Norepinephrine transporter-targeted prodrugs for cancer therapy

Abstract

Provided are macromolecular prodrugs in which 7-hydroxymethylcamptothecin (CPT-MeOH) is covalently bonded to a polymer via ester bonds that are labile under physiological conditions. The CPT-MeOH may be functionalized with a norepinephrine transporter (NET) ligand. Also provided are methods of treating cancer, especially neuroblastoma with the macromolecular prodrugs.

Description

[0001] NOREPINEPHRINE TRANSPORTER-TARGETED PRODRUGS FOR CANCER THERAPY

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application No. 63 / 597,103, filed November 8, 2023, and the contents of which are incorporated herein by reference in their entireties for all purposes.

[0004] FIELD OF THE INVENTION

[0005] Provided are 7-hydroxymethylcamptothecin (CPT-MeOH) based macromolecular prodrugs in which the CPT-MeOH, chemically modified to endow it with affinity for the norepinephrine transporter (NET), is covalently bonded to polymers via ester bonds that are labile under physiological conditions. Also provided are methods of treating cancer, in particular neuroblastoma, with the CPT-MeOH based macromolecular prodrugs.

[0006] BACKGROUND OF THE INVENTION

[0007] Neuroblastoma (NB) remains the most common and deadly solid tumor of childhood accounting for 8-10% of all childhood cancers, and 15% of deaths from cancer in children. Despite improvements in the cure rate for other pediatric neoplasms, the survival rate for patients with NB has lagged behind.

[0008] The intensive, multimodality therapy currently used in the clinic fails in over half of the patients (50-60% of patients experience a relapse with no curative salvage treatment options), with the most formidable therapeutic challenge presented by the non-responder patient subgroup, defined as an "ultrahigh" risk category. High-risk NB with its highly diverse etiology and prevalence of biologically unfavorable variants is currently approached by potent anticancer agents as a first-line treatment, including topoisomerase I inhibitors of the camptothecin family: topotecan and irinotecan . However, their clinical use in the context of aggressive disease remains suboptimal, yielding poor results in relapsed or refractory NB patients due to dose-limiting side effects and acquired drug resistance. Importantly, treatment failure in these patients was shown to be associated with an increase in threshold drug levels required for effectively suppressing NB cell growth by 1-3 orders of magnitude, reaching values not achievable clinically.

[0009] Macromolecular prodrugs, in which camptothecin analogs are covalently bonded to polymers via ester bonds that are labile under physiological conditions, and methods for treating cancer using such macromolecular prodrugs have been reported in U.S. Patent No. 11, 642,414 B2 with 7-ethyl-10-hydroxy-camptothecin (SN38) described as the preferred camptothecin analog. The SN38-based prodrugs have several major limitations. The targeting benzylguanidino moiety of the previously disclosed PEG- [SN38-BG]x is attached to the phenolic hydroxyl of SN38 creating a highly labile ester linkage, 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 generate bioactive SN38. An additional limitation of SN38 as the bioactive agent chosen for constructing the prodrugs in US 11,642,414 B2 is that the hydroxy group at the 10 position makes the compound susceptible to efflux mediated by the ABCG2 transporter, which is found in NB tumors and whose expression correlates with enrichment in drug-resistant tumor stem cells, a more aggressive phenotype, and a lack of durable response to therapy. Additionally, this phenolic hydroxyl promotes inactivation and rapid clearance of SN38 as a water-soluble glucuronide.

[0010] Thus, there remains a need for alternative therapeutic macromolecular prodrugs with enhanced tumor selectivity for strong and durable antitumor effect and markedly extended survival. The embodiments described herein address this need.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention is based on the inventors' surprising discovery of exceptionally strong performance of the prodrugs that are designed with the CPT-MeOH as the bioactive entity demonstrated using an 8-arm PEG-linked prototype prodrug with oxyhexanoyl linker and benzylguanidine (BG) as the targeting ligand. This NET- targeted macromolecular prodrug design using CPT-MeOH as the bioactive agent obviates the significant limitations of SN38-based prodrugs.

[0013] A macromolecular prodrug is provided. In the macromolecular prodrug, 7- hydroxymethylcamptothecin (CPT-MeOH) is covalently bonded to a polymer via ester bonds that are labile under physiological conditions and is functionalized with at least one norepinephrine transporter (NET) ligand.

[0014] The polymer may be a poloxamer. The polymer may be a polyethylene glycol (PEG) polymer. The polymer may be a multi-arm PEG polymer.

[0015] The polymer may be a poloxamine.

[0016] The ester bonds between the CPT-MeOH and the polymer may be oxyacetate ester bonds.

[0017] The at least one NET ligand may be selected from the group consisting of benzylguanidine (BG), phenethylguanidine, and tyramine. The at least one NET ligand may be covalently bonded to the CPT-MeOH via an ester bond that is labile under physiological conditions. The ester bond between the at least one NET ligand and the CPT-MeOH may be selected from the group consisting of an oxyhexanoyl ester, an oxyethoxypropanoyl ester, an oxypropoxyacetyl ester, and an oxyethoxyethoxypropanoyl ester. The macromolecular prodrug may be PEG-[CPT-MeOBG]s having the following structure: wherein : n = 100 in average and

[0018] TP is

[0019] For each of the macromolecular prodrugs of the present invention, a method of treating neuroblastoma in a subject in need thereof is provided, by administering to the subject an effective amount of the macromolecular prodrug. The subject may be a human.

[0020] For each of the macromolecular prodrugs of the present invention, a method of treating a solid tumor in a subject in need thereof is provided, by administering to the subject an effective amount of the macromolecular prodrug. The subject may be a human.

[0021] For each of the macromolecular prodrugs of the present invention, a method of treating a brain tumor in a subject in need thereof is provided, by administering to the subject an effective amount of the macromolecular prodrug. The subject may be a human.

[0022] For each of the macromolecular prodrugs of the present invention, a method of treating cancer in a subject in need thereof is provided, by administering to the subject an effective amount of the macromolecular prodrug. The subject may be a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows comparative drug distribution in animals bearing SK-N-MM xenografts 24 hr after administering PEG-[SN38]s, PEG-[SN38-BG]s or PEG-[CPT- MeOBG]s at doses equivalent to 10 mg drug per kg (n=4).

[0024] FIG. 2 shows antitumor efficacy of PEG-[CPT-MeOBG]s in animals orthotopically xenografted with luciferase-expressing CHLA90 cells. Tumor growth and therapeutic response were monitored by bioluminescent imaging. Treatments were administered over 8 weeks (Irinotecan : twice a week at 30 mg / kg; PEG-linked prodrugs: once a week at 10 mg drug per kg).

[0025] FIG. 3 shows antitumor efficacy of PEG-[CPT-MeOBG]s in animals orthotopically xenografted with luciferase-expressing SK-N-MM cells (performed as above, with treatments administered over 4 weeks).

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates to macromolecular prodrugs for drug delivery to tumors expressing the norepinephrine transporter (NET) and uses thereof. The macromolecular molecules of the present invention may be prepared by using 7- hydroxymethylcamptothecin (CPT-MeOH) as a starting point for chemical derivatization and as the bioactive moiety, and are also referred to as 7-hydroxymethylcamptothecin (CPT-MeOH) based macromolecular prodrugs. The chemistry of CPT-MeOH offers two "handles" for chemical attachment using biodegradable ester linkages. The design of prodrugs based on CPT-MeOH addresses several major limitations of SN38-based prodrugs. For example, the design of PEG-[CPT-MeOBG]xutilizing an aliphatic hydroxyl at the 7 position for the targeting ligand attachment is much better suited for the intended delivery and activation mechanism, with increased selectivity of the drug uptake, retention, and action against the tumor and with reduced dissemination to peripheral (healthy) organs as evidenced by in vivo biodistribution data. In addition, these enhancements in the design of NET-targeted macromolecular prodrugs using CPT-MeOH as a starting point is have resulted in much stronger therapeutic action in comparison to similar constructs based on SN38. The inventors have surprisingly discovered that this relatively small change in the pharmacophore structure greatly enhanced tumor selectivity, which translated into a much more durable antitumor effect and markedly extended survival in models of recurrent multidrug-resistant NGB. This makes the new technology of the present invention highly relevant to the goal of developing clinically viable new treatment modalities for more efficient and safer management of the aggressive disease that presently lacks curative salvage treatment options. The inventors have developed specific examples showing the synthesis of several NET-targeted CPT-MeOH based prodrugs, both as small molecules and in a multi-arm PEG-linked form (e.g., PEG-[CPT-MeOBG]x, wherein x may be 2-10). The small-molecule prodrugs may be conjugates of CPT-MeOH with aralkylguanidino- derivatized acids, and examples of the small-molecule prodrugs include oxyhexanoic, oxyethoxypropanoic, oxypropoxyacetic, and oxyethoxyethoxypropanoic. In particular, the inventors have obtained the key results confirming the superior performance of the prodrugs designed with CPT-MeOH as the bioactive entity using the 8-arm PEG-linked prototype prodrug with oxyhexanoyl linker and benzylguanidine (BF) as the targeting ligand (PEG-[CPT-MeOBG]s, polymer 9a, Scheme 3).

[0028] The present invention provides a macromolecular prodrug. In the macromolecular prodrug, 7-hydroxymethylcamptothecin (CPT-MeOH) is covalently bonded to a polymer via ester bonds that are labile under physiological conditions. The physiological conditions may comprise a temperature of 20-25°C and / or a pH of 7.0- 7.5. The CPT-MeOH may be functionalized with at least one norepinephrine transporter (NET) ligand.

[0029] The polymer may be a poloxamer. Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). The total number of chains of polyoxyethylene may range from 2 to 130. The number of oxypropylene units may range from 15-67. Preferably, the molecular weight of the poloxamers is below the threshold of glomerular filtration, for example, 30-50 kDa. These polymers have a history of safe use in humans and are available as pharmaceutical grade materials (Kolliphor® P). A number of poloxamers have been approved by FDA as excipients and are currently in clinical use for a variety of applications. All of these poloxamers are suitable for the embodiments described herein.

[0030] The biologically relevant properties of poloxamers, such as molecular size and hydrophilic / lipophilic balance, are controlled through adjusting the lengths of the hydrophilic (A) and hydrophobic (B) blocks [A = poly(ethylene oxide) (PEO) and B = poly(propylene oxide) (PPO)], and their molar ratio. Unlike chemically homogeneous poly(ethylene oxides), the ABA triblock poloxamers combining intermediate lengths of the middle PPO blocks with comparatively high hydrophylic / lipophilic balance values are capable of stably associating with cell membranes, which provides an effective mechanism for tumor penetration and for extending intratumoral presence. Examples of poloxamers include Kolliphor® P188, P338 and P407. The polymer may also be a poloxamine. Poloxamines are nonionic surface active polymers similar to poloxamers in chemistry and design but having a branched structure with 4 arms. The polymer may be a polyethylene glycol (PEG) polymer. PEG polymers are well-known in the art. PEG polymers may be linear and represented by the formula H-(O-CH2-CH2)n-OH. The PEG polymer may be a multi-arm polymer. Multi-arm PEG polymers may have 3-10 PEG chains, for example, 4 chains, emanating from a central core group. Examples of the central core groups include a pentaerythritol group, a dipentaerythritol group, a tripentaerythritol group, and a hexaglycerol group. The PEG polymers may have a molecular weight of 1,000 to 100,000 daltons, inclusive of all values and subranges therebetween including 2,000, 5,000, 10,000, 25,000, 35,000, 50,000, 75,000 and 85,000 daltons.

[0031] The ester bonds between the CPT-MeOH and the polymer may be oxyacetate ester bonds. The CPT-MeOH may be bonded to the PEG polymer via a hydroxyl group at the position corresponding to position 20 in camptothecin.

[0032] The NET ligand may be phenethylguanidine, benzylguanidine (BG) or tyramine.

[0033] The NET ligand may be covalently bonded to the CPT-MeOH via an ester bond that is labile under physiological conditions. The physiological conditions may comprise a temperature of 20-25°C and / or a pH of 7.0-7.5.

[0034] The ester bond between the NET ligand and the CPT-MeOH may be an oxyhexanoyl ester, an oxyethoxypropanoyl ester, an oxy propoxya cetyl ester, or an oxyethoxyethoxypropanoyl ester. The ester bond between the NET ligand and the CPT- MeOH may be an oxyhexanoyl ester.

[0035] The polymer may have multi-arms. A multi-arm polymer may have about 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 chains. A multi-arm polymer may have 4, 6 or 8 arms. The arms in a multi-arm polymer may vary in length. Each arm in a multi-arm polymer may have about 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 length. The arms in a multi-arm polymer may be emanating from a central core group. Examples of the central core groups in a multi-arm polymer include a pentaerythritol group, a dipentaerythritol group, a tripentaerythritol group, and a hexaglycerol group. Each arm of the multi-arm polymer may be bound to at least one molecule of the CPT- MeOH. Multiple molecules of the CPT-MeOH may be covalently bonded to the multi-arm polymer. About 3, 4, 5, 6, 7, 8, 9 or 10 molecules of the CPT-MeOH may be bounded to the multi-arm polymer. For example, 8 molecules of the CPT-MeOH may be covalently bonded to the multi-arm polymer. The polymer may be a multi-arm PEG polymer. A multi-arm PEG polymer may have about 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. A multi-arm PEG polymer may have 4, 6 or 8 arms, also referred to as chains. The arms in a multiarm PEG polymer may vary in length. Each arm in a multi-arm PEG polymer may be have about 100-100,000, 1,000-10,000, 1,000-7,500, 1,000-5,000, 1,000-2,500,

[0036] 2.500-10,000, 2,500-7,500, 2,500-5,000, 5,000-10,000, 7,500-10,000, 2,000-3,000,

[0037] 4.500-5,500, 7,000-8,000, or 9,500-10,500 units. The arms in a multi-arm PEG polymer may have the same or different length. The arms in a multi-arm PEG polymer may be emanating from a central core group. Examples of the central core groups in a multi-arm PEG polymer include a pentaerythritol group, a dipentaerythritol group, a tripentaerythritol group, and a hexaglycerol group. Each arm of the multi-arm PEG polymer may be bound to at least one molecule of the CPT-MeOH. Multiple molecules of the CPT-MeOH may be covalently bonded to the multi-arm PEG polymer. About 3, 4, 5, 6, 7, 8, 9 or 10 molecules of the CPT-MeOH may be bounded to the multi-arm PEG polymer. For example, 8 molecules of the CPT-MeOH may be covalently bonded to the multi-arm PEG polymer.

[0038] The CPT-MeOH may be covalently bonded to a poloxamer polymer via ester bonds that are labile under physiological conditions (e.g., 22°C, pH = 7.2). In one embodiment, the ester bonds are oxyacetate ester bonds. The CPT-MeOH may be bonded to the poloxamer polymer via a hydroxyl group at the position corresponding to position 20 in camptothecin.

[0039] The CPT-MeOH may be covalently bonded to a PEG polymer via ester bonds that are labile under physiological conditions (e.g., 22°C, pH = 7.2). In one embodiment, the ester bonds are oxyacetate ester bonds. The CPT-MeOH may be bonded to the PEG polymer via a hydroxyl group at the position corresponding to position 20 in camptothecin.

[0040] In one embodiment, the macromolecular prodrug is PEG-[CPT-MeOBG]x, which is represented by the following structure:

[0041] wherein the polymer may be in the free base or an ionized (salt) form - most typically chloride, sulfate or trifluoroacetate.

[0042] In another embodiment, the macromolecular prodrug is PEG-[CPT-MeOBG]s, which is represented by the following structure: wherein n = 100 in average, and TP is tripentaerythritol core having the structure of

[0043] For each CPT-MeOH based macromolecular prodrug of the present invention, a method of treating neuroblastoma in a subject in need thereof is provided. The neuroblastoma treatment method comprises administering to the subject an effective amount of the CPT-MeOH based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]s). The subject may be a mammal, for example, a human.

[0044] For each CPT-MeOH based macromolecular prodrug of the present invention, a method of treating a solid tumor in a subject in need thereof is provided. The solid tumor treatment method comprises administering to the subject an effective amount of the CPT-MeOH based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]s). The subject may be a mammal, for example, a human.

[0045] For each CPT-MeOH based macromolecular prodrug of the present invention, a method of treating a brain tumor in a subject in need thereof is provided. The brain tumor treatment method comprises administering to the subject an effective amount of the CPT-MeOH based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]s). The subject may be a mammal, for example, a human.

[0046] For each macromolecular prodrug of the present invention, a method of treating cancer in a subject in need thereof is provided. The cancer treatment method comprises administering the subject an effective amount of the CPT-MeOH based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]s). The subject may be a mammal, for example, a human.

[0047] The term "an effective amount" as used herein refers to an amount of the CPT- MeOH based macromolecular prodrug of the present invention (e.g., PEG-[CPT- MeOBG]s) required to achieve a stated goal, for example, treating neuroblastoma, solid tumor, brain tumor and / or cancer in a subject. The effective amount of the CPT-MeOH based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]s) may vary depending upon the stated goal, the physical characteristics of the subject, the nature and severity of the neuroblastoma, solid tumor, brain tumor and / or cancer, existence of related or unrelated medical conditions, the nature of the CPT-MeOH based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]s), the means of administering the CPT-MeOH based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]s) to the subject, and the administration route. A specific dose for a given subject may generally be set by the judgment of a physician. The dosage of the CPT-MeOH based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]s) may be from 0.5 to 200 mg per kg of body weight of the subject per dose. The CPT-MeOH based macromolecular prodrug (e.g., PEG-[CPT- MeOBG]s) may be administered to the subject in one or multiple doses.

[0048] For each treatment method of the present invention, the CPT-MeOH based macromolecular prodrug (e.g., PEG-[CPT-MeOBG]s) may be administered by any method commonly used in the art. Methods of administration include parenteral (e.g., intravenous, intramuscular, and subcutaneous), oral, nasal, ocular, transmucosal (e.g., buccal, vaginal, and rectal), and transdermal routes of administration.

[0049] The term "about" as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate. Example 1. Norepinephrine transporter-targeted derivatives of 7- hydroxymethylcamptothecin 6-[(4-N-Boc-aminomethyl)phenoxy]hexanoic acid (2a, Scheme 1). 4-Hydroxy- N-Boc-benzylamine (la, 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 traces of water. The residue was dissolved in dry 1-methylpyrrolidinone (1-MP, 12 mL) under flow of argon, cooled in ice, and a IM solution of potassium tert-butoxide in tetra hydrofuran (THF) (Sigma-Aldrich, 7.0 mL, 7.0 mmol) was added in 1 min. via syringe. The mixture was stirred under argon in an ice bath for 0.5 h, then at room temperature for 27 h. Aqueous IM H3PO4 (60 mL) and ethyl acetate (30 mL) were added, and after a vigorous stirring, the organic phase was separated, washed with IM H3PO4 (60 mL), with water (4x 10 mL), filtered and dried (RE, 30 °C). The residue (1.151 g) was solidified by trituration with pentane (10 mL), the solid was filtered off, washed with pentane, and purified by precipitation from dichloromethane with pentane. Yield 713 mg (72%).

[0050] 6-[(4-N-Boc-2-aminoethyl)phenoxy]hexanoic acid (2b, Scheme 1). The acid 2b was prepared from N-Boc-tyramine (lb, TCI, >97%) similarly to 2a, yield 87%.

[0051] Scheme 1

[0052] Conjugate of 6-[(4-N-Boc-aminomethyl)phenoxy]hexanoic acid with 7- hydroxymethylcamptothecin, (4a, Scheme 2). 7-Hydroxymethylcamptothecin (3, Scheme 2, ca. 95% pure, 240 mg, 0.60 mmol, prepared as described in [1]), 4-N,N- dimethylaminopyridine tosylate (DPTS-catalyst, 600 mg, 2.04 mmol) and dry pyridine (48 mL) were heated at reflux under argon for a few minutes to a complete dissolution of 3. The solution was rapidly cooled to 35 °C, the acid 2a (324 mg, 0.96 mmol) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Sigma-Aldrich, 612 mg, ca. 3.1 mmol) were added. With the argon protection continued, the mixture was stirred at room temperature for 44 h and dried in vsacuo at 30 °C. Aqueous 1.6M NahhPCk containing 0.08M H3PO4 (180 mL) and ethyl acetate (180 mL) were added. After shaking, the organic layer was separated, washed with 2.5M NaCI (3x200 mL), with water (2x60 mL) and dried (RE, 30 °C). The crude product (594 mg) was purified by flash chromatography (silica gel, chloroform - acetonitrile 5: 1 to 2: 1), yielding 234 mg (56%) of 4a.

[0053] Conjugate of 6-[(4-N,N'-di-Boc-guanidomethyl)phenoxy]hexanoic acid with 7- hydroxymethylcamptothecin, (5a, Scheme 2). The conjugate 4a (234 mg, 0.33 mmol) was dissolved in dry 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 left at room temperature for 0.5 h protected with argon. The solvents were removed in vacuo at room temperature, the residual syrup was co-evaporated as above with benzene (5 mL), with chloroform (5 mL), twice with a mixture of chloroform (3 mL) and THF (3 mL), suspended in a mixture of chloroform (15 mL) and THF (15 mL), and dried on RE at 30 °C. The mostly solid residue (379 mg) was suspended in a mixture of dichloromethane (1.5 mL) and THF (1.5 mL), diisopropylethylamine (DIPEA, Sigma- Aldrich, 99.5%, 0.30 mL, 1.71 mmol) was added followed by l,3-di-Boc-2- (trifluoromethylsulfonyl)guanidine (Acros, 98%, 215 mg, 0.55 mmol). The mixture (homogenizing in ca.0.5 h) was stirred under argon at room temperature for 32 h. The solvents were removed (RE, 30 °C), aqueous 1.6M NaH2PO4 containing 0.08M H3PO4 (30 mL) was added to the residue, the solid material was filtered off, washed with water (35 mL), dried under suction, dissolved in chloroform, filtered and dried (RE, 30 °C). The residue (477 mg) was purified by flash chromatography (silica gel, chloroform - acetonitrile 10: 1 to 4: 1), yielding 240 mg (85%) of 5a.XH NMR (400 MHz, CDCI3) corresponds to the structure of 5a, clearly indicating the attachment of carboxyl to the 7-CH2OH of 3, whereas the 20-OH remains not acylated, noticeable at 3.81 ppm (s).

[0054] Conjugate of 6-[(4-guanidomethyl)phenoxy]hexanoic acid with 7- hydroxymethylcamptothecin, trifluoroacetate salt (6a, Scheme 2). The conjugate 5a (77 mg, 0.092 mmol) was dissolved in dry dichloromethane (1.6 mL). Anisole (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 left at room temperature for 2.5 h protected with argon. The solvents were removed in vacuo at room temperature, the residual syrup was co-evaporated as above with chloroform (3x6 mL) and stirred with hexane (30 mL) until disappearance of turbidity. The supernatant was decanted off, the residue was washed with hexane (3x5 mL) and dried. Methyl tert-butyl ether (MTBE, 5 mL) and trioctylamine (Msynth®plus, >93%, 0.2 mL, ca. 0.42 mmol) were added, the residue was triturated with the solvent, the suspension was left at room temperature to a complete solidification, the solid was filtered off, washed with MTBE (15 mL), with hexane (15 mL) and dried. The crude salt was dissolved in a 5: 1 mixture of chloroform and 2-propanol, the solution was filtered, dried (RE, 30 °C), the residue was solidified by co-evaporations with dichloromethane, suspended in dichloromethane (2 mL). filtered off, washed with dichloromethane (3 mL), with hexane (10 mL) and dried in vacuo. Yield 64 mg (92%).1H NMR (400 MHz, DMSO-de) corresponds to the structure of 6a.

[0055] Scheme 3 Conjugate of 8-arm-PEG with 7-hydroxymethylcamptothecin acylated with 6- [(4-guanidomethyl)phenoxy]hexanoic acid, trifluoroacetate salt (polymer 9a, Scheme 3). Carboxylated 8-arm-PEG (polymer 7, Scheme 3, JenKem Technology, Mn= 37390 Da, 400 mg, 0.085 mmol of carboxylic groups), the conjugate 5a (214 mg, 0.255 mmol), DPTS-catalyst (200 mg, 0.68 mmol) and dichloromethane (4 mL) were stirred for 5 min. to homogenization, and N,N'-dicyclohexylcarbodiimide (DCC, Aldrich, 99%, 303 mg, 1.45 mmol) was added. The mixture was stirred at room temperature under argon for 96 h. The solvent was removed (RE, room temperature), the residue was coevaporated (RE, 30 °C) with benzene (20 mL) and suspended in benzene (10 mL). Undissolved products were 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 with diethyl ether (40 mL) from small volumes of benzene (2 - 3 mL) until TLC (silica gel, chloroform - acetonitrile 3: 1) failed to detect any mobile compounds. Finally, the polymer was dissolved in a 4: 1 mixture of benzene - dichloromethane (30 mL) and washed with 22% aqueous solution of Na?SO4 (3x20 mL) to remove any residual DPTS-catalyst. After drying over Na2SC>4 and filtering from the desiccant, the solution was concentrated (RE, 30 °C) to 3 g, the polymer 8a was solidified as above with diethyl ether, filtered off and dried in vacuo. To remove the protective Boc-groups from the guanidine moieties, the polymer 8a (475 mg) was dissolved in dry dichloromethane (4 mL). Anisole (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, the mixture was left at room temperature for 3.5 h. The volatiles were removed (RE, < 25 °C), the residue was co-evaporated as above with chloroform (2x 10 mL), with benzene (5 mL), and evacuated at room temperature and < 1 mm Hg for 1 h. Diethyl ether (30 mL) and trioctylamine (Msynth®plus, >93%, 0.6 mL, ca. 1.28 mmol) were added, the residue was triturated to solidification, the polymer was filtered off, washed with diethyl ether, dried and precipitated with diethyl ether from a small amount of benzene. After drying in vacuo, yield of 9a was 448 mg.1H NMR (400 MHz, CDCI3) of 9a detected 0.19 mmol / g (14.3% by weight) of bound conjugate 6a. Scheme 3 (2ndvariant)

[0056] Conjugate of (4-N-Boc-aminomethyl)phenylacetic acid with 7- hydroxymethylcamptothecin, (4b, Scheme 4). 7-Hydroxymethylcamptothecin (3, ca. 95%, 100 mg, 0.25 mmol), 4-N,N-dimethylaminopyridine tosylate (DPTS-catalyst, 250 mg, 0.85 mmol) and dry pyridine (20 mL) were heated at reflux under argon for a few minutes to a complete dissolution of 3. The solution was rapidly cooled to 35 °C, (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, ca. 1.3 mmol) were added. With the argon protection continued, the mixture was stirred at room temperature for 47 h and dried in vacuo at 20 - 25 °C. Aqueous 1.6M NahhPCk containing 0.08M H3PO4 (25 mL) and water (7 mL) were added, the residue was well suspended, filtered off, washed with water (25 mL), and dried in vacuo. The crude product (224 mg) was purified by flash chromatography (silica gel, chloroform - acetonitrile 5: 1 to 2: 1), yielding 117 mg (75%) of 4b.

[0057] Conjugate of (4-N,N'-di-Boc-guanidomethyl)phenylacetic acid with 7- hydroxymethylcamptothecin, (5b, Scheme 4). The conjugate 4b (117 mg, 0.18 mmol) was dissolved in dry 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 left at room temperature for 0.5 h protected with argon. The solvents were removed in vacuo at room temperature, the residual syrup was co-evaporated as above with chloroform (5 mL) and dried in vacuo. The residue (215 mg) was suspended in a mixture of dichloromethane (1.1 mL) and THF (1.1 mL), diisopropylethylamine (DIPEA, Sigma- Aldrich, 99.5%, 0.38 mL, 2.17 mmol) was added followed by l,3-di-Boc-2- (trifluoromethylsulfonyl)guanidine (AmBeed, 95%, 152 mg, 0.37 mmol). The mixture (homogenizing in ca.0.5 h) was stirred under argon at room temperature for 44 h. The solvents were removed (RE, 30 °C), aqueous 1.6M NaFhPCk containing 0.08M H3PO4 (25 mL) was added to the residue, the solid material was filtered off, washed with water (35 mL) and dried in vacuo. The residue (272 mg) was purified by flash chromatography (silica gel, chloroform - acetonitrile 10: 1 to 3: 1), yielding 118 mg (83%) of 5b.1H NMR (400 MHz, CDCI3) corresponds to the structure of 5b, with the signal of 20-OH at 3.77 ppm (s).

[0058] Conjugate of (4-guanidomethyl)phenylacetic acid with 7- hydroxymethylcamptothecin, trifluoroacetate salt (6b, Scheme 4). The conjugate 5b (71 mg, 0.092 mmol) was dissolved in dry dichloromethane (1.5 mL). Anisole (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 left at room temperature for 2.5 h protected with argon. The solvents were removed in vacuo at room temperature, the residual syrup was co-evaporated as above with chloroform (3x6 mL) and stirred with hexane (30 mL) for 10 min. The supernatant was decanted off, the residue was washed with hexane (3x5 mL) and dried. Dichloromethane (2.5 mL), trioctylamine (Msynth®plus, >93%, 0.25 mL, ca. 0.53 mmol) and methyl tert-butyl ether (MTBE, 3 mL) were added, the residue was triturated until a complete suspending, the solvents were removed (air flow), the residue was suspended in MTBE (7 mL) and left at room temperature for 3 h. The solid was filtered off, washed with MTBE (10 mL), with hexane (10 mL) and dried. The crude salt was dissolved in a 5: 1 mixture of chloroform and 2-propanol, the solution was filtered, dried (RE, 30 °C), the residue was solidified by co-evaporations with chloroform and dichloromethane, suspended in dichloromethane (3 mL). filtered off, washed with dichloromethane (3 mL), with pentane (10 mL) and dried in vacuo. Yield 59 mg (93%).1H NMR (400 MHz, DMSO-de) corresponds to the structure of 6b.

[0059] Conjugate of 3-(4-(l-Boc-piperidin-4-yl)oxy)phenyl)propanoic acid with 7- hydroxymethylcamptothecin, (4c, Scheme 5). The conjugate 4c was obtained from 7- hydroxymethylcamptothecin (3, ca. 95%, 100 mg, 0.25 mmol) and 3-(4-(l-Boc- piperidin-4-yl)oxy)phenyl)propanoic acid (AmBeed, 95%, 124 mg, 0.40 mmol) similarly to the conjugate 4b. The crude product (251 mg) was purified by flash chromatography (silica gel, chloroform - acetone 10: 1 to 4: 1), yielding 124 mg (70%) of 4c. Di-Boc-guanidine conjugate 5c (Scheme 5). The conjugate 4c (124 mg, 0.17 mmol) was dissolved in dry 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 left at room temperature for 0.5 h protected with argon. The solvents were removed in vacuo at room temperature, the residual syrup was co-evaporated as above with chloroform (5 mL) and dried in vacuo. The residue (203 mg) was suspended in a mixture of dichloromethane (1.1 mL) and THF (1.1 mL), diisopropylethylamine (DIPEA, Sigma- Aldrich, 99.5%, 0.38 mL, 2.17 mmol) was added followed by l,3-di-Boc-2- (trifluoromethylsulfonyl)guanidine (AmBeed, 95%, 152 mg, 0.37 mmol). The mixture was stirred under argon at room temperature for 119 h, failing to become homogeneous. The solvents and the excess of DIPEA were removed in vacuo, the residue was re-suspended in a mixture of dichloromethane (1.5 mL) and 2-propanol (1.0 mL), DIPEA (0.30 mL, 1.71 mmol) was added, the stirring at room temperature was continued for 215 more h. Treatment as for 5b followed by flash chromatography (silica gel, chloroform - acetone 20: 1 to 4: 1) yielded 101 mg (ca. 66%) of 5c, which according to1H NMR contained ca.3% of N,N'-di-Boc-urea. Without an additional purification, the compound was used for the further preparation.

[0060] Guanidine trifluoroacetate conjugate 6c (Scheme 5). The compound 6c was prepared by deprotection of 5c (101 mg, ca. 0.11 mmol) as described for the conjugate 6b. Yield 81 mg (ca. 92%).1H NMR (400 MHz, DMSO-de) corresponds to the

[0061] Conjugate of (4-N-Boc-aminomethyl)phenoxyacetic acid with 7- hydroxymethylcamptothecin, (4d, Scheme 6). The compound 4d was prepared from 7- hydroxymethylcamptothecin (3, ca. 95%, 100 mg, 0.25 mmol) and (4-N-Boc- aminomethyl)phenoxyacetic acid (AmBeed, 95%, 118 mg, 0.40 mmol) similarly to the conjugate 4a . The crude product (219 mg) was purified by flash chromatography (silica gel, chloroform - acetone 8: 1 to 3: 1), yielding 106 mg (66%) of 4d.

[0062] Conjugate of (4-N,N'-di-Boc-guanidomethyl)phenoxyacetic acid with 7- hydroxymethylcamptothecin, (5d, Scheme 4). The conjugate 4d (106 mg, 0.16 mmol) was dissolved in dry 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 left at room temperature for 0.5 h protected with argon. The solvents were removed in vacuo at room temperature, the residual syrup was co-evaporated as above with chloroform (5 mL) and dried in vacuo (< 1 mm Hg). The residue (163 mg) was suspended in a mixture of dichloromethane (3.0 mL) and THF (1.0 mL), diisopropylethylamine (DIPEA, Sigma-Aldrich, 99.5%, 0.38 mL, 2.17 mmol) was added followed by l,3-di-Boc-2- (trifluoromethylsulfonyl)guanidine (AmBeed, 95%, 152 mg, 0.37 mmol). The mixture (homogenizing in ca.l h) was stirred under argon at room temperature for 51 h. The solvents were removed (RE, 30 °C), aqueous 1.6M NaFhPCk containing 0.08M H3PO4 (25 mL) was added to the residue, the solid material was filtered off, washed with 5M aqueous NaCI (35 mL), with water (20 mL) and dried in vacuo. The residue (238 mg) was purified by flash chromatography (silica gel, chloroform - acetone 10: 1 to 5: 1), yielding 93 mg (72%) of 5d.1H NMR (400 MHz, CDCI3) corresponds to the structure of 5d, with the signal of 20-OH at 3.79 ppm (s).

[0063] Conjugate of (4-guanidomethyl)phenoxyacetic acid with 7- hydroxymethylcamptothecin, trifluoroacetate salt (6d, Scheme 6). The conjugate 5d (92 mg, 0.12 mmol) was dissolved in dry dichloromethane (1.3 mL). Anisole (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 left at room temperature for 2.5 h protected with argon. The solvents were removed in vacuo at room temperature, the residual syrup was co-evaporated as above with chloroform (3x6 mL) and stirred with hexane (30 mL) for 10 min. The supernatant was decanted off, the residue was washed with hexane (3x 10 mL) and dried. Dichloromethane (2.5 mL) and trioctylamine (Msynth®plus, >93%, 0.30 mL, ca. 0.63 mmol) were added, the residue was triturated until a complete suspending, the solvent was removed (air flow), the residue was suspended in MTBE (12 mL) and left at room temperature for 3 h. The solid was filtered off, washed with MTBE (15 mL), with 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), the residue was solidified by co-evaporations with dichloromethane, suspended in dichloromethane (5 mL). filtered off, washed with dichloromethane (7 mL), with pentane (12 mL) and dried in vacuo. Yield 79 mg (97%).XH NMR (400 MHz, DMSO-de) corresponds to the

[0064] Conjugate of 6-[(4-N-Boc-2-aminoethyl)phenoxy]hexanoic acid with 7- hydroxymethylcamptothecin, (4e, Scheme 7). The conjugate 4e was obtained from 7- hydroxymethylcamptothecin (3, ca. 95%, 100 mg, 0.25 mmol) and 6-[(4-N-Boc-2- aminoethyl)phenoxy]hexanoic acid_(2b, 141 mg, 0.40 mmol) similarly to the conjugate 4a. The crude product (226 mg) was purified by flash chromatography (silica gel, chloroform - acetone 10: 1 to 4: 1), yielding 125 mg (70%) of 4e.

[0065] Conjugate of 6-[(4-N,N'-di-Boc-guanidoethyl)phenoxy]hexanoic acid with 7- hydroxymethylcamptothecin, (5e, Scheme 7). The conjugate 5e was prepared from the compound 4e (125 mg, 0.17 mmol) as described for preparation of 5a. The crude product was purified by flash chromatography (silica gel, chloroform - acetone 20: 1 to 5: 1), yielding 121 mg (81%) of 5e.XH NMR (400 MHz, CDCI3) corresponds to the structure of 5e, with the signal of 20-OH at 3.76 ppm (s).

[0066] Conjugate of 6-[(4-guanidoethyl)phenoxy]hexanoic acid with 7- hydroxymethylcamptothecin, trifluoroacetate salt (6e, Scheme 7). The compound 6e was prepared by deprotection of 5e (103 mg, 0.12 mmol) as described for the conjugate 6d. Yield 88 mg (94%).XH NMR (400 MHz, DMSO-de) corresponds to the structure of 6e.

[0067] Example 2. Drug biodistribution and tumor uptake

[0068] A study was performed to evaluate intratumoral drug levels and organ distribution (24 hr) of CPT-MeOH delivered as PEG-[CPT-MeOBG]s, in comparison to SN38 administered either as analogously constructed PEG-[SN38-BG]s or PEG-[SN38]s (FIG. 1). The analysis was carried out in an orthotopic model of neuroblastoma. Athymic nude nu / nu') mice (n = 4) were implanted in the suprarenal fat pad with multidrug-resistant, ATRX-mutant SK-N-MM cells stably expressing firefly luciferase (106per animal). Tumors were allowed to reach the size of 1.0±0.4 cm3under the control of bioluminescent imaging. Polymeric conjugates were administered intravenously at doses equivalent to 10 mg / kg of CPT-MeOH or SN38. Tissue samples harvested 24 hr post injection were homogenized at 4°C, and analyzed using a fluorimetric assay after extraction with acetonitrile. Data shown as mean ± SD.

[0069] Example 3. Tumor growth inhibitory activity

[0070] Tumor growth inhibitory activity of PEG-[CPT-MeOBG]s was evaluated in two orthotopic models of recurrent ATRX-mutant neuroblastoma established as above with CHLA90 (FIG. 2) and SK-N-MM cells (FIG. 3), respectively. Treatments were administered intravenously once a week over 8 and 4 weeks, respectively, at a dose equivalent to 10 mg of CPT-MeOH per kg. PEG-[SN38-BG]s (given once a week at the equivalent dose) and irinotecan (given twice a week at 30 mg drug per kg) were included as controls. Tumor-associated signal was monitored by quantitative bioluminescence (data shown as mean ± SD, n = 5). Animal survival over time in respective groups is shown for CHLA90 xenograft-bearing animals in FIG. 2.

[0071] Although the invention is illustrated and described herein with reference to specific embodiments, the invention is not intended to be limited to the details described above. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the invention.

Claims

WHAT IS CLAIMED:

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

2. The macromolecular prodrug of Claim 1, wherein the polymer is a poloxamer.

3. The macromolecular prodrug of Claim 2, wherein the polymer is a polyethylene glycol (PEG) polymer.

4. The macromolecular prodrug of Claim 3, wherein the polymer is a multiarm PEG polymer.

5. The macromolecular prodrug of Claim 1, wherein the polymer is a poloxamine.

6. The macromolecular prodrug of Claims 1-5, wherein the ester bonds between the CPT-MeOH and the polymer are oxyacetate ester bonds.

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

8. The macromolecular prodrug of 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 labile under physiological conditions.

9. The macromolecular prodrug of Claim 8, wherein the ester bond between the at least one NET ligand and the CPT-MeOH is selected from the group consisting of an oxyhexanoyl ester, an oxyethoxypropanoyl ester, an oxypropoxyacetyl ester, and an oxyethoxyethoxypropanoyl ester.

10. The macromolecular prodrug of any one of Claims 1-9, which is PEG- [CPT-MeOBG]s having the following structure:wherein : n = 100 in average andTP is11. A method of treating neuroblastoma in a subject in need thereof, comprising administering to the subject an effective amount of the macromolecular prodrug of any one of Claims 1-10.

12. A method of treating a solid tumor in a subject in need thereof, comprising administering to the subject an effective amount of the macromolecular prodrug of any one of Claims 1-10.

13. A method of treating a brain tumor in a subject in need thereof, comprising administering to the subject an effective amount of the macromolecular prodrug of any one of Claims 1-10.

14. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the macromolecular prodrug of any one of Claims 1-10.

15. The method of any one of Claims 11-14, wherein the subject is a human.