEA, DMF, rt, 3 h, 95%; (iv) TFA, DCM; (v) a DIEA, DCM; b (S)-2-(Fmoc-amino)-6-diazo-5-oxohexanoic acid, EDC, HOBt, DCM, rt, 2 h, 60%; (vi) diethylamine, MeCN; (vii) Fmoc-L-Leu-OSu, MeCN, rt, 3 h, 48%; (viii) Pd(PPh3)4, dimedone, THF, rt, 1 h, 74%.

[0219] Figure 8 is a scheme showing the synthesis scheme for compound VIII: (i) HATU, DIEA, H-Gly-OAll*HCl, DCM, 0°C to rt, 2.5 h, 77%; (ii) diethylamine, DCM, rt, 2.5 h; (iii) (((4-nitrophenoxy)carbonyl)oxy)(phenyl)methyl pivalate, DMF, rt, 4 h, 54% over two steps; (iv) Pd(PPh3)4, phenylsilane, DCM, rt, 0.5 h; (v) HATU, DIEA, H2N-PEG5-COOAll*TFA salt, DMF, 0°C to rt, 4 h; (vi) Pd(PPh3)4, phenylsilane, DCM, rt, 0.5 h.

[0220] 9 is a scheme showing the synthesis scheme for compounds IX and X. (i) LiHMDS, Fmoc-Cl, THF, -78°C to room temperature; (ii) TMS-diazomethane, n-BuLi, THF, -78°C; (iii) piperidine, DMF, room temperature; (iv) dimedone, Pd(PPh3)4, THF; (v) HATU, DCM, DIEA, 0°C; (vi) piperidine, DMF, room temperature; (vii) CH 17 OH, DCC, DMAP, DCM, room temperature; (viii) Pd(PPh3)4, PhSiH3, DCM, room temperature; (ix) HATU, DIEA, DCM, 0 °C; (x) Piperidine, DMF, room temperature; (xi) HO-PEG4-CH2CO2H, HOBt, EDC, DIEA, THF, room temperature.

[0221] 10 is a scheme showing the synthesis scheme for compound XI: (i) 2,2'-((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethan-1-ol), DCC, DMAP, DCM, room temperature; (ii) Pd(PPh3)4, PhSiH3, DCM, 0°C; (iii) compound 40, HATU, DIEA, DCM, 0°C.

[0222] Figure 11 is a scheme showing the synthesis scheme for compound XII: (i) TFA, DCM, 0°C to room temperature, 1 hour; (ii) Fmoc-DON-OH, HATU, DIPEA, DCM, 0°C to room temperature, 2 hours, 98%; (iii) Et2NH, DCM, room temperature, 2 hours; (iv) Fmoc-DON-OH, HATU, DIPEA, DCM, 0°C to room temperature, 2 hours, 42%; (v) Et2NH, DMF, room temperature, 2 hours; (vi) 2,5-dioxopyrrolidin-1-yldimethylglycinate, DMF, room temperature, 2 hours, 53%; (vii) Pd(PPh3)4, dimedone, THF, room temperature, 1 hour, 31%. Reaction conditions: i) TFA, DCM, 0°C to rt, 1 h; ii) Fmoc-DON-OH, HATU, DIPEA, DCM, 0°C to rt, 2 h, 98%; iii) Et2NH, DCM, rt, 2 h; iv) Fmoc-DON-OH, HATU, DIPEA, DCM, 0°C to rt, 2 h, 42%; v) Et2NH, DMF, rt, 2 h; vi) 2,5-dioxopyrrolidin-1-yldimethylglycinate, DMF, rt, 2 h, 53%; vii) Pd(PPh3)4, dimedone, THF, rt, 1 h, 31%.

[0223] (9H-Fluoren-9-yl)methyl(4-hydroxybutyl)carbamate (2) As shown in Figure 1, 4-aminobutanol (967 mg, 1.00 mL, 10.8 mmol, 1.0 equiv) was dissolved in dioxane (30 mL) and the solution was cooled to 0 °C. Saturated NaHCO3 (15 mL) was added dropwise over 5 min, followed by Fmoc chloride (3.08 g, 11.9 mmol, 1.1 equiv) and the reaction mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated NaCl (50 mL). The aqueous phase was extracted with ethyl acetate (3 x 50 mL) and the combined organic layers were washed with distilled water (40 mL), brine (40 mL) and dried over anhydrous MgSO4. The organic solvent was evaporated in vacuum. The residue was chromatographed on silica gel (EtOAc / Hexane 1:1) to give the desired product 2 (2.05 g, 67%) as a colorless solid.

[0224] Isopropyl(2S)-2-(((4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butoxy)(phenoxy)phosphoryl)amino)-6-diazo-5-oxohexanoate (3) As shown in Figure 1, phenyl dichlorophosphate (1.05 mL, 7.05 mmol, 1.5 equiv) was dissolved in anhydrous THF (8.5 mL) under inert atmosphere. The solution was cooled to 0°C and 2,4,6-trimethylpyridine (9.28 mL, 70.3 mmol, 15 equiv) was slowly added followed by a solution of 2 (1.46 g, 4.70 mmol, 1.0 equiv) in anhydrous THF (8.5 mL). The reaction mixture was stirred at 0°C for 20 min. A solution of isopropyl (S)-2-amino-6-diazo-5-oxohexanoate (1.00 g, 4.70 mmol, 1.0 equiv) in anhydrous THF (3.5 mL) was added and the reaction mixture was stirred at 0°C for 30 min and at room temperature for 2 h. The solvent was evaporated in vacuo and the crude product was chromatographed on silica gel (gradient CHCl3 / MeOH 30:1 to 15:1) to give the product 3 (1.26 g, 41%) as a yellow amorphous solid.

[0225] Isopropyl (2S)-2-(((4-aminobutoxy)(phenoxy)phosphoryl)amino)-6-diazo-5-oxohexanoate (Structure I) As shown in Figure 1, compound 3 (340 mg, 0.513 mmol, 1.0 equiv) was dissolved in anhydrous DCM (7 mL) under inert atmosphere. Piperidine (1.00 mL, 10.3 mmol, 20 equiv) was added and the reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated and the residue was chromatographed on silica gel (CHCl3 / MeOH 1:1 + 2% Et3N) to give the desired product I (165 mg, 73%) as a yellowish oil. The prepared product I is very unstable and needs to be stored at -80 °C.

[0226] t-Butyl (11S,14S)-14-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-11-isobutyl-3,9,12-trioxo-2,7-dioxa-4,10,13-triazapentadecan-15-oate (7) As shown in Figure 2, FmocHN-CH2CH2-OCH2COOH (0.24 g, 0.71 mmol, 1.1 equiv.) and HATU (0.27 g, 0.71 mmol, 1.1 equiv.) were dissolved in DMF (5 mL) under inert atmosphere. The reaction mixture was cooled to 0 °C and DIEA (0.37 mL, 2.1 mmol, 3.0 equiv.) was added. Finally, a solution of H-Leu-DON-OtBu 6 (0.22 g, 0.65 mmol, 1.0 equiv.) in DMF (1 mL) was added. The resulting mixture was stirred at 0 °C for 15 min and at room temperature overnight. Then, the solvent was evaporated, EtOAc (20 mL) was added and the organic phase was washed with distilled H2O (15 mL), 10% aqueous KHSO4 (2 x 15 mL), saturated NaHCO3 (2 x 15 mL) and brine (10 mL). The organic phase was then dried over anhydrous MgSO4 and the solvent was evaporated. The residue was purified by column chromatography (DCM / acetone 2:1, R f =0.28) The reaction yielded 0.30 g (70%) of the product as a yellowish amorphous solid.

[0227] t-Butyl (S)-2-((S)-2-(2-(2-aminoethoxy)acetamido)-4-methylpentanamido)-6-diazo-5-oxohexanoate (Structure II) As shown in Figure 2, compound 7 (0.29 g, 0.44 mmol, 1.0 equiv) was dissolved in DCM (2 mL) under inert atmosphere. Then piperidine (0.43 mL, 4.4 mmol, 10 equiv) was added and the resulting mixture was stirred at room temperature for 2 h. The solvent was evaporated and the residue was purified by column chromatography (DCM / MeOH / NH3 100:20:1, R f =0.16). The reaction yielded 0.18 g (93%) of the product as a yellowish amorphous solid.

[0228] t-Butyl (15S,18S)-18-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-15-isobutyl-3,13,16-trioxo-2,7,10-trioxa-4,14,17-triazanonadecan-19-oate (9) As shown in Figure 3, FmocHN-(CH2CH2-O)2CH2CH2COOH (0.29 g, 0.73 mmol, 1.1 equiv.) and HATU (0.28 g, 0.73 mmol, 1.1 equiv.) were dissolved in DMF (5 mL) under inert atmosphere. The reaction mixture was cooled to 0 °C and DIEA (0.37 mL, 2.1 mmol, 3.0 equiv.) was added. Finally, a solution of H-Leu-DON-OtBu 6 (0.23 g, 0.66 mmol, 1.0 equiv.) in DMF (1 mL) was added. The resulting mixture was stirred at 0 °C for 15 min and at room temperature overnight. The solvent was then evaporated, EtOAc (20 mL) was added and the organic phase was washed with distilled H2O (15 mL), 10% aqueous KHSO4 (2 x 15 mL), saturated NaHCO3 (2 x 15 mL) and brine (10 mL). The organic phase was then dried over anhydrous MgSO4 and the solvent was evaporated. The residue was purified by column chromatography (DCM / acetone 2:1, R f =0.19). The reaction yielded 0.32 g (67%) of the product as a yellowish amorphous solid.

[0229] t-Butyl (2S,5S)-15-amino-2-(4-diazo-3-oxobutyl)-5-isobutyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecanoate (Structure III) As shown in Figure 3, compound 9 (65 mg, 0.09 mmol, 1.0 equiv) was dissolved in DCM (1 mL) under an inert atmosphere. Piperidine (90 μL, 0.9 mmol, 10 equiv) was then added and the resulting mixture was stirred at room temperature for 3 h. The solvent was evaporated and the residue was purified by column chromatography (DCM / MeOH 5:1, R f =0.25) The reaction yielded 40 mg (89%) of the product as a yellowish amorphous solid.

[0230] Isopropyl (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-6-diazo-5-oxohexanoate (11) Compound 11 was prepared according to the literature (Rais, R. et al., J. Med. Chem. 2016, 59 (18), 8621-8633).

[0231] Isopropyl (S)-2-((S)-2-amino-4-methylpentanamido)-6-diazo-5-oxohexanoate (12) Compound 12 was prepared according to the literature (Rais, R. et al., J. Med. Chem. 2016, 59 (18), 8621-8633).

[0232] Isopropyl (15S,18S)-18-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-15-isobutyl-3,13,16-trioxo-2,7,10-trioxa-4,14,17-triazanonadecan-19-oate (13) As shown in Figure 4, FmocHN-(CH2CH2-O)2CH2CH2COOH (0.34 g, 0.84 mmol, 1.1 equiv.) and HATU (0.32 g, 0.84 mmol, 1.1 equiv.) were dissolved in anhydrous DMF (5 mL) under inert atmosphere. The reaction mixture was cooled to 0°C and DIEA (0.40 mL, 2.3 mmol, 3.0 equiv.) was added. Finally, a solution of H-Leu-DON-OiPr 12 (0.25 g, 0.77 mmol, 1.0 equiv.) in DMF (1 mL) was added. The resulting mixture was stirred at 0°C for 15 min and at room temperature for 1 h. The solvent was then evaporated, DCM (50 mL) was added and the organic phase was washed with 10% aqueous KHSO4 (25 mL), distilled H2O (25 mL), saturated NaHCO3 (25 mL), distilled H2O (25 mL) and brine (25 mL). The organic phase was then dried over anhydrous MgSO4 and the solvent was evaporated. The residue was purified by column chromatography (DCM / MeOH 40:1, R f =0.18). The reaction yielded 0.47 g (86%) of the product as a yellowish amorphous solid.

[0233] Isopropyl (2S,5S)-15-amino-2-(4-diazo-3-oxobutyl)-5-isobutyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecanoate (Structure IV) As shown in Figure 4, compound 13 (0.41 g, 0.58 mmol, 1.0 equiv) was dissolved in anhydrous DCM (1 mL) under inert atmosphere. Then piperidine (285 μL, 2.89 mmol, 5 equiv) was added and the resulting mixture was stirred at room temperature for 1 h. The solvent was evaporated and the residue was purified by column chromatography (DCM / MeOH 5:1 + 1% EtN, R f =0.44) The reaction yielded 218 mg (78%) of product as a yellowish amorphous solid.

[0234] Isopropyl (11S,14S)-14-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-11-isobutyl-3,9,12-trioxo-2,7-dioxa-4,10,13-triazapentadecan-15-oate (14) As shown in Figure 5, FmocHN-CH2CH2-OCH2COOH (0.29 g, 0.84 mmol, 1.1 equiv.) and HATU (0.32 g, 0.84 mmol, 1.1 equiv.) were dissolved in anhydrous DMF (5 mL) under inert atmosphere. The reaction mixture was cooled to 0°C and DIEA (0.40 mL, 2.30 mmol, 3.0 equiv.) was added. Finally, a solution of H-Leu-DON-OiPr(12) (0.25 g, 0.77 mmol, 1.0 equiv.) in anhydrous DMF (1 mL) was added. The resulting mixture was stirred at 0°C for 10 min and at room temperature for 1 h. The solvent was then evaporated, DCM (50 mL) was added and the organic phase was washed with 10% aqueous KHSO4 (25 mL), distilled H2O (25 mL), saturated NaHCO3 (25 mL), distilled H2O (25 mL) and brine (25 mL). The organic phase was then dried over anhydrous MgSO4 and the solvent was evaporated. The residue was purified by column chromatography (DCM / MeOH 40:1, R f =0.49). The reaction gave 0.44 g (89%) of the product as a yellowish solid.

[0235] Isopropyl (S)-2-((S)-2-(2-(2-aminoethoxy)acetamido)-4-methylpentanamido)-6-diazo-5-oxohexanoate (Structure V) As shown in Figure 5, compound 14 (0.39 g, 0.60 mmol, 1.0 equiv) was dissolved in anhydrous DCM (1 mL) under inert atmosphere. Then piperidine (0.30 mL, 3.12 mmol, 5 equiv) was added and the resulting mixture was stirred at room temperature for 1 h. The solvent was evaporated and the residue was purified by column chromatography (DCM / MeOH 5:1 + 1% Et3N, R f =0.42). The reaction yielded 0.19 g (74%) of the product as a yellowish amorphous solid.

[0236] Allyl 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oate (16) As shown in Figure 6, 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oic acid 15 (1.10 g, 3.0 mmol) and Cs2CO3 (1.47 g, 4.5 mmol) in MeCN (20 mL) were treated with allyl bromide (0.39 mL, 4.5 mmol). The reaction mixture was stirred at room temperature for 1 h, diluted with EtOAc (20 mL), and the precipitated salts were filtered off. The residue was chromatographed on a silica gel column with 5% MeOH / CHCl3. Yield 1.19 g (98%), syrup. Analytical data are available in US Patent Application Publication US2012 / 225089.

[0237] Allyl (S)-6-benzyl-2,2-dimethyl-4,7-dioxo-3,11,14,17,20-pentaoxa-5,8-diaza-tricosane-23-oate (18) As shown in Figure 6, a solution of 16 (1.22 g, 3.01 mmol) in CHCl (6 mL) was treated with TFA (3 mL) at 0°C. The reaction mixture was stirred at room temperature for 30 min, then evaporated and codistilled with toluene (10 mL). The resulting syrup-like trifluoroacetate salt of 17 was kept under vacuum. Meanwhile, a solution of Boc-L-Phe (878 mg, 3.31 mmol) and HATU (1.37 g, 3.61 mmol) in anhydrous DMF (3 mL) was treated with DIPEA (2.62 mL, 15.04 mmol) at 0°C and stirred for 10 min. The previously prepared trifluoroacetate salt of 17 was dissolved in anhydrous DMF (5 mL) and added to the mixture, which was then stirred at room temperature for 3 h. The solution was concentrated and the residue subjected to RP flash chromatography (100 g HP C18Aq column, 50 mL / min, gradient 0% to 100% acetonitrile / water in 25 min, detection at 220 nm). Yield 1.49 g (90%), syrup.

[0238] Allyl(5S,8S)-8-benzyl-5-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-3,6,9-tri-oxo-2,13,16,19,22-pentaoxa-4,7,10-triazapentacosane-25-oate (20) As shown in Figure 6, a solution of 18 (315 mg, 0.57 mmol) in CHCl (3 mL) was treated with TFA (0.5 mL) at 0 °C. The reaction mixture was stirred at room temperature for 30 min, then evaporated and codistilled with toluene (10 mL). The resulting syrup of trifluoroacetate salt of 19 was kept under vacuum. Meanwhile, a mixture of Fmoc-DON (269 mg, 0.68 mmol), HOBt (123 mg, 0.91 mmol), EDC.HCl (175 mg, 0.91 mmol) and DMAP (4 mg, 0.03 mmol) in CHCl (5 mL) was stirred at 0 °C until dissolution was complete. The previously prepared trifluoroacetate salt of 19 was dissolved in CHCl (15 mL) and DIPEA (0.11 mL, 0.63 mmol) and added to the mixture, which was then stirred at room temperature for 2 h. The solvent was evaporated and the residue was subjected to RP flash chromatography (50 g HP C18 Aq column, 30 mL / min, gradient 20% to 100% acetonitrile / water in 20 min, detection 265 nm). Yield 335 mg (71%), lyophilized.

[0239] Allyl(18S,21S)-21-amino-18-benzyl-25-diazo-17,20,24-trioxo-4,7,10,13-tetraoxa-16,19-diazapentacosanoate (21) As shown in Figure 6, a solution of 20 (315 mg, 0.38 mmol) in CHCl (3 mL) was treated with EtNH (0.5 mL) and the resulting mixture was stirred at room temperature for 1 h. The volatiles were evaporated at room temperature and the residue was subjected to RP flash chromatography (50 g HP C18 Aq column, 30 mL / min, gradient 0% to 100% acetonitrile / water in 20 min, detection at 220 nm). Yield 172 mg (75%), lyophilized (light yellow syrup). The corresponding mass of the product was verified by UPLC-MS. The product was used immediately in the next step.

[0240] Allyl (5S,8S,11S)-11-benzyl-8-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-5-isobutyl-3,6,9,12-tetraoxo-2,16,19,22,25-pentaoxa-4,7,10,13-tetraazaoctacosan-28-oate (22) As shown in Figure 6, a solution of Fmoc-L-Leu-OH (110 mg, 0.31 mmol) and HBTU (129 mg, 0.34 mmol) in anhydrous CHCl (3 mL) was treated with DIPEA (0.15 mL, 0.85 mmol) at 0 °C and stirred for 10 min. A solution of 21 (172 mg, 0.28 mmol) in CHCl (2 mL) was then added to the mixture, which was stirred at room temperature for 2 h. The solution was concentrated and the residue was subjected to RP flash chromatography (50 g HP C18 Aq column, 30 ml / min, gradient 20% to 100% acetonitrile / water in 20 min, detection 265 nm). Yield 125 mg (47%), lyophilized.

[0241] (5S,8S,11S)-11-benzyl-8-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-5-isobutyl-3,6,9,12-tetraoxo-2,16,19,22,25-pentaoxa-4,7,10,13-tetraazaoctacosan-28-oic acid (structure VI) As shown in Figure 6, a mixture of 22 (108 mg, 0.12 mmol) and dimedone (80 mg, 0.57 mmol) in THF (2 mL) was treated with a solution of Pd(PPh3)4 (6.6 mg, 0.006 mmol) in THF (0.5 mL). The mixture was stirred at room temperature for 1 h, then concentrated and the residue subjected to RP flash chromatography (50 g HP C18 Aq column, 30 ml / min, gradient 0% to 100% acetonitrile / water in 25 min, detection at 265 nm). Yield 65 mg (63%), lyophilized.

[0242] Allyl 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oate (24) As shown in Figure 7, a mixture of 1-(t-butyloxycarbonyl)amino-3,6,9-trioxadodecanoic-12-oic acid 23 (707 mg, 2.2 mmol) and Cs2CO3 (788 mg, 2.42 mmol) in MeCN (10 mL) was treated with allyl bromide (209 mL, 2.42 mmol). The mixture was heated to 50°C for 1 h, then cooled, diluted with EtOAc (10 mL), and the precipitated salts were filtered off. The residue was chromatographed on a silica gel column with 5% MeOH / CHCl3. Yield 770 mg (97%), syrup.

[0243] Allyl (S)-6-benzyl-2,2-dimethyl-4,7-dioxo-3,11,14,17-tetraoxa-5,8-diazaicosan-20-oate (26) As shown in Figure 7, a solution of 24 (610 mg, 1.69 mmol) in CHCl (6 mL) was treated with TFA (3 mL) at 0 °C. The reaction mixture was stirred at room temperature for 30 min, then evaporated and codistilled with toluene (10 mL). The resulting syrup-like trifluoroacetate salt of 25 was kept under vacuum. Meanwhile, a solution of Boc-L-Phe (447 mg, 1.69 mmol) and HATU (706 mg, 1.86 mmol) in anhydrous DMF (2 mL) was treated with DIPEA (1.2 mL, 6.75 mmol) at 0 °C and stirred for 10 min. The previously prepared trifluoroacetate salt of 25 was dissolved in anhydrous DMF (5 mL) and added to the mixture, which was then stirred at room temperature for 3 h. The solution was concentrated and the residue subjected to RP flash chromatography (100 g HP C18 Aq column, 60 mL / min, gradient 0% to 100% acetonitrile / water in 25 min, detection at 220 nm). Yield 820 mg (95%), syrup.

[0244] Allyl(5S,8S)-8-benzyl-5-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2,13,16,19-tetraoxa-4,7,10-triazadocosane-22-oate (28) As shown in Figure 7, a solution of 26 (790 mg, 1.55 mmol) in CHCl (10 mL) was treated with TFA (5 mL) at 0 °C. The reaction mixture was stirred at room temperature for 30 min, then evaporated and codistilled with toluene (15 mL). The resulting syrup-like trifluoroacetate salt of 27 was kept under vacuum. Meanwhile, a mixture of Fmoc-DON (764 mg, 1.94 mmol), HOBt (393 mg, 2.91 mmol), EDC.HCl (558 mg, 2.91 mmol) and DMAP (4 mg, 0.03 mmol) in CHCl (10 mL) was stirred at 0 °C until dissolution was complete. The previously prepared trifluoroacetate salt of 27 was dissolved in CHCl (15 mL) and DIPEA (540 mL, 3.10 mmol) and added to the mixture, which was then stirred at room temperature for 2 h. The solvent was evaporated and the residue subjected to RP flash chromatography (100 g HP C18 Aq column, 60 mL / min, gradient 20% to 100% acetonitrile / water in 20 min, detection 265 nm). Yield 727 mg (60%), lyophilized.

[0245] Allyl (5S,8S,11S)-11-benzyl-8-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-5-isobutyl-3,6,9,12-tetraoxo-2,16,19,22-tetraoxa-4,7,10,13-tetraazapentacosane-25-oate (30) As shown in Figure 7, a suspension of 28 (718 mg, 0.916 mmol) in MeCN (5 mL) was treated with Et2NH (5 mL) and the resulting solution was stirred at room temperature for 30 min. The solvent was evaporated at room temperature and the residue (crude 29) was co-distilled with MeCN (10 mL) and kept in vacuum for a short time. The residue was dissolved in MeCN (10 mL) and treated with a solution of Fmoc-L-Leu-OSu (371 mg, 0.824 mmol) in MeCN (10 mL). The mixture was stirred at room temperature for 4 h. The reaction was quenched by adding a solution of NH2OH.HCl (14 mg) and NaHCO3 (20 mg) in water (1 mL). The mixture was stirred at room temperature for 40 min, then concentrated and the residue subjected to RP flash chromatography (100 g HP C18 Aq column, 60 mL / min, gradient 20% to 100% acetonitrile / water in 20 min, detection 265 nm). Yield 398 mg (48%), lyophilized.

[0246] (5S,8S,11S)-11-benzyl-8-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-5-isobutyl-3,6,9,12-tetraoxo-2,16,19,22-tetraoxa-4,7,10,13-tetraazapentacosane-25-oic acid (Structure VII) As shown in Figure 7, a mixture of 30 (359 mg, 0.40 mmol) and dimedone (168 mg, 1.2 mmol) in THF (6 mL) was treated with a solution of Pd(PPh3)4 (18 mg, 0.016 mmol) in THF (0.5 mL). The mixture was stirred at room temperature for 1 h, then concentrated and the residue subjected to RP flash chromatography (100 g HP C18 Aq column, 60 mL / min, gradient 0% to 100% acetonitrile / water in 25 min, detection at 265 nm). Yield 255 mg (74%), lyophilized.

[0247] Allyl-L-pyroglutamate (31) Compound 31 shown in Figure 9 was prepared according to the literature (Gang, Fang-li, et al., Bioorganic and Medicinal Chemistry, 2018, vol. 26,16, p. 4644-4649).

[0248] 1-((9H-fluoren-9-yl)methyl)2-allyl(S)-5-oxopyrrolidine-1,2-dicarboxylate (32) As shown in Figure 9, allyl-L-pyroglutamate 31 (11.64 g, 68.79 mmol) was dissolved in anhydrous THF (500 mL) and the solution was cooled to -78 °C. A solution of LiHMDS (1 M in THF, 66 mL, 66 mmol) was added. The resulting mixture was stirred at -78 °C for 15 min and then added via cannula to a solution of Fmoc-Cl (53.4 g, 206.4 mmol) in THF (300 mL). The reaction mixture was stirred at -78 °C for 3 h, then allowed to reach room temperature and left overnight. The reaction was quenched with saturated aqueous NH4Cl (400 mL). The organic layer was separated and the aqueous portion was extracted with EtOAc (2 x 300 mL). The combined organic extracts were dried (MgSO4) and most of the solvent was evaporated. The liquid residue was diluted with Et2O (300 mL) and the solution was allowed to crystallize in the refrigerator overnight. The crystals were filtered off, washed with Et2O, and the filtrate was concentrated. The syrupy residue was chromatographed on a silica gel column, 25-50% EtOAc / Hexane. Yield 26.22 g (97%), syrup.

[0249] Allyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanoate (33) As shown in Figure 9, a solution of trimethylsilyldiazomethane (2 M solution in hexanes, 41.3 mL, 82.6 mmol) in THF (150 mL) was cooled to -78 °C and treated with n-BuLi (2.5 M in hexanes, 33.8 mL, 84.6 mmol). The mixture was stirred at -78 °C for 30 min and then added via cannula to a solution of 32 (26.00 g, 66.08 mmol) in cooled (-78 °C) THF (450 mL). The solution was stirred at -78 °C for an additional 30 min and then quenched with an aqueous solution of saturated NH4Cl (300 mL). The mixture was allowed to reach room temperature, the aqueous layer was separated and extracted with EtOAc (2 x 200 mL). The combined organic portions were dried (MgSO4) and concentrated. The residue was purified on a silica gel column, 40-50% EtOAc / Hexanes. Crystallization from EtOAc / hexanes gave 20.48 g (71%).

[0250] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanoic acid (34) As shown in Figure 9, a solution of allyl ester 33 (20.20 g, 46.60 mmol) and dimedone (19.6 g, 139.8 mmol) in THF (230 mL) was treated with a solution of Pd(PPh3)4 (2.15 g, 1.86 mmol) in THF (70 mL). The mixture was stirred for 90 min, then concentrated, and the residue was chromatographed on a silica gel column with 10-20% MeOH / CH2Cl2 to give 10.95 g (60%).

[0251] 1-((9H-fluoren-9-yl)methyl)2-octyl (S)-5-oxopyrrolidine-1,2-dicarboxylate (36) As shown in Figure 9, to a solution of (S)-octyl 5-oxopyrrolidine-2-carboxylate 35 (10 g, 41.44 mmol) in anhydrous THF (200 mL) was added LiHMDS (39.37 mL, 39.37 mmol, 1 M in THF) dropwise under inert atmosphere at -78 °C and the mixture was stirred for 20 min. The resulting yellow mixture was transferred via cannula to a solution of Fmoc-Cl (12.86 g, 49.73 mmol) in dry THF (150 mL) at -78 °C. The reaction mixture was stirred at -78 °C for 2 h and gradually warmed to room temperature overnight. The reaction was quenched with saturated NH4Cl (100 mL) and water (50 mL). The aqueous phase was extracted with ethyl acetate (3 x 100 mL) and the combined organic layers were washed with brine (200 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure, and the residue was chromatographed on silica gel, 20-100% EtOAc / Hexanes to give 18.23 g (95% yield) of compound 36 as a colorless foam.

[0252] Octyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanoate (37) As shown in Figure 9, a solution of trimethylsilyldiazomethane (23.6 mL, 47.2 mmol, 2 M in diethyl ether) was dissolved in anhydrous THF (210 mL) under argon and cooled to -98 °C (dry ice acetone bath and liquid N2). A solution of n-BuLi (19.35 mL, 48.38 mmol, 2.5 M in hexane) was added dropwise and the solution was stirred at -98 °C for 30 min. The resulting mixture was transferred via cannula to a solution of compound 36 (18.23 g, 39.33 mmol) in dry THF (360 mL) at -98 °C (dry ice acetone bath and liquid N2). The reaction mixture was stirred at -98 °C for 30 min, then gradually warmed to -78 °C and quenched with saturated NH4Cl (200 mL). The aqueous phase was extracted with ethyl acetate (3×100 mL) and the combined organic layers were washed with brine (200 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure. The residue was chromatographed on silica gel with 5% CHCl3 / acetone to give 17.2 g (86% yield) of compound 37 as a pale yellow solid.

[0253] Octyl (S)-2-amino-6-diazo-5-oxohexanoate (38) As shown in Figure 9, to a solution of compound 37 (5.06 g, 10 mmol) in anhydrous DMF (20 mL) was added piperidine (0.94 g, 11 mmol) at room temperature under inert atmosphere. The mixture was stirred for 20 min and quenched with H2O (80 mL). The aqueous phase was extracted with ethyl acetate (3 x 100 mL) and the combined organic layers were washed with brine (100 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure. The residue was chromatographed on silica gel with 0-10% MeOH / DCM containing 0.5% Et3N to give 2.33 g (82% yield) of compound 38 as a yellow solid, which was used immediately in the subsequent step.

[0254] Octyl (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanamide)-6-diazo-5-oxohexanoate (39) To a solution of compound 34 (2.75 g, 6.99 mmol) and 38 (2.38 g, 8.39 mmol) in anhydrous DCM (40 mL) was added N,N-diisopropylethylamine (DIPEA, 1.81 g, 13.98 mmol) and the mixture was cooled to 0 °C under inert atmosphere. HATU (2.93 g, 7.69 mmol) was added in one portion and the mixture was stirred at 0 °C for 1 h and quenched with H2O (80 mL). The aqueous phase was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with brine (100 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure. The residue was chromatographed on silica gel with 0-10% MeOH / DCM to give compound 39 4.0 g (87% yield) as a yellow solid, which was used immediately in the subsequent step.

[0255] Octyl (S)-2-((S)-2-amino-6-diazo-5-oxohexanamide)-6-diazo-5-oxohexanoate (40) To a solution of compound 39 (4.0 g, 6.07 mmol) in anhydrous DMF (20 mL) was added piperidine (0.57 g, 6.68 mmol) at room temperature under inert atmosphere. The reaction was carefully monitored and quenched with H2O (60 mL) once compound 39 had disappeared (approximately 20 min). The aqueous phase was extracted with ethyl acetate (3 x 80 mL) and the combined organic layers were washed with brine (100 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure. The residue was chromatographed on silica gel with 0-10% MeOH / DCM containing 0.5% Et3N to give 1.8 g (68% yield) of compound 40 as a yellow solid, which was used immediately in the subsequent step.

[0256] 5-Allyl 1-octyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (42) To a solution of 41 (3.80 g, 9.29 mmol) in anhydrous DCM (100 mL) was added 1-octanol (1.21 g, 9.29 mmol) and the mixture was stirred at 0 °C. DCC (1.92 g, 9.29 mmol) was added followed by catalytic amount of DMAP under inert atmosphere. The reaction mixture was stirred at room temperature overnight. Precipitated DCU was filtered off and the solvent was removed under reduced pressure. The residue was redissolved in EtOAc (100 mL) and the remaining precipitate of DCU was filtered off. The solvent was removed under reduced pressure. The residue was chromatographed on a silica gel column, 10-100% EtOAc / Hexane to give 3.29 g (68% yield) of compound 42 as a white solid.

[0257] (S)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-(octyloxy)-5-oxopentanoic acid (43) To a solution of 42 (3.29 g, 6.31 mmol) and PhSiH3 (1.37 mg, 12.62 mmol) in anhydrous DCM (100 mL) was added Pd(PPh3)4 (219 mg, 0.19 mmol) at 0 °C under inert atmosphere, and the mixture was gradually warmed to room temperature and stirred for 40 min. A few drops of water were then added and the volatiles were removed under reduced pressure. The residue was chromatographed on a silica gel column, 10-100% EtOAc containing 0.5% AcOH / hexane to give 2.41 g (77% yield) of compound 43 as a white solid.

[0258] Octyl (5S,10S,13S)-10,13-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-5-((octyloxy)carbonyl)-3,8,11-trioxo-2-oxa-4,9,12-triazatetradecane-14-oate (44) To a solution of compound 40 (1.80 g, 4.12 mmol) and 43 (1.99 g, 4.12 mmol) in anhydrous DCM (40 mL) was added N,N-diisopropylethylamine (DIPEA, 1.07 g, 8.25 mmol) and the mixture was cooled to 0 °C under inert atmosphere. HATU (1.73 g, 4.54 mmol) was added in one portion and the mixture was stirred at 0 °C for 40 min and quenched with H2O (80 mL). The aqueous phase was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with brine (100 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure. The residue was chromatographed on silica gel with 0-10% MeOH / DCM to give 3.2 g (86% yield) of compound 44 as a yellow solid.

[0259] Octyl (S)-2-((S)-2-((S)-4-amino-5-(octyloxy)-5-oxopentanamido)-6-diazo-5-oxohexanamido)-6-diazo-5-oxohexanoate (Structure IX) To a solution of compound 44 (1.5 g, 1.67 mmol) in DMF (15 mL) was added piperidine (0.16 g, 1.83 mmol) at room temperature under inert atmosphere. The mixture was stirred for 40 min and quenched with H2O (60 mL). The aqueous phase was extracted with ethyl acetate (3 x 80 mL) and the combined organic layers were washed with brine (100 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure. The residue was chromatographed on silica gel, 0-10% MeOH / CHCl3 containing 0.5% Et3N to give compound IX 0.81 g (72% yield) as a pale yellow solid.

[0260] Octyl (16S,21S,24S)-21,24-bis(4-diazo-3-oxobutyl)-1-hydroxy-16-((octyloxy)carbonyl)-14,19,22-trioxo-3,6,9,12-tetraoxa-15,20,23-triazapentacosane-25-oate (Structure X) To a solution of compound IX (300 mg, 0.44 mmol), hydroxy-PEG-CH2CO2H (122.8 mg, 0.49 mmol) and N,N-diisopropylethylamine (DIPEA, 200 mg, 1.55 mmol) in THF (5 mL) was added 1-hydroxybenzotriazole hydrate (HOBt·H2O, 102 mg, 0.66 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC hydrochloride, 119 mg, 0.62 mmol) at room temperature under inert atmosphere. The mixture was stirred for 3 h and quenched with saturated NaHCO3 (10 mL). The aqueous phase was extracted with ethyl acetate (3×20 mL) and the combined organic layers were washed with brine (10 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure. The residue was purified using reverse-phase preparative HPLC (40% acetonitrile / 60% water, then increasing to 100% acetonitrile over 60 min and washing with 100% acetonitrile for 10 min; flow rate 15 mL / min) to give 142 mg (35% yield) of compound X as a pale yellow sticky solid.

[0261] 5-Allyl 1-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (45) To a solution of 41 (1.0 g, 2.44 mmol) in anhydrous DCM (20 mL) was added 2,2'-((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethan-1-ol) (1.42 g, 7.33 mmol) and the mixture was stirred at 0 °C. DCC (504 mg, 2.44 mmol) was added followed by catalytic amount of DMAP (30 mg, 0.24 mmol) under inert atmosphere. The reaction mixture was stirred at 0 °C for 4 h. Precipitated DCU was filtered off and the solvent was removed under reduced pressure. The solvent was removed under reduced pressure. The residue was chromatographed on a silica gel column, 20-100% EtOAc / Hexane to give 0.8 g (56% yield) of compound 45 as a colorless oil. 1 H NMR (500 MHz, CDCl3): δ 2.00-2.07 (m, 1H), 2.22-2.29 (m, 1H), 2.39-2.51 (m, 2H), 3.58-3.71 (m, 14H), 4.22 (t, J = 6.5 Hz, 1H), 4.28-4.35 (m, 2H), 4.40-4.47 (m, 3H), 4.58 (d, J = 6.0 Hz, 2H), 5.23 (d, J = 10.5 Hz, 1H), 5.33-5.29 (m, 2H), 5.79 (d, J = 8.5 Hz, 1H), 5.95-5.87 (m, 1H), 7.30-7.33 (m, 2H), 7.38-7.41 (m, 2H), 7.59-7.62 (m, 2H), 7.76 (d, J = 7.5 Hz, 2H). ESI MS: 608.3 ([M+Na] + ).

[0262] (S)-14-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-hydroxy-13-oxo-3,6,9,12-tetraoxaheptadecan-17-oic acid (46) To a solution of 45 (0.8 g, 1.37 mmol) and PhSiH3 (296 mg, 2.73 mmol) in anhydrous DCM (15 mL) was added Pd(PPh3)4 (40 mg, 0.035 mmol) under inert atmosphere at 0 °C, and the mixture was gradually warmed to room temperature and stirred for 40 min. A few drops of water were then added and the volatiles were removed under reduced pressure. The residue was chromatographed on a silica gel column, 10-40% MeOH / DCM containing 0.5% AcOH to give 0.49 g (66% yield) of compound 46 as a white solid. ESI MS: 568.3 ([M+Na] + ).

[0263] Octyl (5S,10S,13S)-10,13-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-5-(13-hydroxy-2,5,8,11-tetraoxatridecanoyl)-3,8,11-trioxo-2-oxa-4,9,12-triazatetradecane-14-oate (XI) To a solution of compounds 40 (200 mg, 0.46 mmol) and 46 (250 mg, 0.46 mmol) in anhydrous DCM (15 mL) was added N,N-diisopropylethylamine (DIPEA, 118 mg, 0.92 mmol) and the mixture was cooled to 0° C. under inert atmosphere. HATU (192 mg, 0.50 mmol) was added in one portion and the mixture was stirred at 0° C. for 2 h and quenched with H2O (80 mL). The aqueous phase was extracted with ethyl acetate (3×50 mL) and the combined organic layers were washed with brine (50 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure. The residue was purified using reverse-phase preparative HPLC (20% acetonitrile / 80% water, followed by increasing to 70% acetonitrile over 40 min and washing with 100% acetonitrile over 10 min; flow rate 15 mL / min) to give 145 mg (33% yield) of compound XI as a pale yellow solid. 1H NMR (500 MHz, CDCl3): δ 0.86 (t, J = 6.5 Hz, 3H), 1.23-1.32 (m, 10H), 1.58-1.63 (m, 2H), 1.91-2.24 (m, 6H), 2.33-2.39 (m, 3H), 2.43-2.51 (m, 4H), 3.43 (brs, 1H), 3,55-3.72 (m, 14H), 4.05-4.11 (m, 2H), 4.19-4.28 (m, 2H), 4.33-4.43 (m, 4H), 4.47-4.51 (m, 1H), 5.35-5.39 (m, 2H), 6.24 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 7.0 Hz, 1H), 7.29-7.32 (m, 2H), 7.37-7.40 (m, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.59-7.62 (m, 2H), 7.74 (d, J = 7.5 Hz, 2H). 13 C NMR (125 MHz, CDCl3): δ 14.0, 22.5, 25.7, 26.7, 27.7, 27.9, 28.4, 29.07, 29.09, 31.7, 36.2, 36.4, 47.1, 51.9, 52.5, 53.4, 54.8, 55.1, 61.4, 64.2, 65.7, 66.8, 68.7, 70.1, 70.3, 70.4, 72.5, 119.9, 125.1, 127.0, 127.6, 141.2, 143.7, 143.9, 156.2, 171.3, 171.6, 171.9, 172.3, 193.8, 194.8. ESI MS: 986.4 ([M+Na] + ).

[0264] Allyl (S)-5-(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,10,13,16-tetraoxa-4,7-diazanonadecan-19-oate (47) Fmoc-DON-OH (694 mg, 1.76 mmol, 1.1 equiv) and HATU (701 mg, 1.84 mmol, 1.15 equiv) were suspended in anhydrous DCM (10 mL), cooled to 0° C., and DIPEA (1.12 mL, 6.41 mmol, 4.0 equiv) was added. After stirring for 10 min, a solution of 25 (419 mg, 1.60 mmol, 1.0 equiv) in anhydrous DCM (5 mL) was added dropwise. The reaction mixture was stirred at 0° C. for 30 min and at room temperature for 1.5 h. The volatiles were evaporated in vacuo and the residue was purified on silica gel (DCM / MeOH, 20:1). The desired product 47 (1.0 g, 98%) was observed as a yellowish oil. 1 H NMR (401 MHz, CDCl3): 1.90 - 2.03 (m, 1H), 2.10 - 2.16 (m, 1H), 2.37 - 2.56 (m, 2H), 2.61 (t, J = 6.4 Hz, 2H), 3.18 (q, J = 7.4 Hz, 2H), 3.58 - 3.64 (m, 10H), 3.66 - 3.78 (m, 2H), 4.21 (t, J = 7.1 Hz, 2H), 4.31 - 4.44 (m, 2H), 4.58 (dt, J = 5.7, 1.4 Hz, 2H), 5.15 - 5.37 (m, 2H), 5.80 - 5.97 (m, 2H), 6.86 - 6.94 (m, 1H), 7.27 - 7.44 (m, 5H), 7.60 (d, J = 7.6 Hz, 2H), 7.76 (d, J = 7.5 Hz, 2H).UPLC MS: [M + Na] + (C 33 H 40 N4O9Na): 659.448.

[0265] Allyl (S)-15-amino-19-diazo-14,18-dioxo-4,7,10-trioxa-13-azanonadecanoate (48) Compound 47 (1.0 g, 1.57 mmol, 1.0 equiv.) was dissolved in anhydrous DCM (16 mL), EtNH (1.63 mL, 15.7 mmol, 10.0 equiv.) was added, and the reaction mixture was stirred at room temperature for 2 h. The volatiles were evaporated and the residue 48 was used for further reactions without any purification.

[0266] Allyl (5S,8S)-5,8-bis(4-diazo-3-oxobutyl)-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2,13,16,19-tetraoxa-4,7,10-triazadocosane-22-oate (49) Fmoc-DON-OH (680 mg, 1.73 mmol, 1.1 equiv) and HATU (687 mg, 1.81 mmol, 1.15 equiv) were suspended in anhydrous DM (15 mL), cooled to 0° C., and DIPEA (1.09 mL, 6.29 mmol, 4.0 equiv) was added. After stirring for 10 min, a solution of 48 (651 mg, 1.57 mmol, 1.0 equiv) in anhydrous DCM (7 mL) was added dropwise and the reaction mixture was stirred at 0° C. for 30 min and at room temperature for 1.5 h. The volatiles were evaporated in vacuo and the residue was purified by flash chromatography (gradient of 5% to 100% acetonitrile in H2O, 25 min). The desired product 49 (525 mg, 42%) was observed as a yellowish solid. 1H NMR (401 MHz, DMSO-d6): 1.68 - 1.93 (m, 4H), 2.23 - 2.40 (m, 4H), 2.57 (t, J = 6.2 Hz, 2H), 3.39 (t, J = 5.9 Hz, 2H), 3.43 - 3.52 (m, 10H), 3.63 (t, J = 6.2 Hz, 2H), 3.95 - 4.03 (m, 1H), 4.16 - 4.36 (m, 4H), 4.55 (dt, J = 5.4, 1.6 Hz, 2H), 5.16 - 5.32 (m, 2H), 5.83 - 6.07 (m, 3H), 7.29 - 7.46 (m, 4H), 7.56 (d, J = 7.9 Hz, 1H), 7.68 - 7.77 (m, 2H), 7.89 (d, J = 7.5 Hz, 2H), 7.92 - 8.02 (m, 2H). UPLC MS: [M + Na] + (C 39 H 47 N7O 11 Na): 812.916.

[0267] Allyl (15S,18S)-18-amino-22-diazo-15-(4-diazo-3-oxobutyl)-14,17,21-trioxo-4,7,10-trioxa-13,16-diazadocosanoate (50) Compound 49 (525 mg, 0.665 mmol, 1.0 equiv) was dissolved in anhydrous DMF (5 mL) and EtNH (688 μL, 6.65 mmol, 10.0 equiv) was added. The reaction mixture was stirred at room temperature for 2 h. The volatiles were evaporated in vacuo and the residue 50 was used for further reactions without any purification.

[0268] Allyl (6S,9S)-6,9-bis(4-diazo-3-oxobutyl)-2-methyl-4,7,10-trioxo-14,17,20-trioxa-2,5,8,11-tetraazatricosane-23-oate (51) Compound 50 (377 mg, 0.665 mmol, 1.0 equiv.) and 2,5-dioxopyrrolidin-1-yldimethylglycinate (266 mg, 1.33 mmol, 2.0 equiv.) were suspended in anhydrous DMF (5 mL) and the suspension was stirred at room temperature for 2 h. The volatiles were evaporated in vacuo and the residue was purified on silica gel (DCM / MeOH, 10:1). The desired product 51 (228 mg, 53%) was observed as a yellowish oil. 1 H NMR (401 MHz, CDCl3): 1.95 - 2.21 (m, 4H), 2.32 (s, 6H), 2.35 - 2.58 (m, 4H), 2.91 - 3.09 (m, 2H), 3.39 - 3.47 (m, 2H), 3.52 - 3.58 (m, 2H), 3.58 - 3.66 (m, 10H), 3.77 (t, J = 6.4 Hz, 2H), 4.30 - 4.42 (m, 2H), 4.59 (dt, J = 5.6, 1.5 Hz, 2H), 5.17 - 5.44 (m, 4H), 5.91 (ddt, J = 17.2, 10.4, 5.7 Hz, 1H), 6.85 - 6.92 (m, 1H), 7.48 - 7.59 (m, 1H), 7.80 (d, J = 7.2 Hz, 1H). UPLC MS: [M + H] + (C 28 H 45 N8O 10 ): 653.155.

[0269] (6S,9S)-6,9-bis(4-diazo-3-oxobutyl)-2-methyl-4,7,10-trioxo-14,17,20-trioxa-2,5,8,11-tetraazatricosanoic acid-23-oic acid (XII) (KNM472) Compound 51 (176 mg, 0.269 mmol, 1.0 equiv) and dimedone (113 mg, 0.809 mg, 3.0 equiv) were dissolved in anhydrous THF (3 mL), Pd(PPh3)4 (12 mg, 0.011 mmol, 0.04 equiv) was added, and the reaction mixture was stirred at room temperature for 1 h. The volatiles were evaporated in vacuo, and the residue was purified by flash chromatography (5% to 100% gradient of acetonitrile in H2O, 25 min) and silica gel (DCM / MeOH, 10:1 + 1% Et3N). The desired product XII (51 mg, 31%) was observed as a yellowish oil. 1 H NMR (401 MHz, DMSO-d6): 1.68 - 1.99 (m, 4H), 2.21 (s, 6H), 2.25 - 2.35 (m, 4H), 2.52 - 2.60 (m, 2H), 2.81 - 2.93 (m, 2H), 3.33 - 3.45 (m, 2H), 3.45 - 3.53 (m, 10H), 3.59 (t, J = 6.4 Hz, 2H), 4.15 - 4.23 (m, 1H), 4.26 - 4.35 (m, 1H), 6.02 (bs, 2H), 7.81 (d, J = 8.2 Hz, 1H), 7.98 (t, J = 5.7 Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H). UPLC MS: [M + H] + (C 25 H 41 N8O 10 ): 613.048.

[0270] Liver homogenate studies Liver or EL4 lymphoma tumor tissues were washed, diluted 10-fold in 0.1 M potassium phosphate buffer, and homogenized using a probe sonicator. To assess the stability of the analogs, 1 mL aliquots were made from each matrix and spiked with analogs to a final assay concentration of 20 μm. The spiked samples were incubated at 37° C. for 1 h in an orbital shaker, after which the reaction was quenched in triplicate with 3 volumes of acetonitrile containing an internal standard (IS; losartan: 0.5 μm). The samples were mixed by vortexing for 30 s and centrifuged at 10000 g for 10 min at 4° C. Fifty microliters of the supernatant was diluted with 50 μL of water and transferred to a 250 μL polypropylene vial sealed with a Teflon cap. The disappearance of the compounds was monitored over time using liquid chromatography tandem mass spectrometry (LC-MS / MS).

[0271] DON release assay DON release was assessed in liver or tumor homogenates using a previously described method. Briefly, the supernatant was dried under vacuum at 45°C for 1 h. To each tube, 50 μL of 0.2 M sodium bicarbonate buffer (pH 9.0) and 100 μL of 10 mM dabsyl chloride were added. After vortex mixing, the samples were derivatized by incubation at 60°C for 15 min, followed by centrifugation at 16000g and 4°C for 5 min. One hundred microliters of the supernatant was transferred to a 96-well plate, diluted with 400 μL of water, and injected into the LC-MS / MS. DON was analyzed on a Dionex ultra-performance LC system coupled to a Q Exactive Focus Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). Separation was achieved at 35 °C using an Agilent Eclipse Plus column (100 × 2.1 mm2, id) packed with a 1.8 μm C18 stationary phase. The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The pump was run for 3.5 min at a flow rate of 0.3 mL / min using gradient elution. The mass spectrometer, controlled by Xcalibur software 4.0.27.13 (Thermo Scientific), was run with a heated ESI ion source in positive ionization mode. Quantification was performed in parallel reaction monitoring mode. result [Table 1-1] [Table 1-2] [Table 1-3]

[0272] Example 2 Synthesis of glutamine-antagonist dendrimer conjugates with various linkers All of the glutamine antagonists were synthesized with linkers suitable for conjugation chemistry on the dendrimer surface.

[0273] For conjugation, the hydroxyl groups on the dendrimer surface were partially modified to add a linker with a reactive functional group complementary to that of the antagonist-linker (Figure 9). The antagonist was conjugated onto the surface of the modified dendrimer using an activated acid-amine coupling reaction in the presence of N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). The final conjugate was purified by dialysis to remove solvent and small molecular weight impurities. The intermediate and final dendrimer conjugates were 1 H-NMR, 13 The conjugates were characterized using C-NMR, HPLC, and MALDI-TOF / MS analysis. The conjugates were synthesized with high purity (>95%). The drug loading of the conjugates was assessed using the proton integration method by comparing the integrals of the drug protons versus the internal amide protons of the dendrimer. 1 The drug loading of the conjugates was approximately 5% w / w as calculated by H-NMR. The dendrimer-drug conjugates were water-soluble, with solubilities ranging from 50 to 100 mg / mL. Below is a detailed synthesis protocol for the dendrimer-glutamine antagonist conjugate shown in FIG. 9:

[0274] Synthesis of dendrimer-acid: To a stirred solution of D-OH (1 g, 0.07 mmol) in DMF (20 mL), N,N-diisopropylethylamine (DIPEA; 0.5 mL) was added, followed by glutaric anhydride (95.8 mg, 0.840 mmol). The reaction mixture was stirred at room temperature (RT, approximately 25 °C) for 24 h. The reaction mixture was then diluted with DMF and dialyzed against DMF using a 1 kDa dialysis membrane for 12 h, followed by dialysis against water for another 12 h. During the dialysis process, the solvent was changed every 3-4 h. The aqueous solution was lyophilized to give the D-acid as a white hygroscopic solid. Yield: 88%

[0275] 1H NMR: (500 MHz, DMSO) δ 8.27 - 7.73 (m, D-internal amide H), 4.07 (t, D-ester -CH2), 3.54 - 3.26 (m, D-CH2), 3.25 - 3.00 (m, D-CH2), 2.90 - 2.60 (m, D-CH2 and linker CH2), 2.53 - 2.31 (m, D-CH2 and linker CH2), 2.26 (m, D-CH2), and 1.85 - 1.72 (m, linker -CH2).

[0276] Synthesis of dendrimer-glutamine antagonist conjugate: To a stirred solution of D-acid (200 mg, 0.013 mmol) in DMF (5 mL), DIPEA (0.1 mL) was added followed by NHS (22 mg, 0.195 mmol) and EDC (37.2 mg, 0.195 mmol). The reaction mixture was stirred at room temperature for 30 min. To this was then added glutamine-antagonist (71.5 mg, 0.156 mmol). The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was then diluted with DMF and dialyzed against DMF using a 1 kDa dialysis membrane for 12 h followed by water for another 12 h. During the dialysis process, the solvent was changed every 3-4 h. The aqueous solution was lyophilized to give the dendrimer conjugate as a white hygroscopic solid. Yield: 72%

[0277] 1H NMR (500 MHz, DMSO) δ 8.29 (d, drug-H), 8.16 - 7.69 (m, drug-H and D-internal amide H), 6.04 (drug-H), 4.87 (m, drug-H), 4.34 (m, drug-H), 4.15 (m, drug-H), 4.01 (t, D-ester-CH2), 3.59 (m, drug H), 3.52 - 2.99 (D-CH2 and drug H), 2.66 (m, D-CH2 and drug H), 2.46 - 2.04 (m, D-CH2 and drug H), 2.01 - 1.23 (m, drug H and linker-CH2), 1.17 (d, drug H), and 0.87 (dd, drug H). HPLC: Retention time: Retention time: 11.028 minutes; Purity: 99%.

[0278] result Some exemplary dendrimer and glutamine antagonist conjugates are shown in Table 2 below. [Table 2]

[0279] Example 3 Glutamine antagonist dendrimer conjugate TTM020 (structure VII) demonstrates brain target engagement in a murine model of neuroinflammation method Animals and drug administration Mice were intracranially injected with LPS (2.5 mg / kg) into the right striatum. One hour later, dendrimer-TTM020 or empty dendrimer was administered IP at a dose of 2 mg / kg in DON equivalent. After 12 and 72 hours, mice were sacrificed, the right hemisphere was removed, and CD11b cells were extracted as described below. Glutaminase activity assessment was performed as described below.

[0280] Isolation of brain CD11b+ cells Mice were sacrificed by decapitation and the right striatum was immediately removed. CD11b+ cells were isolated. Briefly, brain tissue was minced in HBSS (catalog number 55021C, Sigma-Aldrich, St. Louis, MO, USA) and dissociated with Neural Tissue Dissociation Kit (P) (catalog number 130-092-628, MACS Militenyi Biotec, Auburn, CA) according to the manufacturer's instructions. After passing through a 70 μm cell strainer, the resulting homogenate was centrifuged at 300 × g for 10 min. The supernatant was removed, the cell pellet was resuspended, and myelin was removed by Myelin Removal Beads II (catalog number 130-096-733, MACS Militenyi Biotec, Auburn, CA) according to the manufacturer's instructions. The myelin-depleted cell pellet was resuspended and incubated with CD11b MicroBeads (catalog no. 130-093-634, MACS Militenyi Biotec, Auburn, CA) for 15 min, loaded onto an LS column, and separated with a quadroMACS magnet. CD11b+ cells were flushed from the LS column, then washed and resuspended in sterile HBSS (catalog no. 55037C, Sigma-Aldrich, St. Louis, MO). The number of viable cells was determined using a hemocytometer and 0.1% trypan blue staining. 5 × 10 cells were harvested from each brain extract. 5 Viable CD11b+ cells were obtained. CD11b+ cells isolated from brain homogenates by this antibody-conjugated microbead method have been demonstrated to be a microglia-enriched population (>95% of isolated cells).

[0281] Glutaminase activity Next, glutaminase activity measurements were performed in CD11b+ cells. Briefly, cells were lysed by sonication in ice-cold potassium phosphate buffer (45 mM, pH 8.2) containing protease inhibitors (catalog number 04693116001, Roche). For CD11b+ and non-CD11b+ cells isolated from the prefrontal cortex, hippocampus, and cerebellum, the lysates were [ 3The mixture was incubated with [3H]-glutamine (0.09 μM, 2.73 μCi) for 180 min at room temperature, and the reaction was carried out in a 50 μl reaction volume in a 96-well microplate. The reaction was then terminated by adding imidazole buffer (20 mM, pH 7). Unreacted [H]-glutamate was separated from the [3H]-glutamate using a 96-well spin column packed with strong anion ion exchange resin (catalog no. 140-1251, Bio-Rad, AG® 1-X2 resin, 200-400 mesh, chloride form). 3 H]-glutamine was isolated. 3 [H]-glutamate was eluted from the column with 0.1N HCl and analyzed for radioactivity using a Perkin Elmer's TopCount instrument in combination with its 96-well LumaPlates (cat. no. 6005173). Total protein measurements were performed using BioRad's Detergent Compatible protein assay kit according to the manufacturer's instructions. Counts per minute were converted to fmol and normalized to total protein content. Data are presented as fmol / mg / hr.

[0282] result The experimental setup is shown in Figure 12. Dendrimer-TTM020 (Figure 13) effectively inhibited glutaminase activity compared to the unconjugated dendrimer at both 24 and 72 hours after drug administration (Figure 14). The dendrimer-conjugated DON analog, TTM020, provided sustained glutaminase inhibition in the brains of these mice.

[0283] Example 4 Dendrimer-CY5 selectively localizes to brain tumors after systemic administration method Tumor inoculation and treatment administration Six- to eight-week-old male and female C57BL / 6 mice were inoculated intracranially with GL261 murine glioblastoma cells obtained from the DTP / DCTD / NCI Tumor Repository (National Cancer Institute, Frederick, MA). GL261 cells were maintained in RPMI containing 10% FBS, 1% P / S, and 1% L-glutamine at 37°C and 5% CO2 atmosphere. Mice were anesthetized with a ketamine (Vedco, St. Joseph, MO) and xylazine (Akorn Animal Health, Lake Forest, IL) cocktail. A scalp incision was made at the midline and a burr hole was drilled 1 mm posterior to the bregma and 2 mm lateral to the midline. A 2 μL Hamilton syringe (Hamilton Company, Reno, NV) was lowered to a depth of 2.5 mm and 2 μL of GL261 cell solution containing 100,000 cells was injected over 10 min using a stereotaxic frame and an automated syringe pump (Stoelting Co., Wood Dale, IL). The syringe was withdrawn at 0.5 mm / min and the incision was sutured (Ethicon Inc., Somerville, NJ).

[0284] To assess the impact of treatment, GL261 brain tumor-bearing mice were treated with dendrimer-KMN045 (FIG. 16) or free DON administered intraperitoneally at a dose of 2 mg / kg (equivalent drug dose for free drug and dendrimer-conjugate) every 4 days starting on day 10 after tumor inoculation. Brains were harvested on day 20 after inoculation (or at time of illness if mice met euthanasia criteria before day 20). Tumors were excised, summarized, and tumor burden was calculated as percent tumor mass of total brain mass.

[0285] result Using confocal microscopy and a rodent tumor-inoculated 9L gliosarcoma model, it was demonstrated that systemic administration of D-Cy5 (24 h) resulted in selective brain tumor uptake (Figure 15). Furthermore, quantification of D-Cy5 in the whole brain showed significant accumulation in the tumor compared to peritumoral and contralateral brain regions. D-Cy5 concentrations were quantified by calculating the area under the curve from 0 to 48 h (AUC0-48h) (p<0.05). Similar selective localization to TAMs was shown in the GL261 mouse model (Zhang F, et al., Biomaterials. 2015;52 and Liaw K, et al., Bioengineering & Translational Medicine. 2020).

[0286] The glutamine antagonist dendrimer conjugate, D-045, showed superior efficacy compared to free DON when directly tested in the GL261 murine model of GBM in C57BL / 6 mice (Figures 17A and 17B). The conjugate released less than 20% of the active drug (Figure 18). These data provided a proof of concept.

[0287] Example 5 Dendrimer-DON (TTM020) shows DON release in tumor homogenates in vitro and complete regression of tumor growth in vivo without any apparent side effects on body weight. method Efficacy studies were performed using C57BL / 6 / EL4 tumor-bearing mice after administration of D-DON via the ip route. Briefly, EL4 cells were injected sc (0.3 × 10 6 Tumor growth was monitored. 3 Animals with a mean tumor volume of 100 mg / kg were randomized into two groups - vehicle and D-DON (DON equivalent dose 2 mg / kg; n=7 / group). Animals were dosed 3 days / week for 2 weeks while tumor volume, body weight, and death were recorded using Vernier calipers (VWR, USA). The study continued until complete regression of the tumor.

[0288] result Dendrimer-DON (TTM020) incubated in tumor homogenates showed a time-dependent release of DON (Figure 19A). DDON treatment not only inhibited tumor growth but also showed complete tumor regression (Figure 19B). No effect on body weight was observed upon treatment, suggesting that dendrimer-DON was well tolerated (Figure 19C).

[0289] It has been established that systemic administration of dendrimer-glutamine antagonist conjugates provides sustained drug levels of glutamine antagonist in activated immune cells, allowing for significant dose reductions and resulting in substantial improvements in the therapeutic index, making this therapeutic strategy viable for clinical development.

[0290] Example 6 Dendrimer-Cy5 is phagocytosed by activated microglia in mice after chronic social defeat stress (CSDS) method Ten days after CSDS (or without), C57BL / 6 mice were injected with 50 mg / kg D-Cy5 and sacrificed 24 h later via transcardial perfusion with PBS. Brains were post-fixed in 4% paraformaldehyde for 24 h, frozen at -80°C, sectioned at 30 μm thickness, and stained for microglia (Iba1), astrocytes (Aldh1l1), and nuclei (DAPI).

[0291] result Fluorescently labeled PAMAM dendrimers (D-Cy5) were synthesized using Cy5 and characterized as previously described (Iezzi, R. et al., Biomaterials. 2012, 33 (3), 979-988; Lesniak, WG et al., Mol Pharm. 2013, 10 (12), 4560-4571) and their brain penetration in mice after CSDS was tested. Activated microglia in mice after CSDS were observed to selectively phagocytose the dendrimers. Positive D-Cy5 signals were observed near the dentate gyrus of the hippocampus of mice after CSDS and overlapped with Iba1 staining, indicating microglial uptake. No D-Cy5 signals were observed in astrocytes (Aldh1l1). No positive signals were observed in brains from non-CSDS mice.

[0292] Example 7 Dendrimer-TTM020 inhibits microglial glutaminase activity in mice subjected to CSDS method After establishing CSDS, mice were orally treated with D-TTM020 (20 mg / kg), and microglia-enriched CD11b+ cells were isolated from the hippocampus 24 h after administration. Proteins were then extracted from these cells, and glutaminase activity was measured.

[0293] result Consistent with our previous report (Zhu, X. et al., Neuropsychopharmacology. 2019, 44 (4), 683-694), mice exposed to CSDS showed a significant increase in glutaminase activity. D-TTM020 attenuated the upregulated glutaminase activity in microglia-enriched CD11b+ cells from CSDS mice (Figure 20), indicating successful functional target engagement.

[0294] Previous reports have shown that glutamine antagonism inhibits stress-induced increases in microglial glutaminase activity, inflammatory cytokine production, and normalizes social avoidance and anhedonia induced by chronic social defeat stress (CSDS), a well-established rodent model used to study stress-induced psychiatric disorders including depression (Zhu, X. et al., Neuropsychopharmacology. 2019, 44 (4), 683-694). Although glutamine antagonism exhibits robust therapeutic efficacy, its chronic dosing is known to result in gastrointestinal toxicity, limiting its translational application. Given its remarkable clinical potential, we addressed this limitation by targeting glutamine antagonists directly to the inflamed brain using a hydroxyl-dendrimer nanoparticle delivery system. Systemic administration of dendrimer-TTM020 (D-TTM020) provides sustained brain drug levels with simultaneous rapid clearance from the periphery, resulting in substantial improvements in its tolerability and maintaining its robust efficacy in treating stress-related psychosocial behavioral deficits.

[0295] 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 the disclosed invention belongs. Publications cited herein and the material cited therein are specifically incorporated by reference.

[0296] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. "AA" refers to amino acids, as follows: 【Chemistry 16】 In the formula, R １ is -OCH ２ CH ３ , -OCH(CH ３ ) ２ -OC(CH ３ ) ３ , -NH ２ , N.H.H. ３ , -NH-AA-COOCH(CH ３ ) ２ , and -NH-AA-COOC(CH ３ ) ３ Selected from: R ２ is -H, -CH ３ , -CH ２ Phenyl, -CH ２ CH (CH ３ ) ２ , -CH ２ (indol-3-yl), and -CH ２ CH ２ C(=O)CH=N≡N; R ３ is -NH ２ and -OH; n is an integer value from 1 to 32. 【Chemistry 17】 In the formula, R １ is -OCH ２ CH ３ , -OCH(CH ３ ) ２ -OC(CH ３ ) ３ , -NH ２ , N.H.H. ３ , -NH-AA-COOCH(CH ３ ) ２ , and -NH-AA-COOC(CH ３ ) ３ Selected from: R ３ is -NH ２ and -OH; n is an integer value from 1 to 16; 【Chemistry 18】 In the formula, R ２ is -H, -CH ３ , -CH ２ Phenyl, -CH ２ CH (CH ３ ) ２ , -CH ２ (indol-3-yl), or -CH ２ CH ２ C(=O)CH=N≡N; R ４ is -H, -CH ３ , -CH ２ Phenyl, or -CH ２ CH (CH ３ ) ２ Selected from: R ５ is -H, -Ac, -Fmoc, dimethylglycine, Ac-AA-C(=O)-, dimethylglycine-NH-AA-C(=O)-, dimethylglycine-NH-DON-C(=O)-, or C ８ H １７ OC(=O)CH(NH ２ ) CH ２ CH ２ C(═O)—NH-6-diazo-5-oxo-L-norleucine-C(═O)—; n is an integer value from 1 to 32; 1. A composition comprising a dendrimer complexed, covalently conjugated, intramolecularly dispersed or encapsulated with one or more 6-diazo-5-oxo-L-norleucine (DON) analogs having one of three general chemical formulas:

2. The DON analogue is 【Chemistry 19-1】 【Chemistry 19-2】 【Chemistry 19-3】 【Chemistry 19-4】 2. The composition of claim 1, wherein the compound has a structure selected from the group consisting of:

3. The composition of claim 1 , wherein the dendrimer is a fourth, fifth, sixth, seventh, or eighth generation dendrimer.

4. The composition of claim 1 , wherein the dendrimer is a poly(amidoamine) (PAMAM) dendrimer.

5. The composition of claim 1 , wherein the dendrimer is a hydroxyl-terminated PAMAM dendrimer.

6. 2. The composition of claim 1, wherein the dendrimer is a fourth, fifth, or sixth generation hydroxyl-terminated PAMAM dendrimer.

7. The composition of claim 1 , wherein the dendrimer is covalently conjugated to the one or more DON analogs, optionally via a linker or spacer moiety.

8. The composition of claim 1 , wherein the dendrimer is further complexed or conjugated to one or more therapeutic, prophylactic or diagnostic agents.

9. The composition of claim 8, wherein the dendrimer is further complexed or conjugated to one or more neuroprotective, anti-inflammatory and / or chemotherapeutic agents.

10. A pharmaceutical composition comprising the composition of any one of claims 1 to 9 and one or more pharma- ceutically acceptable excipients.

11. 11. The pharmaceutical composition of claim 10, formulated for parenteral or oral administration.

12. 11. The pharmaceutical composition of claim 10, formulated in a form selected from the group consisting of a hydrogel, a nanoparticle or microparticle, a suspension, a powder, a tablet, a capsule, and a solution.

13. 11. The pharmaceutical composition of claim 10 for treating one or more of neurological, oncological and immune disorders in a subject in need thereof, wherein the composition is administered to the subject in an effective amount to treat, alleviate and / or prevent one or more symptoms associated with one or more of the neurological, oncological and immune disorders.

14. 14. The pharmaceutical composition of claim 13, wherein the composition is administered in an amount effective to reduce glutaminase activity and / or decrease glutamate release in one or more target cells associated with diseased or damaged tissues of one or more of neurological, oncological, and immune disorders.

15. 14. The pharmaceutical composition of claim 13, wherein the composition is administered in an amount effective to reduce glutaminase activity and / or decrease glutamate release in activated microglia associated with diseased or damaged tissue of one or more of neurological, oncological, and immune disorders.

16. 14. The pharmaceutical composition of claim 13, wherein the neurological disorder is selected from the group consisting of stress-induced mood disorders, cognitive deficits, ischemia, neuroinflammation, Alzheimer's disease, and multiple sclerosis.

17. 14. The pharmaceutical composition of claim 13, wherein the composition is administered in an amount effective to reduce neuroinflammation, improve cognition, or a combination thereof.

18. 14. The pharmaceutical composition of claim 13, wherein the oncological disorder is selected from the group consisting of breast cancer, ovarian cancer, uterine cancer, prostate cancer, testicular germ cell tumors, brain cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, renal cell cancer, colorectal cancer, and pancreatic cancer.

19. 14. The pharmaceutical composition of claim 13, wherein the composition is administered in an amount effective to reduce tumor size.

20. The pharmaceutical composition of claim 13, wherein the composition is administered to the subject in combination with one or more selected from the group consisting of immune checkpoint modulators, chemotherapeutic agents, anti-infective agents, adoptive T cell therapy, cancer vaccines, surgery, and radiation therapy.

21. 21. The pharmaceutical composition of claim 20, wherein the immune checkpoint modulator is selected from the group consisting of a PD-1 antagonist, a PD-1 ligand antagonist, and a CTLA4 antagonist.

22. 14. The pharmaceutical composition of claim 13, wherein the immune disorder is selected from the group consisting of rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), type 1 diabetes, inflammatory bowel disease, and thyroid disease.

23. 11. The pharmaceutical composition of claim 10 for inhibiting glutaminase activity in activated microglia associated with diseased or damaged tissue in a subject, wherein the composition is administered systemically to the subject.

24. 14. The pharmaceutical composition according to claim 13, characterized in that the composition is administered orally or parenterally.

25. 14. The pharmaceutical composition of claim 13, wherein the composition is administered intravenously.

26. below: 【Chemistry 20-1】 【Chemistry 20-2】 【Chemistry 20-3】 【Chemistry 20-4】 A molecule having a structure selected from the group consisting of:

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