Prodrugs targeting norepinephrine transporters for cancer therapy

Abstract

Macromolecular prodrugs are provided in which 7-hydroxymethyl camptothecin (CPT-MeOH) is covalently bonded to a polymer through an ester linkage that is unstable under physiological conditions. The CPT-MeOH can be functionalized with a norepinephrine transporter (NET) ligand. Methods of treating cancer, particularly neuroblastoma, with the macromolecular prodrugs are also provided.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 597,103, filed November 8, 2023, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] Invention Field

[0004] A macromolecular prodrug based on 7-hydroxymethylcamptothecin (CPT-MeOH) is provided, wherein CPT-MeOH is chemically modified to impart affinity for norepinephrine transporter (NET) and covalently bonded to a polymer via an ester bond that is unstable under physiological conditions. A method for treating cancer, particularly neuroblastoma, using the said CPT-MeOH-based macromolecular prodrug is also provided. Background of the Invention

[0006] Neuroblastoma (NB) remains the most common and deadliest solid tumor in childhood, accounting for 8–10% of all childhood cancers and 15% of childhood cancer deaths. Despite improvements in cure rates for other pediatric cancers, survival rates for NB patients remain low.

[0007] Currently, intensive multimodal therapies used in clinical practice fail in more than half of patients (50-60% of patients experience relapse and have no curative salvage therapy options). The most challenging treatment comes from the non-responder subgroup, defined as the "ultra-high-risk" category. High-risk NB, due to its highly diverse etiologies and prevalence of unfavorable biological variants, currently uses potent anticancer agents as first-line treatment, including topoisomerase I inhibitors of the camptothecin family: topotecan and irinotecan. However, their clinical efficacy remains unsatisfactory in invasive disease, with poor efficacy in relapsed or refractory NB patients due to dose-limiting side effects and acquired resistance. Importantly, treatment failure in these patients has been shown to be associated with a 1-3 order of magnitude increase in the threshold drug levels required to effectively inhibit NB cell growth, reaching clinically unattainable levels.

[0008] U.S. Patent No. 11,642,414 B2 discloses a macromolecular prodrug in which a camptothecin analog is covalently bonded to a polymer via an ester bond that is unstable under physiological conditions, and a method of treating cancer using such a macromolecular prodrug, wherein 7-ethyl-10-hydroxycamptothecin (SN38) is described as a preferred camptothecin analog. Several key limitations exist with SN38-based prodrugs. The previously disclosed targeting benzylguanidine moiety of PEG-[SN38-BG]x is linked to the phenolic hydroxyl group of SN38, forming a highly unstable ester link whose hydrolysis rate may not be optimally balanced for the intended sequential release of SN38-BG from the macromolecular carrier, followed by activation of the prodrug (SN38-BG) to produce the biologically active SN38. Another limitation of selecting SN38 as the bioactive agent for constructing the prodrug in US 11,642,414 B2 is that the hydroxyl group at position 10 makes the compound susceptible to efflux mediated by the ABCG2 transporter, which is present in NB tumors and whose expression is associated with enrichment of drug-resistant tumor stem cells, a more aggressive phenotype, and a lack of durable response to treatment. Furthermore, this phenolic hydroxyl group promotes the inactivation and rapid clearance of SN38 as a water-soluble glucuronide.

[0009] Therefore, there remains a need for alternative therapeutic macromolecular prodrugs with enhanced tumor selectivity, potent and durable antitumor effects, and significantly prolonged survival. The implementation scheme described in this article meets this need. Invention Overview

[0011] This invention is based on the inventors' surprising discovery that prodrugs designed with CPT-MeOH as the bioactive entity exhibit very strong performance, as demonstrated by prototype prodrugs using an 8-arm PEG linkage with an oxyhexanoyl linker and benzylguanidine (BG) as the targeting ligand. This design of NET-targeting macromolecular prodrugs using CPT-MeOH as the bioactive agent avoids the significant limitations of SN38-based prodrugs.

[0012] A macromolecular prodrug is provided. In this macromolecular prodrug, 7-hydroxymethylcamptothecin (CPT-MeOH) is covalently bonded to the polymer via an ester bond that is unstable under physiological conditions, and is functionalized by at least one norepinephrine transporter (NET) ligand.

[0013] The polymer may be poloxamer. The polymer may be a polyethylene glycol (PEG) polymer. The polymer may be a multi-arm PEG polymer.

[0014] The polymer may be poloxamine.

[0015] The ester bond between CPT-MeOH and the polymer can be an oxyacetic acid ester bond.

[0016] The at least one NET ligand may be selected from benzylguanidine (BG), phenethylguanidine, and tyramine. The at least one NET ligand may be covalently bonded to CPT-MeOH via an ester bond that is unstable under physiological conditions. The ester bond between the at least one NET ligand and CPT-MeOH may be selected from oxyhexanoyl ester, oxyethoxypropionyl ester, oxypropoxyacetyl ester, and oxyethoxyethoxypropionyl ester.

[0017] The macromolecular prodrug may be PEG-[CPT-MeOBG]8 having the following structure:

[0018]

[0019] in:

[0020] n = average value 100, and

[0021] TP is

[0022] .

[0023] For each macromolecular prodrug of the present invention, a method is provided for treating neuroblastoma in an individual by administering an effective amount of the macromolecular prodrug to that individual in need. The individual may be a human.

[0024] For each macromolecular prodrug of the present invention, a method is provided for treating a solid tumor in an individual by administering an effective amount of the macromolecular prodrug to that individual in need. The individual may be a human.

[0025] For each macromolecular prodrug of the present invention, a method is provided for treating a brain tumor in an individual by administering an effective amount of the macromolecular prodrug to that individual in need. The individual may be a human.

[0026] For each macromolecular prodrug of the present invention, a method is provided for treating cancer in an individual by administering an effective amount of the macromolecular prodrug to that individual in need. The individual may be a human.

[0027] Brief description of the attached figures

[0028] Figure 1 The drug distribution (n=4) was compared in animals carrying SK-N-MM xenografts 24 hours after administration of PEG-[SN38]8, PEG-[SN38-BG]8, or PEG-[CPT-MeOBG]8 at a dose equivalent to 10 mg drug / kg.

[0029] Figure 2This study demonstrated the antitumor efficacy of PEG-[CPT-MeOBG]8 in animals that had undergone orthotopic xenografting of CHLA90 cells expressing luciferase. Tumor growth and treatment response were monitored using bioluminescence imaging. Treatment lasted for 8 weeks (irinotecan: 30 mg / kg twice weekly; PEG-linked prodrug: 10 mg / kg once weekly).

[0030] Figure 3 The antitumor efficacy of PEG-[CPT-MeOBG]8 in animals that have undergone orthotopic xenografting of SK-N-MM cells expressing luciferase was demonstrated (as described above, with treatment lasting 4 weeks).

[0031] Detailed description of the invention

[0032] This invention relates to macromolecular prodrugs for drug delivery to tumors expressing norepinephrine transporter (NET) and their uses. The macromolecules of this invention can be prepared using 7-hydroxymethylcamptothecin (CPT-MeOH) as a starting point for chemical derivatization and as the bioactive moiety, also known as 7-hydroxymethylcamptothecin (CPT-MeOH)-based macromolecular prodrugs. The chemical properties of CPT-MeOH provide two "handles" for chemical linkage using biodegradable esters. Prodrugs designed based on CPT-MeOH address several key limitations of SN38-based prodrugs. For example, PEG-[CPT-MeOBG] utilizes an aliphatic hydroxyl group at position 7 for targeted ligand linkage. x The design is better suited to the intended delivery and activation mechanisms, increasing the selectivity of drug uptake, retention, and antitumor effects, and reducing dissemination to peripheral (healthy) organs, as confirmed by in vivo biodistribution data. Furthermore, these improvements in the design of NET-targeting macromolecular prodrugs using CPT-MeOH as a starting point resulted in stronger therapeutic effects compared to similar constructs based on SN38. The inventors surprisingly found that this relatively small change in pharmacophore structure significantly enhanced tumor selectivity, translating into a more durable antitumor effect and significantly prolonged survival in a relapsed, multidrug-resistant NGB model. This makes the new techniques of this invention highly relevant to the goal of developing clinically viable new therapeutic modalities for more effective and safer management of aggressive diseases for which currently lack curative salvage therapies. The inventors have developed specific embodiments demonstrating the synthesis of various NET-targeting CPT-MeOH-based prodrugs, both as small molecules and in multi-arm PEG-linked forms (e.g., PEG-[CPT-MeOBG]). x(where x can be 2-10). The small molecule prodrug can be a conjugate of CPT-MeOH with an aralkylguanidine-derived acid, examples of which include oxyhexanoyl, oxyethoxypropionyl, oxypropoxyacetyl, and oxyethoxyethoxypropionyl. In particular, the inventors obtained key results demonstrating the superior performance of prodrugs designed with CPT-MeOH as the bioactive entity by using an 8-arm PEG-linked prototype prodrug (PEG-[CPT-MeOBG]8, polymer 9a, scheme 3) having an oxyhexanoyl linker and benzylguanidine (BF) as the targeting ligand.

[0033] This invention provides a macromolecular prodrug. In this macromolecular prodrug, 7-hydroxymethylcamptothecin (CPT-MeOH) is covalently bonded to the polymer via an ester bond that is unstable under physiological conditions. These physiological conditions may include a temperature of 20°C–25°C and / or a pH of 7.0–7.5. The CPT-MeOH may be functionalized with at least one norepinephrine transporter (NET) ligand.

[0034] The polymer may be poloxamer. Poloxamer is a nonionic triblock copolymer composed of a central hydrophobic polyoxypropylene (poly(propylene oxide)) chain and two hydrophilic polyoxyethylene (poly(ethylene oxide)) chains on both sides. The total number of polyoxyethylene chains can be from 2 to 130. The number of oxypropylene units can be from 15 to 67. Preferably, the molecular weight of poloxamer is below the glomerular filtration threshold, for example, 30-50 kDa. These polymers have a history of safe use in humans and can be used as pharmaceutical-grade materials (Kolliphor). ® P) is available. Many poloxamers have been approved by the FDA as excipients and are currently used in various clinical applications. All of these poloxamers are suitable for the implementation methods described herein.

[0035] Biologically relevant properties of poloxamer, such as molecular size and hydrophilic / lipophilic balance, are controlled by adjusting the lengths and molar ratios of the hydrophilic (A) and hydrophobic (B) blocks [A = poly(ethylene oxide) (PEO), and B = poly(propylene oxide) (PPO)]. Unlike chemically homogeneous poly(ethylene oxide), the ABA triblock poloxamer, combining the intermediate length of the central PPO block with a relatively high hydrophilic / lipophilic balance, can stably bind to the cell membrane, providing an effective mechanism for tumor penetration and prolonged intratumoral retention. Examples of poloxamers include Kolliphor® P188, P338, and P407. The polymer can also be poloxamine. Poloxamine is a nonionic surfactant polymer with similar chemical properties and design to poloxamer, but with a four-arm branched structure.

[0036] The polymer may be a polyethylene glycol (PEG) polymer. PEG polymers are well known in the art. PEG polymers may be linear, derived from the formula H-(O-CH2-CH2). n -OH indicates the molecular weight. The PEG polymer can be a multi-arm polymer. A multi-arm PEG polymer can have 3-10 PEG chains, for example, 4 chains, extending from a central core group. Examples of the central core group include pentaerythritol, dipentaerythritol, tripentaerythritol, and hexaglycerol groups. The PEG polymer can have a molecular weight of 1,000 to 100,000 Daltons, including all values ​​and subranges therebetween, including 2,000, 5,000, 10,000, 25,000, 35,000, 50,000, 75,000, and 85,000 Daltons.

[0037] The ester bond between CPT-MeOH and the polymer can be an oxyacetic acid bond. CPT-MeOH can be bonded to the PEG polymer via a hydroxyl group corresponding to the 20th position in camptothecin.

[0038] The NET ligand can be phenethylguanidine, benzylguanidine (BG), or tyramine.

[0039] The NET ligand can be covalently bonded to the CPT-MeOH via ester bonds that are unstable under physiological conditions. These physiological conditions may include a temperature of 20°C–25°C and / or a pH of 7.0–7.5.

[0040] The ester bond between the NET ligand and the CPT-MeOH can be an oxyhexanoyl ester, an oxyethoxypropionyl ester, an oxypropoxyacetyl ester, or an oxyethoxyethoxypropionyl ester. The ester bond between the NET ligand and the CPT-MeOH can be an oxyhexanoyl ester.

[0041] The polymer can have multiple arms. Multi-arm polymers can have approximately 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 arms, also referred to as chains. Multi-arm polymers can have 4, 6, or 8 arms. The arms in a multi-arm polymer can have different lengths. Each arm in a multi-arm polymer may have approximately 100-100,000, 1,000-10,000, 1,000-7,500, 1,000-5,000, 1,000-2,500, 2,500-10,000, 2,500-7,500, 2,500-5,000, 5,000-10,000, 7,500-10,000, 2,000-3,000, 4,500-5,500, 7,000-8,000, or 9,500-10,500 units. The arms in a multi-arm polymer may have the same or different lengths. The arms in a multi-arm polymer may extend from a central core group. Examples of central core groups in multi-arm polymers include pentaerythritol groups, dipentaerythritol groups, tripentaerythritol groups, and hexaglycerol groups. Each arm of the multi-arm polymer can bind at least one CPT-MeOH molecule. Multiple CPT-MeOH molecules can be covalently bonded to the multi-arm polymer. Approximately 3, 4, 5, 6, 7, 8, 9, or 10 CPT-MeOH molecules can be attached to the multi-arm polymer. For example, 8 CPT-MeOH molecules can be covalently bonded to the multi-arm polymer.

[0042] The polymer can be a multi-arm PEG polymer. Multi-arm PEG polymers can have approximately 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 arms. Multi-arm PEG polymers can also have 4, 6, or 8 arms, also referred to as chains. The arms in a multi-arm PEG polymer can have different lengths. Each arm in a multi-arm PEG polymer can have approximately 100-100,000, 1,000-10,000, 1,000-7,500, 1,000-5,000, 1,000-2,500, 2,500-10,000, 2,500-7,500, 2,500-5,000, 5,000-10,000, 7,500-10,000, 2,000-3,000, 4,500-5,500, 7,000-8,000, or 9,500-10,500 units. The arms in a multi-arm PEG polymer can have the same or different lengths. The arms in a multi-arm PEG polymer can extend from a central core group. Examples of central core groups in multi-arm PEG polymers include pentaerythritol, dipentaerythritol, tripentaerythritol, and hexaglycerol groups. Each arm of the multi-arm PEG polymer may bind at least one CPT-MeOH molecule. Multiple CPT-MeOH molecules may be covalently bonded to the multi-arm PEG polymer. Approximately 3, 4, 5, 6, 7, 8, 9, or 10 CPT-MeOH molecules may be attached to the multi-arm PEG polymer. For example, 8 CPT-MeOH molecules may be covalently bonded to the multi-arm PEG polymer.

[0043] CPT-MeOH can be covalently bonded to the poloxamer polymer via an ester bond that is unstable under physiological conditions (e.g., 22 °C, pH = 7.2). In one embodiment, the ester bond is an oxyacetic acid ester bond. CPT-MeOH can also be bonded to the poloxamer polymer via a hydroxyl group corresponding to the 20th position in camptothecin.

[0044] CPT-MeOH can be covalently bonded to the PEG polymer via an ester bond that is unstable under physiological conditions (e.g., 22 °C, pH = 7.2). In one embodiment, the ester bond is an oxyacetic acid bond. CPT-MeOH can also be bonded to the PEG polymer via a hydroxyl group corresponding to the 20th position in camptothecin.

[0045] In one embodiment, the macromolecular prodrug is PEG-[CPT-MeOBG]. x It is represented by the following structure:

[0046]

[0047] The polymer can be in a free base form or an ionized (salt) form - most commonly chloride, sulfate or trifluoroacetate.

[0048] In another embodiment, the macromolecular prodrug is PEG-[CPT-MeOBG]8, which is represented by the following structure:

[0049] ,

[0050] Where n = average value 100, and TP is a tripentaerythritol core with the following structure:

[0051] .

[0052] For each CPT-MeOH-based macromolecular prodrug of the present invention, a method of treating neuroblastoma in an individual of need is provided. The neuroblastoma treatment method comprises administering an effective amount of the CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8) to the individual. The individual may be a mammal, such as a human.

[0053] For each CPT-MeOH-based macromolecular prodrug of the present invention, a method of treating a solid tumor in an individual of need is provided. The solid tumor treatment method comprises administering an effective amount of the CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8) to the individual. The individual may be a mammal, such as a human.

[0054] For each CPT-MeOH-based macromolecular prodrug of the present invention, a method of treating brain tumors in an individual in need is provided. The brain tumor treatment method comprises administering an effective amount of the CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8) to the individual. The individual may be a mammal, such as a human.

[0055] For each macromolecular prodrug of the present invention, a method of treating cancer in an individual in need is provided. The cancer treatment method comprises administering to the individual an effective amount of the CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8). The individual may be a mammal, such as a human.

[0056] As used herein, the term "effective amount" refers to the amount of the present invention's CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8) required to achieve the stated purpose, such as treating an individual with neuroblastoma, solid tumor, brain tumor, and / or cancer. The effective amount of the CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8) can vary depending on the stated purpose, the individual's physical characteristics, the nature and severity of the neuroblastoma, solid tumor, brain tumor, and / or cancer, the presence of relevant or unrelated medical conditions, the nature of the CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8), the manner in which the CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8) is administered to the individual, and the route of administration. The specific dosage for a given individual is typically determined by the physician's judgment. The dosage of the CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8) can be from 0.5 mg to 200 mg / kg of individual body weight / dose. The CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8) can be administered to the individual in one or more doses.

[0057] For each treatment method of the present invention, the CPT-MeOH-based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]8) can be administered by any method commonly used in the art. Administration methods include parenteral (e.g., intravenous, intramuscular, and subcutaneous), oral, nasal, ocular, transmucosal (e.g., oral, vaginal, and rectal) and transdermal routes.

[0058] The term “about” as used herein, when referring to a measurable value (e.g., quantity, percentage, etc.), is intended to cover variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, as such variations are appropriate.

[0059] Example 1. 7-Hydroxymethylcamptothecin derivative targeting norepinephrine transporter

[0060] 6-[(4-N-Boc-aminomethyl)phenoxy]hexanoic acid (2a, Scheme 1). 4-Hydroxy-N-Boc-benzylamine (1a, AmBeed, 96%, 674 mg, 2.90 mmol) and 6-bromohexanoic acid (Sigma-Aldrich, ≥98%, 796 mg, 4.00 mmol) were dissolved in benzene (16 mL) and dried on a rotary evaporator (RE) at 30°C and 15 mm Hg to remove trace amounts of water. The residue was dissolved in anhydrous 1-methylpyrrolidone (1-MP, 12 mL) under an argon atmosphere, cooled with ice, and a 1 M potassium tert-butoxide solution of tetrahydrofuran (THF) (Sigma-Aldrich, 7.0 mL, 7.0 mmol) was added via syringe over 1 minute. The mixture was stirred in an ice bath under argon for 0.5 h, then stirred at room temperature for 27 h. Add 60 mL of 1 M H3PO4 aqueous solution and 30 mL of ethyl acetate, stir vigorously, separate the organic phase, wash successively with 60 mL of 1 M H3PO4 and 4 × 10 mL of water, filter and dry (RE, 30°C). Solidify the residue (1.151 g) by grinding with 10 mL of pentane, filter off the solid, wash with pentane, and purify by precipitation from dichloromethane with pentane. Yield 713 mg (72%).

[0061] 6-[(4-N-Boc-2-aminoethyl)phenoxy]hexanoic acid (2b, scheme 1). Acid 2b was prepared from N-Boc-tyramine (1b, TCI, ≥97%) by a similar method to 2a, with a yield of 87%.

[0062]

[0063] Option 1

[0064] A coupling of 6-[(4-N-Boc-aminomethyl)phenoxy]hexanoic acid with 7-hydroxymethylcamptothecin (4a, scheme 2). 7-hydroxymethylcamptothecin (3, scheme 2, about 95% pure, 240 mg, 0.60 mmol, prepared as described in [1]), 4-N,N-dimethylaminopyridine p-toluenesulfonate (DPTS-catalyst, 600 mg, 2.04 mmol), and anhydrous pyridine (48 mL) were heated under argon and refluxed for several minutes to completely dissolve 3. The solution was rapidly cooled to 35°C, and acid 2a (324 mg, 0.96 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Sigma-Aldrich, 612 mg, about 3.1 mmol) were added. The mixture was stirred at room temperature for 44 hours under argon protection and dried under vacuum at 30°C. Add 180 mL of a 1.6 M NaH2PO4 aqueous solution containing 0.08 M H3PO4 and 180 mL of ethyl acetate. After shaking, separate the organic layer, wash successively with 2.5 M NaCl (3 × 200 mL) and water (2 × 60 mL), and dry (RE, 30°C). The crude product (594 mg) was purified by rapid chromatography (silica gel, chloroform-acetonitrile 5:1 to 2:1) to give 234 mg (56%) of 4a.

[0065] The coupling of 6-[(4-N,N'-di-Boc-guanidinylmethyl)phenoxy]hexanoic acid with 7-hydroxymethylcamptothecin (5a, Scheme 2). Coupling 4a (234 mg, 0.33 mmol) was dissolved in anhydrous dichloromethane (2.2 mL). Dimethyl sulfide (Sigma-Aldrich, ≥99%, 0.2 mL, 2.70 mmol) was added, followed by trifluoroacetic acid (Optima™ LC / MS grade, Fisher Scientific, ≥99.5%, 1.0 mL, 12.8 mmol). The mixture was incubated at room temperature under argon protection for 0.5 hours. The solvent was removed under vacuum at room temperature, and the remaining syrup was co-evaporated sequentially with benzene (5 mL), chloroform (5 mL), and a mixture of chloroform (3 mL) and THF (3 mL) (twice), suspended in a mixture of chloroform (15 mL) and THF (15 mL), and dried on a RE at 30°C. Most of the solid residue (379 mg) was suspended in a mixture of dichloromethane (1.5 mL) and THF (1.5 mL), and diisopropylethylamine (DIPEA, Sigma-Aldrich, 99.5%, 0.30 mL, 1.71 mmol) was added, followed by 1,3-bis-Boc-2-(trifluoromethylsulfonyl)guanidine (Acros, 98%, 215 mg, 0.55 mmol). The mixture was homogenized (for about 0.5 hours) and stirred at room temperature under argon for 32 hours. The solvent was removed (RE, 30°C), and a 1.6 M NaH2PO4 aqueous solution containing 0.08 M H3PO4 (30 mL) was added to the residue. The solids were filtered off, washed with water (35 mL), dried under vacuum, dissolved in chloroform, filtered, and dried (RE, 30°C). The residue (477 mg) was purified by rapid chromatography (silica gel, chloroform-acetonitrile 10:1 to 4:1) to give 240 mg (85%) of 5a. 1 H NMR (400 MHz, CDCl3) corresponds to the structure of 5a, clearly showing that the carboxyl group is connected to the 7-CH2OH of 3, while the 20-OH is still unacylated, with a signal visible at 3.81 ppm (s).

[0066] Option 2

[0067] A conjugate of 6-[(4-guanidinomethyl)phenoxy]hexanoic acid and 7-hydroxymethylcamptothecin, trifluoroacetate (6a, Scheme 2). Conjugate 5a (77 mg, 0.092 mmol) was dissolved in anhydrous dichloromethane (1.6 mL). Anethole (Sigma-Aldrich, 99%, 0.2 mL, 1.82 mmol) was added, followed by trifluoroacetic acid (Optima™ LC / MS grade, Fisher Scientific, ≥99.5%, 1.0 mL, 12.8 mmol). The mixture was incubated at room temperature under argon protection for 2.5 hours. The solvent was removed under vacuum at room temperature, and the remaining syrup was co-evaporated with chloroform (3 × 6 mL) as described above, and stirred with hexane (30 mL) until turbidity disappeared. The supernatant was decanted, and the residue was washed with hexane (3 × 5 mL) and dried. Add methyl tert-butyl ether (MTBE, 5 mL) and trioctylamine (Msynth®plus, ≥93%, 0.2 mL, approx. 0.42 mmol), grind the residue with solvent, allow the suspension to stand at room temperature until completely solidified, filter off the solid, wash successively with MTBE (15 mL) and hexane (15 mL), and dry. Dissolve the crude salt in a 5:1 mixture of chloroform and 2-propanol, filter the solution, dry (RE, 30°C), solidify the residue by co-evaporation with dichloromethane, suspend in dichloromethane (2 mL), filter off, wash successively with dichloromethane (3 mL) and hexane (10 mL), and dry under vacuum. Yield 64 mg (92%). 1 H NMR (400 MHz, DMSO-d6) corresponds to the structure of 6a.

[0068]

[0069] Option 3

[0070] An octahedral-PEG coupling compound with 7-hydroxymethylcamptothecin acylated by 6-[(4-guanidinomethyl)phenoxy]hexanoic acid, trifluoroacetate (polymer 9a, scheme 3). Carboxylated octahedral-PEG (polymer 7, scheme 3, JenKem Technology, M) n= 37390 Da, 400 mg, 0.085 mmol of carboxyl group), coupling compound 5a (214 mg, 0.255 mmol), DPTS-catalyst (200 mg, 0.68 mmol), and dichloromethane (4 mL) were stirred for 5 minutes until homogeneous, and then N,N'-dicyclohexylcarbodiimide (DCC, Aldrich, 99%, 303 mg, 1.45 mmol) was added. The mixture was stirred at room temperature under argon for 96 hours. The solvent was removed (RE, room temperature), the residue was co-evaporated with benzene (20 mL) (RE, 30°C), and suspended in benzene (10 mL). The insoluble product was filtered off, washed with benzene (12 mL), the filtrate was concentrated to a syrup (2 g, RE, 30 °C), suspended in diethyl ether (40 mL), and the crude polymer 8a (Scheme 3) was filtered off. For purification, the polymer was precipitated several times in small volumes of benzene (2-3 mL) with diethyl ether (40 mL) until no mobile compounds were detected by TLC (silica gel, chloroform-acetonitrile 3:1). Finally, the polymer was dissolved in a 4:1 mixture of benzene and dichloromethane (30 mL) and washed with 22% Na₂SO₄ aqueous solution (3 × 20 mL) to remove any residual DPTS-catalyst. After drying with Na₂SO₄ and filtering to remove the desiccant, the solution was concentrated (RE, 30°C) to 3 g. Polymer 8a was cured with diethyl ether as described above, filtered, and vacuum dried. To remove the protective Boc group from the guanidine moiety, polymer 8a (475 mg) was dissolved in anhydrous dichloromethane (4 mL). Anethole (Sigma-Aldrich, 99%, 0.40 mL, 3.64 mmol) and trifluoroacetic acid (Optima™ LC / MS grade, Fisher Scientific, ≥99.5%, 2.0 mL, 25.6 mmol) were added, and the mixture was allowed to stand at room temperature for 3.5 hours. To remove volatile substances (RE, ≤25°C), the residue was co-evaporated sequentially with chloroform (2 × 10 mL) and benzene (5 mL) as described above, and then evacuated under vacuum for 1 hour at room temperature and ≤1 mm Hg. Diethyl ether (30 mL) and trioctylamine (Msynth®plus, ≥93%, 0.6 mL, approximately 1.28 mmol) were added, and the residue was ground until solidified. The polymer was filtered off, washed with diethyl ether, dried, and precipitated from a small amount of benzene with diethyl ether. After vacuum drying, 9a, 448 mg, was obtained. 1 H NMR (400 MHz, CDCl3) detected 0.19 mmol / g (14.3%, by weight) of the bound conjugate 6a.

[0071]

[0072] Option 3 (Variation 2)

[0073] A coupling of (4-N-Boc-aminomethyl)phenylacetic acid and 7-hydroxymethylcamptothecin (4b, Scheme 4). 7-hydroxymethylcamptothecin (3, approx. 95%, 100 mg, 0.25 mmol), 4-N,N-dimethylaminopyridine p-toluenesulfonate (DPTS-catalyst, 250 mg, 0.85 mmol), and anhydrous pyridine (20 mL) were heated under argon and refluxed for several minutes until 3 was completely dissolved. The solution was rapidly cooled to 35°C, and (4-N-Boc-aminomethyl)phenylacetic acid (AmBeed, 98%, 108 mg, 0.40 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Sigma-Aldrich, 254 mg, approx. 1.3 mmol) were added. The mixture was stirred at room temperature for 47 hours under argon protection and then dried under vacuum at 20°C–25°C. Add 25 mL of a 1.6 M NaH2PO4 aqueous solution containing 0.08 M H3PO4 and 7 mL of water to fully suspend the residue, filter, wash with 25 mL of water, and dry under vacuum. The crude product (224 mg) was purified by rapid chromatography (silica gel, chloroform-acetonitrile 5:1 to 2:1) to give 117 mg (75%) of 4b.

[0074] A conjugate of (4-N,N'-di-Boc-guanidinylmethyl)phenylacetic acid and 7-hydroxymethylcamptothecin (5b, scheme 4). Conjugate 4b (117 mg, 0.18 mmol) was dissolved in anhydrous dichloromethane (1.4 mL). Dimethyl sulfide (Sigma-Aldrich, ≥99%, 0.2 mL, 2.70 mmol) was added, followed by trifluoroacetic acid (Optima™ LC / MS grade, Fisher Scientific, ≥99.5%, 1.0 mL, 12.8 mmol). The mixture was incubated at room temperature under argon protection for 0.5 hours. The solvent was removed under vacuum at room temperature, and the remaining syrup was co-evaporated with chloroform (5 mL) as described above and dried under vacuum. The residue (215 mg) was suspended in a mixture of dichloromethane (1.1 mL) and THF (1.1 mL), and diisopropylethylamine (DIPEA, Sigma-Aldrich, 99.5%, 0.38 mL, 2.17 mmol) was added, followed by 1,3-bis-Boc-2-(trifluoromethylsulfonyl)guanidine (AmBeed, 95%, 152 mg, 0.37 mmol). The mixture (homogenized for approximately 0.5 hours) was stirred at room temperature under argon for 44 hours. The solvent (RE, 30°C) was removed, and a 1.6 M NaH2PO4 aqueous solution (25 mL) containing 0.08 M H3PO4 was added to the residue. The solid was filtered off, washed with water (35 mL), and dried under vacuum. The residue (272 mg) was purified by rapid chromatography (silica gel, chloroform-acetonitrile 10:1 to 3:1) to give 118 mg (83%) of 5b. 1 H NMR (400 MHz, CDCl3) corresponds to the 5b structure, with a 20-OH signal at 3.77 ppm (s).

[0075]

[0076] Option 4

[0077] A conjugate of (4-guanidinomethyl)phenylacetic acid and 7-hydroxymethylcamptothecin, trifluoroacetate (6b, scheme 4). Conjugate 5b (71 mg, 0.092 mmol) was dissolved in anhydrous dichloromethane (1.5 mL). Anethole (Sigma-Aldrich, 99%, 0.2 mL, 1.82 mmol) was added, followed by trifluoroacetic acid (Optima™ LC / MS grade, Fisher Scientific, ≥99.5%, 1.0 mL, 12.8 mmol). The mixture was incubated at room temperature under argon protection for 2.5 hours. The solvent was removed under vacuum at room temperature, and the remaining syrup was co-evaporated with chloroform (3 × 6 mL) as described above, stirred with hexane (30 mL) for 10 minutes. The supernatant was decanted, and the residue was washed with hexane (3 × 5 mL) and dried. Add dichloromethane (2.5 mL), trioctylamine (Msynth®plus, ≥93%, 0.25 mL, approx. 0.53 mmol), and methyl tert-butyl ether (MTBE, 3 mL). Grind the residue until completely suspended. Remove the solvent (gas stream). Suspend the residue in MTBE (7 mL) and let it stand at room temperature for 3 hours. Filter the solid, wash successively with MTBE (10 mL) and hexane (10 mL), and dry. Dissolve the crude salt in a 5:1 mixture of chloroform and 2-propanol. Filter the solution and dry (RE, 30°C). Solidify the residue by co-evaporation with chloroform and dichloromethane, and suspend it in dichloromethane (3 mL). Filter, wash with dichloromethane (3 mL) and pentane (10 mL), and dry under vacuum. Yield: 59 mg (93%). 1 H NMR (400MHz, DMSO-d6) corresponds to the 6b structure.

[0078] A conjugate of 3-(4-[(1-Boc-piperidin-4-yl)oxy]phenyl)propionic acid with 7-hydroxymethylcamptothecin (4c, scheme 5). Conjugate 4c was obtained from 7-hydroxymethylcamptothecin (3, about 95%, 100 mg, 0.25 mmol) and 3-(4-[(1-Boc-piperidin-4-yl)oxy]phenyl)propionic acid (AmBeed, 95%, 124 mg, 0.40 mmol) using a method similar to that used for conjugate 4b. The crude product (251 mg) was purified by rapid chromatography (silica gel, chloroform-acetone 10:1 to 4:1) to give 124 mg (70%) of 4c.

[0079] Di-Boc-guanidine conjugate 5c (Scheme 5). Conjugate 4c (124 mg, 0.17 mmol) was dissolved in anhydrous dichloromethane (1.4 mL). Dimethyl sulfide (Sigma-Aldrich, ≥99%, 0.2 mL, 2.70 mmol) was added, followed by trifluoroacetic acid (Optima™ LC / MS grade, Fisher Chemical, ≥99.5%, 1.0 mL, 12.8 mmol). The mixture was incubated at room temperature under argon protection for 0.5 hours. The solvent was removed under vacuum at room temperature, and the remaining syrup was co-evaporated with chloroform (5 mL) as described above and dried under vacuum. The residue (203 mg) was suspended in a mixture of dichloromethane (1.1 mL) and THF (1.1 mL), and diisopropylethylamine (DIPEA, Sigma-Aldrich, 99.5%, 0.38 mL, 2.17 mmol) was added, followed by 1,3-bis-Boc-2-(trifluoromethanesulfonyl)guanidine (AmBeed, 95%, 152 mg, 0.37 mmol). The mixture was stirred at room temperature under argon for 119 hours but failed to become homogeneous. The solvent and excess DIPEA were removed under vacuum, and the residue was resuspended in a mixture of dichloromethane (1.5 mL) and 2-propanol (1.0 mL), and DIPEA (0.30 mL, 1.71 mmol) was added. Stirring was continued at room temperature for 215 hours. Processed according to method 5b, then subjected to rapid chromatography (silica gel, chloroform-acetone 20:1 to 4:1) to obtain 101 mg (approximately 66%) of 5c, according to 1 The compound was analyzed by ¹H NMR and contained approximately 3% N,N'-di-Boc-urea. Without further purification, this compound was used in subsequent preparations.

[0080] Guanidine trifluoroacetate conjugate 6c (Scheme 5). As described for conjugate 6b, compound 6c was prepared by deprotection of 5c (101 mg, about 0.11 mmol). 81 mg (about 92%) was obtained. 1 H NMR (400 MHz, DMSO-d6) corresponds to the 6c structure.

[0081]

[0082] Option 5

[0083] A conjugate of (4-N-Boc-aminomethyl)phenoxyacetic acid and 7-hydroxymethylcamptothecin (4d, scheme 6). Compound 4d was prepared from 7-hydroxymethylcamptothecin (3, about 95%, 100 mg, 0.25 mmol) and (4-N-Boc-aminomethyl)phenoxyacetic acid (AmBeed, 95%, 118 mg, 0.40 mmol) using a method similar to that used for conjugate 4a. The crude product (219 mg) was purified by rapid chromatography (silica gel, chloroform-acetone 8:1 to 3:1) to give 106 mg (66%) of 4d.

[0084] A conjugate of (4-N,N'-di-Boc-guanidinylmethyl)phenoxyacetic acid and 7-hydroxymethylcamptothecin (5d, Scheme 4). Conjugate 4d (106 mg, 0.16 mmol) was dissolved in anhydrous dichloromethane (1.3 mL). Dimethyl sulfide (Sigma-Aldrich, ≥99%, 0.2 mL, 2.70 mmol) was added, followed by trifluoroacetic acid (Optima™ LC / MS grade, Fisher Scientific, ≥99.5%, 1.0 mL, 12.8 mmol). The mixture was incubated at room temperature under argon protection for 0.5 hours. The solvent was removed under vacuum at room temperature, and the remaining syrup was co-evaporated with chloroform (5 mL) as described above and dried under vacuum (<1 mm Hg). The residue (163 mg) was suspended in a mixture of dichloromethane (3.0 mL) and THF (1.0 mL), and diisopropylethylamine (DIPEA, Sigma-Aldrich, 99.5%, 0.38 mL, 2.17 mmol) was added, followed by 1,3-bis-Boc-2-(trifluoromethylsulfonyl)guanidine (AmBeed, 95%, 152 mg, 0.37 mmol). The mixture was homogenized (approximately 1 hour) and stirred at room temperature under argon for 51 hours. The solvent (RE, 30°C) was removed, and 25 mL of a 1.6 M NaH2PO4 aqueous solution containing 0.08 M H3PO4 was added to the residue. The solid was filtered off, washed with 35 mL of 5 M NaCl aqueous solution and 20 mL of water, and dried under vacuum. The residue (238 mg) was purified by rapid chromatography (silica gel, chloroform-acetone 10:1 to 5:1) to give 93 mg (72%) of 5d. 1 HNMR (400 MHz, CDCl3) corresponds to the 5d structure, with a signal of 20-OH at 3.79 ppm (s).

[0085] A conjugate of (4-guanidinomethyl)phenoxyacetic acid and 7-hydroxymethylcamptothecin, trifluoroacetate (6d, scheme 6). The conjugate 5d (92 mg, 0.12 mmol) was dissolved in anhydrous dichloromethane (1.3 mL). Anethole (Sigma-Aldrich, 99%, 0.2 mL, 1.82 mmol) was added, followed by trifluoroacetic acid (Optima™ LC / MS grade, Fisher Scientific, ≥99.5%, 1.0 mL, 12.8 mmol). The mixture was incubated at room temperature under argon protection for 2.5 hours. The solvent was removed under vacuum at room temperature, and the remaining syrup was co-evaporated with chloroform (3 × 6 mL) as described above, followed by stirring with hexane (30 mL) for 10 minutes. The supernatant was decanted, and the residue was washed with hexane (3 × 10 mL) and dried. Dichloromethane (2.5 mL) and trioctylamine (Msynth®plus, ≥93%, 0.30 mL, approximately 0.63 mmol) were added, and the residue was ground until completely suspended. The solvent was removed (by gas flow), and the residue was suspended in MTBE (12... The solid was placed in a 4:1 mixture of chloroform and methanol and allowed to stand at room temperature for 3 hours. The solid was filtered off, washed with MTBE (15 mL) and pentane (20 mL), and dried. The crude salt (90 mg) was dissolved in a 4:1 mixture of chloroform and methanol, the solution was filtered, dried (RE, 30°C), and the residue was solidified by co-evaporation with dichloromethane and suspended in dichloromethane (5 mL). The solid was filtered off, washed with dichloromethane (7 mL) and pentane (12 mL), and dried under vacuum. Yield: 79 mg (97%). 1 H NMR (400 MHz, DMSO-d6) corresponds to the 6d structure.

[0086]

[0087] Option 6

[0088] A conjugate of 6-[(4-N-Boc-2-aminoethyl)phenoxy]hexanoic acid with 7-hydroxymethylcamptothecin (4e, scheme 7). Conjugate 4e was obtained from 7-hydroxymethylcamptothecin (3, about 95%, 100 mg, 0.25 mmol) and 6-[(4-N-Boc-2-aminoethyl)phenoxy]hexanoic acid (2b, 141 mg, 0.40 mmol) using a method similar to that used for conjugate 4a. The crude product (226 mg) was purified by rapid chromatography (silica gel, chloroform-acetone 10:1 to 4:1) to give 125 mg (70%) of 4e.

[0089] A conjugate of 6-[(4-N,N'-di-Boc-guanidinylethyl)phenoxy]hexanoic acid and 7-hydroxymethylcamptothecin (5e, scheme 7). As described with respect to the preparation of 5a, conjugate 5e was prepared from compound 4e (125 mg, 0.17 mmol). The crude product was purified by rapid chromatography (silica gel, chloroform-acetone 20:1 to 5:1) to give 121 mg (81%) of 5e. 1 H NMR (400 MHz, CDCl3) corresponds to the 5e structure, with a 20-OH signal at 3.76 ppm (s).

[0090] A conjugate of 6-[(4-guanidinoethyl)phenoxy]hexanoic acid and 7-hydroxymethylcamptothecin, trifluoroacetate (6e, scheme 7). Compound 6e was prepared by deprotection of 5e (103 mg, 0.12 mmol), as described with respect to conjugate 6d. Yield: 88 mg (94%). 1 H NMR (400 MHz, DMSO-d6) corresponds to the 6e structure.

[0091]

[0092] Option 7

[0093] Example 2. Drug biodistribution and tumor uptake

[0094] The study was conducted to evaluate intratumoral drug levels and organ distribution (24 hours) of CPT-MeOH delivered as PEG-[CPT-MeOBG]8, compared with SN38 administered as a similar construct, PEG-[SN38-BG]8, or PEG-[SN38]8. Figure 1 Analysis was performed in an orthotopic model of neuroblastoma. Athymic nude mice (…) nu / nu (n=4) Multidrug-resistant, ATRX-mutated SK-N-MM cells stably expressing firefly luciferase (10 μL / animal per animal) were implanted into the adrenal fat pad. 6 (Number of tumors). Under the monitoring of bioluminescence imaging, the tumor reached a size of 1.0 ± 0.4 cm. 3 The size of the sample was determined. The polymer conjugate was administered intravenously at a dose equivalent to 10 mg / kg of CPT-MeOH or SN38. Tissue samples harvested 24 hours post-injection were homogenized at 4°C, extracted with acetonitrile, and analyzed using a fluorescence assay. Data are presented as mean ± SD.

[0095] Example 3. Tumor growth inhibitory activity

[0096] The tumor growth inhibitory activity of PEG-[CPT-MeOBG]8 was evaluated in two orthotopic models of recurrent ATRX-mutant neuroblastoma, using CHLA90 ( Figure 2 ) and SK-N-MM cells ( Figure 3 The model was established as described above. Treatment was administered intravenously once weekly for 8 weeks and 4 weeks, respectively, at a dose equivalent to 10 mg of CPT-MeOH / kg. PEG-[SN38-BG]8 (administered at the same dose once weekly) and irinotecan (administered at 30 mg / kg twice weekly) served as controls. Tumor-related signals were monitored by quantitative bioluminescence (data are presented as mean ± SD, n=5). For CHLA90 xenograft animals, the survival rate of animals over time in each group was as follows: Figure 2 As shown.

[0097] Although the invention has been illustrated and described herein by way of example with reference to specific embodiments, the invention is not intended to be limited to the details described above. Rather, various modifications may be made to the details within the scope of the claims and their equivalents without departing from the invention.

Claims

1. A macromolecular prodrug in which 7-hydroxymethylcamptothecin (CPT-MeOH) is covalently bonded to a polymer via an ester bond that is unstable under physiological conditions and is functionalized by at least one norepinephrine transporter (NET) ligand.

2. The macromolecular prodrug according to claim 1, wherein the polymer is poloxamer.

3. The macromolecular prodrug according to claim 2, wherein the polymer is a polyethylene glycol (PEG) polymer.

4. The macromolecular prodrug according to claim 3, wherein the polymer is a multi-arm PEG polymer.

5. The macromolecular prodrug according to claim 1, wherein the polymer is poloxamine.

6. The macromolecular prodrug according to any one of claims 1-5, wherein the ester bond between CPT-MeOH and the polymer is an oxyacetic acid ester bond.

7. The macromolecular prodrug according to any one of claims 1-6, wherein the at least one NET ligand is selected from benzylguanidine (BG), phenethylguanidine and tyramine.

8. The macromolecular prodrug according to any one of claims 1-7, wherein the at least one NET ligand is covalently bonded to the CPT-MeOH via an ester bond that is unstable under physiological conditions.

9. The macromolecular prodrug according to claim 8, wherein the ester bond between the at least one NET ligand and the CPT-MeOH is selected from oxyhexanoyl ester, oxyethoxypropionyl ester, oxypropoxyacetyl ester and oxyethoxyethoxypropionyl ester.

10. The macromolecular prodrug according to any one of claims 1-9, wherein it is PEG-[CPT-MeOBG]8 having the following structure: in: n = average value 100, and TP is 。 11. A method of treating neuroblastoma in an individual in need, comprising administering to said individual an effective amount of a macromolecular prodrug according to any one of claims 1-10.

12. A method for treating a solid tumor in an individual in need, comprising administering to said individual an effective amount of a macromolecular prodrug according to any one of claims 1-10.

13. A method for treating a brain tumor in an individual in need, comprising administering to said individual an effective amount of a macromolecular prodrug according to any one of claims 1-10.

14. A method of treating cancer in an individual in need, comprising administering to said individual an effective amount of a macromolecular prodrug according to any one of claims 1-10.

15. The method according to any one of claims 11-14, wherein the individual is a person.

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