Methods and compositions for synthesis of therapeutic nanoparticles

Abstract

To provide nanoparticles for treating various cancers.SOLUTION: Provided are mucic acid polymer (MAP)-CPT conjugates having the following structure (where, R is an α-carbon atom of one or more amino acids optionally bonded to an amino acid functional group selected from alanine, β-alanine, valine, GABA, and a functional group of dipeptide phenylalanine-glycine; x is a number ranging from 20 to 200; and y is a number ranging from 5 to 200), and nanoparticles including MAP-CPT conjugates.SELECTED DRAWING: None

Description

Cross - Reference to Related Applications

[0001] 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, under 35 U.S.C. § 119(e)(1), and the entire disclosures of both are incorporated herein by reference for all purposes. and

[0002] (Statement Regarding Federally Sponsored Research) Not applicable.

Technical Field

[0003] The present disclosure is in the field of nanoparticles for treating various cancers, said nanoparticles comprising a polymer core and further comprising one or more homing molecules (i.e., targeting molecules) and / or one or more therapeutic molecules. Improved methods for synthesizing such nanoparticles are provided herein.

Background Art

[0004] For the treatment of many brain diseases, such as brain tumors and brain metastases, it is necessary to deliver therapeutic molecules to the brain. Direct delivery of therapeutic agents to the brain poses significant risks to the subject (e.g., skull damage), and continuous administration, such as is required for most chemotherapy treatments, is not practical. However, due to the presence of the blood - brain barrier (BBB), a tightly - bound layer of endothelial cells lining the blood vessels of the brain, systemic delivery (e.g., via the bloodstream) cannot efficiently deliver molecules to the brain. A similar permeation barrier, known as the blood - tumor barrier (BTB), exists in certain solid tumors.

[0005] ​​​​​​​​​​​Breast cancer often metastasizes to the brain, and treating these brain metastases with chemotherapy using molecules would be possible if the therapeutic agent could be delivered to the brain at a sufficient concentration. One such chemotherapeutic drug is camptothecin, an alkaloid that inhibits DNA topoisomerase I and thereby prevents cell division. Camptothecin has problems with solubility in aqueous environments such as the cytoplasm, and therefore derivatives of camptothecin with high solubility have been developed. One such derivative is 7-ethyl-10-hydroxy-camptothecin (SN38 ), which was originally discovered as a metabolite of irinotecan, a camptothecin analogue.

[0006] To address the problem of delivering therapeutic agents across the BBB and BTB, nanoparticles containing a chemotherapeutic molecule and a homing (i.e., targeting) molecule have been developed, where the homing molecule binds to the transferrin receptor present on the surface of brain endothelial cells (and endothelial cells of the tumor vasculature), thereby enabling the nanoparticles to pass through the endothelial cells by transcytosis. See, for example, U.S. Patent Nos. 9,468,681, 10,166,291, and 10,182,986. Each of these patents is hereby incorporated by reference herein for all purposes, or at least for the teachings regarding the properties of the nanoparticles, their methods of manufacture and use, and their modes of operation. use, and their modes of operation.

[0007] Specific steps in the synthesis of such nanoparticles include (1) converting mucic acid to a polymerizable reactive derivative (i.e., a "mucic acid monomer" or MAM), (2) the mucic acid mo The step of polymerizing a nomer to form a mucic acid polymer (MAP), (3) selectively converting the therapeutic molecule into a reactive derivative and (4) one or more of the derivatized therapeutic molecules to MA P binding step, there is.

[0008] Nanoparticles containing camptothecin (CPT) that can pass through the blood-brain barrier and the blood-tumor barrier have been described. For example, U.S. Patent Nos. 9,446,149; 9,468 ,681; 10,166,291 and U.S. Patent Application Publication No. 2019 / 038118 8 (December 19, 2019) (each of which is incorporated herein by reference for all purposes, or at least for the description of the nanoparticles). However However, existing methods for preparing such nanoparticles consume reagents and time, require high temperatures and have low yields. Furthermore, as described above, derivatives of CPT that are more soluble under aqueous conditions are preferred. Therefore, an improved method for synthesizing nanoparticles containing chemotherapeutic agents such as CPT and its derivatives such as SN38, which is rapid, economical with respect to reactants and has a high yield is needed.

[0009] Furthermore, for the treatment of non-CNS malignancies such as non-CNS tumors and hematological malignancies, an improved vehicle for the delivery of chemotherapeutic drugs would be beneficial. SUMMARY OF THE INVENTION

[0010] Provided herein is a mucic acid-polyethylene glycol (PEG) polymer, camptothecin (CPT) and its derivatives and metabolites, such as 7-ethyl-10-hyd roxicanptothecin (SN38), and a therapeutic nanoparticle comprising a chemotherapeutic agent. An improved method therefor. This improvement particularly includes the use of novel intermediates and more stable reactants and the formation of more stable intermediates, faster reaction times, and higher yields of intermediates and products, and novel linkers for conjugating targeting (homing) molecules and therapeutic molecules (e.g., macromolecular therapeutics, e.g., antibodies) to nanoparticles. Derivatized nanoparticles for use in the methods described herein (e.g., including conjugated CPT or SN38 molecules) are also provided, as are sialic acid polymers (MAP) conjugates (e.g., CPT, SN38, transferrin and / or or trastuzumab conjugated MAP) for use in the assembly of therapeutic nanoparticles. Accordingly, provided herein are, in particular, the following embodiments. SN38 molecules) are also provided, as are sialic acid polymers (MAP) conjugates (e.g., CPT, SN38, transferrin and / or or trastuzumab conjugated MAP) for use in the assembly of therapeutic nanoparticles. or trastuzumab conjugated MAP) for use in the assembly of therapeutic nanoparticles.

[0011] Accordingly, provided herein are, in particular, the following embodiments.

[0012] 1. A method for synthesizing sialic acid di(aspartylamine) ditrifluoroacetate from sialic acid diaminochloride, comprising: (a) converting sialic acid diaminochloride having the following structure (a) converting sialic acid diaminochloride having the following structure

Chemical formula

Chemical formula

Chemical formula

[0013] 2. The method according to Embodiment 1, wherein in step (b), (a) the mucic acid bis(aspartyl(O-t-butyl)-Boc) is converted into a mucic acid bis(aspartylamine) neutral species having the following structure [Chemical formula] and is converted into a mucic acid bis(aspartylamine) ditrifluoroacetate having the following structure (b) the mucic acid bis(aspartylamine) neutral species is converted into a mucic acid bis(aspartylamine) di trifluoroacetic acid.

[0014] 3. A method for synthesizing mucic acid bis(aspartylamine) ditrifluoroacetate comprising converting a mucic acid bis(aspartylamine) neutral species having the following structure [Chemical formula] into a mucic acid bis(aspartylamine) ditrifluoroacetate having the following structure having the following structure [Chemical formula] including the step of converting it into a mucic acid bis(aspartylamine) ditrifluoroacetate having the following structure and the method.

[0015] 4. A mucic acid bis(aspartylamine) neutral species having the following structure [Chemical formula] having the following structure

[0016] 5. A method for synthesizing a 10-TBDPS derivative of SN38, comprising heating a mixture of SN38 and tert-butyl(chloro)diphenyl lucirane (TBDPSCl) in a base (e.g., an amine base, e.g., a trialkylamine, e.g., triethylamine) and a solvent (e.g dichloromethane (DCM)), and the product has the following structure [Chemical] A method having

[0017] In certain embodiments, the mixture is heated at a temperature in the range of 15°C to 90°C for a time in the range of 5 minutes to 48 hours. In further embodiments, at the end of the reaction, the reaction mixture is washed successively with dilute acid (e.g., 0.2N HCl), weak base (e.g., saturated NaHCO ), and brine. In further embodiments, the neutralized solution is dried (e.g., with MgSO ). In further embodiments, the dried solution is evaporated in vacuo to give a solid. In further embodiments, the solid is recrystallized (e.g., by dissolving the residue in (e.g., DCM) and precipitating the product with (e.g., hexane)). 3 ) In further embodiments, 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. 4 Combinations of the above embodiments are contemplated as additional embodiments of the invention disclosed herein. 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 reaction product is washed successively with a solvent (e.g., DCM) and / or a coupling agent (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl)) and / or a salt

[0018]

[0019]

[0020] Mix in 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 fractional value therebetween .

[0021] 7. The method according to embodiment 6, wherein the amino acid is selected from the group consisting of glycine (Gly), valine (Val), γ-aminobutyric acid (GABA ), and hexanoic acid (Hex).

[0022] 8. 20-(Boc-Gly)-10-TBDPS-SN38 having the following structure

Chemical formula

[0023] 9. 20-(Boc-GABA)-10-TBDPS-SN38 having the following structure

Chemical formula

[0024] 10. 20-(Boc-Hex)-10-TBDPS-SN38 having the following structure

Chemical formula

[0025] 11. 20-(Boc-Val)-10-TBDPS-SN38 having the following structure

Chemical formula

[0026] 12. A method for synthesizing a 10-OBoc derivative of SN38 (10-OBoc-SN38), comprising SN in the presence of a base (e.g., pyridine) and a solvent (e.g., DCM ). A step of mixing 38 with di-tert-butyl dicarbonate is included, and 10-OBoc- SN38 has the following structure

Chemical formula

[0027] 13. A method for synthesizing a 20-(Boc-aminoacyl), 10-OBoc derivative of SN38, which comprises a step of mixing 10-OBoc-SN3 8 with Boc-amino acid-OH at low temperature in the presence of a solvent (e.g., DCM), a base (e.g., DMAP), and a coupling agent (e.g., EDC.HCl), and 20-(Boc-aminoacyl) -10-OBoc-SN38 has the following structure

Chemical formula

[0028] 14. The method according to 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) .

[0029] 15. 20-(Boc-Gly)-10-OBoc-SN38 having the following structure

Chemical formula

[0030] 16. The following structure

Chemical formula

[0031] 17. The following structure

Chemical formula

[0032] 18. The following structure

Chemical formula

[0033] 19. The following structure

Chemical formula

[0034] 20. The following structure

Chemical formula

[0035] 21. The following structure

Chemical formula

[0036] 22. The following structure

Chemical formula

[0037] 23. The following structure

Chemical formula

[0038] 24. The following structure

Chemical formula

[0039] 25. The following structure

Chemical formula

[0040] 26. The following structure

Chemical formula

[0041] 27. The following structure

Chemical formula

[0042] 28. The following structure

Chemical formula

[0043] 29. The following structure

Chemical formula

[0044] In certain embodiments, x in Compound 16 is a number in the range of 20 to 200, y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, a number in the range of 50 to 100 or 10 to 25. In certain embodiments, x is the PEG moiety of the polymer (i.e., -O-CH 2 -CH 2 -) is selected such that the number average molecular weight is about 500 D a to about 50,000 Da.

[0045] In certain embodiments, the weight average molecular weight of the MAP is 5 to 150 kDa or any integer value therebetween, e.g., in the range of 20 to 120 kDa, and the values of x and y are selected accordingly.

[0046] In additional embodiments, the values of x and y are such that after assembling the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is 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 1 00 nm.

[0047] 30. The following structure

Chemical formula

[0048] 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, a number in the range of 50 to 100 or 10 to 25. In certain embodiments, x is the PEG moiety of the polymer (i.e., -O-CH 2 -CH 2 -) is selected such that the number average molecular weight is about 500 D is selected to be in the range of a~about 50,000 Da.

[0049] In certain embodiments, the weight average molecular weight of the MAP is 5~150 kDa or any integer value therebetween, for example, in the range of 20~120 kDa, and the values of x and y are selected accordingly. For example, in the range of 20~120 kDa, and the values of x and y are selected accordingly. are selected accordingly.

[0050] In additional embodiments, the values of x and y are such that after assembling the MAP or the drug conjugate MAP into nanoparticles, the size of the nanoparticles is 10~900 nm or any integer value therebetween, for example, in the range of 100~800 nm, 200~500 nm, 400~700 nm or 20~100 nm. For example, in the range of 100~800 nm, 200~500 nm, 400~700 nm or 20~100 nm. For example, 100~800 nm, 200~500 nm, 400~700 nm or 20~100 nm. are selected to be in the range of 100~800 nm, 200~500 nm, 400~700 nm or 20~100 nm.

[0051] 31. The following structure

Chemical Formula

[0052] In certain embodiments, x in Compound 18 is a number in the range of 20~200, and y is a number in the range of 5~200 (or any integer value therebetween, for example, in the range of 10~150, 20~120, 50~100 or 10~25). In certain embodiments, x is such that the number average molecular weight of the PEG moiety of the polymer (i.e., -O-CH In certain embodiments, x in Compound 18 is a number in the range of 20~200, and y is a number in the range of 5~200 (or any integer value therebetween, for example, in the range of 10~150, 20~120, 50~100 or 10~25). For example, in the range of 10~150, 20~120, 50~100 or 10~25). In certain embodiments, x is such that the number average molecular weight of the PEG moiety of the polymer (i.e., -O-CH -CH 2 -CH 2 -) is selected to be in the range of about 500 Da~about 50,000 Da. is selected to be in the range of about 500 Da~about 50,000 Da.

[0053] In certain embodiments, the weight average molecular weight of the MAP is 5~150 kDa or any integer value therebetween, for example, in the range of 20~120 kDa, and the values of x and y are selected accordingly. For example, in the range of 20~120 kDa, and the values of x and y are selected accordingly. are selected accordingly.

[0054] In additional embodiments, the values of x and y are such that after assembling the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, e.g., 100-800 nm, 200-500 nm, 400-700 nm or 20-1 00 nm. are selected.

[0055] 32. The following structure

Chemical formula

[0056] In certain embodiments, x in Compound 19 is a number in the range of 20-200, and y is a number in the range of 5-200 (or any integer value therebetween, e.g., 10-150, 20-120, 50-100 or 10-25). In certain embodiments, x is the number average molecular weight of the PEG moiety of the polymer (i.e., -O-CH -CH 2 -CH 2 -) is selected such that it is in the range of about 500 D a to about 50,000 Da.

[0057] In certain embodiments, the weight average molecular weight of the MAP is in the range of 5-150 kDa or any integer value therebetween, e.g., 20-120 kDa, and the values of x and y are selected accordingly.

[0058] In additional embodiments, the values of x and y are such that after assembling the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, e.g., 100-800 nm, 200-500 nm, 400-700 nm or 20-1 00 nm.​ It is selected to be in the range of 00 nm.

[0059] 33. The following structure

Chemical formula

[0060] In certain embodiments, x in Compound 37 is a number in the range of 20 to 200, 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 the po lymer's PEG moiety (i.e., -O-CH 2 -CH 2 -), and is selected such that the number average molecular weight is about 500D a to about 50,000 Da.

[0061] In certain embodiments, the weight average molecular weight of MAP is 5 to 150 kDa or any integer value therebetween, e.g., in the range of 20 to 120 kDa, and the values of x and y are selected accordingly.

[0062] In additional embodiments, the values of x and y are such that after assembling MAP or the drug conjugate MAP into nanoparticles, the size of the nanoparticles is 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 1 00 nm. It is selected to be in the range of 00 nm.

[0063] 34. The following structure

Chemical formula

[0064] In certain embodiments, x in Compound 38 is a number in the range of 20 to 200, 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 a number in the range of 10 to 25. In certain embodiments, x is the po lymer's PEG moiety (i.e., -O-CH 2 -CH 2 -) is selected such that the number average molecular weight is about 500D a to about 50,000 Da.

[0065] In certain embodiments, the weight average molecular weight of MAP is 5 to 150 kDa or any integer value therebetween, e.g., in the range of 20 to 120 kDa, and the values of x and y are selected accordingly.

[0066] In additional embodiments, the values of x and y are such that after assembling MAP or the drug conjugate MAP into nanoparticles, the size of the nanoparticles is 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 1 00 nm.

[0067] 35. The following structure [Chemical formula] A mucic acid polymer-SN38 conjugate (MAP-Leu-SN38) having

[0068] In certain embodiments, x in Compound 39 is a number in the range of 20 to 200, y is 5 to 200 (or any integer value therebetween, for example, 10 to 150, 20 to 120, In certain embodiments, x is a number in the range of 50 to 100 or 10 to 25. The PEG moiety of the polymer (i.e., -O-CH 2 -CH 2 -) has a number average molecular weight of about 500D The molecular weight is selected to be in the range of a to about 50,000 Da.

[0069] In certain embodiments, the weight average molecular weight of the MAP is between 5 and 150 kDa, or any value therebetween. Integer values ​​of x and y are in the range of, for example, 20 to 120 kDa, and the values ​​of x and y are selected accordingly. It is selected.

[0070] In additional embodiments, the values ​​of x and y are determined by the formula: After assembly into nanoparticles, the size of the nanoparticles is 10 to 900 nm or any integer value therebetween, e.g. For example, 100 to 800 nm, 200 to 500 nm, 400 to 700 nm, or 20 to 1 The wavelength is selected to be in the range of 00 nm.

[0071] 36. A method comprising the steps of: Nanoparticles.

[0072] 37. The following structure [ka] 20-(Boc-Gly)-CPT.

[0073] 38. The following structure [ka] 20-(Boc-Val)-CPT having the formula:

[0074] 39. The following structure [Chemical formula] 20-(Boc-Ala)-CPT having the following structure

[0075] 40. The following structure [Chemical formula] 20-(Boc-β-Ala)-CPT having the following structure

[0076] 41. The following structure [Chemical formula] 20-(Boc-GABA)-CPT having the following structure

[0077] 42. The following structure [Chemical formula] 20-(Boc-Phe-Gly)-CPT having the following structure

[0078] 43. The following structure [Chemical formula] 20-(TFA.Gly)-CPT having the following structure

[0079] 44. The following structure [Chemical formula] 20-(TFA.Ala)-CPT having the following structure

[0080] 45. The following structure [Chemical formula] 20-(TFA.β-Ala)-CPT having the following structure

[0081] 46. The following structure [Chemical formula] 20-(TFA.Val)-CPT having

[0082] 47. The following structure

Chemical formula

[0083] 48. The following structure

Chemical formula

[0084] 49. The following structure

Chemical formula

[0085] In certain embodiments, the reaction is carried out at low temperature, for example, in the range of 0° C. to 20° C. or therebetween. Any integer or decimal value of the temperature is then allowed to warm to ambient temperature before acidification and Purification is performed. In certain embodiments, the mixture is acidified to a pH of about 4. In one embodiment, the product is extracted with a solvent (e.g., isopropyl acetate, iPrOAc). The mixture is purified by

[0086] In certain embodiments, the value of n is selected from the group consisting of PEG (i.e., -CH 2 -CH 2 -O- The weight average molecular weight of the ) portion is in the range of about 2 to about 15 kDa, for example, about 2 kDa, about 3 kDa, about 4kDa, about 5kDa, about 6kDa, about 7kDa, about 8kDa, about 9kDa, About 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa or about 15 kDa It is something like Da.

[0087] 50. The following structure [ka] The compound (NPBA-PEG-AA-PFP) has the formula:

[0088] In certain embodiments, n is 2 to 2,000 or any integer value therebetween, such as 10 in the range of 0-300, 20-300, 120-180 and / or 140-160 It is a number.

[0089] In certain embodiments, the value of n is selected from the group consisting of PEG (i.e., -CH 2 -CH 2 -O- ) The weight average molecular weight of the moiety is 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 k Da.

[0090] 51. A method for producing the compound of embodiment 50, comprising the step of mixing the NPBA-PEG-AA of embodiment 49 with bis(pentafluorophenyl) carbonate and a base catalyst (e.g., N-methylmorpholine). A method.

[0091] 52. A method for synthesizing an NPBA-PEG-transferrin conjugate, comprising the step of mixing the NPBA-PEG-AA-PFP of embodiment 50 with holotransferrin (i.e., iron bound transferrin). A method.

[0092] 53. A method for synthesizing an NPBA-PEG-trastuzumab conjugate, comprising the step of mixing the NPBA-PEG-AA-PFP of embodiment 50 with trastuzumab. A method.

[0093] 54. A method for synthesizing an NPBA-PEG-therapeutic polypeptide conjugate, comprising the step of mixing the NPBA-PEG-AA-PFP of embodiment 50 with a therapeutic polypeptide. A method.

[0094] 55. The following structure

Chemical formula

[0095] 56. A 20-Boc-aminoacyl, 10-TBDPS SN38 derivative according to embodiment 55, wherein the amino acid functional group is selected from the functional groups of glycine, valine, γ-aminobutyric acid (GABA) and hexanoic acid.

[0096] 57. The following structure

Chemical formula

[0097] 58. The 20-Boc-aminoacyl, 10-OBoc SN38 derivative according to embodiment 57, wherein the amino acid functional group is selected from the functional groups of glycine, alanine, β-alanine, valine and leucine.

[0098] 59. The following structure

Chemical formula

[0099] 60. The HCl salt according to embodiment 59, wherein the amino acid functional group is selected from the functional groups of glycine and valine.

[0100] 61. The following structure

Chemical formula

[0101] 62. The TFA salt according to embodiment 61, wherein the amino acid functional group is selected from the functional groups of glycine, alanine, β - alanine, valine, GABA, he xanthic acid and leucine.

[0102] 63. The following structure

Chemical formula

[0103] In certain embodiments, x in the MAP-amino acid-SN38 conjugate is 20 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., in the range of 10 to 150, 20 to 120, 50 to 100 or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG moiety of the polymer (i.e., -O-CH 2 -CH 2 -) is in the range of about 500 Da to about 50,000 Da .

[0104] In certain embodiments, the weight average molecular weight of the MAP is 5 to 150 kDa or any integer value therebetween, e.g., in the range of 20 to 120 kDa, and the values of x and y are selected accordingly .

[0105] In additional embodiments, the values of x and y are such that after assembling the MAP or the drug conjugate MAP into nanoparticles, the size of the nanoparticles is 10 to 900 nm or any integer value therebetween, e.g in the range of 100 to 800 nm, 200 to 500 nm, 400 to 700 nm or 20 to 1 00 nm.

[0106] 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, he xanthic acid and leucine.

[0107] 65. A nanoparticle comprising the MAP-amino acid-SN38 conjugate of any of embodiments 63 or 64 .

[0108] 66. The following structure

Chemical formula

[0109] 67. The 20-(Boc-aminoacyl) CPT derivative according to embodiment 66, wherein the amino acid functional group is selected from the functional groups of glycine, valine, alanine, β-alanine, GABA and diphenylalanine-glycine dipeptide

[0110] 68. The following structure

Chemical formula

[0111] 69. The TFA salt according to 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. TFA salt.

[0112] 70. The following structure

Chemical formula

[0113] 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, for example, 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG moiety of the polymer (i.e., -O-CH 2 -CH 2 - ) is in the range of about 500 Da to about 50,000 Da.

[0114] In certain embodiments, the weight average molecular weight of the MAP is 5 - 150 kDa or any integer value therebetween, for example, in the range of 20 - 120 kDa, and the values of x and y are selected accordingly.

[0115] In additional embodiments, the values of x and y are such that after assembling the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is 10 - 900 nm or any integer value therebetween, e.g., in the range of 100 - 800 nm, 200 - 500 nm, 400 - 700 nm, or 20 - 1 00 nm. is selected to be in the range of

[0116] 71. Nanoparticles comprising the MAP - amino acid - CPT conjugate according to embodiment 70 .

DETAILED DESCRIPTION OF THE INVENTION

[0117] The present disclosure may be more readily understood by reference to the following description, which is made in connection with the accompanying examples, all of which form a part of the present disclosure. The present disclosure is not limited to the specific products, methods, conditions or parameters described or shown in this specification, and it should be understood that the terms used in this specification are for the purpose of describing specific embodiments by way of example only and are not intended to limit the claimed invention. Similarly, unless otherwise specified, the possible mechanisms or modes of action or reasons for improvement are not limited to the specific products, methods, conditions or parameters described or shown in this specification, and it should be understood that the terms used in this specification are for the purpose of describing specific embodiments by way of example only and are not intended to limit the claimed invention. Nor are they intended to limit the claimed invention. It will be understood that the present disclosure is not limited to the specific products, methods, conditions or parameters described or shown in this specification, and that the terms used in this specification are for the purpose of describing specific embodiments by way of example only and are not intended to limit the claimed invention. Likewise, unless otherwise specified, the possible mechanisms or modes of action or reasons for improvement Any description is intended to be illustrative only and the disclosure of this specification is not to be construed as being limited by the correctness of any such suggested mechanism or mode of action or reason for improvement throughout this text, the description refers to compositions and methods of making and using said compositions it is recognized that, that is, when the present disclosure describes or claims features or embodiments related to a composition or a method of making or using a composition such description or claim is understood to be intended to extend such features or embodiments to embodiments in each of these contexts (i.e., composition, method of making and method of use).

[0118] In the present disclosure, the singular forms "a", "an" and "the" include references to the plural, and references to a particular numerical value include at least that particular numerical value unless the context clearly indicates otherwise. Thus, for example, a reference to "a material" is a reference to at least one such material and its equivalents known to those skilled in the art and the like.

[0119] When a value is expressed as an approximation using the descriptor "about", it will be understood that the particular value forms another embodiment. Generally, the use of the term "about" is an approximation that may vary depending on the desired characteristics to be obtained by the disclosed subject matter and is to be interpreted in the particular context in which it is used based on its function. One of ordinary skill in the art would be able to interpret this as a matter of course In some cases, the number of significant figures used for a particular value can be one non-limiting way of determining the scope of the word "about". In other cases, the gradual changes used for a series of values are used to determine the term "about" for each value ​​​​​​It is possible to determine the intended scope available for use. If present, all ranges are inclusive and combinable. That is, a reference to a value recited within a range includes all values within that range. For clarity, it will be understood that certain features of the present disclosure described in the context of separate embodiments may also be provided in combination in a single embodiment. That is, unless clearly incompatible or explicitly excluded, individual embodiments are considered combinable with any other embodiment(s), and such combinations are considered another embodiment. Conversely, for brevity, various features of the present disclosure described in the context of a single embodiment may also be provided separately or in any sub-combination. Finally, an embodiment may be described as part of a series of steps or as part of a more general structure, but each such step may also be considered an independent embodiment in itself and combinable with others. The transitional phrases "comprising," "consisting essentially of," and "consisting of" are intended to imply them in their generally accepted meanings in patent terminology. That is, (i) "comprising" 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 elements, steps, or components not specified in the claim. (iii) "consisting essentially of" limits the scope of the claim to the "essential" features of the invention claimed,

[0120] while excluding any non-essential features that materially affect the basic and novel characteristics of the claimed invention.

[0121] limited to materials or steps designated as "not substantially affecting the basic and novel characteristics (if any)". Also, embodiments described in terms of the phrase "comprising" (or its equivalent) are provided as embodiments independently of those described in terms of "consisting of" and "consisting essentially of". For embodiments defined in terms of "consisting essentially of", the basic and novel characteristics (if any) are those of the method of preparing and using the materials of the present invention (and the systems used in such methods and the compositions obtained therefrom) and the ease of operation of the materials themselves, and the method and materials can achieve the characteristics emphasized using only the elements defined in the claims. That is, other materials may be present in the compositions of the present invention, but the presence of these additional materials is not necessary to provide the described advantages 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, when additional steps may be employed in the method, their presence is not necessary to achieve the described effects or advantages and / or they do not impair the described effects or advantages. When a list is presented, unless otherwise specified, each individual element of that list and all combinations of that list are understood to be separate embodiments. For example, a list of embodiments presented as "A, B or C" is construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C" or "A, B or C". Similarly, terms such as C alkyl are, as separate embodiments, C alkyl, C When a list is presented, unless otherwise specified, each individual element of that list and all combinations of that list are understood to be separate embodiments. For example, a list of embodiments presented as "A, B or C" is construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C" or "A, B or C". Similarly, terms such as C alkyl are, as separate embodiments, C alkyl, C When a list is presented, unless otherwise specified, each individual element of that list and all combinations of that list are understood to be separate embodiments. For example, a list of embodiments presented as "A, B or C" is construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C" or "A, B or C". Similarly, terms such as C alkyl are, as separate embodiments, C alkyl, C When a list is presented, unless otherwise specified, each individual element of that list and all combinations of that list are understood to be separate embodiments. For example, a list of embodiments presented as "A, B or C" is construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C" or "A, B or C". Similarly, terms such as C alkyl are, as separate embodiments, C alkyl, C

[0122] When a list is presented, unless otherwise specified, each individual element of that list and all combinations of that list are understood to be separate embodiments. For example, a list of embodiments presented as "A, B or C" is construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C" or "A, B or C". Similarly, terms such as C alkyl are, as separate embodiments, C alkyl, C alkyl, C alkyl, C 1-3 alkyl and the like are, as separate embodiments, C 1 alkyl, C 2Alkyl, C 3 Alkyl, C 1-2 Alkyl and C 2-3 Also includes alkyl .

[0123] Throughout this specification, words shall have the ordinary meaning as understood by those of ordinary skill in the relevant art However, to avoid misunderstanding, the meanings of specific terms are specifically defined or clarified

[0124] References to alcohols, aldehydes, amines, carboxylic acids, ketones or other similarly reactive functional groups also include their protected analogs. For example, references to hydroxy or alcohol include substituents where the hydroxy 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 (P iv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl( triphenylmethyl, Tr), silyl ethers (the most common ones include trimethylsily l (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propyl silyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), ethoxyethyl ether (EE) protected substituents. References to amines include amines where the amine is BOC glycine, carbobenzyloxy (Cbz), p-methoxy benzylcarbonyl (Moz or MeOZ), tert-butyloxycarbonyl (BO and the like. For example, references to amines include amines where the amine is protected by BOC glycine, carbobenzyloxy (Cbz), p-methoxy C), substituents protected by 9-fluorenylmethyloxycarbonyl (FMOC), acetyl (Ac), ben zoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl ( Ts) group or sulfonamide (Nosyl and Nps) groups are also included. Substituents containing a carbonyl group include those in which the carbonyl is protected by an acetal or ketal, a silyl or diathan group. References to substituents containing a carboxylic acid or carboxylate group include those 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 (e.g., 2,6-dimethylphenol, 2,6-diisopropylphenol, 2, 6-di-tert-butylphenol), silyl ester, orthoester or oxaz oline.

[0125] (Abbreviations) AA: Acetic acid ACN: Acetonitrile Ala: Alanine, alanyl ARS: Alizarin red S Asp: Aspartate, aspartic acid β-Ala: β-Alanine, β-alanyl BBB: Blood-brain barrier Boc: tert-Butoxycarbonyl BTB: Blood-tumor barrier CPME: Cyclopentylmethyl ether CPT: Camptothecin CV: Column volume DCM: Dichloromethane DIC: N,N’-Diisopropylcarbodiimide ​DIPEA: N,N'-Diisopropylethylamine DiSPA-PEG: Di(succinimidyl propionate)-PEG DMA: Dimethylacetamide DMAP: 4-Dimethylaminopyridine DMF: Dimethylformamide DMSO: Dimethyl sulfoxide EDC.HCl: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Salt EEDQ: N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline EtOAc: Ethyl acetate GABA: γ-Aminobutyric acid, γ-aminobutyryl Gly: Glycine, glycyl GPC: Gel permeation chromatography HBSS: Hank's balanced salt solution HCl: Hydrochloric acid Hex: 6-Aminohexanoic acid HIC: Hydrophobic interaction chromatography HOPO: Hydroxypyridine N-oxide IPA: Isopropyl alcohol iPrOAc: Isopropyl acetate MAM: Polymerizable mucic acid monomer MAP: Mucic acid polymer MeOH: Methanol MTBE: Methyl tert-butyl ether NHS: N-Hydroxysuccinimide NP(or P): Nanoparticle NPBA: 3-Carboxy-5-nitrophenylboronic acid Mono-NPBA-PEG-Tf: Monomethoxypolyethylene glycol conjugated fraction of transferrin Mono-NPBA-PEG-Tras: Monomethoxypolyethylene glycol conjugated fraction of trastuzumab PEO: Polyethylene oxide PBS: Phosphate buffered saline PEG: Polyethylene glycol PES: Polyethersulfone PFP: Pentafluorophenyl PyAOP: (7-Azabenzotriazol-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate Hexafluorophosphate SN38: 7-Ethyl-10-hydroxy-camptothecin SpP: Strands per particle TBDPS: tert-Butyldiphenylsilane; tert-Butyldiphenylsilyl TBDPSCl: tert-Butyryl(chloro)diphenylsilane TEA: Triethylamine Tf: Holo-transferrin TFA: Trifluoroacetate, Trifluoroacetic acid THF: Tetrahydrofuran Tras: Trastuzumab UF / DF: Ultrafiltration and diafiltration Val: Valine, Valyl

[0126] (Reaction components) The procedures described herein include standard solvents and catalysts known in the art Certain compounds (e.g., acids, bases, coupling agents) are described in this disclosure , but it will be apparent to those skilled in the art that different reagents can be used

[0127] Thus, exemplary solvents include 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 ), etc. ) glycol (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) are included, but not limited thereto.

[0128] Exemplary acids include mineral 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), but not limited thereto.

[0129] Exemplary bases include amino bases (e.g., 1,4-diazabicyclo[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-butyl lithium, 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), but not limited thereto.

[0130] Exemplary coupling agents include carbodiimide reagents (e.g., N,N'-diisoprop ylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride , N,N'-dicyclopentylcarbodiimide), additives for carbodiimide reagents (e.g., 1-hydroxy-7-azabenzotriazole, 6-chloro-1-hydroxybenzotriazole, N-hydroxysuccinimide, 1-hydroxy-2-pyridinone, 6-chloro-N-hydroxy-2-phenylbenzimidazole, ethyl 2-cyano-2- (hydroxyimino)acetate), anhydride-based or forming reagents (e.g., ditert-butyl carbonate, acetic anhydride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ethyl chloroformate), acylazoles (e.g., carbonyldiimidazole), acid halide formation reagents (e.g., thionyl chloride, phosgene, cyanuric chloride, benzyltriethylphosphonium difluoride), phosphonium salt coupling reagents (e.g., benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), tetramethylammonium reagents (e.g., 2-(2-oxo-1(2H)-pyridyl-1,1,3,3-tetramethyluronium tetrafluoroborate), aminium reagents (e.g., 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate), oxymauran salts (e.g., 1-((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)(morpholino)methylene)pyrrolidinium hexafluorophosphate), uranyl antimonate salts (e.g., benzotriazol-1-yloxy-N,N-dimethyl-methaniminium hexachloroantimonate), organic phosphorus reagents (e.g., ), and the like. ), and the like. ), and the like. ), and the like. For example, diethyl cyanophosphonate, 1-oxo-chlorophosphorane, 2-propane phosphoric anhydride), triazine reagents (e.g., 2-chloro-4,6-dimethoxy-1,3, 5-triazine), organic sulfur reagents (e.g., pentafluorophenyl-4-nitrobenzene sulfonate), pyridinium reagents (e.g., 2-chloro-1-methylpyridinium iodide ), polymer-bound reagents (e.g., polymer-supported N-ethoxycarbonyl-2-ethoxy -1,2-dihydroquinoline) are included, but not limited thereto.

[0131] (Synthesis of polymerizable mucic acid monomer (MAM)) In this specification, in particular, a method for synthesizing a polymerizable derivative of mucic acid (the "mucic acid monomer" or MAM ) is provided. In the first step, mucic acid is reacted with methanol (MeOH) in the presence of sulfuric acid to form the 1,6-dimethyldiester of mucic acid (Compound 1). . Next, this diester is reacted with N-Boc -ethylenediamine in the presence of triethylamine (TEA) and MeOH to form N-Boc protected mucic acid ethylenediamine (Compound 2).

[0132] In an alternative method on a preparative scale 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, and then the methoxylated mucic acid is reacted with N-(2-aminoethyl)(tert-butoxy)carboxamide in TEA and MeOH to produce N-Boc protected mucic acid diamine. See Example 24.

[0133] In the conventional method, this first step (i.e., the N-Boc protection of mucic acid to mucic acid ethylenediamine The conversion to amine) was carried out in two separate reactions. First, a diester was formed, the diester was purified, and then the isolated diester was converted to N-Boc protected mucic acid ethylenediamine. For example, see 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 achieved in a single reaction. See Example 1. Also, any combination of two or more of the foregoing embodiments is contemplated as an additional embodiment of the invention disclosed herein. In the next step, the Boc protecting group is removed by reacting N-Boc protected mucic acid ethylenediamine with MeOH and HCl. Thereby, N-Boc protected mucic acid ethylenediamine is converted to mucic acid ethylenediamine chloride (Compound 3). See Example 2. An alternative preparative scale method for preparing mucic acid ethylenediamine chloride (mucic acid diaminochloride) by reaction with MeOH and HCl is provided in Example 25. In the next step, in the presence of hydroxypyridine N-oxide (HOPO), N,N'-diisopropylcarbodiimide (DIC) and acetonitrile (ACN), mucic acid diaminochloride is reacted with Boc-L-aspartic acid 4-tert-butyl ester to convert mucic acid ethylenediamine chloride to mucic acid di(aspartyl(O-t-butyl)-Boc) (Compound 4). See Example 3. The reaction time is in the range of 15 °C to 90 °C.

[0134]

[0135]

[0136] ​​​​​​​​​​​​​​At a temperature that can range from 5 minutes to 48 hours. In the conventional method, ethylene mucate diamine is reacted with di(aspartyl(O-benzyl)-Boc) in the presence of ACN and pyridine to result in di(aspartyl(O-t-butyl )-Boc) protecting groups rather than the di(aspartyl(O-benzyl)-Boc) protecting groups used in the method disclosed herein (for example, U.S. Patent Application Publication No. 2019 / 0381188 (December 19, 2019)). Therefore, the method described herein utilizes new reagents (HOPO and DIC instead of pyridine) and different protecting groups (O-t-butyl instead of O-benzyl ). Compared with the benzyl group used in the conventional method, the advantage of the t-butyl protecting group used in this method is that the t-butyl protecting group can be removed with HCl or trifluoroacetic acid (TFA). Therefore, in the next synthetic step, both protecting groups (t-butyl and Boc) can be removed with one reagent (for example, TFA or H Cl). ). An alternative method for synthesizing mucic acid di(aspartyl(O-t-butyl)-Boc) on a preparative scale is to react Boc-L-aspartic acid 4-tert-butyl ester with ethyl cyano hydroxyiminoacetate (oxime) in dichloromethane (DCM) and proceed while adding 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl). This mixture is mixed with mucic acid di

[0137] aminochloride (Compound 3) in an aqueous solution of sodium carbonate. While distilling off the aqueous phase, ACN is added to concentrate the crude product and crystallize it. See Example 26. (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl)

[0138] ​​​​​Also, any combination of two or more of the foregoing embodiments is contemplated as an additional embodiment of the invention disclosed herein.

[0139] In the final stage of the synthesis of the mucic acid polymerizable monomer, mucic acid di(aspartyl (O-t-butyl)-Boc) is reacted with TFA in the presence of DCM to convert it to mucic acid di(aspartylamine) (Compound 5). See Example 4. The reaction time can range from 5 minutes to 96 hours at a temperature in the range of 15°C to 90°C. Conventional methods (see, for example, US Patent Application Publication No. 2019 / 0381188 (December 19, 2019)) require two steps for this conversion, first converting mucic acid di(Asp(O-benzyl)-Boc) to mucic acid di(Asp(O-benzyl)-amine) and then converting mucic acid di(Asp(O-benzyl)-amine) to mucic acid di(Asp-amine). The use of t-butyl and Boc protecting groups in the method described herein (instead of the aforementioned O-benzyl and Boc protecting groups) enables the removal of two protecting groups in one step in an aqueous or organic solution, avoiding the need for homogeneous or heterogeneous hydrogenation.

[0140] For preparative scale applications, mucic acid di(aspartyl (O-t-butyl)-Boc) is converted to the mucic acid di(aspartylamine) neutral species (Compound 20), which is then converted to the di-TFA salt of mucic acid di(aspartylamine) (Compound 5). In these embodiments, mucic acid di(aspartyl (O-t-butyl)-Boc) is reacted with TFA and triisopropylsilane in DCM and water, and optionally tetrahydrofuran (THF) is used for further re It can crystallize to form the mucic acid monomer neutral species (20). Refer to Example 27. Next, the neutral mucic acid monomer is reacted with TFA in DCM and water, and combined with a solution of the seed crystal of Compound 5 in ether to precipitate the MAM-diTFA salt (5). Refer to Example 28. Refer to Example 28.

[0141] Also, any and all combinations of two or more of the foregoing embodiments are contemplated as additional embodiments of the invention disclosed herein. Refer to Example 28.

[0142] (Synthesis of mucic acid polymer (MAP)) Provided herein is, in particular, a method for synthesizing a polymer of mucic acid (the "mucic acid polymer" or MAP). In certain embodiments, diTFA-mucic acid di(aspartyl ruthenium amine) (MAM, Compound 5) is mixed with di(succinimidyl propionate)-PEG (diSPA-PEG) (e.g., diSPA-PEG diSPA-PEG ) in a solvent (e.g., dimethyl sulfoxide, DMSO), a base (e.g., N,N'-diisopropylethyl amine, DIPEA) is added, and the reaction is carried out at a temperature in the range of 15 °C to 70 °C (e.g., 35 °C) 3.5k for a time in the range of 1 hour to 96 hours (e.g., 66 hours). After the desired polymer length (generally in the range of 10 kDa to 120 kDa) is obtained, the reaction mixture is dialyzed against DMSO, dialyzed against water, and filtered to obtain MAP (Compound 6). Refer to Example 28. Refer to Example 28. Refer to Example 28.

Chemical formula

[0143] The molecular weight of the polymer is controlled by adjusting the molar ratio of MAM to diSPA-PEG. and the highest molecular weight is obtained when the molar ratio of the reactants is 1:1. When the molar ratio of MAM to diSPA- PEG is greater than or less than 1, the molecular weight of the resulting polymer decreases. In the examples of the present inventors, a further decrease in molecular weight correlates with an increase in the ratio of MAM to diSPA-PEG. See Example 29.

[0144] 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, 2 0 to 120, 50 to 100 or 10 to 25). In certain embodiments, x is such that the number average molecular weight of the PEG moiety of the polymer (i.e., -O-CH 2 -CH 2 -) is selected to be in the range of about 500D a to about 50,000 Da.

[0145] In certain embodiments, the weight average molecular weight of MAP is in the range of 5 to 150 kDa or any integer value therebetween, e.g., in the range of 20 to 120 kDa, and the values of x and y are selected accordingly.

[0146] In additional embodiments, the values of x and y are such that after assembling MAP or the drug conjugate 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 1 00 nm.

[0147] In some embodiments, the methods described herein are conjugated to any one of the targeting agents described herein and any one of the chemotherapeutic agents described herein. Performed using target nanoparticles comprising the nanoparticle core described in the book, and the total size of the target nanoparticles is from about 20 nm to about 100 nm. In some embodiments, the size of the target nanoparticles is from about 40 nm to about 100 nm. In some embodiments, the size of the target nanoparticles is from about 40 nm to about 90 nm. In some embodiments, the size of the target nanoparticles is from about 40 nm to about 80 nm. In some embodiments, the size of the target nanoparticles is from about 40 nm to about 70 nm. In some embodiments, the size of the target nanoparticles is about 40 nm to about 60 nm. In some embodiments, the size of the target nanoparticles is about 4 0 nm to about 50 nm. In some embodiments, the size of the target nanoparticles is about 50 nm to about 100 nm. In some embodiments, the size of the target nanoparticles is about 50 nm to about 90 nm. In some embodiments, the size of the target nanoparticles is about 60 n m to about 80 nm. In some embodiments, the size of the target nanoparticles is about 50 nm to about 70 nm. In some embodiments, the size of the target nanoparticles is about 70 nm to about 100 nm.

[0148] In certain embodiments, the solvent is, for example, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) or dimethylacetamide (DMA).

[0149] In certain embodiments, the high temperature is any temperature in the range of 20 °C to 40 °C, i.e., 2 0 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 3 0 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C or 40 °C.

[0150] Dialysis can be performed using membranes of various porosities. For example, membranes with a fractional molecular weight cut-off of 1 kDa, 3 kDa , 5 kDa, 10 kDa, 30 kDa or 50 kDa can be used.

[0151] Also, any and all combinations of two or more of the aforementioned embodiments are contemplated as additional embodiments of the invention disclosed herein.

[0152] (Measurement of Polymer Molecular Weight) The molecular weight of MAP is measured by gel filtration chromatography using a polyethylene oxide (PEO) standard (e.g., Malvern OMNISEC gel permeation chromatography (GPC) system). The chromatography apparatus can include, for example, a solvent supply pump, a degassing device, an automatic sampling injector, a column compartment, a refractive index detector and a light scattering detector. The sample is prepared, for example, by dissolving MAP in phosphate buffered saline (PBS) containing 0.02% sodium azide at a concentration in the range of 1 - 5 mg / mL, and then filtering the sample through, for example,

[0153] a 0.45 micron (pore size 0.45 μm) filter. Along with the PEO molecular weight standard, 100 μL of the filtered sample is injected into a gel filtration or gel permeation column (e.g., 2 × Agilent PL Aquagel-OH 40 column, length 30 0 mm × diameter 7.5 mm, particle size 8 μm) with a mobile phase of PBS + 0.02% sodium azide at a flow rate of, for example, 0.7 mL / min and a column temperature of, for example, 30°C. The peak fractions are detected by differential refractive index and / or light scattering and analyzed, for example, using Malvern OMNISEC software.

[0154] (Camptothecin (CPT) and its derivatives, metabolites, and analogs) The present disclosure provides, in particular, methods and compositions for preparing selectively reactive derivatives of CPT and its derivatives, metabolites, and analogs. Generally, these molecules contain five fused aromatic rings, one or more of which are heteroaromatic and contain one or more pendant hydroxyl groups, as shown in the figure depicting the structure of CPT below. Examples of molecules in these categories include topotecan, irinotecan, silatecan, comitecan, exatecan, larototecan, gimatecan, verotecan, rubitecan, SN38, diflomotecan, carenitecan, namitecan, eromotecan, delimotecan, kimitecan, ZBH-1205, DRF-1042, and FL118. (Binding of derivatized 7-ethyl-10-hydroxy-camptothecin (SN38) to MAP) [Chemical formula]

[0155]

[0156] Nanoparticles for use in the treatment of brain tumors and brain metastases have been conventionally described as utilizing CPT as a therapeutic molecule. However, as described above, more soluble derivatives of CPT, such as SN38, provide a higher therapeutic index and lower toxicity. Thus, the present disclosure provides, in particular, methods for synthesizing nanoparticles having a polymeric mucic acid backbone to which multiple SN38 molecules are attached. For this purpose, the present disclosure provides methods and compositions for synthesizing a number of derivatized variants of SN38 that can covalently bind to the above-described MAP. ​​​​​​​​​​​​​

[0157] Accordingly, provided herein is, inter alia, a method for synthesizing reactive derivatives of the chemotherapeutic molecule SN38 This method involves attaching a number of diverse amino acid-based linker molecules to the SN38 molecule via the SN38 hydroxyl group Other CPT derivatives of SN38 itself have two hydroxyl groups, at the 10 and 20 positions Conjugation at the 20 position stabilizes the active lactone form of these molecules and reduces, or prevents, its conversion to the less active carboxylate form after administration to a subject. However, the 10 hydroxyl is also reactive In the methods described herein, the reactivity of the 10-hydroxyl of SN38 is first blocked (i.e., protected), and then the linker is added to the 20-hydroxyl Exemplary amino acid linkers include glycine, alanine, β-alanine, γ-aminobutyric acid (GABA), valine

[0158] leucine, isoleucine, pentanoic acid, hexanoic acid, arginine, histidine, lysine aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tyrosine tryptophan, as well as dipeptides (e.g., phenylalanine-glycine), tripeptides, and oligopeptides

[0159] Methods for attaching a number of diverse linkers to the 20-hydroxyl group of SN38 are provided herein Each of the various linkers exemplified herein (e.g., glycine, alanine, β-alanine, leucine valine, γ-aminobutyric acid, and 6-aminohexanoic acid, as well as other dipeptides such as phenylalanine-glycine) is attached to the 2 0-hydroxyl of SN38 After conjugation with 0-hydroxy, it reacts with the carboxyl group of MAP (through the amidation reaction between the amine group of the linker and the carboxyl group of the polymer), and the SN38 molecule can be covalently bonded to M AP. Different linkers have different stabilities respectively, affect the properties of the resulting nanoparticles (such as size, density), which in particular affects the rate at which SN38 is released from the nanoparticles after passing through the blood-brain barrier ( BBB) or the blood-tumor barrier (BTB).

[0160] (Synthesis of 10-hydroxy protected SN38 (10-TBDPS-SN38)) In the first step of the synthesis of derivatized SN38, SN38 and tert-butyl (chloro) di phenylsilane (TBDPSCl) are mixed in DCM and triethylamine to add the tert-butyldiphenylsilyl (TBDPS) protecting group to the 10-hydroxy group of SN38 (to block the reactivity at that site). The mixture is refluxed (e.g., 45 °C) for about 16 hours, washed with HCl (e.g., 0.2 N), saturated NaHCO 3 and brine, and finally dried with MgSO 4 and evaporated in vacuo. The product (10- TBDPS-SN38, compound 7) is dissolved in DCM, precipitated with hexane, and the solid is dried .

[0161] The TBDPS protecting group is selective for the 10-hydroxy group of SN38 (the 20-O H group reacts freely in subsequent steps), and the resulting compound is less likely to lose the protecting group spontaneously during storage, providing a more stable intermediate.

[0162] A further advantage of the use of TBDPS for protecting the 10-OH group of SN38 is that it can be removed under the same conditions (1 M to 1 2 M HCl or 1:1 to 1:4 v / v water:TFA) as used for removing the Boc protecting group from the linker attached to the 20- OH group.

[0163] (Synthesis of 10-hydroxy-protected SN38 with a protected 20-hydroxyglycine linker ) Mix 10-TBDPS-SN38 with Boc-Gly-OH in DCM at 0 °C to attach a protected glycine (Gly) linker to the 20- hydroxy position of 10-TBDPS-SN38. Add EDC.HCl and 4-dimethylaminopyridine (DMAP) and stir the mixture at 0 °C. Stirring can be carried out for 0.5 h, 1 h, 1.5 h, 2 h , 2.5 h, 3 h, 3.5 h, 4 h or at any interval in between . After stirring, wash the mixture twice with 0.5% NaHCO 3 , once with water, twice with 0.1 N HCl, and once with brine. Dry this solution (e.g., with MgSO 4 ) and evaporate it in vacuo to obtain 20-(Boc-Gly)-10-TBDPS-SN38 (Compound 8).

[0164] (Synthesis of 20-(HCl.Gly)-SN38) Treat 20-(Boc-Gly)-10-TBDPS-SN38 with HCl (at a concentration in the range of 1 N to 12 N) in the hexane layer at room temperature to deprotect both the 10-hydroxy group and the 20-hydroxy group. This restores the 10-hydroxy group and generates an SN38 derivative (20-( HCl.Gly)-SN38, Compound 12) in which the glycine linker is covalently attached to the 20-hydroxy group.

[0165] (Synthesis of 10-hydroxy-protected SN38 with a protected 20-hydroxy GABA linker ) Mix 10-TBDPS-SN38 with Boc-GABA-OH in DCM at 0 °C to add a protected γ-aminobutyric acid (GABA) linker to the 20-hydroxy position of 10-TBDPS-SN3 8. Add EDC.HCl and DMAP, and stir the mixture at 0 °C. Stirring can be carried out for 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h or at any interval therebetween. After stirring, wash the mixture twice with 0.5% NaHCO 3 , once with water, twice with 0.1 N HCl, and once with brine . Dry this solution (e.g., with MgSO 4 ) and evaporate it in vacuo to obtain 20-(Boc -GABA)-10-TBDPS-SN38 (Compound 9).

[0166] (Synthesis of 20-(TFA.GABA)-SN38 Treat 20-(Boc-GABA)-10-TBDPS-SN38 in the hexane layer at room temperature with TFA at a concentration in the range of 1:1 to 4:1 v / v in water to deprotect both the 10-hydroxy group and the 20-hydroxy group. Thereby, the 10-hydroxy group is restored, and an SN38 derivative (20-(TFA.GABA)-SN38, Compound 13) in which the GABA linker is covalently bonded to the 20-hydroxy group is generated.

[0167] (Synthesis of 10-hydroxy-protected SN38 with a protected 20-hydroxy hexanoic acid linker ) ​Mixing 10-TBDPS-SN38 with Boc-Hex-OH at 0 °C in DCM adds a protected 6-aminohexanoic acid (Hex) linker to the 20-hydroxy position of 10-TBDPS-SN 38. Add EDC·HCl and DMAP and stir the mixture at 0 °C. Stirring can be carried out for 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h or at any interval therebetween. After stirring, wash the mixture twice with 0.5% NaHCO 3 , once with water, twice with 0.1 N HCl, and once with brine to purify. Dry this solution (e.g., over MgSO 4 ) and evaporate in vacuo to obtain 20-(Boc-Hex)-10-TBDPS-SN38 (Compound 10).

[0168] (Synthesis of 20-(TFA·Hex)-SN38) Treat 20-(Boc-Hex)-10-TBDPS-SN38 in the hexane layer at room temperature with TFA at a concentration in the range of 1:1 to 4:1 v / v in water to deprotect both the 10 -hydroxy group and the 20-hydroxy group. Thereby, the 10-hydroxy group is restored and an SN38 derivative (20-(TFA·Hex)-SN38, Compound 14) in which the Hex linker is covalently bonded to the 20-hydroxy group is produced.

[0169] (Synthesis of 10-hydroxy-protected SN38 with a protected 20-hydroxyvaline linker ) Mixing 10-TBDPS-SN38 with Boc-Val-OH at 0 °C in DCM adds a protected valine (Val) linker to the 20-hydroxy position of 10-TBDPS-SN38 . Add EDC·HCl and DMAP and stir the mixture at 0 °C Perform stirring. Stirring can be carried out for 0.5 hour, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours or at any interval therebetween. After stirring, the mixture is washed twice with 0.5% N aHCO 3 and once with water, twice with 0.1 N HCl, and once with brine. This solution is dried (e.g., with MgSO 4 ) and evaporated in vacuo to obtain 20-(Boc-Val) -10-TBDPS-SN38 (Compound 11).

[0170] (Synthesis of 20-(HCl.Val)-SN38) Treat 20-(Boc-Val)-10-TBDPS-SN38 with HCl (at a concentration in the range of 1 N to 12 N) in a hexane layer at room temperature to deprotect both the 10-hydroxy group and the 20-hydroxy group. Thereby, the 10-hydroxy group is restored , and an SN38 derivative (20-(H Cl.Val)-SN38, Compound 15) in which the valine linker is covalently bonded to the 20-hydroxy group is produced.

[0171] (Use of the same protecting group for both the 10- and 20-OH groups of SN38) An alternative route for synthesizing a reactive derivative of SN38 having an amino acid linker is to place a Boc-O protecting group on the 10 -OH group and a Boc-amino acid protecting group on the 20-OH. This compound (20-(Boc-aminoacyl)-10-OBoc-SN38) is reacted with TFA, whereby the 10-OH group is restored and the TFA salt of the amino acid linker is added to the 20 -OH. Using this method, 20-Boc-glycyl, -alanyl , -β-alanyl, -leucyl and -valyl derivatives of SN38 have been synthesized. Hereinafter, Example 33 ​Refer to FIGS. 0 to 35. Other amino acids that can be used in this method include GABA, isoleucine, and he xanthic acid and dipeptides, for example, phenylalanine - glycine. [Chemical formula]

[0172] (Synthesis of Polymer - Drug Conjugates) In this specification, in particular, a method is provided for forming a polymer - drug conjugate (i.e., a MAP - linker - SN38 nanoparticle) by covalently attaching derivatized SN38 to MAP. Many different conjugates are provided where the nature of the linker (i.e., size and chemical properties) between the polymer and the drug is different. Various conjugates are made using SN38 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. In one embodiment, the linker is a β - alanyl (β - Ala) moiety. In one embodiment, the linker is a leucyl (Leu) moiety. To form the polymer - drug conjugate, MAP is dissolved in DMSO, and then E DC.HCl and N - hydroxysuccinimide (NHS) are added. After the addition of EDC.HCl and NHS, derivatized SN38 and DIPEA are added, and the mixture is stirred at room temperature for about 18 hours. The derivatized SN38 is, for example, 20 - (HCl.Gly) - SN38, 2 ... moiety.

[0173] To form the polymer - drug conjugate, MAP is dissolved in DMSO, and then E DC.HCl and N - hydroxysuccinimide (NHS) are added. After the addition of EDC.HCl and NHS, derivatized SN38 and DIPEA are added, and the mixture is stirred at room temperature for about 18 hours. The derivatized SN38 is, for example, 20 - (HCl.Gly) - SN38, 2 0-(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.Va l)-SN38 or 20-(TFA.Leu)-SN38. Next, the reaction mixture is dialyzed against DMSO and then 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).

[0174] (Formation of Nanoparticles) To convert the polymer-drug conjugate (i.e., any one of Compounds 16, 17, 18 or 19, 37, 38 or 39) into nanoparticles, the lyophilized polymer-drug con jugate is dissolved in water at pH 4 at a concentration of 1 - 10 mg / mL (e.g., 4 mg / mL).

[0175] The size of the nanoparticles ranges from about 20 nm to about 60 nm in diameter. Exemplary nanoparticles have diameters of 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 more It is.

[0176] (Amount of SN38 released from nanoparticles in vitro) When 0.1 mg of nanoparticles with a concentration of SN38 / mL is incubated in a humidified oven at 37 °C in PBS at pH 6.5 , 7.0 or 7.4, MAP-Gly-SN38, M AP-GABA-SN38, MAP-Hex-SN38, MAP-Val-SN38, M AP-Ala-SN38, MAP-β-Ala-SN38 and MAP-Leu-SN38 All the releases of SN38 from the nanoparticles showed first-order kinetics. The release rate was strongly dependent on pH It was observed that. When the pH increased from 6.5 to 7.4, the release half-lives decreased in all nanoparticle formulations, suggesting that hydrolysis plays an important role in the release of SN38 was suggested. Nanoparticles with more hydrophobic and sterically hindered linkers had longer half-lives were observed.

[0177] (Derivatized CPT for conjugation to MAP) In certain embodiments, the present disclosure provides nanoparticles comprising CPT as a therapeutic agent. CPT derivatives conjugated to amino acids (e.g., 20-(TFA.Gly)-CPT ) are used in the synthesis of the nanoparticles. Exemplary amino acid linkers include glycine, ala nine, β-alanine, γ-aminobutyric acid (GABA), valine, leucine, isoleucine, pentanoic acid, hexanoic acid and dipeptides (e.g., phenylalanine-glycine), tri peptides and oligopeptides are included.

[0178] 20-(TFA.Gly)-CPT is synthesized in two steps. In the first step, under an inert atmosphere (e.g., under argon), CPT is mixed with Boc-Gly-OH in DMAP and DCM is added to make the reaction solution into a slurry. Next, DIC is added dropwise to the reaction mixture solution over several minutes, and then the mixture is stirred at room temperature for several hours (e.g., 3 hours). Then, about half of the solvent is removed under vacuum, cold MeOH is added to precipitate the product (20-(Boc-Gly)-CPT , compound 21), and this is obtained by filtering. The solid is washed with cold MeOH and cold methyl tert-butyl ether (MTBE) and dried under vacuum. Refer to Example 30 for reference.

[0179] In the second step, TFA is gradually added to a stirred suspension of 20-(Boc-Gly)-CPT in DCM and the mixture is stirred for several hours (e.g., 2 hours). Then, the product (20-(T FA.Gly)-CPT, compound 22) is precipitated with MTBE, filtered, washed, and dried under vacuum for reference. Refer to Example 31 for reference.

[0180] By using other Boc-protected amino acids (or peptides) instead of Boc-Gly-OH , additional CPT derivatives can be produced. For example, when Boc-Ala- OH is used as the starting material, 20-(Boc-Ala)-CPT (compound 41) and 20-(TFA.Ala)-CPT (compound 45) are obtained. When Boc-β-Ala-O H is used as the starting material, 20-(Boc-β-Ala)-CPT (compound 42) and 20-(TFA.β-Ala)-CPT (compound 46) are obtained. When Boc-Val -OH is used as the starting material, 20-(Boc-Val)-CPT (compound 40) and 20-(TFA.Val)-CPT (Compound 47) is obtained. Boc-GABA- Using OH as a starting material, 20-(Boc-GABA)-CPT (Compound 43) and 20-(TFA.GABA)-CPT (Compound 48) are obtained. Boc-Phe- Using Gly-OH as a starting material, 20-(Boc-Phe-Gly)-CPT (Compound 44) and 20-(TFA.Phe-Gly)-CPT (Compound 49) are obtained .

[0181] (Synthesis of CPT-polymer conjugate) Conjugates of CPT and MAP are provided in certain embodiments. These To synthesize these conjugates, MAP and 20-(TFA.aminoacyl)-CPT (see previous section for exemplary amino acid and peptide derivatives of CPT) are dissolved in DMSO under an inert atmosphere (e.g., under argon). In a separate container, (7- Azabenzotriazol-1-yloxy)trispyrrolidinophosphonium hexafluoro rophosphate (PyAOP) is dissolved in DMSO. Next, the PyAOP solution is added to the MAP / 20-(TFA.aminoacyl)-CPT solution, stirred for several minutes (e.g., 2 minutes), then DIPEA is added, and the reaction mixture is stirred 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 lyophilization. See Example 32.

[0182] (Nitrophenylboronic acid (NPBA)-polyethylene glycol (PEG) conjugate )​​ In certain embodiments, a targeting (homing) molecule or a macromolecular therapeutic (e.g., an antibody) is conjugated to the MAP via a linker with nitrophenylboronic acid-polyethylene glycol (NPBA-PEG). Such NPBA-PEG linkers can be synthesized by combining an amine derivative of PEG (e.g., PEG , PEG 3.5k , PEG 5k ) with a carboxyl derivative of NPBA to form an NPBA-PEG-acetic acid (AA) conjugate bonded by an amide bond. In certain embodiments, the PEG derivative has an amino group at one end (for reaction with NPBA) and a carboxyl group at the opposite end that is activated in a subsequent reaction and can react with a macromolecule such as a polypeptide (see Example 36).

Chemical Structure

[0183] In certain embodiments, n is an integer value between 2 and 2,000 or any integer value therebetween, e.g., a number in the range of 100 to 300, 20 to 300, 120 to 180, or 140 to 160.

[0184] In certain embodiments, the value of n is such that the weight average molecular weight of the PEG moiety of the compound (i.e., -CH 2 -CH 2 -O- ) is in the range of about 2 to about 15 kDa, e.g., 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.

[0185] The NPBA-PEG-AA conjugate is activated for reaction with a polypeptide (e.g., transferrin, etc.) by forming a pentafluorophenyl (PFP) derivative . In these embodiments, bis(pentafluorophenyl) carbonate is combined with the NPBA-PEG-AA conjugate to form an NPBA-PEG-penta fluorophenyl ester (e.g., NPBA-PEG -AA-PFP). 5k See Example 37.

Chemical formula

[0186] In certain embodiments, n is an integer value between 2 and 2,000 or any integer therebetween, e.g., in the range of 10 0 - 300, 20 - 300, 120 - 180 and / or 140 - 160 .

[0187] In certain embodiments, the value of n is such that the weight average molecular weight of the PEG (i.e., -CH 2 -CH 2 -O- ) moiety of the compound is in the range of about 2 to about 15 kDa, e.g., 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 k Da.

[0188] (Polymer conjugate containing a homing (targeting) molecule) In certain embodiments, the MAPs described herein contain a homing (or targeting) molecule, instead of or in addition to, a low molecular weight therapeutic agent such as CPT or SN38 . ​Small molecule compounds are known in the art, for example, small molecules (e.g., vitamins, e.g., folic acid), saccharides (e.g., mannose, allose, altrose, glucose, gulose, idose, galactose, talose, disaccharides, trisaccharides, oligosaccharides), peptides (e.g., RGD), polypeptides (e.g., proteins that bind to cell surface receptors such as antibodies, transferrin), nucleic acids, peptoids, and peptide nucleic acids (PNA), etc. For example, MAP (and nanoparticles made from MAP) can contain transferrin, a homing

[0189] molecule that targets polymers (or nanoparticles formed from polymers) to endothelial cells of the blood-brain barrier or blood-tumor barrier. In these embodiments, the homing molecule binds to the NPBA-PEG polymer by reaction with the above-described PFP active ester. Since the primary amine group of the protein reacts with the PFP active ester, binding of holotransferrin to the NPBA-P EG-pentafluorophenyl ester is achieved. The progress of the reaction is monitored by HPLC and purified by hydrophobic interaction chromatography (HIC) based on the difference in binding of mono-PEGylated transferrin to fractions with a higher degree of PEGylation (e.g., di- and tri-PEGylated fractions). See Examples 38 and 39. This method prevents the loss of iron from transferrin during purification as occurs when using conventional methods, thereby maintaining the holotransferrin structure. Binding of ester and holotransferrin is realized by binding holotransferrin to the NPBA-P EG-pentafluorophenyl ester. The progress of the reaction is monitored by HPLC and the mono-PEGylated transferrin product (e.g., mono-NPBA-PEG 5k -T f) is compared with the binding of mono-PEGylated transferrin to fractions with a higher degree of PEGylation (e.g., di- and tri-PEGylated fractions). Based on this difference, it is purified by hydrophobic interaction chromatography (HIC). See Examples 38 and 39. This method prevents the loss of iron from transferrin during purification as occurs when using conventional methods, thereby maintaining the holotransferrin structure. preventing the loss of iron from transferrin during purification as occurs when using conventional methods, thereby maintaining the holotransferrin structure.

[0190] (Polymer conjugate containing a polymeric therapeutic agent) In certain embodiments, the MAPs described herein include polymeric therapeutic agents instead of, or in addition to, small molecule therapeutic agents such as CPT or SN38. Polymeric therapeutic agents are known in the art and include, for example, therapeutic polypeptides (e.g., antibodies, enzymes, and bioactive proteins) and nucleic acids. In certain embodiments, the MAPs described herein include polymeric therapeutic agents instead of, or in addition to, small molecule therapeutic agents such as CPT or SN38. Polymeric therapeutic agents are known in the art and include, for example, therapeutic polypeptides (e.g., antibodies, enzymes, and bioactive proteins) and nucleic acids. Exemplary therapeutic polypeptides include, but are not limited to, trastuzumab (anti-Her2), anti-Her3, anti-Trp2, anti-PSMA, anti-LIV-1, anti-FOLR1, anti-DLL3, anti-PDGF, anti-FRα, 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. Exemplary therapeutic polypeptides include, but are not limited to, trastuzumab (anti-Her2), anti-Her3, anti-Trp2, anti-PSMA, anti-LIV-1, anti-FOLR1, anti-DLL3, anti-PDGF, anti-FRα, 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.

[0191] Exemplary therapeutic polypeptides include, but are not limited to, trastuzumab (anti-Her2), anti-Her3, anti-Trp2, anti-PSMA, anti-LIV-1, anti-FOLR1, anti-DLL3, anti-PDGF, anti-FRα, 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. Exemplary therapeutic polypeptides include, but are not limited to, trastuzumab (anti-Her2), anti-Her3, anti-Trp2, anti-PSMA, anti-LIV-1, anti-FOLR1, anti-DLL3, anti-PDGF, anti-FRα, 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. Exemplary therapeutic polypeptides include, but are not limited to, trastuzumab (anti-Her2), anti-Her3, anti-Trp2, anti-PSMA, anti-LIV-1, anti-FOLR1, anti-DLL3, anti-PDGF, anti-FRα, 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. Exemplary therapeutic polypeptides include, but are not limited to, trastuzumab (anti-Her2), anti-Her3, anti-Trp2, anti-PSMA, anti-LIV-1, anti-FOLR1, anti-DLL3, anti-PDGF, anti-FRα, 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. Exemplary therapeutic polypeptides include, but are not limited to, trastuzumab (anti-Her2), anti-Her3, anti-Trp2, anti-PSMA, anti-LIV-1, anti-FOLR1, anti-DLL3, anti-PDGF, anti-FRα, 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.

[0192] Certain polypeptide therapeutic agents, such as antibodies that target tumor markers as exemplified in the previous paragraph, are also used as target (homing) molecules. Certain polypeptide therapeutic agents, such as antibodies that target tumor markers as exemplified in the previous paragraph, are also used as target (homing) molecules.

[0193] For example, MAPs (and nanoparticles made from MAPs) can include the anti-Her2 antibody trastuzumab. In these embodiments, the antibody binds to the NPBA-PEG polymer by reaction with the PFP active ester described above. For example, MAPs (and nanoparticles made from MAPs) can include the anti-Her2 antibody trastuzumab. In these embodiments, the antibody binds to the NPBA-PEG polymer by reaction with the PFP active ester described above. For example, MAPs (and nanoparticles made from MAPs) can include the anti-Her2 antibody trastuzumab. In these embodiments, the antibody binds to the NPBA-PEG polymer by reaction with the PFP active ester described above.

[0194] Since the primary amine groups of proteins react with PFP active esters, binding of trastuzumab to the NPBA-PEG pentafluorophenyl ester enables binding of the ester to trastuzumab. The progress of the reaction is monitored by HPLC, and the mono-PEGylated trastuzumab product (e.g., mono-NPBA-PEG Since the primary amine groups of proteins react with PFP active esters, binding of trastuzumab to the NPBA-PEG pentafluorophenyl ester enables binding of the ester to trastuzumab. The progress of the reaction is monitored by HPLC, and the mono-PEGylated trastuzumab product (e.g., mono-NPBA-PEG Since the primary amine groups of proteins react with PFP active esters, binding of trastuzumab to the NPBA-PEG pentafluorophenyl ester enables binding of the ester to trastuzumab. The progress of the reaction is monitored by HPLC, and the mono-PEGylated trastuzumab product (e.g., mono-NPBA-PEG Since the primary amine groups of proteins react with PFP active esters, binding of trastuzumab to the NPBA-PEG pentafluorophenyl ester enables binding of 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), P The mono-P compared with fractions having a higher degree of PEGylation (e.g., di- and tri-PEGylated fractions) Based on the difference in the binding of the PEGylated trastuzumab, it is purified by HIC. See Examples 41 and 4 2

[0195] (Measurement of the number of polymer strands per nanoparticle) To measure the number of polymer strands per nanoparticle, divide the average molecular weight of the nanoparticle by the molecular weight of the corresponding polymer-drug (i.e., MAP-SN38) conjugate

[0196] The molecular weight of the nanoparticle is measured 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 is calculated from the molecular weight of the polymer (i.e., MAP) used in the synthesis of the conjugate and adjusted for the drug loading as follows MW コンジュゲート = MW ポリマー / (1 - T) (where MW ポリマー is the molecular weight of the MAP used in the synthesis of the conjugate and T is the ratio of the therapeutic drug loading, expressed as a decimal.)

[0197] The strands per particle (SpP) used herein is the number of sialic acid polymer ("MAP") therapeutic conjugate molecules present in the particle or nanoparticle. In the measurement of SpP the particle or nanoparticle has at least one MAP-therapeutic protein conjugate molecule that behaves as a single unit in any aqueous solution suitable for administration to humans, e.g., water at neutral pH , PBS at pH 7.4, or any formulation administered to the patient entity ​​​​​refers to. In the calculation of the strands per particle, the MAP-therapeutic conjugate molecule is , a single MAP polymer having a covalently attached therapeutic agent.

[0198] The methods disclosed herein provide an evaluation of particles that include one or more MAP-therapeutic conjugate molecules. Generally, the method involves providing a sample that includes a plurality of said particles, measuring a value of the number of MAP-therapeutic conjugates per particle in the sample, and thereby evaluating a preparation of the particles. The value of the particle sample is a function of the values obtained for a plurality of particles.

[0199] As described above, SpP is defined as the number of MAP-therapeutic conjugate molecules that self-assemble into particles or nanoparticles, and thus SpP = [MAP-therapeutic conjugate molecule] / P(or NP) (wherein, [MAP-therapeutic conjugate molecule] is the number of MAP-therapeutic conjugate molecules, and P(or NP) is a single particle (or nanoparticle).).

[0200] In certain embodiments, the method further includes comparing the measured value to a reference value. The comparison can be used in many ways. As an example, depending on the comparison or measurement performed by the method, a determination or step is taken, for example, a manufacturing parameter in the process for making the particles is changed, the sample is classified, selected, accepted or rejected, released or held, processed into a pharmaceutical, shipped, moved to another location, combined with another substance, for example, combined with an excipient, labeled, packaged, commercially sold, or offered for sale or distribution. For example, based on the results of the measurement or based on a comparison to a reference standard, depending on the results of the measurement or based on a comparison to a reference standard, Then, the batch from which the sample was taken can be processed, for example, as described above.

[0201] To calculate the number of strands per particle, the size of the particle, e.g., the molecular weight by light scattering of self-assembled particles, the size of individual polymers, e.g., the molecular weight by light scattering of individual polymers, and the loading amount of the therapeutic agent, e.g., mass%, are determined. Using these values, SpP is calculated as follows. As follows. SpP = MW 粒子 / (MW コンジュゲート ) (where MW 粒子 is the molecular weight of the particle, and MW コンジュゲート is the molecular weight of the MAP-therapeutic agent conjugate molecule and is calculated as follows. As follows. MW コンジュゲート = MW ポリマー / (1 - T % ) (where MW ポリマー is the molecular weight of the MAP, and T % is the loading rate of the therapeutic agent expressed as a decimal, e.g., for a 10% load, T % = 0.1.))

[0202] The measurement of SpP has been demonstrated with MAPs having various linkers and drugs. MAPs of the same molecular weight having CPT and S N38 result in about 1.5 to 2.5 strands per particle. It has been demonstrated with SN38 that when the linker is different, the number of strands per particle changes and the density of the particles changes. With linkers of glycine, γ-aminobutyric acid, hexanoic acid, alanine, β-alanine, leucine, and valine, the number of strands per particle changes from about 1.3 to about 4.6. about 4.6.

[0203] Polymer molecular weight distribution and particle dispersity: MAPs are synthesized to have various molecular weights. ​​When the molecular weight is different, the particle size and the number of strands per particle also differ. The particles may be formed of MAPs (referred to as strands herein) that are smaller or larger than average. The strands may also bind to the maximum size that can be restricted by shear.

[0204] Particle shape: The particle shape is assumed to be approximately spherical. Self-organization is assumed to be driven by the hydrophobic regions created by the therapeutic agent of the MAP-therapeutic agent conjugate molecules.

[0205] (Formulations, Kits, and Routes of Administration) Therapeutic compositions comprising nanoparticles as disclosed herein are also provided. Such compositions typically comprise nanoparticles and a pharmaceutically acceptable carrier. Optional active compounds can also be incorporated into the nanoparticle composition.

[0206] The therapeutic compositions disclosed herein are particularly useful for treating cancer, cancer metastasis, and other disorders of the brain and central nervous system. Thus, a "therapeutically effective amount" of a composition comprising nanoparticles is any amount that alleviates symptoms or, for example, stimulates regression of a tumor. For example, the dosage of nanoparticles can be about 0.1 - 1.0 mg / kg body weight or about 0.5 - 2.0 mg / kg body weight or about 1 - 5 mg / kg body weight or about 1 mg / kg body weight to about 10 mg / kg body weight or more ( or any integer value therebetween), and the dosing frequency can be, for example, once per hour, twice daily, once daily, twice weekly, once weekly, twice monthly, once monthly, depending on, for example, body weight, route of administration, severity of the disease, etc. Thus, a therapeutically effective amount can consist of multiple administrations of the same amount or different amounts of nanoparticles. In certain embodiments, a single administration of nanoparticles is a therapeutically effective amount.

[0207] In certain embodiments, the administration of the nanoparticles is such that it is typical as a dosage of the chemotherapeutic agent to be administered at a dosage of 1 to 20 mg of nanoparticles per square meter of body surface area of the subject (or any integer or decimal value therebetween).

[0208] Techniques for preparing, and using various pharmaceutical compositions, are known to those of skill in the art in light of the present disclosure . A detailed list of suitable pharmaceutical compositions and techniques for their administration can be found in Remington’s Pharmaceutical Sciences , 17th ed. 1985; Brunton et al., “Goodman and Gilman’s The Pharmacological Basi s of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), “Remington: The Science and P ractice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), “Remin gton: The Principles of Pharmacy Practic e,” Lippincott Williams & Wilkins, 2008, and reference may be made to any of these texts

[0209] The nanoparticles described herein are suspended in a physiologically compatible carrier for administration ​​​​It can be done. As used herein, the term "physiologically compatible carrier" refers to a carrier that is compatible with the nanoparticles and any other optional components of the formulation and is not harmful to its recipient. One skilled in the art is proficient in 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 a plurality of electrolyte solutions such as, for example, Plasma-Lyte™ A (Baxter). + / - glucose (HBSS) and a plurality of electrolyte solutions such as, for example, Plasma-Lyte™ A (Baxter).

[0210] The amount of the nanoparticle suspension administered to a subject will vary depending on the site of administration, the treatment goal, and the number of nanoparticles in the solution. Typically, the amount of nanoparticles administered will be a therapeutically effective amount. As used herein, the term "therapeutically effective amount" or "effective amount" means the amount of nanoparticles administered necessary to effectively treat a particular disorder; i.e., to effect a decrease in the amount and / or an improvement in the severity of the symptoms associated with that disorder. For example, in the case of cancer metastasis to the brain, administration of a therapeutically effective amount of nanoparticles will result in a reduction and / or reversal of the symptoms of metastasis; e.g., regression of metastatic tumors. The therapeutically effective amount will vary depending on the type and extent of the brain injury and may also vary depending on the overall condition of the subject.

[0211] The disclosed therapeutic compositions can also include pharmaceutically acceptable materials, compositions, or vehicles such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, i.e., carriers. These carriers can, for example, stabilize the nanoparticles and / or facilitate the in vivo retention of the nanoparticles. Each carrier is compatible with the other components of the formulation and does not harm the subject. ​​​​​​​​​​​In the sense of not being, it is "acceptable". Functioning as a pharmaceutically acceptable carrier Some examples of materials that can include the following: lactose, glucose, and sucrose such as sugars; starches such as corn starch and potato starch; cellulose and its derivatives, for example, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter and suppository such as waxes; excipients; peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, coconut oil and soybean oil; glycols such as propylene glycol and polyethylene glycol ; polyols such as glycerin, sorbitol and mannitol; esters such as ethyl oleate and ethyl laurate ; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; water free of pyrogens; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; other non-toxic compatible substances used in pharmaceutical preparations . Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavors and fragrances, preservatives and antioxidants may also be present in the composition.

[0212] The nanoparticles are in a vehicle containing 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 and 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, etc. Administration of the compounds described herein is within the art ​ It can be carried out using any method known in the art. For example, administration can be transdermal, parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intrathecal , intraperitoneal, intraventricular, intrathecal, intranasal, aerosol, by suppository, or by oral administration and can be. The pharmaceutical composition of the nanoparticles described herein can be for administration by injection or for oral, pulmonary, nasal, transdermal or ocular administration.

[0213] Exemplary formulations include, but are not limited to: parenteral administration, for example, those suitable for intrapulmonary, intravenous, intraarterial, intraocular, intracranial, subarachnoid or subcutaneous administration, and formulations encapsulated in micelles, liposomes or drug delivery capsules (active drugs incorporated within a biocompatible coating 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 composition of the present disclosure varies depending on the degree and severity of the need for treatment, the activity of the composition administered, the general health of the subject and other considerations well known to those skilled in the art.

[0214] In additional embodiments, the compositions described herein are delivered intracranially at or near the site of brain injury or metastasis. Such local delivery allows for non-systemic delivery of the composition, thereby reducing the physical burden of the composition compared to systemic delivery. Local delivery can be achieved, for example, by intracranial injection or by the use of various medical devices, including but not limited to stents and catheters, or by inhalation, drainage or surgery It can be realized by. Methods of coating, implanting, embedding and otherwise binding a desired drug to medical devices such as stents and catheters are well established in the art and are contemplated herein.

[0215] 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 includes, for example, a composition of nanoparticles formulated in a pharmaceutical carrier suitable for administration by injection. For example, the kit includes a composition of nanoparticles formulated in a pharmaceutical carrier suitable for administration by injection.

Examples

[0216] Each example is considered to provide a specific individual embodiment of a composition, a preparation method, and a use method. However, none of the examples should be considered as limiting the more general embodiments described herein. Although each example is considered to provide a specific individual embodiment of a composition, a preparation method, and a use method, none of the examples should be considered as limiting the more general embodiments described herein. None of the examples should be considered as limiting the more general embodiments described herein. In the following examples, efforts have been made to be accurate with respect to the numerical values (e.g., amounts, temperatures, etc.) used, but some experimental errors and deviations should be taken into account. Unless otherwise specified, temperatures are in degrees Celsius and pressures are at or near atmospheric pressure. References to molecular weight refer to number average molecular weight unless otherwise specified.

[0217] In the following examples, efforts have been made to be accurate with respect to the numerical values (e.g., amounts, temperatures, etc.) used, but some experimental errors and deviations should be taken into account. Unless otherwise specified, temperatures are in degrees Celsius and pressures are at or near atmospheric pressure. References to molecular weight refer to number average molecular weight unless otherwise specified. In the following examples, efforts have been made to be accurate with respect to the numerical values (e.g., amounts, temperatures, etc.) used, but some experimental errors and deviations should be taken into account. Unless otherwise specified, temperatures are in degrees Celsius and pressures are at or near atmospheric pressure. References to molecular weight refer to number average molecular weight unless otherwise specified. In the following examples, efforts have been made to be accurate with respect to the numerical values (e.g., amounts, temperatures, etc.) used, but some experimental errors and deviations should be taken into account. Unless otherwise specified, temperatures are in degrees Celsius and pressures are at or near atmospheric pressure. References to molecular weight refer to number average molecular weight unless otherwise specified. References to molecular weight refer to number average molecular weight unless otherwise specified.

[0218] Examples 1-4 describe the conversion of mucic acid to polymerizable mucic acid monomers (MAM). Examples 5 and 6 describe the polymerization of MAM to form mucic acid polymers (MAP). Examples 7-15 describe the conversion of 7-ethyl-10-hydroxy-camptothecin (SN 38) into a form reactive with MAP by four different amino acid-based linkers. 38) into a form reactive with MAP by four different amino acid-based linkers. 38) into a form reactive with MAP by four different amino acid-based linkers. Describes the formation of a conjugated MAP-SN38 conjugate that is conductivized and covalently bonded . Examples 16 - 20 describe the reactions used to synthesize MAP-SN38 conjugates bonded 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 the route for preparative-scale synthesis of MAP. Examples 30 - 32 describe the derivatization of camptothecin (CPT) and its conjugation to MAP. Examples 33 - 35 describe the derivatization of SN38 for conjugation to MAP. Examples 36 and 37 describe the synthesis of nitrophenylboronic acid-polyethylene glycol (NPBA-PEG) polymers having reactive termini suitable for protein conjugates. Examples 38 - 40 describe the conjugation of targeting (homing) molecules to NPBA-PEG polymers and the characterization of the products. Examples 41 - 43 describe the conjugation of macromolecular therapeutics to NPBA-PEG polymers and the characterization of the products.

[0219] (Example 1. Synthesis of N-Boc protected mucic acid diamine (Compound 2)) A 1 L round-bottom flask containing 18 g (86 mmol) of mucic acid and a magnetic stir bar was charged with 400 mL of methanol (MeOH) and 1.4 mL of concentrated sulfuric acid. The mixture was heated under reflux (85 °C) with continuous stirring overnight (about 18 hours). After about 18 hours, 32 mL of triethylamine (TEA) was added to the flask and the mixture was stirred at reflux (85 °C) for 60 minutes. ​​​​​​​​​​​​​Next, 30 g (187 mmol) of N-Boc-ethylenediamine dissolved in 50 mL of MeOH was added to the reaction mixture. The reaction solution was refluxed (85 °C) overnight (about 18 hours) with stirring. The reaction mixture was cooled to room temperature, filtered, and washed with MeOH. Next, the collected solid was taken up in 450 mL of MeOH and refluxed for 1 hour with continuous stirring. The reaction mixture was cooled to room temperature, vacuum filtered, and washed with MeOH. The collected solid was dried under vacuum overnight to obtain 26.1 g (52.8 mmol, 62% yield) of N-Boc-protected mucic acid diamine (2).

Chemical formula

[0220] (Example 2. Synthesis of mucic acid diaminochloride (Compound 3)) 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, 466 mL of 3 M hydrochloric acid (HCl) in MeOH was slowly added with continuous stirring. After slowly adding the HCl, the reaction flask was transferred to an orbital shaker and shaken at room temperature overnight. The reaction slurry was filtered, and the solid was washed with MeOH three times (150 mL). The collected solid was dried under vacuum at 35 °C for overnight and for several hours to obtain 18 .0 g (49 mmol, 94% yield) of mucic acid diaminochloride (3).

Chemical formula

[0221] (Example 3. Synthesis of mucic acid di(Asp(OtBu)-Boc) (Compound 4)) 14.9 g (51 mmol) of Boc-L-aspartic acid 4-tert-butyl ester ​​Tellurium, 6 g (54 mmol) of hydroxypyridine N-oxide (HOPO), and a magnetic stirrer were placed in a 250 mL round-bottom flask, and 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. Next, a suspension of 6.3 g (17 mmol) of mucic acid diaminochloride (3) dissolved in a mixture of water (55 mL) and TEA (5.4 mL) was added to the reaction flask. The reaction mixture was heated (80 °C) with continuous magnetic stirring for about 24 hours . After heating, ACN was removed from the reaction mixture using a vacuum. The concentrated reaction solution was further added with water (150 mL), and the mixture was heated (85 °C) for 1 hour . After cooling, the reaction mixture was collected by vacuum filtration and washed three times (100 mL) with water. The washed solid was returned to the round-bottom flask together with ACN (150 mL). The reaction mixture was heated to reflux with continuous stirring until it dissolved . After cooling, the reaction mixture was collected by filtration and washed three times with cold ACN (100 mL). The collected solid was returned to the round-bottom flask again together with ACN (150 mL), and the reaction mixture was heated to reflux with continuous stirring for about 1 hour . After cooling, the reaction mixture was collected by filtration and washed three times with cold ACN (100 mL). The collected solid was dried under vacuum overnight , and 5.9 g (7 mmol, 41% yield) of mucic acid bis(Asp(OtBu)-Boc) (4) was obtained . (Example 4. Synthesis of mucic acid monomer (MAM) (Compound 5)) 1 g (1 mmol) of mucic acid bis(Asp(OtBu)-Boc) (4) and a magnetic stir bar were placed in a 100 mL round-bottom flask [Chemical formula]

[0222] were placed in a 100 mL round-bottom flask and a magnetic stir bar To a 40 mL vial containing [substance], dichloromethane (DCM, 7.5 mL) was added. While constantly stirring, trifluoroacetic acid (TFA, 7.5 mL, 98 mmol ) was slowly added to this reaction mixture. After stirring at room temperature for 1 hour, methyl tert-butyl ether ( MTBE, 100 mL) was slowly added to precipitate the reaction mixture. The precipitated mixture was centrifuged and subsequently washed once with MTBE (100 mL). The collected solid was dried under vacuum and then dissolved in water (30 mL). The dissolved product was passed through a 0.22 μm filter frozen, and freeze-dried to obtain 0.2767 g (0.384 mmol, 32% yield) of MAM (5). [Chemical formula]

[0223] (Example 5. Synthesis of mucic acid polymer (MAP) (Compound 6)) 224 mg (0.30 mmol) of MAM(5), 1 g of di(succinimidyl propyl onate)-PEG (diSPA-PEG 3.5k )(0.27 mmol) and a magnetic stir bar were placed in a 10 mL round-bottom flask, which was sealed and the two solids were dried under vacuum at room temperature for 4 hours. Next 4.1 mL of anhydrous dimethyl sulfoxide (DMSO) was added to this reaction flask, and the mixture was heated (35 °C). After solubilization, 208 μL of anhydrous N,N'-diisopropylethyl amine (DIPEA, 1.19 mmol) was added to the reaction mixture. The reaction mixture was continuously magnetically stirred and heated for 66 hours (35 °C). The reaction mixture was dialyzed against DMSO and then dialyzed against water using a 10 kDa membrane. Subsequently, the dialyzed product was passed through a 0.22 μm filter frozen, and freeze-dried to obtain 1.03 g (quantitative yield) of MAP(6). [Chemical formula]

[0224] For compound 6 synthesized by this method, x was approximately 80 and y was approximately 16. Thus, when the average molecular weight of compound 6 was determined as the arithmetic mean of the number average molecular weight and the weight average molecular weight, it was approximately 65 kDa. These values vary when different starting materials (e.g., diSPA- PEG ) are used (e.g., for 2 kDa PEG, x is approximately 46 2k ).

[0225] (Example 6. Measurement of the molecular weight of MAP) The molecular weight of MAP(6) was measured using a Malvern Omnisec gel permeation chromatography (GPC) system including a solvent supply pump, a degassing device, an automatic sampling injector, a column compartment, a refractive index detector, and a light scattering detector. Samples were prepared for analysis by dissolving MAP(6) with a known concentration in the range of 1 - 5 mg / mL in phosphate buffered saline (PBS ) + 0.02% sodium azide. After complete dissolution, the samples were passed through a 0.45 μm filter. 100 μL of the filtered sample was injected into a 2 ×PL Aquagel-OH 40 column (Agilent) at a flow rate of 0.7 mL / min to measure the absolute molecular weight. The analysis was performed at 30 °C. The obtained polymer peaks were analyzed using Malvern OMNISEC software.

[0226] (Example 7. Synthesis of 10-TBDPS-SN38 (Compound 7)) A 250 mL round-bottom flask containing 2 g (5.10 mmol) of SN38 and a magnetic stir bar ​​​​100 mL of anhydrous DCM and 4.2 mL (30.58 mmol) of TEA were added. Next, 7.9 mL (30.58 mmol) of tert-butyl(dichloro)diphenylsilane (TBDPSCl) was added to this reaction mixture. The mixture was heated under reflux (50 °C) overnight (about 16 h) with continuous stirring, and then washed with 0.2 N HCl (2 × 50 mL), saturated NaHCO 3 (100 mL) and brine (100 mL). The organic solution was dried over MgSO 4 and evaporated in vacuo. The residue was dissolved in anhydrous DCM and precipitated with hexane. This 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). [Chemical formula]

[0227] (Synthesis of Example 8.20-(Boc-Gly)-10-TBDPS-SN38 (Compound 8)) To a 40 mL vial containing 0.5 g (0.8 mmol) of 10-TBDPS-SN38 (7), 8.9 mL of anhydrous DCM and a magnetic stir bar were added. After addition of the solvent, the reaction solution was cooled to 0 °C . To another vial, 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 were added. Next, the contents of the second vial were transferred to the flask containing 7. After stirring at 0 °C for 3 h, the mixture was washed with 0.5% NaH CO CO pyridine (DMAP) and 3.8 mL of anhydrous DCM were added. Next, the contents of the second vial were transferred to the flask containing 7. After stirring at 0 °C for 3 h, the mixture was washed with 0.5% NaH CO CO 3 ​(2 × 50 mL), water (50 mL), 0.1 N HCl (2 × 50 mL), and brine were washed. The organic solution was dried over MgSO 4 and evaporated in vacuo to give 0.54 g of crude 20-(Boc-Gly)-10-TBDPS-SN38 (8) (0.7 mmol, 86 % yield).

Chemical Structure

[0228] (Example 9. Synthesis of 20-(Boc-GABA)-10-TBDPS-SN38 (Compound 9)) 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 addition of the solvent, the reaction solution was cooled to 0 °C. To another 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. Next, the contents of the second vial were transferred to the flask containing 7. The mixture was stirred at 0 °C for 3 hours and then washed with 0.5% NaHCO 3 (2 × 50 m L), water (50 mL), 0.1 N HCl (2 × 50 mL), and brine. The organic 3 solution was dried over MgSO 4 and evaporated in vacuo to give 1.05 g of crude 20-(Boc- GABA)-10-TBDPS-SN38 (9) (1.28 mmol, quantitative yield). 4

Chemical Structure

[0229] (Example 10. 20-(Boc-Hex)-10-TBDPS-SN38 (Compound 10)​​​​ Synthesis) To a 40 mL vial containing 0.6 g (0.95 mmol) of 10-TBDPS-SN38 (7), 10.4 mL of anhydrous DCM and a magnetic stir bar were added. After adding the solvent, the reaction solution was cooled to 0 °C. To another vial, 0.55 g of Boc-Hex-OH (2.4 mmol), 0.45 g (2.4 mmol) of EDC.HCl, 0.12 g (0.98 mmol) of DMAP and 4.5 mL of anhydrous DCM were added. Next, the contents of the second vial were 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 MgSO and evaporated in vacuo to give 0.88 g of crude 20-(B 3 (2 × 50 mL), water (50 mL), 0.1 N HCl (2 × 50 mL) and brine. The organic solution was dried over MgSO and evaporated in vacuo to give 0.88 g of crude 20-(Boc-Hex)-10-TBDPS-SN38 (10) (1.0 mmol, 111% yield 4 ) was obtained. (Example 11. Synthesis of 20-(Boc-Val)-10-TBDPS-SN38 (Compound 11))

Chemical formula

[0230] (Example 11. Synthesis of 20-(Boc-Val)-10-TBDPS-SN38 (Compound 11)) To a 100 mL round-bottom flask containing 1.0 g (1.6 mmol) of 10-TBDPS-SN38 (7), 17.7 mL of anhydrous DCM and a magnetic stir bar were added. After adding the solvent, the reaction solution was cooled to 0 °C. To another vial, 0.87 g of Boc-Val-OH (4.0 mmol), 0.77 g (4.0 mmol) of EDC.HCl, 0.20 g (1.6 mmol) of DMAP and 7.6 mL of anhydrous DCM were added. Next, the contents of the second vial were transferred to 7 containing the flask. of DMAP and 7.6 mL of anhydrous DCM were added. Next, the contents of the second vial were transferred to the flask containing 7. After stirring at 0 °C for 3 hours, the mixture was washed with 0.5% NaHCO It was transferred to a flask containing [0]. After stirring at 0 °C for 3 hours, the mixture was washed with 0.5% NaHCO 3 (2 ×50 mL), water (50 mL), 0.1 N HCl (2 × 50 mL) and brine. The organic solution was dried over MgSO 4 and evaporated in vacuo to give 1.24 g of crude 20-( (Boc-Val)-10-TBDPS-SN38(11) (1.5 mmol, 93% yield ).

Chemical formula

[0231] (Example 12. Synthesis of 20-(HCl.Gly)-SN38 (Compound 12)) 1.2 g (1.5 mmol) of 20-(Boc-Gly)-10-TBDPS-SN3 8(8) and a magnetic stir bar were placed in a 100 mL round-bottom flask, and 75 mL of 12 N HCl was slowly added while stirring continuously. Hexane (20 mL) was added on top of the HCl solution. After stirring at room temperature overnight (about 16 hours), the hexane layer was removed from the reaction mixture. Next, the reaction mixture was washed three times with hexane (3 × 20 mL). Then, using a vacuum, HCl was removed from the reaction mixture. The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried in vacuo to give 0.44 g (0.91 mmol, 60%) of 20-(HCl.Gly)-S N38(12).

Chemical formula

[0232] (Example 13. Synthesis of 20-(TFA.GABA)-SN38 (Compound 13)) 0.37 g (0.45 mmol) of 20-(Boc-GABA)-10-TBDPS- A 50 mL round-bottom flask containing SN38(9), 8.6 mL of water and a magnetic stir bar was continuously stirred while 8.6 mL of TFA was slowly added. Hexane ([[]] 20 mL) was added from above the TFA solution. After stirring at room temperature overnight (about 16 hours), the hexane layer was removed from the reaction solution . Next, the reaction solution was washed three times with hexane (3 × 20 mL). Then, the reaction mixture was concentrated using a vacuum . The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried under vacuum to obtain 0.16 g (0.27 mmol, 60%) of 20-(TFA.GABA )-SN38(13).

Chemical formula

[0233] (Example 14. Synthesis of 20-(TFA.Hex)-SN38 (Compound 14)) To a 50 mL round-bottom flask containing 0.37 g (0.43 mmol) of 20-(Boc-Hex)-10-TBDPS-S N38(10), 8.3 mL of water and a magnetic stir bar was continuously stirred while 8.3 mL of TFA was slowly added. Hexane ([[]] 20 mL) was added from above the TFA solution. After stirring at room temperature overnight (about 16 hours), the hexane layer was removed from the reaction solution . Next, the reaction solution was washed three times with hexane (3 × 20 mL). Then, the reaction mixture was concentrated using a vacuum . The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried under vacuum to obtain 0.16 g (0.26 mmol, 60%) of 20-(TFA.Hex)- SN38(14).

Chemical formula

[0234] (Synthesis of Example 15. 20-(HCl.Val)-SN38 (Compound 15)) 1.2 g (1.4 mmol) of 20-(Boc-Val)-10-TBDPS-SN3 8(11) and a magnetic stir bar were placed in a 100 mL round bottom flask, and 75 mL of 12 N HC l was slowly added while stirring continuously. Hexane (20 mL) was added from above the HCl solution. After stirring at room temperature overnight (about 16 hours), the hexane layer was removed from the reaction solution. Next, the reaction solution was washed three times with hexane (3×20 mL). Then, using a vacuum, HCl was removed from the reaction mixture. The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried under vacuum to obtain 0.46 g (0.86 mmol, 60% yield) of 20-(HCl.Val )-SN38 (15). [Chemical formula]

[0235] (Synthesis of Example 16. MAP-Gly-SN38 (Compound 16)) 100 mg of MAP (6) and a magnetic stir bar were placed in a 10 mL round bottom flask, and 8 mL of anhydrous DMSO was added. Next, to this reaction mixture, 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of N-hydroxysuccinimide (NHS) were added. Following 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. The mixture was stirred at room temperature overnight (about 18 hours). The reaction mixture was dialyzed against DMSO and then dialyzed against water at pH 4 using a 10 kDa membrane. The dialyzed product was passed through a 0.22 μm filter, frozen, and lyophilized. MAP-Gly-SN38 (16) with a quantitative yield of 98 mg was obtained.

Chemical Structure

[0236] As described in Example 5, for Compounds 6 and 16, x was approximately 80 and y was approximately 16 was.

[0237] (Example 17. Synthesis of MAP-GABA-SN38 (Compound 17)) 8 mL of anhydrous DMSO was added to a 10 mL round-bottom flask containing 100 mg of MAP (6) and a magnetic stir bar. Next, 51.9 mg (0.27 mmol) of EDC·HCl and 24.9 mg (0.22 mmol) of NHS were added to this reaction mixture. Following the addition of EDC·H Cl and NHS, 128.3 mg (0.22 mmol) of 2 0-(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 passed through a 0.22 μm filter, frozen, and freeze-dried to obtain 91 mg (quantitative yield) of MAP -GABA-SN38 (17). μm filter, frozen, and freeze-dried to obtain 91 mg (quantitative yield) of MAP -GABA-SN38 (17).

Chemical Structure

[0238] As described in Example 5, for Compounds 6 and 17, x was approximately 80 and y was approximately 16 was.

[0239] (Example 18. Synthesis of MAP-Hex-SN38 (Compound 18)) To a 10 mL round-bottom flask containing 100 mg of MAP(6) and a magnetic stir bar, 8 mL of anhydrous DMSO was added. Next, 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of NHS were added to this reaction mixture. Following the addition of EDC.H Cl 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 dialyzed against water at pH 4 using a 10 kDa membrane. The dialyzed product was passed through a 0.22 μm filter, frozen, and lyophilized to obtain 97 mg (quantitative yield) of MAP -Hex-SN38(18). As described in Example 5, for Compounds 6 and 18, x was approximately 80 and y was approximately 16 [Chemical formula]

[0240] was.

[0241] (Example 19. Synthesis of MAP-Val-SN38 (Compound 19)) To a 10 mL round-bottom flask containing 100 mg of MAP(6) and a magnetic stir bar, 8 mL of anhydrous DMSO was added. Next, 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of NHS were added to this reaction mixture. Following the addition of EDC.H Cl 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 ​It was analyzed and then dialyzed against water at pH 4 using a 10 kDa membrane. The dialyzed product was passed through a 0.22 μm filter, frozen, and lyophilized to obtain 96 mg (quantitative yield) of MAP -Val-SN38(19).

Chemical formula

[0242] As described in Example 5, for Compounds 6 and 19, x was approximately 80 and y was approximately 16 was.

[0243] (Example 20. Characterization of Drug Loading) The loading of SN38 onto MAP was measured using an Agilent 1100 HPLC system connected to a fluorescence detector with a reverse-phase column (Synergi 4 μm Hydro-R P80Å, Phenomenex) set at 375 / 536 nm (ex / em). ACN / 10 mM potassium phosphate buffer, pH 4 (1:1 v / v) was used as the eluent at a flow rate of 0.5 mL / min.

[0244] For analysis, the polymer-drug conjugates (Compounds 16 - 19) were dissolved in PBS at pH 7.4 at a concentration of 1 mg / mL. First, 10 μL of the sample was mixed with 10 μL of 0.1 N HCl and incubated at room temperature for 30 min to measure the amount of unconjugated SN38 . 6.7 μL of water was added, followed by 73.3 μL of ACN, and the mixture was incubated at room temperature for 3 h . This mixture was centrifuged at 14,000 g for 10 min at 4 °C, and the supernatant was passed through a 0 .45 μm filter. 10 μL of the filtered sample was injected to measure the SN38 concentration . The peak area of the eluted SN38 obtained was compared with the peak area of SN38 at a known concentration .

[0245] To measure the total amount of SN38, 10 μL of the sample was mixed with 6.7 μL of 0.1 N Na OH. This solution was incubated at room temperature for 3.5 hours to release SN38 from the parent polymer . Then, 10 μL of 0.1 N HCl was added (to convert carboxylate SN3 8 form to the lactone form), and this mixture was incubated for 45 minutes. Subsequently , 73.3 μL of ACN was added, and the mixture was incubated at room temperature for 3 hours. Then, the sample was centrifuged and treated as described above. Polymer-bound SN38 was determined from the difference between the total SN38 concentration and the unconjugated SN38 concentration .

[0246] (Example 21. Formation of MAP - linker - SN38 nanoparticles) 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. This solution was passed through a 0.22 μm filter and then frozen for subsequent analysis. The nanoparticles were diluted to 2 mg / mL with water at pH 4 , and the hydrodynamic diameter was measured by dynamic light scattering (DLS) using a ZetaPALS (Brookhaven Instruments Corp oration) apparatus. Five runs were performed for each nanoparticle for 1 minute each. The diameters of all the nanoparticles were approximately 2 5 nm, with a width ranging from 19 - 32 nm .

[0247] (Example 22. In Vitro release test) Experiments were conducted 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 - SN3 8 and MAP - Leu - SN38 nanoparticles was carried out. For the synthesis of MAP-Ala-SN38, MAP-β-Ala-SN38 and MAP-Leu -SN38 nanoparticles, refer to Examples 33-35 below. These studies were performed at 0.1 mg of SN38 / mL in PBS at pH values of 6.5, 7.0 or 7.4.

[0248] PBS medium was pipetted into cuvettes and incubated in a humidified oven at 37 °C for 2 hours to equilibrate. The nanoparticle formulation was mixed into the relevant medium and returned to the oven. Samples were removed at predetermined time points and snap-frozen at -80 °C until the time of analysis. The amount of unconjugated SN38 and the total amount of SN38 were measured as described above in Example 20. The polymer-bound SN38 concentration was determined from the difference between the total SN38 concentration and the unconjugated SN38 concentration.

[0249] As a result, the release of SN38 from all seven types of nanoparticles: MAP-Gly-SN38, MAP-GABA- SN38, MAP-Hex-SN38, MAP-Val-SN38, MAP-Ala-S N38, MAP-β-Ala-SN38 and MAP-Leu-SN38 showed first-order kinetics. It was observed that the release rate was strongly dependent on pH. When the pH increased from 6.5 to 7.4, the release half-life decreased for all nanoparticle formulations, suggesting that hydrolysis played an important role in the release of SN38. The higher the hydrophobicity and steric hindrance of the linker, the longer the half-life of the nanoparticles, with a range of about 30 hours to over 168 hours.

[0250] (Example 23. Determination of the number of strands per particle) The number of strands per nanoparticle (SpP) is the ratio of the molecular weight of the particle to the molecular weight of MAP​​​​ It was determined by calculation. Polyethylene oxide (PEO) standards with nominal molecular weights of 100 kDa and 200 kDa and the target samples were dissolved in water at pH 3 or PBS + 0.02% sodium azide at a concentration of 1 - 5 mg / mL and prepared for GPC analysis (Omnisec GPC, Malvern). The concentration was selected to be close to the concentration expected after particle formation. After dissolution, the samples were passed through a 0.45 μm filter and analyzed at 0.35 mL / min and 30 °C using a Zenix SEC300 or Zenix - C SEC300 column (Sepax, 3 μm). Information on light scattering and differential refractive index was used with Malvern OMNISEC C software or similar molecular weight analysis software to determine the molecular weight of the particles. The molecular weight of the particles was divided by the molecular weight of the MAP - SN38 conjugate molecules to determine the number of strands per particle. The molecular weight of the MAP - SN38 conjugate molecules was calculated from the molecular weight of the polymer adjusted by the drug loading rate according to the following formula. MW = MW

[0251] / (1 - T ) (where MW is the molecular weight of the MAP, and T コンジュゲート is the drug loading rate expressed as a decimal. For example, for a 10% load, T ポリマー = 0.1.)) % ) (In the formula, MW ポリマー is the molecular weight of the MAP, and T % is the drug loading rate of the therapeutic agent expressed as a decimal. For example, for a 10% load, T = 0.1.)) % = 0.1.))

[0252] The measurement of SpP was demonstrated with MAPs having various linkers and drugs. MAPs of the same molecular weight conjugated with CPT or SN3 8 produced nanoparticles with approximately 1.5 - 2.5 strands per particle. Using different linkers for derivatized SN38 results in , the number of strands per particle changes, and as a result, the density of the particles changes. The number of strands per particle varied from about 1.3 to about 4.6 by a linker (glycine, γ-aminobutyric acid, hexanoic acid, alanine, β- alanine, leucine or valine).

[0253] (Example 24. Preparative-scale synthesis of N-Boc protected mucic acid diamine (Compound 2)) To a 15 L reaction vessel mechanically stirred under nitrogen, 450 g (2.1 mol) of mucic acid and MeOH (5.75 L) were added. To this reaction vessel, a solution consisting of MeOH (1 L) and concentrated sulfuric acid (34.2 mL, 0.64 mol) was added. The mixture was heated to an internal temperature of 65 °C for 48 h. The contents of the reaction vessel were cooled to 22 °C, at which point 0.81 L (5.78 mol) of TEA was added over 20 min. The reaction solution was stirred for 1 h, at which point a solution of 0.76 kg (4.71 mol) of N-(2-aminoethyl)( (tert-butoxy)carboxamide in 0.9 L of MeOH was added over 20 min. The reaction solution was heated to an internal temperature of 63 °C, and after 60 min, 1.8 L of MeOH was added to facilitate stirring. After a total of 2 h at 63 °C, the reaction vessel was cooled at 1 °C per minute and held at 20 °C overnight. The reaction slurry was filtered, and the cake was washed with MeOH (3 × 0.5 L). The solid was dried overnight on a vacuum filter to completely remove the liquid. The isolated solid was returned to the reaction vessel with MeOH (9.0 L) and stirred at 63 °C for 1 h. The reaction vessel was set to cool at a rate of 1 °C per minute and held at 20 °C for about 60 h. The slurry was filtered, washed with MeOH (2 × 1.0 L), and the solid was dried overnight in a vacuum oven at 45 °C to give 963 g of N-Boc protected mucic acid diamine (2) ( ( ( ( ( ( ( ( The solvate with MeOH was obtained in a yield of 91%, and the anhydride was obtained in a yield of 82%).

Chem.

[0254] (Example 25. Preparative-scale synthesis of mucic acid diaminochloride (Compound 3)) To a mechanically stirred 15 L reaction vessel, 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 this slurry while maintaining the temperature in the range of 20 °C to 30 °C, 12 M HCl (3. 6 L) was added over 2 hours. The reaction solution was stirred overnight, and at that time the slurry was filtered and washed with 1.8 L of MeOH. The isolated solid was placed in a tray and dried in a vacuum oven at 45 °C The isolated solid was charged into a 15 L reaction vessel together with water (5.5 L) under nitrogen and stirred at 45 °C When 5.5 L of MeOH was added over 30 minutes, crystallization occurred as a result. The reaction vessel was cooled and the reaction contents were cooled to room temperature. The solid was isolated 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 obtain 504 g( 1.37 mol, 76% yield) of mucic acid diaminochloride (3).

Chem.

[0255] (Example 26. Preparative-scale synthesis of mucic acid di(Asp(OtBu)-Boc) (Compound 4)) Synthesis) To a mechanically stirred 15 L reactor, 1.27 kg of Boc-L-aspartic acid 4- tert-butyl ester (4.39 mol), 0.62 kg of oxime (cyanohydrin Ethyl xylyliminoacetate (4.39 mol) and 3.5 L of DCM were added. The reaction mixture was cooled to 5 °C and stirred for 30 minutes. To the reaction mixture, 0.84 kg of EDC .HCl (4.40 mol) was added over 10 minutes. The EDC.HCl was rinsed into the reaction solution with an additional 0.5 L of DCM, and the reaction solution was stirred for about 30 minutes and then cooled to 10 °C. In another 12 L flask, 1.02 kg of sodium carbonate (9.61 mol) and 5.0 L of water were placed. After dissolving the components, it was cooled to room temperature. Over 10 minutes, 504 g (1. 37 mol) of 3 was added to the sodium carbonate solution. This mixture was stirred for 15 minutes, and at this point, it was added to the Boc-L-aspartic acid 4-tert-butyl ester in a 15 L reaction flask over about 3 minutes. The mixed reaction was exothermic, but the cooling jacket was set at 10 °C for 30 minutes , and then at 15 °C for an additional 45 minutes. Then, the reaction jacket was set to 33 °C, and after 1 hour, 8.06 L of water was added and stirred for 5 minutes. Then, stirring was stopped, and after 15 minutes, the reaction phase was separated. The DCM (lower layer) was removed, and 1.0 L of DCM was added to the reactor. This mixture was stirred for 10 minutes and then allowed to settle. The DCM layer was recovered and combined with the initially recovered organic layer , and concentrated to a total volume of about 3 L. 1 L of water was added to the organic mixture to promote further distillation removal of DCM. Next, the organic reaction mixture was added to a 15 L reactor containing 11.1 L of water heated to 40 °C. The stirred mixture was returned to 20 °C to form a slurry. The water was removed, leaving the solid in the reactor. 10.1 L of water was added to the wet solid. The reaction mixture was heated to 37 °C to promote stirring. Then, the reaction solution was cooled to 3 °C overnight and stirred. The solid was filtered, and then returned to the reactor again with an additional 10 L of water. This slurry was at room temperature and stirred. The solid was filtered and then returned to the reactor again with an additional 10 L of water. This slurry was stirred at room temperature It was stirred for 3 hours. The solid was filtered again, washed with 1.0 L of water, and dried to obtain 815 g of the material. 790 g of crude 4 was added to a 5 L round-bottom flask together with 2.4 L of ACN and heated to 60 °C. By azeotropic distillation under partial vacuum and continuous addition of ACN, water was continuously distilled off to a volume of about 1.6 L. 3.1 L of ACN was added thereto to induce crystallization. After the slurry was heated to reflux for a short time, it was cooled to 5 °C and left as it was for 30 minutes. After cooling, the slurry was filtered and washed with ACN (4 × 0.5 L, 5 °C). The solid was heated under vacuum at 50 °C to remove the solvent. 6.3 L of DCM was added to the reaction mixture in two portions, and at that time the slurry was concentrated to distill off ACN. Next, the solid was heated under vacuum at 80 °C to remove DCM and a free-flowing powder was obtained. Next, the solid was placed in a vacuum oven and dried at 80 °C under reduced pressure to obtain 310 g (0.35 mol, 26%) of mucic acid di(Asp(OtBu)-Boc)(4) as a 1:1 molar solvate with ACN.

Chemical formula

[0256] (Example 27. Preparative-scale synthesis of mucic acid monomer neutral species (Compound 20)) To a 5 L round-bottom flask equipped with a mechanical stirrer and a jacket, 300 g of 4 (0.34 mol), 1.2 L of DCM, 0.11 L of water (6.13 mol), and 0.279 L of triisopropylsilane (1.36 mol) were added. The reaction mixture was cooled to 5 °C, and then a slurry was formed. 1.2 L of TFA, which had been cooled in an ice bath for 1 hour in advance, was added to this stirred slurry. This reaction was maintained at an internal temperature of 4 °C overnight. Furthermore, 0.30 TFA of L was added and stirring was continued at 4 °C overnight. When 0.90 L of hexane was added, the reaction solution was stirred at 5 °C for 10 minutes. Next, the reaction mixture was poured into a separatory funnel and the lower phase was separated into a 5 L round bottom funnel. The reaction materials were washed with additional DCM (300 mL) and this was combined with the initially collected fraction. The combined recovered fractions were concentrated at < 30 °C using a rotary evaporator . An additional 0.90 L of DCM was added to the flask and then removed at 25 °C using a rotary evaporator . Next, 0.60 L of toluene was added and concentrated using a rotary evaporator to azeotropically remove water and TFA, yielding an oily substance. This oily substance was passed through a plug of celite and filtered, and washed 4 times with DCM (0.40 L). The DCM was removed at 35 °C using a rotary evaporator . The resulting two-phase mixture was added to a clean 5 L reaction vessel and heated to 23 °C . When 3.0 L of cyclopentyl methyl ether (CPME) was added over 20 minutes, an oily solid formed. The vessel was cooled to 22 °C and stirred overnight to obtain sticky crystals. Using a short path distillation column, 1.5 L of DCM was distilled off at 35 °C. The resulting slurry was filtered and washed with CPME (2 × 0.45 L). Next, the solid was dried in a round bottom flask using a rotary evaporator and vacuum dried at room temperature overnight to obtain 261 g of mucic acid monomer neutral species (208.8 g, 82% yield considering the titer).

[0257] For further recrystallization, 0.24 kg of mucic acid monomer neutral species was added to 1 L of a round bottom flask together with water (0.20 L). This mixture was stirred at room temperature for 1 hour to obtain a solution. This solution was filtered through a filter funnel using additional water (50 mL) and transferred to a 5 L round bottom flask . Transferred to. While stirring 2.2 L of tetrahydrofuran (THF), the flask was added over 30 minutes. Added to the flask. The mixture was stirred for a total of 60 minutes, and at that point, it was concentrated at 35 °C using a rotary evaporator to remove 1 L of distillate. Over 10 minutes, additional THF (0.98 L ) was added. 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), and 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 mucic acid monomer (20). . [Chemical formula]

[0258] (Example 28. Preparative-scale synthesis of MAM (Compound 5)) A 3 L round-bottom flask containing 120 g (0.23 mol) of 20 and DCM (1.2 L) was stirred for 10 minutes, and at that point, water (0.06 L) was added. Stirring was continued for 15 minutes, and at that time, TFA (66.0 mL, 0.86 mol) was added over 5 minutes. The solid was dissolved, and the mixture was stirred at room temperature for 1 hour. This mixture was concentrated at 30 °C using a rotary evaporator . Ether (4.0 L) and MAM seed crystals (5, 12 g) were added to a 5 L round-bottom flask equipped with mechanical stirring. While stirring the 20 / TFA / aqueous solution, it was slowly added to the ether . After 1 hour, the ether was decanted off and fresh additional ether was added ( 3.6 L). 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 dried under vacuum at room temperature and heated at 30 °C for 1 hour to obtain 122 g (75% yield) of MAM (5). . [Chemical formula]

[0259] (Example 29. Preparative-scale synthesis of mucic acid polymer (Compound 6)) MAP(6) was synthesized by sequential polymerization of mucic acid monomer (MAM)(5) and diSPA-PEG 3.5k with 108.52 mg (0.158 mmol) of MAM(5) containing approximately 1.5 equivalents of TFA

[0260] 、579.54 mg of diSPA-PEG 3.5k (0.157 mmol) and a magnetic stir bar were placed in a sealed 20 mL glass vial 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 reaction mixture was solubilized at room temperature. After solubilization, 126 μL of anhydrous DIPEA (0.7 2 mmol) was added to the reaction mixture under argon. The reaction mixture was stirred for 3 hours with continuous magnetic stirring at room temperature

[0261] The reaction mixture was precipitated in cold isopropyl alcohol (IPA, ×10 volumes, 0 - 4 °C) The reactor was washed with DMSO (3 × 1 mL) and then precipitated in cold IPA. The resulting white suspension was stirred at 0 - 4 °C for 30 minutes and centrifuged at 4 °C for 12 minutes. The white flocculent polymer precipitated at the bottom was isolated by decanting IPA. This polymer was washed twice with cold IPA (×5 volumes) and this procedure was repeated. The product was vacuum dried overnight at room temperature on a Schlenk line to remove IPA and then dissolved in MilliQ water (8 mL). The solution in water was frozen and lyophilized to obtain 612.5 mg (quantitative yield) of MAP(6) 。 。 。

[0262] The molecular weight of the polymer was controlled by incorporating a stoichiometric strain between two monomers. At a 1:1 ratio, the molecular weight of MAP(6) was maximized, and the molecular weight decreased as the molar ratio deviated from 1:1. Exemplary stoichiometric strains and experimental molecular weights obtained by GPC are shown in Table 1. Shown in Table 1. [Table 1]

[0263] (Synthesis of Example 30.20-(Boc-Gly)-CPT (Compound 21)) 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 stir bar and a rubber septum. The vessel and its contents were purged with argon for 10 minutes, and at that point anhydrous DCM (1 mL) was added to make the material into a slurry. 280 μL (1.81 mmol) of DIC was added dropwise to the reaction mixture over about 3 minutes via syringe. The reaction solution was stirred at room temperature for 3 hours, and at that point about 50% of the solvent was removed in vacuo. 4 mL of cold MeOH was added to the concentrated slurry. The reaction solution was stirred for several minutes to promote further precipitation, and then filtered through a Buchner funnel. The solid was further washed with cold MeOH and then with cold MTBE. The isolated solid was dried under high vacuum to obtain 110.8 mg (0 .22 mmol, 38%) of 20-(Boc-Gly)-CPT (21).

[0264] In the above procedure, Boc-Gly-OH can be replaced with other Boc-protected amino acids and dipeptides. Boc-Ala-OH, Boc-β-Ala-OH, Boc Replace -Val-OH, Boc-GABA-OH and Boc-Phe-Gly-OH By doing so, 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) were obtained respectively by the same procedure. [Chemical formula]

[0265] (Example 31. Synthesis of 20-(TFA.Gly)-CPT (Compound 22)) To a scintillation vial equipped with a magnetic stir bar and a vent needle, 97.1 mg (0.20 mmol) of 20-(Boc-Gly)-CPT (21) was added. DCM (0.2 m L) was added to generate a stirred suspension. While stirring, TFA (0.2 mL) was slowly added. 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 obtain 82.5 mg (0.16 mmol, 83% yield) of 20-(TFA.Gly)-CPT (22). [Chemical formula]

[0266] Using the above method, when 20-(Boc-Gly)-CPT was replaced with 20-(Boc-Ala)-CP T, 20-(Boc-β-Ala)-CPT, 20-(Boc-Val)-CPT, 20 -(Boc-GABA)-CPT and 20-(Boc-Phe-Gly)-CPT, 20-(TFA.Ala)-CPT (Compound 45), 20-(TFA.β-A ​ la)-CPT (Compound 46), 20-(TFA.Val)-CPT (Compound 47), 2 0-(TFA.GABA)-CPT (Compound 48) and 20-(TFA.Phe-Gly )-CPT (Compound 49) are obtained respectively.

[0267] (Example 32. Synthesis of MAP-Gly-CPT (Compound 23)) 149.2 mg (CO 2 H base, 0.07 mmol) of MAP (6) and 21.5 m g of 20-(TFA.Gly)-CPT (22) (0.04 mmol) were added to a 20 mL scintillation vial containing a magnetic stir bar. The headspace of the container was purged with argon atmosphere 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 with stirring. 166.3 mg of (7-azabenzotriazol-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate (PyAOP, 0.32 mmol, 4.4 equivalents) and a magnetic stir bar were added to an 8 mL scintillation vial and purged with argon atmosphere. The contents of this vessel were dissolved in anhydrous DMSO (about 3.2 mL) with stirring. When all the solids in both vessels had dissolved, 1.6 mL of the PyAOP solution (83.2 mg of PyAOP, 0.1 6 mmol of PyAOP, about 15% of the total reaction volume) was transferred to the polymer-containing vessel. This reaction solution was stirred for about 2 minutes, and at that time about 45 μL (0.26 mmol) of anhydrous DIPE A (target 31.3 μL, 0.18 mmol) was added. The reaction vessel was stirred in the dark at room temperature for about 1 8 hours. After 18 hours, the reaction vessel was removed from the argon atmosphere, and about One-third was transferred to a 50 mL glass centrifuge tube. Cold ethyl acetate (EtOAc) was slowly added to precipitate the reactants (0 - 4 °C). The suspension was stirred at 0 - 4 °C for about 30 minutes and then centrifuged. The EtOAc layer was decanted off, and the next one-third of the reaction medium was added to the same vessel. This procedure was repeated until all of the reaction medium had precipitated into the same tube. The precipitated solid was washed three times in total with cold EtOAc (×5 volumes) while stirring with a vortex, resuspending the solid. After the final wash cycle, the last EtOAc was decanted off and the solid was frozen in the centrifuge tube using liquid nitrogen. The frozen centrifuge tube was exposed to high vacuum for about 3 hours to dry the solid, yielding 115.2 mg (quantitative yield) of MAP-Gly-CPT T(23).

Chemical Structure

[0268] Also, by the same method, instead of 20-(TFA.Gly)-CPT, 20-(TF A.Ala)-CPT, 20-(TFA.β-Ala)-CPT, 20-(TFA.Va l)-CPT, 20-(TFA.GABA)-CPT or 20-(TFA.Phe-Gl y)-CPT was used as the starting material to generate MAP-CPT conjugates, and 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) were generated.

Chemical Structure

Chemical Structure

Chem.

[0269] As described in Example 5, for Compounds 6, 23, and 50 - 54, x was approximately 80 , and y was approximately 16.

[0270] (Synthesis of Example 33. 10 - OBoc - SN38 (Compound 24)) Under an argon atmosphere, 2.0 g (5.10 mmol) of SN38 was placed in a 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 solution was stirred at room temperature for about 16 hours. At that point, the reaction solution was filtered through Celite 545 (filter aid) and washed with 0.5 N HCl and saturated aqueous sodium bicarbonate. The organic phase was dried over magnesium sulfate and concentrated in vacuo. The reaction mixture was diluted with DCM and then precipitated in hexane . The solid was filtered and dried in vacuo to obtain 2.2 g (4.47 mmol, 88% yield) of 10 - OBoc - SN38 (24).

Chem.

[0271] (Synthesis of Example 34. 20 - (Boc - Gly) - 10 - OBoc - SN38 (Compound 25)) Under an argon atmosphere, 502 mg (1.02 mmol) of 1 0 - OBoc - SN38 (24) was placed in a reaction vessel cooled to 0 °C, and 8.8 mL of DCM was added. To this reaction vessel , 3.8 mL of DCM, 447 mg (2.55 mmol) of Boc - Gly - OH, 48 9 mg (2.55 mmol) of EDC·HCl and 125 mg (1.02 mmol) of D A cooling solution containing MAP was added. The reaction solution was stirred at 0 °C for 3 hours. The reaction solution was washed with an aqueous solution of 0.5% sodium bicarbonate, 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 2 0-(Boc-Gly)-10-OBoc-SN38 (25).

[0272] In the above procedure, Boc-Gly-OH can be replaced with other Boc-protected amino acids. By replacing Boc-Ala-OH, Boc-β-Ala-OH, Boc-Val-OH and Boc-Leu-OH as starting materials, 20- (Boc-Ala)-10-OBoc-SN38 (Compound 29), 20-(Boc-β- Ala)-10-OBoc-SN38 (Compound 30), 20-(Boc-Val)-OB oc-SN38 (Compound 31), and 20-(Boc-Leu)-10-OBoc-SN3 8 (Compound 35) were obtained in a similar procedure, respectively. [Chemical formula]

[0273] (Example 35. Synthesis of 20-(TFA·Gly)-SN38 (Compound 26)) To a reaction vessel containing 250 mg (0.38 mmol) of 20-(Boc-Gly)-10-OBoc-SN 38 (25) and 1.88 mL of DCM, 0.47 mL (6.14 m mol) of TFA was added. The reaction solution was stirred at room temperature for 2.5 hours. The reaction mixture was precipitated in cold (4 °C ) MTBE, isolated by centrifugation, washed again with MTBE, and dried in vacuo ​​133 mg (0.24 mmol, 61% yield) of 20-(TFA.Gly)-SN38 (26) was obtained.

Chemical formula

[0274] Using 20-(Boc-Gly)-10-OBoc-SN38 as the starting material, 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 were replaced to obtain 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), respectively.

[0275] Using the TFA salts of the aforementioned amino acid-linked SN38 derivatives, MAP-SN38 conjugates can be formed as described in Examples 17 and 18. (Example 36. Synthesis of NPBA-PEG

[0276] -AA (Compound 27)) 5k 20 g (4 mmol) of NH -PEG 2 -acetic acid (AA) hydrochloride and DIPEA (2. 5k 1 mL, 12 mmol) were placed in a 250 mL round-bottom flask, and anhydrous DCM (40 mL) was added and flushed with argon. This solution was cooled in an ice / water bath. A magnetic stir bar was placed in a 20 mL vial, and 934 mg (5.5 mmol) of 3-carboxy-5-nitrophen nylboronic acid (NPBA) was added in anhydrous DCM / dimethylformamide (DMF) (8 mL, 4 :1 v / v) was dissolved. 1038 mg (5.25 mmol) of N-ethoxycarbonyl -2-ethoxy-1,2-dihydroquinoline (EEDQ) was added to the solution of NPBA . The activated acid was stirred for 15 minutes and then added dropwise to the 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 . Then, sodium dihydrogen phosphate (NaH 2 PO 4 , 2.5 g) was added to adjust the pH to 7 . 1 M HCl (12 mL) was added to further lower the pH of the reaction mixture to 4. The reaction mixture was transferred to a separatory funnel, and isopropyl acetate (iPrOAc, 100 mL) was added. The mixture was shaken well, allowed to precipitate, and a separated phase was obtained. The organic phase was discarded, and extraction with iPrOAc (3 × 100 mL ) was repeated. The aqueous phase was transferred to a 250 mL round-bottom flask and concentrated at room temperature and 20 Torr for 30 minutes using a rotary evaporator . The aqueous solution was lyophilized to obtain a powder. The lyophilized powder was dissolved in DMSO / water (40 mL, 3:1 v / v). This solution was injected into a pre-equilibrated C18Aq Teledyne ISCO column (475 g) with 12 - 15 mL of 95% mobile phase A (water + 0.05% TFA) and 5% mobile phase B (ACN + 0.05% TFA) and purified by hydrophobic interaction chromatography. A gradient from 5% B to 40% B was run, followed by washing the column and subsequent injections. The purification was repeated for the remaining product solution. The fractions containing the product were analyzed by HPLC. Fractions with a purity exceeding 99.5 % were combined, lyophilized, and 14.2 g (2.71 mmol, 68% yield) of NPBA-PEG was obtained . NPBA-PEG5k -AA(27) was obtained.

Chemical formula

[0277] For compound 27 synthesized by this method, n was approximately 114. These values vary when different starting materials (e.g., NH 2 -PEG 3.5k -AA) are used (e.g., for 3.5 kDa PEG, n is approximately 80).

[0278] (Example 37. Synthesis of NPBA-PEG 5k -AA-PFP (Compound 28)) To an 8 mL reaction vessel, 301.8 mg (0.06 mmol) of NPBA-PEG 5k - AA(27) was added together with a magnetic stir bar. The reaction vessel was purged in a triple vacuum and argon cycle. The solid was dissolved in anhydrous DCM (1.2 mL), and then 35 μL of anhydrous N-methylmorpholine (0.35 mmol) was added via syringe. The reaction vessel was briefly uncapped, and 45.3 mg of bis(pentafluorophenyl) carbonate (0.12 mmol) was added. The reaction headspace was purged with argon, and the reaction solution was stirred at room temperature for approximately 2 hours. The reaction solution was added dropwise to a stirred volume of MTBE (25 mL) to promote precipitation. The mixture was stirred under an argon atmosphere for 30 minutes and then centrifuged at 2600 g. The liquid layer was decanted, and at that point, heptane (20 mL) was added to the centrifuge tube. The sample was mechanically stirred and then centrifuged a second time at 2600 g (for 8 minutes). The The solvent was removed by evaporation, and 300 mg (0.055 mmol, 96% yield) of NPBA-PE G 5k -AA-PFP (28) was obtained.

Chemical formula

[0279] As described in Example 36, for Compounds 27 and 28, n was approximately 114.

[0280] (Example 38. Synthesis of NPBA-PEG 5k -Tf crude mixture) 35 g (0.440 mmol) of holotransferrin (Tf) was placed in 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 solubilization, 4.77 g (0.881 mmol) of NPBA-PEG -AA-PFP (28) in 44 mL of DMSO was slowly added to the reaction mixture and mixed well. 5k The reaction mixture was incubated at room temperature for 2 hours. The crude reaction mixture was analyzed on an Agilent 1290 HPLC system equipped with a reversed-phase (RP) column (Zorbax 300SB-CN 4.6 mm ID x 150 cm) 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 column was connected to an absorbance detector set at a wavelength of 280 nm and a reference wavelength of 360 nm. Furthermore, 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) and mobile phase B (ACN / IPA (4:1 v / v) + 0.1% TFA) at a flow rate of 0.7 mL / min. The column was connected to an absorbance detector set at a wavelength of 280 nm and a reference wavelength of 360 nm. Agilent 1290 HPLC system 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 at a wavelength signal of 280 nm and a reference of 360 nm. After incubating for 2 hours, the crude reaction mixture was ready for HIC purification.

[0281] (Example 39. Isolation of Mono-NPBA-PEG 5k -Tf) NPBA-PEG 5k -Tf crude mixture (20 g / L), 1.75 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 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) packed with GE Sepharose Phenyl High Performance HIC resin at a pump flow rate of 200 mL / min. A residence time of approximately 10 minutes was provided for loading the sample mixture onto the HIC column. IPA / 1 0 mM sodium phosphate, pH 7.4 (1:4 v / v, mobile phase B) was used as the eluent. Before the elution step, the column was washed with 2 column volumes (CV) of 20% mobile phase B to remove unbound species. The purified NPBA-PEG -Tf was eluted with a gradient of 20 - 100% mobile phase B over 15 CV. The fraction of mono-PEGylated Tf (mono-NPBA-PEG 5k -Tf) was collected and adjusted for ultrafiltration and diafiltration (UF / DF). The Sartoco n Slice Hydrosart Cassette 30 kDa membrane was used for mono-NPB 5k -T f) and adjusted for ultrafiltration and diafiltration (UF / DF). The Sartoco n Slice Hydrosart Cassette 30 kDa membrane was used for mono-NPB A-PEG 5k was used for UF / DF of -Tf. Mono-NPBA-PEG 5k -Tf conjugate was concentrated to 50 g / L by diafiltration using 7 volumes of PBS (pH 7.4). Then, mono NPBA-PEG -Tf 5k was sterile filtered through a 0.22 μm polyethersulfone (PES) membrane.

[0282] (Example 40. Determination of the number of NPBA per Tf) The number of NPBA per Tf of mono NPBA-PEG 5k5k -Tf was determined by calculating the ratio of the molar concentration of NPBA-PEG -Tf to the molar concentration of Tf using an alizarin red S (ARS) detection assay. To create a standard curve 5k samples of NPBA-PEG 5k -AA(27) were prepared at various concentrations (0 - 0.0098 mM) dissolved in PBS ( pH 7.2). ARS was prepared by dissolving it in water at 0.04 9 mM. Next, the samples were mixed with ARS, and fluorescence was measured using a Varioskan 5k LUX spectrophotometer (ThermoFisher) and SkanIt (Thermo F isher) or similar fluorescence analysis software (excitation wavelength: 46 8 nm, emission wavelength: 572 nm). By linear regression of these data (fluorescence vs. concentration), a calibration curve for determining NPBA in the target samples was obtained. The target samples (e.g., Tf and mono-NPBA-PEG -Tf) were prepared by dissolving them in PBS ( pH 7.2) at 0.007 mM. Mono-NPBA-PEG

[0283] -Tf 5k 5k -Tf ​​of NPBA-PEG 5k The molar concentration of mono-NPBA-PEG 5k -Tf sample was calculated using the following equation from the calibration curve of the fluorescence values of various known concentrations of NPBA-PEG -AA standards (0 to 0.0098 mM 5k ). Fluorescence = ε * C + b N (where ε = slope of the linear regression of the calibration curve, b = intercept of the linear regression of the calibration curve, and C = concentration of NBPA- N PEG (mM). Thus, C (mM) = (Fluorescence - b) / ε.) N (mM) = (Fluorescence - b) / ε.)

[0284] Next, C was divided by the known Tf concentration according to the following equation to calculate the number of NPBA per Tf ( N N ). Tf N =(NPBA-PEG Tf mmol / L) / (Tf mmol / L) 5k

[0285] For the NPBA of mono-NPBA-PEG 5k -Tf, the determination number of NPBA using the ARS detection assay for NPBA was approximately 1 NPBA per Tf for mono-NPBA-PEG . The reaction with ARS, and thus the fluorescence signal, requires NPBA functional groups available for the reaction. Some molecules (e.g., citrate) form complexes with NPBA and reduce the fluorescence signal, 5k changing the measured number of NPBA per mono-NPBA-PEG -Tf. In other cases, excess unreacted NPBA-PEG conjugate may be present in the crude sample, increasing the fluorescence signal and changing the determination number of NPBA per mono-NPBA-PEG -Tf. Exemplary experimental results are shown in Table 2. 5k changing the measured number of NPBA per mono-NPBA-PEG -Tf. In other cases, excess unreacted NPBA-PEG conjugate may be present in the crude sample, increasing the fluorescence signal and changing the determination number of NPBA per mono-NPBA-PEG 5k -Tf. Exemplary experimental results are shown in Table 2. Exemplary experimental results are shown in Table 2.​

Table 2

[0286] (Example 41. Synthesis of NPBA-PEG 5k -Tras crude mixture) 5.5 g (0.038 mmol) of trastuzumab was buffer-exchanged with 50 mM HEPES (pH 7. 8) to adjust the final protein concentration to 10 g / L. After solubilization, NPBA-PEG -AA-PFP (28) 409.07 mg (0 5k .076 mmol) in 3.78 mL of DMSO was slowly added to the reaction mixture and mixed well. The reaction mixture was incubated at room temperature for 2 hours. The crude reaction mixture was analyzed on an Agilent 129 0 HPLC system equipped with a reverse-phase (RP) column (Zorbax 300SB-CN 4.6 mm ID x 150 cm) with mobile phase A (water + 0.1% TFA) and mobile phase B (ACN / IP A (4:1 v / v) + 0.1% TFA) at a flow rate of 0.7 mL / min. The column was connected to an absorbance detector set at a signal of 280 nm and a reference of 360 nm. After incubation for 2 hours, the crude reaction mixture was ready for HIC purification.

[0287] (Example 42. Isolation of mono-NPBA-PEG 5k -Tras) 0.55 L of 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 um, pH 7.4) was added to the crude mixture to bring it to a concentration of 1 M ammonium sulfate, 13.3 mM phosphoric sodium, pH 7.4. After mixing, the sample mixture was applied to a GE Sepha ​A filled with rose Phenyl High Performance HIC resin Loaded onto a xioChrom 140 column (1.7 L) at a flow rate of 200 mL / min. A retention time of about 10 minutes was provided 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 the eluent. Before the elution step, the column was washed with 2 CV of 40% mobile phase B to remove unbound species. The purified NPBA-PEG 5k -Tras was eluted with a gradient of 40 - 100% mobile phase B over 15 CV. The fractions of mono-PEGylated Tras (mono-NPBA-PEG -Tr 5k as) were collected and pooled for UF / DF. A Sartocon Slice Hyd rosart Cassette 30 kDa membrane was used for UF / DF of mono-NPBA-PEG -Tra 5k s. The mono-NPBA-PEG -Tras conjugate was concentrated to 50 g / L by diafiltration using 7 volumes of PBS (pH 7.4). After diafiltration, mono-NPBA-PEG 5k -Tras was sterile filtered through a 0.22 μm PES membrane. 5k

[0288] (Example 43. Determination of the number of NPBA per Tras) The number of NPBA per Tras in mono-NPBA-PEG 5k5k -Tras was determined by calculating the ratio of the molar concentration of NPBA-PEG in mono-N 5k PBA-PEG 5k -Tras to the molar concentration of Tras using the ARS detection assay for NPBA. To generate a standard curve, samples of NPBA-PEG -AA(27) were prepared in PBS (pH 7.2 5k ) ) were prepared at various concentrations (0 - 0.0098 mM) dissolved in . ARS was dissolved in water at 0.049 mM and prepared. Next, the sample and ARS were mixed, and fluorescence was measured using a Varioskan LU X spectrophotometer (ThermoFisher) and SkanIt (Thermo Fishe r) or similar fluorescence analysis software (excitation wavelength: 468 nm, fluorescence wavelength: 572 nm). By linear regression of these data (fluorescence vs. concentration), a calibration curve was obtained to determine NPBA in the target sample.

[0289] The target sample (e.g., Tras and mono - NPBA - PEG 5k - Tras) was prepared by dissolving in PBS (pH 7.2) at 0.007 mM. The molar concentration of NPBA - PEG in mono - NPBA - PEG 5 k - Tras was calculated using the following formula from the average fluorescence value of the mono - NPBA - PEG 5k - Tras sample and the fluorescence values of NPBA - PEG 5k - AA standards at various known concentrations (0 - 0. 0098 mM). Fluorescence = ε * C N + b (where ε = slope of the linear regression of the calibration curve, b = intercept of the linear regression of the calibration curve, C N = concentration of NBPA - PEG in mM). Therefore, C N (mM) = (Fluorescence - b) / ε. )

[0290] Next, according to the following formula, C N was divided by the known Tras concentration to calculate the number of NPB A per Tras (N Tras ). N Tras = (NPBA - PEG mmol / L) / (Tras mmol / L)

[0291] Mono-NPBA-PEG using the NPBA ARS detection assay of NPBA 5k -Tras of NPB The stoichiometry of A is about 1 NPBA per Tras of mono-NPBA-PEG 5k -Tras. As described in Example 40, the number of NPBA per mono-NPBA-PEG -Tras may vary due to unwanted complexation of molecules with NPBA that decreases the fluorescence signal, or 5k the presence of excess unreacted NPBA-PEG conjugate in the crude sample that increases the fluorescence signal. Exemplary experimental results are shown in Table 3.

Table 3

[0292] As will be appreciated by those skilled in the art, in light of these teachings, numerous modifications and variations of the present disclosure are possible, and all of them are contemplated herein. For example, in addition to the embodiments described herein, the present disclosure contemplates inventions arising from combinations of the features of the disclosures cited herein with the features of the cited prior art documents that complement the features of the present disclosure, and claims such combinations. Similarly, it will be understood that any of the described materials, features or articles may be used in combination with any other materials, features or articles, and that such combinations are considered to be within the scope of the present disclosure

[0293]

[0293] The disclosures of each patent, patent application and publication cited or described in this document are hereby incorporated by reference in their entirety for all purposes, or at least for the content relevant to the context in which it is cited into this document. In addition to the documents already mentioned, the present disclosure is directed to 2009 U.S. Patent Application No. 12 / 540,319, filed on August 12, 2009, currently U.S. Patent No. 8,5 57,292; U.S. Patent Application No. 13 / 782,458, filed on March 1, 2013; U.S. Patent Application No. 13 / 782,486, filed on March 1, 2013; U.S. Patent Application No. 13 / 852,303, filed on March 28, 2013; U.S. Patent Application No. 15 / 180,201, filed on June 13, 2016, currently U.S. Patent No. 10,287,4 01; and International Application No. PCT / US2009 / 05 3620, the disclosures of which are incorporated herein by reference for all purposes. incorporated.​​

Claims

1. The following structure: 【Chemistry 1】 where R is the α-carbon atom of one or more amino acids optionally attached to an amino acid functional group. 7-ethyl-10-hydroxy-camptothecin (SN38) 20-(Boc-aminoacyl), 10-TBDPS derivatives (20-(Boc-aminoacyl) Sil)-10-TBDPS-SN38).

2. The amino acid functional groups are glycine, valine, gamma-aminobutyric acid (GABA) and hexane. The 20-(Boc-aminoacyl) of claim 1, selected from the group consisting of acid functional groups. SN38 derivative of TBDPS.

3. The following structure: 【Chemistry 2】 where R is the α-carbon atom of one or more amino acids optionally attached to an amino acid functional group. 20-(Boc-aminoacyl) of SN38, 10-OBoc derivative Conductor (20-(Boc-aminoacyl)-10-OBoc-SN38).

4. The amino acid functional groups are selected from the group consisting of glycine, alanine, β-alanine, valine and leucine. The 20-(Boc-aminoacyl) of claim 3, wherein the 10-OBo SN38 derivative of c.

5. The following structure: 【Chemistry 3】 where R is the α-carbon atom of one or more amino acids optionally attached to an amino acid functional group. The HCl salt of the 20-aminoacyl derivative of SN38 (20-(HC l. Aminoacyl)-SN38).

6. 6. The method of claim 5, wherein the amino acid functional groups are selected from the functional groups of glycine and valine. HCl salt.

7. The following structure: 【Chemistry 4】 where R is the α-carbon atom of one or more amino acids optionally attached to an amino acid functional group. The trifluoroacetate (TF) of the 20-aminoacyl derivative of SN38 having the aryl group A) Salt (20-(TFA.aminoacyl)-SN38).

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

9. The following structure: 【Chemistry 5】 where R is the α-carbon atom of one or more amino acids optionally attached to an amino acid functional group. is an atom, x is a number ranging from 20 to 200; Mucic acid polymer (MAP)-SN38, wherein y is a number ranging from 5 to 200. Conjugate.

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

11. A nanoparticle comprising the MAP-SN38 conjugate of claim 10.

12. The following structure: 【Chemistry 6】 where R is the α-carbon atom of one or more amino acids optionally attached to an amino acid functional group. 20-(Boc-aminoacyl) derivative of camptothecin (CPT) having the aryl group Conductor (20-(Boc-aminoacyl)-CPT).

13. The amino acid functional groups are glycine, valine, alanine, β-alanine, GABA and diaminobutyric acid. 20 according to claim 12, selected from the functional group of the peptide phenylalanine-glycine. -(Boc-aminoacyl)-CPT derivatives.

14. The following structure: 【Chemistry 7】 where R is the α-carbon atom of one or more amino acids optionally attached to an amino acid functional group. The 20-aminoacyl derivative of CPT having the trifluoroacetic acid (TFA) ) salt (20-(TFA.aminoacyl)-CPT).

15. The amino acid functional groups are glycine, alanine, β-alanine, valine, GABA and diaminobutyric acid. The TF of claim 14 is selected from the functional group of the peptide phenylalanine-glycine. A salt.

16. The following structure: 【Chemistry 8】 where R is the α-carbon atom of one or more amino acids optionally attached to an amino acid functional group. The amino acid functional groups are alanine, β-alanine, valine, GABA, and diphenylamine. is selected from the functional groups: x is a number ranging from 20 to 200; and y is a number ranging from 5 to 200. Njugate.

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

18. The following structure: 【Chemistry 9】 (wherein n is a number in the range of 2 to 2,000), G-AA-PFP).

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