Borylated dipeptide amino acid compositions and methods for use in boron neutron capture therapy - Patents.com

Abstract

Disclosed herein are borylated dipeptide amino acid ("Bdi-AA") and dipeptide amino acid (di-AA) compositions and methods for making Bdi-AA and di-AA. Thus, Bdi-AA and di-AA can be administered to a patient as neutron capture agents, providing a method for treating cancer, immune disorders, and other diseases by utilizing a modality of neutron capture therapy. In further embodiments, the present invention includes a method for concentrating boron in a cell, comprising: (i) synthesizing a borylated dipeptide amino acid ("Bdi-AA"); (ii) administering Bdi-AA to a patient, and (iii) irradiating the cell with neutrons.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 361,403, filed December 16, 2021, the contents of which are incorporated by reference in their entirety herein.

[0002] STATEMENT REGARDING RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Not applicable.

[0003] FIELD OF THEINVENTION The invention described herein relates to the field of boron neutron capture therapy (BNCT) and related treatments. In particular, the invention relates to borylated dipeptide amino acid compositions that can be used as vehicles for neutron capture therapy in humans. The invention further relates to the treatment of cancer and other immunological disorders and diseases. [Background technology]

[0004] 2. Background of the Invention Cancer is the second leading cause of death worldwide after coronary artery disease. Millions of people die from cancer each year, with well over half a million people dying from cancer each year in the United States alone, and in 2020, 1.8 million new cases of cancer were diagnosed (American Cancer Society). Deaths from cardiovascular disease have declined significantly, but deaths attributable to cancer in general are increasing. It is predicted that cancer will become the leading cause of death in the early part of the next century, unless medical advances change the current trend.

[0005] Some cancers are noted for having a high mortality rate. In particular, carcinomas of the lung (18.4% of all cancer deaths), breast (6.6% of all cancer deaths), colorectal (9.2% of all cancer deaths), liver (8.2% of all cancer deaths) and stomach (8.2% of all cancer deaths) are the leading causes of cancer deaths in both sexes and at all ages worldwide (GLOBOCAN 2018). These and virtually all other carcinomas share the common fatal feature of metastasizing to sites distant from the primary tumor, and with very few exceptions, metastatic disease is fatal. Moreover, even for cancer patients who initially survive their primary cancer, the general experience indicates that their lives change dramatically. Many cancer patients experience strong anxiety due to the awareness of the possibility of recurrence or treatment failure. Many cancer patients also experience physical debilitation after treatment. Furthermore, many cancer patients experience disease recurrence.

[0006] Although cancer treatment has improved over the past decades and survival rates have increased, new therapeutic strategies utilizing multiple treatment modalities are still needed due to the heterogeneity of cancer. This is especially true for the treatment of solid tumors in anatomically important sites (e.g., glioblastoma, squamous cell carcinoma of the head and neck, and lung adenocarcinoma) that may be limited to standard radiotherapy and / or chemotherapy. Nevertheless, the deleterious effects of these therapies are chemoresistance and radioresistance, which promote locoregional recurrence, distant metastases, and another primary tumor, in addition to severe side effects that reduce the patient's quality of life.

[0007] Neutron capture therapy (NCT) is a promising form of radiation therapy. NCT is a technique that uses boron compounds to selectively kill tumor cells while sparing normal cells. BNCT uses 0.5keV <E n Non-radioactive absorbing epithermal neutrons in the low energy range <30 keV 10 Depending on the tendency of the B isotope, after neutron capture, the boron atom is converted into an α particle and a recoil lithium nucleus ( 7Li). 10 B+n→ 7 Li+ 4 He

[0008] Alpha particles deposit high energy, i.e. 150 keV / μm, along their short path essentially limited to the diameter of a single cell, resulting in double-stranded DNA breaks and subsequent cancer cell death by apoptosis. Thus, BNCT integrates both concepts of chemotherapy, targeted therapy and the macroscopic anatomical localization of traditional radiation therapy.

[0009] Although the conceptual technology of NCT, specifically boron neutron capture therapy (BNCT), is well known, progress has been slowed by the technical limitations associated with this type of treatment. During early investigations using a research reactor at MIT in 1960, dozens of patients were treated using disodium decahydrodecaborate, which was thought to be less toxic than the simpler boron compounds previously used, but capable of delivering more boron to cells. Unfortunately, due to severe brain necrosis in patients undergoing BNCT and the potential hazards of using a reactor, BNCT research was halted in the United States.

[0010] In 1968, Hiroshi Hatanaka reviewed the clinical application of BNCT in Japan using sodium borocaptate (BSH) by directing light beams on surgically exposed intracranial tumors, and reported achieving a 58% 5-year survival rate. In 1987, Japanese clinicians applied BNCT for the treatment of malignant melanoma using boronophenylalanine (BPA) as the boron compound. Thus, a gradual revival of BNCT has occurred, although it is limited to countries with access to research reactor facilities capable of delivering epithermal neutron beams. Currently, NCT treatment methods are being revived due to technical improvements in both (i) injection and delivery of capture compounds that preferably collect within the tumor, and (ii) greater and easier access to neutron beams using cyclotrons.

[0011] Proton-boron fusion reaction is required for BNCT 10 B, but is abundant in nature 11 Depends on the B isotope. Unlike BNCT, it uses protons ( 1 H) and boron ( 11 B) After the fusion reaction between nuclei, three alpha particles are emitted: p+ 11 B->3α. Proton beams have the advantage of Bragg peak characteristics that reduce normal tissue damage and, when combined with proton capture, may improve the efficacy of proton therapy alone.

[0012] Boron carriers have evolved since 1950 and are reviewed in NEDUNCHEZHIAN et al., J. Clin. & Diag. Res., vol. 10(12) pp. ZE01-ZE04 (Dec. 2016). Briefly, the first generation of boron compounds, represented by boric acid and its derivatives, were either toxic or suffered from low tumor accumulation / retention. Both BPA and BSH are considered second generation compounds that emerged in the 1960s. These had significantly lower toxicity as well as better PK and biodistribution. BPA-fructose complexes are considered third generation compounds that have been used since 1994 to treat patients with head and neck (H&N), glioblastoma and melanoma using BNCT. BPA-fructose and BSH are the only compounds in clinical use as boron carriers to date, although both low and high molecular weight biomolecules such as nucleosides, porphyrins, liposomes, nanoparticles and mAbs have been evaluated for tumor targeting in preclinical models. The main drawback of BPA-fructose, in addition to its relatively low solubility, is its rapid excretion, which prevents the achievement of a high blood Cmax, one of the factors that influences tumor uptake.

[0013] From the above description, it will be readily apparent to those skilled in the art that new treatment paradigms are needed in the treatment of cancer and immune diseases. By using modern chemical synthesis and modifying natural amino acids with boron, new disease treatments can be achieved with the overall goal of more effective treatment, reduced side effects, and reduced manufacturing costs. In view of the current shortcomings associated with NCT, it is an object of the present invention to provide new and improved methods of treating cancer(s), immune disorders, and other diseases that utilize borylated amino acids as capture agents in NCT and BNCT treatments. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] NEDUNCHEZHIAN et al., J.Clin.& Diag.Res.,vol.10(12)pp. ZE01-ZE04(Dec.2016) Summary of the Invention

[0015] Summary of the Invention The present invention provides compositions comprising natural dipeptide amino acids borylated via chemical synthesis for use as delivery modalities for treating human diseases such as cancer, immune disorders (including but not limited to rheumatoid arthritis, ankylosing spondylitis), and other cellular diseases (including but not limited to Alzheimer's disease). In certain embodiments, the borylated amino acids are comprised of naturally occurring amino acids such as phenylalanine, tryptophan, tyrosine, histidine, and any other naturally occurring amino acid set forth in Table I.

[0016] In a further embodiment, the invention includes a method for concentrating boron in a cell, comprising: (i) synthesizing a borylated dipeptide amino acid ("Bdi-AA"); (ii) administering the Bdi-AA to a patient; and (iii) irradiating the cell with neutrons.

[0017] In another embodiment, the disclosure teaches a method for synthesizing Bdi-AA'.

[0018] In another embodiment, the present disclosure teaches a method for synthesizing BPA-Ala.

[0019] In another embodiment, the present disclosure teaches a method for synthesizing His-BPA.

[0020] In another embodiment, the present disclosure teaches a method for synthesizing Leu-BPA.

[0021] In another embodiment, the present disclosure teaches a method for synthesizing Ala-BPA.

[0022] In another embodiment, the present disclosure teaches a method for synthesizing BPA-BPA.

[0023] In another embodiment, the present disclosure teaches a method for synthesizing 3-boronobenzyl-BPA.

[0024] In another embodiment, the present disclosure teaches a method for synthesizing 3-BPA-boronopyridine.

[0025] In another embodiment, the present disclosure teaches a method for synthesizing reduced BPA-BPA (denoted as "(Red)-BPA-BPA").

[0026] In another embodiment, the present disclosure teaches a method for synthesizing BPA-Tyr.

[0027] In another embodiment, the present disclosure teaches a method for synthesizing Tyr-BPA.

[0028] In another embodiment, the present disclosure teaches a method for synthesizing BPA-Leu.

[0029] In another embodiment, the present disclosure teaches a method for synthesizing a borylated dipeptide having chemical structure 27 shown in FIG.

[0030] In another embodiment, the present disclosure teaches a method for synthesizing a borylated dipeptide having chemical structure 30 shown in FIG.

[0031] In another embodiment, the present disclosure teaches a method for synthesizing a borylated dipeptide having chemical structure 33 shown in FIG.

[0032] In another embodiment, the present disclosure teaches methods for synthesizing borylated dipeptides having chemical structures 6, 11, 17, 21, 24, 35, 36, 37, and 38 shown in FIG.

[0033] In another embodiment, the present disclosure teaches a method for synthesizing a borylated dipeptide having chemical structure 36 shown in FIG.

[0034] In another embodiment, the present disclosure teaches a method for synthesizing a borylated dipeptide having chemical structure 37 shown in FIG.

[0035] In another embodiment, the present disclosure teaches a method for synthesizing a borylated dipeptide having chemical structure 38 shown in FIG.

[0036] In another embodiment, the disclosure teaches methods of treating cancer, immune disorders and other diseases in humans. [Brief description of the drawings]

[0037] [Figure 1] Chemical synthesis of BPA-Ala.

[0038] [Diagram 2] Purity analysis of BPA-Ala.

[0039] [Diagram 3] Chemical synthesis of His-BPA.

[0040] [Figure 4] Purity analysis of His-BPA.

[0041] [Diagram 5] Chemical synthesis of Leu-BPA.

[0042] [Figure 6] Purity analysis of Leu-BPA.

[0043] [Figure 7] Chemical synthesis of Ala-BPA.

[0044] [Figure 8] Purity analysis of Ala-BPA.

[0045] [Figure 9] BPA - Chemical synthesis of BPA.

[0046] [Figure 10] BPA - Purity analysis of BPA.

[0047] [Figure 11] Chemical synthesis of 3-boronobenzyl-BPA.

[0048] [Figure 12] Chemical synthesis of BPA-3-boronopyridine.

[0049] [Figure 13] Chemical synthesis of (Red)-BPA-BPA.

[0050] [Figure 14-1] Chemical structures of exemplary dipeptide(s). [Figure 14-2] Same as above.

[0051] [Figure 15] Chemical synthesis of BPA-Tyr.

[0052] [Figure 16]Purity analysis of BPA-Tyr.

[0053] [Figure 17] Chemical synthesis of Tyr-BPA.

[0054] [Figure 18] Purity analysis of Tyr-BPA.

[0055] [Figure 19] Chemical synthesis of BPA-Leu.

[0056] [Figure 20] Purity analysis of BPA-Leu.

[0057] [Figure 21] Uptake of borylated dipeptides in multiple cancer cell lines.

[0058] [Figure 22] Correlation of BPA and His-BPA uptake with relative expression of LAT1 and PEPT1 in cell lines.

[0059] [Figure 23] Correlation of BPA and His-BPA uptake with relative expression of LAT1 and PEPT1 in cell lines (continued).

[0060] [Figure 24] Inhibition of His-BPA uptake by Gly-SAR in PEPT1 cells.

[0061] [Diagram 25] Relative uptake of dipeptides in PEPT1 cells.

[0062] [Figure 26-1] Assessment of the degradation of dipeptides to BPA by HPLC. [Figure 26-2] Same as above.

[0063] [Figure 27] Assessment of the degradation of dipeptides to BPA by HPLC (cont.).

[0064] [Figure 28] BPA-BPA Pharmacokinetics.

[0065] [Figure 29] In vivo biodistribution of dipeptides using FaDu cells.

[0066] [Diagram 30] In vivo biodistribution of His-BPA using FaDu cells.

[0067] [Diagram 31] Dose-escalating in vivo boron delivery using borylated dipeptides in multiple xenograft models.

[0068] [Diagram 32] Determination of tumor boron content of multiple dipeptides using a CT26 syngeneic colon cancer model.

[0069] [Figure 33-1] Improved BNCT efficiency using borylated dipeptides compared to BPA. [Figure 33-2] Same as above.

[0070] [Figure 34-1] Tumor regression with BNCT using borylated dipeptides. [Figure 34-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] Detailed Description of the Invention Overview of each section I.) Definition II.) BPA III.) BSH IV.) Boron a. Boron in general V.) Naturally Occurring Amino Acids VI.) Borylated Dipeptide Amino Acids (Bdi-AA) a. BPA-Ala b.His-BPA c.Leu-BPA d.Ala-BPA e.BPA-BPA f.Tyr-BPA g. BPA-Tyr h.BPA-Leu i. one or more dipeptide amino acid compositions i.3-Boronobenzyl-BPA ii. BPA-3-boronopyridine iii. Reduced (RED)-BPA-BPA VII.) Boron Neutron Capture Therapy using Bdi-AA VIII.) Proton-boron fusion therapy using Bdi-AA IX.) Methods for delivering Bdi-AA to cells X.) Kits / Manufactured Products

[0072] I.) Definition: Unless otherwise defined, all technical terms, notations and other scientific or terminological terms used herein are intended to have the meanings commonly understood by those skilled in the art, unless the context clearly indicates otherwise. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0073] When trade names are used herein, reference to a trade name also refers to the product formulation, generic drug and active pharmaceutical ingredients of that trade name product, unless the context dictates otherwise.

[0074] The terms "advanced cancer," "locally advanced cancer," "advanced disease," and "locally advanced disease" refer to cancer that has spread through the relevant tissue capsule and are meant to include stage C disease under the American Urological Association (AUA) system, stages C1-C2 disease under the Whitmore-Jewett system, and stages T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have a substantially worse prognosis compared to patients with clinically localized (organ-confined) cancer.

[0075] "Amino acid" refers to a simple organic compound that contains both a carboxyl (-COOH) and an amino (-NH2) group.

[0076] "Borylation" refers to the reaction of forming organoboron compounds through the functionalization of aliphatic and aromatic C-H bonds.

[0077] "Borylated amino acid" (BAA) refers to a compound comprising a naturally occurring amino acid, such as those shown in Table I, that has undergone a borylation reaction. BAAs can be synthesized in several ways depending on the base amino acid used.

[0078] "Borylated dipeptide amino acid" (Bdi-AA) refers to the borylation of an amino acid paired with either (i) a non-borylated amino acid, or (ii) a second borylated amino acid. For purposes of this definition, the amino acids associated with this definition can be from any of the amino acids shown in Table I, to include amino acid doublings and unique pairings.

[0079] The term "compound" refers to and includes not only the chemical compound (e.g., Bdi-AA) itself, but also, whether or not expressly stated, unless the context makes it clear that the following is to be excluded, the term refers to and includes: amorphous and crystalline forms of the compound, polymorphic forms, which may be part of a mixture or in isolated form; free acid and free base forms of the compound, typically those forms shown in the structures provided herein; isomers of the compound (optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers, as well as mixtures of optical isomers, including racemic and non-racemic mixtures; isomers may be in isolated form or may be a mixture with one or more other isomers; isotopes of the compound, including deuterium-containing and tritium-containing compounds, including compounds that contain radioactive isotopes. and therapeutically and diagnostically effective radioisotopes; multimeric forms of the compounds, including dimeric, trimeric, etc. forms; salts of the compounds, preferably pharma- ceutically acceptable salts, including acid addition salts and base addition salts, including salts with organic and inorganic counterions, including zwitterionic forms, where in the case where the compound is associated with two or more counterions, the two or more counterions may be the same or different; and solvates, hemisolvates, monosolvates, disolvates, etc. of the compounds, including organic solvates and inorganic solvates, said inorganic solvates including hydrates; where in the case where the compound is associated with two or more solvent molecules, the two or more molecules may be the same or different. In some instances, reference herein to the compounds of the invention will include an explicit reference to one of the above forms or, for example, salts and / or solvates. However, this reference is for emphasis only and should not be construed as excluding other of the above forms identified above.

[0080] As used herein, the term "inhibit" or "inhibition of" means to decrease by a measurable amount or to completely suppress.

[0081] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses, and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

[0082] The terms "metastatic cancer" and "metastatic disease" refer to cancer that has spread to regional lymph nodes or distant sites and are meant to include stage D disease by the AUA system and stage TxNxM+ by the TNM system.

[0083] "Molecular recognition" refers to a chemical event by which a host molecule can form a complex with a second molecule (i.e., a guest). This process occurs through non-covalent chemical bonds, including but not limited to hydrogen bonding, hydrophobic interactions, and ionic interactions.

[0084] "Pharmaceutically acceptable" refers to a non-toxic, inert, and / or composition that is physiologically compatible with humans or other mammals.

[0085] The term "neutron capture agent" means a stable, non-reactive chemical isotope that produces alpha particles when activated by a neutron.

[0086] The term "neutron capture therapy" refers to a non-invasive therapeutic modality for treating locally invasive malignancies, such as primary brain tumors and recurrent head and neck cancer, as well as other immune disorders and diseases, by irradiating a neutron capture agent with neutrons.

[0087] As used herein, "treat" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as extending survival, reducing morbidity, and / or reducing side effects that are a by-product of alternative treatment modalities; as will be readily understood in the art, complete eradication of the disease, although preferred, is not a requirement of the act of treatment.

[0088] II.) BPA For reference, ( 10 B)-BPA, L-BPA, or 4-borono-L-phenylalanine (Sigma Aldrich, St. Louis, MO) has the chemical formula: [ka] is a synthetic compound having the formula It is an important boronated compound useful for the treatment of cancer by BNCT. It is a widely known compound with many syntheses being developed (see U.S. Pat. No. 8,765,997, Taiwan Biotech Co., Ltd., Taoyuan City, Taiwan, and U.S. Patent Application Publication No. 2017 / 0015684, Stella Pharma Corp., Osaka Prefecture University, Osaka, Japan).

[0089] III.) BSH Furthermore, BSH, or Sodium Borocaptate, or BSH Sodium Borocaptate, or Sodium Borocaptate B10, or Undecahydrododecaboranethiol, is a synthetic chemical compound having the following chemical formula: [ka] Here, boron atoms are represented by dots at the vertices of an ecosahedron. BSH is used as a capture agent in BNCT. Generally speaking, BSH is injected intravenously and concentrated in tumor cells. The patient then undergoes radiation treatment with atomic particles called neutrons. The neutrons fuse with the boron nuclei in the BSH, producing high-energy alpha particles that kill the tumor cells.

[0090] IV.) Boron (a.) Boron in general Generally speaking, for the purposes of this disclosure, boron is a chemical element with the symbol B and atomic number 5. Natural boron, used primarily in chemical compounds, is composed of two stable isotopes, one of which is boron-10 and the other is boron-11. The boron-10 isotope is useful for capturing epithermal neutrons, making it a promising tool in therapeutic settings using boron neutron capture therapy. Biologically, the borylated compounds disclosed herein are non-toxic to humans and animals. Based on the above, it will be apparent to those skilled in the art that an improved manner for providing high concentrations of boron in cancer cells would be advantageous. It is an object of the present disclosure to provide such advantages.

[0091] V.) Naturally Occurring Amino Acids Generally speaking, for the purposes of this disclosure, naturally occurring amino acids are organic compounds that contain amine (-NH2) and carboxyl (-COOH) functional groups along with a side chain (R group) specific to each amino acid. The essential elements of amino acids are carbon (C), hydrogen (H), oxygen (O) and nitrogen (N), although other elements are found in the side chains of certain amino acids. Approximately 500 naturally occurring amino acids are known (although only 20 appear in the genetic code (Table I)) and can be classified in many ways. They can be classified according to the location of the core structural functional group as alpha- (α-), beta- (β-), gamma- (γ-) or delta- (δ-) amino acids, with other categories relating to polarity, pH level and type of side chain group (aliphatic, acyclic, aromatic, hydroxyl- or sulfur-containing, etc.). In the form of proteins, amino acid residues form the second most abundant component (water is the most abundant) of human muscle and other tissues. Beyond their role as residues in proteins, amino acids are involved in many processes such as neurotransmitter transport and biosynthesis.

[0092] The 20 amino acids directly encoded by the genetic code (see Table I) can be divided into several groups based on their properties. The major factors are charge, hydrophilicity or hydrophobicity, size and functional group. These properties are important for protein structure and protein-protein interactions. Water-soluble proteins tend to have their hydrophobic residues (Leu, Ile, Val, Phe and Trp) buried in the center of the protein, while the hydrophilic side chains are exposed to the aqueous solvent.

[0093] Integral membrane proteins tend to have an exposed outer ring of hydrophobic amino acids that anchor them to the lipid bilayer. In between these two extremes, some peripheral membrane proteins have patches of hydrophobic amino acids on their surface that anchor them on the membrane. Similarly, proteins that must bind positively charged molecules have a surface rich in negatively charged amino acids such as glutamic acid and aspartic acid, while proteins that bind negatively charged molecules have a surface rich in positively charged chains such as lysine and arginine. There are different hydrophobic scales of amino acid residues.

[0094] Some amino acids have special properties, such as cysteine, which can form covalent disulfide bonds to other cysteine ​​residues, proline, which forms rings into the polypeptide backbone, and glycine, which is more flexible than other amino acids.

[0095] VI.) Borylated Dipeptide Amino Acids (Bdi-AA) Based on the above, those skilled in the art have shown that essential amino acid transporter proteins such as LAT1 are responsible for the uptake of certain natural amino acids. See INDIVERI et al., Frontiers in Chem., Vol. 6, Art. 243 (June 2018). In particular, the large neutral amino acid transporter 1 (LAT-1, SLC7a5) is a sodium and pH-independent transporter that supplies cells with essential amino acids (e.g., leucine, phenylalanine). The actual functional transporter is a heterodimeric disulfide-linked complex composed of the multi-membrane-spanning subunit SLC7a5 and the single-membrane-spanning subunit SLC3a2 (CD98). LAT-1 is the main transporter for sending essential amino acids across compartments such as the placenta or the blood-brain barrier. LAT1 also transports thyroid hormones T3 and T4, the dopamine precursor L-DOPA, and amino acid-related exogenous compounds, such as the drugs melphalan and gabapentin. See FRIESEMA et al., Endocrinology, 142(10): pp. 4339-4348 (Oct. 2001) and UCHINO et al., Mol. Pharmacol, 61: 729-737 (2002).

[0096] Moreover, its expression is highly upregulated in several types of human cancers, characterized by a strong requirement of amino acids for metabolism and growth. See SINGH et al., Int. J. Mol. Sci., 19, 1278 (24-April-2018). Furthermore, it has been reported that the nature of the amino acid side chain affects the selectivity of LAT1 for various amino acids in increasing the transport rate in the following order: Phe>Trp>Leu>Ile>Met>His>Tyr>Val. See KANAI et al., J. Bio. Chem., Vol. 273, No. 37, pp. 23629-23632 (Sept. 11, 1998). However, the effect of boron-added modifications on amino acids does not appear to have been taught before.

[0097] Of note, 4-borono-L-phenylalanine (L-BPA) has been used in many clinical studies with BNCT and is approved in Japan for use in the treatment of head and neck squamous cell carcinoma, using a Sumitomo accelerator as a neutron source. L-BPA has been shown to be safe, stable, and easy to manufacture. However, despite these successes, it has some significant drawbacks. These drawbacks include low solubility (1.7 mg / mL) and reliance on fructose formulations to achieve a yield of 30 mg / mL. See Watanabe et al., BMC Cancer 16:859 (2016). It is known that such concentrated solutions are metastable and must be kept ready for use due to the high risk of precipitation.

[0098] Additionally, commercially available dipeptide compositions are available for use in cell culture preparations (GlutaMAX, Thermo-Fisher, Waltham, Massachusetts). With this principle in mind, the present disclosure contemplates the synthesis of natural dipeptide amino acids by borylation reactions to create borylated dipeptide amino acids ("Bdi-AA") with tumor-seeking and tumor-localizing properties for use as neutron capture agents in boron neutron capture therapy ("BNCT") and / or boron proton capture therapy. See, for example, ROBERTS et al., Tetrahedron Letters, vol. 21, Issue 36, pp. 3435-3438 (1980).

[0099] The borylated dipeptide is significantly more soluble than BPA and is taken up by cancer cells both in vitro and in vivo. The mechanism of cellular uptake of the borylated dipeptide remains to be elucidated, but it is likely that it involves, at least in part, proteolytic cleavage by surface peptidases to release L-BPA, which is then transported via LAT-1.

[0100] Alternatively, oligopeptide transporters such as PEPT-1 (SLC15) or PEPT-2 may be involved, as found in the gastrointestinal tract. See GONG et al., Oncotarget, Vol. 8, (No. 25), pp: 40454-40468 (2017) and MIYABE et al., J Pharmacol. Sci., 139(3): 215-222 (2019).

[0101] Based on the above rationale, the present disclosure has evaluated specifically designed borylated oligopeptides for in vitro uptake using various cancer cell lines.Furthermore, the present disclosure teaches that an established subcutaneous xenograft model in mice was used to determine the pharmacokinetics and biodistribution of boron in tumors, blood and other organs.

[0102] The therapeutic potential of BNCT is based on the presence of sufficient amounts of 10 The purpose of this method is to selectively accumulate B. 10 To investigate the ability of borylated dipeptides to deliver boron to cancer cells, the present disclosure teaches a panel of dipeptides that were synthesized and tested over a range of concentrations that represent what are believed to be physiologically relevant amounts of boronophenylalanine (BPA), the boron drug currently most widely studied in BNCT clinical practice.

[0103] Based on the above background and therapeutic rationale, the following Bdi-AAs are disclosed herein and are referenced in more detail in Figure 14. Specific characterization of the chemical structures is provided in more detail below.

[0104] (a) BPA-alanine (i.e., BPA-Ala) In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka]

[0105] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0106] (b) Histidine-BPA (i.e., His-BPA) In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka]

[0107] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0108] (c) Leucine-BPA (i.e., Leu-BPA) In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka]

[0109] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0110] (d) Alanine-BPA (i.e., Ala-BPA) In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka]

[0111] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0112] (e) BPA-BPA In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka]

[0113] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0114] (f) Tyrosine-BPA, i.e., Tyr-BPA In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka]

[0115] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0116] In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka] See also MIYABE et al., J. of Pharmacological Sciences 139 (2019) pp. 215-222.

[0117] (g) BPA-tyrosine, i.e., BPA-Tyr In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka]

[0118] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0119] (h) BPA-leucine, i.e., BPA-Leu In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka]

[0120] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0121] It will be appreciated by those skilled in the art that the synthesis of these compounds can be accomplished by nucleophilic addition to BPA to Boc-protected succinate functionalized BPA, followed by deprotection of the Boc group, peptide coupling of Fmoc-protected BPA with ammonia undecahydrododecaborate, followed by deprotection to reveal the target material.

[0122] The compositions of the present disclosure can be utilized to synthesize Bdi-AAs with functional incorporation into certain complexes to deliver concentrated amounts of boron to cancer or other diseased cells for use in BNCT and / or other cancer treatment modalities.Various branched and aromatic amino acids can be explored based on the ability of certain antigen complexes to upregulate certain amino acids.For purposes of the present disclosure, in one embodiment, the amino acids include valine, leucine, isoleucine, histidine, tryptophan, tyrosine, and any set of amino acids set forth in Table I and / or borylated amino acids from the amino acids shown in Table I.

[0123] This principle can be achieved by side chain manipulation, peptide coupling, and decarboxylation-borylation. See LI et al., Science 356,1045 (2017), and MALAN et al., Synlett 1996(02):167-168; and US Patent Application Publication No. 2018 / 0155368 (Neuboron Medtech, Nanjing, China). The wide variety of useful reactivities specific to boronic acids, such as cross-coupling, oxidation, amination, and homologation, have been shown to guide retrosynthetic analysis. The present disclosure also contemplates manipulation of solubility and lipophilicity by using boronic esters instead of acids. Following one or more modifications disclosed herein, additional antigen complexes and transporters may be engaged by selective borylation of their respective molecular substrates.

[0124] (i) one or more dipeptide compositions In one embodiment, within the scope of the present disclosure is Bdi-AA having the following formula: [ka] (In the formula, A is H, an amino acid, or a boronated amino acid; E is CO2H, B(OH)2, BF3K, CO-amino acid, or CO-boronated amino acid, CO-NHB 12 H 11 and; X is H, B(OH)2, BF3K, or B(OR)2, but can also be in position 2, 3, or 4).

[0125] In a further embodiment, within the scope of the disclosure is a Bdi-AA having the following formula: [ka] (In the formula, A is H, an amino acid, or a borylated amino acid; E is CO2H, B(OH)2, BF3K, CO-amino acid, or CO-borylated amino acid, CO-NHB 12 H 11 and; X is H, B(OH)2, BF3K, or B(OR)2, but can also be in position 2 or 3).

[0126] In a further embodiment, within the scope of the disclosure is a Bdi-AA having the following formula: [ka] (In the formula, A is H, an amino acid, or a borylated amino acid; E is CO2H, B(OH)2, BF3K, CO-amino acid, or CO-borylated amino acid, CO-NHB 12 H 11 and; X is H, B(OH)2, BF3K, or B(OR)2, but can also be present in positions 2, 4, 5, 6, or 7).

[0127] (i) 3-Boronobenzyl-BPA In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka]

[0128] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0129] (ii) BPA-3-boropyridine In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0130] (iii) Reduced (RED)-BPA-BPA In one embodiment, within the scope of the present disclosure is a composition having the following formula: [ka]

[0131] Those skilled in the art will appreciate that the above compositions are synthesized as shown in FIG.

[0132] Those skilled in the art will appreciate that various modifications to the above-described structures may be readily made using methods known in the art. Such modifications or other embodiments may be made without departing from the true scope and spirit of the invention.

[0133] VII.) Boron Neutron Capture Therapy using Bdi-AA One aspect of the present disclosure is the use of Bdi-AA as a modality for boron neutron capture therapy (BNCT) and / or boron proton capture therapy ("BPCT"). Briefly, BNCT is a two-component treatment modality in which neither component alone is lethal or toxic to the tumor. The two components include (i) injection or delivery of a capture compound that preferentially concentrates in the tumor, and (ii) irradiation of the tumor site with neutrons or with protons. In BNCT: 10 In view of the large cross section of thermal neutron interaction with B, the result is that the boron nucleus 4 He 2+ and 7 Li + He 2+ and Li + Given the high ionization capacity of boron and the short distance traveled, cells favorably enriched with boron are killed, while healthy cells are much less damaged due to the lack of high concentrations of boron. In view of this, the advantage of BNCT is that it destroys tumor cells without highly traumatic surgical procedures. However, as will be appreciated by those skilled in the art, success depends on the degree of damage to the tumor cells. 10 This is based on the selective localization of B at high concentrations.

[0134] In one embodiment, 10 B is concentrated in Bdi-AA. The Bdi-AA is then administered to the patient, where it localizes to tumor cells. 10 The B-containing Bdi-AA is concentrated in a tumor, and the tumor is irradiated with epithermal neutrons. The tumor cells are destroyed.

[0135] VIII. Proton-boron fusion therapy using Bdi-AA Another aspect of the present disclosure is the use of Bdi-AA as a modality for proton-boron fusion therapy (PBFT). Briefly, the proton-boron fusion reaction was introduced in the 1960s. 1 H) and boron particles ( 11After the reaction between A and B), three α particles are released. These three α particles damage the tumor cells, similar to the case of α particles in BNCT. Theoretically, for PBFT, the therapeutic efficacy per incident particle is three times (3×) greater than that of BNCT. In addition, proton beams have the advantage of Bragg peak characteristics, so normal tissue damage can be reduced. Generally speaking, many studies have been conducted for tumor treatment with α particles. In order to utilize α particles for dose delivery, two important points should be considered. First, boron uptake should be accurately labeled to the target cells. As mentioned above, α particles are generated at the site where boronized compounds are accumulated. If this occurs in normal tissues near the tumor area, the α particles will damage not only tumor cells but also normal tissues. Secondly, the number of generated α particles is also an important factor for effective treatment. By using PBFT, more effective treatment can be achieved compared to BNCT or conventional proton therapy alone.

[0136] In one embodiment, 10 B and / or 11 B is concentrated in Bdi-AA. The Bdi-AA is then administered to the patient, where it localizes to tumor cells. 10 B and / or 11 The B-containing Bdi-AA is concentrated in a tumor, and the tumor is irradiated with epithermal neutrons. The tumor cells are destroyed.

[0137] IX. Methods for delivering Bdi-AA to cells As will be appreciated by those skilled in the art, it is an advantage of the present invention that high concentrations of boron can be efficiently delivered to cells.

[0138] The Bdi-AA of the present disclosure has been shown to allow safe administration of higher amounts of boron to mammalian cells.In short, the Bdi-AA of the present disclosure is prepared as shown in the present disclosure.The obtained Bdi-AA is taken up into tumor cells by upregulated LAT1 transporter protein and / or upregulated dipeptide transporter protein such as PEPT1.

[0139] X.) Kits / Manufactured Products For use in the laboratory, prognostic, preventive, diagnostic and therapeutic applications described herein, kits are within the scope of the present invention. Such kits can include a carrier, package or container that is compartmentalized to receive one or more containers, such as vials, tubes, each of which contains one of the separate elements to be used in the method, along with a label or insert containing instructions for use, such as those described herein. For example, the container can contain one Bdi-AA or several Bdi-AAs of the present disclosure. The kit can include a container containing a drug unit. The kit can include all or a portion of the Bdi-AA and / or a diagnostic assay for detecting cancer and / or other immune disorders.

[0140] Kits of the present invention typically include the above-mentioned container and one or more other containers associated with the above-mentioned container with equipment desirable from a commercial and user standpoint, such as buffers, diluents, filters, needles, syringes, etc., labels on the carriers, packages, containers, vials and / or tubes listing the contents and / or instructions for use, and package inserts containing the instructions for use.

[0141] A label can be present on or with the container to indicate that the composition is used for a particular therapeutic or non-therapeutic application, such as prognostic, preventive, diagnostic or laboratory application, and can also indicate instructions for either in vivo or in vitro use, such as those described herein. Instructions and / or other information can also be included on an insert or label included with or on the kit. The label can be present on the container or associated with the container. The label can be present on the container if the letters, numbers or other characters forming the label are molded or etched into the container itself, or the label can be associated with the container if the label is present in a receptacle or carrier that also holds the container, such as as a package insert. The label can indicate that the composition is used to diagnose, treat, prevent or prognose a condition, such as cancer or other immune disorder.

[0142] The terms "kit" and "article of manufacture" may be used synonymously.

[0143] In another embodiment of the present invention, one or more articles of manufacture contain the composition of the present disclosure, such as Bdi-AA. The article of manufacture typically includes at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be formed from a variety of materials, such as glass, metal or plastic. The container can hold one or more Bdi-AA and / or one or more therapeutic doses of Bdi-AA.

[0144] Alternatively, the container may hold a composition effective for treating, diagnosing, prognosing or preventing a condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition may be the Bdi-AA of the present disclosure.

[0145] The article of manufacture may further include a second container comprising a pharma- ceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and / or dextrose solution. The article of manufacture may further include other buffers, diluents, filters, stirrers, needles, syringes, and / or other equipment desirable from a commercial and user standpoint, including package inserts with directions and / or instructions for use.

[0146] Exemplary embodiments: 1) The following chemical structure: [ka] A composition comprising: where A is H, an amino acid, or a borylated amino acid; E is CO2H, B(OH)2, BF3K, CO-amino acid, or CO-borylated amino acid, CO-NHB 12 H 11 and; X is H, B(OH)2, BF3K, or B(OR)2, or can be in position 2, 3, or 4; composition.

[0147] 2) The following chemical structure: [ka] A composition comprising: where A is H, an amino acid, or a borylated amino acid; E is CO2H, B(OH)2, BF3K, CO-amino acid, or CO-borylated amino acid, CO-NHB 12 H 11 and; X is H, B(OH)2, BF3K, or B(OR)2, or can be in position 2 or 3; composition.

[0148] 3) The following chemical structure: [ka] A composition comprising: where A is H, an amino acid, or a borylated amino acid; E is CO2H, B(OH)2, BF3K, CO-amino acid, or CO-borylated amino acid, CO-NHB 12 H 11 and; X is H, B(OH)2, or B(OR)2, BF3K, or can be present in position 2, 4, 5, 6, or 7; composition.

[0149] 4) A kit comprising the composition of claim 1.

[0150] 5) A kit comprising the composition according to claim 2.

[0151] 6) A kit comprising the composition according to claim 3.

[0152] 7) A method for producing the composition of claim 1.

[0153] 8) A method for producing the composition according to claim 2.

[0154] 9) A method for producing the composition according to claim 3.

[0155] 10) A unit dosage form comprising the composition of claim 1.

[0156] 11) A unit dosage form comprising the composition of claim 2.

[0157] 12) A unit dosage form comprising the composition of claim 3.

[0158] 13) A composition comprising the following chemical structure: [ka]

[0159] 14) A composition comprising any of the following chemical structures: [ka]

[0160] 15) A composition comprising any of the following chemical structures: [ka]

[0161] 16) A composition comprising any of the following chemical structures: [ka]

[0162] 17) A composition comprising any of the following chemical structures: [ka]

[0163] 18) A composition comprising any of the following chemical structures: [ka]

[0164] 19) A composition comprising any of the following chemical structures: [ka]

[0165] 20) A kit comprising any one or more of the compositions of claim 13.

[0166] 21) A kit comprising any one or more of the compositions of claim 14.

[0167] 22) A kit comprising any one or more of the compositions of claim 15.

[0168] 23) A kit comprising any one or more of the compositions of claim 16.

[0169] 24) A kit comprising any one or more of the compositions of claim 17.

[0170] 25) A kit comprising any one or more of the compositions of claim 18.

[0171] 26) A kit comprising any one or more of the compositions of claim 19.

[0172] 27) A method for producing any of the compositions of claim 13.

[0173] 28) A method for producing any of the compositions of claim 14.

[0174] 29) A method for producing any of the compositions of claim 15.

[0175] 30) A method for producing any of the compositions of claim 16.

[0176] 31) A method for producing any of the compositions of claim 17.

[0177] 32) A method for producing any of the compositions of claim 18.

[0178] 33) A method for producing any of the compositions of claim 19.

[0179] 34) A unit dosage form comprising any of the compositions of claim 13.

[0180] 35) A unit dosage form comprising any of the compositions of claim 14.

[0181] 36) A unit dosage form comprising any of the compositions of claim 15.

[0182] 37) A unit dosage form comprising any of the compositions of claim 16.

[0183] 38) A unit dosage form comprising any of the compositions of claim 17.

[0184] 39) A unit dosage form comprising any of the compositions of claim 18.

[0185] 40) A unit dosage form comprising any of the compositions of claim 19.

[0186] 41) A composition comprising any of the following chemical structures: [ka]

[0187] 42) A composition comprising any of the following chemical structures: [ka]

[0188] 43) A composition comprising any of the following chemical structures: [ka]

[0189] 44) A kit comprising any one or more of the compositions of claim 41.

[0190] 45) A kit comprising any one or more of the compositions of claim 42.

[0191] 46) A kit comprising any one or more of the compositions of claim 43.

[0192] 47) A method for producing any of the compositions of claim 41.

[0193] 48) A method for producing any of the compositions of claim 42.

[0194] 49) A method for producing any of the compositions of claim 43.

[0195] 50) A unit dosage form comprising any of the compositions of claim 41.

[0196] 51) A unit dosage form comprising any of the compositions of claim 42.

[0197] 52) A unit dosage form comprising any of the compositions of claim 43.

[0198] 53) A method of carrying out neutron capture therapy in the treatment of human cancer, comprising the steps of: a. synthesizing a human unit dose of a borylated dipeptide amino acid (Bdi-AA) composition; b. Bdi-AA is injected into a tumor, which results in intracellular accumulation of Bdi-AA; and c. Irradiating Bdi-AA with neutrons A method comprising:

[0199] 54) The method of claim 53, wherein the composition is selected from the group consisting of the compositions of claims 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 41, 42, and 43.

[0200] 55) The method of claim 53, wherein the neutron capture therapy is boron neutron capture therapy.

[0201] 56) The method of claim 53, wherein the irradiation comprises epithermal neutrons.

[0202] 57) A method of performing proton-boron fusion therapy in the treatment of human cancer, comprising: a. synthesizing a human unit dose of a borylated dipeptide amino acid (Bdi-AA) composition; b. Bdi-AA is injected into a tumor, which results in intracellular accumulation of Bdi-AA; and c. Irradiating Bdi-AA with protons A method comprising:

[0203] 58) The method of claim 57, wherein the composition is selected from the group consisting of the compositions of claims 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 41, 42, and 43.

[0204] 59) A composition comprising the following chemical structure: [ka]

[0205] 60) A composition comprising the following chemical structure: [ka]

[0206] 61) A composition comprising the following chemical structure: [ka]

[0207] 62) A kit comprising any one or more of the compositions of claim 59.

[0208] 63) A kit comprising any one or more of the compositions of claim 60.

[0209] 64) A kit comprising any one or more of the compositions of claim 61.

[0210] 65) A method for producing any of the compositions of claim 59.

[0211] 66) A method for producing any of the compositions of claim 60.

[0212] 67) A method for producing any of the compositions of claim 61.

[0213] 68) A unit dosage form comprising any of the compositions of claim 59.

[0214] 69) A unit dosage form comprising any of the compositions of claim 60.

[0215] 70) A unit dosage form comprising any of the compositions of claim 61.

[0216] 71) A method of carrying out neutron capture therapy in the treatment of human cancer, comprising: a. synthesizing a human unit dose of a borylated dipeptide amino acid (Bdi-AA) composition; b. Bdi-AA is injected into a tumor, which results in intracellular accumulation of Bdi-AA; and c. Irradiating Bdi-AA with neutrons A method comprising:

[0217] 72) The method of claim 71, wherein the composition is selected from the group consisting of the compositions of claims 59, 60, and 61.

[0218] 73) The method of claim 71, wherein the neutron capture therapy is boron neutron capture therapy.

[0219] 74) The method of claim 71, wherein the irradiation comprises epithermal neutrons.

[0220] 75) A method of performing proton-boron fusion therapy in the treatment of human cancer, comprising: a. synthesizing a human unit dose of a borylated dipeptide amino acid (Bdi-AA) composition; b. Bdi-AA is injected into a tumor, which results in intracellular accumulation of Bdi-AA; and c. Irradiating Bdi-AA with protons A method comprising:

[0221] 76) The method of claim 75, wherein the composition is selected from the group consisting of the compositions of claims 59, 60, and 61.

[0222] 77) A method of carrying out neutron capture therapy in the treatment of human cancer, comprising: a. synthesizing a human unit dose of a dipeptide amino acid (di-AA) composition; b. di-AA is injected into the tumor, which results in intracellular accumulation of di-AA; and c. Irradiating di-AA with neutrons A method comprising:

[0223] 78) The method of claim 7, wherein the composition is selected from the group consisting of the compositions of claims 41, 42, and 43.

[0224] 79) The method of claim 77, wherein the neutron capture therapy is boron neutron capture therapy.

[0225] 80) The method of claim 77, wherein the irradiation comprises epithermal neutrons.

[0226] 81) A method of performing proton-boron fusion therapy in the treatment of human cancer, comprising: a. synthesizing a human unit dose of a dipeptide amino acid (di-AA) composition; b. di-AA is injected into the tumor, which results in intracellular accumulation of di-AA; and c. Irradiating di-AA with protons A method comprising:

[0227] 82) The method of claim 81, wherein the composition is selected from the group consisting of the compositions of claims 41, 42, and 43. EXAMPLES

[0228] Various aspects of the present invention are further described and illustrated by the following several examples, none of which are intended to limit the scope of the invention.

[0229] Example 1: Synthesis of BPA-Ala.

[0230] BPA-Ala was synthesized as follows. Briefly, to a solution of 20 mL of dioxane and 20 mL of water at room temperature, 2 mL of triethylamine and 2 g of 4-borono-L-phenylalanine are added. After stirring for 15 minutes, di-tert-butyl dicarbonate (2.3 g) is added to the reaction. After 12 hours, the reaction is complete as observed by LCMS. The dioxane is removed under reduced pressure, the aqueous solution is poured into 50 mL of water, and the aqueous solution is washed three times with 50 mL aliquots of ethyl acetate. The aqueous solution is then acidified with 1 M HCl and then back-extracted twice with 50 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material (2) below. [ka]

[0231] Then, to a solution of 13.4 mL of dimethylformamide at room temperature, 3.5 mL of N,N-diisopropylethylamine (DIPEA), 2 grams of Boc-BPA, 0.99 g of DL-alanine methyl ester (Ala-OMe), 1.2 g of hydroxybenzotriazole (HOBt) and 1.49 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are added. After stirring for 12 hours, the reaction is complete as observed by LCMS. The reaction solution is poured into 50 mL of 1 M HCl and the aqueous solution is washed with two 50 mL aliquots of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material (4) below. [ka]

[0232] 2.5 g of Boc-BPA-Ala-OMe is then added to a solution of 54 mL of methanol and 6 mL of water along with 0.227 g of lithium hydroxide. After refluxing at 65° C. for 4 hours, the reaction is complete as observed by LCMS. The methanol is then removed under reduced pressure and the remaining aqueous layer is acidified with 20 mL of 1 M HCl solution. The aqueous solution is then back-extracted with three portions of 30 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is then filtered and the solvent is removed under reduced pressure to reveal the target material (5). [ka]

[0233] Finally, 5 mL of water is added to a flask containing 2.4 g of Boc-BPA-Ala at room temperature. Then, 23.7 mL of 4 M hydrochloric acid in 1,4-dioxane is slowly added. After stirring for 2 hours, the reaction is complete as monitored by LCMS. The solvent is then removed under reduced pressure and placed on preparative HPLC to give the target material (6) below. [ka]

[0234] The resulting synthesis and composition (labeled BPA-Ala) is shown in FIG.

[0235] BPA-Ala was analyzed by LC / MS to confirm the molecular weight and to determine the purity after preparation of aqueous solutions. Briefly, the dipeptide was analyzed using a Luna Omega Polar C18 column (2.1 x 150 mm, Phenomenex) maintained at 40 °C with a flow rate of 0.5 ml / min. The bound specimen was eluted with a gradient of acetonitrile-0.1% TFA. The resulting purity analysis of BPA-Ala is shown in Figure 2.

[0236] Example 3: Synthesis of His-BPA.

[0237] His-BPA was synthesized as follows. Briefly, 1 g of 4-borono-L-phenylalanine was added to a 10 mL methanol solution at 0° C. Then, 0.625 mL of thionyl chloride was added dropwise. After 2 hours, the reaction was complete as monitored by LCMS. The solvent was removed under reduced pressure to give the target material (7). [ka]

[0238] Then, 0.6 g of 4-borono-L-phenylalanine methyl ester (BPA-OMe) and 0.4 mL of triethylamine (TEA) are added to a 10 mL acetonitrile solution at 0° C. After stirring for 10 minutes, 1.34 g of Boc-L-histidine (Boc)-succinimide ester (Boc-His(Boc)-OSu) is added. After stirring for 12 hours, the reaction is complete as observed by LCMS. The acetonitrile is removed under reduced pressure to give the target material (9): [ka]

[0239] Then, to a solution of both 3.4 mL of water and 13.6 mL of methanol, 0.11 g of lithium hydroxide and 1.7 g of Boc-His(Boc)-BPA-OMe are added. The reaction temperature is then increased to reflux at 65° C. After stirring for 4 hours, the reaction is complete as monitored by LCMS. The solvent is removed under reduced pressure to give the target material (10). [ka]

[0240] Finally, 4.5 mL of water is added to a flask containing 1.5 g of Boc-His-BPA at room temperature. Then, 10.3 mL of 4 M hydrochloric acid in 1,4-dioxane is slowly added. After stirring for 2 hours, the reaction is complete as observed by LCMS. The solvent is then removed under reduced pressure and placed on preparative HPLC to give the target material (11) below. [ka]

[0241] The resulting synthesis and composition, designated His-BPA, is shown in FIG.

[0242] His-BPA was analyzed by LC / MS to confirm the molecular weight and to determine the purity after preparation of aqueous solutions. Briefly, the dipeptide was analyzed using a Luna Omega Polar C18 column (2.1 x 150 mm, Phenomenex) maintained at 40 °C at a flow rate of 0.5 ml / min. The bound specimen was eluted with a gradient of acetonitrile-0.1% TFA. The resulting purity analysis of His-BPA is shown in Figure 4.

[0243] Example 3: Synthesis of Leu-BPA.

[0244] Leu-BPA was synthesized as follows. Briefly, to a solution of 10 mL of water and 2.5 mL of dioxane at 20° C., 0.64 g of sodium hydroxide and 1 g of L-leucine are added. After stirring for 15 minutes, 2.5 g of di-tert-butyl dicarbonate are added to the reaction. After 4 hours, the reaction is complete as observed by LCMS. Dioxane is removed under reduced pressure. An additional 10 mL of water is added to the aqueous solution. The aqueous solution is then acidified with 1 M hydrochloric acid and then back-extracted three times with 30 mL of ethyl acetate. The combined organic fractions were washed with 50 mL of brine and then dried over magnesium sulfate. The solution was then filtered and the solvent was removed under reduced pressure to give the following target material (13). [ka]

[0245] Then, to a solution of 38 mL of methylene chloride at room temperature, 1.76 g of Boc-Leu, 1.365 g of N-hydroxyphthalimide (NHP), 0.093 g of 4-dimethylaminopyridine (DMAP) and 1.311 mL of diisopropylcarbodiimide (DIC) are added. After 12 hours, the reaction is complete as observed by LCMS. The solvent is removed under reduced pressure and the concentrate is dissolved in 30 mL of water. The aqueous solution is then extracted three times with 30 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. After filtration, the solvent is removed under reduced pressure and subjected to flash column chromatography. The fractions are combined and the solvent is removed under reduced pressure to reveal the following target material (14). [ka]

[0246] 4-Borono-L-phenylalanine methyl ester (BPA-OMe) was then synthesized as follows. Briefly, 1 g of 4-borono-L-phenylalanine was added to a 10 mL methanol solution at 0° C. Then, 0.625 mL of thionyl chloride was added dropwise. After 2 hours, the reaction was complete as observed by LCMS. The solvent was removed under reduced pressure to give the target material (7). [ka]

[0247] Then, in 10 mL of acetonitrile solution at room temperature, 0.593 g of BPA-OMe and 0.44 mL of triethylamine are added. After stirring for 15 minutes, 1 g of Boc-L-leucine O-phthalimide ester is added. After 4 hours, the reaction is complete as observed by LCMS. The solvent is removed under reduced pressure to reveal the target material (15). [ka]

[0248] To a solution of 24 mL of methanol and 6.5 mL of water, 1.2 g of Boc-Leu-BPA-OMe is then added along with 0.198 g of lithium hydroxide. After refluxing at 65° C. for 4 hours, the reaction is complete as observed by LCMS. The methanol is then removed under reduced pressure and the remaining aqueous layer is acidified with 20 mL of 1 M HCl solution. The aqueous solution is then back-extracted with three portions of 30 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is then filtered and the solvent is removed under reduced pressure to reveal the target material (16). [ka]

[0249] Finally, 3 mL of water is added to a flask containing 1.2 g of Boc-Leu-BPA at room temperature. Then, 14.2 mL of 4 M hydrochloric acid in 1,4-dioxane is slowly added. After stirring for 2 hours, the reaction is complete as observed by LCMS. The solvent is then removed under reduced pressure and placed on preparative HPLC to give the target material (17) below. [ka]

[0250] The synthesis and composition thus obtained (denoted Leu-BPA) are shown in FIG.

[0251] Leu-BPA was analyzed by LC / MS to confirm the molecular weight and to determine the purity after preparation of aqueous solutions. Briefly, the dipeptide was analyzed using a Luna Omega Polar C18 column (2.1 x 150 mm, Phenomenex) maintained at 40 °C at a flow rate of 0.5 ml / min. The bound specimen was eluted with a gradient of acetonitrile-0.1% TFA. The resulting purity analysis of Leu-BPA is shown in Figure 6.

[0252] Example 4: Synthesis of Ala-BPA.

[0253] Ala-BPA was synthesized as follows. Briefly, 1 g of 4-L-boronophenylalanine was added to a 10 mL methanol solution at 0° C. Then, 0.625 mL of thionyl chloride was added dropwise. After 2 hours, the reaction was complete as monitored by LCMS. The solvent was removed under reduced pressure to give the target material (7). [ka]

[0254] Then, 1.5 g of BPA-OMe and 1.125 mL of triethylamine are added to a flask containing 28 mL of acetonitrile solution at room temperature. After stirring for 10 minutes, 1.925 g of Boc-L-Ala-succinimide ester is added. After 4 hours, the reaction is complete as observed by LCMS. The solvent is removed under reduced pressure to reveal the target material (19). [ka]

[0255] 2.6 g of Boc-Ala-BPA-OMe is then added to a solution of 52 mL of methanol and 6.5 mL of water along with 0.237 g of lithium hydroxide. After refluxing at 65° C. for 4 hours, the reaction is complete as observed by LCMS. The methanol is then removed under reduced pressure and the remaining aqueous layer is acidified with 20 mL of 1 M HCl solution. The aqueous solution is then back-extracted with three portions of 30 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is then filtered and the solvent is removed under reduced pressure to reveal the target material (20). [ka]

[0256] Finally, 5 mL of water is added to a flask containing 2.5 g of Boc-Ala-BPA at room temperature. Then, 16.4 mL of 4 M hydrochloric acid in 1,4-dioxane is slowly added. After stirring for 2 hours, the reaction is complete as observed by LCMS. The solvent is then removed under reduced pressure and placed on preparative HPLC to give the target material (21) below.

[0257] The resulting synthesis and composition, designated Ala-BPA, is shown in FIG.

[0258] Ala-BPA was analyzed by LC / MS to confirm the molecular weight and to determine the purity after preparation of aqueous solutions. The dipeptide was analyzed using an ACQUITY BEH C18 column (2.1 x 50 mm, Waters) maintained at 40 °C with a flow rate of 0.5 ml / min. The bound specimen was eluted with a gradient of acetonitrile-0.1% formic acid. The resulting purity analysis of Ala-BPA is shown in Figure 8.

[0259] Example 5: Synthesis of BPA-BPA.

[0260] BPA-BPA was synthesized as follows. Briefly, to a solution of 20 mL of dioxane and 20 mL of water at room temperature, 2 mL of triethylamine and 2 g of 4-borono-L-phenylalanine (BPA) are added. After stirring for 15 minutes, di-tert-butyl dicarbonate (2.3 g) is added to the reaction. After 12 hours, the reaction is complete as observed by LCMS. The dioxane is removed under reduced pressure, the aqueous solution is poured into 50 mL of water, and the aqueous solution is washed three times with 50 mL aliquots of ethyl acetate. The aqueous solution is then acidified with 1 M HCl and then back-extracted twice with 50 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material (2) below. [ka]

[0261] Then, 1 g of 4-borono-L-phenylalanine is added to a 10 mL methanol solution at 0° C. Then, 0.625 mL of thionyl chloride is added dropwise. After 2 hours, the reaction is complete as monitored by LCMS. The solvent is removed under reduced pressure to give the target material (7). [ka]

[0262] Then, to a solution of 15 mL of dimethylformamide at room temperature, 2.4 mL of triethylamine (TEA), 1.89 g of Boc-BPA (2), 1.24 g of BPA-OMe (7), 1.01 g of hydroxybenzotriazole (HOBt), and 1.27 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are added. After stirring for 12 hours, the reaction is complete as observed by LCMS. The reaction solution is poured into 50 mL of 1 M HCl, and the aqueous solution is washed with two 50 mL aliquots of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material (22): [ka]

[0263] Then, 2.2 g of Boc-BPA-BPA-OMe (22) is added to a solution of 36 mL of methanol and 4 mL of water along with 0.151 g of lithium hydroxide. After refluxing at 65° C. for 4 hours, the reaction is complete as observed by LCMS. The methanol is then removed under reduced pressure and the remaining aqueous layer is acidified with 20 mL of 1 M HCl solution. The aqueous solution is then back-extracted three times with 30 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is then filtered and the solvent is removed under reduced pressure to reveal the target material (23). [ka]

[0264] Finally, 2.6 mL of water is added to a flask containing 1.1 g of Boc-BPA-BPA (23) at room temperature. Then, 10.6 mL of 4 M hydrochloric acid in 1,4-dioxane is slowly added. After stirring for 2 hours, the reaction is complete as observed by LCMS. The solvent is then removed under reduced pressure and placed on preparative HPLC to give the target material (24) below. [ka]

[0265] The synthesis and composition thus obtained (labeled BPA-BPA) are shown in FIG.

[0266] BPA-BPA was analyzed by LC / MS to confirm the molecular weight and to determine the purity after preparation of aqueous solutions. Briefly, the dipeptide was analyzed using a Luna Omega Polar C18 column (2.1 x 150 mm, Phenomenex) maintained at 40 °C at a flow rate of 0.5 ml / min. The bound specimen was eluted with a gradient of acetonitrile-0.1% TFA. The resulting purity analysis of BPA-BPA is shown in Figure 10.

[0267] Example 6: Synthesis of 3-boronobenzyl-BPA.

[0268] 3-Boronobenzyl-BPA was synthesized as follows. Briefly, to a 15 mL solution of dimethylformamide at room temperature, 1.7 mL of diisopropylethylamine and 1.0 g of 4-boron-L-phenylalanine methyl ester (7) are added. 3-Boronobenzoic acid (25, 0.82 g) is then added to the reaction. Coupling agents EDC (1.0 g) and HOBt (0.82 g) are then added to the reaction. After 12 hours, the reaction is complete as observed by LCMS. The reaction solution is poured into 100 mL of 1 M HCl and 100 mL of ethyl acetate and the organic layer is separated. The organic layer is washed with 50 mL of saturated sodium bicarbonate, then 50 mL of brine, and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material (26). [ka]

[0269] Then, 0.15 g of LiOH is added to a flask containing 3-boronobenzyl-BPA-OMe (26) dissolved in 12 mL of MeOH and 3 mL of water at room temperature. The reaction is brought to 60 °C. After 2 h, the reaction is complete as monitored by LCMS. This material is purified by preparative LC to reveal the target material (27). The resulting synthesis and composition (labeled 3-boronobenzyl-BPA) are shown in Figure 11. [ka]

[0270] Example 7: Synthesis of BPA-3-boronopyridine.

[0271] BPA-3-boronopyridine was synthesized as follows. Briefly, 0.62 mL of diisopropylethylamine and 0.5 g of Boc-4-boron-L-phenylalanine (2) are added to a 15 mL solution of dimethylformamide at room temperature. 3-boronopyridine (28, 0.36 g) is then added to the reaction. Coupling agents EDC (0.37 g) and HOBt (0.3 g) are then added to the reaction. After 12 hours, the reaction is complete as observed by LCMS. The reaction solution is then poured into 100 mL of 1 M HCl and 100 mL of ethyl acetate and the organic layer is separated. The organic layer is washed with 50 mL of saturated sodium bicarbonate, then 50 mL of brine, and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material (29). [ka]

[0272] Then, 6.6 mL of 4M HCl in dioxane is added to the flask containing Boc-BPA-3-boronopyridine at room temperature. After 30 minutes, the reaction is complete as monitored by LCMS. The solvent is removed under reduced pressure to reveal the target material (30). [ka]

[0273] The synthesis and composition thus obtained (labeled BPA-3-boronopyridine) is shown in FIG.

[0274] Example 8: Synthesis of reduced BPA-BPA ((Red)-BPA-BPA).

[0275] The reduced BPA-BPA (33) was synthesized as follows. Briefly, 3.3 gmL of 4-boron-L-phenylalanine methyl ester (7) was added to a 50 mL solution of tetrahydrofuran at 0 °C. 3.3 g of lithium aluminum hydride was then added in small portions to the reaction. The reaction was complete in 2 h as monitored by LCMS. Following Fieser workup, the intermediate alcohol was resuspended in 75 mL of a dioxane / water mixture at room temperature. To this, 4 mL of triethylamine and 4.5 g of di-tert-butyl dicarbonate were added. The Boc-protected BPA alcohol was purified by ethyl acetate extraction, then dissolved in dichloromethane and the temperature was reduced to -76 °C.

[0276] To this solution, 3 mL of triethylamine, 0.8 mL of dimethyl sulfoxide, and finally 2.5 mL of oxalyl chloride were added. After 20 min, the reaction was extracted to obtain the aldehyde of BPA (31). The BPA aldehyde was dissolved in 45 mL of methanol and 5% acetic acid (v / v). To this, 800 mg of 4-boron-L-phenylalanine methyl ester (7) and 425 mg of sodium cyanoborohydride were added at room temperature. After 2 h, the target secondary amine was isolated by extraction into ethyl acetate. [ka]

[0277] Then, 0.1 g of LiOH is added to a flask containing Boc-BPA-NH-BPA-OMe (32) dissolved in 8 mL of MeOH and 8 mL of water at room temperature. The reaction is brought to 60 °C. After 2 h, the reaction is complete as monitored by LCMS. The material is extracted into acidic water and subsequently lyophilized to remove the boc group in situ. [ka]

[0278] The synthesis and composition thus obtained (denoted as (Red)-BPA-BPA) are shown in FIG.

[0279] Example 9: Synthesis of BPA-Tyr.

[0280] BPA-Tyr (36) was synthesized as follows. Briefly, to a solution of 20 mL of dioxane and 20 mL of water at room temperature, 2 mL of triethylamine and 2 g of 4-borono-L-phenylalanine are added. After stirring for 15 minutes, di-tert-butyl dicarbonate (2.3 g) is added to the reaction. After 12 hours, the reaction is complete as observed by LCMS. The dioxane is removed under reduced pressure, the aqueous solution is poured into 50 mL of water, and the aqueous solution is washed three times with 50 mL aliquots of ethyl acetate. The aqueous solution is then acidified with 1 M HCl and then back-extracted twice with 50 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material: [ka]

[0281] Then, 2 g of L-tyrosine is added to a 20 mL solution of methanol at 0° C. Then, 2.843 mL of thionyl chloride is added dropwise. After 3 hours, the reaction is complete as monitored by LCMS. The solvent is removed under reduced pressure to give the following target material: [ka]

[0282] Then, to a solution of 8.5 mL of dimethylformamide at room temperature is added 2.8 mL of diisopropylethylamine (DIPEA), 1.74 g of Boc-BPA, 1.0 g of Tyr-OMe, 1.04 g of hydroxybenzotriazole (HOBt), and 1.18 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). After stirring for 12 hours, the reaction is complete as observed by LCMS. The reaction solution is poured into 50 mL of 1 M HCl and the aqueous solution is washed with two 50 mL aliquots of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material: [ka]

[0283] 2.5 g of Boc-BPA-Tyr-OMe is then added to a solution of 40 mL of methanol and 2.5 mL of water along with 0.648 g of lithium hydroxide. After refluxing at 65° C. for 4 hours, the reaction is complete as observed by LCMS. The methanol is then removed under reduced pressure and the remaining aqueous layer is acidified with 20 mL of 1 M HCl solution. The aqueous solution is then back-extracted three times with 30 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is then filtered and the solvent is removed under reduced pressure to reveal the following target material: [ka]

[0284] Finally, 3.0 mL of water is added to a flask containing 2.4 g of Boc-BPA-Tyr at room temperature. Then, 24.1 mL of 4 M hydrochloric acid in 1,4-dioxane is slowly added. After stirring for 2 hours, the reaction is complete as observed by LCMS. The solvent is then removed under reduced pressure and placed on preparative HPLC to give the target material (36). [ka]

[0285] The synthesis and composition thus obtained (designated BPA-Tyr) (36) are shown in Figure 15. BPA-Tyr was analyzed by LC / MS to confirm the molecular weight and to determine the purity after preparation of aqueous solutions. Briefly, the dipeptides were analyzed using a BEH C18 column (2.1 x 50 mm, Waters) maintained at 40 °C at a flow rate of 0.5 ml / min. The bound specimens were eluted with a gradient of acetonitrile-0.1% FA. The resulting purity analysis of BPA-Tyr is shown in Figure 16.

[0286] Example 10: Synthesis of Tyr-BPA.

[0287] BPA-Tyr (37) was synthesized as follows. Briefly, to a solution of 23 mL of dioxane and 20 mL of water at room temperature, 2.3 mL of triethylamine and 2 g of L-tyrosine are added. After stirring for 15 minutes, di-tert-butyl dicarbonate (2.65 g) is added to the reaction. After 12 hours, the reaction is complete as observed by LCMS. The dioxane is removed under reduced pressure, the aqueous solution is poured into 50 mL of water, and the aqueous solution is washed three times with 50 mL aliquots of ethyl acetate. The aqueous solution is then acidified with 1 M HCl and then back-extracted twice with 50 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material: [ka]

[0288] Then, 2 g of 4-borono-L-phenylalanine is added to a 20 mL methanol solution at 0° C. Then, 2.88 mL of thionyl chloride is added dropwise. After 2 hours, the reaction is complete as observed by LCMS. The solvent is removed under reduced pressure to give the following target material: [ka]

[0289] Then, to a solution of 6.0 mL of dimethylformamide at room temperature is added 1.9 mL of diisopropylethylamine (DIPEA), 1.0 g of Boc-Tyr, 0.872 g of BPA-OMe, 0.721 g of hydroxybenzotriazole (HOBt), and 0.818 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). After stirring for 12 hours, the reaction is complete as observed by LCMS. The reaction solution is poured into 50 mL of 1 M HCl and the aqueous solution is washed with two 50 mL aliquots of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material: [ka]

[0290] 1.5 g of Boc-Tyr-BPA-OMe is then added to a solution of 24.1 mL of methanol and 1.5 mL of water along with 0.222 g of lithium hydroxide. After refluxing at 65° C. for 4 hours, the reaction is complete as observed by LCMS. The methanol is then removed under reduced pressure and the remaining aqueous layer is acidified with 20 mL of 1 M HCl solution. The aqueous solution is then back-extracted three times with 30 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is then filtered and the solvent is removed under reduced pressure to reveal the following target material: [ka]

[0291] Finally, 11.8 mL of 4 M hydrochloric acid in 1,4-dioxane is slowly added to a flask containing 1.18 g of Boc-Tyr-BPA at room temperature. After stirring for 2 hours, the reaction is complete as monitored by LCMS. The solvent is then removed under reduced pressure and placed on preparative HPLC to give the target material (37). [ka]

[0292] The synthesis and composition thus obtained (designated Tyr-BPA) (37) are shown in Figure 17. Tyr-BPA was analyzed by LC / MS to confirm the molecular weight and to determine the purity after preparation of aqueous solutions. Briefly, the dipeptide was analyzed using a BEH C18 column (2.1 x 50 mm, Waters) maintained at 40 °C at a flow rate of 0.5 ml / min. The bound specimen was eluted with a gradient of acetonitrile-0.1% FA. The purity analysis of the obtained Tyr-BPA is shown in Figure 18.

[0293] Example 11: Synthesis of BPA-Leu.

[0294] BPA-Leu (38) was synthesized as follows. Briefly, to a solution of 20 mL of dioxane and 20 mL of water at room temperature, 2 mL of triethylamine and 2 g of 4-borono-L-phenylalanine are added. After stirring for 15 minutes, di-tert-butyl dicarbonate (2.3 g) is added to the reaction. After 12 hours, the reaction is complete as observed by LCMS. The dioxane is removed under reduced pressure, the aqueous solution is poured into 50 mL of water, and the aqueous solution is washed three times with 50 mL aliquots of ethyl acetate. The aqueous solution is then acidified with 1 M HCl and then back-extracted twice with 50 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material: [ka]

[0295] Then, 2 g of L-leucine is added to a 20 mL solution of methanol at 0° C. Then, 2.88 mL of thionyl chloride is added dropwise. After 2 hours, the reaction is complete as observed by LCMS. The solvent is removed under reduced pressure to give the following target material: [ka]

[0296] Then, to a solution of 11.5 mL of dimethylformamide at room temperature is added 3.7 mL of diisopropylethylamine (DIPEA), 2.3 g of Boc-Tyr, 1.0 g of Leu-OMe, 1.39 g of hydroxybenzotriazole (HOBt), and 1.58 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). After stirring for 12 hours, the reaction is complete as observed by LCMS. The reaction solution is poured into 50 mL of 1 M HCl and the aqueous solution is washed with two 50 mL aliquots of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is filtered and the solvent is removed under reduced pressure to reveal the target material: [ka]

[0297] 3.0 g of Boc-BPA-Leu-OMe is then added to a solution of 48 mL of methanol and 3.0 mL of water along with 0.494 g of lithium hydroxide. After refluxing at 65° C. for 4 hours, the reaction is complete as observed by LCMS. The methanol is then removed under reduced pressure and the remaining aqueous layer is acidified with 20 mL of 1 M HCl solution. The aqueous solution is then back-extracted three times with 30 mL of ethyl acetate. The combined organic layers are washed with 50 mL of brine and then dried over magnesium sulfate. The solution is then filtered and the solvent is removed under reduced pressure to reveal the following target material: [ka]

[0298] Finally, 3.0 mL of water is added to a flask containing 2.9 g of Boc-BPA-Leu at room temperature. Then, 26.1 mL of 4 M hydrochloric acid in 1,4-dioxane is slowly added. After stirring for 2 hours, the reaction is complete as observed by LCMS. The solvent is then removed under reduced pressure and placed on preparative HPLC to give the target material (38). [ka]

[0299] The synthesis and composition thus obtained (designated BPA-Leu) (38) are shown in Figure 19. BPA-Leu was analyzed by LC / MS to confirm the molecular weight and to determine the purity after preparation of aqueous solutions. Briefly, the dipeptide was analyzed on a BEH C18 column (2.1 x 50 mm, Waters) maintained at 40 °C at a flow rate of 0.5 ml / min. The bound specimen was eluted with a gradient of acetonitrile-0.1% FA. The resulting purity analysis of BPA-Leu is shown in Figure 20.

[0300] Example 12: Evaluation of borylated dipeptide uptake in multiple cancer cell lines.

[0301] In this experiment, a panel of dipeptides was evaluated, where each amino acid adjacent to BPA is a putative substrate for LAT-1. The set of amino acids was selected from Leu, His and Ala, and BPA, with the goal of overcoming the shortcomings of BPA by increasing its solubility in solution, and of utilizing alternative uptake mechanisms, ultimately achieving higher boron-10 concentrations in the tumor while maintaining an estimated tumor-to-blood ratio of 3:1 or greater. Utilization of the following materials and methods was provided by the following protocol.

[0302] Indicated cancer cell lines (A) or FaDu cells (B) were harvested from culture and washed twice with PBS. Cell lines were adjusted to 2 million cells / ml in Hanks Balanced Salt Solution (HBSS). BPA or dipeptide boron compounds were added to the cells at a final concentration of 2.5 mM in HBSS, and treated cells were incubated with shaking at 37°C 5% CO2 for 2 hours. Cells were harvested by centrifugation, washed twice with cold PBS, and reconstituted in 1 ml of cold PBS. 50 μl of cell suspension was then removed, pelleted, and lysed using RIPA buffer, and protein concentration was determined using the BCA assay. The remaining 950 μl was used for boron compound uptake measurements using ICP-OES.

[0303] For this purpose, 0.5 mL of concentrated nitric acid was added to each cell pellet in a 15 mL conical tube. The tubes were capped and placed in an oven set at 80 °C for 3 h. 2 mL of water was added to each sample to bring the final volume to 2.5 mL. The boron content in each sample was then measured with an Agilent 5110 ICP-OES using an Agilent SPS4 autosampler for sample introduction.

[0304] Data was analyzed using Agilent's ICP Expert Software, version 7.4.2.10790. Boron was measured axially at a wavelength of 249.772 nm and the internal standard beryllium was measured axially at a wavelength of 313.042 nm. Beryllium was added through a Tee to the solution at a flow rate of 1:5 prior to introduction into the spray chamber. A standard curve with 1000, 100, 10, 1 and 0 ppb of boron was used to calculate the concentration of boron in each sample. Once determined, the boron measurements for each sample were normalized to the protein content and final boron uptake results were expressed as ng boron / mg protein.

[0305] As can be seen in Figure 21(A), the dipeptide BPA-BPA was taken up by several cancer cell lines, to a similar extent in the mouse tumor line CT26 and the human tumor line T98G, but slightly less than the LAT1 substrate BPA in the highly LAT1-expressing FaDu and A431 cancer cell lines. In Figure 21(B), three additional dipeptides, BPA-Ala, Ala-BPA, and Leu-BPA, were taken up by FaDu tumor cells, but also to a lesser extent than BPA.

[0306] Example 13: Evaluation of the correlation between differential uptake and relative expression of BPA and His-BPA in AsPC-1 and FaDu cell lines.

[0307] In this experiment, the differential uptake of BPA and His-BPA was evaluated in cells expressing LAT1 and PEPT1. Figure 22(A) shows the Cancer Cell Line Encyclopedia RNA expression data (RNAseq) for the large neutral amino acid transporter LAT1 (SLC7a5) and the dipeptide transporters PEPT1 (SLC15a1) and PEPT2 (SLC15a2) in the FaDu and AsPC-1 cancer cell lines. See sites.broadinstitute.org / ccle. The results show that FaDu has high expression of LAT1 but not PEPT1, whereas AsPC-1 has high expression of PEPT1 and moderate expression of LAT1. Both cell lines have low expression of PEPT2.

[0308] In another experiment, immunohistochemical analysis of LAT1 and PEPT1 protein expression in FaDu and AsPC-1 cancer cells was evaluated using the following protocol. Briefly, FaDu and ASPC-1 cell pellet samples were fixed in 10% neutral buffered formalin, processed, embedded in paraffin wax, and prepared as 4 μm sections mounted on microscope slides. After deparaffinization and rehydration, cell pellet sections were processed for antigen retrieval. Antigen epitopes of FaDu and ASPC-1 were retrieved by applying heat and pressure in a pressure cooker (Decloaking Chamber, Biocare LLC) by immersion in Diva Decloaker (pH 6.2 heat-induced epitope retrieval buffer, Biocare LLC) for PEPT1 and Borg Decloaker (pH 9.5 heat-induced epitope retrieval buffer, Biocare LLC) for LAT1 at 110°C for 15 min.

[0309] After antigen retrieval, antibodies against PEPT1 (rabbit polyclonal antibody from Sino Biological, catalog number 203418-T10) and LAT1 (rabbit monoclonal antibody EPR17573 from Abcam, catalog number ab208776) were applied to the cell pellet sections, followed by goat anti-rabbit Ig horseradish peroxidase polymer (MACH4 Universal HRP, Biocare LLC) that binds to the PEPT1 or LAT1 rabbit antibodies. The cell pellet sections were then applied with the chromogen 3,3'-diaminobenzidine (DAB), which reacts with horseradish peroxidase to produce a brown product at the site of the antigen. The stained cell pellet sections were scanned and images were taken.

[0310] The results show that the protein expression levels of LAT1 and PEPT1 correlate with the RNA data presented. FaDu shows elevated levels of staining for LAT1, but little staining for PEPT1. In contrast, ASPC-1 shows elevated levels of PEPT1 staining, but little staining for LAT1. (See FIG. 23(B)).

[0311] In another experiment, the uptake of BPA and the dipeptide His-BPA was performed in FaDu and ASPC-1 as described in Figure 21. FaDu cells expressing high levels of LAT1 took up BPA to a much greater extent than His-BPA. In contrast, His-BPA was taken up to a higher level than BPA in AsPC1 cells that highly express PEPT1. Thus, the data support the finding that borylated dipeptides can be efficiently taken up into cancers expressing PEPT1 or other dipeptide transporters. See Figure 16(C).

[0312] Example 14: Evaluation of inhibition of His-BPA uptake by Gly-SAR in PEPt1 cells.

[0313] In this experiment, uptake of BPA and the dipeptide His-BPA (each at 0.8 mM) was performed in FaDu and ASPC-1 in the presence and absence of the indicated concentrations of the PEPT1 dipeptide transporter competitive inhibitor Gly-SAR. Normalized boron uptake was determined as in FIG. 21.

[0314] The results show that Gly-SAR had a dose-dependent inhibitory effect on His-BPA uptake in ASPC-1 cells but not in FaDu cells, supporting the assertion that PEPT1 mediates higher levels of His-BPA uptake in ASPC-1 compared to FaDu cells. Furthermore, Gly-SAR had no effect on BPA uptake in either cell line, supporting the assertion that LAT1 is the main transporter for BPA uptake in both cell lines.

[0315] The resulting finding that His-BPA delivers a boron signal in PEPT1-negative FaDu cells supports the assertion that there is an alternative uptake mechanism for His-BPA in this cell line and potentially other PEPT1-low or -negative cancers. See FIG. 24.

[0316] Example 15: Evaluation of relative uptake of dipeptides in PEPT1 cells.

[0317] In this experiment, the uptake of several dipeptides and dipeptide analogs was performed in FaDu and ASPC-1 cells. Normalized boron uptake was determined as in Figure 21. The results show that all dipeptides and analogs showed higher uptake in ASPC-1 than in FaDu cells, confirming the assertion that PEPT1 is the transporter mediating most of the uptake. However, some dipeptides were still taken up, albeit to a lesser extent, in PEPT1-negative FaDu cells, further confirming the assertion that an uptake mechanism for these dipeptides exists in FaDu cells that is not PEPT1. See Figure 25.

[0318] Example 16: Evaluation of the degradation of dipeptides to BPA by HPLC.

[0319] In this experiment, to investigate whether the dipeptide is converted to BPA and then taken up via its cognate transporter, an assessment of whether the dipeptide remains intact in tissue culture medium in the presence of absence of cancer cells was performed using the following protocol. Briefly, FaDu cells were harvested and counted. Cell numbers were increased to 50 × 10 in HBSS tissue culture medium. 5 The concentration of each test article was adjusted to 1 mL / well. The cells were incubated overnight at 37°C in 5% CO2. Finally, the supernatant was aspirated and the cells were rinsed once with 1 mL of PBS. Each test article was prepared in HBSS medium at a concentration of 2.5 mM and 0.4 mL was added to the cells. After 4 hours at 37°C, the supernatant was collected and analyzed by RP HPLC using an Acquity BEH C18 column 2.1 x 50 mm (Waters) with a matching guard column equilibrated with a 2% acetonitrile in water / 0.1% formic acid mobile phase. The dipeptides were separated in a gradient of 90% acetonitrile / 10% water / 0.1% formic acid up to 20% in 6 min and identified by UV 225 nm. The mass was confirmed by an online QDA mass spectrometer. The degradation of the original dipeptide was quantified by comparison with the control (the same dipeptide incubated in the same manner in the absence of cells). The stability of BPA-BPA, Ala-BPA, and BPA-Ala was compared, and the formation of BPA was measured semiquantitatively by RP-HPLC / MS.

[0320] Results in Figure 26(A) show a representative chromatogram at 225 nm, indicating the formation of BPA (peak 1) from BPA-BPA (peak 2) upon incubation with cells for 4 hours. In the absence of cells, only trace amounts of BPA are detected. See Figure 26(B). Results in Figure 26(C) show MS confirmation of peak assignments. LC / MS confirmation of BPA: [M+H] of BPA is 210 and [M+H] of BPA-BPA is 400. Figure 26(D) shows the conversion of the three dipeptides to BPA in the presence and absence of FaDu cells. The relative conversion in the presence of cells is Ala-BPA>BPA-BPA>BPA-Ala.

[0321] Example 17: Pharmacokinetics of BPA-BPA.

[0322] In this experiment, the pharmacokinetics of BPA-BPA was evaluated using the following protocol. Briefly, 200 mg / mL of each compound was injected into the tail vein of Balb / C non-tumor-bearing male mice (5 mice per group). Blood was collected into EDTA-coated tubes at the following time points: 2, 5, 16, 30, 60, 120, and 240 min. Boron concentrations were measured using ICP OES after 1 h digestion in concentrated nitric acid. Boron concentrations were normalized to 1 mL blood and plotted using GraphPad Prizm. PK parameters (see Figure 28, table) were obtained using PK Solver ver.2.0 using compartmental analysis, with BPA-fructose used as a reference substance.

[0323] The results showed that BPA-BPA had a remarkably similar t 1 / 2The results show that BPA-BPA exhibited biphasic pharmacokinetics with a beta (i.e., elimination phase) and clearance (CL). In general, it takes longer for BPA-BPA to be removed from the system (218 min vs. 195 min for BPA). The volume of distribution at steady state (Vss) is similar to that of BPA, confirming the assertion that the dipeptide has similar blood protein binding and is easily accessible to the excretory organs. The maximum plasma concentrations are similar for both test articles. However, the total drug exposure over time (AUC from 0 to ∞) is slightly lower for BPA-BPA. See Figure 28.

[0324] Example 18: In vivo biodistribution of dipeptides using FaDu cells.

[0325] In this study, the biodistribution of various dipeptides was evaluated in vivo using the following protocol. All animal studies were performed in accordance with the "Guide for the Care and Use of the Laboratory Animals," 8 th The study was carried out in accordance with the Ed. and Animal Welfare Act (USDA). Human hypopharyngeal squamous cell carcinoma FaDu cells were maintained in DMEM supplemented with L-glutamine and 10% FBS.

[0326] Female CB17 SCID mice were inoculated with 2.5 × 10 cells mixed at a 1:1 dilution with Matrigel (Corning Life Sciences) into the right flank. 6 Subcutaneous (sc) tumors were generated by injecting 1000 x 100 cancer cells. Tumor size was determined by caliper measurement and tumor volume was calculated by the range. 2 ×length / 2 (width is the smallest dimension and length is the largest dimension). Tumors were approximately 300 mm 3The mice were allowed to grow untreated until they reached a tumor volume of 100 μL. At that time, animals were randomized and assigned to treatment groups based on tumor volume at the start of treatment to ensure that each group had similar mean tumor size and variability. Each group received a single dose of 200 mg / mg BPA or 800 mg / mg test article via tail vein injection. Injection volumes did not exceed 200 μL per mouse in accordance with veterinary guidelines. Two hours after dosing, blood was collected from the submandibular vein of each mouse into K2 EDTA-coated tubes. Mice were then humanely euthanized and tumors and organs were collected for boron analysis.

[0327] Additionally, all compounds tested were prepared as 80-100 mg / mL stocks, except for BPA, which was prepared as a 20-22 mg / mL solution in fructose. Concentrations were confirmed by ICP OES prior to the study. Blood and tissues (tumor, kidney and pancreas) were collected at the indicated times, weighed, placed in Teflon containers and digested using a CEM microwave oven. Digested tissues were analyzed by ICP OES to measure boron concentrations.

[0328] The results show that due to the much higher solubility of the four dipeptides tested, these dipeptides were administered at concentrations 4 times higher than those used for BPA-F. The amount of boron delivered to the tumor by the dipeptides at 1 hour was 2.1-2.7 times higher than the amount of boron observed in the tumors of mice administered 200 mg / kg BPA-F. Note that the highest boron uptake was observed with Ala-BPA-F (53.9 μg boron / g tumor as shown in the inset). Significantly higher amounts of boron are observed in the kidneys and pancreas of all mice administered the dipeptides compared to BPA-F. See Figure 29.

[0329] Example 19: In vivo biodistribution of His-BPA using FaDu cells.

[0330] In this study, the biodistribution of His-BPA was evaluated in vivo using the following protocol. All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals, 8 th The study was carried out in accordance with the Ed. and Animal Welfare Act (USDA). Human hypopharyngeal squamous cell carcinoma FaDu cells were maintained in DMEM supplemented with L-glutamine and 10% FBS.

[0331] Female CB17 SCID mice were inoculated with 2.5 × 10 cells mixed at a 1:1 dilution with Matrigel (Corning Life Sciences) into the right flank. 6 Subcutaneous (sc or s.c.) tumors were generated by injecting 10 ... 2 ×length / 2 (width is the smallest dimension and length is the largest dimension). Tumors were approximately 300 mm 3 The mice were allowed to grow untreated until they reached a tumor volume of 100 μL. At that time, animals were randomized and assigned to treatment groups based on tumor volume at the start of treatment to ensure that each group had similar mean tumor size and variability. Each group received a single dose of 200 mg / mg BPA or 800 mg / mg test article via tail vein injection. Injection volumes did not exceed 200 μL per mouse in accordance with veterinary guidelines. Two hours after dosing, blood was collected from the submandibular vein of each mouse into K2 EDTA-coated tubes. Mice were then humanely euthanized and tumors and organs were collected for boron analysis.

[0332] Additionally, all compounds tested were prepared as 80-100 mg / mL stocks, except for BPA, which was prepared as a 20-22 mg / mL solution in fructose. Concentrations were confirmed by ICP OES prior to the study. Blood and tissues (tumor, kidney and pancreas) were collected at the indicated times, weighed, placed in Teflon containers and digested using a CEM microwave oven. Digested tissues were analyzed by ICP OES to measure boron concentrations.

[0333] The results show that His-BPA, like the other dipeptides, could be administered at a concentration four times higher than that used for BPA-F. This was due to the higher solubility of this dipeptide. The amount of boron delivered to the tumor by this dipeptide was two times higher than the boron levels in the tumors of mice administered 200 mg / kg BPA-F. This uptake is similar to that observed for the other dipeptides tested. Significantly higher amounts of boron are observed in the kidneys and pancreas of all mice administered His-BPA compared to BPA-F. See Figure 30.

[0334] Example 20: Dose escalation of boron delivery in vivo using borylated dipeptides in multiple xenograft models.

[0335] In this experiment, FaDu and Detroit-562 (head and neck), MeWo (melanoma), HCC-1954 (breast), A549 (lung), and AsPC-1 (pancreatic) human cancer xenograft models implanted in CB17 SCID mice were treated with 200 or 400 mg / kg BPA, or with 1000 mg / kg His-BPA or BPA-BPA due to improved solubility. Tumors were harvested 120 min after treatment and boron content was determined by ICP-OES. Data are mean ± SD of 5 mice per treatment group.

[0336] The results show that both dipeptides could be administered up to 1000mg / kg compared to the maximum dose of BPA at 400mg / kg. BPA-BPA, which contains two boron atoms per molecule, administered at 1000mg / kg, was able to deliver the most boron to all xenografts tested, achieving a maximum of 60ug / g tumor in FaDu xenografts, and a minimum of over 30ug / g in Detroit-562, HCC-1954, and ASPC-1 models, and over 40ug / g tumor in MeWO and A549 models. Additionally, His-BPA administered at 1000mg / kg delivered higher boron levels than standard BPA administered at 200mg / kg, and delivered similar levels compared to the maximum, but non-precipitable, BPA dose of 400mg / kg. See Figure 31.

[0337] Example 21: Evaluation of tumor boron content of multiple dipeptides using the CT26 syngeneic colon cancer model.

[0338] In this experiment, CT26 murine syngeneic colon cancer xenograft models implanted in BALB / c mice were treated with 200 mg / kg BPA, and both His-BPA and BPA-BPA at 200 and 800 mg / kg, respectively. Tumors were harvested 120 min after treatment and boron content was determined by ICP-OES. Data are the mean ± SD of 5 mice per treatment group.

[0339] The results show that His-BPA and BPA-BPA, both dosed at 800mg / kg, delivered 25ug / g boron and 30ug / g tumor, respectively, while BPA, dosed at 200mg / kg, delivered approximately 16ug / g tumor. These results suggest that dipeptide BNCT is more effective than BPA, and optimally the dipeptide is more effective at a lower neutron dose. See Figure 32.

[0340] Example 22: BNCT studies using borylated dipeptides compared to BPA.

[0341] In this experiment, CT26 xenografts in BALB / c mice were administered 200 mg / kg BPA or, due to improved solubility, 800 mg / kg His-BPA or BPA-BPA. After a 1-h treatment period, mice were irradiated for either 12 min (33(A)) or 6 min (33(B)) in the Kyoto University Research Reactor (Kyoto, Japan)1 set at 5 megawatts. Data are the mean ± SD of 3 mice per treatment group. Tumor growth was measured for up to 22 days.

[0342] As seen in Figure 33(A), irradiation alone had a moderate antitumor growth effect, indicating that normal tissues may also be affected at this high irradiation dose. However, all three compounds, BPA, His-BPA, and BPA-BPA, had significant and similar growth inhibition and even regression when monitored for up to 22 days. Therefore, since the dipeptide delivered a higher amount of boron compared to BPA in the tumor biodistribution experiments (Figure 32), and because of the short irradiation time of 6 minutes (lower total neutron dose), the dipeptide should show a significantly higher antitumor effect than BPA.

[0343] The results in Figure 33(B) confirm this. As can be seen, irradiation alone was similar to the non-irradiated control, while BPA with irradiation mediated only a moderate tumor inhibition. However, both dipeptides mediated nearly complete tumor inhibition over the course of 22 days as monitored.

[0344] Example 23: BNCT study showing tumor regression using borylated dipeptides.

[0345] In this experiment, CT26 xenografts in BALB / c mice were administered 400 mg / kg BPA or 900 mg / kg His-BPA or BPA-BPA. After 1 h treatment time, mice were irradiated for 6 min in a Kyoto University Research Reactor (Kyoto, Japan) set at 5 megawatts. Data are the mean ± SD of 3 mice per treatment group. Tumor growth was measured until day 36 and examined for the presence of tumors on day 40. The graph presented in Figure 34(B) is an expanded version of the growth curves of the dipeptide groups presented in Figure 34(A). The BPA and control groups were sacrificed on day 18 due to excessive tumor growth.

[0346] Figure 34 shows a BNCT study comparing the effectiveness of BPA to His-BPA and BPA-BPA using a shorter 6 minute irradiation time (see Example 22). In this study, all control groups and the BPA and irradiation groups continued to grow tumors, necessitating the groups being sacrificed by day 18.

[0347] In contrast, His-BPA and BPA-BPA administered at 900 mg / kg along with irradiation showed significant tumor regression that persisted for up to 36 days of growth monitoring. Visual and histological examination of the tumor area in His-BPA-treated mice showed that the tumor tissue had disappeared and only scar tissue was found at the original tumor site, suggesting that the tumor was indeed cured after BNCT treatment. Figure 33(B).

[0348] Example 24: Human clinical trials for the treatment of human carcinomas using Bdi-AA.

[0349] Bdi-AA is synthesized according to the present invention, accumulates specifically in tumor cells, and is used to treat certain tumors as well as other immune disorders and / or other diseases. In relation to each of these indications, two clinical approaches have been successfully pursued.

[0350] I.) Adjuvant Therapy: In adjuvant therapy, patients are treated with Bdi-AA in combination with chemotherapeutic or biological agents or combinations thereof. The primary cancer target is treated and then irradiated by the addition of Bdi-AA under standard protocols. The protocol design addresses efficacy, as assessed by examples including but not limited to reduction in tumor burden of primary or metastatic lesions, extension of progression-free survival, overall survival, improvement in patient health status, disease stabilization, and the ability to reduce regular doses of standard chemotherapy and other biological agents. These dose reductions allow additional and / or prolonged treatment by reducing dose-related toxicity of chemotherapeutic or biological agents.

[0351] II.) Monotherapy: In relation to the use of Bdi-AA in tumor monotherapy, Bdi-AA is administered to patients without chemotherapy or drugs or biological agents. In one embodiment, monotherapy is clinically implemented in terminal cancer patients with widespread metastatic disease. Protocol design addresses efficacy, which is evaluated by examples including, but not limited to, reduction in tumor burden of primary or metastatic lesions, extension of progression-free survival, overall survival, improvement of patient health status, disease stabilization, and ability to reduce the usual dose of standard chemotherapy and other biological agents.

[0352] Dosage Dosage regimens can be adjusted to provide the optimum desired response. For example, a single Bdi-AA injection can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. As used herein, "dosage unit form" refers to a physically discrete unit suitable as a unitary dosage for the mammalian subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the dosage unit form of the present invention are determined by and directly depend on (a) the unique characteristics of Bdi-AA, the individual mechanism of the irradiation mechanism (reactor) and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the technology of compounding such compounds for the treatment of susceptibility in an individual.

[0353] Clinical Development Plan (CDP) CDP will track and develop the treatment of cancer and / or immune disorders using the Bdi-AA of the present disclosure, which will then be irradiated using neutron capture therapy in conjunction with adjuvant or monotherapy.The study will first demonstrate safety, and then confirm efficacy in repeated administration.The study will be open-label and will compare standard treatment plus Bdi-AA, which will then be irradiated using boron neutron capture therapy, with standard chemotherapy.As will be understood, one non-limiting criterion that can be utilized in conjunction with patient enrollment is the concentration of Bdi-AA in tumors, as determined by standard detection methods known in the art.

[0354] The present invention should not be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any functional equivalents are within the scope of the present invention. Various modifications to the models, methods, and lifecycle methodologies of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and teachings, and are also intended to fall within the scope of the present invention. Such modifications or other embodiments can be made without departing from the true scope and spirit of the invention.

Table 1

Claims

1. The chemical structure below: 【Chemical 77】 A composition comprising: where: A is H, an amino acid, or a borylated amino acid; E is CO ２ H, B(OH) ２ , CO-amino acid, or CO-borylated amino acid, CO-NHB １２ H １１ and X is H, B(OH) ２ , B.F. ３ K, or B (OR) ２ or can be present in the 2-, 3-, or 4-positions, composition.

2. The chemical structure below: 【Transformation 78】 A composition comprising: where: A is H, an amino acid, or a borylated amino acid; E is CO ２ H, B(OH) ２ , CO-amino acid, or CO-borylated amino acid, CO-NHB １２ H １１ and X is H, B(OH) ２ , B.F. ３ K, or B (OR) ２ or can be in the second or third position, composition.

3. The chemical structure below: 【Transformation 79】 A composition comprising: where: A is H, an amino acid, or a borylated amino acid; E is CO ２ H, B(OH) ２ , CO-amino acid, or CO-borylated amino acid, CO-NHB １２ H １１ and X is H, B(OH) ２ , B.F. ３ K, or B (OR) ２ or can be present at position 2, 4, 5, 6, or 7, composition.

4. A kit comprising the composition of claim 1.

5. A kit comprising the composition of claim 2.

6. A kit comprising the composition of claim 3.

7. A method for producing the composition of claim 1.

8. A method for producing the composition of claim 2.

9. A method for producing the composition of claim 3.

10. A unit dosage form comprising the composition of claim 1.

11. A unit dosage form comprising the composition of claim 2.

12. A unit dosage form comprising the composition of claim 3.

13. 1. A borylated dipeptide amino acid (Bdi-AA) composition for use in a method of practicing neutron capture therapy in the treatment of human cancer, said method comprising: a. synthesizing a human unit dose of said Bdi-AA composition; b. injecting the Bdi-AA into a tumor, thereby causing the Bdi-AA to accumulate intracellularly; and c. Irradiating the Bdi-AA with neutrons A composition comprising:

14. 14. The Bdi-AA composition of claim 13, wherein the Bdi-AA composition is selected from the group consisting of the compositions of claims 1, 2, and 3.

15. 14. The Bdi-AA composition of claim 13, wherein said neutron capture therapy is boron neutron capture therapy.

16. 14. The Bdi-AA composition of claim 13, wherein said neutron capture therapy is proton-boron fusion therapy.

17. 16. The Bdi-AA composition of claim 15, wherein said irradiation comprises epithermal neutrons.

18. 14. The Bdi-AA composition of claim 13, wherein the cancer is colon cancer.

19. 14. The Bdi-AA composition of claim 13, wherein the cancer is head and neck cancer.

20. The Bdi-AA composition of claim 13, wherein the cancer is pancreatic cancer.

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