Methods and compositions for synthesizing therapeutic nanoparticles

Abstract

Methods and compositions for the synthesis of therapeutic nanoparticles are provided. The nanoparticles comprise a polymer core, wherein one or more directing molecules and one or more therapeutic agents are attached to the polymer core. Improvements in speed, yield and purity are achieved using the methods disclosed herein.

Description

[0001] This application is a divisional application of a patent application with application number 202080084277.0, application date December 4, 2020, and invention name “Methods and compositions for synthesizing therapeutic nanoparticles”.

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] Pursuant to 35 U.S.C. §119(e)(1), this application claims the benefit of U.S. Provisional Patent Application No. 62 / 943,594, filed on December 4, 2019, and U.S. Provisional Patent Application No. 63 / 110,182, filed on November 5, 2020; the disclosures of both of which are incorporated herein by reference for all purposes.

[0004] Statement on Federal Support

[0005] not applicable. Technical Field

[0006] The present disclosure belongs to the field of nanoparticles for treating various cancers, wherein the nanoparticles comprise a polymer core, further comprising one or more guiding molecules (i.e., targeting molecules) and / or one or more therapeutic molecules. Improved methods for synthesizing such nanoparticles are provided herein. Background Art

[0007] Treatment of many brain diseases (e.g., brain cancer and brain metastases) requires delivery of therapeutic molecules to the brain. Direct delivery of therapeutic agents to the brain poses serious risks to the subject (e.g., damage to the skull) and cannot be performed continuously as required for most chemotherapy treatments. However, due to the presence of the blood-brain barrier (BBB, a layer of tightly connected endothelial cells lining the blood vessels of the brain), systemic delivery (e.g., via the blood) cannot effectively deliver molecules to the brain. A similar permeability barrier exists in some solid tumors, called the blood-tumor barrier (BTB).

[0008] Breast cancer often metastasizes to the brain, and if the therapeutic agent can be delivered to the brain in sufficient concentrations, these brain metastases can be treated with chemotherapeutic molecules for the treatment of breast cancer. One such chemotherapeutic agent is camptothecin (an alkaloid that inhibits DNA topoisomerase I), thereby preventing cell division. Due to the solubility problem of camptothecin in aqueous environments such as cytoplasm, camptothecin derivatives with greater solubility have been developed. One such derivative is 7-ethyl-10-hydroxy-camptothecin (SN38), which was originally discovered as a metabolite of the camptothecin analog irinotecan.

[0009] To address the problem of delivering therapeutic agents across the BBB and BTB, nanoparticles containing chemotherapeutic molecules and guiding (i.e., targeting) molecules have been developed, wherein the guiding molecules bind to transferrin receptors present on the surface of brain endothelial cells (and endothelial cells of tumor vasculature), which enables the nanoparticles to pass through the endothelial cells by endocytosis. See, for example, U.S. Patent Nos. 9,468,681; 10,166,291 and 10,182,986. Each of these patents is incorporated herein by reference for all purposes, or at least for their teachings on the properties of nanoparticles, their preparation and use methods, and their modes of operation.

[0010] Some of the steps in synthesizing such nanoparticles are (1) converting mucic acid into reactive derivatives capable of polymerization (i.e., "mucic acid monomers" or MAMs), (2) polymerizing the mucic acid monomers to form mucic acid polymers (MAPs), (3) converting therapeutic molecules into selective reactive derivatives, and (4) attaching one or more derivatized therapeutic molecules to the MAPs.

[0011] Nanoparticles containing camptothecin (CPT) that are capable of crossing the blood-brain barrier and the blood-tumor barrier have been described. See, for example, U.S. Patent Nos. 9,446,149; 9,468,681; 10,166,291 and U.S. Patent Application Publication No. 2019 / 0381188 (December 19, 2019); each of these patents is incorporated herein by reference for all purposes or at least for the description of the nanoparticles. However, existing methods for preparing such nanoparticles are reagent and time intensive, require elevated temperatures, and have low yields. In addition, as described above, CPT derivatives that are more soluble under aqueous conditions would be preferred. Therefore, there is a need for improved methods for synthesizing nanoparticles containing chemotherapeutic agents such as CPT and its derivatives such as SN38, wherein the methods are rapid, economical in terms of reactants, and provide high yields.

[0012] In addition, the treatment of non-CNS malignancies, such as non-CNS tumors and hematological malignancies, would benefit from improved chemotherapeutic drug delivery vehicles. Summary of the invention

[0013] Provided herein are improved methods for synthesizing therapeutic nanoparticles comprising a mucic acid-polyethylene glycol (PEG) polymer and a chemotherapeutic drug, such as camptothecin (CPT) and its derivatives and metabolites, such as 7-ethyl-10-hydroxy-camptothecin (SN38). These improvements include, among other things, new intermediates, the use of more stable reactants, the formation of more stable intermediates, faster reaction times, higher intermediate and product yields, and new linkers for coupling targeting (guiding) molecules and therapeutic molecules (e.g., macromolecular therapeutic agents; e.g., antibodies) to nanoparticles. Also provided are derivatized nanoparticles (e.g., containing coupled CPT or SN38 molecules) for use in the methods described herein; as well as mucic acid polymer (MAP) conjugates (e.g., MAP coupled to CPT, SN38, transferrin and / or trastuzumab) for assembling therapeutic nanoparticles.

[0014] Therefore, the present invention particularly provides the following embodiments:

[0015] 1. A method for synthesizing mucic acid di(aspartamide) di-trifluoroacetate from mucic acid diamino chloride, the method comprising:

[0016] (a) a diaminomucin chloride having the following structure:

[0017]

[0018] is converted to di(aspartyl(O-tert-butyl)-Boc) mucic acid having the following structure:

[0019] and

[0020] (b) converting mucic acid di(aspartyl(O-tert-butyl)-Boc) into mucic acid di(aspartyl)di-trifluoroacetate having the following structure:

[0021]

[0022] 2. The method of embodiment 1, wherein, in step (b):

[0023] (a) converting di(aspartyl(O-tert-butyl)-Boc) to a neutral substance of di(aspartamide) mucic acid having the following structure:

[0024] and

[0025] (b) converting the neutral substance of mucic acid di(aspartylamine) into mucic acid di(aspartylamine) di-trifluoroacetate.

[0026] 3. A method for synthesizing mucic acid di(asparagine) di-trifluoroacetate, the method comprising preparing a neutral substance of mucic acid di(asparagine) having the following structure:

[0027]

[0028] Converted to mucic acid di(aspartamide) di-trifluoroacetate having the following structure:

[0029]

[0030] 4. Mucic acid di(aspartamide) neutral substance having the following structure:

[0031]

[0032] 5. A method for synthesizing a 10-TBDPS derivative of SN38, the method comprising heating a mixture of SN38 and tert-butyldiphenylsilyl chloride (TBDPSCl) in a base (e.g., an amine base, e.g., a trialkylamine, e.g., triethylamine) and a solvent (e.g., dichloromethane (DCM)), wherein the product has the following structure:

[0033]

[0034] In certain embodiments, the mixture is heated to a temperature in the range of 15°C to 90°C for a period of 5 minutes to 48 hours. In other embodiments, at the end of the reaction, the reaction mixture is washed with dilute acid (e.g., 0.2N HCl), a weak base (e.g., saturated NaHCO3) and brine. In further embodiments, the neutralized solution is dried (e.g., with MgSO4). In other embodiments, the dried solution is evaporated under vacuum to obtain a solid. In further embodiments, the solid is recrystallized; for example, by dissolving residue (e.g., in DCM) and precipitating the product (e.g., with hexane).

[0035] In a further embodiment, a derivative or analog of SN38 or CPT having a 20-OH group and / or a 10-OH group is used as a starting material instead of SN38.

[0036] Combinations of the above-described embodiments are also considered additional embodiments of the invention disclosed herein.

[0037] 6. A method for synthesizing a 20-Boc-aminoacyl-10-TBDPS derivative of SN38, the method comprising mixing 10-TBDPS-SN38 with Boc-amino acid-OH in the presence of a solvent, a base, and a coupling agent. In certain embodiments, the reactants are mixed in a solvent (e.g., DCM) and / or a coupling agent (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl)) and / or a base (e.g., 4-dimethylaminopyridine (DMAP)). In certain embodiments, the reaction is carried out at a temperature of 0°C to 20°C or any integer or decimal value therebetween.

[0038] 7. The method of embodiment 6, wherein the amino acid is selected from glycine (Gly), valine (Val), γ-aminobutyric acid (GABA) and hexanoic acid (Hex).

[0039] 8. 20-(Boc-Gly)-10-TBDPS-SN38 having the following structure:

[0040] 9. 20-(Boc-GABA)-10-TBDPS-SN38 having the following structure:

[0041] 10. 20-(Boc-Hex)-10-TBDPS-SN38 having the following structure:

[0042] 11. 20-(Boc-Val)-10-TBDPS-SN38 having the following structure:

[0043] 12. A method for synthesizing a 10-OBoc derivative of SN38 (10-OBoc-SN38), the method comprising combining SN38 with di-tert-butyl dicarbonate in the presence of a base (e.g., pyridine) and a solvent (e.g., DCM), wherein 10-OBoc-SN38 has the following structure:

[0044]

[0045] 13. A method for synthesizing a 20-(Boc-aminoacyl)-10-OBoc derivative of SN38, the method comprising combining 10-OBoc-SN38 with Boc-amino acid-OH in the presence of a solvent (e.g., DCM), a base (e.g., DMAP), and a coupling agent (e.g., EDC.HCl) at a reduced temperature, wherein the 20-(Boc-aminoacyl)-10-OBoc-SN38 has the following structure:

[0046]

[0047] wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group. In certain embodiments, the reaction is carried out at a temperature of 0°C to 20°C or any integer or fractional value therebetween.

[0048] 14. The method of embodiment 12, wherein the amino acid functional group is selected from the functional groups of glycine (Gly), alanine (Ala), β-alanine (β-Ala), valine (Val) and leucine (Leu).

[0049] 15. 20-(Boc-Gly)-10-OBoc-SN38 having the following structure:

[0050]

[0051] 16. 20-(Boc-Ala)-10-OBoc-SN38 having the following structure:

[0052] 17. 20-(Boc-β-Ala)-10-OBoc-SN38 having the following structure:

[0053] 18. 20-(Boc-Val)-10-OBoc-SN38 having the following structure:

[0054] 19. 20-(Boc-Leu)-10-OBoc-SN38 having the following structure:

[0055] 20. 20-(HCl.Gly)-SN38 having the following structure:

[0056] 21. 20-(TFA.GABA)-SN38 having the following structure:

[0057] 22. 20-(TFA.Hex)-SN38 having the following structure:

[0058] 23. 20-(HCl.Val)-SN38 having the following structure:

[0059] 24. 20-(TFA.Gly)-SN38 having the following structure:

[0060] 25. 20-(TFA.Ala)-SN38 having the following structure:

[0061] 26. 20-(TFA.β-Ala)-SN38 having the following structure:

[0062] 27. 20-(TFA.Val)-SN38 having the following structure:

[0063] 28. 20-(TFA.Leu)-SN38 having the following structure:

[0064]

[0065] 29. A mucic acid polymer-SN38 conjugate (MAP-Gly-SN38) having the following structure:

[0066]

[0067] In certain embodiments, x in compound 16 is a number in the range of 20 to 200, and y is a number in the range of 5 to 200 (or any integer value therebetween; e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected to provide a number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer in the range of about 500 Da to about 50,000 Da.

[0068] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integral value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0069] In other embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug-conjugated MAP into nanoparticles, the size of the nanoparticles is in the range of 10 to 900 nm or any integer value therebetween, e.g., 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 100 nm.

[0070] 30. A mucic acid polymer-SN38 conjugate (MAP-GABA-SN38) having the following structure:

[0071]

[0072] In certain embodiments, x in compound 17 is a number in the range of 20 to 200, and y is a number in the range of 5 to 200 (or any integer value therebetween; e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected to provide a number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer in the range of about 500 Da to about 50,000 Da.

[0073] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integral value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0074] In other embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug-conjugated MAP into nanoparticles, the size of the nanoparticles is in the range of 10 to 900 nm or any integer value therebetween, e.g., 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 100 nm.

[0075] 31. A mucic acid polymer-SN38 conjugate (MAP-Hex-SN38) having the following structure:

[0076]

[0077] In certain embodiments, x in compound 18 is a number in the range of 20 to 200, and y is a number in the range of 5 to 200 (or any integer value therebetween; e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected to provide a number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer in the range of about 500 Da to about 50,000 Da.

[0078] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integral value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0079] In other embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug-conjugated MAP into nanoparticles, the size of the nanoparticles is in the range of 10 to 900 nm or any integer value therebetween, e.g., 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 100 nm.

[0080] 32. A mucic acid polymer-SN38 conjugate (MAP-Val-SN38) having the following structure:

[0081]

[0082] In certain embodiments, x in compound 19 is a number in the range of 20 to 200, and y is a number in the range of 5 to 200 (or any integer value therebetween; e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected to provide a number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer in the range of about 500 Da to about 50,000 Da.

[0083] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integral value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0084] In other embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug-conjugated MAP into nanoparticles, the size of the nanoparticles is in the range of 10 to 900 nm or any integer value therebetween, e.g., 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 100 nm.

[0085] 33. A mucic acid polymer-SN38 conjugate (MAP-Ala-SN38) having the following structure:

[0086]

[0087] In certain embodiments, x in compound 37 is a number in the range of 20 to 200, and y is a number in the range of 5 to 200 (or any integer value therebetween; e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected to provide a number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer in the range of about 500 Da to about 50,000 Da.

[0088] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integral value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0089] In other embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug-conjugated MAP into nanoparticles, the size of the nanoparticles is in the range of 10 to 900 nm or any integer value therebetween, e.g., 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 100 nm.

[0090] 34. A mucic acid polymer-SN38 conjugate (MAP-β-Ala-SN38) having the following structure:

[0091]

[0092] In certain embodiments, x in compound 38 is a number in the range of 20 to 200, and y is a number in the range of 5 to 200 (or any integer value therebetween; e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected to provide a number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer in the range of about 500 Da to about 50,000 Da.

[0093] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integral value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0094] In other embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug-conjugated MAP into nanoparticles, the size of the nanoparticles is in the range of 10 to 900 nm or any integer value therebetween, e.g., 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 100 nm.

[0095] 35. A mucic acid polymer-SN38 conjugate (MAP-Leu-SN38) having the following structure:

[0096]

[0097] In certain embodiments, x in compound 39 is a number in the range of 20 to 200, and y is a number in the range of 5 to 200 (or any integer value therebetween; e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected to provide a number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer in the range of about 500 Da to about 50,000 Da.

[0098] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integral value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0099] In other embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug-conjugated MAP into nanoparticles, the size of the nanoparticles is in the range of 10 to 900 nm or any integer value therebetween, e.g., 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 100 nm.

[0100] 36. A nanoparticle comprising the MAP-SN38 conjugate of any one of embodiments 29-35.

[0101] 37. 20-(Boc-Gly)-CPT having the following structure:

[0102]

[0103] 38. 20-(Boc-Val)-CPT having the following structure:

[0104]

[0105] 39. 20-(Boc-Ala)-CPT having the following structure:

[0106]

[0107] 40. 20-(Boc-β-Ala)-CPT having the following structure:

[0108]

[0109] 41. 20-(Boc-GABA)-CPT having the following structure:

[0110] 42. 20-(Boc-Phe-Gly)-CPT having the following structure:

[0111] 43. 20-(TFA.Gly)-CPT having the following structure:

[0112] 44. 20-(TFA.Ala)-CPT having the following structure:

[0113] 45. 20-(TFA.β-Ala)-CPT having the following structure:

[0114] 46. ​​20-(TFA.Val)-CPT having the following structure:

[0115]

[0116] 47. 20-(TFA.GABA)-CPT having the following structure:

[0117]

[0118] 48. 20-(TFA.Phe-Gly)-CPT having the following structure:

[0119]

[0120] 49. A method for synthesizing a nitrophenylboronic acid-polyethylene glycol conjugate (NPBA-PEG-AA) having the following structure:

[0121]

[0122] The method comprises:

[0123] (a) NH2-PEG-acetic acid (AA) is suspended in a base (e.g., N,N'-diisopropylethylamine, DIPEA) and a solvent (e.g., DCM);

[0124] (b) dissolving 3-carboxy-5-nitrophenylboronic acid in a solvent mixture (e.g., DCM and dimethylformamide (DMF));

[0125] (c) adding a coupling agent (e.g., N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, EEDQ) to the solution of (b);

[0126] (d) mixing the solutions of (a) and (c); and

[0127] (e) acidifying the mixture,

[0128] wherein n is a number in the range of 2 to 2,000 or any integer value therebetween; for example, 100 to 300, 20 to 300, 120 to 180, and / or 140 to 160.

[0129] In certain embodiments, the reaction is carried out at a reduced temperature; for example, at a temperature in the range of 0°C to 20°C or any integer or decimal value therebetween, followed by warming to ambient temperature prior to acidification and purification. In certain embodiments, the mixture is acidified to a pH of about 4. In further embodiments, the product is purified by extraction with a solvent (e.g., isopropyl acetate, iPrOAc).

[0130] In certain embodiments, the value of n is such that the PEG (i.e., -CH2-CH2-O-) portion of the compound has a weight average molecular weight in the range of about 2 to about 15 kDa; for example, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0131] 50. A compound having the following structure (NPBA-PEG-AA-PFP):

[0132]

[0133] In certain embodiments, n is a number in the range of 2 to 2,000 or any integer value therebetween; for example, 100 to 300, 20 to 300, 120 to 180, and / or 140 to 160.

[0134] In certain embodiments, the value of n is such that the PEG (i.e., -CH2-CH2-O-) portion of the compound has a weight average molecular weight in the range of about 2 to about 15 kDa; for example, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0135] 51. A method for preparing a compound of embodiment 50, the method comprising:

[0136] The NPBA-PEG-AA of Embodiment 49 is combined with bis(pentafluorophenyl) carbonate and a base catalyst (eg, N-methylmorpholine).

[0137] 52. A method for synthesizing a NPBA-PEG-transferrin conjugate, the method comprising:

[0138] The NPBA-PEG-AA-PFP of embodiment 50 is conjugated to holo-transferrin (ie, iron-bound transferrin).

[0139] 53. A method for synthesizing a NPBA-PEG-trastuzumab conjugate, the method comprising:

[0140] The NPBA-PEG-AA-PFP of embodiment 50 is conjugated to trastuzumab.

[0141] 54. A method for synthesizing a NPBA-PEG-therapeutic polypeptide conjugate, the method comprising:

[0142] The NPBA-PEG-AA-PFP of embodiment 50 is conjugated to a therapeutic polypeptide.

[0143] 55. A 20-Boc-aminoacyl-10-TBDPS derivative of SN38 (20-(Boc-aminoacyl)-10-TBDPS-SN38) having the following structure:

[0144]

[0145] wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group. The term "R is one or more α-carbon atoms of an amino acid" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker may contain more than one amino acid; i.e., the linker may be a dipeptide, a tripeptide, or an oligopeptide.

[0146] 56. The 20-Boc-aminoacyl-10-TBDPS SN38 derivative of embodiment 55, wherein the amino acid functional group is selected from the functional groups of glycine, valine, γ-aminobutyric acid (GABA) and hexanoic acid.

[0147] 57. A 20-Boc-aminoacyl-10-Oboc derivative of SN38 (20-(Boc-aminoacyl)-10-OBoc-SN38) having the following structure:

[0148]

[0149] wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group. The term "R is one or more α-carbon atoms of an amino acid" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker may contain more than one amino acid; i.e., the linker may be a dipeptide, a tripeptide, or an oligopeptide.

[0150] 58. The 20-Boc-aminoacyl-10-Oboc SN38 derivative of embodiment 57, wherein the amino acid functional group is selected from the functional groups of glycine, alanine, β-alanine, valine and leucine.

[0151] 59. The HCl salt of a 20-aminoacyl derivative of SN38 (20-(HCl.aminoacyl)-SN38) having the following structure:

[0152]

[0153] wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group. The term "R is one or more α-carbon atoms of an amino acid" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker may contain more than one amino acid; i.e., the linker may be a dipeptide, a tripeptide, or an oligopeptide.

[0154] 60. The HCl salt of embodiment 59, wherein the amino acid functional group is selected from the functional groups of glycine and valine.

[0155] 61. A trifluoroacetic acid (TFA) salt of a 20-aminoacyl derivative of SN38 having the following structure (20-(TFA.aminoacyl)-SN38):

[0156]

[0157] wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group. The term "R is one or more α-carbon atoms of an amino acid" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker may contain more than one amino acid; i.e., the linker may be a dipeptide, a tripeptide, or an oligopeptide.

[0158] 62. The TFA salt of embodiment 61, wherein the amino acid functional group is selected from the functional groups of glycine, alanine, β-alanine, valine, GABA, hexanoic acid and leucine.

[0159] 63. A mucic acid polymer (MAP)-amino acid-SN38 conjugate having the following structure:

[0160]

[0161] wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group. The term "R is one or more α-carbon atoms of an amino acid" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker may contain more than one amino acid; i.e., the linker may be a dipeptide, a tripeptide, or an oligopeptide.

[0162] In certain embodiments, x in the MAP-amino acid-SN38 conjugate is a number in the range of 20 to 200, and y is a number in the range of 5 to 200 (or any integer value therebetween; e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected to provide a number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer in the range of about 500 Da to about 50,000 Da.

[0163] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integral value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0164] In other embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug-conjugated MAP into nanoparticles, the size of the nanoparticles is in the range of 10 to 900 nm or any integer value therebetween, e.g., 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 100 nm.

[0165] 64. The MAP-amino acid-SN38 conjugate of embodiment 63, wherein the amino acid functional group is selected from the functional groups of glycine, alanine, β-alanine, valine, GABA, hexanoic acid and leucine.

[0166] 65. Nanoparticles comprising the MAP-amino acid-SN38 conjugate of any one of embodiments 63 or 64.

[0167] 66. A 20-Boc-aminoacyl derivative of camptothecin (20-(Boc-aminoacyl)-CPT) having the following structure:

[0168]

[0169] wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group. The term "R is one or more α-carbon atoms of an amino acid" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker may contain more than one amino acid; i.e., the linker may be a dipeptide, a tripeptide, or an oligopeptide.

[0170] 67. The 20-(Boc-aminoacyl)CPT derivative of embodiment 66, wherein the amino acid functional group is selected from the functional group of glycine, valine, alanine, β-alanine, GABA and the dipeptide phenylalanine-glycine.

[0171] 68. A trifluoroacetic acid (TFA) salt of a 20-aminoacyl derivative of CPT (20-(TFA.aminoacyl)-CPT) having the following structure:

[0172]

[0173] wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group. The term "R is one or more α-carbon atoms of an amino acid" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker may contain more than one amino acid; i.e., the linker may be a dipeptide, a tripeptide, or an oligopeptide.

[0174] 69. The TFA salt of embodiment 68, wherein the amino acid functional group is selected from the functional groups of glycine, alanine, β-alanine, valine, GABA and the dipeptide phenylalanine-glycine.

[0175] 70. A mucic acid polymer (MAP)-amino acid-CPT conjugate having the following structure:

[0176]

[0177] wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group. The term "R is one or more α-carbon atoms of an amino acid" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker can contain more than one amino acid; i.e., the linker can be a dipeptide, a tripeptide, or an oligopeptide. In certain embodiments, the amino acid functional group is selected from the functional groups of alanine, β-alanine, valine, GABA, and the dipeptide phenylalanine-glycine.

[0178] In certain embodiments, x in the MAP-amino acid-CPT conjugate is a number in the range of 20 to 200, and y is a number in the range of 5 to 200 (or any integer value therebetween; e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected to provide a number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer in the range of about 500 Da to about 50,000 Da.

[0179] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integral value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0180] In other embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug-conjugated MAP into nanoparticles, the size of the nanoparticles is in the range of 10 to 900 nm or any integer value therebetween, e.g., 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 100 nm.

[0181] 71. A nanoparticle comprising the MAP-amino acid-CPT conjugate of embodiment 70. DETAILED DESCRIPTION

[0182] By reference to the following description in conjunction with the accompanying examples, the disclosure can be more easily understood, all of which constitute a part of the disclosure. It should be understood that the disclosure is not limited to the specific product, method, condition or parameter described or shown herein, and the terms used herein are only used for the purpose of describing specific embodiments by example, and are not intended to limit any claimed invention. Similarly, unless otherwise specifically stated, any description about possible mechanisms of action or modes or reasons for improvement is only illustrative, and the disclosure herein is not limited by the correctness or incorrectness of the mechanisms of action or modes or reasons for improvement of any such suggestions. Throughout the text, it should be recognized that descriptions relate to compositions and methods for preparing and using the compositions. That is, in the case of the features or embodiments related to compositions or methods for preparing or using the compositions described in the disclosure or claiming, it should be understood that such descriptions or claims are intended to extend these features or embodiments to the embodiments (that is, compositions, preparation methods and methods of use) in each of these contexts.

[0183] In this disclosure, the singular forms "a", "an", and "the" include plural references, and references to a particular value include at least that particular value unless the context clearly dictates otherwise. Thus, for example, a reference to "a material" is a reference to at least one of such materials and equivalents thereof known to those skilled in the art.

[0184] When a value is expressed as an approximation by using the descriptor "about", it will be understood that the particular value forms another embodiment. In general, the use of the term "about" represents an approximation that can vary depending on the desired properties that the disclosed subject matter seeks to obtain, and will be interpreted based on its function in the specific context in which it is used. Those skilled in the art will be able to interpret this as a convention. In some cases, the number of significant figures used for a particular value may be a non-limiting method for determining the scope of the term "about". In other cases, the rank used in a series of values ​​can be used to determine the expected range of the term "about" available for each value. Where present, all ranges are inclusive and combinable. That is, references to the values ​​described in the range include each value within the range.

[0185] It should be understood that, for the sake of clarity, certain features of the present disclosure described herein in the context of a single embodiment may also be provided in combination in a single embodiment. That is, unless clearly incompatible or explicitly excluded, each individual embodiment is considered to be combinable with any other embodiment, and such combination is considered to be another embodiment. Conversely, for the sake of brevity, various features of the present disclosure described in the context of a single embodiment may also be provided individually or in any sub-combination. Finally, although an embodiment may be described as part of a series of steps or part of a more general structure, each of the steps itself may also be considered an independent embodiment that may be combined with other embodiments.

[0186] The transitional terms "comprising," "consisting essentially of," and "consisting of" are intended to encompass their generally accepted meanings in patent jargon; that is, (i) "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to the specified materials or steps and "those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention." Embodiments described in terms of the phrase "comprising" (or its equivalent) also provide those independently described in terms of "consisting of" and "consisting essentially of" as embodiments. For those embodiments provided as "consisting essentially of," the basic and novel characteristics are the ease of operation of the methods of making and using the materials of the invention (as well as the systems used in these methods and the compositions derived therefrom), and the materials themselves, wherein the methods and materials are capable of providing the outstanding properties using only the elements provided in the claims. That is, while other materials may also be present in the compositions of the invention, the presence of these additional materials is not necessary to provide the benefits of those compositions (i.e., the effects may be additive) and / or these additional materials do not impair the performance of the product composition. Similarly, where additional steps may also be used in the methods, their presence is not necessary to achieve the effects or benefits and / or they do not impair the effects or benefits.

[0187] Unless otherwise indicated, when a list is presented, it should be understood that each individual element of the list and each combination of the list is a separate embodiment. For example, a list of embodiments expressed as "A, B, or C" will be interpreted to include embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C". Similarly, as separate embodiments, terms such as C 1-3 Alkyl also includes C1 alkyl, C2 alkyl, C3 alkyl, C 1-2 Alkyl and C 2-3 alkyl.

[0188] As will be understood by those skilled in the relevant art, throughout this specification, words will be given their normal meanings. However, in order to avoid misunderstanding, the meanings of certain terms will be specifically defined or clarified.

[0189] Reference to alcohols, aldehydes, amines, carboxylic acids, ketones or other similar reactive functional groups also includes their protected analogs. For example, reference to hydroxyl or alcohol also includes those substituents in which the hydroxyl is protected by acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl, MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ether (the most commonly used include trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), triisopropylsiloxymethyl (TOM) and triisopropylsilyl (TIPS) ether), ethoxyethyl ether (EE). Mention of amines also includes those substituents in which the amine is protected by BOC glycine, benzoyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butoxycarbonyl (BOC), 9-fluorenylmethoxycarbonyl (FMOC), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), toluenesulfonyl (Ts) group or sulfonamide (Nosyl & Nps) group. Mention of substituents containing a carbonyl group also includes those substituents in which the carbonyl group is protected by acetal or ketal, acetyl acetal (acylal) or diathane. Mention of substituents containing a carboxylic acid or carboxylate group also includes those substituents in which the carboxylic acid or carboxylate group is protected by: its methyl ester, benzyl ester, tert-butyl ester, ester of 2,6-disubstituted phenol (for example 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), silyl ester, orthoester or oxazoline.

[0190] abbreviation

[0191] AA: Acetic acid

[0192] ACN: Acetonitrile

[0193] Ala: alanine, alanyl

[0194] ARS: Alizarin Red S

[0195] Asp: Aspartate, aspartic acid

[0196] β-Ala: β-alanine, β-alanyl

[0197] BBB: blood-brain barrier

[0198] Boc: tert-Butyloxycarbonyl

[0199] BTB: blood tumor barrier

[0200] CPME: Cyclopentyl Methyl Ether

[0201] CPT: Camptothecin

[0202] CV: column volume

[0203] DCM: dichloromethane

[0204] DIC: N,N'-diisopropylcarbodiimide

[0205] DIPEA: N,N'-diisopropylethylamine

[0206] diSPA-PEG: bis(succinimidyl propionate)-PEG

[0207] DMA: dimethylacetamide

[0208] DMAP: 4-dimethylaminopyridine

[0209] DMF: dimethylformamide

[0210] DMSO: dimethyl sulfoxide

[0211] EDC.HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0212] EEDQ: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

[0213] EtOAc: ethyl acetate

[0214] GABA: γ-aminobutyric acid, γ-aminobutyryl

[0215] Gly:glycine, glycyl

[0216] GPC: Gel Permeation Chromatography

[0217] HBSS: Hank's balanced salt solution

[0218] HCl: hydrochloric acid

[0219] Hex: 6-aminohexanoic acid

[0220] HIC: Hydrophobic Interaction Chromatography

[0221] HOPO: Hydroxypyridine N-oxide

[0222] IPA: Isopropyl alcohol

[0223] iPrOAc: isopropyl acetate

[0224] MAM: polymerizable mucic acid monomer

[0225] MAP: Mucic acid polymer

[0226] MeOH: Methanol

[0227] MTBE: Methyl tert-butyl ether

[0228] NHS: N-hydroxysuccinimide

[0229] NP (or P): nanoparticles

[0230] NPBA: 3-Carboxy-5-nitrophenylboronic acid

[0231] Mono-NPBA-PEG-Tf: Mono-PEGylated portion of transferrin

[0232] Mono-NPBA-PEG-Tras: Mono-PEGylated portion of trastuzumab

[0233] PEO: Polyethylene oxide

[0234] PBS: Phosphate buffered saline

[0235] PEG: polyethylene glycol

[0236] PES: Polyethersulfone

[0237] PFP: Pentafluorophenyl

[0238] PyAOP: (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate

[0239] SN38: 7-Ethyl-10-hydroxy-camptothecin

[0240] SpP: number of chains per particle

[0241] TBDPS: tert-butyldiphenylsilane; tert-butyldiphenylsilyl

[0242] TBDPSCl: tert-butyldiphenylchlorosilane

[0243] TEA: triethylamine

[0244] Tf: holotransferrin

[0245] TFA: trifluoroacetate, trifluoroacetic acid

[0246] THF: Tetrahydrofuran

[0247] Tras: trastuzumab

[0248] UF / DF: Ultrafiltration and Diafiltration

[0249] Val:valine, valyl

[0250] Reaction components

[0251] The procedures described herein include standard solvents and catalysts known in the art. Although specific compounds (eg, acids, bases, coupling agents) are recited in this disclosure, it will be apparent to one skilled in the art that different reagents may be used.

[0252] To this end, exemplary solvents include, but are not limited to, chlorinated solvents (e.g., dichloromethane, chloroform, 1,2-dichloroethane), ethers (e.g., diethyl ether, tert-butyl methyl ether, cyclopentyl methyl ether, diglyme, 1,4-dioxane, 2-methyltetrahydrofuran), alcohols (e.g., methanol, ethanol, isopropanol, tert-butanol), alkanes (e.g., pentane, hexane, heptane), glycols (e.g., ethylene glycol, polyethylene glycol), polar aprotic solvents (e.g., dimethylacetamide, acetonitrile, dimethyl sulfoxide, dimethylformamide, acetone, N-methyl-2-pyrrolidone), and polar protic solvents (e.g., water, ethanol, acetic acid, propionic acid).

[0253] Exemplary acids include, but are not limited to, inorganic acids (e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid) and organic acids (e.g., acetic acid, malonic acid, methanesulfonic acid, propionic acid, thioacetic acid, p-toluenesulfonic acid, tribromoacetic acid, trichloroacetic acid, trifluoroacetic acid).

[0254] Exemplary bases include, but are not limited to, amide bases (e.g., 1,4-diazabicyclo[2.2.2]octane, diethylamine, triethylamine, N,N-diisopropylethylamine, lithium amide, lithium bis(trimethylsilyl)amide, morpholine, piperidine), alkoxides (e.g., barium tert-butoxide, lithium tert-butoxide, sodium methoxide), hydroxides (e.g., tetrabutylammonium hydroxide, sodium hydroxide, potassium hydroxide), organometallic bases (e.g., n-butyllithium, tert-butyllithium, butylmagnesium chloride), pyridines (e.g., 4-dimethylaminopyridine, 2,6-lutidine, pyridine), carbonates (e.g., lithium carbonate, sodium carbonate, magnesium carbonate, potassium carbonate), and hydrides (e.g., sodium hydride, calcium hydride, potassium hydride).

[0255] Exemplary coupling agents include, but are not limited to, carbodiimide reagents (e.g., N,N'-diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclopentylcarbodiimide), carbodiimide reagent additives (e.g., 1-hydroxy-7-azabenzotriazole, 6-chloro-1-hydroxybenzotriazole, N-hydroxysuccinimide, 1-hydroxy-2-pyridone, 6-chloro-N-hydroxy-2-phenylbenzimidazole, ethyl 2-cyano-2-(hydroxyimino)acetate), anhydride-based or forming reagents (e.g., di-tert-butyl carbonate, acetic anhydride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ethyl chloroformate), acylazoles (e.g., carbonyl di imidazole), acyl halide generating reagents (e.g., thionyl chloride, phosgene, cyanuric chloride, benzyltriphenylphosphonium dihydrogen trifluoride), phosphonium salt coupling reagents (e.g., benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), tetramethylaminium reagents (e.g., 2-(2-oxo-1(2H)-pyridinyl-1,1,3,3-tetramethyluronium tetrafluoroborate), aminium reagents (e.g., 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate), oxyma uranium salts (e.g., 1-((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)(morpholino)methylene)pyrrolidinium hexafluorophosphate), antimonate uranium) salts (e.g., benzotriazol-1-yloxy-N,N-dimethyl-formimidinium hexachloroantimonate), organophosphorus reagents (e.g., diethyl cyanophosphate, 1-oxo-chlorophosphane, 2-propanephosphonic anhydride), triazine-based reagents (e.g., 2-chloro-4,6-dimethoxy-1,3,5-triazine), organosulfur reagents (e.g., 4-nitrobenzenesulfonic acid pentafluorophenyl ester), pyridinium reagents (e.g., 2-chloro-1-methylpyridinium iodide), polymer-bound reagents (e.g., polymer-supported N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline).

[0256] Synthesis of polymerizable mucic acid monomer (MAM)

[0257] Specifically provided herein are methods for synthesizing polymerizable derivatives of mucic acid ("mucic acid monomers" or MAMs). In the first stage, mucic acid is reacted with methanol (MeOH) in the presence of sulfuric acid to form a 1,6-dimethyl diester of mucic acid (Compound 1). The diester is then reacted with N-Boc-ethylenediamine in the presence of triethylamine (TEA) and MeOH to form N-Boc-protected mucic acid ethylenediamine (Compound 2).

[0258] In an alternative method for the preparative scale production of N-Boc-protected mucic acid ethylenediamine, mucic acid is reacted with MeOH in the presence of sulfuric acid to produce a methoxylated mucic acid derivative, which is then reacted with N-(2-aminoethyl)(tert-butoxy)formamide in TEA and MeOH to produce N-Boc-protected mucic acid diamine. See Example 24.

[0259] Previous methods performed the first stage (i.e., conversion of mucic acid to N-Boc-protected mucic acid ethylenediamine) in two separate reactions; first forming the diester, purifying the diester, and then converting the separated diester to N-Boc-protected mucic acid ethylenediamine. See, for example, U.S. Patent No. 10,166,291; and U.S. Patent Application Publication No. 2019 / 0381188 (December 19, 2019). In the method described herein, the conversion of mucic acid to N-Boc-protected mucic acid ethylenediamine is completed in a single reaction. See Example 1.

[0260] Any and all combinations of two or more of the foregoing embodiments are also contemplated as additional embodiments of the invention disclosed herein.

[0261] In the next stage, the Boc protecting group is removed from the N-Boc-protected ethylenediamine mucic acid by reacting with MeOH and HCl. This converts the N-Boc-protected ethylenediamine mucic acid to ethylenediamine mucic acid chloride (Compound 3). See Example 2. An alternative manufacturing scale method for preparing ethylenediamine mucic acid chloride (ethylenediamine mucic acid chloride) by reacting with MeOH and HCl is provided in Example 25.

[0262] In the next stage, the ethylenediamine chloride is converted to bis(aspartyl(O-tert-butyl)-Boc) (compound 4) by reacting ethylenediamine chloride with Boc-L-aspartic acid 4-tert-butyl ester in the presence of hydroxypyridine N-oxide (HOPO), N,N'-diisopropylcarbodiimide (DIC) and acetonitrile (ACN). See Example 3. The reaction time can be from 5 minutes to 48 hours at a temperature ranging from 15°C to 90°C. In previous methods, ethylenediamine chloride is reacted with di(aspartyl(O-benzyl)-Boc) in the presence of ACN and pyridine, resulting in the addition of a di(aspartyl(O-benzyl)-Boc) protecting group (see, e.g., U.S. Patent Application Publication No. 2019 / 0381188 (December 19, 2019)), rather than the di(aspartyl(O-tert-butyl)-Boc) protecting group used in the methods disclosed herein. Therefore, the method described herein uses new reagents (HOPO and DIC instead of pyridine) and different protecting groups (O-tert-butyl instead of O-benzyl). The advantage of the tert-butyl protecting group used in this method over the benzyl used in the previous method is that the tert-butyl protecting group can be removed with HCl or trifluoroacetic acid (TFA). Therefore, in the next stage of the synthesis, both protecting groups (tert-butyl and Boc) can be removed with a single reagent (e.g., TFA or HCl).

[0263] Another method for synthesizing mucic acid di(aspartyl(O-tert-butyl)-Boc) on a preparative scale is to react Boc-L-aspartic acid 4-tert-butyl ester with ethyl cyanohydroxyiminoacetate (Oxyma) by inducing the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) in dichloromethane (DCM). The mixture is then combined with mucic acid diamino chloride (Compound 3) in aqueous sodium carbonate. The crude product is concentrated and crystallized by adding ACN while distilling off the aqueous phase. See Example 26.

[0264] Any and all combinations of two or more of the foregoing embodiments are also contemplated as additional embodiments of the invention disclosed herein.

[0265] In the final stage of the synthesis of the mucic acid polymerizable monomer, mucic acid di(aspartyl(O-tert-butyl)-Boc) is converted to mucic acid di(aspartamide) (Compound 5) by reacting mucic acid di(aspartyl(O-tert-butyl)-Boc) with TFA in the presence of DCM. See Example 4. The reaction time can be in the range of 5 minutes to 96 hours at a temperature range of 15°C to 90°C. Previous methods (see, for example, U.S. Patent Application Publication No. 2019 / 0381188 (December 19, 2019)) require two steps to perform this conversion; first, mucic acid di(Asp(O-benzyl)-Boc) is converted to mucic acid di(Asp(O-benzyl)-amine); then, mucic acid di(Asp(O-benzyl)-amine) is converted to mucic acid di(Asp-amine). The use of tert-butyl and Boc protecting groups in the methods described herein; instead of O-benzyl and Boc protecting groups as previously described, allows for the removal of both protecting groups in one step in aqueous or organic solution; and avoids the need for homogeneous or heterogeneous hydrogenation.

[0266] For the application of preparation scale, mucic acid two (aspartyl (O-tert-butyl)-Boc) is converted into mucic acid two (asparagine) neutral substance (compound 20), and then it is converted into the di-TFA salt (compound 5) of mucic acid two (asparagine). In these embodiments, mucic acid two (aspartyl (O-tert-butyl)-Boc) is reacted with TFA and triisopropylsilane in DCM and water, and tetrahydrofuran (THF) can be optionally used to further recrystallize to form the mucic acid monomer neutral substance (20). See Example 27. Then in DCM and water, neutral mucic acid monomer is reacted with TFA, and then combined with the solution of crystal seeds of compound 5 in ether to precipitate MAM-two TFA salt (5). See Example 28.

[0267] Any and all combinations of two or more of the foregoing embodiments are also contemplated as additional embodiments of the invention disclosed herein.

[0268] Synthesis of Mucic Acid Polymers (MAP)

[0269] In particular, methods for synthesizing polymers of mucic acid ("mucic acid polymers" or MAPs) are provided herein. In certain embodiments, di-TFA-mucic acid di(aspartamide) (MAM, Compound 5) is reacted with di(succinimidyl propionate)-PEG (diSPA-PEG) (e.g., diSPA-PEG) in a solvent (e.g., dimethyl sulfoxide, DMSO). 3.5k) is combined, a base (e.g., N,N'-diisopropylethylamine, DIPEA) is added, and the reaction is carried out at a temperature range of 15°C to 70°C (e.g., 35°C) for 1 hour to 96 hours (e.g., 66 hours). After obtaining the desired polymer length (usually in the range of 10 kDa to 120 kDa), the reaction mixture is dialyzed with DMSO, dialyzed with water, and filtered to obtain MAP (Compound 6).

[0270]

[0271] The molecular weight of the polymer is controlled by adjusting the molar ratio of MAM to diSPA-PEG; the highest molecular weight is obtained at a 1:1 reactant molar ratio. As the molar ratio of MAM to diSPA-PEG becomes greater or less than 1, the molecular weight of the resulting polymer becomes lower. In our examples, further reductions in molecular weight are associated with increases in the ratio of MAM to diSPA-PEG. See Example 29.

[0272] In certain embodiments, x in compound 6 is a number in the range of 20 to 200, and y is a number in the range of 5 to 200 (or any integer value therebetween; e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected to provide a number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer in the range of about 500 Da to about 50,000 Da.

[0273] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integral value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0274] In other embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug-conjugated MAP into nanoparticles, the size of the nanoparticles is in the range of 10 to 900 nm or any integer value therebetween, e.g., 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 100 nm.

[0275] In some embodiments, the methods described herein are carried out using targeted nanoparticles comprising a nanoparticle core as described herein, the nanoparticle core being coupled to any one of the targeting agents described herein and any one of the chemotherapeutic agents described herein, wherein the total size of the targeted nanoparticle is from about 20nm to about 100nm. In some embodiments, the size of the targeted nanoparticle is from about 40nm to about 100nm. In some embodiments, the size of the targeted nanoparticle is from about 40nm to about 90nm. In some embodiments, the size of the targeted nanoparticle is from about 40nm to about 80nm. In some embodiments, the size of the targeted nanoparticle is from about 40nm to about 70nm. In some embodiments, the size of the targeted nanoparticle is from about 40nm to about 60nm. In some embodiments, the size of the targeted nanoparticle is from about 40nm to about 50nm. In some embodiments, the size of the targeted nanoparticle is from about 50nm to about 100nm. In some embodiments, the size of the targeted nanoparticle is from about 50nm to about 90nm. In some embodiments, the size of the targeted nanoparticle is from about 60nm to about 80nm. In some embodiments, the size of the targeted nanoparticle is from about 50nm to about 70nm. In some embodiments, the targeting nanoparticles have a size of about 70 nm to about 100 nm.

[0276] In certain embodiments, the solvent is, for example, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or dimethylacetamide (DMA).

[0277] In certain embodiments, the elevated temperature is any temperature within the range of 20°C to 40°C; i.e., 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0278] Dialysis can be performed using membranes of various porosities, for example, membranes having a molecular weight cut-off of 1 kDa, 3 kDa, 5 kDa, 10 kDa, 30 kDa or 50 kDa can be used.

[0279] Any and all combinations of two or more of the foregoing embodiments are also contemplated as additional embodiments of the invention disclosed herein.

[0280] Determination of polymer molecular weight

[0281] Molecular weight determination of MAP is performed by gel filtration chromatography (e.g., Malvern OMNISEC gel permeation chromatography (GPC) system) using polyethylene oxide (PEO) standards. The chromatography apparatus may include, for example, a solvent delivery pump, a degasser, an autosampler syringe, a column compartment, a refractive index detector, and a light scattering detector.

[0282] For example, the sample is prepared by dissolving MAP in phosphate buffered saline (PBS) with 0.02% sodium azide at a concentration of 1 to 5 mg / mL, and then filtering the sample through, for example, a 0.45 micron (0.45 μm pore size) filter. 100 μL of the filtered sample is injected into a gel filtration or gel permeation column (e.g., 2×Agilent PL Aquagel-OH 40 columns; 300 mm long×7.5 mm diameter; 8 μm particle size) along with a PEO molecular weight standard, with a mobile phase of PBS+0.02% sodium azide, a flow rate of, for example, 0.7 mL / min, and a column temperature of, for example, 30° C. Peak fractions are detected by differential refractive index and / or light scattering, and analyzed using, for example, MalvernOMNISEC software.

[0283] Camptothecin (CPT) and its derivatives, metabolites and analogs

[0284] The present disclosure provides, inter alia, methods and compositions for preparing selective reactive derivatives of CPT and derivatives, metabolites and analogs thereof. Typically, these molecules contain five fused aromatic rings, one or more of which are heteroaromatic rings, and contain one or more pendant hydroxyl groups, as shown in the following figure, which shows the structure of CPT.

[0285]

[0286] Exemplary molecules in these classes include topotecan, irinotecan, silatacan, cosotecan, exatecan, lortecan, gimmitecan, belotecan, rubitecan, SN38, diflutecan, karenitecan, namitecan, ilotecan, dermotecan, chimmitecan, ZBH-1205, DRF-1042, and FL118.

[0287] Derivatized 7-ethyl-10-hydroxy-camptothecin (SN38) for attachment to MAP

[0288] Previously described nanoparticles for treating brain cancer and brain metastases utilize CPT as a therapeutic molecule. However, as described above, more soluble CPT derivatives, such as SN38, provide a higher therapeutic index and lower toxicity. Therefore, the present disclosure provides, among other things, methods for synthesizing nanoparticles having a polymeric mucic acid backbone to which multiple SN38 molecules are attached. To this end, the present disclosure provides methods and compositions for synthesizing a variety of derivative variants of SN38 that are capable of covalently attaching to the above-mentioned MAP.

[0289] Thus, the present invention specifically provides a method for synthesizing reactive derivatives of the chemotherapeutic molecule SN38. The method involves attaching a number of different amino acid-based linker molecules to the SN38 molecule via the SN38 hydroxyl group. SN38 and other CPT derivatives have two hydroxyl groups: a 10-hydroxyl group and a 20-hydroxyl group. The conjugation of the 20-hydroxyl group stabilizes the active lactone form of these molecules; reduces or prevents their conversion to the less active carboxylate form after administration to a subject; however, the 10-hydroxyl group is also reactive.

[0290] In the method described herein, the 10-hydroxyl group of SN38 is first blocked from reactivity (i.e., protected); then a linker is added to the 20-hydroxyl group. Exemplary amino acid linkers include glycine, alanine, β-alanine, γ-aminobutyric acid (GABA), valine, leucine, isoleucine, valeric acid, caproic acid, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tyrosine, tryptophan, and dipeptides (e.g., phenylalanine-glycine), tripeptides, and oligopeptides.

[0291] Provided herein are methods for attaching a number of different linkers to the 20-hydroxyl group of SN38. After coupling to the 20-hydroxyl group of SN38, each of the different linkers exemplified herein (e.g., glycine, alanine, β-alanine, leucine, valine, γ-aminobutyric acid, and 6-aminocaproic acid; and others such as the dipeptide phenylalanine-glycine) can react with the carboxyl group of MAP (via an amidation reaction between the amine group of the linker and the carboxyl group of the polymer), thereby covalently attaching the SN38 molecule to MAP. Each different linker has a different stability and affects the properties of the resulting nanoparticles (e.g., size, density), which particularly affects the rate at which SN38 is released from the nanoparticles after crossing the blood-brain barrier (BBB) ​​or the blood-tumor barrier (BTB).

[0292] Synthesis of 10-Hydroxy Protected SN38 (10-TBDPS-SN38)

[0293] In the initial step of the synthesis of derived SN38, a tert-butyldiphenylsilyl (TBDPS) protecting group is added to the 10-hydroxyl group of SN38 (to block the reactivity of this site) by mixing SN38 and tert-butyldiphenylsilyl chloride (TBDPS) in DCM and triethylamine. The mixture is refluxed (e.g., at 45° C.) with stirring for approximately 16 hours, then washed with HCl (e.g., 0.2N), saturated NaHCO3, and brine; and finally dried over MgSO4 and evaporated in vacuo. The product (10-TBDPS-SN38, compound 7) is dissolved in DCM, precipitated with hexane, and the solid is dried.

[0294] The TBDPS protecting group is selective for the 10-hydroxyl group of SN38 (leaving the 20-OH group free to react in the subsequent step), and the resulting compound is less likely to spontaneously lose the protecting group during storage; thereby providing a more stable intermediate.

[0295] Another advantage of using TBDPS to protect the 10-OH group of SN38 is that it can be removed under the same conditions as those used to remove the Boc protecting group from the linker attached to the 20-OH group (1 M-12 M HCl or water:TFA between 1:1 and 1:4 v / v).

[0296] Synthesis of 10-hydroxy-protected SN38 using protected 20-hydroxyglycine linker

[0297] A protected glycine (Gly) linker is added to the 20-hydroxy position of 10-TBDPS-SN38 by combining 10-TBDPS-SN38 with Boc-Gly-OH in DCM at 0°C. EDC.HCl and 4-dimethylaminopyridine (DMAP) are added and the mixture is stirred at 0°C. The stirring may be continued for 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, or any interval therebetween. After stirring, the mixture is washed twice with 0.5% NaHCO3, once with water, twice with 0.1N HCl, and once with brine. The solution is dried (e.g., with MgSO4) and evaporated under vacuum to generate 20-(Boc-Gly)-10-TBDPS-SN38 (Compound 8).

[0298] Synthesis of 20-(HCl.Gly)-SN38

[0299] The 10-hydroxyl and 20-hydroxyl groups of 20-(Boc-Gly)-10-TBDPS-SN38 were deprotected at room temperature by treatment with HCl (concentrations ranging from 1 N to 12 N) under a layer of hexane. This resulted in a SN38 derivative in which the 10-hydroxyl group was restored and a glycine linker was covalently attached to the 20-hydroxyl group (20-(HCl.Gly)-SN38, compound 12).

[0300] Synthesis of 10-hydroxy protected SN38 using protected 20-hydroxy GABA linker

[0301] A protected γ-aminobutyric acid (GABA) linker is added to the 20-hydroxy position of 10-TBDPS-SN38 by combining 10-TBDPS-SN38 with Boc-GABA-OH in DCM at 0°C. EDC.HCl and DMAP are added and the mixture is stirred at 0°C. The stirring may be continued for 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours or any interval therebetween. After stirring, the mixture is washed twice with 0.5% NaHCO3, once with water, twice with 0.1N HCl, and once with brine. The solution is dried (e.g., with MgSO4) and evaporated under vacuum to generate 20-(Boc-GABA)-10-TBDPS-SN38 (Compound 9).

[0302] Synthesis of 20-(TFA.GABA)-SN38

[0303] The 10-hydroxyl and 20-hydroxyl groups of 20-(Boc-GABA)-10-TBDPS-SN38 were deprotected by treatment with an aqueous solution of TFA (concentration range 1:1 to 4:1 v / v) under a layer of hexane at room temperature, thereby generating a SN38 derivative in which the 10-hydroxyl group was restored and the GABA linker was covalently linked to the 20-hydroxyl group (20-(TFA.GABA)-SN38, compound 13).

[0304] Synthesis of 10-hydroxy-protected SN38 using protected 20-hydroxyhexanoic acid linker

[0305] A protected 6-aminohexanoic acid (Hex) linker is added to the 20-hydroxy position of 10-TBDPS-SN38 by combining 10-TBDPS-SN38 with Boc-Hex-OH in DCM at 0°C. EDC.HCl and DMAP are added and the mixture is stirred at 0°C. The stirring may be continued for 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, or any interval therebetween. After stirring, the mixture is washed twice with 0.5% NaHCO3, once with water, twice with 0.1N HCl, and once with brine. The solution is dried (e.g., with MgSO4) and evaporated under vacuum to generate 20-(Boc-Hex)-10-TBDPS-SN38 (Compound 10).

[0306] Synthesis of 20-(TFA.Hex)-SN38

[0307] The 10-hydroxyl and 20-hydroxyl groups of 20-(Boc-Hex)-10-TBDPS-SN38 were deprotected by treatment with an aqueous solution of TFA (concentrations ranging from 1:1 to 4:1 v / v) under a layer of hexane at room temperature, thereby generating a SN38 derivative in which the 10-hydroxyl group was restored and the Hex linker was covalently linked to the 20-hydroxyl group (20-(TFA.Hex)-SN38, compound 14).

[0308] Synthesis of 10-hydroxy-protected SN38 using protected 20-hydroxyvaline linker

[0309] A protected valine (Val) linker is added to the 20-hydroxy position of 10-TBDPS-SN38 by combining 10-TBDPS-SN38 with Boc-Val-OH in DCM at 0°C. EDC.HCl and DMAP are added and the mixture is stirred at 0°C. The stirring may be continued for 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, or any interval therebetween. After stirring, the mixture is washed twice with 0.5% NaHCO3, once with water, twice with 0.1N HCl, and once with brine. The solution is dried (e.g., with MgSO4) and evaporated under vacuum to generate 20-(Boc-Val)-10-TBDPS-SN38 (Compound 11).

[0310] Synthesis of 20-(HCl.Val)-SN38

[0311] The 10-hydroxyl and 20-hydroxyl groups of 20-(Boc-Val)-10-TBDPS-SN38 were deprotected at room temperature by treatment with HCl (concentrations ranging from 1 N to 12 N) under a layer of hexane. This resulted in a SN38 derivative in which the 10-hydroxyl group was restored and a valine linker was covalently attached to the 20-hydroxyl group (20-(HCl.Val)-SN38, compound 15).

[0312] The same protecting group was used on the 10- and 20-OH groups of SN38

[0313] Another approach to synthesizing reactive derivatives of SN38 with amino acid linkers is to place a Boc-O protecting group on the 10-OH group and a Boc-amino acid protecting group on the 20-OH. When the compound (20-(Boc-aminoacyl)-10-OBoc-SN38) reacts with TFA, the 10-OH group is restored, and the TFA salt of the amino acid linker is connected to the 20-OH. 20-Boc-glycyl, -alanyl, -β-alanyl, -leucyl and -valyl derivatives of SN38 were synthesized using this method. See below and Examples 33-35. Other amino acids that can be used in this method include GABA, isoleucine and caproic acid, and dipeptides such as phenylalanine-glycine.

[0314]

[0315]

[0316] Synthesis of polymer-drug conjugates

[0317] In particular, provided herein are methods for forming polymer-drug conjugates (i.e., MAP-linker-SN38 nanoparticles) by covalently linking a derivatized SN38 to a MAP. A number of different conjugates are provided that differ in the nature of the linker between the polymer and the drug (i.e., size and chemical nature). Different conjugates are prepared by using SN38 that has been derivatized with various types of linkers. In one embodiment, the linker is a glycyl (Gly) moiety. In one embodiment, the linker is a valyl (Val) moiety. In one embodiment, the linker is a γ-aminobutyric acid (GABA) moiety. In one embodiment, the linker is a 6-aminohexanoic acid (Hex) moiety. In one embodiment, the linker is an alanyl (Ala) moiety. In one embodiment, the linker is a β-alanyl (β-Ala) moiety. In one embodiment, the linker is a leucyl (Leu) moiety.

[0318] To form the polymer-drug conjugate, MAP is dissolved in DMSO, and then EDC.HCl and N-hydroxysuccinimide (NHS) are added. After the addition of EDC.HCl and NHS, the derivatized SN38 and DIPEA are added, and the mixture is stirred at room temperature for about 18 hours. The derivatized SN38 can be, for example, 20-(HCl.Gly)-SN38, 20-(HCl.Val)-SN38, 20-(TFA.GABA)-SN38, 20-(TFA.Hex)-SN38, 20-(TFA.Gly)-SN38, 20-(TFA.Ala)-SN38, 20-(TFA.β-Ala)-SN38, 20-(TFA.Val)-SN38, or 20-(TFA.Leu)-SN38. The reaction mixture is then dialyzed against DMSO and then against water at pH 4 using a 10 kDa membrane. The dialyzed product is filtered (e.g., through a 0.22 μm filter), frozen and lyophilized. Depending on the derivatized SN38 used, the product is MAP-Gly-SN38 (Compound 16); MAP-GABA-SN38 (Compound 17), MAP-Hex-SN38 (Compound 18), MAP-Val-SN38 (Compound 19), MAP-Ala-SN38 (Compound 37), MAP-β-Ala-SN38 (Compound 38) or MAP-Leu-SN38 (Compound 39).

[0319] Nanoparticle formation

[0320] To convert the polymer-drug conjugate (i.e., any of compounds 16, 17, 18 or 19, 37, 38 or 39) into nanoparticles, the lyophilized polymer-drug conjugate is dissolved in water at pH 4 at a concentration of 1-10 mg / mL (e.g., 4 mg / mL). The solution is then filtered and can be frozen until further use.

[0321] The size range of nanoparticles is about 20 to about 60 nm in diameter. Exemplary nanoparticle diameters are 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm or larger.

[0322] Release of SN38 from nanoparticles in vitro

[0323] The release of SN38 from MAP-Gly-SN38, MAP-GABA-SN38, MAP-Hex-SN38, MAP-Val-SN38, MAP-Ala-SN38, MAP-β-Ala-SN38, and MAP-Leu-SN38 nanoparticles all exhibited first-order kinetics when the nanoparticles were incubated at a concentration of 0.1 mg SN38 / mL in PBS at 37°C in a humidified oven at pH 6.5, 7.0, or 7.4. A strong dependence of the release rate on pH was observed. The release half-life decreased for all nanoparticle formulations as the pH increased from 6.5 to 7.4, indicating that hydrolysis plays an important role in the release of SN38. Longer half-lives were observed for nanoparticles with more hydrophobic and sterically hindered linkers.

[0324] Derived CPT for connecting MAP

[0325] In certain embodiments, the present disclosure provides nanoparticles containing CPT as a therapeutic agent. CPT derivatives coupled to amino acids (e.g., 20-(TFA.Gly)-CPT) are used to synthesize the nanoparticles. Exemplary amino acid linkers include glycine, alanine, β-alanine, γ-aminobutyric acid (GABA), valine, leucine, isoleucine, valeric acid, caproic acid, and dipeptides (e.g., phenylalanine-glycine), tripeptides, and oligopeptides.

[0326] 20-(TFA.Gly)-CPT is synthesized in two steps. In the first step, CPT is combined with Boc-Gly-OH in DMAP under an inert atmosphere (e.g., under argon), and the reactants are slurried by adding DCM. DIC is then added dropwise to the reaction mixture over a few minutes, and the mixture is subsequently stirred at room temperature for several hours (e.g., 3 hours). About half of the solvent is then removed under vacuum, and frozen MeOH is added to precipitate the product (20-(Boc-Gly)-CPT, compound 21), which is obtained by filtration. The solid is washed with frozen MeOH and frozen methyl tert-butyl ether (MTBE) and dried under vacuum. See Example 30.

[0327] In the second step, TFA is added stepwise to a stirred suspension of 20-(Boc-Gly)-CPT in DCM, and the mixture is stirred for several hours (e.g., 2 hours). The product (20-(TFA.Gly)-CPT, compound 22) is then precipitated with MTBE, filtered, washed and dried under vacuum. See Example 31.

[0328] Other CPT derivatives can be prepared by using other Boc-protected amino acids (or peptides) instead of Boc-Gly-OH. For example, using Boc-Ala-OH as a starting material, 20-(Boc-Ala)-CPT (Compound 41) and 20-(TFA.Ala)-CPT (Compound 45) are obtained. Using Boc-β-Ala-OH as a starting material, 20-(Boc-β-Ala)-CPT (Compound 42) and 20-(TFA.β-Ala)-CPT (Compound 46) are obtained. Using Boc-Val-OH as a starting material, 20-(Boc-Val)-CPT (Compound 40) and 20-(TFA.Val)-CPT (Compound 47) are obtained. Using Boc-GABA-OH as a starting material, 20-(Boc-GABA)-CPT (Compound 43) and 20-(TFA.GABA)-CPT (Compound 48) are obtained. Using Boc-Phe-Gly-OH as the starting material, 20-(Boc-Phe-Gly)-CPT (Compound 44) ​​and 20-(TFA.Phe-Gly)-CPT (Compound 49) were obtained.

[0329] Synthesis of CPT-polymer conjugates

[0330] In certain embodiments, conjugates of CPT and MAP are provided. To synthesize these conjugates, MAP and 20-(TFA.aminoacyl)-CPT (see the previous section for exemplary amino acid and peptide derivatives of CPT) are dissolved in DMSO in an inert atmosphere (e.g., under argon). In a separate container, (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) is dissolved in DMSO. The PyAOP solution is then added to the MAP / 20-(TFA.aminoacyl)-CPT solution and stirred for a few minutes (e.g., 2 minutes), followed by addition of DIPEA and stirring the reaction mixture at room temperature in the dark for 12-24 hours (e.g., 18 hours). The product is precipitated with cold (0-4°C) ethyl acetate (EtOAc) (optionally in several batches) and washed with cold EtOAc. The product can be further isolated by freeze drying. See Example 32.

[0331] Nitrophenylboronic acid (NPBA)-polyethylene glycol (PEG) conjugate

[0332] In certain embodiments, a macromolecule such as a targeting (guiding) molecule or a macromolecular therapeutic agent (e.g., an antibody) is linked to a MAP via a nitrophenylboronic acid-polyethylene glycol (NPBA-PEG) linker. Such an NPBA-PEG linker can be formed by attaching an amine derivative of PEG (e.g., PEG 3.5k ,PEG5k ) is combined with a carboxyl derivative of NPBA to form a NPBA-PEG-acetic acid (AA) conjugate linked by an amide bond. In certain embodiments, the PEG derivative comprises an amino group at one end (for reaction with NPBA) and a carboxyl group at the opposite end (in a subsequent reaction, it can be activated to react with a macromolecule such as a polypeptide). See Example 36.

[0333]

[0334] In certain embodiments, n is a number in the range of 2 to 2,000 or any integer value therebetween; for example, 100 to 300, 20 to 300, 120 to 180, or 140 to 160.

[0335] In certain embodiments, the value of n is such that the PEG (i.e., -CH2-CH2-O-) portion of the compound has a weight average molecular weight in the range of about 2 to about 15 kDa; for example, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0336] The NPBA-PEG-AA conjugate is activated to react with a polypeptide (e.g., transferrin) by forming a pentafluorophenyl (PFP) derivative. In these embodiments, bis(pentafluorophenyl) carbonate is combined with the NPBA-PEG-AA conjugate to form NPBA-PEG-pentafluorophenyl ester (e.g., NPBA-PEG 5k -AA-PFP). See Example 37.

[0337]

[0338] In certain embodiments, n is a number in the range of 2 to 2,000 or any integer value therebetween; for example, 100 to 300, 20 to 300, 120 to 180, and / or 140 to 160.

[0339] In certain embodiments, the value of n is such that the PEG (i.e., -CH2-CH2-O-) portion of the compound has a weight average molecular weight in the range of about 2 to about 15 kDa; for example, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0340] Polymer conjugates containing targeting molecules

[0341] In certain embodiments, a MAP as described herein comprises a targeting (or directing) molecule instead of or in addition to a small molecule therapeutic (e.g., CPT or SN38). Targeting molecules are known in the art and include, for example, small molecules (e.g., vitamins such as folic acid), carbohydrates (e.g., mannose, allose, altrose, glucose, gulose, idose, galactose, talose, disaccharides, trisaccharides, oligosaccharides), peptides (e.g., RGD), polypeptides (e.g., antibodies, proteins that bind to cell surface receptors such as transferrin), nucleic acids, peptoids, and peptide nucleic acids (PNAs).

[0342] For example, MAP (and nanoparticles made from MAP) can include a targeting molecule, transferrin, that targets the polymer (or nanoparticles formed from the polymer) to endothelial cells of the blood-brain or blood-tumor barrier. In these embodiments, the targeting molecule is linked to the NPBA-PEG polymer by reacting with a PFP active ester as described above. Since the primary amine groups in the protein react with the PFP active ester, the connection of the full iron transferrin to the NPBA-PEG-pentafluorophenyl ester is achieved by combining the ester with the full iron transferrin. The progress of the reaction is monitored by HPLC, and the mono-PEGylated transferrin product (e.g., Mono-NPBA-PEG 5k -Tf) was purified by hydrophobic interaction chromatography (HIC) based on the differential binding of mono-PEGylated transferrin compared to fractions containing a higher degree of PEGylation (e.g., di- and tri-PEGylated fractions). See Examples 38 and 39. This approach prevents iron loss from transferrin during purification, as occurs using previous methods, thereby maintaining the holo-transferrin structure.

[0343] Polymer conjugates containing macromolecular therapeutic agents

[0344] In certain embodiments, a MAP as described herein comprises a macromolecular therapeutic agent in place of or in addition to a small molecule therapeutic agent (e.g., CPT or SN38). Macromolecular therapeutic agents are known in the art and include, for example, therapeutic polypeptides (e.g., antibodies, enzymes, and biologically active proteins) and nucleic acids.

[0345] Exemplary therapeutic polypeptides include, but are not limited to, antibodies such as trastuzumab (anti-Her2), anti-Her3, anti-Trop2, anti-PSMA, anti-LIV-1, anti-FOLR1, anti-DLL3, anti-PDGF, anti-FRalpha, anti-PTK7, anti-mesothelin, anti-c-MET, anti-MUC1, anti-CD70, anti-CD74, anti-CD30, anti-CD33, anti-FLT3, anti-CD22, anti-CD20, and anti-CD19.

[0346] Certain polypeptide therapeutics, such as antibodies directed against tumor markers, such as those exemplified in the preceding paragraph, can also be used as targeting molecules.

[0347] For example, MAP (and nanoparticles prepared from MAP) may comprise the anti-Her2 antibody trastuzumab. In these embodiments, the antibody is linked to the NPBA-PEG polymer by reaction with a PFP active ester as described above.

[0348] Since the primary amine groups in proteins react with PFP active esters, the connection of trastuzumab to NPBA-PEG-pentafluorophenyl ester is achieved by conjugating the ester to trastuzumab. The progress of the reaction is monitored by HPLC, and the mono-PEGylated trastuzumab product (e.g., Mono-NPBA-PEG 5k -Tras) by HIC purification based on the differential binding of mono-PEGylated trastuzumab compared to fractions containing a higher degree of PEGylation (e.g., di- and tri-PEGylated fractions). See Examples 41 and 42.

[0349] Determination of the number of polymer chains per nanoparticle

[0350] To determine the number of polymer chains per nanoparticle, the average molecular weight of the nanoparticles was divided by the molecular weight of the corresponding polymer-drug (ie, MAP-SN38) conjugate.

[0351] The molecular weight of the nanoparticles was determined by gel permeation chromatography (GPC) using PEO standards with nominal molecular weights of 100 kDa and 200 kDa. The molecular weight of the polymer-drug conjugate was calculated from the molecular weight of the polymer (i.e., MAP) used in the conjugate synthesis, and the drug loading was adjusted as follows:

[0352] MW 缀合物 =MW 聚合物 / (1-T)

[0353] MW 聚合物 is the molecular weight of MAP used in the conjugate synthesis and T is the partial loading of the therapeutic agent expressed as a decimal part.

[0354] As used herein, the number of chains per particle (SpP) is the number of mucic acid polymer ("MAP") therapeutic agent conjugate molecules present in a particle or nanoparticle. For the purpose of determining SpP, a particle or nanoparticle is an entity having at least one MAP-therapeutic agent-protein conjugate molecule that behaves as a single unit in any aqueous solution (e.g., water at neutral pH, PBS at pH 7.4, or any formulation for administration to a patient) at a concentration suitable for administration to a human. For the purpose of calculating the number of chains per particle, a MAP-therapeutic agent conjugate molecule is a single MAP polymer with a covalently attached therapeutic agent.

[0355] Methods disclosed herein are provided for evaluating particles, wherein the particles comprise one or more MAP-therapeutic agent conjugate molecules. Typically, the method entails providing a sample comprising a plurality of said particles and determining a value for the quantity of MAP-therapeutic agent conjugate per particle in the sample, thereby evaluating the preparation of the particles. The value for the particle sample will be a function of the value obtained for the plurality of particles.

[0356] As mentioned above, SpP is defined as the number of MAP-therapeutic agent conjugate molecules that self-assemble into particles or nanoparticles, thus

[0357] SpP = [MAP-therapeutic agent conjugate molecule] / P (or NP)

[0358] where [MAP-therapeutic agent conjugate molecules] is the number of MAP-therapeutic agent conjugate molecules, and P (or NP) is a single particle (or nanoparticle).

[0359] In certain embodiments, the method further comprises comparing the determined value with a reference value. The comparison can be used in a variety of ways. For example, in order to accommodate the comparison or determination performed in the method, a decision is made or a step is taken, for example, a production parameter is changed in the process for preparing the particles, the sample is sorted, selected, accepted or discarded, released or detained, processed into a drug, transported, transferred to a different location, formulated (e.g., formulated with another substance (e.g., excipient)), labeled, packaged, put on the market, or sold or priced for sale. For example, based on the assay results, or when compared with a reference standard, the batch from which the sample was extracted can be processed, for example, as just described.

[0360] To calculate the number of chains per particle, determine the size of the particle (e.g., molecular weight determined by light scattering of the self-assembled particle), the size of the individual polymers (e.g., molecular weight determined by light scattering of the individual polymers), and the loading of the therapeutic agent (e.g., mass %). Using these values, calculate SpP as follows:

[0361] SpP=MW 颗粒 / (MW 缀合物 )

[0362] MW 颗粒 is the molecular weight of the particle, and MW 缀合物 is the molecular weight of the MAP-therapeutic agent conjugate molecule, which is calculated as follows:

[0363] MW 缀合物 =MW 聚合物 / (1-T ％ )

[0364] MW 聚合物 is the molecular weight of MAP, and T ％ is the percentage loading of the therapeutic agent, expressed as a decimal, e.g. a 10% loading results in T ％ =0.1.

[0365] Determination of SpP was demonstrated using MAPs with different linkers and drugs. MAPs with the same molecular weight of CPT and SN38 resulted in approximately 1.5-2.5 chains per particle. Different linkers altered the chains per particle, thus changing the density of the particles, as demonstrated in SN38. The linkers glycine, γ-aminobutyric acid, hexanoic acid, alanine, β-alanine, leucine, and valine varied the chains per particle from approximately 1.3 to up to 4.6 chains per particle.

[0366] Polymer molecular weight distribution and particle dispersity: MAPs are synthesized so that they have a range of molecular weights. Molecules of different molecular weights provide different contributions to particle size and chains per particle. Particles can be formed with MAPs (here considered chains) that are smaller or larger than the average. Chains may also be associated with a maximum size that may be shear limited.

[0367] Particle shape: The particle shape was assumed to be approximately spherical. Self-assembly was assumed to be driven by the hydrophobic regions created by the therapeutic agent of the MAP-therapeutic agent conjugate molecule.

[0368] Preparations, kits and routes of administration

[0369] Also provided are therapeutic compositions comprising nanoparticles as disclosed herein. Such compositions generally comprise nanoparticles and a pharmaceutically acceptable carrier. Supplementary active compounds may also be incorporated into the nanoparticle compositions.

[0370] The therapeutic compositions disclosed herein are particularly useful for treating cancer, cancer metastasis, and other diseases of the brain and central nervous system. Therefore, the "therapeutically effective amount" of the composition comprising nanoparticles is any amount that alleviates symptoms or, for example, stimulates tumor regression. For example, the dosage of nanoparticles can be from about 0.1-1.0 mg / kg body weight, or from about 0.5-2.0 mg / kg body weight, or from about 1-5 mg / kg body weight, or from about 1 mg / kg body weight to about 10 mg / kg body weight or more (or any integer value therebetween); the frequency of administration is, for example, hourly, twice a day, once a day, twice a week, once a week, twice a month, once a month, depending on, for example, body weight, route of administration, severity of the disease, etc. Therefore, the therapeutically effective amount can include multiple administrations of the same or different amounts of nanoparticles. In certain embodiments, a single administration of nanoparticles is a therapeutically effective amount.

[0371] In certain embodiments, the nanoparticles are administered at a dose of 1-20 mg of nanoparticles per square meter of body surface area of ​​the subject (or any integer or fractional value therebetween), which is typical for dosages of chemical drugs.

[0372] In view of the present disclosure, various pharmaceutical compositions and their preparation and use techniques are known to those skilled in the art. For a detailed list of suitable pharmacological compositions and their administration techniques, reference may be made to the following texts: for example, Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics," McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), "Remington: The Principles of Pharmacy Practice," Lippincott Williams & Wilkins, 2008.

[0373] The nanoparticles described herein can be suspended in a physiologically compatible carrier for administration. As used herein, the term "physiologically compatible carrier" refers to a carrier that is compatible with the nanoparticles and any other components of the formulation and is harmless to the recipient. Those skilled in the art are familiar with physiologically compatible carriers. Examples of suitable carriers include water (e.g., water at pH 4), phosphate buffered saline, Hank's balanced salt solution + / - glucose (HBSS), and various electrolyte solutions such as Plasma-Lyte TM A (Baxter).

[0374] The volume of the nanoparticle suspension administered to the subject will vary depending on the site of administration, the therapeutic target, and the number of nanoparticles in the solution. Typically, the amount of nanoparticles administered will be a therapeutically effective amount. As used herein, a "therapeutically effective amount" or "effective amount" refers to the amount of nanoparticles administered required to achieve the treatment of a specific disease (i.e., reduce the number and / or severity of symptoms associated with the disease). For example, in the case of cancer metastasis to the brain, administration of a therapeutically effective amount of nanoparticles results in a reduction and / or reversal of metastatic symptoms; for example, regression of metastatic tumors. The therapeutically effective amount varies depending on the type and extent of brain damage, and may also vary depending on the overall condition of the subject.

[0375] The disclosed therapeutic compositions may also include pharmaceutically acceptable materials, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, i.e., carriers. For example, these carriers can stabilize the nanoparticles and / or promote the retention of the nanoparticles in vivo. Each carrier is "acceptable" in the sense that it is compatible with the other ingredients of the formulation and is not harmful to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol and polyethylene glycol; polyols such as glycerol, sorbitol, and mannitol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0376] Nanoparticles can be administered to a subject by any suitable route (including but not limited to inhalation, topical, nasal, oral, parenteral (e.g., intravenous, intraperitoneal, intravesical or intrathecal) or rectal) in a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical properties of the compound, the selected route of administration and standard practice. Any method known in the art can be used to administer the compounds described herein. For example, administration can be transdermal, parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intraventricular, intrathecal, intranasal, atomized, by suppository or by oral administration. The pharmaceutical compositions of nanoparticles described herein can be used for administration by injection, or for oral, pulmonary, nasal, transdermal or ocular administration.

[0377] Exemplary formulations include, but are not limited to, those suitable for parenteral administration (e.g., intrapulmonary, intravenous, intraarterial, intraocular, intracranial, submeningeal or subcutaneous administration), including formulations encapsulated in micelles, liposomes or drug release capsules (active agents incorporated into biocompatible coatings designed for sustained release); ingestible formulations; formulations for topical use, such as eye drops, creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays. The dosage of the compositions of the present disclosure will vary depending on the degree and severity of the treatment needs, the activity of the administered composition, the general health of the subject, and other considerations well known to those skilled in the art.

[0378] In other embodiments, the compositions described herein are delivered intracranial at or near the site of brain injury or metastasis. This local delivery allows for non-systemic delivery of the composition, thereby reducing the body burden of the composition compared to systemic delivery. Local delivery can be achieved, for example, by intracranial injection or by using various medical implant devices including but not limited to stents and catheters, or can be achieved by suction, phlebotomy or surgery. Methods for coating, implanting, embedding and otherwise adsorbing the desired medicament to medical devices such as stents and catheters have been established in the art and are considered herein.

[0379] Another aspect of the disclosure relates to a kit for administering nanoparticles, optionally in combination with another therapeutic agent, to a subject. In one embodiment, the kit comprises a nanoparticle composition formulated in a pharmaceutical carrier suitable for administration, for example, by injection.

[0380] Example

[0381] The following examples are provided to illustrate some of the concepts described in this disclosure. While each example is considered to provide a specific embodiment of the compositions, methods of preparation, and methods of use, no one example should be considered a limitation of the more general embodiments described herein.

[0382] In the following examples, efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, temperatures are in degrees Celsius and pressures are at or near atmospheric pressure. Unless otherwise indicated, any reference to molecular weight is intended to refer to number average molecular weight.

[0383] Examples 1-4 describe the conversion of mucic acid to polymerizable mucic acid monomers (MAMs). Examples 5 and 6 describe the polymerization of MAMs to form mucic acid polymers (MAPs). Examples 7-15 describe the derivatization of 7-ethyl-10-hydroxy-camptothecin (SN38) with four different amino acid-based linkers into a form capable of reacting with MAP to form a covalent MAP-SN38 conjugate. Examples 16-20 describe reactions for the synthesis of MAP-SN38 conjugates linked by amino acid-based linkers. Example 21 describes a method for converting MAP-SN38 conjugates into nanoparticles. Examples 22 and 23 describe the characterization of MAP-SN38 nanoparticles. Examples 24-29 describe a preparative-scale synthetic route for MAP. Examples 30-32 describe the derivatization of camptothecin (CPT) and its attachment to MAP. Examples 33-35 describe the derivatization of SN38 for attachment to MAP. Examples 36 and 37 describe the synthesis of nitrophenylboronic acid-polyethylene glycol (NPBA-PEG) polymers with reactive termini suitable for protein coupling. Examples 38-40 describe the coupling of targeting (guiding) molecules to NPBA-PEG polymers and the characterization of the products. Examples 41-43 describe the coupling of macromolecular therapeutic agents to NPBA-PEG polymers and the characterization of the products.

[0384] Example 1. Synthesis of N-Boc-protected mucic acid diamine (Compound 2)

[0385] 400mL methanol (MeOH) and 1.4mL concentrated sulfuric acid were added to a 1L round-bottom flask equipped with 18g (86mmol) of mucic acid and a magnetic stirring bar. The mixture was heated to reflux (85°C) overnight (about 18 hours) under constant stirring. After about 18 hours, 32mL triethylamine (TEA) was added to the flask, and the mixture was stirred at reflux (85°C) for 60 minutes. Then 30g (187mmol) of N-Boc-ethylenediamine dissolved in 50mL MeOH was added to the reaction mixture. The reaction was stirred at reflux (85°C) overnight (about 18 hours). The reaction mixture was cooled to room temperature, filtered and washed with MeOH. The collected solid was then dissolved in 450mL MeOH and refluxed for 1 hour under constant stirring. The reaction mixture was cooled to room temperature, vacuum filtered, and washed with MeOH. The collected solid was dried under vacuum overnight to give 26.1 g (52.8 mmol, 62% yield) of N-Boc-protected mucic acid diamine (2).

[0386]

[0387] Example 2. Synthesis of diaminomucin chloride (Compound 3)

[0388] To a 500 mL round bottom flask containing 25.9 g (52 mmol) of N-Boc-protected mucic acid diamine (2) and a magnetic stir bar was slowly added 466 mL of 3 M aqueous hydrochloric acid (HCl) in MeOH under constant stirring. After the slow addition of HCl, the reaction flask was moved to an orbital shaker and shaken overnight at room temperature. The reaction slurry was filtered and the solid was washed 3 times with MeOH (150 mL). The collected solid was dried overnight at 35 ° C under vacuum for several hours to obtain 18.0 g (49 mmol, 94% yield) of mucic acid diamino chloride (3).

[0389]

[0390] Example 3. Synthesis of Dimucic Acid (Asp(OtBu)-Boc) (Compound 4)

[0391] To a 250 mL round bottom flask containing 14.9 g (51 mmol) of 4-tert-butyl Boc-L-aspartate, 6 g (54 mmol) of hydroxypyridine N-oxide (HOPO) and a magnetic stirring bar, 140 mL of acetonitrile (ACN) and 8.5 mL (54 mmol) of N,N'-diisopropylcarbodiimide (DIC) were added. The reaction mixture was stirred for 10 minutes. A suspension of 6.3 g (17 mmol) of mucic acid diamino chloride (3) dissolved in a mixture of water (55 mL) and TEA (5.4 mL) was then added to the reaction flask. The reaction mixture was heated (80 ° C) for about 24 hours under constant magnetic stirring. After heating, ACN was removed from the reaction mixture using vacuum. Additional water (150 mL) was added to the concentrated reaction, and the mixture was heated (85 ° C) for 1 hour. After cooling, the reaction mixture was collected by vacuum filtration and washed with water 3 times (100 mL). The washed solid was returned to the round bottom flask with ACN (150 mL). The reaction mixture was heated to reflux under constant stirring until dissolved. After cooling, the reaction mixture was collected by filtration and washed 3 times with frozen ACN (100 mL). The collected solid was returned to the round bottom flask again with ACN (150 mL), and the reaction mixture was heated to reflux under constant stirring for about 1 hour. After cooling, the reaction mixture was collected by filtration and washed 3 times with frozen ACN (100 mL). The collected solid was dried under vacuum overnight to obtain 5.9 g (7 mmol, 41% yield) of Asp(OtBu)-Boc (4).

[0392]

[0393] Example 4. Synthesis of Mucic Acid Monomer (MAM) (Compound 5)

[0394] Dichloromethane (DCM, 7.5 mL) was added to a 40 mL vial containing 1 g (1 mmol) of Asp(OtBu)-Boc (4) and a magnetic stirring bar. Trifluoroacetic acid (TFA, 7.5 mL, 98 mmol) was slowly added to the reaction mixture under constant stirring. After stirring at room temperature for 1 hour, the reaction mixture was precipitated by slowly adding methyl tert-butyl ether (MTBE, 100 mL). The precipitated mixture was centrifuged and then washed once with MTBE (100 mL). The collected solid was dried under vacuum and then dissolved in water (30 mL). The dissolved product was then passed through a 0.22 μm filter, frozen and lyophilized to obtain 0.2767 g (0.384 mmol, 32% yield) of MAM (5).

[0395]

[0396] Example 5. Synthesis of Mucic Acid Polymer (MAP) (Compound 6)

[0397] A mixture containing 224 mg (0.30 mmol) of MAM (5), 1 g of di(succinimidyl propionate)-PEG (diSPA-PEG 3.5k A 10 mL round-bottom flask containing 5-nitropropanediol (2-nitropropanediol) (0.27 mmol) and a magnetic stirring bar was sealed, and the two solids were dried under vacuum at room temperature for 4 hours. 4.1 mL of anhydrous dimethyl sulfoxide (DMSO) was then added to the reaction flask, and the mixture was heated (35°C). After dissolution, 208 μL of anhydrous N,N'-diisopropylethylamine (DIPEA, 1.19 mmol) was added to the reaction mixture. The reaction mixture was heated (35°C) for 66 hours under constant magnetic stirring. The reaction mixture was dialyzed against DMSO and then dialyzed against water using a 10 kDa membrane. The dialyzed product was then passed through a 0.22 μm filter, frozen and lyophilized to obtain 1.03 g (quantitative yield) of MAP (6).

[0398]

[0399] For compound 6 synthesized by this method, x is about 80 and y is about 16. Therefore, compound 6, determined as the arithmetic mean of the number average molecular weight and the weight average molecular weight, has an average molecular weight of about 65 kDa. 2k ) (e.g., for 2 kDa PEG, x is about 46), these values ​​will vary.

[0400] Example 6. Determination of MAP molecular weight

[0401] MAP (6) molecular weight was determined on a Malvern Omnisec gel permeation chromatography (GPC) system, which includes a solvent delivery pump, degasser, autosampler syringe, column compartment, refractive index detector and light scattering detector. Samples were prepared for analysis by dissolving MAP (6) in phosphate buffered saline (PBS) + 0.02% sodium azide at known concentrations in the range of 1-5 mg / mL. After complete dissolution, the sample was passed through a 0.45 μm filter. The absolute molecular weight was determined by injecting 100 μL of the filtered sample into a 2×PL Aquagel-OH 40 column (Agilent) at a flow rate of 0.7 mL / min. The analysis was performed at 30°C. The resulting polymer peaks were analyzed using Malvern OMNISEC software.

[0402] Example 7. Synthesis of 10-TBDPS-SN38 (Compound 7)

[0403] A 250 mL round bottom flask containing 2 g (5.10 mmol) of SN38 and a magnetic stir bar was charged with 100 mL of anhydrous DCM and 4.2 mL (30.58 mmol) of TEA. To the reaction mixture was then added 7.9 mL (30.58 mmol) of tert-butyldiphenylsilyl chloride (TBDPSCl). The mixture was heated to reflux (50 °C) overnight (about 16 hours) with constant stirring and then washed with 0.2 N HCl (2×50 mL), saturated NaHCO3 (100 mL) and brine (100 mL). The organic solution was dried over MgSO4 and evaporated under vacuum. The residue was dissolved in anhydrous DCM and precipitated with hexane. The precipitation was repeated to further remove excess TBDPSCl. The solid was dried in vacuo to give 1.2 g (1.9 mmol, 37% yield) of 10-TBDPS-SN38 (7).

[0404]

[0405] Example 8. Synthesis of 20-(Boc-Gly)-10-TBDPS-SN38 (Compound 8)

[0406] To a 40 mL vial containing 0.5 g (0.8 mmol) of 10-TBDPS-SN38 (7) was added 8.9 mL of anhydrous DCM and a magnetic stir bar. After the solvent was added, the reaction was cooled to 0°C. To a separate vial were added 0.35 g of Boc-Gly-OH (2.0 mmol), 0.39 g (2.0 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl), 0.1 g (0.8 mmol) of 4-dimethylaminopyridine (DMAP) and 3.8 mL of anhydrous DCM. The contents of the second vial were then transferred to the flask containing 7. After stirring at 0°C for 3 hours, the mixture was washed with 0.5% NaHCO (2×50 mL), water (50 mL), 0.1 N HCl (2×50 mL) and brine. The organic solution was dried over MgSO4 and evaporated in vacuo to afford 0.54 g of crude 20-(Boc-Gly)-10-TBDPS-SN38(8) (0.7 mmol, 86% yield).

[0407]

[0408] Example 9. Synthesis of 20-(Boc-GABA)-10-TBDPS-SN38 (Compound 9)

[0409] To a 40 mL vial containing 1 g (1.6 mmol) of 10-TBDPS-SN38 (7) was added 17.7 mL of anhydrous DCM and a magnetic stir bar. After the addition of solvent, the reaction was cooled to 0°C. To a separate vial were added 0.82 g (4.0 mmol) of Boc-GABA-OH, 0.77 g (4.0 mmol) of EDC.HCl, 0.20 g (1.6 mmol) of DMAP, and 7.6 mL of anhydrous DCM. The contents of the second vial were then transferred to the flask containing 7. After stirring at 0°C for 3 hours, the mixture was washed with 0.5% NaHCO3 (2×50 mL), water (50 mL), 0.1 N HCl (2×50 mL), and brine. The organic solution was dried over MgSO4 and evaporated in vacuo to give 1.05 g of crude 20-(Boc-GABA)-10-TBDPS-SN38 (9) (1.28 mmol, quantitative yield).

[0410]

[0411] Example 10. Synthesis of 20-(Boc-Hex)-10-TBDPS-SN38 (Compound 10)

[0412] To a 40 mL vial containing 0.6 g (0.95 mmol) 10-TBDPS-SN38 (7) was added 10.4 mL of anhydrous DCM and a magnetic stirring bar. After the addition of solvent, the reaction was cooled to 0°C. To a separate vial were added 0.55 g Boc-Hex-OH (2.4 mmol), 0.45 g (2.4 mmol) EDC.HCl, 0.12 g (0.98 mmol) DMAP and 4.5 mL of anhydrous DCM. The contents of the second vial were then transferred to a flask containing 7. After stirring at 0°C for 3 hours, the mixture was washed with 0.5% NaHCO (2×50 mL), water (50 mL), 0.1 N HCl (2×50 mL) and brine. The organic solution was dried over MgSO4 and evaporated in vacuo to afford 0.88 g of crude 20-(Boc-Hex)-10-TBDPS-SN38(10) (1.0 mmol, 111% yield).

[0413]

[0414] Example 11. Synthesis of 20-(Boc-Val)-10-TBDPS-SN38 (Compound 11)

[0415] To a 100 mL round bottom flask containing 1.0 g (1.6 mmol) 10-TBDPS-SN38 (7) was added 17.7 mL of anhydrous DCM and a magnetic stirring bar. After the addition of solvent, the reaction was cooled to 0°C. To a separate vial were added 0.87 g Boc-Val-OH (4.0 mmol), 0.77 g (4.0 mmol) EDC.HCl, 0.20 g (1.6 mmol) DMAP and 7.6 mL of anhydrous DCM. The contents of the second vial were then transferred to the flask containing 7. After stirring at 0°C for 3 hours, the mixture was washed with 0.5% NaHCO (2×50 mL), water (50 mL), 0.1 N HCl (2×50 mL) and brine. The organic solution was dried over MgSO4 and evaporated in vacuo to afford 1.24 g of crude 20-(Boc-Val)-10-TBDPS-SN38(11) (1.5 mmol, 93% yield).

[0416]

[0417] Example 12. Synthesis of 20-(HCl.Gly)-SN38 (Compound 12)

[0418] 75 mL of 12N HCl was slowly added to a 100 mL round bottom flask containing 1.2 g (1.5 mmol) 20-(Boc-Gly)-10-TBDPS-SN38 (8) and a magnetic stirring bar under constant stirring. After the solvent was added, the reaction was cooled to 0°C. 0.87 g Boc-Val-OH (4.0 mmol), 0.77 g (4.0 mmol) EDC.HCl, 0.20 g (1.6 mmol) DMAP and 7.6 mL anhydrous DCM were added to a separate vial. Hexane (20 mL) was added to the top of the HCl solution. After stirring at room temperature overnight (about 16 hours), the hexane layer was removed from the reaction. The reaction was then washed with hexane in triplicate (3×20 mL). HCl was then removed from the reaction mixture using vacuum. The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried in vacuo to afford 0.44 g (0.91 mmol, 60% yield) of 20-(HCl.Gly)-SN38 (12).

[0419]

[0420] Example 13. Synthesis of 20-(TFA.GABA)-SN38 (Compound 13)

[0421] To a 50 mL round bottom flask containing 0.37 g (0.45 mmol) 20-(Boc-GABA)-10-TBDPS-SN38 (9), 8.6 mL water and a magnetic stir bar was slowly added 8.6 mL of TFA under constant stirring. Hexane (20 mL) was added on top of the TFA solution. After stirring overnight (approximately 16 hours) at room temperature, the hexane layer was removed from the reaction. The reaction was then washed with hexane in triplicate (3×20 mL). The reaction mixture was then concentrated using vacuum. The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried under vacuum to give 0.16 g (0.27 mmol, 60% yield) of 20-(TFA.GABA)-SN38 (13).

[0422]

[0423] Example 14. Synthesis of 20-(TFA.Hex)-SN38 (Compound 14)

[0424] To a 50 mL round bottom flask containing 0.37 g (0.43 mmol) 20-(Boc-Hex)-10-TBDPS-SN38 (10), 8.3 mL water and a magnetic stirring bar was slowly added 8.3 mL of TFA under constant stirring. Hexane (20 mL) was added on top of the TFA solution. After stirring overnight (approximately 16 hours) at room temperature, the hexane layer was removed from the reaction. The reaction was then washed with hexane in triplicate (3×20 mL). The reaction mixture was then concentrated using vacuum. The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried under vacuum to give 0.16 g (0.26 mmol, 60% yield) of 20-(TFA.Hex)-SN38 (14).

[0425]

[0426] Example 15. Synthesis of 20-(HCl.Val)-SN38 (Compound 15)

[0427] To a 100 mL round bottom flask containing 1.2 g (1.4 mmol) of 20-(Boc-Val)-10-TBDPS-SN38 (11) and a magnetic stir bar was slowly added 75 mL of 12N HCl under constant stirring. Hexane (20 mL) was added on top of the HCl solution. After stirring overnight (approximately 16 hours) at room temperature, the hexane layer was removed from the reaction. The reaction was then washed with hexane in triplicate (3×20 mL). HCl was then removed from the reaction mixture using vacuum. The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried under vacuum to give 0.46 g (0.86 mmol, 60% yield) of 20-(HCl.Val)-SN38 (15).

[0428]

[0429] Example 16. Synthesis of MAP-Gly-SN38 (Compound 16)

[0430] A 10 mL round bottom flask containing 100 mg of MAP (6) and a magnetic stir bar was charged with 8 mL of anhydrous DMSO. Then, 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of N-hydroxysuccinimide (NHS) were added to the reaction mixture. After the addition of EDC.HCl and NHS, 105.2 mg (0.22 mmol) of 20-(HCl.Gly)-SN38 (12) and anhydrous DIPEA (0.22 mmol) were added to the reaction mixture. The mixture was stirred at room temperature overnight (about 18 hours). The reaction mixture was dialyzed against DMSO and then against pH 4 water using a 10 kDa membrane. The dialyzed product was then passed through a 0.22 μm filter, frozen and lyophilized to obtain 98 mg (quantitative yield) of MAP-Gly-SN38 (16).

[0431]

[0432] As described in Example 5, for compounds 6 and 16, x is about 80, and y is about 16.

[0433] Example 17. Synthesis of MAP-GABA-SN38 (Compound 17)

[0434] A 10 mL round bottom flask containing 100 mg MAP (6) and a magnetic stirring bar was charged with 8 mL of anhydrous DMSO. Then 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of NHS were added to the reaction mixture. After the addition of EDC.HCl and NHS, 128.3 mg (0.22 mmol) of 20-(TFA.GABA)-SN38 (13) and anhydrous DIPEA (0.22 mmol) were added to the reaction mixture. The mixture was stirred at room temperature overnight (about 18 hours). The reaction mixture was dialyzed with DMSO and then dialyzed with water at pH 4 using a 10 kDa membrane. The dialyzed product was then passed through a 0.22 μm filter, frozen and lyophilized to obtain 91 mg (quantitative yield) of MAP-GABA-SN38 (17).

[0435]

[0436] As described in Example 5, for compounds 6 and 17, x is about 80, and y is about 16.

[0437] Example 18. Synthesis of MAP-Hex-SN38 (Compound 18)

[0438] A 10 mL round bottom flask containing 100 mg of MAP (6) and a magnetic stir bar was charged with 8 mL of anhydrous DMSO. Then, 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of NHS were added to the reaction mixture. After the addition of EDC.HCl and NHS, 134.4 mg (0.22 mmol) of 20-(TFA.Hex)-SN38 (14) and anhydrous DIPEA (0.22 mmol) were added to the reaction mixture. The mixture was stirred at room temperature overnight (about 18 hours). The reaction mixture was dialyzed against DMSO and then against water at pH 4 using a 10 kDa membrane. The dialyzed product was then passed through a 0.22 μm filter, frozen and lyophilized to obtain 97 mg (quantitative yield) of MAP-Hex-SN38 (18).

[0439]

[0440] As described in Example 5, for compounds 6 and 18, x is about 80, and y is about 16.

[0441] Example 19. Synthesis of MAP-Val-SN38 (Compound 19)

[0442] A 10 mL round bottom flask containing 100 mg of MAP (6) and a magnetic stir bar was charged with 8 mL of anhydrous DMSO. Then, 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of NHS were added to the reaction mixture. After the addition of EDC.HCl and NHS, 114.3 mg (0.22 mmol) of 20-(HCl.Val)-SN38 (15) and anhydrous DIPEA (0.22 mmol) were added to the reaction mixture. The mixture was stirred at room temperature overnight (about 18 hours). The reaction mixture was dialyzed against DMSO and then against water at pH 4 using a 10 kDa membrane. The dialyzed product was then passed through a 0.22 μm filter, frozen and lyophilized to obtain 96 mg (quantitative yield) of MAP-Val-SN38 (19).

[0443]

[0444] As described in Example 5, for compounds 6 and 19, x is about 80, and y is about 16.

[0445] Example 20. Drug loading properties

[0446] The loading of SN38 onto MAP was determined on an Agilent 1100 HPLC system with a reverse phase column (Synergi 4 μm Hydro-RP Phenomenex) with the fluorescence detector set to 375 / 536 nm (ex / em). ACN / 10 mM potassium phosphate buffer, pH 4 (1:1 v / v) was used as the eluent with a flow rate of 0.5 mL / min.

[0447] For analysis, polymer drug conjugates (compounds 16-19) were dissolved in PBS at pH 7.4 at a concentration of 1 mg / mL. The amount of uncoupled SN38 was determined by first mixing 10 μL of sample with 10 μL of 0.1N HCl and incubating at room temperature for 30 minutes. 6.7 μL of water was then added, followed by 73.3 μL of ACN, and the mixture was incubated at room temperature for 3 hours. The mixture was centrifuged at 14,000g for 10 minutes at 4°C, and the supernatant was then passed through a 0.45 μm filter. The SN38 concentration was determined by injecting 10 μL of the filtered sample. The resulting peak area of ​​the eluted SN38 was compared with the peak area of ​​SN38 of known concentration.

[0448] To measure the total amount of SN38, 10 μL of sample was mixed with 6.7 μL of 0.1N NaOH. The solution was incubated at room temperature for 3.5 hours to release SN38 from the parent polymer. 10 μL of 0.1N HCl was then added (to convert the carboxylate SN38 form to the lactone form) and the mixture was incubated for 45 minutes. 73.3 μL of ACN was then added and the mixture was incubated at room temperature for 3 hours. The sample was then centrifuged and processed as above. The polymer-bound SN38 was determined by the difference between the total SN38 and uncoupled SN38 concentrations.

[0449] Example 21. Formation of MAP-Linker-SN38 Nanoparticles

[0450] The polymer drug conjugates (compounds 16-19) were dissolved in water at pH 4 at a concentration of 4 mg / mL to form MAP-linker-SN38 nanoparticles. The solution was then passed through a 0.22 μm filter and frozen for subsequent analysis. The nanoparticles were diluted to 2 mg / mL in water at pH 4 and the hydrodynamic diameter was measured by dynamic light scattering (DLS) using a ZetaPALS (Brookhaven Instruments Corporation) instrument. The nanoparticles were operated five times for 1 minute each. The diameter of all nanoparticles was close to 25 nm, ranging from 19 to 32 nm.

[0451] Example 22. In vitro release study

[0452] Experiments were performed to evaluate the release of SN38 from MAP-Gly-SN38, MAP-GABA-SN38, MAP-Hex-SN38, MAP-Val-SN38, MAP-Ala-SN38, MAP-β-Ala-SN38, and MAP-Leu-SN38 nanoparticles. See Examples 33-35 below for the synthesis of MAP-Ala-SN38, MAP-β-Ala-SN38, and MAP-Leu-SN38 nanoparticles. These studies were performed in PBS at 0.1 mg SN38 / mL at pH 6.5, 7.0, or 7.4.

[0453] The PBS medium was pipetted into a cuvette and incubated in a humidified oven at 37°C for 2 hours to reach equilibrium. The nanoparticle formulation was mixed into the relevant medium and returned to the oven. Samples were removed at predetermined time points and immediately frozen at -80°C until the time of analysis. The amount of unconjugated SN38 and the total amount of SN38 were determined as described in Example 20 above. The polymer-bound SN38 concentration was determined by the difference between the total SN38 and unconjugated SN38 concentrations.

[0454] The results showed that the release of SN38 from all seven of the following nanoparticles exhibited first-order kinetics: MAP-Gly-SN38, MAP-GABA-SN38, MAP-Hex-SN38, MAP-Val-SN38, MAP-Ala-SN38, MAP-β-Ala-SN38, and MAP-Leu-SN38. A strong dependence of the release rate on pH was observed. The release half-life decreased for all nanoparticle formulations as the pH increased from 6.5 to 7.4, indicating that hydrolysis plays an important role in the release of SN38. Longer half-lives were observed for nanoparticles with more hydrophobic and sterically hindered linkers, ranging from approximately 30 hours to >168 hours.

[0455] Example 23. Determination of the number of chains per particle

[0456] The number of chains per nanoparticle (SpP) was determined by calculating the ratio between the molecular weight of the particle and the molecular weight of the MAP. Polyethylene oxide (PEO) standards and target samples with nominal molecular weights of 100 kDa and 200 kDa were prepared for GPC analysis (Omnisec GPC, Malvern) by dissolving in water at pH 3 or PBS + 0.02% sodium azide to a concentration of 1-5 mg / mL. The selected concentration was similar to the concentration expected after particle formation. After dissolution, the sample was passed through a 0.45 μm filter and analyzed on a Zenix SEC 300 or Zenix-C SEC 300 column (Sepax, 3 μm) at 0.35 mL / min at 30 ° C. Light scattering and differential refractive index information were used with Malvern OMNISEC software or similar molecular weight analysis software to determine the molecular weight of the particles.

[0457] The molecular weight of the particle was divided by the molecular weight of the MAP-SN38 conjugate molecule to determine the number of chains per particle. The molecular weight of the MAP-SN38 conjugate molecule was calculated by adjusting the molecular weight of the polymer for the drug loading percentage according to the following formula:

[0458] MW 缀合物 =MW 聚合物 / (1-T ％ )

[0459] MW 聚合物 is the molecular weight of MAP, T ％ is the percentage loading of the therapeutic agent, expressed as a decimal, e.g., a 10% loading results in T ％ =0.1.

[0460] Determination of SpP was demonstrated using MAP with different linkers and drugs. MAP of the same molecular weight coupled to CPT or SN38 produced nanoparticles with approximately 1.5-2.5 chains per particle. Using different linkers on the derivatized SN38 changed the number of chains per particle and thus the density of the resulting particles. The number of chains per particle varied from about 1.3 to about 4.6, depending on the linker (glycine, γ-aminobutyric acid, hexanoic acid, alanine, β-alanine, leucine, or valine).

[0461] Example 24. Preparative Scale Synthesis of N-Boc-Protected Mucic Acid Diamine (Compound 2)

[0462] Under nitrogen, 450 g (2.1 mol) of mucic acid and MeOH (5.75 L) were added to a mechanically stirred 15-liter reaction vessel. A solution consisting of MeOH (1 L) and concentrated sulfuric acid (34.2 mL, 0.64 mol) was added to the reaction vessel. The mixture was heated to an internal temperature of 65° C. for 48 hours. The contents of the reaction vessel were cooled to 22° C., at which time 0.81 L (5.78 mol) of TEA was added over the course of 20 minutes. The reaction was stirred for 1 hour, at which time a solution of 0.76 kg (4.71 mol) of N-(2-aminoethyl) (tert-butoxy) formamide in 0.9 L of MeOH was added over the course of 20 minutes. The reaction was heated to an internal temperature of 63° C., and 1.8 L of MeOH was added after 60 minutes to facilitate stirring. After a total of 2 hours at 63° C., the reaction vessel was cooled at a rate of 1° C. per minute and kept at 20° C. overnight. The reaction slurry was filtered and the filter cake was washed with MeOH (3×0.5 L). The solid was dried on a vacuum filter overnight to ensure complete removal of the liquid. The separated solid was loaded back into the reaction vessel with MeOH (9.0 L) and stirred at 63 ° C for 1 hour. The reaction vessel was set to cool at a rate of 1 ° C per minute and maintained at 20 ° C for about 60 hours. The slurry was filtered and washed with MeOH (2×1.0 L), and the solid was dried in a vacuum oven at 45 ° C overnight to obtain 963 g (91% yield, as a solvate with MeOH, 82% anhydrous yield) of N-Boc-protected mucic acid diamine (2).

[0463]

[0464] Example 25. Preparative Scale Synthesis of Mucic Acid Diamino Chloride (Compound 3)

[0465] 890 g of N-Boc-protected mucic acid diamine (2) (1.80 mmol), 3.6 L of water and 7.1 L of MeOH were added to a mechanically stirred 15-liter reaction vessel. 12 M HCl (3.6 L) was added to the slurry over 2 hours while maintaining the temperature in the range of 20° C. to 30° C. The reaction was stirred overnight, at which point the slurry was filtered and washed with 1.8 L of MeOH. The separated solid was dried in a vacuum oven at 45° C. in a tray. The separated solid was loaded into a 15 L reaction vessel with water (5.5 L) under nitrogen and stirred at 45° C. 5.5 L of MeOH was added over the course of 30 minutes to cause crystallization. The reaction vessel was frozen to cool the reaction contents to room temperature. The solid was separated by filtration and washed with MeOH (2×1.0 L). The solid was collected and dried in a vacuum oven at 40°C overnight to give 504 g (1.37 mol, 76% yield) of mucic acid diamino chloride (3).

[0466]

[0467] Example 26. Preparative Scale Synthesis of Di(Asp(OtBu)-Boc) Mucic Acid (Compound 4)

[0468] 1.27 kg of Boc-L-aspartic acid 4-tert-butyl ester (4.39 mol), 0.62 kg of Oxyma (ethyl cyanohydroxyiminoacetate) (4.39 mol) and 3.5 L of DCM were added to a mechanically stirred 15 L reactor. The reaction mixture was cooled to 5 ° C and stirred for 30 minutes. 0.84 kg of EDC.HCl (4.40 mol) solid was added to the reaction mixture over the course of 10 minutes. EDC.HCl was flushed into the reaction with an additional 0.5 L of DCM, the reaction was stirred for about 30 minutes, and then refrigerated to 10 ° C. 1.02 kg of sodium carbonate (9.61 mol) and 5.0 L of water were charged to a separate 12 L flask. The components were dissolved and then cooled to room temperature. Over the course of 10 minutes, 504 g (1.37 mol) of 3 was added to the sodium carbonate solution. The mixture was stirred for 15 minutes, at which time it was added to the Boc-L-aspartic acid 4-tert-butyl ester in the 15 L reaction flask over the course of about 3 minutes. The mixed reaction was exothermic, but the cooling jacket was set to 10°C for 30 minutes and then to 15°C for another 45 minutes. The reaction jacket was then set to 33°C for 1 hour, and then 8.06L of water was added under stirring for 5 minutes. Stirring was then stopped and the reaction phases were separated after 15 minutes. The DCM (bottom layer) was removed and an additional 1.0L of DCM was added to the reactor. The mixture was then stirred for 10 minutes and then allowed to settle. The DCM layer was collected and combined with the organic layer initially collected and concentrated to a total volume of about 3L. 1 liter of water was added to the organic mixture to help further distill off the DCM. The organic reaction mixture was then added to a 15L reactor containing 11.1L of water heated to 40°C. The stirred mixture was returned to 20°C and a slurry was formed. Water was removed and the solids were left in the reactor. 10.1 liters of water were added to the wet solid. The reaction mixture was heated to 37°C to facilitate stirring. The reaction was then cooled to 3°C overnight and stirred. The solid was filtered and then loaded back into the reactor with an additional 10L of water. The slurry was stirred at room temperature for 3 hours. The solid was filtered again, washed with 1.0L water, and dried to obtain 815g of material. 790 grams of crude product 4 was added to a 5L round-bottom flask together with 2.4L of ACN and heated to 60°C. Water was continuously distilled off by azeotropic distillation under partial vacuum, and ACN was continued to be added to a volume of about 1.6L. 3.1L ACN was added thereto to induce crystallization. The slurry was briefly heated to reflux, then cooled to 5°C and kept for 30 minutes. After cooling, the slurry was filtered and washed with ACN (4×0.5L, 5°C). The solid was heated to 50°C under vacuum to remove the solvent. 6.3L of DCM was added to the reaction mixture in two batches, at which time the slurry was concentrated to distill off ACN. The solid was then heated to 80°C under vacuum to remove DCM and provide a free-flowing powder.The solid was then placed in a vacuum oven and dried at 80°C under reduced pressure to give 310 g (0.35 mol, 26% yield) of mucic acid bis(Asp(OtBu)-Boc) (4) as a 1:1 molar solvate with ACN.

[0469]

[0470] Example 27. Preparative Scale Synthesis of Neutral Mucic Acid Monomer (Compound 20)

[0471] 300g of 4 (0.34mol), 1.2L DCM, 0.11L water (6.13mol) and 0.279L triisopropylsilane (1.36mol) are loaded into a 5L jacketed round-bottomed flask equipped with mechanical stirring. The reaction mixture is cooled to 5°C and subsequently forms a slurry. 1.2L TFA that was previously cooled in an ice bath for 1 hour is added to the stirred slurry. The reaction is maintained at an internal temperature of 4°C overnight. Additional 0.30L TFA is added and continued to stir overnight at 4°C. After adding 0.90L hexane, the reaction is stirred at 5°C for 10 minutes. The reaction mixture is then poured into a separating funnel and the bottom phase is separated into a 5L round-bottomed funnel. The reaction mass is rinsed with additional DCM (300mL) and merged with the fraction initially collected. The collected fractions merged are concentrated on a rotary evaporator lower than 30°C. The additional 0.90L DCM is added to the flask and subsequently removed using a rotary evaporator at 25°C. 0.60L toluene is then added and concentrated on a rotary evaporator to remove water and TFA azeotropically to obtain an oil. The oil is filtered through a diatomaceous earth plug and washed four times with DCM (0.40L). DCM is removed by a rotary evaporator at 35°C. The resulting biphasic mixture is added to a clean 5L reaction vessel and heated to 23°C. 3.0L cyclopentyl methyl ether (CPME) is added over the course of 20 minutes to form an oily solid. The container is cooled to 22°C and stirred overnight to provide sticky crystals. 1.5L DCM is distilled out using a short-path distillation column at 35°C. The resulting slurry is filtered and washed with CPME (2×0.45L). The solid was then dried in a round bottom flask on a rotary evaporator and then dried under vacuum at room temperature overnight to yield 261 g of the neutral material of the mucic acid monomer (208.8 g, 82% potency yield calculation).

[0472] For further recrystallization, 0.24 kg of the neutral substance of the mucic acid monomer was added to a 1 L Erlenmeyer flask together with water (0.20 L). The mixture was stirred at room temperature for 1 hour to provide a solution. The solution was filtered into a 5 L round-bottom flask through a filter funnel with the aid of additional water (50 mL). During 30 minutes, 2.2 L of tetrahydrofuran (THF) was added to the flask under stirring. The mixture was stirred for a total of 60 minutes, at which time it was concentrated on a rotary evaporator at 35 ° C to remove 1 L of distillate. Additional THF (0.98 L) was added within 10 minutes. The slurry was cooled to room temperature and stirred for 20 minutes. The cooled slurry was filtered and washed with THF (2×0.25 L), then dried in a vacuum oven at room temperature for 2.5 days to obtain 119 g (0.23 mol, 66% yield) of the neutral form of the mucic acid monomer (20).

[0473]

[0474] Example 28. Preparative Scale Synthesis of MAM (Compound 5)

[0475] A 3 L round bottom flask containing 120 g (0.23 mol) of 20 and DCM (1.2 L) was stirred for 10 minutes, at which time water (0.06 L) was added. Stirring was continued for 15 minutes, at which time TFA (66.0 mL, 0.86 mol) was added over 5 minutes. The solid dissolved and the mixture was stirred at room temperature for 1 hour. The solid dissolved and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated on a rotary evaporator at 30 °C. To a 5 L round bottom flask equipped with mechanical stirring was added ether (4.0 L) and MAM seeds (5,12 g). The 20 / TFA / water solution was slowly added to the ether with stirring. After 1 hour, the ether was decanted and additional fresh ether (3.6 L) was added. The mixture was stirred at room temperature until the MAM-diTFA salt precipitated. The solid was filtered and washed once with ether (0.2 L). The solid was filtered and washed once with ether (0.2 L). The solid was dried under vacuum at room temperature and then heated at 30°C for 1 hour to afford 122 g (75% yield) of MAM (5).

[0476]

[0477] Example 29. Preparative Scale Synthesis of Mucic Acid Polymer (Compound 6)

[0478] MAP (6) is a poly(amic acid) monomer (MAM) (5) and diSPA-PEG 3.5k The synthesis is carried out by a stepwise polymerization reaction.

[0479] A solution containing about 1.5 equivalents of TFA, 579.54 mg of diSPA-PEG 3.5kA 20 mL glass vial of 108.52 mg (0.158 mmol) of MAM (5) with 1% dextrin (0.157 mmol) and a magnetic stirring bar was sealed, and the two solids were vacuum dried at room temperature for 1 hour. 3.43 mL of anhydrous DMSO was added to the reaction flask under argon. The reactants were dissolved at room temperature. After dissolution, 126 μL of anhydrous DIPEA (0.72 mmol) was added to the reaction mixture under argon. The reaction mixture was stirred at room temperature for 3 hours under constant magnetic stirring.

[0480] The reaction mixture was precipitated in cold isopropanol (IPA, ×10 volumes, 0-4°C). The reactor was washed with DMSO (3×1 mL) and then precipitated in cold IPA. The obtained white suspension was stirred at 0-4°C for 30 minutes and centrifuged at 4°C for 12 minutes. The white fluffy polymer precipitated at the bottom was separated by decanting the IPA. The polymer was washed twice with cold IPA (×5 volumes) and the operation was repeated. The product was dried under vacuum on a Schlenk line at room temperature overnight to remove the IPA and then dissolved in milli-Q water (8 mL). The aqueous solution was frozen and lyophilized to obtain 612.5 mg (quantitative yield) of MAP (6).

[0481] The molecular weight of the polymer is controlled by introducing a stoichiometric deviation between the two monomers. At 1:1, the molecular weight of MAP (6) is the highest, and as the molar ratio deviates from 1:1, the molecular weight decreases. Exemplary stoichiometric deviations and experimental molecular weights obtained by GPC are shown in Table 1.

[0482] Table 1. Stoichiometric deviations in controlled MAP (6) synthesis

[0483] <![CDATA[MAM:diSPA-PEG 3.5k Ratio]]> Molecular weight (kDa) 1.01 77.49 1.04 62.44 1.10 55.87

[0484] Example 30. Synthesis of 20-(Boc-Gly)-CPT (Compound 21)

[0485] 200.4 mg (0.58 mmol) of CPT, 302.7 mg (1.73 mmol) of Boc-Gly-OH and 140.6 mg (1.15 mmol) of DMAP were added to a 10 mL round bottom flask equipped with a magnetic stirring bar and a rubber septum. The container and its contents were purged with argon for 10 minutes, at which time the material was slurried by adding anhydrous DCM (1 mL). Over the course of approximately three minutes, 280 μL (1.81 mmol) of DIC was added dropwise to the reaction mixture via a syringe. The reaction was stirred at room temperature for 3 hours, at which time approximately 50% of the solvent was removed under vacuum. 4 mL of frozen MeOH was added to the concentrated slurry. The reaction was stirred for a few minutes to promote further precipitation, then filtered into a Buchner funnel. The solid was washed with more frozen MeOH, then with frozen MTBE. The isolated solid was dried under high vacuum to afford 110.8 mg (0.22 mmol, 38% yield) of 20-(Boc-Gly)-CPT (21).

[0486] In the above operation, other Boc-protected amino acids and dipeptides can be used to replace Boc-Gly-OH. By replacing with Boc-Ala-OH, Boc-β-Ala-OH, Boc-Val-OH, Boc-GABA-OH and Boc-Phe-Gly-OH, the same operation was used to obtain 20-(Boc-Ala)-CPT (Compound 41), 20-(Boc-β-Ala)-CPT (Compound 42), 20-(Boc-Val)-CPT (Compound 40), 20-(Boc-GABA)-CPT (Compound 43) and 20-(Boc-Phe-Gly)-CPT (Compound 44), respectively.

[0487]

[0488] Example 31. Synthesis of 20-(TFA.Gly)-CPT (Compound 22)

[0489] To a scintillation vial equipped with a magnetic stirrer and an orifice needle was added 97.1 mg (0.20 mmol) of 20-(Boc-Gly)-CPT (21). A stirred suspension was generated by the addition of DCM (0.2 mL). TFA (0.2 mL) was gradually added under stirring. The reaction mixture was stirred for 2 hours. After 2 hours, the product was precipitated with MTBE, filtered, and washed. The isolated sample was dried under high vacuum to give 82.5 mg (0.16 mmol, 83% yield) of 20-(TFA.Gly)-CPT (22).

[0490]

[0491] In the above method, 20-(Boc-Ala)-CPT, 20-(Boc-β-Ala)-CPT, 20-(Boc-Val)-CPT, 20-(Boc-GABA)-CPT and 20-(Boc-Phe-Gly)-CPT were used instead of 20-(Boc-Gly)-CPT to obtain 20-(TFA.Ala)-CPT (Compound 45), 20-(TFA.β-Ala)-CPT (Compound 46), 20-(TFA.Val)-CPT (Compound 47), 20-(TFA.GABA)-CPT (Compound 48) and 20-(TFA.Phe-Gly)-CPT (Compound 49), respectively.

[0492] Example 32. Synthesis of MAP-Gly-CPT (Compound 23)

[0493] 149.2 mg (0.07 mmol, based on CO2H) of MAP (6) and 21.5 mg of 20-(TFA.Gly)-CPT (22) (0.04 mmol) were added to a 20 mL scintillation vial equipped with a magnetic stirrer. The headspace of the container was evacuated with argon for about 30 minutes and then sealed under argon. After adding anhydrous DMSO (about 9 mL, about 85% of the total reaction volume), the contents of the vial were dissolved under stirring. An 8 mL scintillation vial containing 166.3 mg of (7-azabenzotriazole-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyAOP, 0.32 mmol, 4.4 eq) and a magnetic stirrer was purged under an argon atmosphere. The contents of the container were dissolved in anhydrous DMSO (about 3.2 mL) under stirring. Once all the solids in both containers are dissolved, 1.6 mL of PyAOP solution (83.2 mg PyAOP, 0.16 mmol PyAOP, approximately 15% of the total reaction volume) is transferred to the container containing the polymer. The reaction is stirred for approximately 2 minutes, at which time approximately 45 μL (0.26 mmol) of anhydrous DIPEA (target 31.3 μL, 0.18 mmol) is added. The reaction vessel is stirred at room temperature in the dark for approximately 18 hours. After 18 hours, the reaction vessel is removed from the argon atmosphere and approximately 1 / 3 of the reaction contents are transferred to a 50 mL glass centrifuge tube. The reaction is precipitated by slowly adding cold ethyl acetate (EtOAc) (0-4°C). The suspension is stirred at 0-4°C for approximately 30 minutes and then centrifuged. The EtOAc layer is decanted and the lower 1 / 3 of the reaction medium is added to the same container. Repeat this operation until all the reaction medium has been precipitated into the same tube. The precipitated solid was washed a total of 3 times with chilled EtOAc (×5 volumes) while vortexing to resuspend the solid. After the final wash cycle, the final EtOAc was decanted and the solid was frozen in a centrifuge tube using liquid nitrogen. The cooled centrifuge tube was exposed to high vacuum for approximately 3 hours to dry the solid and provide 115.2 mg (quantitative yield) of MAP-Gly-CPT (23).

[0494]

[0495] MAP-CPT conjugates were also prepared by the same method using 20-(TFA.Ala)-CPT, 20-(TFA.β-Ala)-CPT, 20-(TFA.Val)-CPT, 20-(TFA.GABA)-CPT or 20-(TFA.Phe-Gly)-CPT as starting materials instead of 20-(TFA.Gly)-CPT to produce MAP-Ala-CPT (Compound 50), MAP-β-Ala-CPT (Compound 51), MAP-Val-CPT (Compound 52), MAP-GABA-CPT (Compound 53) and MAP-Phe-Gly-CPT (Compound 54).

[0496]

[0497]

[0498]

[0499] As described in Example 5, for compounds 6, 23, and 50-54, x is about 80, and y is about 16.

[0500] Example 33. Synthesis of 10-OBoc-SN38 (Compound 24)

[0501] Under argon atmosphere, 2.0 g (5.10 mmol) of SN38 was added to the reaction vessel. 204 mL of anhydrous DCM was added to the reaction vessel, followed by 12.2 mL (151 mmol) of anhydrous pyridine and 1.5 mL (6.53 mmol) of di-tert-butyl dicarbonate. The reaction was stirred at room temperature for about 16 hours, at which time the reaction was filtered through Celite 545 (filter aid) and washed with 0.5 N HCl and saturated sodium bicarbonate solution. The organic phase was dried over magnesium sulfate and concentrated in vacuo. The reaction mixture was diluted into DCM and then precipitated into hexane. The solid was filtered and dried in vacuo to give 2.2 g (4.47 mmol, 88% yield) of 10-OBoc-SN38 (24).

[0502]

[0503] Example 34. Synthesis of 20-(Boc-Gly)-10-OBoc-SN38 (Compound 25)

[0504] To a reaction vessel cooled to 0°C containing 502 mg (1.02 mmol) of 10-OBoc-SN38 (24) was added 8.8 mL of DCM under an argon atmosphere. A frozen solution containing 3.8 mL of DCM, 447 mg (2.55 mmol) of Boc-Gly-OH, 489 mg (2.55 mmol) of EDC.HCl and 125 mg (1.02 mmol) of DMAP was added to the reaction vessel. The reaction was stirred at 0°C for 3 hours. The reaction was washed with 0.5% sodium bicarbonate solution, water, 0.1 N HCl and brine. The organic phase was dried over magnesium sulfate and evaporated in vacuo to give 639 mg (0.98 mmol, 97% yield) of 20-(Boc-Gly)-10-OBoc-SN38 (25).

[0505] In the above operation, other Boc-protected amino acids can be used to replace Boc-Gly-OH. By replacing Boc-Ala-OH, Boc-β-Ala-OH, Boc-Val-OH and Boc-Leu-OH as starting materials, the same operation was used to obtain 20-(Boc-Ala)-10-OBoc-SN38 (Compound 29), 20-(Boc-β-Ala)-10-OBoc-SN38 (Compound 30), 20-(Boc-Val)-OBoc-SN38 (Compound 31) and 20-(Boc-Leu)-10-OBoc-SN38 (Compound 35), respectively.

[0506]

[0507] Example 35. Synthesis of 20-(TFA.Gly)-SN38 (Compound 26)

[0508] To a reaction vessel containing 250 mg (0.38 mmol) 20-(Boc-Gly)-10-OBoc-SN38 (25) and 1.88 mL DCM was added 0.47 mL (6.14 mmol) TFA. The reaction was stirred at room temperature for 2.5 hours. The reaction mixture was precipitated into chilled (4° C.) MTBE, separated by centrifugation, washed again with MTBE, and dried under vacuum to give 133 mg (0.24 mmol, 61% yield) 20-(TFA.Gly)-SN38 (26).

[0509]

[0510] Using 20-(Boc-Ala)-10-OBoc-SN38, 20-(Boc-β-Ala)-10-OBoc-SN38, 20-(Boc-Val)-10-OBoc-SN38 and 20-(Boc-Leu)-10-OBoc-SN38 as the starting material instead of 20-(Boc-Gly)-10-OBoc-SN38, 20-(TFA.Ala)-SN38 (compound 32), 20-(TFA.β-Ala)-SN38 (compound 33), 20-(TFA.Val)-SN38 (compound 34) and 20-(TFA.Leu)-SN38 (compound 36) were obtained respectively.

[0511] As described in Examples 17 and 18, TFA salts of the aforementioned amino acid-linked SN38 derivatives can be used to form MAP-SN38 conjugates.

[0512] Example 36. NPBA-PEG 5k Synthesis of -AA (Compound 27)

[0513] Anhydrous DCM (40 mL) was charged into a 20 g (4 mmol) NH2-PEG 5k-acetic acid (AA) hydrochloride, DIPEA (2.1 mL, 12 mmol) in a 250 mL round bottom flask and purged with argon. The solution was cooled in an ice / water bath. In a 20 mL vial containing a magnetic stir bar, 934 mg (5.5 mmol) of 3-carboxy-5-nitrophenylboronic acid (NPBA) was dissolved in anhydrous DCM / dimethylformamide (DMF) (8 mL, 4:1 v / v). 1038 mg (5.25 mmol) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added to the NPBA solution. The activated acid was stirred for 15 minutes and then added dropwise to a pre-cooled solution of PEG-amine in DCM. The reaction mixture was stirred at 0 ° C for 20 minutes. Deionized water (100 mL) was added to the reaction mixture, stirred vigorously and warmed to room temperature. At this stage, the pH of the reaction mixture was 9. Subsequently, monosodium phosphate (NaH2PO4, 2.5g) was added to make the pH 7. 1M HCl (12mL) was added to further reduce the pH value of the reaction mixture to 4. The reaction mixture was transferred to a separatory funnel and isopropyl acetate (iPrOAc, 100mL) was added. The mixture was fully shaken and allowed to settle to obtain a separated phase. The organic phase was discarded and repeatedly extracted with iPrOAc (3×100mL). The aqueous phase was transferred to a 250mL round-bottom flask and concentrated on a rotary evaporator at room temperature at 20Torr for 30 minutes. The aqueous solution was freeze-dried to provide a powder. The freeze-dried powder was dissolved in DMSO / water (40mL, 3:1v / v). The solution was purified by hydrophobic interaction chromatography on a C18Aq Teledyne ISCO column (475 g), which was pre-equilibrated with 95% mobile phase A (water + 0.05% TFA) and 5% mobile phase B (ACN + 0.05% TFA), with an injection volume of 12-15 mL. Before washing the column and performing subsequent injections, the gradient was run from 5% B to 40% B. The remaining product solution was purified repeatedly. The fractions containing the product were analyzed by HPLC. Fractions with a purity of >99.5% were combined and lyophilized to obtain 14.2 g (2.71 mmol, 68% yield) of NPBA-PEG 5k -AA(27).

[0514]

[0515] For compound 27 synthesized by this method, n is about 114. If a different starting material (e.g., NH2-PEG 3.5k -AA), these values ​​will vary (e.g., for 3.5 kDa PEG, n is about 80).

[0516] Example 37. NPBA-PEG 5kSynthesis of -AA-PFP (Compound 28)

[0517] Add 301.8 mg (0.06 mmol) NPBA-PEG to the 8 mL reaction vessel. 5k -AA (27) and a magnetic stirring bar. The reaction vessel is purged three times with vacuum and argon circulation. The solid is dissolved in anhydrous DCM (1.2mL), and 35 μL of anhydrous N-methylmorpholine (0.35mmol) is then added by syringe. The reaction vessel is briefly opened and 45.3mg of bis (pentafluorophenyl) carbonate (0.12mmol) is added. The reaction head space is purged with argon, and the reaction is stirred at room temperature for about 2 hours. The reaction solution is added dropwise to the MTBE (25mL) of the stirring volume to promote precipitation. The mixture is stirred for 30 minutes under an argon atmosphere, and then centrifuged at 2600g. The liquid layer is decanted, and heptane (20mL) is now added to a centrifuge tube. Before a second round of centrifugation at 2600g (8 minutes), the sample is mechanically stirred. Liquid is poured out from the container to leave remaining wet solid. The vessel was briefly cooled in liquid nitrogen and then pumped using a Schlenk line to remove the solvent, yielding 300 mg (0.055 mmol, 96% yield) of NPBA-PEG 5k -AA-PFP(28).

[0518]

[0519] As described in Example 36, n in compounds 27 and 28 is about 114.

[0520] Example 38. NPBA-PEG 5k Synthesis of Crude Mixture of -Tf

[0521] 35 g (0.440 mmol) of holotransferrin (Tf) was loaded into a reaction vessel, and 1.75 L of 50 mM HEPES (pH 7.8) was slowly added to dissolve Tf at a concentration of 20 g / L. After dissolution, 4.77 g (0.881 mmol) of NPBA-PEG in 44 mL of DMSO was added. 5k-AA-PFP (28) is slowly added to the reaction mixture and fully mixed. The reaction mixture is incubated at room temperature for 2 hours. Thick reaction mixture is analyzed on the Agilent1290HPLC system equipped with reversed phase (RP) column (Zorbax 300SB-CN 4.6mm ID x 150cm), mobile phase A (water+0.1% TFA) and mobile phase B (ACN / IPA (4:1v / v)+0.1%TFA), flow velocity is 0.7mL / min. Chromatographic column is connected to the absorbance detector that is set to 280nm wavelength and 360nm reference wavelength. In addition, the crude reaction mixture was analyzed on an Agilent 1290 HPLC system equipped with a hydrophobic interaction chromatography (HIC) column (TSK gel Butyl-NPR 4.6 mm ID x 3.5 cm) with mobile phase A (20 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0) and mobile phase B (20 mM sodium phosphate, pH 7.0 / IPA (4:1 v / v)) at a flow rate of 0.8 mL / min. The column was connected to an absorbance detector set to a 280 nm wavelength signal and a 360 nm reference. After incubation for 2 hours, the crude reaction mixture was ready for HIC purification.

[0522] Example 39. Mono-NPBA-PEG 5k -Isolation of Tf

[0523] 1.75 L of NPBA-PEG 5k -Tf crude mixture (20 g / L) was added to the reaction vessel. 7 L of mobile phase A (1.5 M ammonium sulfate, 20 mM sodium phosphate, pH 7.4) was added to the crude mixture to obtain a concentration of 1.2 M ammonium sulfate, 16 mM sodium phosphate, pH 7.4. After mixing, the sample mixture was loaded onto an AxioChrom 140 column (1.7 L) filled with GE Agarose Phenyl High Performance HIC resin with a pump flow rate of 200 mL / min. Approximately 10 minutes of residence time was allowed to load the sample mixture onto the HIC column. IPA / 10 mM sodium phosphate, pH 7.4 (1:4 v / v, mobile phase B) was used as eluent. Prior to the elution step, the column was washed with 20% mobile phase B for 2 column volumes (CV) to remove unbound material. The purified NPBA-PEG 5k -Tf was eluted with a 15CV gradient of 20-100% mobile phase B. Mono-PEGylated Tf (Mono-NPBA-PEG 5k -Tf) fractions and combined for ultrafiltration and diafiltration (UF / DF). Sartocon Slice Hydrosart Cassette 30 kDa membrane was used for Mono-NPBA-PEG 5k-Tf UF / DF. Mono-NPBA-PEG was filtered using 7 volumes of PBS pH 7.4. 5k -Tf conjugate was concentrated to 50 g / L. Then, Mono-NPBA-PEG 5k -Tf was sterile filtered.

[0524] Example 40. Determination of the number of NPBA per Tf

[0525] Calculation of Mono-NPBA-PEG by Alizarin Red S (ARS) detection using NPBA 5k NPBA-PEG in -Tf 5k The ratio of the molar concentration of Mono-NPBA-PEG to the molar concentration of Tf was used to determine 5k -Number of NPBA per Tf in Tf. To prepare the standard curve, NPBA-PEG was prepared at different concentrations (0-0.0098 mM) in PBS at pH 7.2. 5k -AA (27) sample. ARS was prepared by dissolving in water at 0.049 mM. The sample was then mixed with ARS and fluorescence (Ex: 468 nm, Em: 572 nm) was measured using a Varioskan LUX spectrophotometer (ThermoFisher) and SkanIt (Thermo Fisher) or similar fluorescence analysis software. Linear regression of these data (fluorescence versus concentration) provides a calibration curve for determining NPBA in the target sample.

[0526] Prepare samples of interest (e.g., Tf and Mono-NPBA-PEG) at 0.007 mM in PBS, pH 7.2. 5k -Tf). Mono-NPBA-PEG 5k -NPBA-PEG in Tf 5k The molar concentration of Mono-NPBA-PEG 5k Average fluorescence values ​​of -Tf samples and different known concentrations of NPBA-PEG 5k -The fluorescence value calibration curve of AA standard (0-0.0098mM) was calculated using the following equation:

[0527] Fluorescence = ε*C N +b

[0528] where ε = the slope of the linear regression of the calibration curve, b = the intercept of the linear regression of the calibration curve, and C N = NBPA-PEG concentration (mM). Therefore, C N (mM) = (fluorescence-b) / ε.

[0529] Then, according to the following equation, C N Divide by the known Tf concentration to calculate the number of NPBA per Tf (N Tf ):

[0530] N Tf =(NPBA-PEG 5k mmol / L) / (Tf mmol / L)

[0531] Mono-NPBA-PEG was determined using the ARS assay of NPBA 5k -The amount of NPBA in Tf is Mono-NPBA-PEG 5k -Tf is about 1 NPBA per Tf. The reaction with ARS and thus the fluorescence signal requires NPBA functional groups available for the reaction. Some molecules (such as citrate) complex with NPBA, reduce the fluorescence signal and change the fluorescence signal of each Mono-NPBA-PEG 5k In other cases, there may be excess unreacted NPBA-PEG conjugates in the crude sample, increasing the fluorescence signal and altering the fluorescence intensity of each Mono-NPBA-PEG 5k The exemplary experimental results are shown in Table 2.

[0532] Table 2. Mono-NPBA-PEG 5k -Number of NPBA per Tf of Tf samples

[0533] sample <![CDATA[C N (mM)]]> <![CDATA[N Tf =(number of NPBA per Tf)]]> <![CDATA[Mono-NPBA-PEG 5k -Tf(+citrate)]]> 6.40E-03 0.91 <![CDATA[Mono-NPBA-PEG 5k -Tf(-citrate)]]> 7.34E-03 1.05

[0534] Example 41. NPBA-PEG 5k Synthesis of -Tras crude mixture

[0535] 5.5 g (0.038 mmol) of trastuzumab was buffer exchanged with 50 mM HEPES (pH 7.8) and the final protein concentration was adjusted to 10 g / L. After dissolution, 409.07 mg (0.076 mmol) of NPBA-PEG in 3.78 mL of DMSO was added. 5k-AA-PFP (28) was slowly added to the reaction mixture and mixed thoroughly. The reaction mixture was incubated at room temperature for 2 hours. The crude reaction mixture was analyzed on an Agilent 1290HPLC system equipped with a reverse phase (RP) column (Zorbax 300SB-CN 4.6mm ID x 150cm), with mobile phase A (water + 0.1% TFA) and mobile phase B (ACN / IPA (4:1 v / v) + 0.1% TFA) at a flow rate of 0.7 mL / min. The chromatographic column was connected to an absorbance detector set to 280 nm signal and 360 nm reference. After incubation for 2 hours, the crude reaction mixture was ready for HIC purification.

[0536] Example 42. Mono-NPBA-PEG 5k -Separation of Tras

[0537] 0.55L NPBA-PEG 5k -Tras crude mixture (10 g / L) was added to the reaction vessel. 1.1 L of mobile phase A (1.5 M ammonium sulfate, 20 mM sodium phosphate, pH 7.4) was added to the crude mixture to obtain a concentration of 1 M ammonium sulfate, 13.3 mM sodium phosphate, pH 7.4. After mixing, the sample mixture was loaded onto an AxioChrom 140 column (1.7 L) filled with GE Agarose Phenyl High Performance HIC resin at a flow rate of 200 mL / min. Approximately 10 minutes of residence time was allowed to load the sample mixture onto the HIC column. IPA / 10 mM sodium phosphate, pH 7.4 (1:4 v / v, mobile phase B) was used as eluent. Prior to the elution step, the column was washed with two CVs of 40% mobile phase B to remove unbound material. The purified NPBA-PEG 5k -Tras was eluted with a 15CV gradient of 40-100% mobile phase B. Mono-PEGylated Tras (Mono-NPBA-PEG 5k -Tras) fractions were pooled and used for UF / DF. Sartocon Slice Hydrosart Cassette 30 kDa membrane was used for Mono-NPBA-PEG 5k -Tras UF / DF. Mono-NPBA-PEG was filtered using 7 volumes of PBS pH 7.4. 5k -Tras conjugate was concentrated to 50 g / L. After diafiltration, Mono-NPBA-PEG 5k -Tras were sterile filtered.

[0538] Example 43. Determining the number of NPBAs per Tras

[0539] Calculation of Mono-NPBA-PEG by ARS detection using NPBA 5k -Tras NPBA-PEG 5k The molar concentration ratio of Mono-NPBA-PEG 5k -The amount of NPBA per Tras in Tras. To prepare the standard curve, NPBA-PEG was prepared at different concentrations (0-0.0098 mM) in PBS at pH 7.2. 5k -AA (27) sample. ARS was prepared by dissolving in water at 0.049 mM. The sample was then mixed with ARS and fluorescence (Ex: 468 nm, Em: 572 nm) was measured using a Varioskan LUX spectrophotometer (ThermoFisher) and SkanIt (Thermo Fisher) or similar fluorescence analysis software. Linear regression of these data (fluorescence versus concentration) provides a calibration curve for determining NPBA in the target sample.

[0540] Prepare samples of interest (e.g., Tras and Mono-NPBA-PEG) at 0.007 mM in PBS, pH 7.2. 5k -Tras). Mono-NPBA-PEG 5k The molar concentration of NPBA-PEG in -Tras is determined by Mono-NPBA-PEG 5k Average fluorescence values ​​of -Tras samples and different known concentrations of NPBA-PEG 5k -The fluorescence value calibration curve of AA standard (0-0.0098mM) was calculated using the following equation:

[0541] Fluorescence = ε*C N +b

[0542] where ε = the slope of the linear regression of the calibration curve, b = the intercept of the linear regression of the calibration curve, and C N = NBPA-PEG concentration (mM). Therefore, C N (mM) = (fluorescence-b) / ε.

[0543] Then, according to the following equation, C N Divide by the known Tras concentration to calculate the number of NPBA per Tras (N Tras ):

[0544] N Tras =(NPBA-PEG mmol / L) / (Tras mmol / L)

[0545] Mono-NPBA-PEG was determined using the ARS assay of NPBA 5k -The amount of NPBA in Tras is Mono-NPBA-PEG 5k -Tras contains about 1 NPBA per Tras. As described in Example 40, each Mono-NPBA-PEG 5k The amount of NPBA in -Tras can be altered by undesired complexation of the molecule with NPBA, which would reduce the fluorescence signal, or by the presence of excess unreacted NPBA-PEG conjugate in the crude sample, which would increase the fluorescence signal. Exemplary experimental results are shown in Table 3.

[0546] Table 3. Mono-NPBA-PEG 5k -Number of NPBA per Tras in Tras sample

[0547] sample <![CDATA[C N (mM)]]> <![CDATA[N Tras =(number of NPBAs per Tras)]]> <![CDATA[Mono-NPBA-PEG 5k -Tras(small gift)]]> 1.15E-02 1.64 <![CDATA[Mono-NPBA-PEG 5k -Tras(Purified)]]> 7.05E-03 1.01

[0548] As will be appreciated by those skilled in the art, many modifications and variations of the present disclosure are possible in light of these teachings, and all of these are contemplated herein. For example, in addition to the embodiments described herein, the present disclosure also contemplates and claims those inventions resulting from combinations of the disclosed features cited herein and those features of the prior art references that supplement the features of the present disclosure. Similarly, it should be understood that any described material, feature, or article may be used in combination with any other material, feature, or article, and such combinations are considered to be within the scope of the present disclosure.

[0549] The disclosure of each patent, patent application, and publication cited or described in this document is incorporated herein by reference in its entirety for all purposes, or at least for the context in which it is cited. In addition to the references already mentioned, this disclosure also contains subject matter related to the following documents: U.S. Patent Application Serial Nos. 12 / 540,319, filed on August 12, 2009, now U.S. Patent 8,557,292; 13 / 782,458, filed on March 1, 2013; 13 / 782,486, filed on March 1, 2013; 13 / 852,303, filed on March 28, 2013; 15 / 180,201, filed on June 13, 2016, now U.S. Patent 10,287,401; and International Application No. PCT / US2009 / 053620, filed on August 12, 2009, the contents of which are incorporated by reference for all purposes.

Claims

1. 20-(Boc-aminoacyl) and 10-TBDPS derivatives of 7-ethyl-10-hydroxy-camptothecin (SN38) (20-(Boc-aminoacyl)-10-TBDPS-SN38) having the following structure: wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group.

2. The 20-(Boc-aminoacyl)-10-TBDPS derivative of SN38 of claim 1, wherein the amino acid functional group is a functional group selected from glycine, valine, γ-aminobutyric acid (GABA) and hexanoic acid.

3. A 20-(Boc-aminoacyl)-10-OBoc derivative of SN38 (20-(Boc-aminoacyl)-10-OBoc-SN38) having the following structure: wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group.

4. The 20-(Boc-aminoacyl)-10-OBoc derivative of SN38 of claim 3, wherein the amino acid functional group is a functional group selected from glycine, alanine, β-alanine, valine and leucine.

5. The HCl salt of a 20-aminoacyl derivative of SN38 (20-(HCl.aminoacyl)-SN38) having the following structure: wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group.

6. The HCl salt of claim 5, wherein the amino acid functional group is a functional group selected from glycine and valine.

7. A trifluoroacetic acid (TFA) salt of a 20-aminoacyl derivative of SN38 (20-(TFA.aminoacyl)-SN38) having the following structure: wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group.

8. The TFA salt of claim 7, wherein the amino acid functional group is a functional group selected from the group consisting of glycine, alanine, β-alanine, valine, GABA, hexanoic acid and leucine.

9. A mucic acid polymer (MAP)-SN38 conjugate having the following structure: wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group, x is a number in the range 20 to 200; and y is a number in the range of 5 to 200.

10. The MAP-SN38 conjugate of claim 9, wherein the amino acid functional group is a functional group selected from the group consisting of glycine, alanine, β-alanine, valine, GABA, hexanoic acid and leucine.

11. Nanoparticles comprising the MAP-SN38 conjugate of claim 10.

12. A 20-(Boc-aminoacyl) derivative of camptothecin (CPT) (20-(Boc-aminoacyl)-CPT) having the following structure: wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group.

13. The 20-(Boc-aminoacyl)-CPT derivative of claim 12, wherein the amino acid functional group is a functional group selected from the group consisting of glycine, valine, alanine, β-alanine, GABA, and the dipeptide phenylalanine-glycine.

14. A trifluoroacetic acid (TFA) salt of a 20-aminoacyl derivative of CPT (20-(TFA.aminoacyl)-CPT) having the following structure: wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group.

15. The TFA salt of claim 14, wherein the amino acid functional group is a functional group selected from the group consisting of glycine, alanine, β-alanine, valine, GABA, and the dipeptide phenylalanine-glycine.

16. A mucic acid polymer (MAP)-CPT conjugate having the following structure: wherein R is one or more α-carbon atoms of an amino acid optionally bonded to an amino acid functional group, wherein the amino acid functional group is a functional group selected from alanine, β-alanine, valine, GABA and the dipeptide phenylalanine-glycine, x is a number in the range 20 to 200; and y is a number in the range of 5 to 200.

17. Nanoparticles comprising the MAP-CPT conjugate of claim 16.

18. A compound having the following structure (NPBA-PEG-AA-PFP): Where n is a number in the range of 2 to 2000.

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