Scalable thyrointegrin antagonist compositions with improved blood-brain barrier penetration and retention in brain tumors

Abstract

Chemical compounds / compositions, methods for their synthesis, and methods of use are provided. The compounds / compositions are directed to thyrointegrin antagonists conjugated to polymers. The compounds / compositions further have additional substituents conjugated to the polymer. The compounds / compositions demonstrate enhanced uptake across the blood-brain barrier and retention within tumors with enhanced retention. The compounds / compositions may also include improved scalability, improved purity, improved aqueous solubility, solid forms, or intermediates. The compounds / compositions demonstrate improved antiangiogenic efficacy and improved efficacy against diseases, such as glioblastoma (GBM), which are particularly susceptible to blood-brain barrier permeability.

Description

[Technical Field]

[0001] The present disclosure generally relates to improved thyroid hormone receptor antagonist (referred to as "thyrointegrin antagonists") compounds, as well as compositions comprising the same, methods of using these compounds and compositions to treat disease, and synthetic methods. More specifically, the disclosure relates to compounds comprising an alpha-V-beta-3 (αvβ3) integrin thyroid hormone receptor antagonist conjugated to a polymer, where the polymer is also conjugated to an additional substituent or functional group. The disclosed compounds and compositions utilizing the compounds have improved blood-brain barrier permeability and retention, improved synthetic scalability, aqueous solubility, and / or solid product or intermediates. The compounds are also amenable to readily scalable purification, for example, by normal-phase chromatography. Due to their enhanced permeability across the blood-brain barrier and retention in brain tumors, the disclosed compositions and compounds are particularly effective in treating certain diseases, such as glioblastoma, glioma, astrocytoma, CNS lymphoma, medulloblastoma, meningioma, metastatic brain tumors, pituitary tumors, primitive neuroectodermal tumors (PNETs), and other brain-related diseases. [Background technology]

[0002] Integrins are a superfamily of cell surface adhesion receptors that regulate cell attachment to both the extracellular matrix (ECM) and other cells in the solid extracellular environment. Adhesion is essential for cells, providing anchorage, cues for migration, and signals for proliferation and differentiation. Integrins are directly involved in numerous normal and pathological conditions, making them important targets for therapeutic intervention. Integrins are integral transmembrane proteins and heterodimers whose binding specificity varies depending on which of their 14 α chains are combined with which of their 8 β chains. Integrins are classified into four overlapping subfamilies, containing β1, β2, β3, or αv chains. Cells can express several different integrins from each subfamily. Over the past few decades, integrins have been shown to be suitable targets for therapeutic intervention because they are the primary receptors involved in cell adhesion. Integrin αvβ3 regulates cell proliferation and survival, as ligation of this receptor can induce apoptosis in tumor cells under certain circumstances. Disruption of cell adhesion with anti-αvβ3 antibodies, RGD peptides, and other integrin antagonists (such as the cyclic peptide cilengitide, which failed in phase 3 clinical trials in glioblastoma due to limited blood-brain barrier permeability and brain tumor retention) has been shown to slow tumor growth.

[0003] Applicant has previously disclosed compounds and compositions comprising non-cleavable polymers conjugated to αβ integrin antithyroid agents, and related methods, for example, in U.S. Patent Application No. 15 / 616,637 (now U.S. Patent No. 10,201,616) and U.S. Patent Application No. 16 / 223,176, the entire contents of both of which are incorporated herein by reference.

[0004] Additionally, Applicant has previously disclosed compounds, compositions, and methods comprising an αβ integrin antithyroid agent and targeting the norepinephrine transporter or catecholamine transporter (e.g., benzylguanidine or derivative), and related methods, e.g., in U.S. Patent Application No. 15 / 950,870 (now U.S. Patent No. 10,328,043) and U.S. Patent Application No. 16 / 398,342, the entire contents of both of which are incorporated herein by reference.

[0005] While the compounds, compositions, and methods disclosed in these prior applications and issued patents represent improvements over the existing state of the art, such compounds and compositions may suffer from one or more drawbacks, including low permeability through the blood-brain barrier, poor synthetic scalability, lack of aqueous solubility, and lack of formation of solid products or intermediates. Purification can also currently be difficult. The disclosed compounds, and compositions containing these compounds, represent an improvement in the field and demonstrate unexpected efficacy in the treatment of glioblastoma, other brain tumors, and similar diseases.

[0006] Blood-brain barrier permeability is important for targeting certain diseases, such as glioma, meningioma, pituitary adenoma, vestibular schwannoma, and medulloblastoma. Glioblastoma (glioblastoma multiforme or GBM) is a specific example of a disease that requires blood-brain barrier permeability for effective treatment. It is well known in the art that drug delivery methods with improved blood-brain barrier permeability would be advantageous (see, for example, Bhowmik A, Khan R, Ghosh MK. Blood Brain Barrier: A Challenge for Effective Therapy of Brain Tumors. BioMed Research International, Volume 2015; Upadhyay RK. Drug Delivery Systems, CNS Protection, and the Blood Brain Barrier. BioMed Research International, Volume 2014). The improved compounds, compositions, and methods described herein demonstrate improved blood-brain barrier permeability and exhibit significantly improved efficacy for this type of disease. Furthermore, the improved compounds, compositions, and methods described herein demonstrate improved retention in the brain, particularly at the site of tumors located within the brain. This improved retention provides further enhanced efficacy in treating such diseases. The improved compounds, compositions, and methods described herein also demonstrate improved scalability and solubility, and may result in solid products or intermediates.

[0007] Compounds or compositions as described herein, including αβ integrin thyroid hormone receptor antagonists (thyrointegrin antagonists), and having the described improved blood-brain barrier penetration and retention properties would be appreciated in the art, as would methods of treatment using such compounds and / or compositions. Compounds or compositions having such improved blood-brain barrier penetration properties, along with one or more of the described improved synthetic scalability, aqueous solubility, and / or formation of solid products or intermediates, would also be appreciated in the art. Summary of the Invention [Means for solving the problem]

[0008] According to one aspect of the invention, a compound comprises a thyrointegrin antagonist, a non-biodegradable polymer, a linker covalently attaching the thyrointegrin antagonist to the non-biodegradable polymer via a non-cleavable covalent bond, and a substituent A attached to the non-biodegradable polymer.

[0009] According to another aspect, the compound comprises the general formula: [ka] In the formula, n1≧0, n2 is 5 to 200, and R1 to R4 and R9 are H, Me, Et, iPr, nPr, nBu, iBu, secBu, tBu, C5-C 12 wherein R10-R13 are each independently selected from the group consisting of hydrogen, iodine, and an alkane group; and wherein Y is selected from the group consisting of: n-alkyl, cyclopentyl, cyclohexyl, phenyl, F, Cl, Br, I, CN, CF3, OCF3, CHF2, OCHF2, SO2Me, NO2, -O-alkyl, -O-aryl, -CH2-O-alkyl, -CH2-O-aryl, ester, and amide; [ka]

[0010] According to another embodiment, the compound comprises a thyrointegrin antagonist conjugated to a polymer and a substituted benzyl conjugated to said polymer, wherein said compound penetrates and is absorbed across the blood-brain barrier.

[0011] According to another aspect, a method of treatment includes providing a compound having a thyrointegrin antagonist and a substituted benzyl linked by a polymer and administering a therapeutically effective amount of the compound to a patient in need thereof.

[0012] The patent or application file contains at least one drawing executed in color. Copies of such patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] Some embodiments will be described in detail with reference to the following drawings, in which like designations refer to like elements. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows the general formula of an exemplary compound according to an embodiment of the present invention. [Figure 2] 1 shows a more detailed general formula of an exemplary compound according to an embodiment of the present invention. [Figure 3] 1 shows a more detailed general formula of an exemplary compound according to an embodiment of the present invention. [Figure 4] Exemplary compound 2 is shown below. [Figure 5] Exemplary compound 3 is shown below. [Figure 6] Exemplary compound 4 is shown below. [Figure 7] Exemplary compound 1 is shown below. [Figure 8] An exemplary compound 5 is shown below. [Figure 9A] 2 illustrates an embodiment of Permutation A from FIG. 1, in accordance with an embodiment of the present invention. [Figure 9B] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 9C] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 10A] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 10B] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 10C] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 11] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 12A] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 12B] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 13A] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 13B] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 13C] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 13D] 2 illustrates another embodiment of Permutation A from FIG. 1, according to an embodiment of the present invention. [Figure 14] 1 shows an exemplary synthetic route for compound 1, according to an embodiment of the present invention. [Figure 15] 1 shows an exemplary synthetic route for exemplary compound 2, according to an embodiment of the present invention. [Figure 16] 1 shows an exemplary synthetic route for exemplary compound 3, according to an embodiment of the present invention. [Figure 17] 1 shows an exemplary synthetic route for exemplary compound 4, according to an embodiment of the present invention. [Figure 18] 1 shows an exemplary synthetic route for exemplary compound 5, according to an embodiment of the present invention. [Figure 19]1 shows the expression levels of integrin αvβ3 in glioblastoma cancer cells analyzed by flow cytometry. [Figure 20] 1 shows the results of artificial membrane permeability assay (PAMPA) for exemplary compounds 1-4. [Figure 21] 1 shows the fluorescence intensity of exemplary compound 5 in various organs and brains with and without glioblastoma. [Figure 22] 1 shows the levels of exemplary compound 2 present in brain tissue over time following subcutaneous administration to mice. [Figure 23] Cynomolgus monkeys were given subcutaneous injections of 15 mg / Kg daily for 14 days, and brain tissue was removed and blood samples were taken for analysis by LC / MS / MS to show the plasma and brain levels of exemplary compound 2 over time after subcutaneous administration. [Figure 24] 1 shows the anti-angiogenic effect of exemplary compound 2 in the presence of various growth factors. [Figure 25] The anti-angiogenic effect of the known compound Avastin® on VEGF alone, and its lack on other growth factors, is shown in the presence of various growth factors. [Figure 26] 1 shows the bioluminescent signal of GBM tumors in the brain. [Figure 27] 1 shows the blood-brain barrier uptake of exemplary compound 5 in the brains of GBM tumor-bearing and GBM tumor-free mice. [Figure 28A] 1 shows the blood-brain barrier uptake of exemplary compound 5 in the brains of mice with and without GBM tumors, where exemplary compound 5 was administered with compounds used in humans that could potentially compete for uptake and retention. [Figure 28B] 1 shows the blood-brain barrier uptake of exemplary compound 5 in brain regions with and without GBM tumors where exemplary compound 5 is administered together with compounds used in humans that may potentially compete for uptake and retention. [Figure 29]1 shows the uptake of exemplary compound 5 in the brain of tumor-bearing and tumor-free mice, as well as the accumulation or lack thereof of the drug in other organs. [Figure 30] 1 shows the effect of various doses of exemplary compound 2 on tumor weight in mice bearing GBM xenografts. [Figure 31] 1 shows the effect of various doses of exemplary compound 2 on the intensity of tumor cell luminescent signal in mice bearing GBM xenografts. [Figure 32] 1 shows the effect of exemplary compound 2 at a dose of 6 mg / kg compared to the known compound cilengitide at a dose of 75 mg / kg on tumor weight in mice with GBM xenografts. [Figure 33] 1 shows the effect of exemplary compound 2 at a dose of 6 mg / kg compared to the known compound cilengitide at a dose of 75 mg / kg on tumor cell luminescent signal intensity in mice with GBM xenografts. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed descriptions of the hereafter described embodiments of the disclosed compositions and methods are presented herein, by way of example and not limitation, with reference to the drawings. While particular embodiments are shown and described in detail, it should be understood that various changes and modifications can be made without departing from the scope of the appended claims. The scope of the present disclosure is not limited to the number of components, their materials, their shapes, their colors, their relative arrangements, etc., which are disclosed merely as examples of embodiments of the present disclosure. A more complete understanding of the present embodiments and their advantages will be obtained by reference to the following description in conjunction with the accompanying drawings in which like reference numerals indicate like features.

[0016] Before describing in detail, it should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise. overview

[0017] Embodiments of the present disclosure describe novel chemical compounds, compositions containing the novel chemical compounds, methods for their synthesis, and methods of treatment using such compounds and compositions.

[0018] The compounds disclosed herein (including, but not limited to, exemplary compounds such as Compound 2, Compound 3, Compound 4, and Compound 5, described in detail below, as well as compositions prepared from such compounds) demonstrate improved blood-brain barrier penetration and retention in brain tumors. Additionally, these compounds and their respective compositions show unexpectedly enhanced efficacy for brain tumors and other diseases, such as glioblastoma (GBM).

[0019] The unexpected enhanced efficacy against these diseases may be due to a combination of factors, including active transport across the blood-brain barrier, overexpression of integrin αvβ3 in GBM and similar diseases, and the effect of additional substituents on thyrointegrin antagonists on uptake / retention.

[0020] First, the compounds and compositions described herein include thyrointegrin antagonists. Thyrointegrin antagonists such as those described herein can be subject to active transport across the blood-brain barrier by thyroid-binding proteins. A review of the transport of thyroid hormones and their analogs in the brain can be found in Wirth EK, Schwiezer U, Kohrle J. Transport of thyroid hormone in the brain. Frontiers in Endocrinology. June 2014, Volume 5, Article 98. While achieving drug delivery methods with improved blood-brain barrier permeability can be difficult, the compounds and compositions disclosed by the applicant are actively transported across the blood-brain barrier and thus reach their intended target sites for therapeutic activity.

[0021] Second, the compounds and compositions described herein may be retained within the blood-brain barrier due to overexpression of integrin αvβ3 in GBM and related diseases. For example, αvβ3 expression in GBM can reach levels of 80-97%, as shown in Figure 19. Similar to other thyrointegrin antagonists, the disclosed compounds may bind to this integrin binding site. Thus, in addition to being transported into the brain, the described compounds and compositions may bind to tumor cells, retain within the blood-brain barrier, and remain at their intended target site, thereby reducing unintended effects on non-tumor tissue.

[0022] Third, as described in more detail below, the compounds and compositions described herein contain additional functional groups. These additional functional groups may be conjugated to a polymer. For example, in embodiments with a linear polymer, the additional functional group may be conjugated to the opposite side of the polymer rather than to the thyrointegrin antagonist. Nonlinear polymers may also be used. The additional functional groups may further improve blood-brain barrier permeability and retention, and may also improve scalability and / or solubility. For example, analysis by passive transport artificial membrane permeability assay (PAMPA) (shown below in Figure 20) shows low permeability for all derivatives in the absence of thyroid-binding protein, suggesting that the enhanced uptake is not mediated by passive transport. Instead, the additional functional groups enhance active transport, for example, by making the thyrointegrin antagonist (transporter target) more accessible. PAMPA was performed on PMT36 and related compounds 2-4, all of which exhibited low permeability (less than 1.5 × E-6 cm / s). Similarly, low permeabilities of less than 1.5 E-6 cm / s were demonstrated for compound 5 and P-bi-TAT (a compound discussed in U.S. Patent Application No. 15 / 616,637 (now U.S. Patent No. 10,201,616) and U.S. Patent Application No. 16 / 223,176). The results suggest that these compounds do not penetrate the blood-brain barrier by passive diffusion, clearly suggesting that BBB permeability is primarily facilitated by an active transport system, using thyroid-binding proteins in the blood, such as transthyretin (TTR), to deliver the bound complex across the blood-brain barrier.

[0023] Exemplary compounds will now be discussed in more detail, along with additional background information regarding potential thyrointegrin antagonists and polymers that may be used in embodiments of the present invention.

[0024] As discussed in U.S. Patent Application No. 15 / 616,637 (now U.S. Patent No. 10,201,616) and U.S. Patent Application No. 16 / 223,176, both incorporated by reference above, compounds or compositions comprising αvβ3 integrin thyroid hormone receptor antagonists may include an antiangiogenic thyroid hormone or a derivative thereof conjugated to a polymer via a non-cleavable linker, forming a single chemical entity that can be considered a small molecule or a macromolecule (depending on the size of the polymer or its derivative covalently attached to the thyroid hormone). The size of the single chemical entity and the strength of the non-cleavable covalent bond may be advantageous in preventing the entry of thyroid hormone or its derivative into cells containing cell surface receptors for integrin αvβ3 variants. Due to the size of the attached polymer and the inability of the cellular environment to cleave the strong non-cleavable covalent bond of thyroid hormone from the polymer, internalization of the thyroid hormone portion of the described chemical entity may not be possible within the nucleus of the cell with which the thyroid hormone or its derivative may interact. In this way, the thyroid hormone moiety can interact with the cell non-genomically, avoiding genomic interactions that may be caused by thyroid hormone or its derivatives entering the cell and interacting with nuclear receptors in the cell nucleus.

[0025] As discussed in U.S. Patent Application No. 15 / 616,637 (now U.S. Patent No. 10,201,616) and U.S. Patent Application No. 16 / 223,176, incorporated by reference above, compounds or compositions comprising an αvβ3 integrin thyroid hormone receptor antagonist may be synthesized to include, but are not limited to, entities including non-biodegradable polymers such as polyethylene glycol (PEG) (1,000-15,000 daltons, e.g., 4,000-8,000 daltons), α, β, or γ cyclodextrin, chitosan, alginic acid, or hyaluronic acid, conjugated via a non-cleavable linker containing a short chain of PEG (100-800 molecular weight) free amine or triazole linkages to the αvβ3 antithyroid agent. Embodiments of antithyroid drugs conjugated to a polymer may include tetraiodothyroacetic acid (tetrac), triiodothyroacetic acid (triac), derivatives thereof, and variants thereof. One or more variants of thyroid hormone antagonists, including tetrac and triac, may in some embodiments include diaminotetrac (DAT) or diaminotriac (DATri) (hereinafter interchangeably referred to as "DAT"), monoaminotetrac (MAT) or monoaminotriac (MATri) (hereinafter interchangeably referred to as "MAT"), triazoletetrac (TAT) or triazoletriac (TATri) (hereinafter interchangeably referred to as "TAT"), derivatives thereof, or other antithyroid drugs known to those skilled in the art.

[0026] As discussed in U.S. Patent Application No. 15 / 616,637 (now U.S. Patent No. 10,201,616) and U.S. Patent Application No. 16 / 223,176, both of which are incorporated by reference above, compounds or compositions containing αvβ3 integrin thyroid hormone receptor antagonists have been further synthesized and characterized as DAT, MAT, or TAT conjugated to polyethylene glycol of various molecular weights (1,000-15,000 daltons). We have scaled up the relatively most soluble embodiments, PEG-DAT (P-Mono-DAT, P-bi-DAT) and PEG-TAT (P-Mono-TAT, P-bi-TAT), for biological characterization in various in vitro and in vivo biological systems. Chemical labeling of DAT or TAT and PEG-DAT or PEG-TAT, as well as C-DAT and C-TAT, for imaging and cellular dynamics. Our data reveal that polymer conjugation to DAT or TAT results in limited nuclear uptake of the polymer-conjugated DAT or TAT, as opposed to robust nuclear uptake of DAT or TAT. This unique cellular distribution outcome translates into a lack of genomic activity for polymer-conjugated DAT, MAT, or TAT compared to unconjugated DAT, MAT, or TAT. Other polymers, such as hyaluronic acid, alginate, and chitosan, conjugated to DAT, MAT, or TAT, with or without short-chain PEG (100-1,000 daltons), are also described. Additional polymer conjugations to DAT, MAT, or TAT were synthesized using bifunctional or tetrafunctional PEG, but may also include other branched PEGs with up to eight chains.

[0027] As discussed in U.S. Patent Application No. 15 / 616,637 (now U.S. Patent No. 10,201,616) and U.S. Patent Application No. 16 / 223,176, both incorporated by reference above, compounds or compositions containing αvβ3 integrin thyroid hormone receptor antagonists may have multiple utilities for treating a variety of conditions modulated by angiogenesis or its inhibition. Given the presence of antithyroid agents present in the described compositions, each of the compositions may have affinity for targeting the αvβ3 integrin receptor located on a variety of cells found throughout the human body and various animal bodies. For example, the compositions may be useful for treating angiogenesis-mediated diseases, such as human or mammalian cancers (both solid and liquid tumors). Cancers may include glioblastoma, pancreatic cancer, ovarian cancer, breast cancer, prostate cancer, bladder cancer, lung cancer, and liver cancer. Liquid tumors may also include acute myeloid leukemia, multiple myeloma, lymphoma, and chronic lymphocytic leukemia. The compositions may further treat ophthalmic diseases (diabetic retinopathy and age-related macular degeneration), inflammatory diseases (arthritis, osteoarthritis), atherosclerotic lesions, and dermatological diseases (rosacea, psoriasis, skin cancer), each of which may be mediated by or dependent on the generation of new blood cells through angiogenesis for survival. Treatments may slow or eliminate the angiogenic pathway, depending on the formation of new blood vessels and antagonizing it.

[0028] The compounds and compositions disclosed herein improve upon Applicant's previously disclosed compounds and compositions, achieving one or more of effective blood-brain barrier penetration and retention, good synthetic scalability, good aqueous solubility, and capable of yielding solid products suitable for scalable purification.

[0029] References herein to specific thyrointegrin compounds, e.g., tetrac, triac, etc., include derivatives of such compounds, even if such derivatives are not specifically described, in accordance with the full teachings of this disclosure.

[0030] Referring to the drawings, Figure 1 shows an embodiment of general formula 100 comprising a thyrointegrin antagonist 110 joined via a linker 130 to a substituent 120 (generally designated "A"). The substituent may hereafter be referred to as substituent A, substituent 120, or substituent A 120. Figure 1, like many other figures in this application, shows the carboxylic acid form of general formula 100. As would be apparent to one of skill in the art, salts (e.g., sodium salts) of general formula 100 may also be used.

[0031] In the illustrated embodiment, linker 130 includes spacer 132 and polymer 131. Linker 130 resists biodegradation so that the linker remains uncleaved under physiological conditions. In one embodiment, spacer 132 includes a CH unit and adjacent repeating methylene (CH) units, which may be defined by n1 repeats, where n1 is an integer greater than or equal to 0 (≧0). In other embodiments, n1 may be greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3 (≧1, ≧2, ≧3). Linker 130 further includes moiety "Y." In some embodiments, moiety "Y" may be an amine. For example, moiety Y in the general formula may be a divalent alkane having one amine group or a divalent alkane having two amine groups, as known from applicant's previous applications. In another embodiment, moiety Y may be a triazole, as shown in the example of general formula 102 shown in FIG. 3. Polymer 131 may include a polyether, such as polyethylene glycol (PEG). Other polymers may be used, including chitosan, alginate, hyaluronic acid, and other polymers. In embodiments using PEG as polymer 131, the polymer may have a molecular weight of 200 to 4,000 g per mole.

[0032] The term thyrointegrin antagonist describes a compound capable of inhibiting or antagonizing one or more thyroid hormone receptors known to those skilled in the art, for example, the integrin family of thyroid hormone receptors, such as the thyroid hormone cell surface receptor αvβ3. The thyrointegrin antagonist 110 may be an anti-angiogenic thyroid hormone or thyroid hormone receptor antagonist. For example, the thyrointegrin antagonist 110 may be an alpha-V-beta-3 (αvβ3) integrin thyroid hormone receptor antagonist.

[0033] Particular embodiments of the thyrointegrin antagonist 110 may include tetraiodothyroacetic acid (tetrac), triiodothyroacetic acid (triac), derivatives thereof, and variants thereof. One or more variants of thyrointegrin antagonists, including tetrac and triac, may, in some embodiments, include diaminotetrac (DAT) or diaminotriac (DATri) (hereinafter interchangeably referred to as "DAT"), monoaminotetrac (MAT) or monoaminotriac (MATri) (hereinafter interchangeably referred to as "MAT"), triazoletetrac (TAT) or triazoletriac (TATri) (hereinafter interchangeably referred to as "TAT"), derivatives thereof, or other antithyroid agents known to those of skill in the art. The thyrointegrin antagonist may be of the type described in U.S. Patent Application Nos. 15 / 616,637 (now U.S. Patent No. 10,201,616) and 16 / 223,176, the entire contents of which are incorporated herein by reference, and / or U.S. Patent Application Nos. 15 / 950,870 (now U.S. Patent No. 10,328,043) and 16 / 398,342, the entire contents of which are incorporated herein by reference. As described in these documents, in some embodiments of the thyrointegrin antagonist 110, the variables designated as R10, R11, R12, and R13 may each be independently substituted with a molecule such as hydrogen, iodine, or an alkane. In some embodiments, the alkane has four or fewer carbons.

[0034] In embodiments of the present invention, the substituent A120 may be or include an aryl group and / or an aromatic group. For example, in embodiments, the substituent A120 may include a benzyl group, a phenyl group, etc. In some embodiments, the substituent A120 may include a substituted benzyl group. In further embodiments, a heterobenzyl group may be used. Also, five-membered heteroaryls, fused heteroaryls, quinolines, and indoles may be used. The heteroaryl may include heteroarylmethyl. In further embodiments, the substituent A120 may include esters and amides.

[0035] 2 shows general formula 101, according to an embodiment, where substituent A120 is shown as including an aromatic ring. Substituent A120 including an aromatic ring may be, for example, a substituted benzyl group. In embodiments, substituent A120 including an aromatic ring may be substituted with one or more of R1, R2, R3, R4, and R9. In some embodiments of substituent A120 including an aromatic ring, the variables shown as R1, R2, R3, R4, and R9 may each independently be substituted with a molecule such as hydrogen, iodine, fluorine, bromine, a methoxy group, a nitro group, an amine group, or a nitrile group. For example, in some embodiments of substituent A120 containing an aromatic ring, the variables designated as R1, R2, R3, R4, and R9 may each independently be substituted with a molecule such as hydrogen, iodine, fluorine, bromine, a methoxy group, a nitro group, an amine group, or a nitrile group, as described in Table 2 of U.S. Patent Application No. 15 / 950,870 (now U.S. Patent No. 10,328,043) and U.S. Patent Application No. 16 / 398,342. The variables designated as R1, R2, R3, R4, and R9 may also be substituted with an alkyl, aryl, halo, amido, or the like.

[0036] As shown in Figure 3, in some embodiments comprising the depicted general formula 102, the variables R1, R2, R3, and R4 may be substituted with hydrogen atoms, while R9 may be substituted with a different atom or group ("Z" in the depicted embodiment). Thus, substituent A120 may comprise an aromatic ring as discussed above, or more specifically, may comprise a substituted benzyl group 122 in which atom or group Z is substituted with R9 for hydrogen. Alternatively, in some embodiments, as shown in Figure 6, the variables R1 and R2, or other variables, may be substituted in place of R9.

[0037] Each exemplary compound shown in Figures 3-8 (including exemplary compounds 1-5) contains tetrac as the relevant thyrointegrin αvβ3 receptor antagonist, polyethylene glycol as the relevant linker, and triazole as the included Y moiety.

[0038] More specifically, exemplary compounds 2-5 may be broadly referred to as X-PTAT, where the substituent A120 in X-PTAT is specified herein as a substituted benzyl group (e.g., substituted benzyl group 122 shown in Figure 3, fluorobenzyl or chlorobenzyl shown in Figures 4 and 5, respectively, or a different substituted benzyl group as shown in Figure 6), and is designated X. P is a polymer or polyethylene glycol, and TAT refers to triazole tetrac. Exemplary compounds 2-5 may also be referred to as X-PMTAT, where the substituent A120 in X-PMTAT is specified herein as a substituted benzyl group, and is designated X. P is a polymer or polyethylene glycol, and MTAT refers to monotriazole tetrac. Exemplary compounds 2-5 may also be referred to as X-PMT, where the substituent A in X-PMT is specified herein as a substituted benzyl group, and is designated X. P is a polymer or polyethylene glycol, and MT again refers to monotriazole tetrac.

[0039] These names are in contrast to the compound shown in Figure 7, i.e., Compound 1. This Compound 1 comprises a specific embodiment of a thyrointegrin antagonist conjugated to a polymer as described in U.S. Patent Application No. 15 / 616,637 (now U.S. Patent No. 10,201,616) and U.S. Patent Application No. 16 / 223,176. This compound may be referred to as Compound 1, or may be referred to as PTAT, PMT, or PMTAT. As noted above, these names indicate the inclusion of a polymer conjugated to (mono)triazole tetrac, in this case conjugated only to the methyl group, without conjugation to a functional group such as substituent A120 above (or more specific examples such as a substituted benzyl group).

[0040] Returning to the embodiments of the invention disclosed thus far, as shown in Figures 4 and 5, the variable R9 may be substituted with a halogen. For example, R9 may be substituted with a fluorine molecule as shown in Figure 4. This structure is referred to as Compound 2 and may also be referred to as fluorobenzyl conjugated to triazole tetrac by polyethylene glycol, alternatively as fb-PTAT, fb-PMTAT, or fb-PMT. In another embodiment, R9 may be substituted with a chlorine atom as shown in Figure 5. This structure is referred to as Compound 3 and may also be referred to as chlorobenzyl conjugated to triazole tetrac by polyethylene glycol, alternatively as cb-PTAT, cb-PMTAT, or cb-PMT.

[0041] In a further embodiment shown in Figure 6, the variables R1 and R2 may be substituted. For example, R1 and R2 may be substituted with tert-butyl groups. This structure is referred to as Compound 4, and may also be referred to as di-tbutylbenzyl conjugated to triazole tetrac by polyethylene glycol, or alternatively as Dtbb-PTAT, Dtbb-PMTAT, or Dtbb-PMT.

[0042] Further embodiments may include dyes, markers, labels, etc., for example, for imaging purposes. The dye, marker, or label may be on the substituted benzyl in some embodiments. For example, Figure 8 shows a labeled polymer-conjugated monotetrac (PMT) derivative, designated Compound 5. In this example, substituent A120 includes a dye marker, e.g., BODIPY. Compound 5 may be designated BODIPY-PMT.

[0043] As noted above, additional embodiments of the substituent A120 may be contemplated. For example, as shown in Figures 9A, 9B, and 9C, additional cyclic structures are disclosed for the substituent A120. In these examples, R8 is equal to H, Me, Et, etc. R1-R7 are equal to H, Me, Et, iPr, nPr, nBu, iBu, secBu, tBu, C5-C 12 They may be independently selected from n-alkyl, cyclopentyl, cyclohexyl, phenyl, F, Cl, Br, I, CN, CF3, OCF3, CHF2, OCHF2, SO2Me, NO2, -O-alkyl, -O-aryl, -CH2-O-alkyl, -CH2-O-aryl, ester, amide, etc. Ester substitutions may be selected from: [ka] The amide substitution may be selected from the following: [ka] wherein R9 and R10 are independently selected from H, alkyl, aryl, etc.

[0044] Also as noted above, further embodiments of substituent A120 may include heterobenzyl as shown in Figures 10A-10C. Additionally, five-membered heteroaryls, fused heteroaryls, quinolines, indoles, and the like may be used. R1-R5, and R8 may be substituted as described above.

[0045] A phenoxy group, as shown in Figure 11, may be used as an embodiment of substituent A120, where again R1-R5 may be substituted as described above.

[0046] In further embodiments, substituent A120 may include an amide as shown in Figures 12A and 12B. R9 and R10 may be substituted.

[0047] Esters may be used as substituent A120 in embodiments, for example, esters such as those shown in Figures 13A-13D may be used.

[0048] As described above, various types of polymers and polymers of various molecular weights may be used in embodiments. In embodiments, monodisperse polymers may be used relative to polydisperse polymers for improved ease of analysis and scalability. As shown in exemplary compounds 2-4, embodiments may also include relatively large polymers, such as PEG36. Large, monodisperse polymers, such as monodisperse PEG36, contribute to the solubility and analysis of exemplary compounds. Furthermore, such large, monodisperse polymers may enhance scalability by producing relatively large solid products suitable for purification. Thus, embodiments including PEG36 conjugated to monotriazole tetrac may offer simplified synthesis and scalability compared to other embodiments. In some embodiments, the polymer may have a molecular weight of approximately 4,000 daltons, e.g., 4,000±10%. These large, monodisperse polymers may also contribute to enhanced active transport of compounds, for example, by making thyrointegrin antagonists (transporter targets) more accessible.

[0049] The synthesis of certain exemplary compounds described herein (Compounds 1-5) is demonstrated below. The synthetic descriptions are provided by way of example only and are not intended to limit the present disclosure. In these examples, propargylated tetrac (PGT) is used. The preparation of PGT or its derivatives from tetrac is described in U.S. Patent Application Serial No. 15 / 616,637 (now U.S. Patent No. 10,201,616).

[0050] Example 1: Synthesis of Compound 1 (PMT) [ka]

[0051] Compound 1 and similar compounds / compositions are described in U.S. Patent Application Serial No. 15 / 616,637 (now U.S. Patent No. 10,201,616) (see, e.g., Figures 7c and 8 (Compound 730)) and may be prepared as described therein. However, applicant also provides the following sample methods.

[0052] Figure 14 shows an overview of the synthetic route for Compound 1. The individual steps of the synthetic scheme for Compound 1 are described in further detail below.

[0053] Step 1: 1 g (0.625 mmol) of monodispersed MeoPEG 36 OH (PurePEG, San Diego, CA) and 0.285 ml (4 eq) of TEA were dissolved in 10 ml of DCM. 238 mg (2 eq) of TosCl was added in small portions over 10 min with stirring and stirred overnight. 10 ml of DCM was added, and the mixture was washed twice with 5 ml of saturated ammonium chloride, twice with 5 ml of saturated sodium bicarbonate, and once with 5 ml of saturated brine, and the solvent was stripped under vacuum. The solid was dissolved in 10 ml of hot THF and an equal volume of hot hexane was added. The liquid was decanted from a small amount of insoluble material, and the product was allowed to precipitate overnight at -20 °C. The product was filtered, washed with hexane, and dried under vacuum to yield 995 mg (90%) of product.

[0054] Step 2: 990 mg of MeOPEG 36 OTs (0.565 mM) was dissolved in 5 mL of CH3CN. 110 mg (3 equiv.) of sodium azide was added, and the mixture was heated to 70 °C overnight with stirring. The reaction was then cooled, and most of the acetonitrile was removed under reduced pressure. The residue was partitioned between 10 mL of DCM and water. The aqueous layer was extracted three times with 5 mL portions of DCM, and the combined organic layers were washed with 5 mL of water and saturated brine. The solvent was stripped under reduced pressure, and the material was precipitated from THF / hexane using a procedure similar to the previous step to yield 832 mg (89%) of product.

[0055] Step 3: 830 mg (505 mmol) of MeO-PEG 36N3, 474 mg of (4-{3,5-diiodo-4-[(2-prop-2-yn-1-yl)oxy]-phenoxy]-3,5-diiodophenyl)acetic acid (1.2 equiv.), and 13.7 mg of TBTA (5%) were dissolved in 20 mL of THF. 12.6 mg (0.1 equiv.) of copper sulfate hydrate and 60 mg (0.6 equiv.) of sodium ascorbate were dissolved in 5 mL of water and added to the THF solution. After stirring under N2 for 16 h, the liquid was decanted from a small amount of blue solid at the bottom of the flask. The THF was stripped from the solution under reduced pressure, 10 mL of water was added, and the solution was acidified to pH 3 with dilute HCl and extracted 3 x 40 mL with DCM. The combined organic layers were washed three times with 5 mL of saturated EDTA solution, followed by 5 mL of saturated brine. The DCM was stripped under reduced pressure, and the residue was dissolved in 20 mL of warm THF. 20 mL of hot hexane was added and warmed until nearly everything dissolved. After cooling to room temperature, the liquid was decanted from a small amount of solid and oil at the bottom of the vial. The mixture was allowed to settle overnight at -20 °C, and the solid was removed by filtration and washed with cold hexane. The remaining white solid (685 mg) was dried under reduced pressure. 2.5 mL of 1 M NaOH and 1 mL of saturated sodium chloride were dissolved in 6.5 mL of HO. 2 × 25 mL of the solution was washed with hexane:DCM (3:2). 1 mL more of saturated NaCl was added, and the solution was washed with another 2 × 25 mL of hexane:DCM (3:2). The aqueous layer was acidified to pH 2.0 with dilute HCl and extracted 3 × 25 mL with DCM. The combined organic layers were washed with saturated sodium chloride, and the solvent was removed under reduced pressure. The residue was precipitated from 20 mL of THF and 20 mL of hexane to give 491 mg of product. 1 H-NMR(600MHz,DMSO-D6,)d(PPM):8.258(s,1H), 7.850(s,2H), 7.195(s,2H), 5. 015(s,2H), 4.581(br.s,2H), 3.851(br.s,2H), 3.6-3.3(m,144H), 3.239(S,3H). MSm / z 2452.2(M+Na), 1215.4(M+2H), 810.2(M+3H), 608.2(M+4H).

[0056] Example 2: Synthesis of Compound 2 (fb-PMT) [ka]

[0057] Figure 15 shows an overview of the synthetic route for compound 2. The individual steps of the synthetic scheme for compound 2 are described in further detail below.

[0058] Step 1: 250 mg of HO-PEG36-azide (0.155 mmol, PurePEG, San Diego, CA) was added to 19 mg of 60% NaH (3 equiv.) in 5 mL of THF. The mixture was stirred for 30 min, and then 58 μL of 4-(fluorobenzyl) bromide (Aldrich) (3 equiv.) in 2 mL of THF was added dropwise. The mixture was stirred for 18 h, and then 2 mL of saturated sodium bicarbonate was added. The THF was stripped under vacuum, 10 mL of saturated brine was added, and the mixture was extracted three times with 15 mL portions of DCM. The combined organic layers were washed with 5 mL of saturated brine, and the solvent was stripped under vacuum. The material was then chromatographed on 24 g of silica using 0–10% MeOH in DCM to yield 170 mg of material (>99% pure by HPLC).

[0059] Step 2: 170 mg (0.147 mmol) of 4-fluorobenzyl PEG azide, 138 mg (0.176 mmol) of (4-{3,5-diiodo-4-[(2-prop-2-yn-1-yl)oxy]-phenoxy]-3,5-diiodophenyl)acetic acid, and 3 mg of TBTA were dissolved in 8 mL of THF. 3 mg of CuSO hydrate and 23 mg of Na ascorbate were added to 2 mL of water and stirred under N for 4 h. The THF was stripped under vacuum, followed by the addition of 5 mL of saturated brine and 0.5 mL of 1 M HCl. Extraction was performed with 3 x 10 mL of DCM, followed by 3 x 5 mL of saturated EDTA washes, followed by one 5 mL wash with saturated brine, and the solvent was stripped under vacuum. The residue was dissolved in 10 mL of warm THF and hexane was added until the mixture just began to turn cloudy. The product was allowed to precipitate overnight at -20° C. The solid was removed by filtration to give 180 mg of product. 1 H-NMR (600 MHz, DMSO-D6) d(PPM): 8.246 (s, 1H), 7.879 (s, 2H), 7.361 (dd, 2H), 7.167 (m, 4H), 5.013 (s, 2H), 4.575 (m, 2H), 4.466 (s, 2h), 3.847 (m, 2H), 3.640 (s, 2H), 3.55-3.4 (m, 144H). MS m / z 1262.8 (M+2H), 842.5 (M+3H), 632.2 (M+4H). This product can be further purified by normal phase silica gel chromatography.

[0060] Example 3: Synthesis of Compound 3 (cb-PMT) [ka]

[0061] Figure 16 outlines the synthetic route for compound 3. The individual steps of the synthetic scheme for compound 3 are described in further detail below.

[0062] Step 1: 250 mg of PEG azide (0.155 mmol) was added to 19 mg of 60% NaH (3 equiv.) in 5 mL of THF. After stirring for 30 min, 95.5 mg of 4-chlorobenzyl bromide (Aldrich) (3 equiv.) was added dropwise to the THF. After stirring for 18 h, saturated sodium bicarbonate solution was added, the THF was stripped off under vacuum, 10 mL of saturated brine was added, and the mixture was extracted three times with 15 mL portions of DCM. The combined organic layers were washed with saturated brine, the solvent was stripped off under vacuum, and the mixture was chromatographed on silica gel using 0-10% MeOH in DCM. (190 mg)

[0063] Step 2: 190 mg (0.111 mmol) of chlorobenzyl PEG azide, 131 mg of (4-{3,5-diiodo-4-[(2-prop-2-yn-1-yl)oxy]-phenoxy]-3,5-diiodophenyl)acetic acid (1.5 equiv.), and 3 mg of TBTA were dissolved in 8 mL of THF. 3 mg of CuSO4 hydrate and 23 mg of Na ascorbate were added to 2 mL of water and stirred for 4 h. The THF was stripped under vacuum, followed by the addition of 5 mL of saturated brine and 0.5 mL of 1 M HCl. This was extracted three times with DCM, washed three times with saturated EDTA, and once with brine, and the solvent was stripped under vacuum. The resulting mixture was dissolved in 10 mL of warm THF and hexane was added until the mixture just began to cloud. The material was precipitated in a -20 °C freezer, yielding 180 mg of product. 1 H-NMR (800 MHz, D20) d (PPM): 8.369 (s, 0.3H), 8.147 (s, 0.7H), 7.758 (s, 2H), 7.261 (m, 6H), 4.993 (s, 2H), 4.541 (m, 2H), 4.451 (s, 2H), 3.849 (m, 2H), 3.640 (s, 2H), 3.65-3.54 (m, 14H), 3.346 (s, 2H). MS m / z 1281.2 (M+2H), 854.9 (M+3H), 641.4 (M+4H). This product can be further purified by chromatography on normal phase silica gel.

[0064] Example 4: Synthesis of Compound 4 (Dtbb-PMT) [ka]

[0065] Figure 17 outlines the synthetic route for compound 4. The individual steps of the synthetic scheme for compound 4 are described in further detail below.

[0066] Step 1: 250 mg of HO-PEG36 azide (0.155 mmol) was added to 19 mg of 60% NaH (3 equiv.) in 5 mL of THF. After stirring for 30 min, 131 mg of bromide (Aldrich) (3 equiv.) was added dropwise to the THF. After stirring for 18 h, saturated sodium bicarbonate was added, the THF was stripped under vacuum, 10 mL of saturated brine was added, and the mixture was extracted three times with 15 mL portions of DCM. The combined organic layers were washed with saturated brine, the solvent was stripped under vacuum, and the mixture was chromatographed on silica gel using 0-20% MeOH in DCM. (270 mg)

[0067] Step 2: 270 mg (0.147 mmol) of di-t-butylbenzyl PEG azide, 171 mg of (4-{3,5-diiodo-4-[(2-prop-2-yn-1-yl)oxy]-phenoxy]-3,5-diiodophenyl)acetic acid, and 4 mg of TBTA were dissolved in 8 mL of THF. 4 mg of CuSO4 hydrate and 34 mg of Na ascorbate were added to 2 mL of water and stirred for 4 h. The THF was stripped under vacuum, followed by the addition of 5 mL of saturated brine and 0.5 mL of 1 M HCl. This was extracted three times with DCM, washed three times with saturated EDTA, and once with brine, and the solvent was stripped under vacuum. Chromatography was performed on silica gel using 0-10% MeOH in DCM. (310 mg) 1H-NMR(600MHz,DMSO-D6,d(PPM):8.248(s,1H), 7.842(s,2H), 7.300(s,1H), 7.188(s,2H), 7.130(s,2 MSm / z. 1309.3(M+2H), 873.4(M+3H), 655.8(M+4H).

[0068] Example 5: Synthesis of Compound 5 (BODIPY-PMT) [ka]

[0069] Figure 18 outlines the synthetic route for compound 5. The individual steps of the synthetic scheme for compound 5 are described in further detail below.

[0070] Step 1: 250 mg of NH2-PEG36-N3 was treated with 1.5 equivalents of BOC anhydride and 3 equivalents of triethylamine in 5 mL of DCM. The mixture was stirred at room temperature for 18 hours, then diluted with 20 mL of DCM and washed with 0.1 M HCl, followed by saturated sodium bicarbonate and saturated brine. The solvent was removed under reduced pressure, and the residue was dissolved in 5 mL of warm THF, and hexane was added until the mixture just began to turn cloudy. The mixture was allowed to stand overnight before filtering and washing with hexane. 240 mg of product was recovered.

[0071] Step 2: 235 mg of the product from Step 1, 171 mg of (4-{3,5-diiodo-4-[(2-prop-2-yn-1-yl)oxy]-phenoxy]-3,5-diiodophenyl)acetic acid, and 4 mg of TBTA were dissolved in 8 mL of THF. 4 mg of CuSO4 hydrate and 34 mg of Na ascorbate were added to 2 mL of water and stirred for 18 h. The THF was stripped under vacuum, followed by the addition of 5 mL of saturated brine and 0.5 mL of 1 M HCl. Extraction was performed three times with DCM, washing three times with saturated EDTA, and once with brine, and the solvent was stripped under vacuum. The residue was dissolved in 5 mL of warm THF, and hexane was added until the mixture began to turn cloudy. The mixture was allowed to stand overnight before filtering and washing with hexane. 220 mg of product was recovered.

[0072] Step 3: 215 mg of the product from Step 2 was dissolved in 2 mL of DCM and 2 mL of 5 M HCl in dioxane was added. The mixture was stirred for 18 h, the solvent was removed under reduced pressure, and the product was used for the next step.

[0073] Step 4: 18 mg of the product from the last step (0.0076 mmol) was dissolved in 1 ml of DCM along with 10 μL of triethylamine. 5 mg of BODIPY 630 / 650 NHS ester (Thermo Fisher) dissolved in 100 μL of DCM was added. The mixture was shaken for 18 hours, the solvent was removed under reduced pressure, and the residue was chromatographed on silica gel using 0-20% methanol in DCM. 4.5 mg was recovered. 1H-NMR(800MHz,CDCl3)d(PPM):8.221(s,1H), 8.058(s,1H), 7.850(s,2H), 7.642(m,3H), 7. 513(d,1H), 7.237(m,3H), 7.075(m,1H), 7.021(m,1H), 6.993(m,3H), 6.828(m,1H), 6.729(m , 1H), 5.218 (s, 2H), 4.619 (m, 2H), 4.570 (s, 2H), 3.836 (m, 2H), 3.75-3.6 (m, 140H), 3.545 (m, 2H), 3.416 (m, 2H), 3.310 (m, 2H), 2.223 (m, 2H), 1.688 (m, 2H), 1.603 (m, 2H), 1.359 (m, 2H). The product had a retention time of 34.05 min on an HPLC system using a Pursuit XRs 3 C18 column and mobile phase A (water with 0.1% formic acid and 5% acetonitrile) and methanol (B). The flow rate was 1.0 mL / min, the gradient was linear from 50% B at 0 min to 95% B at 40–45 min, and the column temperature was 25°C.

[0074] Again, other synthetic routes in addition to those described above may be used to generate the exemplary compounds, and additional compounds modified as necessary for each substitution may be generated using the techniques described above. Methods of Use / Treatment

[0075] As described above, the compounds and compositions described herein have enhanced brain uptake across the blood-brain barrier. Table 1 below demonstrates this enhanced uptake by showing the average brain concentrations for each of the exemplary compounds 1-4. Concentrations are recorded and shown 3 hours after administration. [Table 1]

[0076] The data in Table 1 were generated using the following study. C57BL / 6 mice were subcutaneously administered 10 mg / kg of Compositions 1-4. Each group contained four mice. The mice were sacrificed 3 hours after administration, and brain tissue was excised for bioanalytical measurement of Compounds 1-4 in the brain. The average concentrations are shown above, and each of Compounds 2-4 demonstrated enhanced uptake compared to Compound 1.

[0077] Compounds 2-4 also demonstrated enhanced uptake compared to BG-P-TAT. BG-P-TAT refers to benzylguanidine conjugated to tetrac via the polymer PEG, and was described in U.S. Patent Application No. 15 / 950,870 (now U.S. Patent No. 10,328,043) and U.S. Patent Application No. 16 / 398,342, along with other compounds and compositions containing αβ integrin antithyroid agents and targeting the norepinephrine transporter or catecholamine transporters. PEG36 was used in tested embodiments of BG-P-TAT. As can be seen, BG-P-TAT does not cross the blood-brain barrier, with no detectable levels in the brain 3 hours after administration. Again, this is in stark contrast to compounds disclosed herein, such as exemplary compounds 2-4, which demonstrate high brain concentrations.

[0078] The enhanced uptake of the compounds disclosed herein is believed to be due to active transport rather than passive permeability. For example, as shown in Figure 20, analysis by passive transport artificial membrane permeability assay (PAMPA) demonstrated low permeability for each of compounds 1-4. Thus, without thyroid-binding proteins, little permeability across the blood-brain barrier is achieved. Because passive permeability is unaffected and each of compounds 1-4 has the same transporter recognition element (the thyrointegrin antagonist, triazole tetrac), these compounds are each substrates for the thyroid hormone transporter and would be expected to have the same or similar uptake into the brain.

[0079] However, as demonstrated in Table 1 above, exemplary compounds 2-4 exhibit significant and unexpected enhancements in brain concentration levels over both compound 1 and BG-P-TAT. Moreover, as discussed in more detail below, this unexpected enhancement in brain uptake and concentration results in similarly unexpected enhanced efficacy in the treatment of diseases requiring blood-brain penetration, including, for example, glioblastoma.

[0080] Also shown in Figure 21, uptake is substantially enhanced when the tumor is in the brain. As discussed above, this is due, at least in part, to binding of the compound / composition to the high expression of αvβ3 by GBM tumors. Also, uptake is predominantly concentrated in the brain, to the exclusion of other organs as shown. In the data shown in Figure 21, uptake was measured using compound 5 (BODIPY-PMT).

[0081] Figure 22 also shows this initial uptake into the brain. As shown, exemplary compound 2 (fb-PMT) demonstrates uptake and retention over 24 hours. Again, a single subcutaneous injection was used in mice. The dose was 10 mg / Kg. The brain tissue in this example does not contain tumor cells.

[0082] Plasma and brain levels for exemplary Compound 2 (fb-PMT) in cynomolgus monkeys are shown in Figure 23. As can be seen, plasma concentrations peak between 1 and 4 hours. Brain levels are also included, and Compound 2 was measured using a validated LC / MS / MS method, showing 72.3 ng / g and 80.5 ng / g (in male and female cynomolgus monkeys, respectively) 24 hours after the last dose of a 14-day treatment regimen (15 mg / kg, SC QD 14 days).

[0083] In addition to good initial uptake, the disclosed compounds also have good anti-angiogenic effects. For example, exemplary compound 2 (fb-PMT) demonstrates broad-spectrum anti-angiogenic effects against various growth factors, as shown in Figure 24. In particular, when administered in the presence of the following growth factors: bFGF, VEGF, VEGF + bFGF, and bFGF + VEGF + HGF, compound 2 is effective in reducing the percentage of angiogenesis present in the CAM model. Each of these growth factors produces a 250% or greater increase in angiogenesis in the CAM model. However, administration of 1.0 μg of compound 2 (fb-PMT) significantly reduces this increase to just slightly above baseline.

[0084] This broad-spectrum antiangiogenic effect is in contrast to existing therapeutic compounds / compositions such as AVASTIN® (bevacizumab). As shown in Figure 25, AVASTIN® is primarily effective against VEGF alone, but does not significantly reduce the percent angiogenesis associated with bFGF or HGF. Furthermore, AVASTIN® does not demonstrate favorable effects when bFGF or bFGF and HGF are present together with VEGF. Referring again to Figure 24, exemplary compound 2 (fb-PMT) demonstrates inhibition of angiogenesis against all three growth factors, two alone, and in combination.

[0085] Referring again to Figure 21, exemplary compound 5 (BODIPY-PMT) exhibits substantially enhanced uptake when tumors (GBM) are present in the brain. Uptake is also predominantly concentrated in the brain, to the exclusion of other organs, as shown. The investigational protocol is further described below.

[0086] Athymic female mice with and without GBM in the brain were used. Mice with GBM received orthotopic implantation of U87-luc cells (1 million cells) into the brain. 3 mg / kg of Compound 5 (BODIPY-PMT) (a far-red fluorescent dye) was injected subcutaneously. Fluorescent signals were detected (Ex / Em 630 nm / 650 nm). The table below shows all treatment groups. [Table 2] *L-T4 (thyroxine for hypothyroidism) and phenytoin (antiepileptic) bind to thyroid-binding proteins

[0087] The fluorescent signal of compound 5 was imaged after 1, 2, 6, and 24 hours. After termination, the ex vivo fluorescent signal was imaged in the brain and organs. Following termination and imaging, luciferase substrate was added to detect tumor luminescence signals in the brain.

[0088] FIG. 26 shows the bioluminescence signal of GBM (U870-luc) tumors in the brain for different treatment groups.

[0089] Figure 27 shows the presence of Compound 5 (administered subcutaneously at 3 mg / kg) in the brain at 1 hour, 2 hours, 6 hours, and 24 hours. As shown, Compound 5 is taken up by the blood-brain barrier and is retained at all intervals. Also shown, Compound 5 is present at significantly higher levels in animals with GBM tumors, providing further evidence of uptake across the blood-brain barrier and retention at the tumor site.

[0090] Figure 28A shows the blood-brain barrier uptake and retention of exemplary compound 5 (3 mg / Kg) after 24 hours. Again, compound 5 is present at significantly higher levels in animals with GBM, providing further evidence of uptake across the blood-brain barrier and retention at the tumor site. Compound 5 is present and retained when administered alone and also when administered with other drugs that penetrate the blood-brain barrier, such as the thyroid hormone L-T4 and phenytoin. Thus, the disclosed compounds / compositions demonstrate good uptake and retention in the brain, even in the presence of drugs that may compete for uptake / binding.

[0091] Figure 28B also shows the intensity of the fluorescent signal for Compound 5 in brain regions with and without GBM tumors. Again, Compound 5 was administered alone in the presence of L-T4 and phenytoin. As can be seen, uptake is substantially enhanced in the presence of tumor. Furthermore, the presence of L-T4 or phenytoin does not mitigate the enhancement in uptake. Thus, the disclosed compounds demonstrate good brain uptake, as well as high-affinity binding and retention at tumor sites.

[0092] Figure 29 shows the brain uptake of a single dose (3 mg / kg, SC) of exemplary compound 5 (fb-PMT) in tumor-bearing and tumor-free mice. As can be seen, animals with implanted tumors demonstrate substantially enhanced levels of uptake into the brain. Figure 29 also shows accumulation levels in other organs, including the heart and lungs, liver, and kidneys. Drug accumulation is present in the liver in both test groups. However, while tumor-free animals demonstrate accumulation in the kidneys, tumor-bearing animals demonstrate no kidney accumulation. This provides further evidence of retention at the tumor site. Figure 29 also shows the luminescence signal in the GBM of tumor-bearing animals compared to the absence of luminescence signal in tumor-free animals.

[0093] 21-29 above demonstrate the enhanced blood-brain barrier uptake and retention of exemplary compounds. Thus, the disclosed compounds and compositions containing these compounds can penetrate the blood-brain barrier and be delivered to tumor sites, particularly those located in the brain. The compounds may also be used to target such tumors while minimizing effects on healthy tissue.

[0094] The efficacy of these compounds / compositions on GBM tumors will now be described with reference to Figures 30-33. The study protocol was as follows: Nude mice bearing U87-luc xenografts were treated with various doses of Compound 2 (fb-PMT) for three weeks. Efficacy was assessed by tumor weight and luminescence signal intensity compared to the control group. The therapeutic effect was also evaluated in comparison with cilengitide, a known potential treatment. Efficacy was assessed again after three weeks without additional treatment.

[0095] Figure 30 shows the effect of exemplary compound 2 (fb-PMT) on tumor weight after 3 weeks of treatment, as well as on tumor weight after 3 weeks of treatment and 3 weeks without treatment. Results are shown for doses of 1 mg / kg, 3 mg / kg, 6 mg / kg, and 10 mg / kg versus the control group. After 3 weeks, the tumor weight in the control group was approximately 600 mg. All treatment groups demonstrated a dose-dependent reduction in tumor weight to less than 100 mg.

[0096] Tumor weights were also compared after 6 weeks of treatment (3 weeks of treatment followed by 3 weeks without additional treatment). The control group showed an increased tumor weight of approximately 750 mg. All treatment groups showed a further decrease in tumor weight over the 3 weeks without treatment.

[0097] Figure 31 shows the effect of Compound 2 (fb-PMT) on the luminescence signal intensity after 3 weeks of treatment, as well as on the luminescence signal intensity after 3 weeks of treatment without treatment. Results are shown for doses of 1 mg / kg, 3 mg / kg, 6 mg / kg, and 10 mg / kg versus the control group. The luminescence signal intensity for the control group after 3 weeks was approximately 600,000 p / s. All treatment groups showed a dose-dependent decrease in luminescence signal intensity to less than 100,000 p / s.

[0098] Signal intensities were also compared after 6 weeks (3 weeks of treatment followed by 3 weeks without further treatment). The control group showed an increase in signal intensity to over 800,000 p / s. All treatment groups showed a further decrease in luminescence signal intensity over the 3 weeks without treatment.

[0099] Figure 32 shows the effect of Compound 2 (fb-PMT) on tumor weight after three weeks of treatment, as well as on tumor weight after three weeks of treatment and three weeks without treatment. Results are shown for a 6 mg / kg dose and compare both the control group and the group treated with 75 mg / kg cilengitide. Again, tumor weight in the control group was approximately 600 mg after three weeks. Both treatment groups showed a reduction in tumor weight after three weeks of treatment. However, the group treated with Compound 2 showed a greatly enhanced reduction when compared to the group treated with cilengitide. For example, as can be seen, the group treated with cilengitide showed a reduction in tumor weight to approximately 300 mg, while the group treated with Compound 2 showed a reduction to less than 50 mg.

[0100] Tumor weights were also compared after 6 weeks (3 weeks of treatment followed by 3 weeks without further treatment). Again, the control group showed an increase in tumor weight of approximately 750 mg after 3 weeks without treatment. After an additional 3 weeks without treatment, the cilengitide group showed an increase in tumor weight compared to the 3-week treatment, with a final tumor weight exceeding 400 mg. Thus, even after 3 weeks of treatment at 75 mg / kg, tumors remained active and proliferating relative to the cilengitide group. In contrast, the compound 2 group showed a further decrease in tumor weight even after an additional 3 weeks without treatment.

[0101] Figure 33 shows the effect of Compound 2 on luminescence signal intensity after three weeks of treatment, as well as after three weeks of treatment without treatment. Results are shown for a dose of 6 mg / kg Compound 2 (fb-PMT) and are compared to both the control group and the group treated with 75 mg / kg cilengitide. Again, the luminescence signal intensity in the control group after three weeks was approximately 600,000 p / s. Both treatment groups showed a decrease in signal intensity after three weeks of treatment. However, the group treated with Compound 2 showed a greatly enhanced decrease compared to the group treated with cilengitide. For example, as can be seen, the group treated with cilengitide showed a decrease in signal intensity to approximately 300,000 p / s, while the group treated with Compound 2 showed a negligible decrease.

[0102] Signal intensities were also compared after 6 weeks (3 weeks of treatment followed by 3 weeks without further treatment). Again, the control group showed increased signal intensity of over 800,000 p / s. After an additional 3 weeks without treatment, the cilengitide group showed increased signal intensity compared to the 3-week treatment, with a final signal intensity exceeding 500,000 p / s. Thus, even after 3 weeks of treatment with 75 mg / kg, tumors remained active and proliferating in the cilengitide group. In contrast, the compound 2 group showed a further decrease in luminescence signal activity, even after an additional 3 weeks without treatment.

[0103] As demonstrated in the studies and shown in these figures, the described compounds have enhanced therapeutic efficacy against glioblastoma (GBM) when compared to controls and known therapeutic compounds / compositions with limited blood-brain barrier permeability. As discussed above, this enhanced therapeutic efficacy may be attributable to a combination of factors, including active transport across the blood-brain barrier by the thyrointegrin antagonist portion of the compound, retention in the brain, particularly at the tumor site, due to binding of the thyrointegrin antagonist portion of the compound to integrin αvβ3, which is present and overexpressed in brain tumors such as GBM, and the effect of substituent A on uptake across the blood-brain barrier, e.g., due to enhanced accessibility of transporter targets in some embodiments. These features contribute to enhanced initial uptake and retention in the brain and desired treatment site, resulting in enhanced therapeutic efficacy.

[0104] The disclosed compounds also have enhanced scalability, solubility, and can yield solid products or intermediates. Synthetic scalability allows for the efficient and cost-effective production of patient-use compounds and compositions. Furthermore, compounds and compositions must be synthesized with a sufficient level of purity for therapeutic use. The availability of a compound or composition in solid form offers improved purification options. This contrasts with other compounds, such as P-Bi-TAT, which yield oil products. The solid exemplary compounds described herein are also readily purified by normal-phase chromatography on silica gel, which is not feasible for many other pegylated molecules, including P-Bi-TAT. For example, P-Bi-TAT requires reverse-phase chromatography, which is not easily scalable. Similarly, aqueous solubility facilitates certain means of administration, such as injection methods, such as subcutaneous injection. These characteristics are therefore often important for the successful production of compounds / compositions and for the successful implementation of effective treatments using the compounds / compositions. The disclosed compounds are particularly useful as potential treatment options for glioblastoma and other diseases, and can be produced in sufficient quantities for therapeutic doses.

[0105] The compounds may also be prepared as compositions comprising the disclosed compounds. Furthermore, the compounds and / or compositions may be used to treat a disease, such as GBM, by administering a therapeutically effective amount of the compound and / or composition to a patient in need thereof, e.g., a patient suffering from the disease.

[0106] The compositions may be used for imaging cancer cells / tumors. For example, the compositions described herein may be used to image tumors in the brain, such as glioblastoma. Imaging may be desirable for diagnosis and / or treatment monitoring. Furthermore, the compositions may be used for simultaneous treatment and imaging. For example, the compositions may exhibit enhanced retention in targeted cancer cells / tumors, thereby improving treatment.

[0107] While the description of various embodiments of the present invention has been presented for illustrative purposes, the description is not intended to be exhaustive or to be limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein has also been selected to best explain the spirit of the embodiments, practical applications or technical improvements to the technology recognized in the market, and to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A compound comprising: a thyrointegrin antagonist; a non-biodegradable polymer; a linker covalently attaching the thyrointegrin antagonist to the non-biodegradable polymer via a non-cleavable covalent bond; a substituent A attached to the non-biodegradable polymer; Including, said substituent A is a substituted benzyl; A is selected from the group consisting of: 【Chemistry 10】 wherein R 1 -R 7 are independently selected from the group consisting of H, Me, Et, iPr, nPr, nBu, iBu, secBu, tBu, C 5 -C 12 n-alkyl, cyclopentyl, cyclohexyl, phenyl, F, Cl, Br, I, CN, CF 3 , OCF 3 , CHF 2 , OCHF 2 , SO 2 Me, NO 2 , -O-alkyl, -O-aryl, -CH 2 -O-alkyl, -CH 2 -O-aryl, ester, and amide; and R 8 is selected from H, Me, and Et; compound.

2. The alkyl is Me, Et, iPr, nPr, nBu, iBu, secBu, tBu, C ５ -C １２ The compound of claim 1 , wherein the alkyl group is selected from the group consisting of n-alkyl, cyclopentyl, and cyclohexyl.

3. The aryl is Phenyl and Alkyl, F, Cl, Br, I, CN, CF ３ , OCF ３ , CHF ２ , OCHF ２ , S.O. ２ Me, and NO ２ and a phenyl substituted with one of selected from the group consisting of The compound of claim 1.

4. 2. The compound of claim 1, wherein the ester is selected from the group consisting of: 【Chemistry 11】

5. The amide is selected from the group consisting of: 【Chemistry 12】 wherein R9 and R10 are independently selected from at least one of H, alkyl, and aryl. The compound of claim 1.

6. 2. The compound of claim 1, wherein the thyrointegrin antagonist is selected from the group consisting of triiodothyroacetic acid, triiodothyroacetic acid derivatives, tetraiodothyroacetic acid, and tetraiodothyroacetic acid derivatives.

7. 2. The compound of claim 1, wherein the polymer is polyethylene glycol (PEG).

8. The compound of claim 1 , wherein the substituted benzyl comprises a halogen.

9. having the general formula: 【Chemistry 16】 wherein n=5 to 200. The compound of claim 1.

10. A compound comprising: a thyrointegrin antagonist; a non-biodegradable polymer; a linker covalently attaching the thyrointegrin antagonist to the non-biodegradable polymer via a non-cleavable covalent bond; a substituted benzyl bonded to the non-biodegradable polymer; Including, having the general formula: 【Chemistry 17】 wherein n1≧0; In the formula, n2 is 5 to 200; wherein Z is a halogen; compound.

11. A compound having the general formula: [Chemistry 18] wherein n1≧0; In the formula, n2 is 5 to 200; In the formula, R1 to R4 and R9 are H, Me, Et, iPr, nPr, nBu, iBu, secBu, tBu, C ５ -C １２ n-Alkyl, cyclopentyl, cyclohexyl, phenyl, F, Cl, Br, I, CN, CF ３ , OCF ３ , CHF ２ , OCHF ２ , S.O. ２ Me, NO ２ , —O-alkyl, —O-aryl, —CH ２ —O-alkyl, —CH ２ independently selected from the group consisting of —O-aryl, ester, and amide; wherein R10 to R13 are each independently selected from the group consisting of hydrogen, iodine, and an alkane group; wherein Y is selected from the group consisting of: 【Chemistry 19】

12. A compound comprising: a thyrointegrin antagonist conjugated to a polymer; a substituted benzyl conjugated to the polymer, The compound penetrates the blood-brain barrier and is absorbed, The compound has the general formula: 【Chemistry 20】 wherein n1≧0; In the formula, n2 is 5 to 200; wherein Z is a halogen; compound.

13. 13. The compound of claim 12, wherein the thyrointegrin antagonist is selected from the group consisting of triiodothyroacetic acid, triiodothyroacetic acid derivatives, tetraiodothyroacetic acid, and tetraiodothyroacetic acid derivatives.

14. 13. The compound of claim 12, wherein the polymer is polyethylene glycol (PEG).

15. The compound of claim 12 , wherein the substituted benzyl comprises a halogen.

16. 16. The compound of claim 15, wherein the halogen is at least one of fluorine and chlorine.

17. The compound of claim 12, wherein the substituted benzyl is fluorobenzyl.

18. The compound of claim 12 , wherein the compound comprises a dye for imaging.

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