Dendrimer conjugates and methods of use thereof

Abstract

Aspects of the present disclosure provide dendrimer conjugates, compositions comprising dendrimer conjugates, and methods of using dendrimer conjugates and compositions thereof. In some aspects, the present disclosure provides dendrimer conjugates comprising a dendrimer conjugated to at least one agent. In some embodiments, the dendrimer conjugates comprise one or more agents useful in therapy, imaging, and / or targeted delivery. In some aspects, methods are provided for synthesizing functionalized dendrimers, comprising reacting a first dendrimer with one or more amines.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 327,301, filed April 4, 2022, and U.S. Provisional Patent Application No. 63 / 209,348, filed June 10, 2021, each of which is hereby incorporated by reference in its entirety. [Background technology]

[0002] background Advances in the field of nanotechnology have produced many new materials and devices with widespread applications. Nanotechnology has emerged as a wide range of materials and particles, e.g., fullerenes and dendrimers. However, in vivo applications of nanoparticles suffer from the challenge of rapid clearance from circulation due to their interactions with biological systems. Alternative strategies for nanoparticle development may allow the advantageous properties of nanoparticles to be better utilized in the context of clinical applications, diagnostics, and biomedical research. Summary of the Invention [Means for solving the problem]

[0003] overview In some aspects, the disclosure provides therapeutic and / or diagnostic compounds comprising a dendrimer conjugated to an agent via a terminal ether or amide bond. In some embodiments, the dendrimer comprises a terminal hydroxy group that is optionally substituted with an agent. In some embodiments, the agent is a therapeutic agent or an imaging agent. In some aspects, the present disclosure provides a dendrimer conjugate of formula (I): [ka] [wherein D is a dendrimer, X is O or NH, and Y 1is optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, or a covalent bond; Y 2 is selected from the group consisting of secondary amides, tertiary amides, sulfonamides, secondary carbamates, tertiary carbamates, carbonates, ureas, carbinols, disulfides, hydrazones, hydrazides, ethers, carbonyls, and combinations thereof; Z is a therapeutic agent or an imaging agent; L is a linker; m is an integer between 16 and 4096, inclusive; and n is an integer between 1 and 100, inclusive. In some embodiments, the ratio of m to (m+n) is at least 0.5. In some embodiments, the ratio of m to (m+n) is between about 0.50 and about 0.99.

[0004] In some aspects, the present disclosure provides a dendrimer conjugate of formula (II) [ka] wherein D is a dendrimer; each instance of X is independently O or NH; and Y 1 Each instance of is independently an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, or a covalent bond; 2 Each instance of is independently selected from the group consisting of secondary amides, tertiary amides, sulfonamides, secondary carbamates, tertiary carbamates, carbonates, ureas, carbinols, disulfides, hydrazones, hydrazides, ethers, carbonyls, and combinations thereof; Z 1 and Z 2 is independently a therapeutic agent, a targeting agent, or an imaging agent, with the proviso that Z 1 and Z 2 is different, L 1 and L 2 is independently a linker; m is an integer between 16 and 4096, inclusive; and each instance of n is independently an integer between 1 and 100, inclusive.

[0005] In some embodiments, the dendrimer of the therapeutic compound or dendrimer conjugate of the present disclosure is selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof.

[0006] In some embodiments, the linker of the therapeutic compound or dendrimer conjugate comprises a polymer. In some embodiments, the polymer is a polymeric polyol, a polypeptide, or an unsubstituted alkyl chain. In some embodiments, the polymer is a polymeric polyol selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, and polyvinyl alcohol. In some embodiments, the polymer is a polypeptide comprising at least 2 and up to 25 amino acids. In some embodiments, the polymer is an unsubstituted C2-30 alkyl chain. In some embodiments, the linker comprises at least one moiety selected from the group consisting of 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, and 1,4-dihydropyridazyl. In some embodiments, the linker comprises a polymer and at least one moiety. In some embodiments, the linker is non-hydrolyzable under physiological conditions.

[0007] In some embodiments, the therapeutic compound or therapeutic agent of the dendrimer conjugate is an angiotensin II receptor blocker, a farnesoid X receptor agonist, a death receptor 5 agonist, a sodium-glucose cotransporter type 2 inhibitor, a lysophosphatidic acid 1 receptor antagonist, an endothelin-A receptor antagonist, a PPAR delta agonist, an AT1 receptor antagonist, a CCR5 / CCR2 antagonist, an antifibrotic agent, an anti-inflammatory agent, an antioxidant, a STING agonist, a CSF1R inhibitor, an AXL inhibitor, a c-Met inhibitor, a PARP inhibitor, a receptor tyrosine kinase inhibitor, a MEK inhibitor, a group I p21-activated kinase (PAK1) inhibitor, a glutaminase inhibitor, a TIE inhibitor, a vasopressin ... II antagonists, CXCR2 inhibitors, CD73 inhibitors, arginase inhibitors, PI3K inhibitors, TLR4 agonists, TLR7 agonists, SHP2 inhibitors, chemotherapeutic agents, STING antagonists, and JAK1 inhibitors.

[0008] In some embodiments, the therapeutic compound or the therapeutic agent of the dendrimer conjugate is a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD325901, PD035901, PD032901, and TAK-733. In some embodiments, the therapeutic compound or the therapeutic agent of the dendrimer conjugate is a receptor tyrosine kinase inhibitor. In some embodiments, the receptor tyrosine kinase inhibitor is selected from the group consisting of sunitinib, sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, dasatinib, bemcentinib (R428), duvelmatinib (TP-0903), nintedanib, cabozantinib, and motesanib. In some embodiments, the therapeutic compound or the therapeutic agent of the dendrimer conjugate is a PAK1 inhibitor. In some embodiments, the PAK1 inhibitor is Frax-1036 (6-[2-chloro-4-(6-methyl-2-pyrazinyl)phenyl]-8-ethyl-2-[[2-(1-methyl-4-piperidinyl)ethyl]amino]-pyrido[2,3-d]pyrimidin-7(8H)-one).

[0009] In some embodiments, the imaging agent of the therapeutic compound or dendrimer conjugate is selected from the group consisting of dyes, fluorescent dyes, near-infrared dyes, single-photon emission computed tomography (SPECT) imaging agents, positron emission tomography (PET) imaging agents, magnetic resonance imaging (MRI) contrast agents, and radionuclides.

[0010] In some embodiments, the therapeutic compound or dendrimer conjugate of the present disclosure comprises at least one targeting agent conjugated to a dendrimer. In some embodiments, the targeting agent is tribranched-N-acetylgalactosamine (GalNAc).

[0011] In some aspects, the present disclosure provides a method for preparing a dendrimer comprising the steps of: [ka] wherein D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; X is NH; and Y 1 is an optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, or a covalent bond; m is an integer from 16 to 4096, inclusive; n is an integer from 1 to 100, inclusive; and the polydispersity value of the functionalized dendrimer in the composition is less than or equal to 1.10. A composition comprising:

[0012] In some aspects, the present disclosure provides a functionalized dendrimer of formula (IA) [ka] [In the formula, D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; X is NH; Y is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; 1 is an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, or a covalent bond, m is an integer from 16 to 4096, inclusive, and n is an integer from 1 to 100, inclusive, comprising: [ka] wherein D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof, and t is an integer from 16 to 4096, inclusive, with one or more amines, wherein each amine is a group having the formula HNR 1 and R 1 is an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, or a covalent bond. In some embodiments, a functionalized dendrimer as described herein, or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, isotopically enriched form (e.g., isotopically labeled derivative) thereof is provided.

[0013] In some embodiments, n is approximately 3 or 10 (e.g., 3 or 10). In some embodiments, m is 64 minus n. In some embodiments, n is 3 and m is 61. In some embodiments, n is 10 and m is 54. In some embodiments, m is approximately 61 or 54 (e.g., 61 or 54).

[0014] In some embodiments, the dendrimer of formula (II-A) has the formula [ka] It is a dendrimer.

[0015] In some embodiments, the functionalized dendrimer of formula (IA) has the formula [ka] [ka] is a functionalized dendrimer.

[0016] In yet another aspect, the present disclosure provides a functionalized dendrimer of formula (IA) synthesized by the methods described herein. In some aspects, a dendrimer of formula (II-A) [ka] is provided.

[0017] In some aspects, the disclosure provides compositions of therapeutic compounds comprising dendrimers conjugated to a therapeutic agent via a terminal ether or terminal amide bond. In some embodiments, the dendrimer comprises a high density of terminal hydroxy groups that are optionally substituted with a therapeutic agent. In some embodiments, the therapeutic compound comprising a dendrimer conjugated to a therapeutic agent is 10-20% by weight of the therapeutic agent. In some embodiments, the terminal ether or terminal amide bond is conjugated to the therapeutic agent via a linker.

[0018] In some embodiments, the therapeutic compound is about 10% to about 15% by weight of the therapeutic agent. In some embodiments, the therapeutic compound is about 15% to about 20% by weight of the therapeutic agent. In some embodiments, at least 50% of the terminal sites on the dendrimer comprise terminal hydroxy groups. In some embodiments, at least 50% to 99% (e.g., 50-95%, 50-90%, 50-80%, 50-70%, 50-60%, 60-80%, 70-90%) of the terminal sites on the dendrimer comprise terminal hydroxy groups.

[0019] In some embodiments, the therapeutic agent has increased water solubility compared to an unconjugated compound comprising the therapeutic agent in the absence of the dendrimer. In some embodiments, the water solubility is increased by at least 10% compared to the unconjugated compound. In some embodiments, the water solubility is increased by about 10% to about 100% compared to the unconjugated compound. In some embodiments, the water solubility is increased by at least about 2-fold compared to the unconjugated compound. In some embodiments, the water solubility is increased by about 2-fold to about 10-fold compared to the unconjugated compound. In some embodiments, the water solubility is solubility under physiological conditions. In some embodiments, the water solubility is solubility in water having a pH between about 7.0 and about 8.0. In some embodiments, the therapeutic agent is present at a concentration at which the unconjugated compound is insoluble under physiological conditions.

[0020] In some aspects, the disclosure provides a method of treating or imaging a disease or disorder of the brain or central nervous system in a subject in need thereof, comprising administering to the subject a composition comprising a dendrimer conjugate described herein in an amount effective to treat or image the disease or disorder of the brain or central nervous system in the subject.

[0021] In some embodiments, the dendrimer conjugate is selectively taken up by activated microglia and / or activated macrophages in the brain or central nervous system of the subject. In some embodiments, the activated macrophages are resident macrophage cells of the central nervous system. In some embodiments, the dendrimer conjugate crosses the blood-brain barrier of the subject. In some embodiments, the disease or disorder is a brain or central nervous system tumor. In some embodiments, the tumor is a benign or malignant tumor associated with neurofibromatosis. In some embodiments, the disease or disorder is neurofibromatosis (e.g., neurofibromatosis type 1 (NF1)). Thus, in some embodiments, the subject has or is suspected of having neurofibromatosis (e.g., NF1). In some embodiments, the dendrimer conjugate is selectively taken up by tumor-associated macrophages of the tumor of the subject. In some embodiments, the tumor is a brain cancer or a central nervous system cancer. In some embodiments, the tumor is a brain cancer selected from the group consisting of neoplasia and hyperplasia. In some embodiments, the tumor is a central nervous system cancer selected from the group consisting of glioma, glioblastoma, astrocytoma, oligodendroglioma, meningioma, medulloblastoma, ganglioneuroma, and schwannoma. In some embodiments, the dendrimer conjugate is not conjugated to a targeting moiety. In some embodiments, the composition is administered to the subject systemically, intravenously, or orally. In some embodiments, the dendrimer conjugate comprises a therapeutic agent, and the composition has an increased therapeutic index compared to a composition comprising the therapeutic agent in the absence of the dendrimer. In some embodiments, the therapeutic agent is a MEK inhibitor (e.g., trametinib, cobimetinib, binimetinib, selumetinib, PD325901, PD035901, PD032901, or TAK-733). In some embodiments, the therapeutic agent is a receptor tyrosine kinase inhibitor (e.g., sunitinib, sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, dasatinib, bemcentinib (R428), duvelmatinib (TP-0903), nintedanib, cabozantinib, or motesanib).In some embodiments, the therapeutic agent is a PAK1 inhibitor (e.g., Frax-1036 (6-[2-chloro-4-(6-methyl-2-pyrazinyl)phenyl]-8-ethyl-2-[[2-(1-methyl-4-piperidinyl)ethyl]amino]-pyrido[2,3-d]pyrimidin-7(8H)-one)).

[0022] In some aspects, the disclosure provides a method of treating or imaging an ocular disease or disorder in a subject in need thereof, comprising administering to the subject a composition comprising a dendrimer conjugate described herein in an amount effective to treat or image the ocular disease or disorder in the subject.

[0023] In some embodiments, the dendrimer conjugate is selectively taken up by activated microglia and / or activated macrophages in the eye of the subject. In some embodiments, the dendrimer conjugate crosses the blood-retinal barrier of the subject. In some embodiments, the dendrimer conjugate is not conjugated to a targeting moiety. In some embodiments, the composition is administered systemically, intravenously, or orally to the subject. In some embodiments, the dendrimer conjugate comprises a therapeutic agent, and the composition has an increased therapeutic index compared to a composition comprising a therapeutic agent in the absence of the dendrimer.

[0024] In some aspects, the disclosure provides a method of treating or imaging a proliferative disease in a subject in need thereof, comprising administering to the subject a composition comprising a dendrimer conjugate described herein in an amount effective to treat or image the proliferative disease in the subject.

[0025] In some embodiments, the proliferative disease is neurofibromatosis. In some embodiments, the proliferative disease is selected from neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), and schwannomatosis. In some embodiments, the proliferative disease is NF1. In some embodiments, the dendrimer conjugate is not conjugated to a targeting moiety. In some embodiments, the composition is administered to the subject systemically, intravenously, or orally. In some embodiments, the dendrimer conjugate comprises a therapeutic agent, and the composition has an increased therapeutic index compared to a composition comprising the therapeutic agent in the absence of the dendrimer. In some embodiments, the therapeutic agent is a MEK inhibitor (e.g., trametinib, cobimetinib, binimetinib, selumetinib, PD325901, PD035901, PD032901, or TAK-733). In some embodiments, the therapeutic agent is a receptor tyrosine kinase inhibitor (e.g., sunitinib, sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, dasatinib, bemcentinib (R428), duvelmatinib (TP-0903), nintedanib, cabozantinib, or motesanib). In some embodiments, the therapeutic agent is a PAK1 inhibitor (e.g., Frax-1036 (6-[2-chloro-4-(6-methyl-2-pyrazinyl)phenyl]-8-ethyl-2-[[2-(1-methyl-4-piperidinyl)ethyl]amino]-pyrido[2,3-d]pyrimidin-7(8H)-one)).

[0026] The details of certain embodiments of the present disclosure are set forth in the detailed description, as set forth below. Other features, objects, and advantages of the present disclosure will become apparent from the examples, figures, and claims.

[0027] The accompanying drawings, which form a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention. [Brief description of the drawings]

[0028] [Figure 1-1] FIG. 1 is a schematic diagram showing the chemical structure of the dendrimer conjugate (compound D-4517.2). [Figure 1-2] Same as above.

[0029] [Diagram 2] FIG. 2 is a reaction scheme illustrating one synthetic strategy for N,N-didesethylsunitinib azide using an amide linkage.

[0030] [Diagram 3] FIG. 3 is a reaction scheme showing the synthesis of a dendrimer conjugate (D-4517.2) in which N,N-didesethylsunitinib is conjugated to a dendrimer using an ether linkage for enhanced in vivo stability.

[0031] [Figure 4] FIG. 4 is a bar graph showing the percentage drug release by weight (0.0%-0.50%) of D-didesethylsunitinib conjugate, D-4517.2, in human, mouse, and rat plasma conditions at 4, 24, and 48 hour time points, respectively.

[0032] [Diagram 5] FIG. 5 is a reaction scheme showing the synthesis of a dendrimer-N-acetyl-L-cysteine ​​methyl ester conjugate.

[0033] [Figure 6] FIG. 6 is a reaction scheme showing the synthesis of a β-GalNAc-triantennary-PEG3-azide building block for conjugation to dendrimers.

[0034] [Figure 7] Figure 7 is a chromatograph showing an exemplary integration performed using Method A. Example calculation:

number

[0035] [Figure 8]FIG. 8 is a chromatograph showing an exemplary integration performed using Method A. Example calculation: Hk+Hl=3×Hm

number

[0036] [Figure 9] FIG. 9 is a calibration data plot for FID vs. ethanolamine (mg / mL) and FID vs. PEG-alkyne (mg / mL).

[0037] [Figure 10] FIG. 10 is a plot showing the average alkyne arm number versus PEG-alkyne loading for experiment DP07-55-1.

[0038] [Figure 11] FIG. 11 is a plot showing the average alkyne arm number versus PEG-alkyne loading for experiments AA08-85, AA08-88, and DP07-55-1.

[0039] [Figure 12] FIG. 12 is a plot showing the average alkyne arm number versus PEG-alkyne loading for experiments DP07-51 and DP07-60.

[0040] [Figure 13] FIG. 13 is a calibration data plot for FID vs. ethanolamine (mg / mL) and FID vs. PEG-alkyne (mg / mL).

[0041] [Figure 14A] Figures 14A-C are ASTRA reports for PAMAM G3.5 dendrimer. Concentration: 7.080 mg / mL. [Figure 14B] Figures 14A-C are ASTRA reports for PAMAM G3.5 dendrimer. Concentration: 7.080 mg / mL. [Figure 14C]Figures 14A-C are ASTRA reports for PAMAM G3.5 dendrimer. Concentration: 7.080 mg / mL.

[0042] [Figure 15A] Figures 15A-C are ASTRA reports for PAMAM G4. Concentration: 7.080 mg / mL. [Figure 15B] Figures 15A-C are ASTRA reports for PAMAM G4. Concentration: 7.080 mg / mL. [Figure 15C] Figures 15A-C are ASTRA reports for PAMAM G4. Concentration: 7.080 mg / mL.

[0043] [Figure 16A] Figures 16A-C are ASTRA reports for PAMAM G3.5. Concentration: 7.080 mg / mL. [Figure 16B] Figures 16A-C are ASTRA reports for PAMAM G3.5. Concentration: 7.080 mg / mL. [Figure 16C] Figures 16A-C are ASTRA reports for PAMAM G3.5. Concentration: 7.080 mg / mL.

[0044] [Figure 17] Figure 17 shows the 1H-NMR data for DP07-51-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=5.78 Hz, 248 H) 3.50 - 3.74 (m, 191 H) 4.20 (d, J=2.49 Hz, 27 H) 4.18 -4.20 (m, 1 H).

[0045] [Figure 18]Figure 18 is the 1H-NMR data for DP07-51-2. 1H NMR (300 MHz, methanol-d4) δ ppm 2.23 - 2.49 (m, 248 H) 3.61 (s, 194 H) 4.16 - 4.24 (m, 31 H).

[0046] [Figure 19] Figure 19 is the 1H-NMR data for DP07-51-3. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=5.78 Hz, 248 H) 3.50 - 3.74 (m, 191 H) 4.20 (d, J=2.49 Hz, 27 H) 4.18 -4.20 (m, 1 H).

[0047] [Figure 20] Figure 20 is the 1H-NMR data for DP07-51-4. 1H NMR (300 MHz, methanol-d4) δ ppm 2.23 - 2.49 (m, 248 H) 3.61 (s, 194 H) 4.16 - 4.24 (m, 31 H).

[0048] [Figure 21A] Figures 21A-C are the ASTRA report for DP07-51-1. [Figure 21B] Figures 21A-C are the ASTRA report for DP07-51-1. [Figure 21C] Figures 21A-C are the ASTRA report for DP07-51-1.

[0049] [Figure 22A] Figures 22A-C are the ASTRA report for DP07-51-2. [Figure 22B] Figures 22A-C are the ASTRA report for DP07-51-2. [Figure 22C]Figures 22A-C are the ASTRA report for DP07-51-2.

[0050] [Figure 23A] Figures 23A-C are the ASTRA report for DP07-51-3. [Figure 23B] Figures 23A-C are the ASTRA report for DP07-51-3. [Figure 23C] Figures 23A-C are the ASTRA report for DP07-51-3.

[0051] [Figure 24A] Figures 24A-C are the ASTRA report for DP07-51-4. [Figure 24B] Figures 24A-C are the ASTRA report for DP07-51-4. [Figure 24C] Figures 24A-C are the ASTRA report for DP07-51-4.

[0052] [Diagram 25] Figure 25 is the 1H-NMR data for DP07-60-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.25 - 2.49 (m, 248 H) 3.50 - 3.74 (m, 145 H) 4.20 (d, J=2.34 Hz, 6 H).

[0053] [Figure 26] Figure 26 is the 1H-NMR data for DP07-60-2. 1H NMR (300 MHz, methanol-d4) δ ppm 2.26 - 2.50 (m, 248 H) 3.61 (t, J=5.63 Hz, 150 H) 4.20 (d, J=2.49 Hz, 6 H).

[0054] [Figure 27]Figure 27 is 1H-NMR data for DP07-60-3. 1H NMR (300 MHz, methanol-d4) δ ppm 2.26 - 2.49 (m, 248 H) 3.61 (t, J=5.63 Hz, 143 H) 4.18 - 4.22 (m, 6 H).

[0055] [Figure 28] Figure 28 is the 1H-NMR data for DP07-60-4. 1H NMR (300 MHz, methanol-d4) δ ppm 2.28 - 2.48 (m, 248 H) 3.61 (t, J=5.63 Hz, 148 H) 4.18 - 4.22 (m, 7 H).

[0056] [Figure 29A] Figures 29A-C are the ASTRA report for DP07-60-1. [Figure 29B] Figures 29A-C are the ASTRA report for DP07-60-1. [Figure 29C] Figures 29A-C are the ASTRA report for DP07-60-1.

[0057] [Figure 30A] Figures 30A to 30C are the ASTRA report for DP07-60-2. [Figure 30B] Figures 30A to 30C are the ASTRA report for DP07-60-2. [Figure 30C] Figures 30A to 30C are the ASTRA report for DP07-60-2.

[0058] [Figure 31A] Figures 31A to 31C are the ASTRA report for DP07-60-3. [Figure 31B] Figures 31A to 31C are the ASTRA report for DP07-60-3. [Figure 31C]Figures 31A to 31C are the ASTRA report for DP07-60-3.

[0059] [Figure 32A] Figures 32A-C are the ASTRA report for DP07-60-4. [Figure 32B] Figures 32A-C are the ASTRA report for DP07-60-4. [Figure 32C] Figures 32A-C are the ASTRA report for DP07-60-4.

[0060] [Figure 33A] Figures 33A-C are the ASTRA report for DP07-74-1. [Figure 33B] Figures 33A-C are the ASTRA report for DP07-74-1. [Figure 33C] Figures 33A-C are the ASTRA report for DP07-74-1.

[0061] [Figure 34-1] Figure 34 is the 1H-NMR data for DP07-74-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.27 - 2.48 (m, 243 H) 2.53 - 2.67 (m, 121 H) 2.68 - 2.95 (m, 255 H) 3.31 (s, 46 H) 3.61 (t, J=5.71 Hz, 143 H) 4.20 (d, J=2.34 Hz, 6 H). [Figure 34-2] Same as above.

[0062] [Figure 35A] 35A-35B show the GC data for DP07-74-1. [Figure 35B] 35A-35B show the GC data for DP07-74-1.

[0063] [Figure 36A] 36A-36B show GC data for an IPA blank. [Figure 36B] 36A-36B show GC data for an IPA blank.

[0064] [Figure 37A] Figures 37A-C are the ASTRA report for DP07-68-1. [Figure 37B] Figures 37A-C are the ASTRA report for DP07-68-1. [Figure 37C] Figures 37A-C are the ASTRA report for DP07-68-1.

[0065] [Figure 38A] Figures 38A-B show 1H-NMR data for DP07-68-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=6.07 Hz, 248 H) 2.50 - 2.67 (m, 138 H) 2.71 - 2.87 (m, 241 H) 2.94 (s, 36 H) 3.18 - 3.44 (m, 39 H) 3.49 - 3.73 (m, 178 H) 4.20 (d, J=2.34 Hz, 21 H). [Figure 38B-1] Figures 38A-B show 1H-NMR data for DP07-68-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=6.07 Hz, 248 H) 2.50 - 2.67 (m, 138 H) 2.71 - 2.87 (m, 241 H) 2.94 (s, 36 H) 3.18 - 3.44 (m, 39 H) 3.49 - 3.73 (m, 178 H) 4.20 (d, J=2.34 Hz, 21 H). [Figure 38B-2] Same as above.

[0066] [Figure 39A]FIG. 39A-FIG. 39B are GC data for DP07-68-1. [Figure 39B] FIG. 39A-FIG. 39B are GC data for DP07-68-1.

[0067] [Diagram 40] Figure 40 is the 1H-NMR data for DP07-82-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.37 (br s, 248 H) 3.71 - 3.74 (m, 84 H) 6.05 - 6.08 (m, 198 H).

[0068] [Figure 41A] Figures 41A-C are the ASTRA report for DP07-82-1. [Figure 41B] Figures 41A-C are the ASTRA report for DP07-82-1. [Figure 41C] Figures 41A-C are the ASTRA report for DP07-82-1.

[0069] [Diagram 42] Figure 42 is the 1H-NMR data for DP07-82-1 alkyne. 1H NMR (300 MHz, methanol-d4) δ ppm 2.25 - 2.48 (m, 247 H) 2.36 - 2.36 (m, 5 H) 3.50 - 3.71 (m, 140 H) 3.60 - 3.61 (m, 3 H) 4.20 (d, J=2.49 Hz, 5 H) 4.20 - 4.20 (m, 1 H).

[0070] [Figure 43-1]Figure 43 is the 13C-NMR data for DP07-82-1. 13C NMR (75 MHz, methanol-d4) δ ppm 33.32 (s, 1 C) 41.61 (s, 1 C) 47.87 (s, 1 C) 49.80 (s, 1 C) 60.23 (s, 1 C) 173.75 (s, 1 C). [Figure 43-2] Same as above.

[0071] [Diagram 44] Figure 44 is the 1H-NMR data for DP07-85-1. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br s, 242 H) 6.04 - 6.08 (m, 218 H).

[0072] [Figure 45A] Figures 45A-C are the ASTRA report for DP07-85-1. [Figure 45B] Figures 45A-C are the ASTRA report for DP07-85-1. [Figure 45C] Figures 45A-C are the ASTRA report for DP07-85-1.

[0073] [Diagram 46] Figure 46 is the 1H-NMR data for DP07-85-1 alkyne. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=6.14 Hz, 244 H) 2.36 - 2.36 (m, 9 H) 3.49 - 3.73 (m, 167 H) 4.16 - 4.22 (m, 20 H).

[0074] [Figure 47A]Figures 47A-B show the C-NMR data for DP07-85-1. C NMR (75 MHz, methanol-d4) δ ppm 33.31 (s, 1 C) 41.63 (s, 1 C) 47.87 (s, 1 C) 60.23 (s, 1 C) 173.74 (s, 1 C). [Figure 47B] Figures 47A-B show the C-NMR data for DP07-85-1. C NMR (75 MHz, methanol-d4) δ ppm 33.31 (s, 1 C) 41.63 (s, 1 C) 47.87 (s, 1 C) 60.23 (s, 1 C) 173.74 (s, 1 C).

[0075] [Figure 48] Figure 48 is 1H-NMR data for DP07-82-1 (Lot No. DP07-82-2). 1H NMR (300 MHz, methanol-d4) δ ppm 2.37 (br s, 248 H) 3.71 - 3.74 (m, 84 H) 6.05 - 6.08 (m, 198 H).

[0076] [Figure 49A] Figures 49A to 49C are ASTRA reports for DP07-82-1 (lot number DP07-82-2). [Figure 49B] Figures 49A to 49C are ASTRA reports for DP07-82-1 (lot number DP07-82-2). [Figure 49C] Figures 49A to 49C are ASTRA reports for DP07-82-1 (lot number DP07-82-2).

[0077] [Figure 50]Figure 50 is the 1H-NMR data for DP07-82-1 (Lot No. DP07-82-2) alkyne. 1H NMR (300 MHz, methanol-d4) δ ppm 2.25 - 2.48 (m, 247 H) 2.36 - 2.36 (m, 5 H) 3.50 - 3.71 (m, 140 H) 3.60 - 3.61 (m, 3H) 4.20 (d, J=2.49 Hz, 5 H) 4.20 - 4.20 (m, 1 H).

[0078] [Figure 51A] Figures 51A-51B show the C-NMR data for DP07-82-1 (Lot No. DP07-82-2). C NMR (75 MHz, methanol-d4) δ ppm 33.32 (s, 1 C) 41.61 (s, 1 C) 47.87 (s, 1 C) 49.80 (s, 1 C) 60.23 (s, 1 C) 173.75 (s, 1 C). [Figure 51B] Figures 51A-51B show the C-NMR data for DP07-82-1 (Lot No. DP07-82-2). C NMR (75 MHz, methanol-d4) δ ppm 33.32 (s, 1 C) 41.61 (s, 1 C) 47.87 (s, 1 C) 49.80 (s, 1 C) 60.23 (s, 1 C) 173.75 (s, 1 C).

[0079] [Figure 52] Figure 52 is the 1H-NMR data for DP07-85-1 (Lot No. DP07-85-3). 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br s, 242 H) 6.04 - 6.08 (m, 218 H).

[0080] [Figure 53A] Figures 53A to 53C are ASTRA reports for DP07-85-1 (lot number DP07-85-3). [Figure 53B]Figures 53A to 53C are ASTRA reports for DP07-85-1 (lot number DP07-85-3). [Figure 53C] Figures 53A to 53C are ASTRA reports for DP07-85-1 (lot number DP07-85-3).

[0081] [Figure 54] Figure 54 is the 1H-NMR data for DP07-85-1 (Lot No. DP07-85-3) alkyne. 1H NMR (300 MHz, methanol-d4) δ ppm 2.38 (br t, J=6.14 Hz, 244 H) 2.36 - 2.36 (m, 9 H) 3.49 - 3.73 (m, 167 H) 4.16 - 4.22 (m, 20 H).

[0082] [Figure 55A] Figures 55A-55B are 13C-NMR data for DP07-85-1 (Lot No. DP07-85-3). 13C NMR (75 MHz, methanol-d4) δ ppm 33.31 (s, 1 C) 41.63 (s, 1 C) 47.87 (s, 1 C) 60.23 (s, 1 C) 173.74 (s, 1 C). [Figure 55B] Figures 55A-55B are 13C-NMR data for DP07-85-1 (Lot No. DP07-85-3). 13C NMR (75 MHz, methanol-d4) δ ppm 33.31 (s, 1 C) 41.63 (s, 1 C) 47.87 (s, 1 C) 60.23 (s, 1 C) 173.74 (s, 1 C).

[0083] [Figure 56A] Figures 56A-56C show results from the synthesis and characterization of dendrimers conjugated to the following PAK1 inhibitor: Frax-1036. [Figure 56B-1]Figures 56A-56C show results from the synthesis and characterization of dendrimers conjugated to the following PAK1 inhibitor: Frax-1036. [Figure 56B-2] Same as above. [Figure 56B-3] Same as above. [Figure 56C-1] Figures 56A-56C show results from the synthesis and characterization of dendrimers conjugated to the following PAK1 inhibitor: Frax-1036. [Figure 56C-2] Same as above. [Fig. 56C-3] Same as above.

[0084] [Figure 57A] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Figure 57B-1] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57B-2] Same as above. [Fig. 57C-1] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57C-2] Same as above. [Fig. 57D] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Figure 57E-1]Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Figure 57E-2] Same as above. [Figure 57E-3] Same as above. [Fig. 57F-1] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57F-2] Same as above. [Fig. 57F-3] Same as above. [Figure 57G-1] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Figure 57G-2] Same as above. [Fig. 57G-3] Same as above. [Figure 57G-4] Same as above. [Figure 57G-5] Same as above. [Fig. 57G-6] Same as above. [Fig. 57H-1] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57H-2] Same as above. [Fig. 57H-3] Same as above. [Fig. 57H-4] Same as above. [Fig. 57I-1] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57I-2] Same as above. [Fig. 57I-3] Same as above. [Fig. 57J] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57K-1] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57K-2] Same as above. [Fig. 57K-3] Same as above. [Figure 57L-1] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57L-2] Same as above. [Figure 57M] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57N] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57O-1] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57O-2] Same as above. [Fig. 57O-3] Same as above. [Fig. 57O-4] Same as above. [Figure 57P] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57Q] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57R] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57S] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57T-1] Figures 57A-57T show results from the synthesis and characterization of dendrimers conjugated to the following MEK inhibitors: selumetinib (Figures 57A-57D), trametinib (Figures 57E-57P), and cobimetinib (Figures 57Q-57T). [Fig. 57T-2] Same as above. [Fig. 57T-3] Same as above. [Fig. 57T-4] Same as above.

[0085] [Figure 58A]Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Figure 58B-1] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Fig. 58B-2] Same as above. [Figure 58C-1] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Fig. 58C-2] Same as above. [Fig. 58D-1] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Fig. 58D-2] Same as above. [Figure 58E-1] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Figure 58E-2] Same as above. [Figure 58E-3] Same as above. [Figure 58E-4] Same as above. [Figure 58E-5] Same as above. [Figure 58E-6] Same as above. [Fig. 58F] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Figure 58G-1] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Figure 58G-2] Same as above. [Fig. 58H] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Fig. 58I] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Fig. 58J-1] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Fig. 58J-2] Same as above. [Fig. 58J-3] Same as above. [Figure 58K-1]Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Figure 58K-2] Same as above. [Fig. 58K-3] Same as above. [Figure 58L-1] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Figure 58L-2] Same as above. [Figure 58M] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Figure 58N] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Fig. 58O] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Figure 58P-1]Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Figure 58P-2] Same as above. [Figure 58P-3] Same as above. [Figure 58Q] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Fig. 58R-1] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Fig. 58R-2] Same as above. [Fig. 58S-1] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). [Fig. 58S-2] Same as above. [Fig. 58T] Figures 58A-58T show results from the synthesis and characterization of dendrimers conjugated to the following receptor tyrosine kinase inhibitors: dasatinib (Figures 58A-58H), bemcentinib (Figures 58I-58N), duvelmatinib (Figures 58O-58P), and cabozantinib (Figures 58Q-58T). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] Detailed Description Among other aspects, the present disclosure provides dendrimer conjugates, compositions comprising dendrimer conjugates, and methods of using the dendrimer conjugates and compositions thereof. In some embodiments, the dendrimer conjugates comprise a dendrimer conjugated to at least one agent. In some embodiments, the dendrimer conjugates comprise one or more agents useful in therapy, imaging, and / or targeted delivery.

[0087] In some aspects, the present disclosure relates to therapeutic compounds comprising dendrimers conjugated to therapeutic agents. The inventors have recognized and understood that certain therapeutic agents with adverse in vivo profiles may be modified by conjugation to dendrimers to provide therapeutic compounds exhibiting reduced off-target toxicity, more selective uptake, and sustained intracellular effects. The inventors have further recognized and understood that such therapeutic compounds are highly tunable in the dendrimer moiety, and thus the hydrophilicity of the therapeutic compound may be tailored to enable targeted delivery of certain therapeutic agents to biological targets that would otherwise be difficult to reach by the therapeutic agent. In another aspect, a method of synthesizing a functionalized dendrimer of formula (IA) is provided, comprising: reacting a dendrimer of formula (II-A) with one or more amines (wherein each amine is of the formula HNR) under conditions suitable to form a functionalized dendrimer of formula (IA). 1 In another aspect, there is provided a functionalized dendrimer of formula (IA) synthesized by the methods described herein.

[0088] In some aspects, the present disclosure provides a method of treating or imaging a disease or disorder in a subject in need thereof, comprising administering to the subject a composition comprising a dendrimer conjugate as described herein. In some embodiments, the dendrimer conjugate of the present disclosure can target reactive immune cells in the absence of any targeting moiety. For example, in some embodiments, the dendrimer conjugate described herein can cross the blood-brain barrier in the central nervous system of a subject, where the dendrimer conjugate is selectively taken up by activated microglia and / or activated macrophages. In some embodiments, the dendrimer conjugate described herein can cross the blood-retina barrier in the eye of a subject, where the dendrimer conjugate is selectively taken up by activated microglia and / or activated macrophages. Dendrimer Conjugates

[0089] In some embodiments, dendrimer conjugates refer to dendrimers conjugated to at least one agent, as described herein. In some embodiments, the dendrimer is covalently conjugated (e.g., covalently bonded) to at least one agent. In some embodiments, dendrimer conjugates include dendrimers that can be described as having a molecular structure with 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, with the outermost generation having terminal functional groups.

[0090] In some embodiments, the terminal functional group of the dendrimer comprises one or more hydroxyl groups, one or more amine groups, and / or one or more carboxyl groups. In some embodiments, the terminal functional group of the dendrimer provides a binding site for at least one agent to be conjugated to form a dendrimer conjugate. Thus, in some embodiments, at least one agent is conjugated to the dendrimer through an ether bond, an amide bond, or an ester bond formed by conjugation to the terminal functional group of the dendrimer. In some embodiments, at least one agent is conjugated to the dendrimer through an ether bond or an amide bond. In some embodiments, at least one agent is conjugated to the dendrimer through an ether bond.

[0091] In some embodiments, the number of terminal sites on a dendrimer may depend on a particular dendrimer scaffold and its generation.For example, in some embodiments, a dendrimer is based on a PAMAM dendrimer scaffold of generation 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, which scaffolds have 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096 terminal sites, respectively.However, it should be understood that different dendrimer scaffolds with different numbers of terminal sites in each generation can be used according to the present disclosure.

[0092] In some embodiments, all terminal sites of the dendrimer include hydroxy groups. In some embodiments, each terminal site of the dendrimer includes either a hydroxy group or an amine group. In some embodiments, each terminal site of the dendrimer conjugate includes either a hydroxy group, an amine group, or an agent conjugated to the dendrimer via an ether or amide bond. In some embodiments, each terminal site of the dendrimer conjugate includes either a hydroxy group, or an agent conjugated to the dendrimer via an ether bond.

[0093] In some embodiments, at least 50% of the terminal sites on the dendrimer conjugate comprise hydroxy groups (e.g., at least 50% of the terminal sites comprise neither an amine group nor an agent). For example, in some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the terminal sites on the dendrimer conjugate comprise hydroxy groups. In some embodiments, about 50-99%, about 60-99%, about 70-99%, about 80-99%, about 90-99%, about 95-99%, about 98-99%, about 70-95%, about 70-90%, about 80-95%, or about 80-90% of the terminal sites on the dendrimer conjugate comprise hydroxy groups.

[0094] In some embodiments, one or more terminal sites on the dendrimer conjugate comprise an agent. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or more terminal sites on the dendrimer conjugate comprise an agent. In some embodiments, at least 1% of the terminal sites on the dendrimer conjugate comprise an agent. For example, in some embodiments, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% of the terminal sites on the dendrimer conjugate comprise an agent. In some embodiments, about 1-50%, about 1-40%, about 1-25%, about 1-10%, about 5-50%, about 5-40%, about 5-25%, about 5-10%, about 10-50%, about 10-40%, or about 10-25% of the terminal sites on the dendrimer conjugate comprise an agent. In some embodiments, about 1%, about 2%, about 3%, about 4%, or about 5% of the terminal sites on the dendrimer comprise an agent. In some embodiments, less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75% of the terminal sites on the dendrimer comprise an agent. In some embodiments, the dendrimer conjugate has an effective amount of terminal functional groups (e.g., terminal hydroxy groups) to target a particular cell type while at the same time having an effective amount of agent to treat and / or image as described herein. In some embodiments, the terminal sites of the dendrimer conjugate are capable of measuring the proton nuclear magnetic resonance ( 1 The percentage of terminal sites bearing a drug and / or terminal functional group can be assessed using 1 H NMR, or other analytical methods known in the art to determine the percentage of terminal sites bearing a drug and / or terminal functional group.

[0095] In some embodiments, the desired drug loading may depend on certain factors, including the choice of drug, the structure and size of the dendrimer, and the cell or tissue to be treated. In some embodiments, the dendrimer conjugate (e.g., therapeutic compound) is about 0.01% to about 45% (m / m) by weight of the drug (e.g., therapeutic agent). In some embodiments, the dendrimer conjugate (e.g., therapeutic compound) is about 10% to about 20% by weight of the drug (e.g., therapeutic agent). In some embodiments, the dendrimer conjugate is 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, about 3% to about 10% by weight of the drug.

[0096] As described herein, in some embodiments, the dendrimer conjugates can be characterized in terms of the mass percentage of the drug (e.g., mass (m / m)%). In some embodiments, the mass percentage refers to the molecular weight (Da) percentage of the drug in the dendrimer conjugate. In some embodiments, the mass percentage can be expressed by the general formula: (M of drug) W ) / (M of the conjugate W ) × 100. For example, in some embodiments, the M W ) can be determined by calculating or estimating the molecular weight of the agent as a single molecule or compound (conjugated or unconjugated) and multiplying this value by the number of terminal sites of the dendrimer conjugate in which the agent is present. In some embodiments, (the M W ) can be determined by calculating or estimating the sum of the atomic masses of all the atoms that form the drug of the dendrimer conjugate. W ) is the total molecular weight of the dendrimer conjugate (the M W) and multiplied by 100 to provide the mass percentage. In some embodiments, the mass percentage can be determined by experimental or empirical means. For example, in some embodiments, the mass percentage can be determined by proton nuclear magnetic resonance ( 1 1 H NMR) or other analytical methods known in the art.

[0097] In some embodiments, the dendrimer has a diameter between about 1 nm and about 50 nm. For example, in some embodiments, the diameter is 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. In some embodiments, the diameter is between about 1 nm and about 2 nm. In some embodiments, dendrimers conjugated to relatively large agents (e.g., large proteins, e.g., antibodies) can have diameters that are approximately 5-15 nm greater than these values ​​compared to unconjugated dendrimers. In some embodiments, the dendrimer has a molecular weight between about 500 Daltons (Da) and about 100,000 Da (e.g., between about 500 Da and about 50,000 Da, or between about 1,000 Da and about 20,000 Da).

[0098] In some embodiments, the dendrimer of the conjugates described herein is a poly(amidoamine) (PAMAM) dendrimer, a polypropylamine (POPAM) dendrimer, a 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) dendrimer, a polyethyleneimine dendrimer, a polylysine dendrimer, a polyester dendrimer, an iptycene dendrimer, an aliphatic poly(ether) dendrimer, an aromatic polyether dendrimer, or a combination thereof.

[0099] In some embodiments, the dendrimer conjugate comprises a PAMAM dendrimer. In some embodiments, the PAMAM dendrimer comprises a different core with amidoamine building blocks. In some embodiments, the PAMAM dendrimer comprises any generation of carboxylic acid, amine, and / or hydroxyl end groups, including but not limited to generation 1, generation 2, generation 3, generation 4, generation 5, generation 6, generation 7, generation 8, generation 9, or generation 10 PAMAM dendrimers. In some embodiments, the PAMAM dendrimer is a generation 4, generation 5, generation 6, generation 7, or generation 8 hydroxyl-terminated PAMAM dendrimer.

[0100] In some embodiments, the dendrimer comprises multiple hydroxy groups. Some exemplary high density hydroxy 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, generation 4), dendritic polyglycerol. In some embodiments, the high density hydroxy group-containing dendrimers are oligoethylene glycol (OEG)-like dendrimers. For example, generation 2 OEG dendrimers (D2-OH-60) can be synthesized using highly efficient, robust, atom-economical chemical reactions, such as Cu(I)-catalyzed alkyne-azide click and photocatalyzed thiol-ene click chemistry. Very low generation, very high density polyol dendrimers with minimal reaction steps can be achieved by using an orthogonal hypermonomer and hypercore strategy, as described, for example, in WO2019094952. In some embodiments, the dendrimer backbone has non-cleavable polyether linkages throughout the structure (e.g., is non-biodegradable) to avoid degradation of the dendrimer in vivo and allow such dendrimers to be eliminated from the body as a single entity.

[0101] In some embodiments, the dendrimer conjugate comprises a dendrimer conjugated to one or more therapeutic agents, one or more imaging agents, and / or one or more targeting agents. In some embodiments, it should be understood that "at least one" agent, "one or more" agents, and similar terms refer to a particular agent, and not necessarily the amount of a particular agent conjugated to the dendrimer. For example, in some embodiments, a dendrimer conjugate comprising two agents refers to a dendrimer having a first agent at one or more terminal positions and a second agent at one or more different terminal positions, where the first and second agents are different (e.g., chemically different). In some embodiments, the first and second agents may be useful for similar purposes (e.g., both agents are therapeutic agents), or the first and second agents may be useful for different purposes (e.g., the first agent is a therapeutic agent and the second agent is a targeting agent). The first and second agents, when used for a similar purpose, can be chemically different and thus provide different functions, e.g., different therapeutic agents that target different receptors or biological pathways, or different imaging agents with different spectral properties.

[0102] In some embodiments, the agent (e.g., therapeutic agent, imaging agent, targeting agent) of the dendrimer conjugate is a peptide, a protein, a sugar, a carbohydrate, an oligonucleotide, a nucleic acid, a lipid, a small molecule compound, or a combination thereof. In some embodiments, the agent is an antibody or an antigen-binding fragment of an antibody. In some embodiments, the agent is a nucleic acid or an oligonucleotide that codes for a protein, such as a DNA expression vector or an mRNA. In some embodiments, the agent is an RNA silencing agent, such as an siRNA, shRNA, or microRNA.

[0103] In some embodiments, the drug is a small molecule compound, such as a small molecule organic compound, organometallic compound, or inorganic compound. In some embodiments, the drug is a small molecule compound having a molecular weight of less than 2,000 Daltons (Da), less than 1,500 Da, less than 1,000 Da, or less than 500 Da. In some embodiments, the drug is a small molecule compound having a molecular weight of between about 100 and about 2,000 Da. For example, in some embodiments, the small molecule compound has a molecular weight of between about 100 and about 1,500 Da, between about 100 and about 1,000 Da, between about 500 and about 2,000 Da, or between about 300 and about 700 Da.

[0104] In some aspects, the non-releasable form of the dendrimer conjugates described herein provides enhanced therapeutic efficacy compared to the releasable form of the same conjugate.Thus, in some embodiments, the drug is conjugated to the dendrimer via a linker that is non-releasably linked to the dendrimer and the drug (e.g., by an ether bond and / or an amide bond).In some embodiments, the linker has a composition that is minimally releasable (e.g., minimally cleavable) under physiological conditions.

[0105] In some embodiments, the dendrimer is conjugated to the drug via a covalent bond that is stable under in vivo conditions. In some embodiments, the covalent bond is minimally cleavable when administered to a subject and / or excreted intact from the body. For example, in some embodiments, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, or less than 0.1% of the total dendrimer conjugate has a drug that is cleaved within 24 hours, or within 48 hours, or within 72 hours after in vivo administration to a subject. In some embodiments, the covalent bond comprises an ether bond. In some embodiments, the covalent bond between the dendrimer and the drug is not a hydrolytically or enzymatically cleavable bond, such as an ester bond.

[0106] In some aspects, the present disclosure provides a dendrimer conjugate of formula (I): [ka] [wherein D is a dendrimer, X is O or NH, and Y 1 is the first group, and Y 2 is a second group, Z is an agent, L is a linker, m is an integer between 16 and 4096, inclusive, and n is an integer between 1 and 100, inclusive.

[0107] In some embodiments, D is a dendrimer selected from the group consisting of a poly(amidoamine) (PAMAM) polymer, a polypropylamine (POPAM) polymer, a polyethyleneimine polymer, a polylysine polymer, a polyester polymer, an iptycene polymer, an aliphatic poly(ether) polymer, an aromatic polyether polymer, a 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymer, and combinations thereof.

[0108] In some embodiments, Y 1 is non-hydrolyzable under physiological conditions. 1 is an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, or a covalent bond. 1 is replaced as necessary 1~20 In some embodiments, Y is an alkylene. 1 is unsubstituted C 1~10 It is alkylene.

[0109] In some embodiments, Y 1 is an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, or a covalent bond. 1 is an optionally substituted alkylene (e.g., optionally substituted C 1~20In some embodiments, Y is 1 is optionally substituted ethylene. In some embodiments, Y 1 is optionally substituted methylene. In some embodiments, Y 1 is the formula [ka] wherein q is 1, 2, 3, 4, 5, or 6 (e.g., q is 1, 2, or 3). In some embodiments, q is 2. In some embodiments, q is 1. In some embodiments, Y 1 is an unsubstituted alkylene (e.g., unsubstituted C 1~10 In some embodiments, Y is 1 is the formula [ka] It is of the following.

[0110] In some embodiments, Y 2 is selected from the group consisting of secondary amides, tertiary amides, sulfonamides, secondary carbamates, tertiary carbamates, carbonates, ureas, carbinols, disulfides, hydrazones, hydrazides, ethers, carbonyls, and combinations thereof. 2 -CONH-, -CONR A -,-SO2NR A -, -OCONH-, -NHCOO-, -OCONR A -, -NR A COO-, -OC(=O)O-, -NHCONH-, -NR A CONH-, -NHCONR A -,-NRCONR A -, -CHOH-, -CR A OH-, -C(=O)-, and -C(=O)R A -, R A is an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heterocyclic group.

[0111] In some embodiments, Y 2 In some embodiments, Y comprises a polymer. 2 is an alkylene chain (e.g., C 1~100,000 alkylene), the chain being the shortest path between L and Z excluding hydrogen atoms and substituents. 2 The chain of Y contains 5,000-7,000, 7,000-9,000, 9,000-10,000, 10,000-12,000, 100,000-120,000, 120,000-150,000, or up to 150,000-200,000 consecutive covalently bonded atoms or lengths, excluding hydrogen atoms and substituents. 2 is the shortest path between L and Z, excluding hydrogen atoms and substituents, and is an all-carbon substituted or unsubstituted C 1~200,000 is a hydrocarbon chain. In some embodiments, Y 2 Any of the atoms in may be substituted. 2 None of the atoms in Y are substituted. 2 None of the carbon atoms in Y are substituted. 2 At least one chain atom of the hydrocarbon chain is independently selected from -C(=O)-, -O-, -NR b -, -S-, or a cyclic moiety; R b are independently hydrogen, substituted or unsubstituted C 1~6 In some embodiments, Y is an alkyl, or a nitrogen protecting group. 2 At least one chain atom of the hydrocarbon chain of is independently replaced with an amide, hydroxamate, ether, n-alkyl, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane, oxime, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, and / or optionally substituted heteroarylene. 2At least one chain atom of the hydrocarbon chain of is independently replaced with an amide, hydroxamate, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane, and / or oxime. 2 is an alkylene moiety (e.g., of the formula [ka] [wherein q is an integer between 1 and 100, inclusive]. In some embodiments, Y 2 is a polyethylene glycol moiety (e.g., [ka] [wherein q is an integer between 1 and 100, inclusive]. In some embodiments, Y 2 comprises an alkylene or polyethylene glycol moiety and a hydrocarbon chain. 2 At least one chain atom of the hydrocarbon chain is independently replaced with an amide, hydroxamate, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane, and / or oxime.

[0112] In some embodiments, Z is a therapeutic agent, an imaging agent, or a targeting agent as described herein. In some embodiments, Z is a therapeutic agent or an imaging agent. In some embodiments, the dendrimer conjugate of formula (I) further comprises at least one targeting agent conjugated to the dendrimer.

[0113] In some embodiments, Z comprises a PAK1 inhibitor. In some embodiments, Z comprises Frax-1036. In some embodiments, Z comprises a compound of the formula [ka] It is of the following.

[0114] In some embodiments, Z comprises a MEK inhibitor. In some embodiments, Z comprises selumetinib. In some embodiments, Z comprises a compound of the formula [ka] In some embodiments, Z comprises trametinib. In some embodiments, Z is of the formula [ka] In some embodiments, Z comprises cobimetinib. In some embodiments, Z is of the formula [ka] In some embodiments, Z is of the formula [ka] In some embodiments, Z is of the formula [ka] It is of the following.

[0115] In some embodiments, Z comprises a receptor tyrosine kinase inhibitor. In some embodiments, Z comprises dasatinib. In some embodiments, Z comprises the formula [ka] In some embodiments, Z comprises bemcentinib (R428). In some embodiments, Z is of the formula [ka] In some embodiments, Z comprises duvelmatinib (TP-0903). In some embodiments, Z is of the formula [ka] In some embodiments, Z comprises cabozantinib. In some embodiments, Z is of the formula [ka] It is of the following.

[0116] In some embodiments, L is a linker comprising a polymer and at least one moiety. In some embodiments, the polymer is a polymeric polyol, a polypeptide, or an unsubstituted alkyl chain. In some embodiments, the polymer is a polymeric polyol selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, and polyvinyl alcohol. In some embodiments, the polymer is a polypeptide having at least two amino acids. In some embodiments, the polymer is a polypeptide having between about 2 and about 40 amino acids (e.g., 2-25, 5-30, 10-25, or 5-15 amino acids). In some embodiments, the polymer is an unsubstituted alkyl chain. In some embodiments, the polymer is an unsubstituted C 2~50 In some embodiments, the polymer is an unsubstituted C 2~30 In some embodiments, the polymer is an unsubstituted C 5~25 is an alkyl chain. In some embodiments, the polymer is a polymer as described elsewhere herein.

[0117] In some embodiments, at least one moiety of L is a moiety resulting from a click reaction. In some embodiments, at least one moiety is a 5-membered heterocyclic ring resulting from an electrocyclic reaction (e.g., a 3+2 cycloaddition, or a 4+2 cycloaddition) between a reactive click chemistry handle (e.g., an azide and a terminal or strained alkyne, a diene and a dienophile, a thiol and an alkene) used to generate the conjugate. In some embodiments, at least one moiety is a diradical comprising 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, or 1,4-dihydropyridazyl.

[0118] In some embodiments, L is of the formula [ka] and a hydrocarbon chain of the formula: wherein q is an integer between 1 and 100, inclusive. In some embodiments, at least one chain atom of the hydrocarbon chain of L is independently replaced with an amide, hydroxamate, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane, and / or oxime. In some embodiments, q is an integer between 1 and 50, inclusive. In some embodiments, q is an integer between 1 and 10, inclusive. In some embodiments, q is an integer between 1 and 8, inclusive. In some embodiments, L is a group of the formula: [ka] wherein M is a diradical comprising 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, or 1,4-dihydropyridazyl, and q is an integer between 1 and 100, inclusive. In some embodiments, q is an integer between 1 and 50, inclusive. In some embodiments, q is an integer between 1 and 10, inclusive. In some embodiments, q is an integer between 1 and 8, inclusive. In some embodiments, L is of the formula [ka] where q is an integer between 1 and 100, inclusive. In some embodiments, q is an integer between 1 and 50, inclusive. In some embodiments, q is an integer between 1 and 10, inclusive. In some embodiments, q is an integer between 1 and 8, inclusive.

[0119] In some aspects, the present disclosure provides a dendrimer conjugate of formula (II) [ka] [In the formula, each example of D, m, and n, each example of X, Y 1 Each example of, and Y 2 Each instance of is independently as defined for formula (I); 1 and L 2 are independently a linker as defined for formula (I), Z 1 and Z 2 are different drugs.

[0120] In some embodiments, Z 1 and Z 2 is independently a therapeutic agent, a targeting agent, or an imaging agent, with the proviso that Z 1 and Z 2 are different (e.g., chemically distinct). 1 and Z 2 are different therapeutic agents. 1 and Z 2 are different therapeutic agents that target different biological pathways involved in a common pathology. 1 and Z 2 are different therapeutic agents, and the dendrimer conjugate of formula (II) further comprises at least one targeting agent conjugated to the dendrimer. 1 and Z 2 are different imaging agents. 1 is a therapeutic agent, and Z 2 is a targeting agent. 1 is the contrast agent, Z 2 is a targeting agent. Synthesis method

[0121] In some aspects, the present disclosure relates to the discovery of new techniques for synthesizing dendrimers (e.g., functionalized dendrimers, e.g., dendrimers functionalized with a specific number of PEG-alkynes that allow for further introduction of drugs via click conjugation chemistry, e.g., azide-alkyne cycloaddition). Advantageously, the methods and compositions provided herein can enable large, multigram-scale synthesis (e.g., greater than 10 grams, greater than 40-50 grams, greater than 100 grams) of dendrimers (e.g., dendrimers functionalized with a specific number of PEG-alkynes) with high product uniformity (low polydispersity, e.g., less than 1.05) using milder conditions with tailored stoichiometry relative to past techniques.

[0122] In some aspects, the present disclosure provides a functionalized dendrimer of formula (IA) [ka] [In the formula, D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; X is NH; Y 1 is an optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, or a covalent bond; m is an integer between 16 and 4096, inclusive; and n is an integer from 1 to 100, inclusive, Dendrimer of formula (II-A) [ka] [In the formula, D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; t is an integer between 16 and 4096, inclusive. with one or more amines (wherein each amine is of the formula HNR 1 is an amine of R 1 is an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, or a covalent bond. The method includes reacting a compound represented by the formula (I) with a compound represented by the formula (I).

[0123] In some embodiments, in the dendrimer of formula (IA) or (II-A), D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof.

[0124] In some embodiments, Y 1 is an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, or a covalent bond. 1 is an optionally substituted alkylene (e.g., optionally substituted C 1~20 In some embodiments, X is -NH and Y 1is optionally substituted ethyl. In some embodiments, X is -NH and Y 1 is ethyl substituted with a PEG-alkyne group. In some embodiments, X is -NH and Y 1 is the formula [ka] wherein q is 1, 2, 3, 4, 5, or 6 (e.g., q is 1, 2, or 3). In some embodiments, X is -NH and Y 1 is the formula [ka]

[0023] wherein q is 2. In some embodiments, Y 1 is an unsubstituted alkylene (e.g., unsubstituted C 1~10 alkylene).

[0125] In some embodiments, m is an integer between 16 and 4096, inclusive. In some embodiments, m is an integer between 16 and 20, 20 and 40, 40 and 50, 50 and 60, 60 and 70, 70 and 80, 80 and 90, 90 and 100, 100 and 110, 110 and 120, 120 and 130, 130 and 140, 140 and 160, 160 and 180, 180 and 200, 200 and 250, 250 and 300, 300 and 350, 350 and 400, 400 and 450, 450 and 500, inclusive. , 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4100, or 4100-4200. In some embodiments, m is about 61 or 54 (e.g., 61 or 54).

[0126] In some embodiments, n is an integer from 1 to 100, inclusive. In some embodiments, n is an integer from 1 to 3, 3 to 5, 1 to 5, 5 to 7, 7 to 9, 9 to 10, 10 to 12, 12 to 14, 14 to 16, 16 to 18, 18 to 20, 20 to 22, 22 to 24, 24 to 26, 26 to 28, 28 to 30, 30 to 32, 32 to 34, 34 to 36, 36 to 38, 38 to 40, 40 to 42, 42 to 44, 44 to 46, 46 to 48, 48 to 5, inclusive. In some embodiments, n is an integer between 0, 50 and 52, 52 and 54, 54 and 56, 56 and 58, 58 and 60, 60 and 62, 62 and 64, 64 and 66, 66 and 68, 68 and 70, 70 and 72, 72 and 74, 74 and 76, 76 and 78, 78 and 80, 80 and 82, 82 and 84, 84 and 86, 86 and 88, 88 and 90, 90 and 92, 92 and 94, 94 and 96, 96 and 98, or 98 and 100. In some embodiments, n is about 3 or 10 (e.g., 3 or 10). In some embodiments, m is 64 minus n. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In some embodiments, n is between about 1-2, 2-4, 4-5, 5-6, 6-8, 8-10, or 10-12, or between about 2-11 (e.g., 3, 4, 5, 8, 10). In some embodiments, n is about 3 or 10 (e.g., 3 or 10). In some embodiments, n is 3 and m is 61. In some embodiments, n is 10 and m is 54. In some embodiments, m is about 61 or 54 (e.g., 61 or 54).

[0127] In some embodiments, in the synthetic methods described herein, a dendrimer of formula (II-A) [ka] wherein D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; t is an integer between 16 and 4096, inclusive. one or more amines, where each amine has the formula HNR 1 is an amine of R 1 is an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, or a covalent bond. is provided.

[0128] In some embodiments, in formula (II-A), D is a dendrimer as described herein. In some embodiments, the synthesis method includes reacting a dendrimer of formula (II-A), or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, or isotopically enriched form (e.g., isotopically labeled derivative) thereof. Formula (II-A) includes a substituent t. In some embodiments, t is an integer between 16 and 4096, inclusive. In some embodiments, t is 16 to 20, 20 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 160, 160 to 180, 180 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, inclusive. , 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4100, or 4100-4200. In some embodiments, n is t. In some embodiments, t is n. In some embodiments, in the dendrimer of formula (II-A), t is the same as n. In some embodiments, in the dendrimer of formula (II-A), t is 64. In some embodiments, t is an integer between 20 and 40, between 40 and 50, between 50 and 60, between 60 and 70, or between 70 and 80, inclusive.

[0129] In some embodiments, the synthesis method comprises reacting a dendrimer of formula (II-A) with one or more amines. In some embodiments, the synthesis method comprises reacting a dendrimer of formula (II-A) with one or more amines, wherein at least one instance of the one or more amines is a compound of formula (Z) [ka] or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, isotopically enriched form (e.g., isotopically labeled derivative) thereof, wherein L A is the linker, R 1A is halogen, optionally substituted acyl, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted acetylene, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, -CH(=N)(OH)R D1 , -CN, -NO2, -OR D1 , -N(R D1a )2, -SO2OR D1 , or -SR D1 and R D1 and R D1a is as defined herein. In some embodiments, the linker L A In some embodiments, the linker L A is an alkylene chain (e.g., C 1~100,000 alkylene), the chain is made up of -NH2 and R excluding hydrogen atoms and substituents. 1A In certain embodiments, the linker L A The chain of L comprises 5,000-7,000, 7,000-9,000, 9,000-10,000, 10,000-12,000, 100,000-120,000, 120,000-150,000, or up to 150,000-200,000 consecutive covalently bonded atoms or lengths, excluding hydrogen atoms and substituents. A -NH2 and R excluding hydrogen atoms and substituents 1A All carbon substituted or unsubstituted C as the shortest path between 1~200,000 In certain embodiments, L A Any of the atoms in may be substituted. In certain embodiments, the linker L AIn certain embodiments, none of the atoms in the linker are substituted. In certain embodiments, none of the carbon atoms in the linker are substituted. A At least one chain atom of the hydrocarbon chain is independently selected from -C(=O)-, -O-, -NR b -, -S-, or a cyclic moiety; R b are independently hydrogen, substituted or unsubstituted C 1~6 In certain embodiments, L is an alkyl, or a nitrogen protecting group. A At least one chain atom of the hydrocarbon chain of is independently replaced with an amide, hydroxamate, ether, n-alkyl, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane, oxime, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, and / or optionally substituted heteroarylene. In certain embodiments, L A At least one chain atom of the hydrocarbon chain of is independently replaced with an amide, hydroxamate, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane, and / or oxime. In certain embodiments, the linker L A is a polyethylene glycol moiety (e.g., [ka] [wherein q is an integer between 1 and 100,000 inclusive], L A At least one chain atom of the hydrocarbon chain of is independently replaced with an amide, hydroxamate, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane, and / or oxime. In certain embodiments, the linker L Acomprises a moiety resulting from a click reaction. In some embodiments, at least one moiety resulting from a click reaction is a 5-membered heterocyclic ring resulting from an electrocyclic reaction (e.g., a 3+2 cycloaddition, or a 4+2 cycloaddition) between the reactive click chemistry handles (e.g., an azide and a terminal or strained alkyne, a diene and a dienophile, a thiol and an alkene) used to generate the conjugate. In some embodiments, at least one moiety resulting from a click reaction is a diradical comprising 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, or 1,4-dihydropyridazyl. In certain embodiments, the linker L A is the formula [ka] [In the formula, l n represents a bond to -NH2 in the compound of formula (Z), and l1 represents R 1A In some embodiments, the synthesis method comprises reacting a dendrimer of formula (II-A) with one or more amines, wherein at least one instance of the one or more amines is a compound of formula (A): [ka] or a salt, solvate, hydrate, stereoisomer, polymorph, tautomer, isotopically enriched form (e.g., isotopically labeled derivative) thereof, wherein p, q, r, W, and R 1A is as defined herein. In some embodiments, the synthesis method comprises reacting a dendrimer of formula (II-A) with one or more amines, wherein at least one instance of the one or more amines is a compound of formula (A): [ka] [In the formula, R 1Ais halogen, optionally substituted acyl, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted acetylene, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, -CH(=N)(OH)R D1 , -CN, -NO2, -OR D1 , -N(R D1a )2, -SO2OR D1 , or -SR D1 and R D1 are independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or, if attached to an oxygen atom, an oxygen protecting group, or, if attached to a sulfur atom, a sulfur protecting group; R D1a each occurrence is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group, or optionally R D1a two instances of, taken together with their intervening atoms, form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaryl ring, W is -O- or -CH2-, if valence allows, p is 0, 1, 2, or 3, q ​​is an integer from 1 to 100,000, inclusive, and r is 0, 1, 2, 3, 4, 5, or 6. 1A is halogen (e.g., F, Cl, Br, or I). In certain embodiments, R 1Ais optionally substituted acyl (e.g., -C(=O)Me). In certain embodiments, R 1A is an optionally substituted alkyl (e.g., substituted or unsubstituted C 1~6 In certain embodiments, R 1A is substituted or unsubstituted methyl. In certain embodiments, R 1A is substituted or unsubstituted ethyl. In certain embodiments, R 1A is substituted or unsubstituted propyl. In certain embodiments, R 1A is an optionally substituted alkenyl (e.g., substituted or unsubstituted C 2~6 alkenyl). In certain embodiments, R 1A is an optionally substituted alkynyl (e.g., substituted or unsubstituted C 2~6 alkynyl). In certain embodiments, R 1A is an optionally substituted carbocyclyl (e.g., a substituted or unsubstituted 3- to 7-membered monocyclic carbocyclyl containing 0, 1, or 2 double bonds in the carbocyclic ring system). 1A is an optionally substituted heterocyclyl (e.g., a substituted or unsubstituted 5-10 membered monocyclic or bicyclic heterocyclic ring, where one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). 1A is optionally substituted aryl (e.g., substituted or unsubstituted 6-10 membered aryl). In certain embodiments, R 1A is benzyl. In certain embodiments, R 1A is substituted or unsubstituted phenyl. In certain embodiments, R 1Ais an optionally substituted heteroaryl (e.g., a substituted or unsubstituted 5-6 membered monocyclic heteroaryl where 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur, or a substituted or unsubstituted 9-10 membered bicyclic heteroaryl where 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, R 1A is -CH(=N)(OH)R D1 , -CN, -NO2, -OR D1 , -N(R D1a )2, -SO2OR D1 , or -SR D1 and R D1 and R D1a is as defined herein. In certain embodiments, R 1A is -CH(=N)(OH)R D1 (e.g., -CH(=N)(OH) (optionally substituted C 1~6 In certain embodiments, R 1A is -CH(=N)(OH) (C optionally substituted with a polyethylene glycol linker) 1~6 In certain embodiments, R 1A is -CN. In certain embodiments, R 1A is -NO2. In certain embodiments, R 1A -OR D1 (e.g., -OH or -OMe). In certain embodiments, R 1A is -N(R D1a )2 (e.g., -NMe2). In certain embodiments, R 1A -SO2OR D1 (e.g., -SO(optionally substituted alkyl)). In certain embodiments, R 1A -SR D1 (e.g., -SMe). In certain embodiments, R 1A is -CH(=N)(OH)R D1 , -CN, -NO2, -OR D1 , -N(RD1a )2, -SO2OR D1 , or -SR D1 and R D1 are independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or, if attached to an oxygen atom, an oxygen protecting group, or, if attached to a sulfur atom, a sulfur protecting group; R D1a each occurrence is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group, or optionally R D1a Two instances of, taken together with their intervening atoms, form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaryl ring. In certain embodiments, R 1A is bromo, alkyne, acetylene, alkene, aldehyde, amine, COOH, hydroxyl, carboxyl (e.g., dibenzocyclooctyne or DBCO), thiol, sulfonate, or -CN. 1A is a click reaction handle partner (e.g., a click chemistry handle in Table A or shown in Schemes 1-19 of Example 6). In certain embodiments, click handle, click partner, and click reaction handle partner are used interchangeably. In certain embodiments, the click reaction is Cu(I)-catalyzed alkyne-azide click and photocatalyzed thiol-ene click chemistry, or a cyclic electrochemical click reaction (e.g., a 3+2 cycloaddition, or a 4+2 cycloaddition). [Table 43] (a) ketone / aldehyde condensation, (b) Staudinger reaction, (c) 1,3 dipolar cycloaddition (top: copper-catalyzed alkyne-azide cycloaddition (CuAAC), bottom: strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC)), (d) inverse electron demand Diels-Alder reaction. In certain embodiments, R 1A and R 2 One of the groups is -N3, and the other is R 1A and R 2 The other of R is dibenzocyclooctyne. 1A and R 2 On the other hand, [ka] and R 1A and R 2 The other is -SH. In certain embodiments, R 1A and R 2 One of them is tetrazine, and the other is R 1A and R 2 and the other is trans-cyclooctene. In certain embodiments, R 1A and R 2 On the other hand, [ka] and R 1A and R 2 The other is, [ka] In certain embodiments, R 1A and R 2 One of the groups is -SH, and the other is R 1A and R 2 The other is, [ka] In certain embodiments, R 1A and R 2 One of the groups is -SH, and the other is R 1A and R2 The other is, [ka] In certain embodiments, R 1A and R 2 One of the groups is -SH, and the other is R 1A and R 2 The other is, [ka] It is.

[0130] In certain embodiments, R D1 is hydrogen. In certain embodiments, R D1 is optionally substituted acyl (e.g., -C(=O)Me). In certain embodiments, R D1 is an optionally substituted alkyl (e.g., substituted or unsubstituted C 1~6 In certain embodiments, R D1 is substituted or unsubstituted methyl. In certain embodiments, R D1 is substituted or unsubstituted ethyl. In certain embodiments, R D1 is substituted or unsubstituted propyl. In certain embodiments, R D1 is an optionally substituted alkenyl (e.g., substituted or unsubstituted C 2~6 alkenyl). In certain embodiments, R D1 is an optionally substituted alkynyl (e.g., substituted or unsubstituted C 2~6 alkynyl). In certain embodiments, R D1 is an optionally substituted carbocyclyl (e.g., a substituted or unsubstituted 3- to 7-membered monocyclic carbocyclyl containing 0, 1, or 2 double bonds in the carbocyclic ring system). D1is an optionally substituted heterocyclyl (e.g., a substituted or unsubstituted 5-10 membered monocyclic or bicyclic heterocyclic ring, where one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). D1 is optionally substituted aryl (e.g., substituted or unsubstituted 6-10 membered aryl). In certain embodiments, R D1 is benzyl. In certain embodiments, R D1 is substituted or unsubstituted phenyl. In certain embodiments, R D1 is an optionally substituted heteroaryl (e.g., a substituted or unsubstituted 5-6 membered monocyclic heteroaryl (wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur) or a substituted or unsubstituted 9-10 membered bicyclic heteroaryl (wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, R D1 is an oxygen protecting group when attached to an oxygen atom. In certain embodiments, R D1 is a sulfur protecting group when attached to a sulfur atom.

[0131] In certain embodiments, R D1a At least one instance of R is hydrogen. D1a At least one instance of is optionally substituted acyl (e.g., -C(=O)Me). In certain embodiments, at least one R D1a is an optionally substituted alkyl (e.g., substituted or unsubstituted C 1~6 In certain embodiments, R D1a At least one instance of is substituted or unsubstituted methyl. In certain embodiments, R D1a At least one instance of is substituted or unsubstituted ethyl. In certain embodiments, R D1a At least one instance of is substituted or unsubstituted propyl. In certain embodiments, RD1a At least one example of an optionally substituted alkenyl (e.g., substituted or unsubstituted C 2~6 alkenyl). In certain embodiments, R D1a At least one example of an optionally substituted alkynyl (e.g., substituted or unsubstituted C 2~6 alkynyl). In certain embodiments, R D1a At least one instance of is an optionally substituted carbocyclyl (e.g., a substituted or unsubstituted 3- to 7-membered monocyclic carbocyclyl containing 0, 1, or 2 double bonds in the carbocyclic ring system). D1a At least one instance of is an optionally substituted heterocyclyl (e.g., a substituted or unsubstituted 5-10 membered monocyclic or bicyclic heterocyclic ring, where one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). D1a At least one instance of is optionally substituted aryl (e.g., substituted or unsubstituted 6-10 membered aryl). In certain embodiments, R D1a At least one instance of is benzyl. In certain embodiments, R D1a At least one instance of is substituted or unsubstituted phenyl. In certain embodiments, R D1a At least one instance of is an optionally substituted heteroaryl (e.g., a substituted or unsubstituted 5-6 membered monocyclic heteroaryl (wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur) or a substituted or unsubstituted 9-10 membered bicyclic heteroaryl (wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur)). In certain embodiments, R D1aAt least one instance of R is a nitrogen protecting group (e.g., benzyl (Bn), t-butyl carbonate (BOC or Boc), benzyl carbamate (Cbz), 9-fluorenylmethyl carbonate (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl, or p-toluenesulfonamide (Ts)). In certain embodiments, R D1a Two examples of the following, taken together with their intervening atoms, form an optionally substituted heterocyclic ring (e.g., a substituted or unsubstituted 5-10 membered monocyclic or bicyclic heterocyclic ring, where one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur), or an optionally substituted heteroaryl ring (e.g., a substituted or unsubstituted 5-6 membered monocyclic heteroaryl, where one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur, or a substituted or unsubstituted 9-10 membered bicyclic heteroaryl, where one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur).

[0132] In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3.

[0133] In some embodiments, W is -O-. In some embodiments, W is -O- and q is an integer between 1 and 100,000, inclusive. In some embodiments, W is -CH2-. In some embodiments, W is -CH2- and q is an integer between 1 and 10,000, inclusive.

[0134] In some embodiments, q is an integer between 1 and 50, inclusive. In some embodiments, q is an integer between 1 and 100, inclusive. In some embodiments, q is an integer between 1 and 5,000, inclusive. In some embodiments, q is an integer between 1 and 10,000, inclusive. In some embodiments, q is an integer between 1 and 50,000, inclusive. In some embodiments, q is an integer between 1 and 100,000, inclusive. In some embodiments, q is 1 to 25, 25 to 50, 50 to 75, 75 to 100, 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to 225, 225 to 250, 250 to 275, 275 to 300, 300 to 325, 325 to 350, 350 to 375, 375 to 400, 400 to 425, 400 to 450, 450 to 500, 500 to 550, 550 to 600, 600 to 650, 650 to 700, 700 to 750, 750 to 800, 800 to 850, 850 to 900, 900 to It is an integer between 1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-6000, 6000-7000, 7000-8000, 8000-9000, 9000-10,000, 10,000-12,000, 100,000-120,000, 120,000-150,000, 150,000-175,000, or 175,000-200,000.

[0135] In some embodiments, the synthesis method comprises reacting a dendrimer of formula (II-A) with one or more amines, each amine being of the formula HNR 1 and R 1 is as defined herein. In some embodiments, R 1 At least one instance of is optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, or a covalent bond. 1 At least one example of optionally substituted alkylene (e.g., optionally substituted C1~20 In some embodiments, R 1 Each instance of Y is one or more amines. 1 where each amine has the formula HNR 1 In some embodiments, R 1 At least one instance of R is optionally substituted ethyl. 1 At least one example of R is ethyl substituted with a PEG-alkyne group. 1 At least one instance of the formula [ka] wherein q is 1, 2, 3, 4, 5, or 6 (e.g., q is 1, 2, or 3). In some embodiments, R 1 At least one instance of the formula [ka]

[0023] wherein q is 2. In some embodiments, R 1 At least one example of the formula is unsubstituted alkylene (e.g., unsubstituted C 1~10 alkylene).

[0136] In some embodiments, the amine has the formula [ka] wherein q is 1, 2, 3, 4, 5, or 6. In some embodiments, the amine is a PEG-alkyne of the formula [ka] In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, q is 5. In some embodiments, q is 6. In some embodiments, at least one instance of the one or more amines is of the formula [ka] In some embodiments, at least one instance of the one or more amines is a PEG-alkyne of the formula [ka] In some embodiments, the synthetic methods described herein include two different amines. In some embodiments, one of the one or more amines is an ethanolamine of the formula [ka] where q is 1, 2, 3, 4, 5, or 6; and the other amine is an ethanolamine (e.g., [ka] In some embodiments, one of the one or more amines is of the formula [ka] and other amines include ethanolamines (e.g., [ka] In some embodiments, in the synthetic methods described herein, the dendrimer of formula (II-A) is a dendrimer having ethanolamine and the formula [ka] The PEG-alkyne can be reacted with both

[0137] In some embodiments, a first amine (e.g., ethanolamine) is substituted with a second amine (e.g., a compound of the formula [ka] In some embodiments, the ratio of ethanolamine to PEG-alkyne is about 10:1, 9.8:1, 9.5:1, 9.4:1, 9.3:1, 9.2:1, 9.1:1, or 9:1. [ka] (for example, [ka] ) to PEG-alkyne is 9.4:1. In some embodiments, the ethanolamine has the formula [ka] to PEG-alkyne ratio is 9.42:1.

[0138] In some embodiments, a first amine (e.g., ethanolamine) is substituted with a second amine (e.g., a compound of the formula [ka] In some embodiments, the ratio of ethanolamine to PEG-alkyne is about 3:1, 3.0:1, 2.8:1, 2.6:1, 2.4:1, 2.3:1, 2.2:1, or 2.1:1, or 2.0:1. [ka] (for example, [ka] ) to PEG-alkyne is 2.2:1. In some embodiments, the ratio of ethanolamine to PEG-alkyne is [ka] to PEG-alkyne is 2.23:1. In some embodiments, the ethanolamine has the formula [ka]

[0033] where q is 1, 2, 3, 4, 5, or 6. [ka] The ratio of PEG to PEG-alkyne is 2.2:1 or 9.4:1.

[0139] In some embodiments, one amine (e.g., a compound of the formula [ka] In some embodiments, the ratio of one amine (e.g., PEG-alkyne of formula (II-A) to a dendrimer of formula (II-A) is approximately 150:1. [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] where D is PAMAM and t is an integer between about 50-60, 60-70, or 70-80, inclusive) is about 150:1. [ka] The ratio of ethanolamine to PEG-alkyne is approximately 150:1, the ratio of ethanolamine to PEG-alkyne is 9.42:1, and n is 3.

[0140] In some embodiments, one amine (e.g., a compound of the formula [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] where D is PAMAM and t is an integer between about 50-60, 60-70, or 70-80, inclusive) is about 152:1, 150:1, 149:1, 148:1, 145:1, 142:1, 140:1, or 138:1. In some embodiments, the ratio of one amine (e.g., of the formula [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] wherein D is PAMAM and t is an integer between about 50-60, 60-70, or 70-80, inclusive) is about 152:1, 150:1, 149:1, 148:1, 145:1, 142:1, 140:1, or 138:1, and n is 3. In some embodiments, the ratio of one amine (e.g., a amine of the formula [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] wherein D is PAMAM and t is an integer between about 50-60, 60-70, or 70-80, inclusive) is about 152:1, 150:1, 149:1, 148:1, 145:1, 142:1, 140:1, or 138:1, the ratio of ethanolamine to PEG-alkyne is 9.42:1, and n is 3. In some embodiments, one amine (e.g., a PEG-alkyne of the formula [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] wherein D is PAMAM, t is an integer between approximately 50-60, 60-70, or 70-80, inclusive, the ratio of ethanolamine to PEG-alkyne is approximately 150:1, the ratio of ethanolamine to PEG-alkyne is 9.42:1, and n is 3.

[0141] In some embodiments, D is PAMAM. In some embodiments, D is PAMAM of generation 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, or 9.5, where the PAMAM dendrimer has one carboxymethyl group. In some embodiments, one amine (e.g., a compound of formula [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] ) is approximately 500:1. In some embodiments, the ratio of one amine (e.g., [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] ) is approximately 500:1, the ratio of ethanolamine to PEG-alkyne is 2.2:1, and n is 10. In some embodiments, one amine (e.g., [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] ) is 495:1. In some embodiments, the ratio of one amine (e.g., [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] wherein D is PAMAM and t is an integer between about 50-60, 60-70, or 70-80, inclusive) is about 505:1, 503:1, 502:1, 501:1, 500:1, 499:1, 498:1, 497:1, 496:1, 495:1, 494:1, 493:1, 492:1, 491:1, or 490:1. [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] where D is PAMAM and t is an integer between about 50-60, 60-70, or 70-80, inclusive) is about 495:1. [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] wherein D is PAMAM and t is an integer between about 50-60, 60-70, or 70-80, inclusive) is about 505:1, 503:1, 502:1, 501:1, 500:1, 499:1, 498:1, 497:1, 496:1, 495:1, 494:1, 493:1, 492:1, 491:1, or 490:1 (e.g., about 495:1), and n is 10. In some embodiments, the ratio of one amine (e.g., a amine of formula [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] wherein D is PAMAM and t is an integer between about 50-60, 60-70, or 70-80, inclusive) is about 495:1, and n is 10. In some embodiments, the ratio of one amine (e.g., of the formula [ka] PEG-alkyne) of the dendrimer of formula (II-A) ( [ka] wherein D is PAMAM, t is an integer between approximately 50-60, 60-70, or 70-80, inclusive, the ratio of ethanolamine to PEG-alkyne is approximately 500:1, the ratio of ethanolamine to PEG-alkyne is 2.2:1, and n is 10.

[0142] In some embodiments, the molecular weight of the dendrimer of formula (II-A) is about 12,500 g / mol, about 12,425 g / mol, about 12,420 g / mol, or about 12,418 g / mol, or about 12,415 g / mol. In some embodiments, the molecular weight of the dendrimer of formula (II-A) is between about 12,000 and 12,500 g / mol.

[0143] In some embodiments, the dendrimer of formula (II-A) has the formula [ka] It is a dendrimer.

[0144] In some embodiments, the functionalized dendrimer of formula (IA) has a low polydispersity value, for example, from about 1.00 to about 1.05 (e.g., about 1.03). In some embodiments, the functionalized dendrimer of formula (IA) has a polydispersity value of about 1.03.

[0145] In some embodiments, more than 10-20 grams, 20-30 grams, 30-40 grams, 40-50 grams, 50-60 grams, 60-70 grams, 70-80 grams, 80-90 grams, 90-100 grams, 100-110 grams, 110-120 grams, 120-130 grams, 130-140 grams, 150-160 grams, 160-180 grams, 180-200 grams of functionalized dendrimer of formula (IA) are synthesized by the synthesis methods described herein. In some embodiments, more than 30 grams, or more than 50-100 grams (e.g., 40 grams, 160 grams) of functionalized dendrimer of formula (IA) are synthesized by the synthesis methods described herein.

[0146] In some embodiments, in the method of synthesizing a functionalized dendrimer of formula (IA), the method comprises: [ka] with one or more amines (wherein each amine is of the formula HNR 1(an amine of the formula (I)), suitable conditions include a reaction solvent and reacting the reactants at room temperature (e.g., from about 19° C. to about 23° C.). In certain embodiments, suitable conditions include a reaction solvent that is a protic solvent. For example, the reaction solvent can be a protic solvent or a mixture of protic and aprotic solvents. In certain embodiments, the protic solvent is an alcohol, such as methanol, ethanol, or isopropanol. In certain embodiments, the protic solvent is methanol. In certain embodiments, the alcohol can be anhydrous or contain water. In certain embodiments, the reaction solvent can include dichloromethane.

[0147] In certain embodiments, suitable conditions for synthesizing a functionalized dendrimer of formula (IA) include reacting the reactants at about 19° C. to about 23° C., such as about 20° C. to about 22° C. In certain embodiments, suitable conditions include reacting the reactants at about 18-19° C., 19-20° C., 20-21° C., 21-22° C., or 22-23° C. Suitable conditions include a reaction solvent of methanol and reacting the reactants at about 20° C.

[0148] In certain embodiments, suitable conditions for synthesizing a functionalized dendrimer of formula (IA) include reacting one or more amines (wherein each amine is of the formula HNR 1 (which is an amine of formula (II-A)) with a dendrimer of formula (II-B) and a reaction solvent (e.g., an alcohol, e.g., methanol) at 0° C. for about 1-5 hours (e.g., 2 hours), followed by a second step of stirring at about 19° C. to about 23° C. (e.g., 20° C.) for about 3-8 days (e.g., 6 days). In certain embodiments, the synthesized functionalized dendrimer of formula (IA) has the formula [ka] [ka] is a functionalized dendrimer.

[0149] In some embodiments, the method for synthesizing a functionalized dendrimer of formula (IA) comprises the step of: [ka] [In the formula, R 2 is halogen, optionally substituted acyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted acetyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, -N3, -CH(=N)(OH)R D1 , -CN, -NO2, -OR D1 , -N(R D1a )2, -SO2OR D1 , or -SR D1 and R D1 are independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or, if attached to an oxygen atom, an oxygen protecting group, or, if attached to a sulfur atom, a sulfur protecting group; R D1a each occurrence is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group, or optionally R D1a Two instances of are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaryl ring, with the proviso that R 1A and R2 is the reaction partner and L B is an alkylene linker, where one or more chain atoms of the hydrocarbon chain are independently replaced with an amide, ester, hydroxamate, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane or oxime moiety, or a cyclic moiety, and T is a therapeutic agent. The method further comprises reacting with

[0150] In some embodiments, the linker L B In some embodiments, the linker L B is an alkylene chain (e.g., C 1~100,000 alkylene), the chain of which is R excluding hydrogen atoms and substituents. 2 and T. In certain embodiments, the linker L B The chain of L comprises 5,000-7,000, 7,000-9,000, 9,000-10,000, 10,000-12,000, 100,000-120,000, 120,000-150,000, or up to 150,000-200,000 consecutive covalently bonded atoms or lengths, excluding hydrogen atoms and substituents. B R excluding hydrogen atoms and substituents 2 All-carbon substituted or unsubstituted C as the shortest path between and T 1~200,000 In certain embodiments, L B Any of the atoms in may be substituted. In certain embodiments, the linker L B In certain embodiments, none of the atoms in the linker are substituted. In certain embodiments, none of the carbon atoms in the linker are substituted. B At least one chain atom of the hydrocarbon chain is independently selected from -C(=O)-, -O-, -NR b -, -S-, or a cyclic moiety; R b are independently hydrogen, substituted or unsubstituted C 1~6In certain embodiments, L is an alkyl, or a nitrogen protecting group. B At least one chain atom of the hydrocarbon chain of is independently replaced with an amide, hydroxamate, ether, n-alkyl, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane, oxime, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, and / or optionally substituted heteroarylene. In certain embodiments, L B At least one chain atom of the hydrocarbon chain of is independently replaced with an amide, hydroxamate, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane, and / or oxime. In certain embodiments, the linker L B is an alkylene moiety (e.g., of the formula [ka] wherein q is an integer between 1 and 10,000 or between 1 and 100,000, inclusive. B is a polyethylene glycol moiety (e.g., [ka] [wherein q is an integer between 1 and 100,000 inclusive], L B At least one chain atom of the hydrocarbon chain of is independently replaced with an amide, hydroxamate, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane, and / or oxime. In certain embodiments, the linker L Bcomprises a moiety resulting from a click reaction. In some embodiments, at least one moiety resulting from a click reaction is a five-membered heterocyclic ring resulting from an electrocyclic reaction (e.g., a 3+2 cycloaddition, or a 4+2 cycloaddition) between the reactive click chemistry handles (e.g., an azide and a terminal or strained alkyne, a diene and a dienophile, a thiol and an alkene) used to generate the conjugate. In some embodiments, at least one moiety resulting from a click reaction is a diradical comprising 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, or 1,4-dihydropyridazyl.

[0151] In certain embodiments, R 2 is halogen (e.g., F, Cl, Br, or I). In certain embodiments, R 2 is optionally substituted acyl (e.g., -C(=O)Me). In certain embodiments, R 2 is an optionally substituted alkyl (e.g., substituted or unsubstituted C 1~6 In certain embodiments, R 2 is substituted or unsubstituted methyl. In certain embodiments, R 2 is substituted or unsubstituted ethyl. In certain embodiments, R 2 is substituted or unsubstituted propyl. In certain embodiments, R 2 is an optionally substituted alkenyl (e.g., substituted or unsubstituted C 2~6 alkenyl). In certain embodiments, R 2 is an optionally substituted alkynyl (e.g., substituted or unsubstituted C 2~6 alkynyl). In certain embodiments, R 2 is optionally substituted acetyl. In certain embodiments, R 2is an optionally substituted carbocyclyl (e.g., a substituted or unsubstituted 3- to 7-membered monocyclic carbocyclyl containing 0, 1, or 2 double bonds in the carbocyclic ring system). 2 is an optionally substituted heterocyclyl (e.g., a substituted or unsubstituted 5-10 membered monocyclic or bicyclic heterocyclic ring, where one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). 2 is optionally substituted aryl (e.g., substituted or unsubstituted 6-10 membered aryl). In certain embodiments, R 1A is benzyl. In certain embodiments, R 2 is substituted or unsubstituted phenyl. In certain embodiments, R 2 is an optionally substituted heteroaryl (e.g., a substituted or unsubstituted 5-6 membered monocyclic heteroaryl (wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur) or a substituted or unsubstituted 9-10 membered bicyclic heteroaryl (wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, R 2 is -CH(=N)(OH)R D1 , -CN, -NO2, -OR D1 , -N(R D1a )2, -SO2OR D1 , or -SR D1 and R D1 and R D1a is as defined herein. In certain embodiments, R 2 is -CH(=N)(OH)R D1 (e.g., -CH(=N)(OH) (optionally substituted C 1~6 In certain embodiments, R 2 is -CH(=N)(OH) (C optionally substituted with a polyethylene glycol linker) 1~6 In certain embodiments, R 2is -CN. In certain embodiments, R 2 is -NO2. In certain embodiments, R 2 -OR D1 (e.g., -OH or -OMe). In certain embodiments, R 1A is -N(R D1a )2 (e.g., -NMe2). In certain embodiments, R 2 -SO2OR D1 (e.g., -SO(optionally substituted alkyl)). In certain embodiments, R 2 -SR D1 (e.g., -SMe). In certain embodiments, R 2 is -CH(=N)(OH)R D1 , -CN, -NO2, -OR D1 , -N(R D1a )2, -SO2OR D1 , or -SR D1 and R D1 are independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or, if attached to an oxygen atom, an oxygen protecting group, or, if attached to a sulfur atom, a sulfur protecting group; R D1a each occurrence is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group, or optionally R D1a Two instances of, taken together with their intervening atoms, form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaryl ring. In certain embodiments, R 2is bromo, alkyne, acetylene, alkene, aldehyde, amine, COOH, hydroxyl, carboxyl (e.g., dibenzocyclooctyne or DBCO), thiol, sulfonate, or -CN. 2 is a click reaction handle (e.g., a click chemistry handle in Table A or shown in Schemes 1-19 of Example 6). In certain embodiments, R 1A and R 2 is a reaction partner (e.g., a bioconjugation reaction partner). In certain embodiments, R 1A and R 2 is a bioconjugation reaction partner (e.g., a click reaction partner). In certain embodiments, R 1A and R 2 is a click reaction partner from Table A. In certain embodiments, L B is an alkylene linker, where one or more chain atoms of the hydrocarbon chain are independently replaced with an amide, ester, hydroxamate, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane or oxime moiety, or a cyclic moiety.

[0152] In certain embodiments, at least one instance of T is a therapeutic agent, a targeting agent, or an imaging agent as defined herein. In certain embodiments, at least one instance of T is a therapeutic agent as defined herein. In certain embodiments, at least one instance of T is a biomolecule. In certain embodiments, at least one instance of T is a proteolytic targeting chimeric (PROTAC) drug. In certain embodiments, at least one instance of T is a biological therapeutic agent (e.g., a protein, a peptide, a nucleic acid, or an antibody). In certain embodiments, at least one instance of T is a peptide, a nucleic acid, or an antibody. In certain embodiments, the nucleic acid is an oligonucleotide, DNA, or RNA (e.g., siRNA, mRNA). In certain embodiments, at least one instance of T is a gene, a protein, a peptide, an oligonucleotide, a carbohydrate, DNA, or RNA. In certain embodiments, each instance of T is different. In certain embodiments, each instance of T is the same.

[0153] In certain embodiments, the intermediate dendrimer of formula (II-A) is [ka] wherein D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; t is an integer between 16 and 4096, inclusive. is provided.

[0154] In certain embodiments, provided herein is an intermediate dendrimer in the synthetic methods described herein, the intermediate dendrimer having the formula [ka] It is of the following. Compositions Comprising Functionalized Dendrimers

[0155] In some aspects, the present disclosure provides a functionalized dendrimer of formula (IA) [ka] [In the formula, D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; X is NH; Y 1 is an optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, or a covalent bond; m is an integer between 16 and 4096, inclusive; n is an integer between 1 and 100, inclusive. A composition comprising:

[0156] In some embodiments, the polydispersity value of the functionalized dendrimer in the composition is less than or equal to 1.10. In some aspects, the present disclosure provides a method for preparing a carrier and a functionalized dendrimer of formula (IA). [ka] [In the formula, D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; X is NH; Y 1 is an optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, or a covalent bond; m is an integer between 16 and 4096, inclusive; n is an integer between 1 and 100, inclusive. A composition comprising:

[0157] In some embodiments, the polydispersity value of the functionalized dendrimer in the composition is equal to or less than 1.10. In certain embodiments, the substituents D, X, Y in the functionalized dendrimer of formula (IA) 1The definitions of , m, and n are as above. In certain embodiments, the polydispersity value of the functionalized dendrimer in the composition is equal to or less than 1.10 (e.g., equal to or less than 1.09, equal to or less than 1.08, equal to or less than 1.07, equal to or less than 1.06, equal to or less than 1.05, equal to or less than 1.04, equal to or less than 1.03, equal to or less than 1.02). In certain embodiments, the polydispersity value of the functionalized dendrimer in the composition is equal to or less than 1.05, equal to or less than 1.04, equal to or less than 1.03, equal to or less than 1.02, equal to or less than 1.01, or equal to or less than 1.00. In certain embodiments, the composition comprises at least 10 grams of functionalized dendrimer. In certain embodiments, the composition comprises at least 10 grams, at least 25 grams, at least 50 grams, at least 150 grams, or between 10-100 grams, 100-150 grams, 150-200 grams, 20-200 grams, 50-200 grams, or 100-200 grams of functionalized dendrimer (e.g., a functionalized dendrimer of formula (IA)). In some embodiments, the composition comprises greater than 10-20 grams, 20-30 grams, 30-40 grams, 40-50 grams, 50-60 grams, 60-70 grams, 70-80 grams, 80-90 grams, 90-100 grams, 100-110 grams, 110-120 grams, 120-130 grams, 130-140 grams, 150-160 grams, 160-180 grams, 180-200 grams of functionalized dendrimer (e.g., functionalized dendrimer of formula (IA)). In some embodiments, the composition comprises greater than 30 grams, or greater than 50-100 grams (e.g., 40 grams, 160 grams) of functionalized dendrimer (e.g., functionalized dendrimer of formula (IA)).

[0158] In certain embodiments, the compositions comprise a suitable carrier (eg, a solid or liquid carrier) as described herein. Therapeutic Agents

[0159] In some embodiments, the disclosure provides dendrimer conjugates comprising a dendrimer having at least one therapeutic agent at one or more terminal positions of the dendrimer. In some embodiments, such dendrimer-drug conjugates have an increased therapeutic index compared to the unconjugated drug (e.g., the therapeutic agent in the absence of the dendrimer). In some embodiments, the dendrimer-drug conjugates have a therapeutic index greater than 10% of the therapeutic index of the unconjugated drug. In some embodiments, the dendrimer-drug conjugates have a therapeutic index greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the therapeutic index of the unconjugated drug.

[0160] The therapeutic index (TI) of a therapeutic agent is the amount of the therapeutic agent that causes a therapeutic effect compared to the amount of the therapeutic agent that causes toxicity. In some embodiments, the therapeutic index is the LD 50 / ED 50 Expressed as a ratio, ED 50 corresponds to a therapeutically effective dose in 50% of the population, and LD 50 corresponds to the dose that is lethal in 50% of the population. In some embodiments, the therapeutic efficacy and toxicity of drugs and dendrimer-drug conjugates can be determined in cell cultures or experimental animals by standard pharmaceutical procedures.

[0161] In some embodiments, dendrimers are complexed or conjugated with two or more different classes of therapeutic agents, resulting in simultaneous delivery with different or independent release kinetics at target sites.For example, in some embodiments, STING agonists and CSF1R inhibitors are conjugated to dendrimers for delivery to target cells or tissues.In some embodiments, dendrimer conjugates each having a different class of therapeutic agent are administered simultaneously for combination treatment.In some embodiments, a generation 4 or generation 6 PAMAM dendrimer is conjugated to sunitinib and a CXCR2 inhibitor, or an analog thereof.In some embodiments, a generation 4 or generation 6 PAMAM dendrimer is conjugated to vincristine and sunitinib, or an analog thereof.

[0162] In some embodiments, the therapeutic agent is any of the following compounds, or any of the pharma- ceutically acceptable derivatives, analogs, or prodrugs of any of the following compounds. A prodrug is a compound that undergoes conversion to a compound with the desired pharmacological activity when metabolized in vivo. Prodrugs can be prepared by replacing the appropriate functional group present in the therapeutic agent with a "promoiety" as described in the art (see, for example, H. Bundgaar, Design of Prodrugs (1985)). Examples of prodrugs include ester, ether, or amide derivatives of the therapeutic agents described herein, polyethylene glycol derivatives of the therapeutic agents described herein, N-acylamine derivatives, dihydropyridine pyridine derivatives, amino-containing derivatives conjugated to polypeptides, 2-hydroxybenzamide derivatives, carbamate derivatives, N-oxide derivatives that are biologically reduced to active amines, and N-Mannich base derivatives. For further discussion of prodrugs, see, for example, Rautio, J. et al. Nature Reviews Drug Discovery. 7:255-270 (2008).

[0163] In some embodiments, the dendrimer conjugates of the present disclosure are selected from the group consisting of angiotensin II receptor blockers, farnesoid X receptor (FXR) agonists, death receptor 5 agonists, sodium-glucose cotransporter type 2 (SGLT2) inhibitors, lysophosphatidic acid 1 receptor antagonists, endothelin-A receptor antagonists, peroxisome proliferator-activated receptor delta (PPARδ) agonists, AT1 receptor antagonists, CCR5 / CCR2 antagonists, antifibrotic agents, anti-inflammatory agents, antioxidants, stimulator of interferon genes (STING) agonists, colony-stimulating factor 1 receptor (CSF1R) inhibitors, AXL inhibitors, c-Met inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors, receptor tyrosine kinase inhibitors, MEK inhibitors, PAK1 inhibitors, glutaminase inhibitors, TIE inhibitors, and the like. II antagonists, chemokine receptor 2 (CXCR2) inhibitors, CD73 inhibitors, arginase inhibitors, phosphatidylinositol-3-kinase (PI3K) inhibitors, toll-like receptor 4 (TLR4) agonists, toll-like receptor 7 (TLR7) agonists, Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) inhibitors, chemotherapeutic agents, STING antagonists, and JAK1 inhibitors.

[0164] In some embodiments, the therapeutic agent is an immunomodulatory agent. In some embodiments, an immunomodulatory agent refers to an agent that elicits a particular effect on the recipient's immune system. Immunomodulation may include, in some embodiments, suppression, reduction, enhancement, prolongation, or stimulation of one or more physiological processes of the innate or adaptive immune response to an antigen compared to a control. In some embodiments, the immunomodulatory agent can modulate the immune microenvironment for a desired immunological response (e.g., increased anti-tumor activity, or increased anti-inflammatory active sites in autoimmune diseases that require increased anti-inflammatory active sites) by targeting one or more immune cells or cell types at the target site. In some embodiments, the immunomodulatory agent is delivered to kill, inhibit, or reduce the activity or amount of suppressive immune cells, such as tumor-associated macrophages, for enhanced anti-tumor response at the tumor site. In other embodiments, the immunomodulatory agent is delivered to kill, inhibit, or reduce the activity or amount of pro-inflammatory immune cells (e.g., M1-type macrophages) for reduced pro-inflammatory immune environment at the pathogenic site associated with autoimmune disease.

[0165] Examples of immunomodulatory agents for use in accordance with the present disclosure include, but are not limited to, STING agonists, STING antagonists, Janus kinase 1 (JAK1) inhibitors, CSF1R inhibitors, AXL inhibitors, c-Met inhibitors, PARP inhibitors, receptor tyrosine kinase inhibitors, MEK inhibitors, PAK1 inhibitors, glutaminase inhibitors, TIE II antagonists, CXCR2 inhibitors, CD73 inhibitors, arginase inhibitors, PI3K inhibitors, TLR4 agonists, TLR7 agonists, SHP2 inhibitors, anti-inflammatory agents, and combinations thereof.

[0166] In some embodiments, the therapeutic agent is a STING agonist selected from the cyclic dinucleotides GMP-AMP and DMXAA.In some embodiments, the therapeutic agent is a STING antagonist selected from C-178, C-176, C18, astin C, NO2-CLA, H-151, and alpha-mangostin.In some embodiments, the therapeutic agent is a JAK1 inhibitor selected from tofacitinib, ruxolitinib, baricitinib, peficitinib, decernotiniba, filgotinib, solcitinib, itacitinib, SHR0302, upadacitinib, PF-04965842, Target-007, and Target-006. In some embodiments, the therapeutic agent is a CSF1R inhibitor selected from PLX3397, PLX108-01, ARRY-382, PLX7486, BLZ945, JNJ-40346527, and GW 2580. In some embodiments, the therapeutic agent is a PARP inhibitor selected from olaparib, veliparib, niraparib, and rucaparib. In some embodiments, the therapeutic agent is a receptor tyrosine kinase inhibitor of vascular endothelial growth factor receptor (VEGFR) or epidermal growth factor receptor (EGFR). In some embodiments, the therapeutic agent is an AXL inhibitor (e.g., bemcentinib (R428), duvelmatinib (TP-0903)). In some embodiments, the therapeutic agent is a c-Met inhibitor (e.g., cabozantinib). In some embodiments, the therapeutic agent is a receptor tyrosine kinase (e.g., VEGFR, CSF1R, AXL, and / or c-Met) inhibitor selected from sunitinib, sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, dasatinib, bemcentinib (R428), duvelmatinib (TP-0903), nintedanib, cabozantinib, and motesanib. In some embodiments, the therapeutic agent is a MEK inhibitor selected from trametinib, cobimetinib, binimetinib, selumetinib, PD325901, PD035901, PD032901, and TAK-733. In some embodiments, the therapeutic agent is a PAK1 inhibitor.In some embodiments, the PAK1 inhibitor is Frax-1036 (6-[2-chloro-4-(6-methyl-2-pyrazinyl)phenyl]-8-ethyl-2-[[2-(1-methyl-4-piperidinyl)ethyl]amino]-pyrido[2,3-d]pyrimidin-7(8H)-one). In some embodiments, the therapeutic agent is a glutaminase inhibitor selected from bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), azaserine, acivicin, and CB-839. In some embodiments, the therapeutic agent is a CXCR2 inhibitor selected from navarixin, SB225002, and SB332235. In some embodiments, the therapeutic agent is a CD73 inhibitor selected from APCP, quercetin, and tenofovir. In some embodiments, the therapeutic agent is an arginase inhibitor, such as 2-(S)-amino-6-boronohexanoic acid. In some embodiments, the therapeutic agent is a PI3K inhibitor selected from alpelisib, ceravelisib, piralalisib, WX-037, dactolisib, plexasertib, voxtalisib, PX-866, ZSTK474, buparlisib, pictilisib, and copanlisib.

[0167] In some embodiments, the therapeutic agent is an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent reduces inflammation, which may include steroidal and non-steroidal drugs. Examples of steroidal drugs include, but are not limited to, glucocorticoids, progestins, mineralocorticoids, and corticosteroids. Examples of nonsteroidal anti-inflammatory drugs (NSAIDs) include, but are not limited to, mefenamic acid, aspirin, diflunisal, salsalate, ibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, sulindac, etodolac, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, firocoxib, sulfonanilides, nimesulide, niflumic acid, and licofelone. Further examples of anti-inflammatory agents include, but are not limited to, triamcinolone acetonide, fluocinolone acetonide, methylprednisolone, prednisolone, prednisone, dexamethasone, loteprendol, fluorometholone, ibuprofen, aspirin, naproxen, cyclosporine, tacrolimus, rapamycin, and metformin. In some embodiments, the therapeutic agent is triamcinolone acetonide, prednisone, or dexamethasone.

[0168] In some embodiments, the therapeutic agent is a cytotoxic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of cytotoxic agents for use in accordance with the present disclosure include amsacrine, bevacizumab, 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, innotecan, leucovorin, daunorubicin, lomustine, riboflavin ... In some embodiments, the therapeutic agent may be a medicament for treating rheumatoid arthritis. In some embodiments, the therapeutic agent may be a medicament for treating rheumatoid arthritis. In some embodiments, the therapeutic agent may be a medicament for treating rheumatoid arthritis. In some embodiments, the therapeutic agent may be a medicament for treating rheumatoid arthritis. In some embodiments, the therapeutic agent may be a medicament for treating rheumatoid arthritis.

[0169] In some embodiments, the therapeutic agent is an anti-cancer agent, such as a cytotoxic agent described herein. In some embodiments, the therapeutic agent is a histone deacetylase (HDAC) inhibitor, such as vorinostat. In some embodiments, the therapeutic agent is a topoisomerase I and / or II inhibitor, such as etoposide or camptothecin. Further examples of anti-cancer agents include, but are not limited to, irinotecan, exemestane, octreotide, carmofur, clarithromycin, zinostatin, tamoxifen, tegafur, toremifene, doxifluridine, nimustine, vindensine, nedaplatin, pirarubicin, flutamide, fadrozole, prednisone, medroxyprogesterone, mitotane, mycophenolate mofetil, and mizoribine.

[0170] In some embodiments, the therapeutic agent is an anti-angiogenic agent. Examples of antiangiogenic agents include bevacizumab (AVASTIN®), rhuFAb V2 (ranibizumab, LUCENTIS®), aflibercept (EYLEA®), MACUGEN® (pegaptanim sodium, anti-VEGF aptamer or EYE001), pigment epithelium-derived factor (PEDF), celecoxib (CELEBREX®), rofecoxib (VIOXX®), interferon alpha, interleukin-12 (IL-12), thalidomide (THALOMID®), lenalidomide (REVLIMID®), squalamine, endostatin, angiostatin, ANGIOZYME® (Sirna Therapeutics), NEOVASTAT® (AE-941) (Aeterna Laboratories, Quebec City, Canada), sunitinib (SUTENT®), sorafenib (Nexavar®), erlotinib (Tarceva®), panitumumab (VECTIBIX®), and cetuximab (ERBITUX®). Further examples of antiangiogenic agents include members of the platelet-derived growth factor family, epidermal growth factor family, fibroblast growth factor family, transforming growth factor-β superfamily (TGF-β1, activin, follistatin and bone morphogenetic protein), angiopoietin-like family, galectin family, integrin superfamily, as well as pigment epithelium-derived factor, hepatocyte growth factor, angiopoietin, endothelin, hypoxia-inducible factor, insulin-like growth factor, cytokines, matrix metalloproteases and their inhibitors, and agents that target glycosylated proteins.

[0171] In some embodiments, the therapeutic agent is an agent that can be used to treat one or more conditions or diseases associated with the liver and / or related diseases or conditions, such as infection, sepsis, diabetic complications, hypertension, obesity, hypertension, heart failure, kidney disease, and cancer. Examples of such therapeutic agents include, but are not limited to, angiotensin II receptor blockers, FXR agonists, death receptor 5 agonists, SGLT2 inhibitors, lysophosphatidic acid 1 receptor antagonists, endothelin-A receptor antagonists, PPAR delta agonists, AT1 receptor antagonists, CCR5 / CCR2 antagonists, insulin sensitizers, pioglitazone, antifibrotic agents, antioxidants, antiangiogenic agents, anti-excitotoxic agents (e.g., valproic acid, D-aminophosphonovalerate, D-aminophosphonoheptanoate), glutamate formation / release inhibitors (e.g., baclofen, NMDA receptor antagonists, ranibizumab, anti-VEGF agents), and immunomodulatory and cytotoxic agents described herein.

[0172] In some embodiments, the therapeutic agent is an angiotensin II receptor blocker, such as telmisartan, telmisartan-amide derivatives, or telmisartan-ester derivatives. In some embodiments, the therapeutic agent is an FXR agonist, such as chenodeoxycholic acid, chenodeoxycholic acid-amide derivatives, or chenodeoxycholic acid-ester derivatives. In some embodiments, the therapeutic agent is an SGLT2 inhibitor selected from phlorizin, T-1095, canagliflozin, dapagliflozin, ipragliflozin, tofogliflozin, empagliflozin, luseogliflozin, ertugliflozin, and remogliflozin etabonate. In some embodiments, the therapeutic agent is a PPARδ agonist, such as GW0742, GW0742-amide derivatives, or GW0742-ester derivatives. In some embodiments, the therapeutic agent is an antioxidant, such as vitamin E.

[0173] In some embodiments, the therapeutic agent is N-acetyl-L-cysteine. In some embodiments, N-acetyl-L-cysteine ​​is conjugated to the hydroxyl-terminated dendrimer by a non-cleavable linkage so as to release minimal free N-acetyl-cysteine ​​in vivo after administration. In some embodiments, the non-cleavable form of the dendrimer / N-acetyl-cysteine ​​conjugate provides enhanced therapeutic efficacy compared to the releasable or cleavable form of the dendrimer / N-acetyl-cysteine ​​complex.

[0174] In some embodiments, the therapeutic agent is polysialic acid (e.g., low molecular weight polySia with an average degree of polymerization of 20 (polySia avDP20)), a translocator protein ligand (e.g., diazepam binding inhibitor (DBI)), interferon-β (IFN-β), or minocycline.

[0175] In some embodiments, the therapeutic agent is an anti-infective agent. Examples of anti-infective agents include, but are not limited to, antiviral agents, antibacterial agents, antiparasitic agents, and antifungal agents. In some embodiments, the therapeutic agent is selected from moxifloxacin, ciprofloxacin, erythromycin, levofloxacin, cefazolin, vancomycin, tigecycline, gentamicin, tobramycin, ceftazidime, ofloxacin, gatifloxacin, amphotericin, voriconazole, and natamycin.

[0176] Therapeutic agents suitable for use in accordance with the present disclosure are described in further detail in co-pending International Application Nos. PCT / US2020 / 063332, PCT / US2020 / 063347, PCT / US2020 / 063342, and PCT / US2021 / 029139, the relevant contents of each of which are incorporated by reference herein in their entireties. Contrast agents

[0177] In some embodiments, the present disclosure provides a dendrimer conjugate comprising a dendrimer having at least one imaging agent at one or more terminal positions of the dendrimer. In some embodiments, the dendrimer conjugate comprising an imaging agent can be used for diagnostic, therapeutic, or labeling purposes. In some embodiments, the imaging agent is a paramagnetic molecule, a fluorescent compound, a magnetic molecule, a radionuclide, an X-ray contrast agent, or a contrast agent. In some embodiments, the contrast agent is a radiopaque gas or gas-emitting compound. In some embodiments, the dendrimer conjugate comprising an imaging agent can be used to determine the location of an administered composition. Imaging agents useful for this purpose include, but are not limited to, fluorescent tags, radionuclides, and contrast agents. Examples of imaging agents useful for diagnostic purposes include, but are not limited to, dyes, fluorescent dyes, near-infrared dyes, SPECT contrast agents, PET contrast agents, and radioisotopes. Examples of dyes include carbocyanine, indocarbocyanine, oxacarbocyanine, thiocarbocyanine 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 Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye These include, but are not limited to, 700DX, ADS780WS, ADS830WS, and ADS832WS.

[0178] In some embodiments, the dendrimer conjugate comprises a radionuclide reporter suitable for imaging by scintigraphy, single photon emission computed tomography (SPECT), or positron emission tomography (PET). In some embodiments, the dendrimer conjugate comprises a radionuclide suitable for radiotherapy. In some embodiments, the dendrimer conjugate comprises a contrast agent for imaging by magnetic resonance imaging (MRI). In some embodiments, the dendrimer conjugate comprises a chelator or MRI contrast agent for a radionuclide useful for diagnostic imaging, and a chelator useful for radiotherapy. Thus, in some embodiments, a single dendrimer / imaging agent conjugate can simultaneously treat and diagnose a disease or condition at one or more locations in the body. In some embodiments, the dendrimer conjugate comprises a radiolabeled SPECT or scintigraphy imaging agent with a suitable amount of radioactivity.

[0179] Suitable contrast agent can be selected based on specific imaging method.For example, in some embodiments, contrast agent is near-infrared fluorescent dye for optical imaging, gadolinium chelate for MRI imaging, radionuclide for PET or SPECT imaging, or gold nanoparticle for CT imaging.

[0180] In some embodiments, the dendrimer conjugate comprises one or more imaging agents, e.g., one or more radionuclides, for PET imaging, a technique that uses special cameras and computers to detect small amounts of radioactive tracers or radiopharmaceuticals in vivo to assess organ and tissue function (e.g., to detect the early onset of disease).

[0181] PET has a half-life of approximately 110 minutes. 18 F, with a half-life of approximately 20 minutes 11 C. Half-life of approximately 10 minutes 13 N, and a half-life of approximately 2 minutes 15The present invention includes the use of coincidence detection to detect gamma rays in the form of annihilation photons from short-lived positron emitting radioisotopes, including, but not limited to, O. Thus, in some embodiments, exemplary imaging agents for use in PET imaging include various positron emitting metal ions, e.g. 51 Mn, 52 Fe, 60 Cu, 68 Ga, 72 As, 94 mTc, or 110 In some embodiments, the imaging agent includes, but is not limited to, one or more of: 18 F, 124 I, 125 I, 131 I, 123 I, 77 Br, and 76 Examples of metal radionuclides for scintigraphy or radiotherapy include: 99m Tc, 51 Cr, 67 Ga, 68 Ga, 47 Sc, 51 Cr, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 90 Y, 88 Y, 153 Sm, 166 Ho, 165 Dy, 166 Dy, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 117 mSn, 149 Pm,161 Tb, 177 Lu, 225 Ac, 198 Au and 199 Examples of suitable radionuclides include, but are not limited to, Au. The choice of metal will be determined based on the desired therapeutic or diagnostic application. For example, for diagnostic purposes, in some embodiments, useful radionuclides include: 64 Cu, 67 Ga, 68 Ga, 99m Tc, and 111 For therapeutic purposes, in some embodiments, useful radionuclides include: 64 Cu, 90 Y, 105 Rh, 111 In, 117 mSn, 149 Pm, 153 Sm, 161 Tb, 166 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 225 Ac, 186 / 188 Re, and 199 Contains Au.

[0182] In some embodiments, the imaging agent is Technetium-99m ( 99m In some embodiments, 99m Tc is useful for diagnostic applications due to its low cost, availability, imaging properties, and high specific activity. 99m The nuclear and radioactive properties of Tc make this isotope useful for scintigraphic imaging. This isotope has a single photon energy of 140 keV and a radioactive half-life of about 6 hours. 99 Mo- 99m Radionuclides useful for PET imaging include: 18 F, 4-[ 18 F] Fluorobenzaldehyde ( 18 FB), Al [ 18 F]-NOTA, 68 Ga-DOTA, and 68In some embodiments, the present invention includes 153 Sm can be used as a contrast agent together with a chelating agent, such as ethylenediaminetetramethylenephosphonic acid (EDTMP) or 1,4,7,10-tetraazacyclododecanetetramethylenephosphonic acid (DOTMP).

[0183] MRI can be used to assess brain disease, spinal cord disorders, angiography, cardiac function, and musculoskeletal injuries, among other uses. MRI does not require the use of ionizing radiation and can perform scans in any selected stereotaxy. MRI offers full three-dimensional capabilities, high soft tissue contrast, high spatial resolution, and is ideal for morphological and functional imaging. Thus, in some embodiments, the dendrimer comprises one or more contrast agents for MRI, such as one or more MRI contrast agents. Examples of MRI contrast agents are known in the art and include, but are not limited to, Gd, Mn, BaSO4, iron oxide, and iron platinum. Targeting Agents

[0184] In some embodiments, the dendrimer comprises one or more tissue targeting or tissue binding moieties to target the dendrimer to a specific location in vivo and / or to extend the in vivo residence time at a desired location in the body. For example, in some embodiments, the dendrimer is captured or bound to one or more separate tissues or organs after local or systemic administration in the body. Thus, the presence of a targeting or binding moiety can enhance the delivery of the drug to the target site compared to the dendrimer and the drug without the targeting or binding moiety. The conjugation of the dendrimer to one or more targeting or binding moieties can be performed by a spacer and a link between the spacer and the dendrimer, and / or the spacer and the targeting agent can be designed to provide a dendrimer-targeting agent complex in a releasable or non-releasable form.

[0185] An exemplary targeting agent is alendronate, which binds to hydroxyapatite on the surface of bone, extending the residence time of the dendrimer complex in bone. Alendronate is a small molecule targeting moiety that selectively binds to hydroxyapatite, a component of bone. Thus, in some embodiments, the dendrimer is conjugated to alendronate to selectively target the dendrimer to bone. In some embodiments, the conjugation between the alendronate and the dendrimer is performed by a reversible (non-covalent) linker. In other embodiments, the conjugation between the alendronate and the dendrimer is performed by a non-cleavable or minimally cleavable linker. In some embodiments, the targeting agent also has a therapeutic effect at the targeting site. In some embodiments, the dendrimer is conjugated to alendronate to target the dendrimer complex to bone and provide a therapeutic effect at the site of bone inflammation. In some embodiments, the alendronate-linked dendrimers are conjugated to one or more drugs for selective delivery of the drugs to sites of bone inflammation.

[0186] It has been established that dendrimers conjugated or complexed with the carbohydrate triantennary N-acetylgalactosamine (GalNAc) selectively accumulate in hepatocytes. Described herein are compositions of dendrimers modified by the addition of triantennary N-acetylgalactosamine (GalNAc) to the dendrimer surface.

[0187] The asialoglycoprotein receptor (ASGPR), abundantly expressed on hepatocytes, can selectively recognize galactose and N-acetylgalactosamine (GalNAc) through their carbohydrate recognition domains (CRDs), which bind tightly to the receptor. Efficient binding of carbohydrate moieties to the ASGPR receptor allows selective internalization into hepatocytes by receptor-mediated endocytosis. The low pH in endosomes disrupts the tetravalent calcium-chelation between the sugar ligand and the ASGPR receptor, thereby releasing the ligand into the hepatocyte. Once released, the receptor complex is recycled, allowing large amounts of the ligand to be internalized into hepatocytes without saturation effects. Binding of GalNAc to ASGPR occurs at the sinusoidal surface of hepatocytes, which contain approximately 500,000 ASGPR receptors per cell, of which approximately 5%-10% are present on the cell surface at any one time. Previous studies have shown that ligand binding to ASGPR depends on the type of sugar (GalNAc>Gal) and the number of sugars (4>3>2>1). The X-ray crystal structure of the extracellular domain of ASGPR revealed a shallow carbohydrate-binding pocket, which explains the requirement for multivalency. Thus, multivalent binding was explored, and the binding affinity of trivalent and tetravalent carbohydrate constructs to ASGPR is 100-1000 times stronger compared to monovalent ligands, due to the glycan clustering effect.

[0188] We demonstrated that biantennary and triantennary GalNAc ligands conjugated to siRNAs produced significantly higher levels of GalNAc-siRNA in the liver of C57BL / 6 mice upon subcutaneous administration, with 94% of GalNAc-siRNA localized to hepatocytes. Furthermore, these siRNA conjugates mediated efficient gene silencing. Further studies reported that antisense oligonucleotides (ASOs) linked to triantennary GalNAc were up to 10-fold more potent than parental ASOs in mouse models.

[0189] Carbohydrate-protein interactions play an important role in biological processes, such as receptor-mediated endocytosis, and have been applied in cell recognition studies and biomedical materials design. Carbohydrate-terminated dendrimers (glycodendrimers) have enhanced binding affinity with relevant receptors, which enables them to interact with specific cell types with avidity and selectivity for targeted drug delivery. The introduction of carbohydrate moieties in drug delivery platforms also confers biocompatibility and increases the water solubility of dendrimer complexes.

[0190] Triantennary-GalNAc provides effective multivalent binding to ASGPR on hepatocytes. Thus, in some embodiments, dendrimers are modified with one or more triantennary-GalNAc groups at one or more surface terminal groups (e.g., -OH).

[0191] Triantennary GalNAc modification of a dendrimer results in a set of three GalNAcs at each surface end group. In some embodiments, three β-GalNAc molecules are grafted onto a building block by one or more linkers to provide an AB3 building block (i.e., a triantennary GalNAc dendron) suitable for conjugation to a surface functional group of a dendrimer.

[0192] In some embodiments, three β-GalNAc molecules are grafted onto a propargylated pentaerythritol building block by one or more linkers to provide an AB3 building block suitable for conjugation to surface functional groups of a dendrimer, as shown below. [ka]

[0193] In some embodiments, conjugation of triantennary-GalNAc via one or more surface groups occurs with about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30% of the total available surface functional groups, e.g., hydroxy groups, of the dendrimer prior to conjugation. In other embodiments, conjugation of triantennary-β-GalNAc occurs with less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, or less than 50% of the total available surface functional groups of the dendrimer prior to conjugation. In some embodiments, the dendrimer is conjugated to an effective amount of triantennary-β-GalNAc to bind to ASGPR and / or target hepatocytes, and is simultaneously conjugated to an effective amount of an agent to treat, prevent, and / or image a liver disease or disorder. composition

[0194] In some aspects, the disclosure provides a composition comprising one or more dendrimer conjugates described herein. In some embodiments, the composition is a pharmaceutical composition. The pharmaceutical composition comprising one or more dendrimer conjugates can 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 preparation that can be used pharma- ceuticals. The appropriate formulation depends on the selected route of administration. In some embodiments, the composition is formulated for parenteral delivery. In some embodiments, the composition is formulated for intratumoral injection. In some embodiments, the composition can be formulated in a sterile saline or buffer solution for injection into the tissue or cells to be treated. The composition can be stored lyophilized in a single-use vial for rehydration immediately prior to use.

[0195] In some embodiments, the pharmaceutical composition comprises one or more dendrimer conjugates and one or more pharma- ceutically acceptable excipients. Examples of excipients include solvents, diluents, pH modifiers, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, isotonicity agents, stabilizers, and combinations thereof. Suitable pharma-ceutically acceptable excipients can be selected from materials that are generally recognized as safe (GRAS) and can be administered to subjects without causing undesirable biological side effects or undesirable interactions.

[0196] In some embodiments, pharma- ceutically acceptable salts can be prepared by reacting the free acid or base form of the compound with a stoichiometric amount of a suitable base or acid in water or an organic solvent (e.g., non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile). Pharmaceutically acceptable salts can include salts of the compound derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts, as well as salts formed by reacting the compound 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.

[0197] In some embodiments, the composition is formulated into a unit dosage form for ease of administration and uniformity of dosage. In some embodiments, unit dosage form refers to a physically discrete unit of the conjugate appropriate for the subject to be treated. Therapeutic effective doses can be estimated initially in cell culture assays or animal models, such as mice, rabbits, dogs, or pigs. Animal models can also be used to achieve a desired concentration range and route of administration. Such information can be used to determine useful doses and routes for administration in humans. The therapeutic efficacy and toxicity of the conjugates, such as ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal in 50% of the population), can be determined in cell cultures or experimental animals by standard pharmaceutical procedures. The dose ratio of the toxic therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50.

[0198] In some embodiments, the composition is administered locally, for example by direct injection into the site to be treated. In some embodiments, the composition is injected, applied locally, or otherwise administered directly to the vasculature on vascular tissue at or adjacent to the site of injury, surgery, or implant. For example, in some embodiments, the composition is applied locally to vascular tissue exposed during surgery or implantation, or transplantation procedures. Pharmaceutical compositions formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), enteral, and topical routes of administration are described.

[0199] In some embodiments, the dendrimer conjugate is formulated for parenteral administration. The phrases "parenteral administration" and "administered parenterally" are art-recognized terms and include methods of administration other than enteral and topical administration, such as injection, which may include intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In some embodiments, the composition is administered parenterally, for example, by subdural, intravenous, intrathecal, intraventricular, intraarterial, intraarticular, intrasynovial, intraamniotic, intraperitoneal, or subcutaneous routes.

[0200] For liquid preparations, the pharma- ceutically acceptable carrier may be, for example, an aqueous or non-aqueous solution, a suspension, an emulsion, or an 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 oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and injectable organic esters, for example, ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions, or suspensions, including saline and buffered media. The composition can also be administered in emulsions, for example, water-in-oil emulsions. Examples of oils include oils of 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, and corn oil. Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, isostearic acid, ethyl oleate, and isopropyl myristate.

[0201] In some embodiments, compositions suitable for parenteral administration include aqueous and non-aqueous sterile suspensions that can 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 can also include fluid and nutrient replenishers, as well as 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. Injectable pharmaceutical carriers for injectable compositions are known 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)).

[0202] In some embodiments, the dendrimer conjugate is formulated for enteral administration. The carrier or diluent can be a solid carrier or diluent for solid formulation, a liquid carrier or diluent for liquid formulation, or a mixture thereof. For liquid formulation, the pharma- ceutically acceptable carrier can be, for example, an aqueous or non-aqueous solution, a suspension, an emulsion, or an 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. Examples of oils and fatty acids are as described for formulating the composition for parenteral administration.

[0203] Vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Formulations include aqueous and non-aqueous isotonic sterile injection solutions that may contain, for example, antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In general, water, saline, aqueous dextrose, and related sugar solutions can be used as liquid carriers. These can also be formulated with proteins, fats, saccharides, and other components of infant formula.

[0204] In some embodiments, the dendrimer conjugate is formulated for oral administration. The oral formulation can be in the form of chewing gum, gel strip, tablet, capsule or lozenge. Encapsulating materials for preparing enteric coated oral formulations include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose 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.

[0205] In some embodiments, the dendrimer conjugate is formulated for local administration. Local administration may include, for example, direct application to exposed tissue, vasculature, mucosa, or tissue or artificial joint during surgery. A preferred tissue for local administration is a tumor. Therapeutic Use

[0206] In some embodiments, the dendrimer complexes are used to treat cancer. In other embodiments, the dendrimer complexes are used to treat autoimmune diseases. The method typically includes administering to a subject in need of modulating the immune microenvironment an effective amount of a composition including a dendrimer and one or more therapeutic agents for modulating the immune microenvironment to reduce an autoimmune response or increase an anti-tumor response.

[0207] In general, the compositions and methods of treatment thereof are useful in the context of cancer, including tumor therapy.The compositions can also be used to treat other diseases, disorders and injuries, including inflammatory diseases, including but not limited to ulcerative colitis, Crohn's disease, and rheumatoid arthritis.

[0208] In some embodiments, the subject to be treated is a human.All of the methods described can include a step of identifying and selecting a subject who needs treatment or who will benefit from administering the composition.Thus, in some embodiments, the composition of dendrimer conjugated or complexed with one or more immunomodulatory agents and / or additional therapeutic or diagnostic agents is administered to a subject who needs immunomodulation in the context of treating cancer or other diseases, disorders and injuries, including inflammatory diseases, such as ulcerative colitis, Crohn's disease, rheumatoid arthritis, and bone disease.

[0209] In some embodiments, a composition of dendrimers conjugated or complexed with one or more immunomodulatory agents and / or additional therapeutic or diagnostic agents is administered to a subject with a proliferative disease, e.g., a benign or malignant tumor. In some embodiments, the subject being treated has been diagnosed with stage I, stage II, stage III, or stage IV cancer. In some embodiments, the proliferative disease is neurofibromatosis. Neurofibromatosis refers to a group of genetic disorders that cause tumors to form on nerve tissue. These tumors can develop anywhere in the nervous system, including the brain, spinal cord, and nerves. There are three types of neurofibromatosis: neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), and schwannomatosis. NF1 is usually diagnosed in childhood, while NF2 and schwannomatosis are usually diagnosed in early adulthood. The tumors in these disorders are usually non-cancerous (benign), but can sometimes become cancerous (malignant). Thus, in some embodiments, the subject being treated has or is suspected of having a proliferative disease, such as NF1, NF2, and / or schwannomatosis. In some embodiments, the subject being treated has or is suspected of having NF1.

[0210] Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome characterized by the gradual development of slow-growing tumors called plexiform neurofibromas. These tumors involve cranial and large peripheral nerves, are initiated by loss of NF1 heterozygosity in Schwann cells, contain high levels of collagen, and are infiltrated by inflammatory cells. Because neurofibromas are highly inoperable, pharmacological therapy is the primary strategy to target these neoplasms. Selumetinib (Koselugo™), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, is the only FDA-approved drug for NF1, and even though this important inhibitor promotes partial responses in both children and adults, greater shrinkage and persistence are still required. However, the overall toxicity of combination agents is a limiting factor in the treatment of neurofibromas, thereby reducing the ability of the drugs to shrink tumors or attenuate progression.

[0211] The compositions and methods are useful for treating subjects with benign or malignant tumors by slowing or inhibiting tumor growth in the subject, reducing tumor growth or size, inhibiting or reducing tumor metastasis, and / or inhibiting or reducing symptoms associated with tumor development or growth. For example, in some embodiments, the present disclosure provides compositions and methods for treating subjects with tumors associated with (e.g., caused by) a proliferative disease, such as neurofibromatosis (e.g., NF1).

[0212] The types of cancer that can be treated with the compositions and methods include, but are not limited to, vascular cancers, such as bone, bladder, brain, breast, cervical, colorectal, esophageal, renal, liver, lung, nasopharyngeal, pancreatic, prostate, skin, stomach, and uterine cancers, such as multiple myeloma, adenocarcinoma, and sarcoma. In some embodiments, the compositions are used to treat multiple cancer types simultaneously. The compositions can also be used to treat metastases or tumors in multiple locations.

[0213] In some embodiments, the composition of dendrimers conjugated or complexed with one or more immunomodulatory agents and / or additional therapeutic or diagnostic agents is administered to a subject with an autoimmune or inflammatory disease or disorder. Autoimmune diseases occur when the body's natural defense system cannot effectively distinguish between the body's own cells and foreign cells, causing the body to mistakenly attack normal cells. There are more than 80 types of autoimmune diseases that affect a wide range of body parts. Common autoimmune diseases include rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), type 1 diabetes, inflammatory bowel disease, and thyroid disease.

[0214] In some embodiments, the composition is used to treat an autoimmune or inflammatory disease or disorder, such as, for example, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, anklosing 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, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome-immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, dermatomyositis, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, grey's disease, glaucoma ... It can also be used for the treatment of Busu's 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, polyglancular 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.

[0215] In some embodiments, the compositions and methods can also be used for the treatment of autoimmune or inflammatory diseases or disorders involving the bones and joints, including infectious diseases and immune-mediated local and systemic diseases.

[0216] The composition and method are suitable for treating one or more diseases or disorders of the eye.The composition and method are suitable for relieving one or more symptoms associated with one or more diseases or disorders of the eye, such as discomfort, pain, dryness, excessive tear production, injury, infection, burns, and gradual blindness.

[0217] In some embodiments, the eye disorder treated is a posterior eye disease, such as diabetic eye disease, symptomatic vitreomacular adhesion / vitreomacular traction (sVMA / VMT), and wet (neovascular) or dry AMD (age-related macular degeneration). In some embodiments, the eye disorder treated is one or more retinal and choroidal vascular diseases (e.g., AMD, retinopathy of prematurity, diabetic macular edema, retinal vein occlusion, retinopathy associated with chemotherapy toxicity, such as MEK retinopathy). In some embodiments, the eye disorder treated is age-related macular degeneration (AMD). Age-related macular degeneration (AMD) is a neurodegenerative neuroinflammatory disease of the macula that causes loss of central vision. The pathogenesis of age-related macular degeneration involves chronic neuroinflammation in the choroid (vascular layer under the retina), retinal pigment epithelium (RPE), cell layers under the neurosensory retina, Bruch's membrane, and the neurosensory retina itself.

[0218] In other embodiments, the eye disorder to be treated is ocular inflammatory disease, i.e., eye disease associated with inflammation of eye tissue, including, for example, AMD, retinitis pigmentosa, optic neuritis, sarcoid, retinal detachment, temporal arteritis, retinal ischemia, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, Stargardt's disease (also known as Stargardt's macular dystrophy or juvenile macular degeneration), geographic atrophy, neuromyelitis optica, and also includes, for example, angiogenic disease, including retinal neovascularization and choroidal neovascularization.Other conditions may also cause inflammation and / or angiogenesis in the eye, such as infection, sickle cell disease, hypotension, etc.

[0219] Further examples of eye disorders that may be treated include amebic keratitis, fungal keratitis, bacterial keratitis, viral keratitis, onchocercal keratitis, bacterial keratoconjunctivitis, viral keratoconjunctivitis, corneal dystrophic disease, Fuchs' endothelial dystrophy, meibomian gland dysfunction, anterior and posterior blepharitis, conjunctival hyperemia, conjunctival necrosis, cicatrical scarring and fibrosis, punctate epithelial keratopathy, filamentous keratitis, corneal erosions, thinning, ulcers and perforations, Sjogren's syndrome, Stevens-Johnson syndrome, autoimmune dry eye disease, environmental dry eye disease, and corneal keratitis. Eye diseases, corneal neovascularization diseases, prevention and treatment of corneal transplant rejection, autoimmune uveitis, infectious uveitis, anterior uveitis, posterior uveitis (including toxoplasmosis), panuveitis, vitreous or retinal inflammatory diseases, prevention and treatment of endophthalmitis, macular edema, macular degeneration, age-related macular degeneration, proliferative and non-proliferative diabetic retinopathy, hypertensive retinopathy, autoimmune diseases of the retina, primary and metastatic intraocular melanoma, other intraocular metastatic tumors, open-angle glaucoma, angle-closure glaucoma, pigmentary glaucoma, and combinations thereof. Other disorders include corneal injury, burns or abrasions, cataracts, and age-related degeneration of the eye or vision associated therewith.

[0220] In some embodiments, a triantennary-GalNAc modified dendrimer complexed or conjugated to one or more agents for treating, preventing, and / or diagnosing one or more liver disorders and / or diseases is administered to a subject to treat, prevent, and / or diagnose one or more symptoms of one or more liver disorders and / or diseases in the subject.

[0221] The dendrimer-triantennary-β-GalNAc compositions are effective in treating or ameliorating one or more symptoms of a liver disease or disorder, such as acute or chronic liver disease. Exemplary indications that may be treated include, but are not limited to, acute liver failure (acute hepatitis, fulminant hepatitis), e.g., resulting from neoplastic infiltration, acute Budd-Chiari syndrome, heat stroke, mushroom ingestion, metabolic diseases, e.g., Wilson's disease, or those associated with viral liver disease, e.g., caused by herpes simplex virus, cytomegalovirus, Epstein-Barr virus, parvovirus, hepatitis viruses (e.g., Hepatitis A, Hepatitis E, Hepatitis D+B infections), or drug-induced liver injury, including rifampicin-induced hepatotoxicity, acetaminophen-induced hepatotoxicity, recreational drug-induced toxicity, e.g., from 3,4-methylenedioxy-N-methylamphetamine (MDMA, also known as ecstasy), or cocaine-induced toxicity, acute ischemic hepatocellular injury, or hypoxic hepatitis, or those resulting from traumatic liver injury. The methods can treat and prevent any hyperacute, acute and subacute liver disease defined by the development of encephalopathy, coagulopathy and jaundice in individuals with previously normal livers.

[0222] Symptoms and clinical symptoms of acute liver disease include jaundice and encephalopathy, and liver dysfunction (e.g., metabolic function loss, decreased gluconeogenesis leading to hypoglycemia, decreased lactate clearance leading to lactic acidosis, decreased ammonia clearance leading to hyperammonemia, and decreased synthetic ability leading to coagulopathy).Acute liver disease and injury are often associated with multiple systemic manifestations, including immunoparesis, which contributes to high-risk sepsis; systemic inflammatory response with high energy consumption or catabolic rate; portal hypertension; renal insufficiency; myocardial injury; pancreatitis (especially in acetaminophen-related disease); inadequate glucocorticoid production in the adrenal gland, which contributes to hypotension; and acute lung injury, which leads to acute respiratory distress syndrome.

[0223] All of the methods described may also include a step of identifying and selecting a subject in need of treatment or who would benefit from administration of the composition. In some embodiments, the subject has been medically diagnosed with acute liver disease or disorder by exhibiting clinical (e.g., physical) symptoms of the disease. In other embodiments, the subject has been medically diagnosed with subacute or chronic liver disease or disorder by exhibiting clinical (e.g., physical) symptoms indicating an increased risk or likelihood of developing acute liver disease. Thus, in some embodiments, a formulation of the disclosed dendrimer composition is administered to the subject prior to clinical diagnosis of acute liver disease.

[0224] In some embodiments, the methods treat or prevent non-alcoholic steatohepatitis, liver fibrosis associated with non-alcoholic steatohepatitis, primary biliary cholangitis.

[0225] In some embodiments, the dendrimer conjugates can be administered in combination with one or more additional therapeutically active agents known to be capable of treating the conditions or diseases discussed above. EXAMPLES

[0226] Example 1 Conjugation of didesethylsunitinib via a non-cleavable linkage Overexpression of vascular endothelial growth factor (VEGF) has been implicated in several diseases associated with angiogenesis. Sunitinib is a receptor tyrosine kinase inhibitor that blocks VEGF receptors and has excellent antiangiogenic activity, and has been approved by the FDA for use in various types of cancer. Didesethyl sunitinib is the active metabolite of sunitinib. Despite the excellent therapeutic value of sunitinib and its analogs, their clinical development is hindered by associated toxicity. The dendrimer-didesethyl sunitinib conjugate aims to overcome the dose-related toxicity of sunitinib by attaching sunitinib to a hydroxyl-terminated dendrimer. The chemical structure of the dendrimer conjugate synthesized in this example is shown in Figure 1.

[0227] Synthesis and characterization of N,N-didesethylsunitinib amide azide

[0228] The reaction scheme for the synthesis of N,N-didesethylsunitinib azide using an amide linkage is shown in FIG.

[0229] Step 1: Synthesis of 5-fluoro-2,3-dihydro-1H-indol-2-one (compound 2)

[0230] To a stirred solution of 5-fluoro-2,3-dihydro-1H-indole-2,3-dione (6.0 g, 1.0 equiv.) in n-butanol (10 V) was added triethylamine (6.12 mL, 1.2 equiv.) followed by hydrazine hydrate (3.56 mL, 2.0 equiv.) at room temperature. The resulting solution was stirred at 100° C. for 16 h. The progress of the reaction was monitored by TLC (50% ethyl acetate in hexanes). Once the reaction was deemed complete, the reaction mass was directly evaporated to dryness under vacuum at 45° C. to give a dark brown solid. The resulting solid was quenched with water (20 V), extracted with ethyl acetate (30 V) and the organic layer was washed with water. The organic layer was concentrated to dryness on a rotary evaporator. The crude product was purified by recrystallization using ethyl acetate to give a fluffy grey solid (4.0 g, 72% yield). Compound 2, shown in FIG. 2, was prepared by: 1 Confirmation was achieved by 1 H NMR, liquid chromatography, and mass spectrometry.

[0231] Step 2: Synthesis of 5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (compound 4)

[0232] To a stirred solution of 5-fluoro-2,3-dihydro-1H-indol-2-one (compound 2) (4.0 g, 1.0 equiv.) and 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (compound 3) (4.41 g, 1.0 equiv.) in ethanol (10 V) was added pyrrolidine (4.42 mL, 2.0 equiv.) at room temperature. The resulting solution was stirred at 80° C. for 3 h. The progress of the reaction was monitored by TLC (10% methanol in DCM). Once the reaction was deemed complete, the reaction mass was cooled to room temperature and 2M HCl solution was added until pH=3. A brownish red precipitate formed which was filtered. The resulting solid was washed with ethanol (20 V) followed by hexane (30 V) and filtered to give a reddish orange solid (6.6 g, 82% yield). Compound 4, shown in FIG. 2, was prepared by: 1 Confirmed by 1 H NMR.

[0233] Step 3: Synthesis of tert-butyl N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}carbamate (Compound 6)

[0234] To a solution of 5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (compound 4) (6.5 g, 1.0 equiv.) in DMF was added triethylamine (6.08 mL, 2.0 equiv.), EDC.HCl ((8.68 g, 2.1 equiv.), HOBT (3.94 g, 1.35 equiv.) and tert-butyl N-(2-aminoethyl)carbamate (4.16 g, 1.2 equiv.) at 0° C. The reaction was stirred at room temperature for 16 h. The reaction mixture was diluted with water (20.0 V), stirred for 10 min to precipitate and filtered to give a brown solid. The resulting solid was purified by elution with ethyl acetate (15.0 V), followed by hexanes. (15.0V), filtered and dried to give a brownish orange solid as tert-butyl N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}carbamate (compound 6) (7.5 g, 78% yield). Compound 6, shown in FIG. 1 Confirmed by 1 H NMR.

[0235] Step 4: Synthesis of N-(2-aminoethyl)-5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxamide (compound 7)

[0236] To a solution of tert-butyl N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}carbamate (compound 6) (9.0 g, 1.0 equiv.) in DCM (10.0 V) was added trifluoroacetic acid (3.0 V) at 0-5° C. The reaction was stirred at room temperature for 12 h. The reaction mass was evaporated to dryness under vacuum at 45° C. to give a dark brown solid. The solid obtained was washed with diethyl ether (15.0 V), filtered and dried to give an orange-yellow solid (crude 6.0 g). Compound 7, as shown in FIG. 2, was prepared by the following procedure: 1 Confirmation was achieved by 1 H NMR, liquid chromatography, and mass spectrometry.

[0237] Step 5: Synthesis of N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}-3-[2-(2-propoxyethoxy)ethoxy]propanamide (compound 9)

[0238] To a solution of 3-[2-(2-propoxyethoxy)ethoxy]propanoic acid (8) (5.95 g, 1.0 equiv.) in DMF (10.0 V), DIPEA (8.40 mL, 2.0 equiv.), EDC.HCl (6.90 g, 1.5 equiv.), HOBT (0.65 g, 0.2 equiv.), N-(2-aminoethyl)-5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxamide (compound 7) (11.0 g, 1.0 equiv.) and DMAP (0.294 g, 0.1 equiv.) were added at 0-5 °C. The reaction was stirred at room temperature for 3 h. The progress of the reaction was monitored by TLC (10% MeOH in DCM). The reaction mixture was diluted with water (20.0 V) and stirred for 10 min, forming a brown precipitate which was filtered. The resulting solid was purified by reverse phase column chromatography to give N,N-didesethylsunitinib amide azide as an orange solid (5.2 g, 37% yield). Compound 9 was1 Confirmation was achieved by 1 H NMR, liquid chromatography, and mass spectrometry.

[0239] Synthesis of dendrimer conjugates via non-cleavable ether linkages on dendrimers

[0240] The synthesis began by constructing a bifunctional dendrimer. In dendrimer generation 3.5, seven alkyne functional groups were introduced using a polyethyl glycol (PEG) linker with an amine at one end and a hexyne at the other to produce a generation 4 bifunctional dendrimer (compound 1 in Figure 3) with seven alkyne arms and 57 hydroxy groups on the surface. The structure of the dendrimer was confirmed by 1H NMR spectroscopy.

[0241] A clickable didesethylsunitinib analog (compound 2 in Figure 3, AVT-4517) containing three ethylene glycol (PEG3) spacers and a terminal azide was synthesized to participate in a click reaction with an alkyne group on the dendrimer surface. The active drug, compound 2, is prepared using a five-step synthesis as shown in Figure 2 and described previously.

[0242] Finally, AVT-4517 (compound 2 in Figure 3) is reacted with a bifunctional dendrimer bearing hexyne groups (compound 1 in Figure 3) by copper(I)-catalyzed alkyne-azide click chemistry to obtain D-4517.2 (compound 3 in Figure 3) with the overall structure shown in Figure 1. After conjugation of the analog to the dendrimer, D-4517.2 is purified by tangential flow filtration (TFF) to remove any impurities and allow purification into the final formulation.

[0243] D-4517.2 conjugate 1 H-NMR analysis

[0244] Formation of product D-4517.2 1 The conjugate was confirmed by H NMR. 1The H NMR spectrum clearly shows peaks corresponding to the dendrimer, the drug and the linker attached to it. The drug loading was calculated by comparing these peaks with the aid of proton integration method. The internal amide protons from the dendrimer are present between δ 8.5-7.5 ppm when the spectrum is recorded in deuterated DMSO. These amide peaks become the reference standard for the rest of the peaks. The -NH peaks from the drug appear at δ 13.6 ppm and 10.8 ppm. There are four protons from the drug and one triazole proton formed after the click reaction, which is integrated with the internal amide peak and falls between δ 8.5-7.5 ppm. In addition, two aromatic protons from sunitinib located next to the fluorine group appear at δ 6.95-6.85 ppm. A sharp triazole peak at δ 7.7 ppm, which is a characteristic peak of the click transformation, is observed when the NMR solvent is switched from deuterated DMSO to CD3OD. After clicking, CH2 is next to the azide shielded in the low field and can be observed at δ 4.4 ppm. NMR is also used to quantify the number of drug molecules conjugated to the hydroxyl dendrimer. Drug loading was calculated by comparing the protons of the dendrimer interior amide protons with the drug protons by the proton integration method.

[0245] HPLC analysis to assess the purity of D-4517.2

[0246] The purity of the dendrimer drug conjugate, intermediates and drug linker was assessed using HPLC. The final conjugate is >99% pure by HPLC. The dendrimer G4-OH and dendrimer hexine intermediates are visible in the 210 nm channel by HPLC, and didesethylsunitinib is visible at 430 nm. The retention time of compound 2 is about 16.9 min, but when the hydrophobic drug molecule is bound to the dendrimer, the peak of the final conjugate shifts to the right to about 27 min, thereby confirming the binding of the hydrophobic drug to the dendrimer construct. When the drug is bound to the dendrimer, the corresponding peak can be observed in both the 210 nm (dendrimer absorption wavelength) and 430 nm (drug absorption wavelength) channels, thereby further confirming the formation of the product. The drug loading of the dendrimer conjugate is about 12.6% wt / wt, which corresponds to 7 drug molecules bound per dendrimer molecule.

[0247] Size and Zeta Potential

[0248] The size and zeta potential distribution of D-4517.2 are determined using a Zetasizer Nano ZS instrument. For size measurements, samples were prepared by dissolving the dendrimer in deionized water (18.2Ω) to make a solution with a final concentration of 0.5 mg / mL. The solution was then directly filtered through a 0.2 μm syringe filter (Pall Corporation, 0.2 μm HT Tuffryn membrane) into a cell (UV-transparent disposable cuvette, dimensions: 12.5×12.5×45 mm). For zeta potential measurements, samples were prepared at a concentration of 0.2 mg / mL in 10 mM NaCl using the procedure mentioned above. A Malvern Zetasizer Nanoseries disposable folded capillary cell was used for the measurements. The size of D-4517 was 5.5±0.5 nm and the zeta potential was slightly positive (+5.4±0.4 mV).

[0249] Size Exclusion Chromatography Multi-Angle Laser Scattering (SEC-MALS)

[0250] The molar mass of D-4517.2 is determined by size exclusion chromatography multi-angle laser scattering (SEC-MALS).

[0251] result

[0252] D-4517 has nanomolar affinity for VEGFR2 and does not require the release of the active drug, AVT-4517. To further increase the stability of the conjugate under physiological conditions and further reduce the release of drug from the conjugate observed in buffer and plasma stability studies of D-4517, the cleavable ester linkages on the dendrimer surface were replaced with non-cleavable linkages, as demonstrated in the structure of D-4517.2 (Figure 1). No cleavable bonds are present in the structure of D-4517.2.

[0253] D-4517.2 is a covalent conjugate of a generation 4 hydroxyl-terminated PAMAM dendrimer (chemical formula: C) containing an ethylenediamine (EDA) core, amidoamine repeating units [CH2CH2CONHCH2CH2N], and 64 hydroxyl end groups, in which a didesethyl sunitinib analog (AVT-4517) has been conjugated to the dendrimer by a highly efficient click chemistry approach. 622 H 1184 N 186 O 188 ). Hydroxyl, generation 4 PAMAM dendrimers are monodisperse and produced with high compositional purity (>95%). For preparation of D-4517.2, 7 of the 64 hydroxy groups on the dendrimer are modified to attach AVT-4517 (approximately 12.6% of the total mass).

[0254] Stability studies in human, mouse and rat plasma

[0255] The in vitro stability of dendrimer-diseethyl sunitinib conjugates D-4517 and D-4517.2 in human, mouse and rat plasma was evaluated in physiological conditions. The results are presented in Figure 4. Compared to D4517 (2% (weight percentage) released in human plasma and 4% (weight percentage) released in rat plasma), the plasma stability of D4517.2 is significantly improved. At 48 hours, less than 0.5% of the drug (by weight) was released from the dendrimer-drug conjugate in all three plasmas.

[0256] binding affinity

[0257] The comparative kinase binding affinities of D-4517 and D-4517.2 were evaluated and the results are presented in Table 1. [Table 1]

[0258] The IC50 results for D-4517.2 were lower than D-4517 in all assays tested, indicating stronger binding of D4517.2 to the tyrosine kinase receptor. Example 2 Conjugation of N-acetyl-L-cysteine ​​via a non-cleavable linkage

[0259] Dendrimers conjugated with N-acetyl-L-cysteine ​​via a non-cleavable linker were synthesized. The synthetic route for the non-releasable (i.e. non-cleavable) form of the dendrimer / N-acetyl-cysteine ​​conjugate is shown in FIG. 5. As shown, N-acetyl-L-cysteine ​​was conjugated to the hydroxyl-terminated PAMAM dendrimer via a non-cleavable linkage to minimize the release of free N-acetyl-cysteine ​​in vivo after administration. The non-releasable form of the dendrimer / N-acetyl-cysteine ​​conjugate provides enhanced therapeutic efficacy compared to the releasable or cleavable form of the dendrimer / N-acetyl-cysteine ​​conjugate. Example 3 Conjugation of targeting agents via non-cleavable linkages

[0260] Triantennary GalNAc-based hydroxyl dendrimers were evaluated for targeting and delivering drugs to hepatocytes in a site-specific manner. Surface GalNAc sugars have been shown to provide a multivalent binding effect on ASGPR, allowing the dendrimers to selectively target and internalize hepatocytes in vivo in the STAM model of nonalcoholic steatohepatitis.

[0261] The synthesis scheme of β-GalNAc-triantennary-PEG3-azide (AB3 building block) is shown in Figure 6. Reagents and conditions: (i) scandium triflate, DCE, 3 hours, 80°C, (ii) propargyl bromide, toluene, sodium hydroxide, water, TBAB, (iii) pyridine, thionyl chloride, chloroform, 65°C, 2 hours, (iv) tetrabutylammonium hydrogen sulfate, 50% NaOH, 16 hours, room temperature, (v)(iii) CuSO4.5H2O, Na ascorbate, THF, water, 10 hours, (vi) DMF, tetrabutylammonium iodide, NaN3, 80°C, 5 hours, (vii) sodium methoxide, dry methanol, 30°C, 3 hours.

[0262] Triantenary building blocks were prepared. Three molecules of beta-GALNAc-PEG3 azide were grafted onto the propargylated pentaerythritol building block to obtain an orthogonal building block of AB3 type. The synthesis started with the glycosylation reaction of β-D-GalNAc pentaacetate (1, Figure 6) with 2-[2-(2-azidoethoxy)ethoxy]ethan-1-ol (2) in the presence of scandium triflate in dichloroethane to give peracetylated β-GalNAc-PEG3-azide (3). On the other hand, pentaerythritol 4 was selectively modified with three propargyl arms according to literature methods in the presence of sodium hydroxide and tetrabutylammonium bromide in DMSO to give tripropargyl pentaerythritol (5). The single remaining hydroxy group on compound (5) was reacted with bis-chlorotetraethylene glycol (7) using sodium hydroxide and TBAB in DMSO to give intermediate compound (8). In the next synthetic step, peracetylated β-GalNAc-PEG3-azide was clicked with AB3 building block (8) using conventional CuAAC click reaction conditions (copper(II) sulfate pentahydrate and sodium ascorbate in THF:water) to give compound (9). A successful click reaction was confirmed by 1 Confirmed by 1 H NMR, HRMS and HPLC. 1 In the H NMR, a sharp singlet characteristic of triazole at δ 7.9 ppm was observed. Other characteristic peaks were the acetate peak between δ 2.0 and 1.74 ppm, the GalNAc protons between δ 5.2 and 3.2 ppm, and the NH of GALNAC at δ 7.78 ppm. In the next synthetic step, the terminal chloride group of compound (9) was converted to an azide by nucleophilic substitution in the presence of sodium azide and tetrabutylammonium iodide in DMF to give compound (10). The final step was a transesterification using Zemplen conditions, where the reaction was carried out using sodium methoxide in methanol to give the deacetylated β-GalNAc-triantetennary-PEG3 azide (11) building block.

[0263] Successful completion of the reaction is 1 This is confirmed by 1 H NMR, where the peak corresponding to O-acetate has completely disappeared and all sugar protons have shifted upfield. The entire synthetic sequence is 1 Characterization using 1 H NMR, HPLC, and HRMS confirmed the desired compound.

[0264] Dendrimer-β-GalNAc conjugates are prepared as described in Examples 1 and 2. Example 4 Preparation of PEG-alkyne functionalized PAMAM-G4-OH.

[0265] PAMAM G4-OH-alkyne 7~8 was produced in a multi-step process, which was successfully scaled up to provide 1 kg and 0.5 kg lots of this material. [ka] [ka]

[0266] A manufacturing process for poly(amidoamine) generation 4 hydroxyl-terminated dendrimers (PAMAM-G4-OH) was developed for targeted agent / drug delivery applications. Azide modification of PAMAM G4-OH was used to enable conjugation of drugs using azide-alkyne cycloaddition (click) conjugation technology. One PAMAM G4-OH synthetic route follows a diverse strategy for the preparation of PAMAM dendrimers. The final step of the process is the addition of ethanolamine and PEG-alkyne amine, resulting in G4 dendrimers with 56-57 alcohol functional groups and 7-8 PEG-alkyne functional groups. [ka]

[0267] Analysis method

[0268] For higher generation PAMAM dendrimers (≧G4), several analytical methods were developed and are outlined below.

[0269] UPLC method (ASHV001O)

[0270] A reversed-phase UPLC method for separating each PAMAM dendrimer generation and starting material was developed using a modification of a literature method (Table 1-1). See Cason, CA, et al. Journal of Nanomaterials. 2008, 1-7. DOI:10.1155 / 2008 / 456082. [Table 1-1]

[0271] The method was developed using PAMAM core obtained by synthesis and samples G0-G4 obtained from Sigma Aldrich, Inc. A summary of sample retention times and UPLC reports for the final two generations is provided below in Table 2. [Table 2]

[0272] SEC-MALLS method

[0273] A size-exclusion chromatography method using multi-angle laser light scattering (MALLS) and differential refractive index (dRI) detection was developed adapted from a method described in the literature. Mullen, DG, et. al. Macromolecules. 2012, 45, 5316-5320. A summary of the method is provided in the appendix. Peaks of each PAMAM generation could be resolved using this method. A summary of the elution times and measured polydispersities for the final two generations is provided in Table 3 below. [Table 3]

[0274] quantitative 1 H NMR

[0275] quantitative 1 H NMR spectroscopy was performed with reference to Sigma Aldrich TraceCERT® grade internal standard. These methods were used to quantify the potency of the PAMAM dendrimer solutions and isolated products. The method was further used to assay the quality of the PEG-alkyne material (2-[2-(propargyloxy)ethoxy]ethylamine). 1,3,5-trimethoxybenzene was used as the solvent, d 4 - in methanol was used as an internal standard.

[0276] Protocol for analysis of the average number of PEG-alkyne arms

[0277] Determination of the degree of functionalization of PAMAM G4-OH with PEG-alkyne 1 The integrals were determined by H-NMR spectroscopy using the average values ​​provided by two methods, designated Method A and Method B. Each method utilizes the integrals observed for PAMAM-G4-64-OH and the theoretical integrals that would be observed for 100% PEG-alkyne functionalized PAMAM-G4-64-PEG-alkyne. The respective integrals for these two materials are provided below in Tables 4 and 5. [Table 4] [Table 5]

[0278] Method A. Method A is a method for the synthesis of 100% alkyne-functionalized PAMAM-G4. m Compared with the integral of m This is based on the calculation of the percentage of the actual integral of the proton Ha and H f was determined by setting the integral for the signal corresponding to to its theoretical value of 248 (this signal is predicted to be 248 for both PAMAM-G4-64-OH and PAMAM-G4-64-PEG-alkyne). An example calculation of this method is seen in FIG.

[0279] Method B. Method B uses the proton H from the ethanolamine terminal arm. e Proton H from the PEG-alkyne terminal arm m The calculation was based on the ratio of the protons H of the ethanolamine arms. e and H of the PEG-alkyne terminal arm. k and H l Due to overlap with the protons, the integrals of these PEG-alkyne protons must be subtracted (H m (The integrals of the protons are used to determine the integrals.) This ratio can then be used to calculate the average number of alkyne-functionalized arms. An example calculation is shown in Figure 8.

[0280] Manufacturing Overview

[0281] Synthesis of PAMAM core [ka] [Table 44]

[0282] The synthesis of the PAMAM dendrimer core was carried out according to what has been described in the literature, as shown in the scheme above. A solution of ethylenediamine in methanol was added dropwise to a solution of excess methyl acrylate in methanol at 0° C. After 1 h, the solution was warmed to room temperature and stirred for 24 h. The solution was then concentrated under reduced pressure at 20° C. The resulting oil was then diluted with MeOH (1 L) and concentrated again under reduced pressure. This was repeated once more to give the PAMAM core as a colorless oil in quantitative yield (17.5 grams).1 H-NMR and GC analysis showed that the PAMAM core was produced in high purity. A summary of the process metrics is shown in Table 6 below. [Table 6]

[0283] Synthesis of full-generation amine-terminated PAMAM dendrimers

[0284] Full generations of PAMAM dendrimers were prepared according to literature conditions. Under these conditions, a solution of ethylenediamine (86 w / w% in methanol, 25 equivalents per ester on the PAMAM starting material) was cooled to 0° C. under a nitrogen atmosphere. A solution of half-generation PAMAM dendrimer or PAMAM core (10 w / w% in methanol) was then added over a period of 2 hours. After the addition, the reaction mixture was stirred at 0° C. for 1 hour. After this time, the reaction mixture was warmed to 20° C. and stirred for 5 days. The crude product was then concentrated by vacuum distillation while maintaining the temperature of the crude product below 25° C. Residual ethylenediamine was then removed by one of two methods: Method 1: 1. Dissolve the crude product residue in methanol (2.5 volumes). 2. Dilute with toluene (24 volumes). 3. Concentrate by vacuum distillation. This process was repeated four times or analyzed by GC and 1 Repeat until no more residual ethylenediamine was detected in the crude product or distillate by H-NMR analysis. Method 2: 1. Dissolve the crude residue in pentanol to make a 10 wt% dendrimer solution. 2. Concentrate by distillation using a wiped film evaporator. This process was repeated four more times or analyzed by GC and 1 Repeat until no more residual ethylenediamine was detected in the crude product or distillate by H-NMR analysis. Method 1 was used for PAMAM G0 and G1, and Method 2 was used for PAMAM G2 and G3. A summary of the results during the synthesis of each full generation of amine-terminated PAMAM dendrimers is provided below in Table 7. [Table 7]

[0285] Synthesis of half-generation ester-terminated PAMAM dendrimers

[0286] Half-generation PAMAM dendrimers were prepared according to literature conditions. Under these conditions, a solution of methyl acrylate (55% w / w in methanol, 3 equivalents per terminal amine on the dendrimer starting material) was cooled to 0° C. under a nitrogen atmosphere. A solution of full-generation PAMAM dendrimer (10% w / w in methanol) was then added over a period of 2 hours. After the addition, the reaction mixture was stirred at 0° C. for 1 hour. After this time, the reaction mixture was warmed to 20° C. and the crude product was 1 The mixture was stirred for 3-4 days until H-NMR analysis showed complete conversion of the starting material. The crude product was then concentrated by vacuum distillation while maintaining the temperature of the crude product below 25 °C. Residual methyl acrylate was then removed by azeotroping with methanol three times by vacuum distillation (removal of methyl acrylate was performed using a 100% ethanol distillation flask). 1 (As demonstrated by H-NMR, GC, and / or UPLC analysis.) A summary of the results during the synthesis of each half-generation PAMAM dendrimer is provided below in Table 8. [Table 8]

[0287] PAMAM G4-OH-alkyne 7~8 Generation of [ka] [ka]

[0288] A jacketed vessel is charged with ethanolamine and PEG-alkyne in a 3:1 molar ratio, followed by methanol. The vessel is purged with N2 (gas) and cooled to 0°C. PAMAM G3.5, 10 wt% in methanol, is then added to the reactor over 2 hours. The solution is then stirred at 0°C for 4 hours, before being warmed to 20°C. The solution is then stirred at 20°C for 6 days. After this time, the solution is diluted to half strength with water and subjected to purification by ultrafiltration to provide the product as an aqueous solution. For production batch AA10-065, the aqueous solution is then subjected to purification using the largest pore size membrane to remove dimer impurities. The solvent is then replaced with methanol by ultrafiltration using 10 diafiltration volumes of methanol. A summary of the results for each scale-up is provided in Table 9 below. [Table 9]

[0289] conclusion

[0290] PAMAM G4-OH-alkyne 7~8 A process was successfully scaled up and developed to produce more than 1 kg of the compound. The process was performed at 500 gram and 2 kg scales and produced products of reproducible quality and yield. This demonstrates that product quality can be increased and that the dimer impurity can be removed during downstream purification by ultrafiltration. This allowed for the removal of a significant amount of the dimer impurity produced upstream in the pathway leading to lot AA10-065.

[0291] experiment

[0292] Synthesis of PAMAM core [ka] [Table 45]

[0293] The synthesis of the PAMAM dendrimer core was carried out according to that already described in the literature. A solution of ethylenediamine in methanol was added dropwise to a solution of excess methyl acrylate in methanol at 0° C. After 1 h, the solution was warmed to room temperature and stirred for 24 h. The solution was then concentrated under reduced pressure at 20° C. The resulting oil was then diluted with MeOH (1 L) and concentrated again under reduced pressure. This was repeated once more to give the PAMAM core as a colorless oil in quantitative yield (17.5 grams). 1 H-NMR and GC analyses showed that the PAMAM core was produced in high purity. A summary of the process metrics is shown in Table 10. [Table 10]

[0294] Synthesis of PAMAM G0.0 [ka] [Table 46]

[0295] A 5 L jacketed vessel was charged with a solution of ethylenediamine in methanol under a nitrogen atmosphere. The reaction solution was cooled to 0° C. and the solution of PAMAM cores was added over 2 hours. After the addition, the reaction mixture was stirred at this temperature for 1 hour. After this time, the reaction mixture was warmed to room temperature and stirred for 5 days. A substantial portion of the methanol and ethylenediamine was then removed by vacuum distillation while maintaining the reactor contents below 25° C. Residual ethylenediamine was then removed by azeotropic distillation five times with 21 volumes of 1:9 methanol / toluene. Complete removal was confirmed by evaporating 100% methanol / toluene at 37° C. for 1 hour. 1 After complete removal of ethylenediamine, residual toluene was removed by azeotropic distillation from 3 volumes of methanol three times to give 600 grams of PAMAM G0.0 as a 35.61 w / w% solution in methanol.

[0296] Synthesis of PAMAM G0.5 [ka] [Table 11]

[0297] A 10 L reactor was charged with the methanolic solution of methyl acrylate and the methanolic solution of PAMAM core was added over a period of 2 hours at 0° C. The solution was stirred at 0° C. for an additional hour, then warmed to 20° C. and stirred for 3 days. Completion of the reaction was determined by the Kaiser test for residual primary amines. 1 This was further verified by H-NMR spectroscopy. Volatiles were then removed by distillation under reduced pressure with the reactor jacket controlled at 20°C. Residual methyl acrylate was then removed by azeotroping twice with methanol (3 volumes) after which GC and NMR of the crude material showed complete removal of methyl acrylate. The product was obtained as a viscous oil, 2359.9 g. Quantitative. 1 H-NMR analysis showed the oil to be 21.16 wt% PAMAM G0.5 and was obtained in an assay yield of 499.35 g (100.11% yield). A summary of process metrics is provided in Table 12. [Table 12]

[0298] Synthesis of PAMAM G1.0 [ka] [Table 13]

[0299] A 20 L jacketed reactor was purged with N2 (g) and charged with the ethylenediamine solution. The PAMAM G0.5 solution was added dropwise at 0° C. over a period of about 2 hours. After stirring at 0° C. for about an additional 4 hours, the solution was warmed to 20° C. and stirred for 5 days, after which 1 H-NMR analysis showed complete consumption of the methyl ester of PAMAM G0.5. Volatiles were removed under reduced pressure with the reactor jacket controlled at 20° C. Residual ethylenediamine was then removed by azeotropic distillation with 1:9 MeOH / toluene (23 volumes) seven times. Removal of ethylenediamine was monitored by NMR of the crude reaction mixture. Residual toluene was then removed by concentration under reduced pressure from methanol three times. The product was obtained as an oil. Quantitative H-NMR analysis showed that the oil was 23.5 wt% PAMAM G1.0 and was obtained in an assay yield of 563.03 g (95% yield). A summary of the process metrics is shown in Table 14. [Table 14]

[0300] Synthesis of PAMAM G1.5 [ka] [Table 15]

[0301] A 10 L reactor was charged with a methanolic solution of methyl acrylate and a methanolic solution of PAMAM G1.0 was added over a period of 2 hours at 0° C. The solution was stirred at 0° C. for an additional 2 hours, then warmed to 20° C. and stirred for 3 days. Completion of the reaction was confirmed by 1The reaction mixture was verified by H-NMR spectroscopy. Volatiles were then removed by distillation under reduced pressure with the reactor jacket controlled at 20° C. Residual methyl acrylate was then removed by azeotroping with methanol (3 volumes) three times, after which GC and NMR of the crude material showed complete removal of methyl acrylate. The product was obtained as a viscous oil, 2359.9 g. Quantitative. 1 H-NMR analysis showed the oil to be 32.89 wt% PAMAM G1.5 and was obtained in an assay yield of 1025 g (91% yield). A summary of process metrics is provided in Table 16 below. [Table 16]

[0302] Synthesis of PAMAM G2.0 [ka] [Table 17]

[0303] The jacketed reactor was purged with N2 (gas) and charged with the ethylenediamine solution. The PAMAM G1.5 solution was added dropwise at 0° C. over a period of about 2 hours. After stirring at 0° C. for about an additional 4 hours, the solution was warmed to 20° C. and stirred for 5 days, after which 1 H-NMR analysis showed complete consumption of the methyl ester of PAMAM G1.5. Volatiles were removed under reduced pressure. Residual ethylenediamine was then removed by distillation. Removal of ethylenediamine was monitored by NMR of the crude reaction mixture. The product was obtained as an oil. Quantitative H-NMR analysis showed that the oil was 9.5 wt% PAMAM G2.0. A summary of process metrics is shown in Table 18. [Table 18]

[0304] Synthesis of PAMAM G2.5 [Table 19]

[0305] A 20 L jacketed reactor was charged with the methanolic solution of methyl acrylate and the methanolic solution of PAMAM G2.0 was added over a period of 2 hours at 0° C. The solution was stirred at 0° C. for an additional 2 hours, then warmed to 20° C. and stirred for 4 days. Completion of the reaction was confirmed by 1 The reaction was verified by H-NMR spectroscopy. Volatiles were then removed by distillation under reduced pressure with the reactor jacket controlled at 20°C. Residual methyl acrylate was then removed by azeotroping with methanol (3 volumes) three times, after which GC and NMR of the crude material showed complete removal of methyl acrylate. The product was obtained as a viscous oil, 7610 g. Quantitative. 1 H-NMR analysis showed the oil to be 15.97 wt% PAMAM G2.5, with an assay yield of 1215.3 g (100% yield). A summary of process metrics is provided in Table 20. [Table 20]

[0306] Synthesis of PAMAM G3.0 [ka] [Table 21]

[0307] The jacketed reactor was purged with N2 (gas) and charged with the ethylenediamine solution. The PAMAM G2.5 solution (HA04-064) was added via a diaphragm pump at 0°C over a period of about 2 hours. After stirring at 0°C for about an additional 4 hours, the solution was warmed to 20°C and stirred for 5 days, after which it was cooled to 20°C. 1H-NMR analysis indicated complete consumption of the methyl ester of PAMAM G2.5. Volatiles were removed under reduced pressure. Residual ethylenediamine was then removed by distillation using a wiped film evaporator. Removal of ethylenediamine was monitored by NMR of the crude reaction mixture. The product was obtained as an oil. Quantitative H-NMR analysis showed that the oil was 21.5 wt% PAMAM G3.0. A summary of process metrics is shown in Table 22. [Table 22]

[0308] Synthesis of PAMAM G3.5 [Table 23]

[0309] A 20 L jacketed reactor was charged with a methanolic solution of methyl acrylate and a methanolic solution of PAMAM G3.0 (AA08-086) was added over a period of 2 hours at 0° C. The solution was stirred at 0° C. for an additional 2 hours and then warmed to 20° C. and stirred for 4 days. Completion of the reaction was confirmed by 1 The reaction was verified by H-NMR spectroscopy. Volatiles were then removed by distillation under reduced pressure with the reactor jacket controlled at 20°C. Residual methyl acrylate was then removed by azeotroping with methanol (3 volumes) three times, after which GC and NMR of the crude material showed complete removal of methyl acrylate. The product was obtained as a viscous oil, 7610 g. Quantitative. 1 H-NMR analysis showed the oil to be 15.97 wt% PAMAM G2.5 and was obtained in an assay yield of 2360 g (99% yield). A summary of process metrics is provided in Table 24. [Table 24]

[0310] PAMAM G4-OH-alkyne7~8 Synthesis of - Lot AA10-064 [ka] [ka] [Table 25]

[0311] The jacketed reactor was purged with N2 (gas) and charged with the ethylenediamine solution. The PAMAM G3.5 solution was added via a diaphragm pump at 0°C over a period of about 2 hours. After stirring at 0°C for about an additional 4 hours, the solution was warmed to 20°C and stirred for 6 days. After this time, the solution was diluted with H2O (11 kg). The solution was then subjected to purification by ultrafiltration using a regenerated cellulose membrane with 5 kDa MWCO to obtain the product as an aqueous solution, 1 H-NMR showed complete removal of ethanolamine and PEG-alkyne. The solvent was then exchanged with methanol by successive diafiltration to give the product PAMAM G4 as a methanol solution (3230.9 g). Quantitative H-NMR analysis showed that the solution contained 16.6 wt% PAMAM G4-OH-alkyne. 7~8 (yield 93%). A summary of process metrics is shown in Table 26. [Table 26]

[0312] PAMAM G4-OH-alkyne 7~8 Synthesis of - Lot AA10-065 [ka] [ka] [Table 47]

[0313] The jacketed reactor was purged with N2 (gas) and charged with the ethylenediamine solution. The PAMAM G3.5 solution was added via a diaphragm pump at 0°C over a period of about 2 hours. After stirring at 0°C for about an additional 4 hours, the solution was warmed to 20°C and stirred for 6 days. After this time, the solution was diluted with H2O (42 kg). The solution was then subjected to purification by ultrafiltration using a regenerated cellulose membrane with 5 kDa MWCO to obtain the product as an aqueous solution, 1 H-NMR showed complete removal of ethanolamine and PEG-alkyne. The dimer impurity was then removed by ultrafiltration using a regenerated cellulose membrane with a 30 kDa MWCO, which allowed the desired product to pass through to the permeate. The solvent was then exchanged with methanol by continuous diafiltration to give the product PAMAM G4 as a methanol solution (8205 g). Quantitative H-NMR analysis showed that the solution contained 19.5 wt% PAMAM G4-OH-alkyne. 7~8 (71.8% yield). A summary of process metrics is provided in Table 27 below. [Table 27]

[0314] Analysis method

[0315] UPLC method (ASHV001O) A. Reagents 1. Water: HPLC grade 2. Acetonitrile: HPLC grade 3. Trifluoroacetic acid: HPLC grade B. Solution 1. Diluent: Milli-Q water 2. Mobile phase: A. 0.05% trifluoroacetic acid in water B. 0.05% trifluoroacetic acid in acetonitrile 3. Sample solution: Sample solution with a target concentration of approximately 2 mg / mL. 4. Blank: Diluent. C. Chromatography Conditions Column: ACE Excel 3 Super C18, LC column, 50 x 3 x 3 μm Temperature: 40℃ Flow rate: 1.27mL / min Detection: A: UV at 210 nm, 4.8 nm bandwidth B: UV at 220nm, 4.8nm bandwidth Injection volume: 10μL Implementation time: 12.3 minutes [Table 48] D. Calculation

number

[0316] GC method (ASV003F) A. Chromatography Conditions Column: Agilent CP-Volamine (part number CP7447) Inlet temperature: 250℃ Inlet pressure: 14.3psi Inlet total flow rate: 37.3mL / min Inlet split ratio: 10:1 Injection volume: 5μL Column flow rate: 3.1 mL / min FID temperature: 300℃ FID H2 flow rate: 40.0mL / min FID air flow rate: 360.0mL / min FID make-up flow rate: 25mL / min FID ignition offset: 0.5pA Duration: 20.33 minutes [Table 49] D. Calculation

number

[0317] To determine the impurities using this method, standards of ethanolamine and PEG alkyne were prepared at 14.928 and 10.332 mg / mL, respectively, in isopropyl alcohol and injected into the GC. This resulted in a retention time of approximately 6.88 minutes for ethanolamine, while the PEG alkyne had a retention time of 14.1 minutes. These solutions were combined in a 1:1 ratio. This combined solution was used as a stock solution for a 1 / 2 serial dilution using isopropyl alcohol to reach concentrations of 0.007 and 0.005 mg / mL for ethanolamine and PEG alkyne, respectively. The results of the calibration are shown below. [Table 50]

[0318] A calibration curve for ethanolamine was used to calculate the concentration of ethanolamine in the samples. Since the signal for PEG-alkyne was below the LOD, the lowest value reached in the calibration was reported as the concentration.

[0319] SEC-MALLS method A. Reagents 1. Water: HPLC grade 2. Citric acid: ACS reagent, ≥99.5% B. Solution 1. Diluent: Water, 0.1M citric acid, pH 2.7 2. Mobile phase: A. Water, 0.1M citric acid, pH 2.7 3. Sample solution: Sample solution with a target concentration of approximately 2 mg / mL. 4. Blank: Diluent. C. Chromatography Conditions Column: TosoHaas TSK-Gel Guard PHW 0662 (75mm x 7.5mm, 12μm), G2000 PW 05761 (300mm x 7.5mm, 10μm, 125Å), G 3000 PW 05762 (300mm x 7.5mm, 10μm, 200Å), G4000 PW (300mm×7.5mm, 17μm, 500Å) Temperature: 25℃ Flow rate: 1mL / min Detection: A: Wyatt Optilab refractive index detector operating at 658 nm B: Wyatt miniDAWN multi-angle laser light scattering detector operating at 658 nm with a 120 mW laser. Injection volume: 100μL Duration: 40 minutes

[0320] References for Example 4 1.Cason, C. A, et al. Journal of Nanomaterials. 2008, 1-7. DOI:10.1155 / 2008 / 456082 2.Mullen, DG, et. al. Macromolecules. 2012, 45, 5316-5320. Example 5 Preparation of PEG-alkyne functionalized PAMAM-G4-OH

[0321] A process was developed to incorporate three PEG-alkyne (PAMAM G4-OH-alkyne 3) linker arms and ten PEG-alkyne (PAMAM G4-OH-alkyne 3) linker arms into the G4.0 PAMAM dendrimer. These functionalized dendrimers were generated by adjusting the stoichiometry of the PEG-alkyne relative to ethanolamine to account for differences in reactivity. The process was successfully scaled up to provide 40 g of PAMAM G4-OH-alkyne 3 and 160 g of PAMAM G4.0-50-OH-10-PEG-alkyne. In both cases, products with low polydispersity of approximately 1.03 were obtained.

[0322] In addition, a process for the preparation of poly(amidoamine) generation 4 hydroxyl-terminated dendrimers (PAMAM-G4-OH) was developed for targeted drug delivery applications. Azide modification of PAMAM G4-OH was used to enable the conjugation of active substances using azide-alkyne cycloaddition (click) conjugation technology. A process was developed for the preparation of PAMAM G4 with an average of seven alkyne functional groups. The current PAMAM G4-OH synthetic route follows a diverse strategy for the preparation of PAMAM dendrimers. The final step of the process was the addition of ethanolamine, resulting in a G4 dendrimer with 64 alcohol functional groups. A process was developed for the preparation of two G4-OH analogs with n approximately equal to 3 and 10, respectively, for conjugation. [ka]

[0323] Analysis method

[0324] Due to the complexity of PAMAM dendrimers, the product quality characteristics were characterized by SEC-MALLS (polydispersity), 1 H NMR (identification, potency, average linker loading), 13It was characterized using several analytical methods including C NMR (identification) and GC (residual ethanolamine and PEG alkyne monomer).

[0325] SEC-MALLS method

[0326] A size-exclusion chromatography method using multi-angle laser light scattering (MALLS) and differential refractive index (dRI) detectors was developed, adapted from a literature method. See Mullen, DG, et. al. Macromolecules. 2012, 45, 5316-5320. The peaks of each PAMAM generation could be resolved using this method. A summary of the elution times and measured polydispersities per generation is provided in Table 28. [Table 28]

[0327] quantitative 1 H NMR

[0328] quantitative 1 H NMR spectroscopy was performed with reference to Sigma Aldrich TraceCERT® grade internal standard. These methods were used to quantify the potency of the PAMAM dendrimer solutions and isolated products. The method was further used to assay the quality of the PEG-alkyne material (2-[2-(propargyloxy)ethoxy]ethylamine). 1,3,5-trimethoxybenzene was used as the solvent, d 4 - in methanol was used as an internal standard.

[0329] Process Development

[0330] PAMAM Generation 3.5 Source Material

[0331] A solution of PAMAM generation 3.5 in methanol (29.4 w / w%) was made up prior to use. The quality attributes for this input material are provided in Table 29. [Table 29]

[0332] Reaction of PAMAM G3.5 with PEG-alkyne / ethanolamine under high amine excess conditions

[0333] A series of experiments were performed exploring variation of the PEG-alkyne / ethanolamine molar ratio under high amine excess conditions (1600 equivalents total amine, 25 equivalents per terminal ester). First, a solution of PEG-alkyne and ethanolamine (approximately 1600 equivalents) in methanol was cooled to 0° C. Then, a solution of PAMAM G3.5 (10 w / w%) in methanol was added dropwise over 2 hours. After stirring at 0° C. for an additional 2 hours, the solution was warmed to 20° C. and stirred for 6 days. For purification, the material was processed by TFF to provide an aqueous solution of the product. For analysis, an aliquot of the solution was taken, then the water was removed by vacuum, and it was 1 The results were analyzed by H-NMR. Following the same protocol, three other reactions were set up to explore the PEG-alkyne / ethanolamine ratio to reach 10 and 14 alkyne chains. Similar studies are currently underway to optimize the conditions for a degree of functionalization of an average of three alkyne arms.

[0334] Protocol for analysis of the average number of PEG-alkyne arms

[0335] Determination of the degree of functionalization of PAMAM G4-OH with PEG-alkyne 1 The integrals were determined by H-NMR spectroscopy using the average values ​​provided by two methods, designated Method A and Method B. Each method utilizes the integrals observed for PAMAM-G4-64-OH and the theoretical integrals that would be observed for 100% PEG-alkyne functionalized PAMAM-G4-64-PEG-alkyne. The respective integrals for these two materials are provided in Tables 30 and 31. [Table 30] [Table 31]

[0336] Results for experiments varying the PEG-alkyne / ethanolamine ratio

[0337] Methods A and B were used to calculate the average number of PEG-alkyne arms on the dendrimers obtained in experiment DP07-51, which used 1600 equivalents of total amines relative to the dendrimer (25 equivalents relative to the end groups). These results are summarized in Table 32. The relationship between the average number of PEG-alkyne arms and the mol % of PEG-alkyne relative to the total amine content is shown in FIG. 10. [Table 32]

[0338] The data, combined with previously developed data, are listed in Table 33 and plotted in Figure 11. These data further verify the reproducible linear correlation between the average number of PEG-alkyne arms and the mol % of PEG-alkyne relative to the total amine content. [Table 33]

[0339] Results for experiments varying the PEG-alkyne / ethanolamine ratio for n=3

[0340] A series of experiments were performed to fine-tune the PEG-alkyne / ethanolamine molar ratio to tailor the target average linker n=3 (1600 equivalents total amine, 25 equivalents per terminal ester). First, a solution of PEG-alkyne and ethanolamine (approximately 1600 equivalents) in methanol was cooled to 0° C. Then, a solution of PAMAM G3.5 (10 w / w%) in methanol was added dropwise over 2 hours. After stirring at 0° C. for another 2 hours, the solution was warmed to 20° C. and stirred for 6 days. For purification, the material was processed by TFF to provide an aqueous solution of the product. For analysis, a portion of the solution was taken, then the water was removed by vacuum, and it was 1 The results were analyzed by H-NMR.

[0341] These results are summarized in Table 34. The relationship between the average number of PEG-alkyne arms and the mol% of PEG-alkyne relative to the total amine content is shown below in Figure 12. Based on the linear equation, 9.6 mol% of PEG-alkyne is required for n=3 and 31 mol% is required for n=10. [Table 34] [Table 35]

[0342] 5g validation run to generate PAMAM G4.0 (DP07-74-1) with n=3 alkyne arms

[0343] Experiment DP07-74-1 was carried out on a 5 gram scale to allow further validation of the PEG-alkyne / ethanolamine ratio already established by the small scale reaction screen. The reaction was set up with 9.6 mol% PEG-alkyne as previously described and stirred at 20° C. for 6 days. The crude reaction mass was diluted 2-fold with water and purified by TFF using a 5 kDa ultrafiltration membrane, which allows the removal of impurities. After this purification, the water was removed by lyophilization. Quantitative 1H-NMR analysis showed this material to be 97 w / w% PAMAM G-4 material for an adjusted isolated yield of 74%. An analytical summary for this process is provided below in Table 36. The reduced isolated yield compared to the range of previous studies is not currently understood and is likely simply due to mechanical losses during TFF purification. [Table 36]

[0344] Validation of 5g to generate PAMAM G4.0 (DP07-68-1) with n=10 alkyne arms

[0345] Experiment DP07-68-1 was carried out on a 5 gram scale to allow further validation of the PEG-alkyne / ethanolamine ratio obtained from the reaction screen for the synthesis of n=10 alkyne arms. The reaction was set up with 31 mol% PEG-alkyne as previously described and stirred at 20° C. for 6 days. The crude reaction mass was diluted 2-fold with water and purified by TFF using a 5 kDa ultrafiltration membrane, which allows for the removal of impurities. After this purification, water was removed by lyophilization. An analytical summary for this process is provided in Table 37. As mentioned above, the reduced isolated yield compared to past research and development is believed to be due to mechanical losses in the filtration equipment. [Table 37]

[0346] Scale-up to produce 40 grams of PAMAM G4.0 (DP07-82-1) with 3.0 alkyne arms [ka] [ka]

[0347] Based on the results of the 5 g validation run, the process was further scaled up to produce 40 g of dendrimer functionalized with n=3 PEG-alkyne groups. A 1 liter jacketed vessel was charged with ethanolamine and PEG-alkyne in a 9.42:1 molar ratio, followed by methanol. The vessel was purged with N2 (gas) and cooled to 0°C. PAMAM G3.5, 10 wt% in methanol, was then added to the reactor over a period of 2 hours. The solution was then stirred at 0°C for 2 hours before warming to 20°C. The solution was then stirred at 20°C for 6 days. After this time, the solution was diluted to half strength with water and subjected to purification by ultrafiltration to provide the product as an aqueous solution. The solution was frozen and the water was removed by lyophilization to give the product as a light yellow foam. A small sample of the foam was removed by lyophilization to give the product as a light yellow foam. 1 H-NMR and 13 A 15.3 wt % methanol solution (Lot No. DP07-82-2) was made. A summary of the results for scale-up is provided in Table 38. [Table 38]

[0348] Scale-up to produce 160 grams of PAMAM G4.0 (DP07-85-1) with 10.0 alkyne arms [ka] [ka]

[0349] Similarly, the synthesis of PAMAM G4.0 was scaled up to produce a dendrimer functionalized with n=10 PEG-alkyne groups, as previously described. A 5 liter jacketed vessel was charged with ethanolamine and PEG-alkyne in a 2.23:1 molar ratio, followed by methanol. The vessel was purged with N2 (gas) and cooled to 0°C. PAMAM G3.5, 10 wt% in methanol, was then added to the reactor over a period of 2 hours. The solution was then stirred at 0°C for 2 hours before warming to 20°C. The solution was then stirred at 20°C for 6 days. After this time, the solution was diluted to half concentration with water and subjected to purification by ultrafiltration. The solution was frozen and the water was removed by lyophilization, yielding the product as a light yellow foam. A small sample of the foam was then added to the reactor over a period of 2 hours. 1 H-NMR and 13 A solid was taken for analysis by C-NMR to determine the degree of alkyne functionalization using methods A and B described in Example 4. The remaining solid was dissolved in methanol to make a 15.3 wt % methanol solution (Lot No. DP07-85-3). A summary of the results for scale-up is provided in Table 39. [Table 39]

[0350] conclusion

[0351] More than 40 g of PAMAM G4-OH-alkyne 3 and more than 224 g of PAMAM G4-OH-alkyne 10 A process for producing has been successfully developed, with product quality attributes within expected control limits.

[0352] experiment

[0353] Synthesis of PAMAM G4-OH-alkyne 3 - Lot DP07-82-2 [ka] [ka] [Table 51]

[0354] A 1 liter jacketed vessel was charged with ethanolamine and PEG-alkyne (Ambeed, Lot No. 100902012-00223 0BFY) in a 9.42:1 molar ratio, followed by methanol. The vessel was purged with N2 (gas) and cooled to 0°C. PAMAM G3.5 (Dentritech, Lot No. 0121-02-E3.5-LD-A), 10 wt% in methanol, was then added to the reactor over a period of 2 hours. The solution was then stirred at 0°C for 2 hours before warming to 20°C. The solution was then stirred at 20°C for 6 days. After this time, the solution was diluted to half strength with water and subjected to purification by ultrafiltration to provide the product as an aqueous solution. The solution was frozen and the water was removed by lyophilization to give the product as a light yellow foam. A small sample of the foam was removed by lyophilization to give the product as a light yellow foam. 1 H-NMR and 13 A solid was taken for analysis by C-NMR to determine the degree of alkyne functionalization using methods A and B described in Example 4. The remaining solid was dissolved in methanol to make a 15.3 wt % methanol solution (Lot No. DP07-82-2). A summary of the results for scale-up is provided in Table 40. [Table 40]

[0355] PAMAM G4-OH-alkyne 10 Synthesis of - Lot DP07-85-3 [ka] [ka] [Table 52]

[0356] A 5 liter jacketed vessel was charged with a 2.23:1 molar ratio of ethanolamine and PEG-alkyne (Ambeed, Lot No. 100902012-00223 0BFY), followed by methanol. The vessel was purged with N2 (gas) and cooled to 0°C. PAMAM G3.5 (Dentritech, Lot No. 0121-02-E3.5-LD-A), 10 wt% in methanol, was then added to the reactor over a period of 2 hours. The solution was then stirred at 0°C for 2 hours before warming to 20°C. The solution was then stirred at 20°C for 6 days. After this time, the solution was diluted to half strength with water and subjected to purification by ultrafiltration. The solution was frozen and the water was removed by lyophilization to give the product as a light yellow foam. A small sample of the foam was added to 1 H-NMR and 13 A 15.3 wt % methanol solution (Lot No. DP07-85-3) was made. A summary of the results for scale-up is provided in Table 41. [Table 41]

[0357] Analysis method

[0358] GC method (ASV003F) A. Chromatography Conditions Column: Agilent CP-Volamine (part number CP7447) Inlet temperature: 250℃ Inlet pressure: 14.3psi Inlet total flow rate: 37.3mL / min Inlet split ratio: 10:1 Injection volume: 5μL Column flow rate: 3.1 mL / min FID temperature: 300℃ FID H2 flow rate: 40.0mL / min FID air flow rate: 360.0mL / min FID make-up flow rate: 25mL / min FID ignition offset: 0.5pA Duration: 20.33 minutes [Table 53] D. Calculation

number

[0359] To determine the impurities using this method, standards of ethanolamine and PEG alkyne were prepared at 14.928 and 10.332 mg / mL, respectively, in isopropyl alcohol and injected into the GC. This resulted in a retention time of approximately 6.88 minutes for ethanolamine, while the PEG alkyne had a retention time of 14.1 minutes. These solutions were combined in a 1:1 ratio. This combined solution was used as a stock solution for a 1 / 2 serial dilution using isopropyl alcohol to reach concentrations of 0.007 and 0.005 mg for ethanolamine and PEG alkyne, respectively. The results of the calibration are shown below in Table 42. [Table 42]

[0360] Data from the above dilution series was used to generate a calibration curve (Figure 13).

[0361] A calibration curve for ethanolamine was used to calculate the concentration of ethanolamine in the samples. Since the signal for PEG-alkyne was below the LOD, the lowest value reached in the calibration was reported as the concentration.

[0362] SEC-MALLS method A. Reagents 1. Water: HPLC grade 2. Citric acid: ACS reagent, ≥99.5% B. Solution 1. Diluent: Water, 0.1M citric acid, pH 2.7 2. Mobile phase: A. Water, 0.1M citric acid, pH 2.7 3. Sample solution: Sample solution with a target concentration of approximately 2 mg / mL. 4. Blank: Diluent. C. Chromatography Conditions Column: TosoHaas TSK-Gel Guard PHW 0662 (75mm x 7.5mm, 12μm), G2000 PW 05761 (300mm x 7.5mm, 10μm, 125Å), G 3000 PW 05762 (300mm x 7.5mm, 10μm, 200Å), G4000 PW (300mm×7.5mm, 17μm, 500Å) Temperature: 25℃ Flow rate: 1mL / min Detection: A: Wyatt Optilab refractive index detector operating at 658 nm B: Wyatt miniDAWN multi-angle laser light scattering detector operating at 658 nm with a 120 mW laser. Injection volume: 100μL Duration: 40 minutes Example 6 Exemplary PAMAM dendrimers conjugated to drugs

[0363] Schemes 1-19 below show exemplary reactions for functionalizing a PAMAM dendrimer (e.g., a PAMAM carboxymethyl-functionalized dendrimer) with a linker attached to a functional group R (where R is an amine, alkyne, acetylene, COOH, hydroxy, bromo, DBCO, thiol, alkene, aldehyde, hydroxyl, sulfonate, nitrile). In Schemes 1-19, the polyethylene glycol (PEG) linker is [ka] Exemplary reaction partners for step 1 of Schemes 1-19 include: [ka] wherein the length of the linker may vary from 1 to 100,000 units of CH2 and ethylene glycol (PEG), and the linker can include PEG linkers having various molecular weights.

[0364] Other exemplary linkers are [ka] It is.

[0365] [ka]

[0366] [ka]

[0367] Other types of click reactions for functionalizing dendrimers include the following (shown in Schemes 3-19): [ka]

[0368] Thiol Maleimide Click [ka]

[0369] [ka]

[0370] Tetrazine Ligation [ka]

[0371] [ka]

[0372] Isoxazole formation from alkynes and oximes. [ka]

[0373] [ka]

[0374] Thiol-yne

[0375] [ka]

[0376] Thiol-ene

[0377] [ka]

[0378] Thiol - Michael Crick

[0379] [ka]

[0380] [ka]

[0381] [ka]

[0382] [ka]

[0383] Three different moieties were synthesized on one dendrimer in one pot using the following method:

[0384] [ka]

[0385] Two drugs on one dendrimer in one pot using the following method

[0386] [ka]

[0387] One drug and one antibody on one dendrimer

[0388] [ka]

[0389] One antibody and one oligonucleotide on one dendrimer

[0390] [ka]

[0391] Peptide and drug on one dendrimer in one pot

[0392] [ka]

[0393] Targeting peptide and PROTAC on one dendrimer in one pot

[0394] [ka]

[0395] Drug and RNA or DNA on one dendrimer

[0396] [ka]

[0397] Three different drugs on one dendrimer in one pot

[0398] [ka] Example 7 Dendrimer-PAK1 inhibitor conjugates

[0399] Dendrimers conjugated to PAK1 inhibitors (Frax-1036 analogs) were synthesized and characterized (Figures 56A-56C). Figure 56A shows an exemplary synthetic scheme for preparing a dendrimer-PAK1 conjugate, D4-5100 (D4-Frax-1036 analog). Binding of the PAK1 kinome was assessed for PAK1, and the results are shown in Figure 56A (insert table). Figures 56B-56C show results from characterization of D4-5100. D4-5100 was determined to be >99% pure by HPLC (Figure 56B, top panel), 1 H NMR confirmed the conjugation of six molecules (Figure 56B, bottom panel) and loading was determined to be 15%. D4-5100 was further evaluated by plasma stability studies in human plasma (Figure 56C, left panel), mouse plasma (Figure 56C, center panel), and rat plasma (Figure 56C, right panel), which demonstrated that D4-5100 appears stable for 72 hours in various plasma sources. Example 8 Dendrimer-MEK inhibitor conjugates

[0400] Dendrimers conjugated to MEK inhibitors (selumetinib, trametinib, or cobimetinib) were synthesized and characterized (Figures 57A-57T).

[0401] Figure 57A shows an exemplary synthetic scheme for preparing the dendrimer-selumetinib conjugate D4-5111 (D4-selumetinib). Figures 57B-57D show results from the characterization of D4-5111.

[0402] Figures 57E-57H show an exemplary synthetic scheme for preparing a dendrimer-trametinib conjugate, D4-5116 (D4-trametinib-amide analog), and results from characterization of D4-5116. Figure 57I shows an exemplary synthetic scheme for preparing a dendrimer-trametinib conjugate, D4-5119 (D4-trametinib-disulfide analog), and results from characterization of D4-5119. Figures 57J-57M show an exemplary synthetic scheme for preparing a dendrimer-trametinib conjugate, D4-5121 (D4-trametinib-disulfide), and results from characterization of D4-5121. Figures 57N-57P show an exemplary synthetic scheme for preparing the dendrimer-trametinib conjugate, D4-5124 (D4-trametinib-ester), and results from the characterization of D4-5124.

[0403] Figure 57Q shows an exemplary synthetic scheme for preparing dendrimer-cobimetinib conjugate D4-5123. Figures 57R-57T show an exemplary synthetic scheme for preparing dendrimer-cobimetinib conjugate D4-5120, and results from characterization of D4-5120. Example 9 Dendrimer-receptor tyrosine kinase inhibitor conjugates

[0404] Dendrimers conjugated to receptor tyrosine kinase inhibitors (dasatinib, bemcentinib (R428), duvelmatinib (TP-0903), or cabozantinib) were synthesized and characterized (Figures 58A-58T).

[0405] Figure 58A shows an exemplary synthetic scheme for preparing dendrimer-dasatinib conjugate D4-5113 (D4-dasatinib-thiolated). Figures 58B-58E show results from characterization of D4-5113. Figures 58F-58G show chemical structures and results from characterization of synthesized dendrimer-dasatinib conjugate D4-4531 (D4-dasatinib ester analog). Figure 58H shows exemplary chemical structures of dasatinib analogs for chemical conjugation via the terminal azide group.

[0406] Figure 58I shows an exemplary synthetic scheme for preparing D4-R428, a dendrimer-bemcentinib (R428) conjugate. Figures 58J-58L show results from characterization of D4-R428. Figure 58M shows an exemplary synthetic scheme for preparing D4-R428-thiolated, a dendrimer-bemcentinib (R428) conjugate. Figure 58N shows results from characterization of D4-R428-thiolated.

[0407] Figure 58O shows an exemplary synthetic scheme for preparing the dendrimer-duvelmatinib (TP-0903) conjugate D4-5132 (D4-TP-0903 analog). Figure 58P shows the results from the characterization of D4-5132.

[0408] Figure 58Q shows an exemplary synthetic scheme for preparing dendrimer-cabozantinib conjugates D4-4595 and D4-4598. Figures 58R-58T show results from the characterization of D4-4595 and D4-4598. Equivalents and Scope

[0409] In the claims, articles such as "a," "an," and "the" may mean one or more than one, unless stated to the contrary or otherwise clear from the context. A claim or description containing "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process, unless stated to the contrary or otherwise clear from the context. The invention includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of the members of a group are present in, employed in, or otherwise relevant to a given product or process.

[0410] Furthermore, the present invention encompasses all alternatives, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim can be amended to include one or more limitations found in any other claim that is dependent on the same base claim. When elements are presented as a list, for example in Markush group format, each subgroup of elements is also disclosed, and any element(s) can be excluded from the group. In general, when the invention or aspects of the invention are referred to as including certain elements and / or features, it should be understood that certain embodiments of the invention or aspects of the invention consist of or consist essentially of such elements and / or features. For purposes of simplicity, those embodiments have not been specifically described in these terms herein.

[0411] In the present specification and claims, the phrase "and / or" as used herein should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may be present as needed, whether or not related to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used with open-ended language such as "comprising", may refer in one embodiment to only A (including elements other than B as needed), in another embodiment to only B (including elements other than A as needed), in yet another embodiment to both A and B (including other elements as needed), etc.

[0412] As used herein, "or" in the specification and claims should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one of the elements or elements of the list, but also including more than one, and including additional items not listed as necessary. Terms clearly indicating the contrary, such as "only one of" or "exactly one of," or "consisting of" only when used in the claims, refer to the inclusion of exactly one element of the elements or elements of the list. In general, the term "or" as used herein shall be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") only when preceded by a phrase indicating exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0413] As used herein, the phrase "at least one" in the specification and claims with reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, and does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for elements to be present, if desired, other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one A (and optionally including more than one element other than B), with no B present; in another embodiment to at least one B (and optionally including more than one element other than A), with no A present, with optionally more than one element other than A; in yet another embodiment to at least one A, optionally including more than one element, and at least one B (and optionally including other elements), with optionally more than one element other than A; and so forth.

[0414] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

[0415] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., meaning including but not limited to. As set forth in the United States Patent Office Manual of Patent Examining Procedure, Section 2111.03, only the transitional phrases "consisting of" and "consisting essentially of" are intended to be closed or semi-closed transitional phrases, respectively. It should be understood that embodiments described in this document using open-ended transitional phrases (e.g., "comprising") are also contemplated in alternative embodiments as "consisting of" and "consisting essentially of" the features described by the open-ended transitional phrase. For example, if the application describes "a composition comprising A and B," the application also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B."

[0416] When ranges are given, the endpoints are included. Furthermore, unless otherwise specified or otherwise clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can envisage any specific value or subrange within the range described in different embodiments of the invention, to the tenth of the lower limit of that range, unless otherwise clearly indicated by the context.

[0417] This application refers to various issued patents, patent application publications, academic papers, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall prevail. In addition, any particular embodiment of the present invention that is within the scope of the prior art may be expressly excluded from any one or more of the claims. Such an embodiment may be excluded even if this exclusion is not expressly stated herein, since it is deemed to be known to those skilled in the art. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether related to the existence of prior art or not.

[0418] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments of the invention described herein is not intended to be limited to the above description, but rather as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the invention, as defined in the following claims.

[0419] The recitation of a list of chemical groups in any definition of a variable herein includes that definition of the variable as any single group or combination of the listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. In one embodiment, for example, the following items are provided: (Item 1) Supports and functionalized dendrimers of formula (IA) [ka] [In the formula, D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, iptycene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof; X is NH; Y 1 is an optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, or a covalent bond; m is an integer between 16 and 4096, inclusive; n is an integer from 1 to 100, inclusive; The polydispersity value of said functionalized dendrimer in the composition is less than or equal to 1.10. A composition comprising: (Item 2) 2. The composition according to claim 1, wherein the polydispersity value of the functionalized dendrimer in the composition is less than or equal to 1.05. (Item 3) 2. The composition according to claim 1, wherein the polydispersity value of the functionalized dendrimer in the composition is less than or equal to 1.04. (Item 4) 2. The composition according to claim 1, wherein the polydispersity value of the functionalized dendrimer in the composition is less than or equal to 1.03. (Item 5) 5. The composition according to any one of claims 1 to 4, wherein the composition comprises at least 10 grams of the functionalized dendrimer. (Item 6) 5. The composition according to any one of claims 1 to 4, wherein the composition comprises at least 50 grams of the functionalized dendrimer. (Item 7) 5. The composition according to any one of items 1 to 4, wherein the composition comprises at least 100 grams of the functionalized dendrimer. (Item 8) 5. The composition according to any one of claims 1 to 4, wherein the composition comprises at least 150 grams of the functionalized dendrimer. (Item 9) 5. The composition according to any one of items 1 to 4, wherein the composition comprises 10 to 200 grams of the functionalized dendrimer. (Item 10) 5. The composition according to any one of items 1 to 4, wherein the composition comprises 20 to 200 grams of the functionalized dendrimer. (Item 11) 5. The composition according to any one of items 1 to 4, wherein the composition comprises 50 to 200 grams of the functionalized dendrimer. (Item 12) 5. The composition according to any one of items 1 to 4, wherein the composition comprises 100 to 200 grams of the functionalized dendrimer. (Item 13) Functionalized dendrimers of formula (IA)

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Claims

1. A carrier and a functionalized dendrimer of formula (I-A) 【Chemical Formula 153】 [In the formula,[[]]END] D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, eptisene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof, X is NH, Y １ is an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, or a covalent bond, m is an integer from 16 to 4096 including both ends, n is an integer from 1 to 100 including both ends, the value of the polydispersity of the functionalized dendrimer in the composition is equal to or less than 1.10] A composition comprising the same.

2. (i) the value of the polydispersity of the functionalized dendrimer in the composition is equal to or less than 1.05, equal to or less than 1.04, or equal to or less than 1.03; and / or, (ii) the composition comprises at least 10 grams, at least 50 grams, at least 100 grams, at least 150 grams, 20 to 200 grams, 50 to 200 grams, or 100 to 200 grams of the functionalized dendrimer, the composition according to claim 1.

3. A functionalized dendrimer of formula (I-A) 【Chemical Formula 154】 [In the formula,[[]]END] D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, eptisene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof, X is NH, Y １ is alkylene optionally substituted, alkenylene optionally substituted, alkynylene optionally substituted, or a covalent bond, m is an integer from 16 to 4096 inclusive, n is an integer from 1 to 100 inclusive] A method of synthesizing The dendrimer of formula (II-A) 【Chemical formula 155】 [Wherein, D is a dendrimer selected from the group consisting of poly(amidoamine) (PAMAM) polymers, polypropylamine (POPAM) polymers, polyethyleneimine polymers, polylysine polymers, polyester polymers, eptisene polymers, aliphatic poly(ether) polymers, aromatic polyether polymers, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) polymers, and combinations thereof, t is an integer from 16 to 4096 inclusive] Under conditions suitable for forming the functionalized dendrimer of formula (I-A), one or more amines [where each amine is of the formula H ２ NR １ amine of, R １ is alkylene optionally substituted, alkenylene optionally substituted, alkynylene optionally substituted, or a covalent bond] Reacting with. A method.

4. The method according to claim 3, wherein n is 1, n is 2, n is 3, n is 4, or n is 10.

5. The method according to claim 3, wherein m is 61 or 54.

6. R １ The method according to claim 3, wherein at least one instance of R is an alkylene optionally substituted.

7. At least one instance of said one or more amines is a compound of formula (A) 【Chemical 156】 [wherein, R １Ａ is halogen, acyl optionally substituted, alkenylene optionally substituted, alkynylene optionally substituted, acetylene optionally substituted, carbocyclic optionally substituted, heterocyclic optionally substituted, aryl optionally substituted, heteroaryl optionally substituted, -CH(=N)(OH)R Ｄ１ , -CN, -NO ２ , -OR Ｄ１ , -N(R Ｄ１ａ ), ２ , -SO ２ OR Ｄ１ , or -SR Ｄ１ and R Ｄ１ is independently hydrogen, acyl optionally substituted, alkyl optionally substituted, alkenyl optionally substituted, alkynyl optionally substituted, carbocyclic optionally substituted, heterocyclic optionally substituted, aryl optionally substituted, heteroaryl optionally substituted, or an oxygen protecting group when bonded to an oxygen atom, or a sulfur protecting group when bonded to a sulfur atom, R Ｄ１ａEach time it appears, independently, it is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally R Ｄ１ａ Two examples of Ｄ１ａ , together with their intervening atoms, form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaryl ring, When possible by valence, W is -O- or -CH ２ -, p is 0, 1, 2, or 3, q is an integer between 1 and 100,000 including both ends, r is 0, 1, 2, 3, 4, 5, or 6] The method according to claim 3, wherein

8. (i) W is -O-, and optionally, W is -O-, and q is an integer between 1 and 100,000 including both ends; or, (ii) W is -CH ２ -, and q is an integer between 1 and 10,000 including both ends, the method according to claim 7.

9. (i) At least one example of said one or more amines is of the formula 【Chemical Formula 157】 [wherein q is 1, 2, 3, 4, 5, or 6] is a PEG-alkyne; (ii) At least one example of said one or more amines is of the formula 【Chemical Formula 158】 is a PEG-alkyne; (iii) At least one example of said one or more amines is ethanolamine; and / or, (iv) The dendrimer of formula (II-A) is ethanolamine and the formula 【Chemical 159】 The method according to claim 3, which is reacted with both of the PEG-alkynes, and optionally, the ratio of the ethanolamine to the PEG-alkyne is 9.42:1 or 2.2:

1.

10. (i) The ratio of the PEG-alkyne to the dendrimer of the formula (II-A) is approximately 150:1, and optionally, the ratio of the PEG-alkyne to the dendrimer of the formula (II-A) is approximately 150:1, the ratio of the ethanolamine to the PEG-alkyne is 9.42:1, and n is 3; (ii) The ratio of the PEG-alkyne to the dendrimer of the formula (II-A) is approximately 500:1, and optionally, the ratio of the PEG-alkyne to the dendrimer of the formula (II-A) is approximately 500:1, the ratio of the ethanolamine to the PEG-alkyne is 2.2:1, and n is 10; or (iii) The ratio of the PEG-alkyne to the dendrimer of the formula (II-A) is 495:1, the method according to claim 9.

11. The method according to claim 3, wherein D is PAMAM.

12. The dendrimer of the formula (II-A) is of the formula 【Chemical 160】 The method according to claim 3, which is a dendrimer.

13. The value of the polydispersity of the functionalized dendrimer of the formula (I-A) is from about 1.00 to about 1.05, and optionally, the value of the polydispersity of the functionalized dendrimer of the formula (I-A) is about 1.03, the method according to claim 3.

14. (i) The suitable conditions include a protic solvent and reacting at approximately 19°C to approximately 23°C, and optionally, the protic solvent is an alcohol, and further optionally, the protic solvent is methanol; (ii) the suitable conditions include reacting at approximately 20°C to approximately 22°C; and / or, (iii) the suitable conditions include reacting at approximately 20°C in the presence of methanol, the method according to claim 3.

15. The functionalized dendrimer of formula (I-A) is of the formula 【Chemical Formula 161-1】 【Chemical Formula 161-2】 the functionalized dendrimer of, the method according to claim 3.

16. A compound of formula (B) 【Chemical Formula 162】 [wherein, R ２ is halogen, optionally substituted acyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted acetyl, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, -N ３ -, -CH(=N)(OH)R Ｄ１ -, -CN, -NO ２ -, -OR Ｄ１ -, -N(R Ｄ１ａ ) ２ -, -SO ２ OR Ｄ１ -, or -SR Ｄ１ and R Ｄ１ is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group when bonded to an oxygen atom, or a sulfur protecting group when bonded to a sulfur atom, R Ｄ１ａEach time it appears, independently, it is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclic, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally R Ｄ１ａ Two examples of Ｄ１ａ , together with their intervening atoms, form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaryl ring, provided that R １Ａ and R ２ are reaction partners, L Ｂ is an alkylene linker, where one or more chain atoms of the hydrocarbon chain are independently replaced by an amide, ester, hydroxamate, ether, carbonate, carbamate, hydrazone, thioether, thioester, disulfide, orthoester, urethane or oxime moiety, or a cyclic moiety, and T is a therapeutic agent] The method according to any one of claims 3 to 15, further comprising the step of reacting with .

17. (i) R １Ａ and R ２ are bioconjugation reaction partners; and / or, (ii) R １Ａ and R ２ are click reaction partners, and optionally, R １Ａ and R ２ are click reaction partners from Table A, the method according to claim 16.

18. (i) One of R １Ａ and R ２ is -N ３ and the other of R １Ａ and R ２ is dibenzocyclooctyne; (ii) One of R １Ａ and R ２ is, 【Chemical 163】 and one of R １Ａ and R ２ is -SH; (iii) One of R １Ａ and R ２ is tetrazine, and the other of R １Ａ and R ２ is trans-cyclooctene; (iv) One of R １Ａ and R ２ is 【Chemical 164】 and the other of R １Ａ and R ２ is 【Chemical 165】 ; (v) One of R １Ａ and R ２ is -SH, and the other of R １Ａ and R ２ is 【Chemical 166】 ; (vi) One of R １Ａ and R ２ is -SH, and the other of R １Ａ and R ２ is 【Chemical 167】 ; or (vii) One of R １Ａ and R ２ is -SH, and the other of R １Ａ and R ２ is 【Chemical 168】 The method according to claim 16.

19. (i) At least one instance of T is a proteolysis-targeting chimera (PROTAC) drug or a biological therapeutic agent, and optionally, the biological therapeutic agent is a peptide, nucleic acid, or antibody; and / or (ii) The method according to claim 16, wherein each instance of T is different. **Claim 20** The formula **Chemical Formula 169** of the dendrimer.

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